-

The cell culture laboratoryCAROLINE B. WIGLEY

1. Introduction-animal cell culture: past,present, and future

The history of animal cell culture dates from the turn of the twentleth century(fable /), with Ross Harrison's demonstration that explants of neural tissuefrom frog embryos, sealed into a chamber containing frog lymph, generatedflbres which extended into the lymph clot. He thus showed, for the first time,thnt nerve axons are formed by the outgrowth ofprocesses from the neuronalcell body. These processes lengthen as a result of the amoeboid movement ofthcir distal tip, now called the grgwth cone., This observation finally resolvedone of the long-standing controversies in neurobiology, showing clearly thatcuch nerve fibre is the outgrowth of a single nerve cell.

Even earlier than this, mammalian cells (bull spermatozoa) had been frozenand stored for extended periods and recovered in a viable state for artificiallnsemination and selective breeding of cattle. This technology was to provelmportant once the first continuous cell lines were.developed in the 1940s byllurle and others, who were thus able to store cells indefinitely in liquidttltrogen; at -L96'C there is imperceptible deterioration in cell viability andcclls can be released from 'suspended animation' on thawing and reculturing.As early as 1885, Roux had succeeded in maintaining embryonic chick cellsalivc in a saline solution outside the body for short lengths of time. Althoughllris cannot be described as tissue culture, Roux's experiments opened thewuy for Harrison's pioneering work, where cells were not only kept alive, butglew as they would do in vivo.

lly the early 1970s, methods were being developed for the growth ofrpecific cell types in chemically defined media. In particular, Gordon Sato andlris colleagues published a series of papers on the specific requirements ofdll'lbrent cell types for protein growth factors, attachment factors such as highrnolccular weight glycoproteins of the extracellular matrix, and hormonesruch as insulin or the insulin-like growth factors. The mixture of supplementsrcquired for the serum-free culture of neural cells, defined by Bottenstein andSrto in 1979 (10), still forms the basis of many serum-free supplements for awidc variety of ccll types. Most of the additives in these supplements had

rCoroline B. WigJey

otherwise been provided by ilr-defined and variabre biologicar fluid mediumsupplements such as serum, which is stilr a comp"""ir-.t'.any routineculture media today.over the next decade, tissue culture became, as well as an experimentalresearch field in its own right, a technorogy useo uy marrf biotogists andbiotechnologists in the newlyistabrished fieldif m"l;";iili;i;gy. Hybridomatechnology alrowed the mass production of monoclonar antibodies from ceilculture medium; viral vaccine production depended on the mass culture ofhost cells; recombinant DNA technology made use of curtured celrs, either asa source of

'RNA or gene sequence or as an expression vector for recom-binant DNA. In many areas ofrbiomedicar research, tissue curture methodswere developed which replaced much of the former n""J io, unirnal experi_mentation. For instance, in-vitro pharmacotoxicology became an establisheddiscipline for the investigation or drug activities "*iir," J"rig"

"f new formsof therapy.

A recent development ig the cet curture field which points the way forfuture progress, has been tiie introduction of methods tor nisiotypic culture,using filter well inserts in culture vessels. This allows for the co-culture of twoor more purified and defined populations of cells, ,"purut"Jurr=t in the samemedium so that interactions -"diut"d by sorubre factors can occur. Forexample, the expression of differentiated claracteristi". noio"o'onstrated bya particular celr type in.isoration may be induced r, rri*rvpi. culture. It ishoped that such technology wilr furiher our understanding of the complexcellular interactions that oiiur within tissues in vivo,ro, orr] Juring develop_ment, but also those which regulate tissue homeostasis and normal function-ing throughout life. Many diseise processes are thought to invorve a perturba_tion of cellular interactions (cancer is an obviour""*u-pt"j and the newmethods now becoming availaLle for the study of cellular interactions in vitrowill undoubtedly lead to progress in researctrin;; il;;;ii.,iuii"r""t. *ti"r,cause or contribute to disease processes.In the biomedicar field, theri are hopes that tissue culture technorogy wilrcontribute more and more to clinicaf treatments involving transprants ofvarious kinds. cultured skin ceils are already in use

", *o; or even assuperior wound dressings in patients with burns and .kin".rtcers; curturedurothelium has been used to correct congenital defects in the urogenitarsystem; transplanted ceils carrying geneticaily engineered oNe ,"qu".r"",may be used in the future to correct Jefects in aiuuEti. puilrrt.,'puti"nts withcystic fibrosis or haemophilia, parkinsonian patients, #;-;;;some of therare enzyme deficiency syndromes. There aie likely to be a ,r,.iob", of situa_tions where it is preferable to correct a defective r.in"tion ,niu ffiunt"d cellsrather than by conventionar.drug therapy. Even so, to, -ori situations,therapy will stlll involve administlring oiug, conventiona[y. In the case ofcomplcx protcln 'r hormone therapy] these-drugs wifl increasingry be pro-duced from lurge'ncure ccil curtures, .ittrr or the appropriate ceri type or of

1: The cell culture loborotory

Trble 1. The early years of cell and tissue culture

Lrto 19th century Methods established for the cryopreservation of semen for theaalcctive breeding of livestock for the farming industrytl07 Ross Harrison (1) published experiments showing frog embryo nerve fibregrowth in vitrolll2 Alexis Carrel (2) cultured connective tissue cells for extended periods andlhowed heart muscle tissue contractility over 2-3 monthstlaS Katherine Sanford et al. l3l were the first to clone-from L-cellsll02 George GeV et al. (4) established HeLa from a cervical carcinoma-the firsthuman cell linel164 Abercrombie and Heaysman (5) observed contact inhibition betweenf lbroblasts-the beginnings of quantitative cell culture experimentationttl6 Harry Eagle (6) and, later, others developed defined media and described6tt6chment factors and feeder layersll0l Hayflick and Moorhead (7) described the finite lifespan of normal human diploidosllrIt6:l Buonassisi et al. l8l published methods for maintaining differentiated cells (oflumour origin)llt8 David Yaffe (9) studied the differentiation of normal myoblasts in vitro

eclls cxpressing recombinant genes, in order to avoid some of the drawbacksol' microbial expression vectors. Biotechnology is still in its infancy and willrurrdoubtedly develop rapidly in the years running up to the centenary, in2(107, of tissue culture's first appearance as a new methodology.

2, The laboratory2.1 Design concepts and layout't'issue culture laboratories differ from general purpose biomedical researchInlxrratories in that their most important function is to allow the sterilehnndling of cultured cells. It is practically impossible to work in a totallygcrrn-free environment, so that provision must be made to ensure that culturesslrrl culture media are maintained free from contaminating micro-organisms.'l'ltcsc will flourish in the cell culture environment and will rapidly overgrow,arrtl in many cases kill, the animal cells by release of toxins or depletion of thernrlrient medium.

ln almost all modern laboratories, sterile handling of cell cultures is carriedortl in laminar flow cabinets or 'hoods', where only the operator's arms enterllrc stcrile work area. All other areas in the laboratory must be kept scrupu-krusly clean, with surfaces kept clear of unnecessary clutter and swabbedtkrwn with an antiseptic cleansing solution at regular intervals. In addition tolhe sterile handling facility, all tissue culture laboratories must incorporaterurcus for a variety of other activities; these may be in one room or arranged as

7Coroline B. Wigley

a suite of rooms according to the scale of the operation, available space and,of course, budget.

The sterile handling area itself should be positioned so that there is minimalmovement of people past or through the clean area. There should be adequateseparation of the clean area for sterile handling of cells from the u.ei fo,'dirty' operations, including disposal of used culture vessels and a washing-upfacility for re-usable

.qlalsware and equipment. In between, at increa-singdistance from the sterile handling area to produce a ,sterility gradient' withinthe laboratory, should be the other facilities essential to cet'i culture work.These are, in order

(u) laminar flow cabinets(b) incubators (CO2 and, if required, dry, ungassed)(") storage space for sterile equipment and solutions(d) instrument and equipment benches(") media fridges (sterile working solutions only)(f) fud,ezer for culture reagents requiring storage at -20"C or below(g) pteparation area for media and other solutions(h) general cold storage facility for chemicals and non-sterile reagents

Where possible, in a separate room or rooms:

(i) liquid nitrogen freezers for frozen cell stocks(j) general preparation bench(k) storage area for unopened plasticware(l) sterilization oven and autoclave(m) drying oven(n) water purification system

(o) washing-up area with sinks, soak-tanks, pipette washer, and automaticwashing machine

Plans of two possible arrangements are given in Figures I and 2. The first is asingle, self-contained tissue culture laboratory whilst the second is for a biggergroup, with a clean culture laboratory and a separate, adjacent, washin!--upand sterilization facility.

In addition to the essential facilities outlined above, several desirablefeatures of a cell culture laboratory suite should be considered, according tothe type and scale of the work to be carried out:

(a) a warm room at 37"cfor large-scale cultures in e.g. roller bottles, time-lapse photographic or video equipment, etc.

(b) a laminar flow cabinet away from the sterile handling area for the initialdissection of tissucs fur primary culture work

7: The cell culture loborotory

Glass-fronted cupboards for sterile glassware above b€nch

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COZ incubators

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llgure 1. Self-contained tissue culture laboratory, suitable for two or three people.

(c)rrnelectroniccellcounterforlaboratorieshandlinglargenumbersofcelllines or for those involved in growth kinetics research

(tl)airconditioning(thiswillbenecessaryfor'laboratoriesinwarmclimatesand where many heat-generating items of equipment' e'g' laminar flow

cabinets, are concentrui"d irrto a small space. Incubators may overheat if

theambienttemperatureistooclosetotheiroperatingtemperature)(c) a cold room at 4'C for storage of media and for liquid nitrogen

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(l) some form of containment facility for incoming cell lines and potentially

hazardous material

A containment facility is advisable for laboratories which receive biological

ilnrrrples, including ir 1i""., from elsewhere' Any potentially pathogenic

tttutcrialwillalsoneedtobehandledhereaccordingtoregulationsforthel,,,rr,rd they pose. A containment facility should comprise a sep-arate room'

without direct access to trr" r"st of the cuiture suite, containing a laminar flow

Iuxrd of the appropriui".ut"goty, e'g' class II' a dedicated incubator which

c'n occommoOut" ."ufuti", gi.ruUt" iontainers to isolate cells in quarantine-,

ilncl flll back_up "q;;;;;;;eeded to handle the cells (centrifuge, small

,l"irig;roi.rr, etc.), ceh lines new to the laboratory, unless obtained directly

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from a reputable cell culture collection which guarantees them free fromg()ntaminants, should be isolated and handled as potential carriers of myco-plasma, for instance, until shown to be clear. For further treatment of thiswhole topic, see Chapter 8.

t,2 Services'l'lruue culture laboratories have some of their service requirements in com-Itton with more general laboratories, for example, water supply to sinks, gas

lnpr lbr Bunsen burners, and above and below bench power sockets. OtherFrvices are less widely used outside the culture laboratory.

1,2.1 cozMost incubators for cell culture work depend on a supply of CO2 to maintainn llxcd CO2 tension in the humidified air within the incubator (see below).lrlenlly, CO2 cylinders should be outside the main cell culture laboratory andlltc gas piped through, to maintain cleanliness in the sterile handling area.lllnce this may not always be possible, Figures I and 2 show cylinders along-rldc the incubators. A bank of CO2 cylinders should be secured to a rack

;tlaeed near the incubators and the gas fed via a reduction valve on theeylinder head, through pressure-tubing to the incubator intake port.

( irntrol of the CO2 level in gassed incubators is achieved through theIttr:lusion of CO2 monitors, which increase the initial costs of an incubator butrepny this with lower CO2 usage and better control and reliability. As long as

llte incubator remains closed, usage is minimal and, in the event of a cylinderrunning out unexpectedly, the gas phase remains within tolerable limits forquile a long time. It is a useful precaution to have more than one cylinder onllrrc, with an automatic switch-over unit, operated by the fall in pressure as alylirrder empties.

'l'hc effect of a leak in the CO2 system is worth consideration. If this is large:'rrorrgh and is left unchecked over a holiday period, for example, everythingr'orrnected to that supply will be affected. Thus it may be worth having twotorrrpletely separate supplies, each to a different bank of incubators. Valuablerloek cultures can then be duplicated in independently supplied incubators tosvoid their complete loss in the event of a major leak in one of the CO2lines.lrrt'vcntion is always better than cure, however, and a little care in using thecorrect pressure-tubing and securing and testing all connections should avoidrrrort problems. A wash-bottle filled with soap solution (household washing-rrp licluid is ideal), squirted around connections, makes a cheap and effectivelenk rletector. In particular, newly connected cylinders should be tested in thiswnyl dirt on either face of the connection can cause significant leakage.

2.2.2 tJltrapure waterWutcr is used in the culture laboratory for several different purposes whichtlsnand a particularly high level of purity. Water is used:

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1: The cell culture Ioborotory

inlcrvals e.g. monthly, with a proprietary solution containing formaldehyde,thc nrcmbrane will have a lifespan of several years. There is an automated€lerrrrsing cycle on most models e.g. Fisons Fistreem RO 60, which requiresrRlttirnum effort and cost to run. The initial cost is soon repaid in muchfetlrrccd power consumption, in the saving on replacement of elements etc.,gntl in time spent on cleaning and descaling a glass still.

3, Equipping the laboratoryg,l Maior items of equipment and instrumentation9,1.1 Laminar flow hoodsIt wus common practice in the early days of tissue culture to carry out sterileploccdures under a sheet of glass or Perspex, clamped horizontally about0,,1 ttt above a bench in a still-air room. Bench and glass sheet were swabbeddrrwrr with 70o/o ethanol and, since most microbial contamination falls fromgltovc or is wafted into an open, sterile vessel from a non-sterile source,Gf,lclul manipulation of sterile material under the plate glass was often suc-

€elslul. Nowadays, however, most tissue culture laboratories rely on the useuf lrrminar flow hoods for all sterile handling work. The alternative is torupply whole rooms or a suite of individual cubicles with sterile-filtered air.T'ltc rooms are kept under slight positive pressure to ensure that non-sterilealt' is not drawn in. This arrangement may be necessary for some large-scaleptoccdures where cost is not a major consideration.

t,rrrninar flow hoods, then, are widely used in cell culture laboratories.Tltcy operate by filter-sterilizing the air taken in, so excluding particleslltcluding bacterial and fungal organisms. Air thus sterilized then passes in a

verlical downflow on to the work surface. Some other types of cabinetpt'rltluce a laminar flow of sterile air which is directed horizontally towards the.tl)clator, but these do not offer protection to the operator from the potentialIrnzrrrds of some cultured cells.

l,irminar flow cabinets are classified according to the degree of safety theyol'l'cl the user and it will depend on the work to be undertaken, which is mostappropriate (see Chapter 2). The principles behind the design of most laminarflrrw cabinets are shown in Figures 3 and 4. Figure 3 represents a standardlattrinar downflow cabinet, and Figure 4 shows a cabinet with Class II safetyrlrrrrtlards, suitable for handling primate or human material, some virallylitlcctcd cells, certain carcinogenic reagents, etc. Both types of cabinet pro-vitlc similar control of air-borne microbial contamination. In addition, the('lrrss II cabinet offers greater protection to the operator by:

(rr) provision of a front window (screen) with a lower edge which givesminimum turbulence to air drawn in from outside, whilst allowing adequateflccess for the operator's arms. The air taken in at the front acts as a safetycurtain, prevcnting acrosols of potentially hazardous material from

o for the final rinsing steps in washing up re-usable glassware which will beused for the preparation, storage, a"ndianOting of culture mediaFor a small laboratory with limited resources, it may be more cost_effective tobuv readv-prepared,

,rt:til:, ri"gt"-.tt"ig;h'mediJ il ilil;ents from asupplier. It wil be advisabre ats]o to uuy"rrttrup.rr" ,nui"i-iJriil o""urionatpreparation of solutions of reagents which cannot be purchased reacy to use.In this situation' water from u Jonu"ntioiul double distillation apparatus maybe adequate for glasswa^re rinsing urrO otn", general purposes.Ideally, a water purification slstem situut"o in the washing-up area canserve as a continuous lupnlv.or-ultrapure water for utt prrrpo."slirris witt atsohelp in the long-term tominimize the costs or media iro oti"i sierile, tissueculture reagents. Double glass distillation i, adequate ro, tt

" ni.i".tage in thepurification of tap water but tras.oow-l-u-rgely,been superseded by reverseotTo:t (Ro)' This process typically ,"-ou", about 9g% of water contamin-ants' Tap water fed to an Ro unit stroun nrst be passed ,rri."gr, " "rnventionalwater softener cartridge in hard water areas, to protect the Ro ;;#;;.The ourput from the- Ro unit

"u.t u" ,tr"o (ror a rimiteJ time) in anintermediate tank which shourd b; ;;;; with hydrophobic srerilizationfilters on the air inlets.

water from this tank can be used directly to feed the second-stage, urtra-purification system, which-is compriseo of a series ,f ;;;ilg"s for ion-exchange and the remoya!_or organic ;dT;;** Tr"iiir'"1;"" throughcarbon and other types of nrter. daier puri,y is monit#ed;rh "

resistivitymeter which should reach about lg Mb/cm. IJltrapurification systems aresupplied by companies such as Millipore (Milli_e ,Vrr"_lo_ i.isons (Nano_pure system)' Finally, ultrapure water is p)ssed tr,rougf, ;',ni"ropor" filter toexclude residual particulate matter, ln"i fulng micro_organisms.In the choice of water urtrapurihcation systems, the rate of pure watergeneration is important, particularly if rarge volumes are needed fQr awashing machine, for instance. vou Jrouti arso be sure that the unit wirl run'l'l'tap water at hieh pressure and will not require an intermeaiate storagelircility' 'l'hc steririiation cycre rn."ro"u" .lm-iautomatic and easy to operate(ree bclow), rrherwise its impreme"i,i." *il b".b;;;;iiilotn". auy,by buny lechniciuns and membra"" ;;;;;" may result.waror sh.urcr rro cotected uno uuto.iuuEl imm"oiatery for ster'e use. Theirrtcrnrcttlarc Hro*rgc t,nk can ,'- L;;;;;ed to feed; il;;;;r" washingnr'chlrrc [rr'rhe rrrrrrr,'trisritcd water'rinrl

"y.r"* (see berow). Tanks andIuhlng llurultl bc ,liglrt-tighr'

fo prcvenr-"ig"l growth.Alth,ugh the c'rir ,r' tirc Ro unit i,, o ,,"uu.tuntiar initiar outray, providedlltrt c're is r'ken r'ereurrse rrrtr slcririze the unit and its ,"rt riin'" at regurarI

Coroline B. WigJey 7: The cell culture laborotory

Air exhaust through outlet filter

llgure 4. Class ll laminar flow cabinet, for sterile work requiring higher operator safetyStanrlurds than provided by an open-fronted sterile work cabinet lsee Figure 3l..

{c) rrn air flow monitor and alarm system which warns when the rate fallsbclow or rises above safe levels

'l'ht:se and other types of cabinet are discussed further in Chapter 2.All cabinets should be inspected regularly according to current regulations

gnrl rnnintained through good laboratory practice; in particular, all spillagesrltorrltl be cleared up immediately and the surfaces wiped down with 70%ellrirrrol (it is useful to keep a wash-bottle of this close at hand). The cabinet:trurrld also be cleaned out completely at regular intervals, by removing theelelneltuble work surface.

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reaehing the operator. In an empty hood, this curtain of air is drawn im-medlately to exhaust, sodoes noi .'o-pro-ise the steriliffi any materialinnicle tlrc working area. However, the operator,s arms disturb the air flowand eaune eddies. N.on-sterire air may then enter the cabinet and it is wiseto avoiel uairrg the fr,nt r0 cm of work surface for sterile rrananng.(b) exhauetlng air lrowing ncross the curtures to the raboratory only afterpessing. it througrr sn uutrer firter; most i, ,";y;i;l ;;;;ii'e cauinet asnhown in the dlcgram

Main H.E.P.A (high efficienCyparticulate air) filter

Laminar downflow of sterile air

10 l7

Coroline B. Wigley

when choosing a laminar flow cabinet (or hood), a number of factorsshould be taken into consideration. The first, of course, is that the hoodshould be of the appropriate class for the work which will be carried out in it(for further discussion, see chapter 2). For a general purpose tissue culturelaboratory, a class II hood is generally the most upprop.iute, providing adegree of protection to both worker and cultures, so plrmitting tire handlingof all but the most hazardous cell lines. Such hoods aie availab-ie in a variet|of sizes (usually based on nominal width) and can vent either to the labora-tory or can be ducted to the outside. Ducting to the outside need only beconsidered if there is good reason to do so, e.g. regular use of higher risk celllines or the frequent need to fumigate the hood. Most tissue

"iltur" labor-

atories will find a 4 feet (120 cm) wide hood adequate for most purposes,although certain procedures, particularry at large- oipilot-scal", -uy be moreeasily carried out in one that is 6 feet (1g0 cm) wide. It should be noted thatthe working height within such hoods varies between manufacturers (animportant thing to consider if frequently dealing with large bottles or spinnerflasks) and the width is only a very rough guidi to the riseful working area.The actual internal dimensions of two class II hoods from different manufac-turers, both nominally 720 cm wide, are given below.

1; The cell culture laboratory

I lslllPcratureI gtu phase

I hurrridity

The tcmperature required for most mammalian cells to grow optimally is37"(', lncubators should be set half a degree below this for safety and a cut-EBt ogrcrated at 39oC, above which cells rapidly die. Avian cells prefer a

dlghtly higher temperature, reflecting the higher body temperature of birds,*hlh,. rnammalian skin cells, for instance, prefer a slightly lower temperature.Eellr liom cold-blooded animals are generally incubated at a constantt€m;trrature within their normal exposure range.

Mrlnl cell culture work requires that cells are incubated in an atmosphere*hlclr contains an elevated CO2 tension. Culture medium is thus maintained att phyriologicalpH (7 .2-7.4) by the equilibrium established between dissolved€O1 nrrd the bicarbonate buffer in the medium (see Chapter 3). The gas phase

€€n hc ldjusted in culture flasks by introducing the correct gas mixture fromE eylirrrlcr via a plugged pipette and then closing the cap tightly. Alternatively,Ep€tt vcssels can be incubated in a sealable chamber, which can be humidified,ga$crl, und closed. Several suppliers market such chambers e.g. ICN Biomedi-€Blr, lrut a good workshop should be able to produce an equally effectivevcrrhrrr l'rom Perspex, with a removable front fitted with a silicone gasket andbUtterlly screws to rnake a tight seal when assembled. Inlet and outlet portsWltlr tubing that can be clamped permit the introduction of a gas mixture. Such€hantbcrs then allow the use of less expensive, dry-air incubators.

Monl tissue culture laboratories, however, use CO2 incubators as the mosteeGuullc and reliable wayof providing the right incubation conditionsfor cells inV€tttctl vcssels. Indeed, for certain critical, sensitive work, a CO2 incubator maybe eencntial. Even in tightly capped flasks, gas-phase leaks can occur. Whilst inBthet' irrcubators this can lead to a (sometimes devastating) rise in pH of themedirrrn due to loss of CO2, this is not a problem in a CO2 incubator.

('( )., incubators can regulate the CO2 tension in a number of ways. In older-rlylc irrcubators, a constant flow of a CO2lair mixture was utilized, with eachgar lreirrg supplied separately from a diff€rent cylinder and mixed accordingto rrl proportions by gas burettes. The major drawbacks to this arrangement$ert' lhc cxtravagant use of CO2 and the likelihood that, if the CO, ran outunexpcctcdly, the air supply on its own would rapidly flush out the incubatorarrrl rrllow the pH of media to rise.

'l'lrc rrtust efficient and widely used method involves the use of an instru-mettl wlrich measures the CO2 level in the incubator and activates a valvetu rltrrw on pure CO2 from a cylinder, whenever the gas phase falls below aael vuluc.'l'his is generally 5%, although media containing high levels ofbh,allrolrute, such as Dulbecco's modified Eagle's medium (DMEM), particu-let'ly wlren used for serum-free cultures, should be incubated in a lOY" CO,Btntorltlture (see Chapter 3). Simple CO2 calibration devices, e.g. the

Width (cm)Depth (cm)Height (cm)

Manufacturer A720.559.582.5

Manufacturer B105.550.068.5

clearly, the hood from manufacturer A is much the larger, with a workingarea 36o/" greater and a working volume 640/o greater than that from manu-facturer B.

Other factors which may be considered are listed below.(a) Does the hood require an internal power point and/or gas tap? (These can

be fitted on request by most manufacturirs if not s.rppli"d^u. standard.)(b) Is the inside of the hood easy to clean? There should be no difficult,

inaccessible spots and the work surface should be easy to remove andreplace.

(c) will it be necessary to open the front of the hood regularly to introducelarge vessels? If so, the front glass, once opened, stroua remain open byitsell'so that both of the worker's hands are free to handle the vessel.

(tl) woulcl it bc useful for the height of the working surface of the hood to berudjustnhle? lf so, some manufacturers can supply height-adjustablenlrrnrh,

11.1.2 Incubulorallrcubatorn lirr eell cullurc work shuuld provide an,environment in which thelirllowing nro contrullod:

t2 13

Coroline B. Wigley

Fyrite test kit marketed by Shawcity Ltd, can be purchased to check themonitor readings, but for most purposes, the colour of medium in a dishwithout cells is an accurate guide to the experienced worker.

Humidity is maintained by including a water tray in the bottom of theincubator over which the air is circulated (most incubators have fans to ensureeven temperature and humidity throughout). Water trays should be pre-vented from harbouring micro-organisms by the inclusion of a non-volatilecytostatic reagent such as thimerosal (but not azide, which reacts with metals)or a low concentration (1%) of a disinfectant detergent such as Roccal,supplied by Sterling Medicare. The water level should be topped up regularly(using deionized or RO water) and condensation aspirated from other areasof the incubator base. There is, nevertheless, a potential risk offungal growthin particular, in humidified incubators. The interior should therefore becleaned out and wiped over with antiseptic at regular intervals; it is importantto check when buying a new incubator that the interior can be dismantled toallow adequate cleaning. LEEC Ltd make simple, reliable, and easily cleanedCO2 incubators at the lower end of the price range.

When choosing an incubator, it is nevertheless worth considering whetherthe extra expense of a model with an automatic sterilization cycle (e.g.Heraeus Cytoperm), is justified. This may be a distinct advantage if the tissueculture laboratory has a high number of trainees or where there are otherfactors which make contamination more likely. If the whole culture labora-tory is to be fumigated periodically, care should also be taken that the CO2monitor can be removed from the incubator or is of a type that will not bedamaged by aldehyde fumigants.

For special applications, CO2 incubators are available in which the partialpressure of oxygen can also be controlled. As well as CO2, such incubatorsrequire an oxygen and nitrogen supply. There are also CO2 incubators thatcan be used in high ambient temperature conditions (e.g. laboratories withoutair-conditioning or that are constantly exposed to the sun or other heatsources). These use heating or cooling, as necessary, to maintain their setincubation temperature.

3.1..3 CentrifugeThe main use of a centrifuge in a tissue culture laboratory is to spin down cellsduring tissue disaggregation or in harvesting cell lines for subculture, freez-ing, analysis, etc. A very simple benchtop centrifuge which will reach 80-lfi) g, preferably with a variable braking system, is generally all that isneeded. Various considerations may indicate the need for a more sophisti-cated piece of equipment. The volumes of cell suspensions to be handled in asingle opcration will dictate the bucket size and adaptability needed, forinstancc the capucity to take four swing-out buckets each holding five 50 mltubes in onc run, tbllowed by n mixture of 15 ml and 50 ml tubes. If primaryculturcg urc antlcipnted, il ntuy bc an advantage to have u ctmlcd centrifuge

1: The cell culture loborotory

Wlth a timer for long runs with sensitive cells which have been exposed to

ffJteotytic .nry-"r.hor some laboratories, a benchtop microfuge is a useful

lgai,iun for high-speed centrifugation of small volumes of reagents which

m€y gcnerate Jprecipitate' e.g. after thawing from the freezer'

9,1,4 Microscope

An esscntial instrument for any culture laboratory is a good quality inverted

mia,.,r.op", preferably with phase-contrast optics and a photographic facility'

b-e!l nlorittoiogy-ttte degrei of spreading, granularity, membrane blebbing,

t-G pr,rpfrtion"tf multinricleates,-vacuolation, and so on-should be moni-

ior*.f ,"g"furly for signs of stress in cells. Cell morphology is a sensitive

lndh,lrtor of problems with culture conditions'

Eilrty signs of microbial contamination can also be detected with a good

phArc contrast microscope. Regular checking of cultures under the micro-

leup. .un help to avoid catast.optric losses of irreplaceable material by allow-

ing'n proUte- to b" noticed at in early stage. Also, cells which are used for

tfperitn"nts in a less than healthy state may give variable or erroneous

ieiutr*. When choosing a microscope, select the long or extra-long working

dktnlrcc condenser so i-hat flasks and even roller bottles can be viewed' There

ir urur'lly no need for objectives above 20x; their depth of field is often too

Ittw 16 ,ibtui.r a sharp image of all but the very flattest cells. A_ good, low-

fu**r, wide-field oblective, e.g. the Nikon 4x, is very useful for scanning

Lulttrrcs for foci or colonies, for instance'

8,1,6 l'ridges and freezers

FEI rnost laboratories, domestic larder-type fridges (with no ice box) are

ederguate. Media storage requires considerable space and i1 -uy be more

ggxyl:nient to store ,rnop"n"d bottles in a nearby cold room, if available' The

irldgc in the tissue culiure laboratory can then be reserved for media in

U,Uricrrt use, with each opened bottle designated for one individual's work

wltlr orrc cell line. Ideally, separate fridges should be used for sterile culture

lnerlirr lnd for non-sterile solutions, chemical stocks, etc'

lircczers at -20"C and, ideally, -70"C, will be needed for storage of sera,

lriluli0ns, and reagents which ire unstable at higher temperatures' At the

lowt r, tcmperature, sera and proteins such as collagenase, which is prone to

tlegrrrtlation even at -20oC, can be stored for extended periods. Some of

llreEc lower temperature freezers are available with liquid co2 or liquid

iitt,,,1,"n back-up facilities that permit temperature to, be maintained even in

lhe rvc,nt of an eiectrical supply or compressor failure. Liquid nitrogen freezers

f,r lrrrg-term cryopreservition of cells will be considered in Chapter 4.

S,l.ll Miscellaneous small items of equipment

A wltcr bath, set at 36.5 oC, will be needed to warm media etc. before use, to

thaw l'rozen aliquots of reagents or cells, and to carry out enzyme incubations.

74 16

Coroline B. Wigley

Care should be taken to change the water regularly (e.g. at weekly intervals)and to add a cytostatic reagent such as thimerosal (but not azide) to preventmicrobial growth" A submersible magnetic stirrer is a useful accessory. Someworkers prefer not to use water baths because of a perceived risk of contami-nation from the water and associated aerosols. In this case a small dryincubator at the same temperature fulfils a similar function but has thedisadvantage that heat dispersal is less efficient.

In the area for preparation of reagents for culture use, there will need to bea balance, a pH meter and one or more magnetic stirrers. The balance shouldbe capable of weighing accurately in the milligram range. Many reagents forculture work are active in the microgram or nanogram range and stocksolutions are usually prepared at 100-1000x. An osmometer is a useful, butnot essential, piece of equipment, especially if media are prepared frompowder or basic ingredients or if a number of additives are used whichwill affect osmolality. The Fiske One-Ten osmometer supplied by AstellScientific, for example, measures small volume samples (10 pl) and is quickand easy to use.

Spent medium can be drawn off into an aspirator jar, most convenientlysituated underneath the laminar flow hood, with a pump to draw a vacuum.Figure 5 shows such an assembly, which can be adapted to serve two or threelaminar flow cabinets by using a two- or three-way connection on the aspir-ator line and an in-line valve to close off lines not in use. A simple diaphragmpump will pull a vacuum sufficient for one aspirator line but a more powerfulpump, such as an oil vane vacuum pump, may be needed if two or more linesare open simultaneously.

The aspirator jar should contain a small amount of detergent such as Decon(Decon Laboratories Ltd), and a small volume of Chloros (hypochlorite)

DisposablePasleur pipette

Flgutr l, Arpltator lrr mrrmbly for withdrewing spent medlum.

10

1: The cell culture loborotorY

l€lulion, kept in a wash-bottle standing on a plastic surface (since- it corrodes

m*tut;, rtro"to be drawn through the aspirator line at the end of each work

reaci0rr and between handling different cell lines. This will help to avoid both

Fierobial contamination attd cross-contamination between cell lines via the

lfpln,tor titt". The aspirator jarcontentsshouldbedisinfectedbeforeemptying,

tret'eraUty into a sink in a separate room. As an alternative to an aspirator

iar u**"-'Uty, if there is a suitable water supply at sufficient pressure nearby,

L-':lnrpte tap syphon is a cheaper and more convenient way of aspirating

fpent'rnediu-,- iinc" waste is drawn directly into the mains drainage.

1,3 Culture plasticware and associated smallconsumable items

8,8.1 Tissue culture PlasticwareThlr is supplied by a number of specialist companies; the. top four or five

Cenrp,,ni"r produce very comparable ranges of sterile plastics (usually poly-

iiyrl',"y, in terms of boih price and quality, but may offer different modifica-

iienr.ttto basic design or iniroduce new, specialist ranges. The most commonly

Eted ilcms of Plasticware are:

ia) lirr growing cells (most are 'tissue culture' treated, to produce an electro-

It.tically clarged'surface for wettability and cell adhesion):

r llat-bottomed flasks

o Pctri dishes

o ntulti-well dishes

r conical flasks (for suspensions)

r rollcr bottles

r lubes

{b) tirr handling solutions and cell suspensions:

r volumetric PiPettesr plastic Pasteur PiPettesr rrricropipette tips

r ccntrifuge tubes

(e) lor storage:

r sttmple tubes

r lippendorf tubes

o cryotubeso hrgcr volume screw-capped bottles

( )ttr ttl'the most useful vessels for growing up stocks of cells in culture is the

flgrh , which is widely available with uiable sittace areas of 25, 75, and \75 cmz .

Flgrkr huve cither itraight or canted necks and are supplied with a conven-

tlprt,,t. two-position, sciew-threaded cap for vented or closed incubations'

77

Coroline B. Wigley 7: The cell culture laborutory

llltcring small volumes of tissue culture solutions. The pore size should be0,2 pm or even 0.1 pm in order to be sure of excluding mycoplasma as well as

other micro-organigms. For most purposes however, 0.2 pm is the standardpore size for liquid sterilization. It may be necessary to use an assembly with aprelilter of e.g. 0.8 pm, if solutions are likely to block a low pore size filtertsrdily, e.g. a semi-purified enzyme preparation or a biological fluid.

Scveral features should be considered before choosing the appropriateflltcr. Some filter membranes e.g. those made of polysulphone, are lowprotcin binding and essential for proteins where the concentration of thef,llcred solution is critical, particularly if the molecule is highly charged, as areilrmc of the polypeptide growth factors. Very small volumes of valuablerotqlonts should be filtered through units where the 'dead' space (hold-upVolume) is minimal. It is cost-effective, therefore, to have some of these more€rpcnsive filters reserved for special purposes. Larger volumes can be filterllorilized using either pre-assembled, disposable units which attach to a

vncuum line (see Section 3.1.6) or, more economically, a washable, auto-elnvuble unit in which the filter can be replaced.

l,nrge tissue culture facilities may consider production and sterilization offtotlia 'in house', in which case a stainless steel pressure vessel, connected to anlllogcn line, may be useful. However, this is outside the requirements ofmotl tissue culture laboratories. Filter sterilization is considered in furthertlslnil in Chapter 2.

E,l.ll Pipettes

'l'hosc needed for all culture laboratories are of four basic types, with differ-€lll ttscs:

I ttticropipettes with disposable, autoclavable tips

I trrrplugged Pasteur pipettes

t plrrgged Pasteur pipettes

I volrunetric pipettes

Plnntic micropipette tips are supplied by a number of companies to fit thevttt'lt'ty of micropipettes on the market (see below). These will be of severallyltcs, tn suit volumes in the 1-20, 20-200, and 100-1000 pl ranges and up to5 trrl. ln addition, extended, fine tips are available for use with small volumeslil rrru row diameter tubes. Tips also come with or without a circumferentialt,lrlp,r' part way along their length, so that they can be supported in anurrlocluvable tip carrier and easily attached to the micropipette without hand-llttp, Scvcral companies also market tips with an integral filter for extra safetyItr xlt'rilc handling, but these may have to be purchased pre-sterilized, whichurhhi lo their cost. Autoclaving, but not irradiation (which is in any case notI'erurible lbr most laboratories), causes the filters to deteriorate.

l)ir;rosuble plastic Pasteur pipettes (l ml, with an integral bulb) and

Alternatively' some suppriers (e.g. costar, Nunc, and Greiner) may offerflasks with a sterile, hydrophobic firter in a perforated cap so that gas ex-change can occur without Lven the slight risk of microbial contaminationassociated with a loosened cap.

Tissue culture dishes are widely used and are available in 35, 60, 90, 100and 145 mm diameters with a viriety of modifications possibll, such as aseries of internal wells and.grids. Genlraily, the lids are vented for adequategas exchange but designed for minimum Lvaporation. Multiwell dishes aresupplied in various sizes, but 96-well (flat or round bottomed for differentapplications) and 24-well are the most widely used. Four- and six-well platesmay have specialist applications, such as the accommodation of filter well

For large-scale adherent cell cultures,.prastic roller bottres can be used,which require a special apparatus within a cabinet or warm room to turn thebottles at a constant rate, thus utilizing a large surface area for growth in arelatively small volume of mediu-. in"r"uringly, this typ" "r large-scaleculture is achieved in other ways, such as the us=e bt,oi".o"urrier ueads (e.g.those supplied by pharmacia 'or

Sera-Lab;, o,utri""r-"in*r"g'r"growth ofcells, or meshes of various types to increase the available culture surface area.cells grown in suspension "un

b" curtured either in rpinn", nurrc, where themedium is constantly circurated by a magnetic stirrer, in "o.ri"ui

flasks on arotating platform (kept in a warm room Jr dedicated in""ou-t-o4, or in staticculture on non-adherent plasticware. There are a number of other newdevelopments in the design of curture vessels for particula, ,r."r, such aschamber slides (e.g. Labtek, from Nunc) for growing ce's to Le used forimmunocytochemistry, or tripre flasks (Nunc;, "wtricrr ;;;" ;;ee stackedsurface areas for cell growth within a conventionally sized n"*t,

", a solutionto the problem of limited incubator space. Supplieis of tissrre crrtture plasticswill advise on new or more specialist items on ih"i, li.ts and most cataloguesgive detailed information on the specification and uses of their pioducts.

For some cells, the tissue curture surface, even when treated to enhance celladhesion, does not provide an adequately adhesive surface. some ceils havean.absolute .requirement, especialfu in serum-free medium, for an extra-ccllulur,matrix protein substrite. consideration of such ,p""ih" ,rurtrates istlertll with in chapters.4 and 6.It may be sufficient to coat the tissue culture*rrliree

_with a highry charged porymei such as porylysine o, potyornithine atrulr'ul l0 pg/nrl. 'l'his is also useful for encouraging attachment oicem wnichttttuully grerw in suspe nsi.n, for e .g. Hoechst staining for mycoplasma testing.

inserts (e.g. those supplied by costar) which alow oin"r"niiypis of cells tobe.grown^separately, on one or more membrane ,upport, *iitin the samevolume of culture medium.

3.2.2 l'lltcrr ltlr rtorilizing tissue culture solutionssovcrsl ,rr,rppllc*r r|. nre,ririzing filters (Miilipore, German, etc.) produce dis-punrble, eterllo urrlltl I'o' nttnchment to a syringe, which are'suitable for

18 1g

volumetric pipettes of all volumes (1-50 ml) can be purchased from mtissue culture plasticware suppliers in pre-sterilized packs. However, this isavoidable expense if substantial numbers of pipettes are used and there i

facilities to wash and sterilize glass pipettes. Generally, glass pasteur pipetare supplied non-sterile but ready to use after dry heit sterilization. Bothplugged and unplugged glass Pasteur pipettes will be needed and are intendedfor disposal after a single use. Extra-long unplugged pasteur pipettes arepreferable for reaching to the corners of large flasks for aspiration of spentmedium. Standard length, plugged pasteur pipettes ur" u."d for any small.scale, non-volumetric pipetting of tissue culture solutions and for the intro-duction of gasses into e.g. a flask or sealable chamber, where the cottonplug ensures sterility.

Glass volumetric pipettes should be of good quality borosilicate glass,graduated to the tip, preferably with the maximum volume graduation at thetop. A relatively wide-bored tip is advisable for fast delivef. A selection ofvolumes from 5 to 25 ml is generally required; below this, micropipettes aremore accurate and convenient. The most frequently used pipettJs are 5 andL0 ml volumes and there should be at least nve timei the num-ber in daily use,to allow for those out of circulation in soak, wash, or sterilization. Re-usablevolumetric pipettes will have to be unplugged for washing and repluggedbefore sterilization, care being taken to discard any thaiare cracked orbroken (see below).

Coroline B. Wigliy

3.2.4 Pipetting aidsA number of micropipettes are available on the market to suit most

7: The cell culture loborotory

ggg, but this is not usually a problem in practice, provided that high qualityb€rolilicate glass is used and that washing-up facilities are good. Glassware

hEE a g.eutet propensity to adsorb substances such as alkaline detergent on toIt! turl'ace, than plastics such as polypropylene. Whatever the choice, most

tebcrutories will require a selection of volumetric cylinders, flasks, and beakers

€f prcparation and bottles for storage of sterile solutions. Schott bottles

fBehott Glass Ltd) are particularly suited to this. They are robust and willItand the pressure created by solutions autoclaved with a tightly closed cap(€,g, hicarbonate). The cap is deep and will therefore ensure a good depth ofIt€tlle outer screw thread surface and greater safety on the few occasions

then pouring cannot be avoided.

911,0 Miscellaneous small itemsI plpctte cans

I luhc racks

! heenrocytometers

I lntlrument tins

I plpcttc bulbs

Fgr tlrc preparation area:

I Buloclave tape

I evcn tape

I Erowne sterilizer control tubes (for oven and autoclave, from Solmedia)

I Cotlon 'rope' for pipette plugging

! Bttltrclave bags (at least two sizes)

lf rc-usable volumetric pipettes are used, it is convenient to sterilize themin clttinless steel, cylindrical cans or aluminium cans with a square cross-

l€€tion, according to preference. Aluminium cans are less expensive, but willEildizc over a period of time. There should be at least one can per worker forEBelt pipette volume-enough so that individual workers can keep a can, oncegperrcd, for their exclusive use. Shorter cans are needed for Pasteur pipettes.Elverr-stcrilizable, tightJidded flat tins are convenient for sterilizing instru.ments for dissection, sterile handling of coverslips, etc.

4. Washing re-usable tissue culture equipmentAll tissue culture equipment which can be washed, sterilized, and recycledrhorrlcl go through the following general process, either manually or through a

tlrurrc culture-dedicated automatic washing machine with both acid-rinse anddlalilled water-rinse facilities.

100-1000 pl, and 1-5 ml will be adequate, perhaps *ith orr"'o, two fixedvolume micropipettes, such as a 50 or 100 pi for itequently used volumes.Depending on the number of regular users, sets -uy n"id to te replicated. Inaddition, it is useful to have a multichannel micropipette; this is essential ifmuch work is done with 96-well plates. For many liboratories, an automatedaid to pipetting larger volumes is also a high prioiity. several companies make,

and needs. Some are autoclavable for additional safety for sterile procedures.Formostlaboratories,arangeofmicropipettescoveringe.g. t-zop,i,zo-200p,1,

lightweight, hand-held, battery- or mains-operated units for this purpose,with loading and dispensing push-button controls and an air filter toriteritity.For those on a tight budget, conventional rnanual pipette bulbs will beadequate; these are available in a range of volumes. una-er no circumstancesshould mouth pipetting be considered for tissue culture work.

3.2.5 Glaegwaro for tissue culture useLaborotories diffcr in their preferences for glass or plastic for handling andstorage of solutions for tissue curture use.' There ii no doubt that p'iasticcontainers are lers prone to lenching trace elements which might be deleteri-

20 2t

Caroline B. Wigley

Protocol 1. washing and sterilization of re-usable labware

Eguipment und ,e"gents. Ultrapure watero Chloros (hypochlorite) solution for soakingr Detergent [e.g. Micro (lnternational producti

Corp.) for manual washing; low_foam foimach i ne, as recom m ended bl m a nufi"tr."ii

Method

1. Soak items either in Chloros solution (except metals), or directly indetergent.

2. Wash with detergent (by soaking or machine).3. Perform tap water rinses (continuous or seguential).4. Rinse with acid (except for metals).5. Repeat tap water rinses as step 3 above.6. Rinse with distilled/reverse osmosis/ultrapure water (two or moretimes).

7. Dry with hot air.

8. Store temporarily, capped or covered, e.g. on preparation bench,9. Prepare for sterilization (see Chapter 2).

10. Sterilize by autoclaving or in dry oven.ll. Store for use (in dedicated cupboards or racks).

o AnalaR. (or similar high purity) HCI formanual washing; formic or aceiic acid formachines

e Rigid plastic soak tanks, cylinders, andoeakers (tor small items and instrumentsl

Problems with inadequateJy y?:h"g equipment are common, but mostlyavoidable by attention to the following.'

4.1 SoakingMany tissue culture sorutions.contain protein. If glassware is left unrinsed andunwashed for any length of time aftei use o, i, illo*"J ;;;;;r, there areseveral consequences:

r residual medium will support microbial growth and act as a source of air-borne contaminants in t-he laborutory-- -driccl<ln protein wiil be difficult to remove by normal washing procedures

il I 1xili, l:l1,,;,vi11: l"yT." : _*_h"" n sterlize d, *il ;";;;;mi n ating

llli:]lll f ll1-::il j:li:,tj:0""1 or r,"i_oenatured proteins, ",:llfrH:c$rbonlt) into new solutions

a

a

l'hc folkrwing pnrccdure$ ure thereforesituIted near lhc wurhlng_up arear one

advisable. Soak tanks should befor heavy-duty glussware such as

22 2S

1: The cell culture loboratory

f,odical flats, bijou bottles, etc. and one for fine glassware such as beakersEntl cylinders which are easily broken. The soak tanks can be filled with taptvater but must contain Chloros or an equivalent sterilizing solution. Thisihould be changed regularly. Presept tablets (Johnson & Johnson Ltd) pro-Vlde a convenient way of making up this sterilizing solution. If the washing-upllgu is outside the main laboratory, it is acceptable to collect glassware in, for€Iutnple, a bucket kept beside the work station, provided that it is taken totha soak tank as soon as possible at the end of a session. Glassware forEBlking should:

I he rinsed under a cold tap

I hnvc any tape removed

! ltnvc marker pen labelling removed with acetone (a carefully labelled wash-Itottle kept by the tank is useful)

I lrc immersed completely in the soak tank

l,l WashinglVhen the tanks contain a load for washing, glassware can be taken straightfttiln the tank. Different laboratory washing machine manufacturers, e.g.Lgttcer, recommend different detergents, usually liquid and often their ownbrnlrtl, and acids for acid-rinsing, e.g. 507o acetic acid, which is furtherdlluteC in the machine. When loading the machine, care should be taken to€flrure that all items, especially caps and small bottles, are stable in the 'openlldc down' position and can be rinsed adequately.

lf gl:rssware (or dissection instruments, for instance) are to be washed byhntttl, they should first be soaked overnight in a detergent solution. It does

Fol nrrrttcr, in this case, if the detergent is not low-foam, but it should rinse off€ontplctcly under running tap water. Some products e.g. Micro (InternationalFfltrlucts Corporation) are advertized for their suitability for culture work orpfee ision equipment use, particularly where protein-contaminated material islllely, ll'possible, for non-metallic equipment, an acid-rinse step should be

inclutlccl to neutralize residual alkalinity (although some detergents are

Ehlrlccl to have a neutral pH so should not require acid-rinsing). Further tap*[l* rinsing should be followed by two to three changes of double-distilled,R() or', better still, ultrapure water.

(llnssware must then be dried in a hot air oven, set at about 100"C (some

ilnnlrirrg machines incorporate this facility), before re-sterilization. Cleanllettts which cannot be sterilized immediately must be kept covered or in-Velle(l on a clean surface until they can be prepared for sterilization.

{,il Washing pipettesRe.rraublc glass volumetric pipettes can be soaked in a plastic cylinder con-te lrrilrg ( )hloros solution, kept beside each work station. Suppliers of laboratory

Coroline B. Wigley

plastics, e.g. Nalgene, from Nalge Company, market all the items requiredfor the procedures described below. In principle, the steps involved are thoseoutlined for general glassware above, but several points should be notedwhich are special to pipette-washing. After a load has accumulated in thesoak cylinder, the protocol below should be used, unless a pipette-washingfacility is available in the automatic washing machine, when supplier's instruc-tions should be followed,

Protocol 2. Washing glass volumetric pipettes

1. Unplug the pipettes using forceps or a high pressure air or water jetapplied to the opposite (tip) end.

2. Place them, tip upwards, in a plastic pipette carrier.

3. lnsert the carrier into an outer cylinder containing detergent solutionand leave overnight to soak.

4. Transfer the carrier to an automatic, water siphon-operated pipettewasher."

5. After 3-4 h, add about 50 ml of concentrated acid (HCl or acetic) at thefilling stage of the cycle and turn off the tap when full,

6. Leave for about 30 min to remove residual traces of detergent.

7. Run the tap water rinse cycle again for at least t h.

8. lmmerse the pipettes in their carrier in two or three changes of ultra-pure water, in a clean plastic cylinder kept for the purpose.

9. Drain for a few minutes and transfer the carrier to a drying oven.

' Pipette washers operate on tap water, the flow rate of which must be within certain limits, setempirically. The container fills and empties repeatedly, by a siphon mechanism.

The pipettes should still be tip upwards to dry; the excess water willevaporate more easily from fine-bore tips this way. When volumetric pipettesare dry and ready to be replugged, this is a good time to check for any that arebroken at the tip or which have a crack or chip at the other end. It is crucial,for reasons of safety, that these are discarded. One of the most common,serious accidents in tissue culture laboratories occurs when a manual pipettehulb is fitted to a volumetric pipette. Using too much force and/or an unsafemethod of attaching the bulb, the end of the.pipette may break off and jaggedglass cause serious injury to the palm of the hand, with a high probability ofsevering a tendon. This is especially likely when the glass is already weakenedby a crack. Tendons can be difficult to repair, leaving the victim with apermanent disubility.

All re-usable glunswure will have to be sterilized after washing and drying.'l'his is one of the topiur covorod in Chapter 2.

1: The cell culture loborotorY

!, General care and maintenance of the tissue

culture laboratorY

Bontc aspects of laboratory care and mainten.ance {ft t-1 "t]:-t"^|: ^t**:::iil;,;;;;;;;;i;t# shourd be made.here' rhe ?TryI.:i: :::3::t:; : il ;#";;i"" ;"';h;';;;;' an d cre anrin" * "i :l-" 1i!:.1"t".'{::."" :111 "fiilffifi;I" "'p**uv'o

i1 .u.

tissue cultu'" l?!:it::v: tl"ll,"::::L'::lrrrl'r'rtcrtr' "-'*t-"il'u.r., *ti"r, must be completed on a daily, weekly,Opentte a check-list ol

monthly, etc., basis''i1iil"i,i,"iil; ril; """

be used as a guide but can, of course, be modified

tnd cxtended to suit the individual laboratory'

Enlly checkJist:

I fcc.rd incubator temPerature' '

I clteck CO2 cYlinder Pressures

I ttllt uP water baths

a etnpty PiPette soak cYlinders

t elteck glassware soak tanks for washload

I tlttw Chloros through aspirator lines

I rrplcnish stocks of sterile glassware, solutions' etc'

Thln is Only a brief list of essential points; further details and additional items

Bfe covcred in Chapter 4, Section 4'1'

Wpel,t.v check-list:

! wttlh down all work surfaces (if not done daily)

i check under laminar flow cabinet work surface for media spills

I eltnrrgc water in water baths

I lop tr;r humidifier trays in incubators

I ltttltirttc condensation from incubator bases

I clrcck stocks of routine reagents, plasticware' etc'

i ettrgrty aspirator jars as necessary (if not done daily)

f lop up liquid nitrogen in cell freezers (or twice weekly)

llt tttt ltl y <:heck-list :

I r'k ttttsc and sterilize reverse osmosis unit

I r'lrcek water ultrapurification cartridges

I t'lrrck whether equipment services/safety tests are due

I rlelrost I'reezers (as necessary)

I t'slibt'tttc instruments and monitors

I rllip down and cleanse/sterilize insides of incubators

2t

Coroline B. Wigley

If the laboratory is a murti-user facility, then one or more individuals,perhaps in rotation, should be assigned the task of overseeinjihe runnirrg ofthe laboratory and ensuring that thJ users abide by an agreeJJoJe otpractice.Examples of good practice include:

o clearing away promptly-after finishing sterile work, i.e. vacating raminarflow cabinet after swabbing down the work surface with alcohol and draw-ing Chloros through the aspirator line

o dealing promptly with used glassware etc. to be soakedo labelling opened sterile equipment and solutions, for re-use onry by the

same named individualo keeping good communal records e.g. of shared cell or reagent stockso checking incubated ceils regularly for early signs of contaminationo checking incubators regularly for unused cell cultures and discarding

surplus (or contaminated) stoiks according to approved safety procedures(e.g. after autoclaving, soaking in Chloros, etc.)-More than in most multi-user raboratories (other than where specific

hazards, e.g. radiation, are involved), workers in ihe tissue culture Iaboratorymust follow local rules. Failure to do so is perhaps more than usually likely toaffect the work of colreagues, as well ur yo.r, own. Shared sterile materials orneglected microbial contamination can iesult in persistent or recurrent prob-Iems for the whole laboratory, which are much easier to pr"u"ntlrrun to cure.

AcknowledgementsThanks are due to Peter wigrey for help in preparation of the illustrations andto Andrew Kent for helpful commenti onin" first draft of the manuscript.

References1.2.J.

9,I0.

Biol. Med., 4, 140.

4.5.6.7.It.

28

usA,76, 574.

SterilizationPETER L. ROBERTS

l, lntroductionEffectivc sterilization techniques are essential pre-requisites for practising thelft tlf ccll culture. Cell culture provides an ideal environment for the growth€l Fle ro-organisms including viruses, and it is only by excluding such agentsthft cett* can be successfully cultured and meaningful experimentation can beHfflerl out. In this chapter the various methods for sterilization will beFnglde rcd including some theoretical background but concentrating on prac-tlBel arpccts of their use. For additional background information iee refer-tF€es I -3, Guidance on relevant aspects of good laboratory practice andpod nrtnufacturing practice is given in reference 4 and in Chapter 9.

1,1 What is sterilization?fre nrethods used for sterilization have a long history. Pasteur first used heatF prcncrve wine (1864), and even before this heat was used in the canninglnduntry (c. 1700) although with no understanding of the microbial agentslnVolver,l. Other sterilizationtechniques such as filtration were first used in thelrte lll(ltls.

Bterilization is defined as a process which removes all living things. Inpfnelk'c we are really concerned with micro-organisms i.e. protozoa, somefilRgl , ltrcteria, mycoplasma, viruses, and unconventional agents [e.g. theFlctltn clusing scrapie and bovine spongiform encephalopathy (BSE)], all ofthL.h rrrc invisible to the unaided eye. Although sterility is an absolute term,ifl prncticc methods are used that give ahighprobability that an item is sterile.Elet'llizntion methods vary in their severity and the properties of the itemF€erllng to be sterilized also need to be considered. Micro-organisms also varyln their scnsitivity/suitability for different methods of inactivation or removal.

1,1 'l'he importance of sterility in cell cultureWhetr uprplied to a cell culture, sterility means the absence of any contaminat-1fl3 ot gnnism apart from the cells of interest. Such sterile cultures are essentialhtnlE irr rcsearch and diagnostics, and also for the production of various

Peter L. Roberts

biological products. The presence of contamination can have a wide range ofdeleterious effects. Examples include:

o complete destruction of the cell culture in the worst case

o problems and artefacts in research or diagnostic studieso effects on cell-derived products in terms of yield, stability, etc.. compromised safety of cell-derived products for therapeutic useo contamination of other cultures also being handled in the laboratory

1.3 The use of antibioticswhen a cell culture is free of contaminating micro-organisms, sterility can bemaintained by the use of good aseptic technique as outlined in this and otherchapters. This represents the first and best line of defence, but the use ofantibiotics may also have a role (see Chapters 5 and g).

2. Basic principles

2: Sterilizotion

*rElght line. From this the time required to kill any desired number of€ffnrrisms can be determined. The efficiency of different sterilizationFgtlrocls can be compared by determining whether large numbers e.g. >1 xl0l, crrn be inactivated or removed and in what time.

t, Wet heat at up to 100'Cflgnl lcpresents one of the most commonly used methods for sterilizinglQUlpnrcnt and media. Heat can be used in the dry state but is much morelffcctivc in the wet state and is at its most effective at temperatures of 121'C€l jt'crrtcr. In this section the use of wet heat at temperatures of up to 100 "C is

3€[rldcrcd.

t,l 'l'heoryMiny rnicro-organisms, including bacteria and viruses, are relatively easily

lfllcllvntod at temperatures of 60-80'C in the wet state. In fact, temperatures

ef t.t 60'C are often adequate. However, there are micro-organisms that are

FOfc resistant to heat, such as the spore-forming bacteria and the unconven-tlttttnl ngcnts. In addition, even for any susceptible type of micro-organism,

lflUlntrts or variants may exist that are less heat-sensitive. The exact mode ofintcllvttion will depend on the micro-organism and conditions involved, butdEltnlur:rtion and coagulation of protein are thought to be important. How-!Y€l', olhcr targets-such as nucleic acid-may be involved in some situa-

llOnl, Wittt some agents, including the viruses, inactivation kinetics often give

fhe to n curve with two (or more) components, e.g. an initial phase of rapidlEtcllvltion followed by a period when the rate of inactivation is lower, This

Fltt xuggest that two or more targets are involved in inactivation or thatllSt'cgntcs or genetic variation exist within the population. For the highlyfEtlrlnrrt bacterial endospores, the target of wet heat is believed to be theFN A , cc ll membrane, or cell wall precursors and the mechanism of resistance

Itt ltent irrvolves dehydration of the cells. The stability of micro-organisms(lnclrrtling viruses) to heat can be influenced by pH, and by levels of salts,pt'ttlclrr, irnd sugar.

Itt plrrctice the use of wet heat at temperatures of up to 100'C has only a

f?w llrrritcd applications in the cell culture laboratory and it is preferable,lhet'c possible, to use other more effective techniques such as autoclaving(FFt'lkrn 4) or dry-heating at high temperature (Section 5) for more effectivellct lllzrrtion,

Hevcrrrl points should be noted when considering the use of wet heat belowlllll'(' lirr sterilization:

{B) 'l'lre tlegree of sterilization will depend on temperature (and time).

(hl tiporc-ftrrming bacteria and unconventional agents are resistant to all butllrc rnrlst severc conditions, i.e. autoclaving.

Sterilization techniques are designed to kill or remove a wide range of micro-organisms including, in descending order of size, the protozoa and fungi,bacteria, mycoplasma, and finally viruses. In all cases ihe agents are com-posed of single cells, of varying complexity, except for the viruses which arebasically composed of only nucleic acid surrounaea uy a protein coat and insome cases a lipid-containing envelope. In addition to these, even simpleragents of disease are known: the so-called'unconventional agents' or prions,which cause slow degenerative encephalopathies in man and u.rimuis e.g.creutzfeldt-Jakob disease, scrapie, and BSE ('mad cow disease'). Howevei,the exact nature of these agents is still controversial; one theoryis that theyare infectious proteins (5).

The principal target(s) on which a method of inactivation has its effectvaries with the technique and with the micro-organism concerned but mayinclude the nucleic acid, proteins, or membranes. Micro-organisms also varywith regard to their sensitivity to the sterilizing technique 6eing used:

o bacterial endospores are resistant to heato unconventional agents are resistant to heat, irradiation, and detergento non-enveloped viruses are resistant to organic solvents and detergentso mycoplnsrnu, viruses, and unconventional agents, are not removed by con-

vonlionnl 0,2 pm storilizing filters

'ltr unclerntanrl lirlly thc cl'ficiency of any sterilization method that dependson lnsetivailon, it ir irnporlunt to know the kinetics of the process for relevantmicro-organiamn, pnrlicultrly those that are resistant. A graph of the log-nrithm of the numhor ol' surviving organisms against tiile often gives a

28 2g

Peter L. Roberts

It can be useful for heat-sensitive materials that cannot withstand moresevere heat-treatment.

It can be used to inactivate viruses in heat-sensitive products (see below),

Pasteurization

2: Sterilization

(c)

(d)

3.2

?fbtr t. Standard autoclave cycles

Time Pressure(min) (barl"

Survivalb

in 104in 108in 1017

in 1032

Equivalent timec{min}

Heat-treatment at temperatures of about 60-80'c has long been used infood industry. For example, milk is treated at 63.C for 30 min or at72"C15 sec. These procedures will kill vegetative micro-organisms and virusesnot bacterial spores. Pasteurization at 60"c for 10 h is used in the pharma-ceutical industry for certain products derived from human plasma as a metof inactivating any viral contaminants, such as the human immunodeficiencyvirus and hepatitis B and c, that might be present. Some of these productssuch as human albumin and transferrin can be used as supplements for cellculture. Before heat-treatment, stabilizers may need to be added to protectthe product, but these must not compromise the effectiveness of viraf inacti-vation. For example, in the case of human albumin, sodium octanoate can beused as a specific stabilizer. Another process that uses wet heat, combinedwith detergents, is that employed in the washing machines that are used fordecontaminating re-usable equipment particularly in hospitals.

3.3 100'CBoiling or steaming at 100'c for 5-10 min represents a simpre and effectimeans of sterilizing equipment and fluids. It is very effective and will evdestroy some bacterial endospores. However, some types of endosporealso unconventional agents are resistant to boiling and require more seveconditions for inactivation. It is possible to inactivate endoipores by the uof several 100'C cycles. This method, known as Tyndallization, involusing three cycles of heat-treatment with intervening periods during whichspores germinate. This type of procedure can be used in the cell cultulaboratory for decontaminating incubators that have a heat sterilization option

4, Wet heat above 100 "C and autoclaving4.1 TheoryBoiling water can be a very effective sterilizing agent, but in practicelteverc sterilization methods that are capable of inactivating bacterial endo-Epore$ nre requircd for many applications in the laboratory. using water attelnperuture ol'ubout l2loc, i.e. as steam under pressure, is such a method.'lir proeluee slennr under these conditions special equipment is required i.e.an autoclave. By hnving a knowledge of the inactivation kinetics forreclrtent traeterial errdoepores, suitable autoclave cycles have been devethat give a htgh arnurance of sterility. Some of the standard autoclave tem.perature:time cyelea that nrc commonly used are given in Tahle L It can be

jl har ' los Pa.

lfCr a relatively heat-resistant bacterial endospore such as BacrTlus stearothermophilus,ETima required to give endospore survival of 1 in 108, i.e. time equivalent to 121 "C for 15 min.

E€€n tllut the various cycles are not in fact equivalent and that, when survival is

€€mltatcd with the widely used cycle of I21"C for 15 min, other recommended€J€lec rrrc either less severe (115"C) or more severe (126" or 134"C).

Qneonventional agents are very resistant to heat-treatment and require theEBgt extreme conditions. Recommended conditions are 132"C for t h in a

pfevlty displacement autoclave or 134-138"C for 18 min in a more efficientp€Four-load autoclave (2).

1,3 SleamFBf rtelm to be fully effective it must be at its maximum water-holding€Epaeity, but with no droplets to wet wrapped items and it must not be

lEperltelted i.e. in an over-dry state. This aspect of steam quality can be

€hgekerl by an autoclave engineer determining the steam-dryness value. Thepf€lenc:c of too much air, i.e. non-condensable gas in the steam supply, willBllc rrduce the effective steam pressure in the chamber. This too can be

Fhcehetl by an autoclave engineer in a non-condensable gas test and maytsBulrc rnodifications to the boiler or pipework for improvement. The pres-

EBee ol'air in the chamber, either from non-condensable gas in the steamSUpply or because of poor air-removal, will lead to a lower temperature in the€henrbcr. For instance, if only half the chamber contained steam then, undereEndilirrns of pressure for which a temperature of I2l,"C was expected, only1lE'(' would actually be reached. If localized pockets of air were to remain,E,g, lrr packaged goods or in infectious laboratory discards, this effect could be

Fede r:ven worse. Adequate quantities of steam must be available so that noRBte llrirn u 10"/' drop in the supply pressure occurs when the machine is oper-Btlng, ltor pharmaceutical applications, 'clean-steam' is used which involvespfuviding the boiler with purified water of a conductivity of less than 15 pS.

!l,B 'l'ypes of autoclaveThe type s of autoclave available vary widely in size, complexity, and versatility.Thev rrrnge l'rom small, simple pressure cookers, which generate their own

60154.70.8

301510

3

lrFt:tr3€li1

0.71.01.42.0

30 31

Peter L. Roberts

steam, to large machines which are fully automated and may possess multiplecycle options. The nature of the load is important for determining which cycleis most appropriate.

4.3.1 Portable bench-top autoclavesi. Upright modelsThese are essentially pressure cookers as used in the kitchen at home. Steamis generated in the chamber base, either by an external or internal heatsource, and an air-steam mixture is discharged from the chamber throughhole in the top. A pressure gauge, possibly a temperature gauge, a pcontrol valve, and a safety valve are present. In more sophisticated modelsthere may be an automatic timer, safety features that seal the lid until tcycle has been completed and the contents have cooled to 80"C,cycle-stage indicators. A guide to the operation of a basic model is given inProtocol L

ii. Horizontol modelsThese are essentially similar to the upright models described above but hmore features such as cycles at both lzl"C and 134"C, a drying optionheat (but not vacuum), safety features such as a temperature lock,indicators, and automatic timers. However, steam is generated in an identway to thc upright models.

iii. Usee cnd linritotionsSomc of the poinln thnl nccd to be considered when using these small typesuutoclnvo nro:

2: Stedlization

(ll 'Fhey have poor air removal and are therefore not suitable for porous

Iontls such as wrapped items or laboratory discards.

[b) T't,. use of wet steam and no, or limited, drying capability can lead to wetItettts.

(Cl 'l-trr tcmperature in the load is not monitored.

[€) Tlr"t" is no cycle record i.e. print-out or trace.

(31 'l'he krad only cools slowly after sterilization.

[f) Eafety features may be limited or absent.

(l) 'l'tre ir use is limited to small items that have not been wrapped and bottledfluirls with loosened caps.

*t,l Gravity displacement arrtoclaves

ilhlle tlrcre exists a range of larger capacity (e.g. 100 litres) floor-standing

l$lttglavcs, which are either top or front loading, these are often essentially

hflet vcrsions of the bench-top models already described, although more

l€Fhlrlicltcd types exist. The next major advance in design involves the use

Ff iR external steam supply. In these, air is displaced from the bottom of the

€henfhcr by the less dense steam entering at the top. These machines also

Stllerr rr iacket for water or steam, or spray-cooling systems in some bottled-

F$ldr nulrrclaves, and thus cool the chamber contents more rapidly. The use

€f the .inckct, combined with a simple vacuum system, can be used to aid in

ihc tttvi"g of the load. This type oi autoclave is:

i Urefrrl lirr items that are easily exposed to steam, e.g. glass or plasticware

I rultsble ltrr bottled fluids

I llttl rruilirble for porous loads and wrapped items because the air removal

ffielllrrl is not adequate

ftt,S Multicycle porous-load autoclaves

?gftttts krird autoclaves were developed because of the problem of air re-

Httval lront wrapped items and other loads; these use a vacuum which, when

Hferl lrr prrlses combined with steam, efficiently removes air from the load and

Ehanther'. lt thus helps the penetration of steam into the load. The use of al€r,rrrriir rrlso aids in the final drying stage. Such machines tend to be large,

illllt a rrrinimum overall size of about 4 m3 and a chamber of at least 0.5 m3,

6fltl expr:lrsive. Some machines used in hospitals or the pharmaceutical in-

d1tlty ,,r'e far larger and materials can be directly wheeled in, or loaded via

detllt,trtetl trolley systems. In the laboratory a multicycle machine is commonly

Uterl, willr ir number of different cycles (Frgure 1) available for use depending

Ell llrc rrrrlrrrc of the load; this increases versatility in a multi-user situation.

FHlntttt.lcrs such as temperature and time can be pre-programmed into the

F€ulrirrt: rrnd are often not user-adjustable. In addition, a cycle may be

€YHllrrtrle irr which any comhination of these parameters can be set by the user

Protocol 1. Operation of a basic upright bench-top autoclave

1. Consult manufacturer's instructions before using instrument. The fol-lowing is for outline guidance only.

2. Remove lid and fill to required depth with water.

3. Place the items to be sterilized into the machine on shelves or in a

basket.

4. Replace lid and seal; heat water to boiling.

5. Allow steam to escape for several minutes before closing the pressurevalve (if appropriate).

6. Start timer when the required pressure and temperature are reached.

7. At cycle end, turn off heat and allow pressure and temperature to fall.

8. Open the steam valves and allow to cool to 80 "C and atmosphericpressure before removing the contents.

32 lts

Air-removal Sterilizing Drying

Peter L. Roberts

A+o

+B

o

C

+

o

D+

o

Figure 1. Typical cycles found on a representative multicycle porous-loadPressures within the machine, whether positive (+) or negative (-). are indicated. Cyclewhich involves both negative and positive pulsing, is used for fabrics, assembledunits, and discard loads from which it is particularly difficult to remove air. Cycle B usessingle negative pulse followed by positive pulsing and is used for laboratory pland glassware. Cycle C, which uses a series of negative pulses, can be used for dloads. However, in validation studies, cycle A has proved more effective fordiscard loads. Cycle D is a bottled-fluid cycle which uses air displacement to remove

immediately before use. Pressure and temperature during the run arecated by dials or digital meters. In addition a printer and/or chart-recorderfitted to allow the temperature throughout the cycle to be recorded. Arccordcr is morc easily checked by the user. On more recent models an adetcctor is usuully ['itted to confirm that all the air has been extracted from tchantber. Some points to consider with a porous-load autoclave are:

(a) Thero in goocl rtteflm penetrution of porous loads, allowing wrappedanel ditficult itemn to be sterilized

2: Stefilization

Dil'l'erent cycles are available allowing flexibility of use.

1'he chamber has a large capacity e.g. )0.5 m3, and will accommodate€,!1. 40 x 500 ml bottles, several large 5 or L0 litre vessels, or bags ofdlncards.

Sterilized items are free of moisture due to the presence of a vacuumdrying stage at the end of the cycle.

Eeenuse of the wide range of options and cycles available, this type ofEptgcluvc is usually made to order. Thus the user must carefully consider the

Sppllcntions and loads for which the machine will be used, in consultation*th a sterilization engineer and the manufacturer.

l,* Preparation of the load and operation of themachine

Ffotocol 2. Preparation of the autoclave load

A, Equipment

t, Wrap items in aluminium foil or place in sterilization bags using auto-clave tape. Do not seal completely.

t, Loosen the caps of bottles and other containers, or wrap up the opentops.

l, $ome items, e.g. pipettes or micropipette tips, may conveniently beplaced in containers such as tins or beakers which are then coveredwith aluminium foil or a sterilization bag. Looseh the tops of tins.

l, Micropipette tips are best sterilized in the boxed racks supplied by themanufacturer.

t, The necks of autoclave bags containing mixed discard loads must belrlosened to ensure good steam penetration.

l. Add a piece of autoclave-sensitive tape to the items in the load.

H, Fluids

L For a fluid cycle, bottles may be either sealed or left unsealed.

L e heck caps for their ability to withstand autoclaving. With some typesof cap the rubber liner may be sucked into the bottle.

I, Add autoclave-sensitive tape to the items in the load.

[lrlirlc routine use, the sterilization of typical loads should be validated as

€€cr,r ibed in Section 4.5, The heat-scnsitivity of the items should be considered

{blte)

* bgstc outline procedure for preparing material to be autoclaved is given infutat\il 2.

34 35

Peter L. Roberts

and tested before use. Many plastics can withstand 12roc, butstyrene cannot. Fluids such as water, salt sorutions, and buffers can alsoautoclaved. The heat-sensitivity of more complex formulations containicomponents such as vitamins, growth factors, antibiotics, or heat-sensiamino acids, e.g. glutamine, should be considered or tested. cell culr

use on every item allows a clear distinction between items that havesterilized and those that have not. Such indicators are essential with thebasic types of autoclave considered earlier which do not record details ofcycle' The simplest system is the use of autoclave-sensitive tape, but arclave tubes [Browne's tubes (solmedia)] or indicator strips can be used. Sotypes of autoclave bag possess integral indicators. only an approximindication that sterilizing conditions liave been met is given by zuch indicators. Nevertheless, they do provide a measure of assurance. Ii there is an,

2: Sterilization

On completion the cycle-end indicator will come on.

Check the chart paper or print-out for a satisfactory run.

Open the door and remove items using heat-resistant gloves to pre-vent injury.

Check autoclave tape or other in-load indicators. Label each item toindicate sterility if autoclave tape has not been applied to every item.

Complete log book record.

l,i Autoclave testingFgr nrr autoclave to perform satisfactorily and to ensure the sterility of thelgltl, it is necessary for tests to be carried out by the user and by the

lfrlllzution engineer, and for records to be kept. This is also essential in€fdet to meet good laboratory practice and/or good manufacturing practice

$ldelincs (see Chapter 9). Such testing goes hand in hand with a regularpflverrtive maintenance programme. An indication of the sort of tests thatihrrulrl lrc performed (6) is given in Protocol 4.

llotocol 4. User tests and records for the autoclave

l, Hocord details of all runs, including pre-use warm-up and leak-ratetEcts, in a log book with details of date, load, cycle, result (pass or fail),op€rator, and other relevant information.

l, Carry out leak-rate test, preceded by a warm-up run, at least weekly.

f, At the end of every run, check the chart and/or digital recorder for a

ratisfactory cycle. Relevant parameters include temperature, time, and

lrrrlsing profiles.

At regular intervals, e.g. daily or weekly, carry out a Bowie-Dick testlaae Protocol 5l or similar test to assess steam penetration into a

ctandardized difficult load (7).

Arrange for a sterilization engineer to carry out servicing at regularIrrtervals, e.g. every 3-6 months, and keep records of test results andwork performed.

llofore the machine is first used and at regular intervals thereafter, e.g.tl nronthly, arrange for the sterilization of standard/typical loads to bevalldated by thermocouple studies carried out by sterilization/qualityeeBUranco personnel.

I,t.

10,

lr,

tt.

problem with the wetness of porous loads at the end of the cycle, considetion should be given to modifying the length of the dry-cooling stage or simto repeating this stage at the end of the run. proiocolj o-uttiries trowautoclave should be operated.

Protocol 3. operation of a murti-cycre porous-load autocrave1. Follow the manufacturer's instructions and standard operating pro-

cedures.

2. carry out routine or weekry tasks such as cleaning the door-sear, re-moving debris/broken grass from the chamber, and creaning the drainfilter.

3. Perform warm-up, leak-rate, or performance tests as necessary (seeProtocol 4l-.

4. Load the chamber but do not overfill.5. Fill in dotails of run in the log book (see protocol 4il.

6. Place the wander-probe into the load. ln the case of a bottled-fluidscyclo, place tho probo into an identical control bottle with liquid whichcen bc dltcardod later.

7. Scloot approprlatc cycle and start the run.

97

Protocol 5. Bowie-Dick test for porous_load autoclavesThis test is designed to confirm that steam penetration into a test pack oftowels is both rapid and even (7). lt is performed after an initial warm_uprun.

1. Fold cotton huckaback towels (g50 x 500 mm) in half three times.These towels should be clean, dry, and not over_compressed. Openthem and allow to dry between tests.

2. Make a test-pack from the forded towers to give a height of about 270mm (25-36 towers). rf more than 36 towers are required to reach thisheight, dispose of the old towels and use new ones.

3. Place a sheet of A4 paper with a cross made of autoclave tape (e.g. 3Mno. 12221 in the centre of the pack. The tape must not be more than 1year past the manufacturer's date stamp. speciar ready-prepared testsheets can be used.

4' Tape the pack together without compressing it further and/or prace it ina wire basket to prevent it failing over. place it carefuily in the chamberon the longitudinal centre line and c. 100 mm above the chamber floor,and carry out a standard porous_load cycle.

5. At the end of the run, check that the hord-time at steririzing temperaturewas within the specified limits for the cycle used.

6. check the autocrave tape. rf the machine has been operating correctry,the colour change arong the tape wiil be even. rf an area wiih a rightercolour is found, this indicates a cord-spot where poor air-removar hasoccurred' An autocrave engineer must be contacted and the machinemust not be used until the fault has been rectified.

Peter L. Roberts 2: Sterilization

!, Dry heat

Hfntlng in the dry state can also be used for sterilization; however, because

thh lt not as effective as wet heat, higher temperatures and longer times must

bi uncU. Under these conditions the inactivation of micro-organisms is

pflmarity due to oxidation.

trl Incinerationln tt * ccll culture laboratory there are a few examples of the use of this

Aftho(l lbr destroying micro-organisms. For instance, during routine aseptic

ffhniquc, the openings of glass bottles and other containers may be brieflytfrfmett' i.e. placed in the heat of a Bunsen burner (although this may not be

fiffmnrcnded when working in a laminar flow cabinet-see Chapter 4). This

Ei lle carried out even with plastic flasks and tubes if care is taken to keep

S! ex;t,rrur" time to a minimum. In the microbiology laboratory a metal loop

b ute.l l'or transferring cultures and this must be completely sterilized be-

Hicn usos. This is carried out by heating to red-heat in a Bunsen flame.

Tltel itcms and scissors, used for example during the preparation of primary

tsll eultures, may simply be re-sterilized between manipulations by being

Sppe,t irr cthanol which is then ignited and allowed to burn off.lneltrcration is often used for the safe disposal of laboratory waste. However,

*hlle this is an effective method for use with microbiologically contaminated

Ftterlnl, it is recommended that such material should be autoclaved orHfted hy chemical methods in the worker's own department prior to inciner-

ItlCn, 't'hus any problems due to transportation, inefficient incinerators, orinndeepr,,t" direct supervision are avoided. Incineration may be an acceptable

€ffpo*rt route for routine cell culture materials but, if contamination is known

€f tscltcctcd e.g. in accidentally contaminated cultures, or cell lines trans-

Ffmetl with viruses, the above two-step procedure should be carried out. For

EF lncllr(.rator to operate efficiently it should reach a temperature of 350"C

Bfld ptclcrably have after-burners fitted to incinerate any unburnt material

lhel rrrr,y bc found in the smoke.

!'fl !kll-air ovensHentirrp. itcms in the dry state provides a commonly used alternative to the

HIE of nlc:irm for heat-sterilization. Some points to consider when using this

Fethorl itrclude the following:

{al lt ix sirnpler and cheaper than using a porous-load autoclave but longerIrrrrllncnt times and higher temperatures are required e.g. > t h at 170'C(erelutling load warm-up).

lb) lt"rrrs do not get wet during the process.

[el !t in nrutincly used with glassware and metal objects.

During validation of standard/typicar roads, the parameters that shouldtested, and the limits expected, include:

(a) porous-load thermocouple tests

ii' other relevant loads should be tested including worst-case/difficuloads and items e.g. discards, filters, and wrappJd items.

(h) liquid loud rcsrs

i. 'l'h-crmocouplc tests on bottled fluids should show ll5_117"c for all.5nc'cycle, ur rzr-rz4'c for a'r}'roc cycre. Ail probes in the roadshoukl ilgrce lo within t.C,

30 3g

r

occurring during cooling. The required temperature and time is set and tactual temperature monitored by an inbuilt digitat or dial thermometer aror a thermometer placed through a port into the chamber. Some timers aronly triggered when the required temperature is reached in the chamber. T

Peter L. Roberts

(d) some plasticware can be treated but requires a lower temperature eI?O"C for > 18 h.

A hot-air oven comprises an insulated box with electrical heaters. Heatdistributed by simple convection or more effectively by a fan. For mocritical applications, e-g. in the pharmaceutical industry, u Rtt", may beto the air vent to prevent any possible recontaminaiion of the load

monitor the temperature continuously a chart-recorder may be included. Flarge volume sterilization on an industrial scale, tunnel-sterilizers are usedwhich items are continuously fed by conveyor through a hot-air tunnel.

various temperature-time combinations can be r^"d fo, sterilization.most commonly used conditions, and those generally recognized byregulatory authorities that control medical products, are-160 "cio r 2h, i7ofor t h, and 180"c for 0.5 h. These excludelhe time required for the oven aload to warm up to the required temperature. other conditions can be urwhere items cannot withstand these high temperatures e.g. 150.c for 2.5140'c for 3 h, or r20"c for trg h. The latter conditi6ns are useful fsterilizing certain types.of plastic, e.g. poryallomer and polycarbonate (used in centrifuge tubes) or polypropylene (as used in micropipette tipsboxes). However, they are not suitibie for porystyrene. the'supplier,s rlogue should be consulted for further detaili on ttt" heat-sensitivity ofplasticware.

5.3 Load preparation and oven useThe basic steps involved in preparing items for oven sterilization andcarrying out the process are given in protocol 6.

Protocol 6. Sterilization using a hot-air oven

1' wrap items in aluminium foir. containers need onry have their openingwrapped or capped (check heat-sensitivity of the cap).

2. Add heat-sensitive tape, or other temperature indicator, preferabry toevery item in the load.

3. Fill the oven by placing items on shelves. To aid circulation of air and topromote warming up, do not over-fill.

4. sat tlmer, temperature, and chart-recorder controls. Add on additionaltlm€ to allow for oven and/or load warm_up as necessary.

5. On complrtlon, allow ovsn to cool. Some ovens have an automatic

2: Sterilizution

l6fety device which prevents the door being opened until the tempera-ture has dropped to a preset level.

f, Check chart and in.load sterilization indicators for a satisfactory cycle!nd then remove the load. Mark all items as sterile.

Routine monitoring and testingindicators can be used as simple checks to confirm that the load has

heut-treated. Because most of these are just indicators of the temperaturennd not the time, they do not indicate sterility. However, they do

ru useful indication as to which items have been in the oven when all itemsilied with heat-sensitive tape. The most commonly used and cheapestis the use of heat-sensitive tape on which dark stripes appear when

falures of 160 oC are reached. Alternatively, indicatorstripswhich changeovcr the range of e.g. 116-154'C or 160-199oC, are available.

rure less commonly subjected to extensive routine validation com-wllh autoclaves, partly because they are much simpler to operate and

llttle or no routine servicing. However, official standards do exist and itblc to test the performance and instrumentation of an oven after

purchase and on a regular basis, particularly where an oven is forturc in the hospital or pharmaceutical area. Checks should include:

Ullng t hermocouples placed in contact with the oven temperature indicatorI warnr-up <135 min

' I ovcrshoot <2oCI rlpplc <1'CI drlll <2"C

Eeattlngs of these terms are illustrated in Figure 2.

It Jrrtlgcd by thermocouples placed around the load, the load tempera-lgre nhould be within + 5 oC of the oven temperature indicator

* Irrrrrliation,*-1 Ultroviolet light.X$tfntl,tto, (tJV) light at a wavelength of about 260 nm will inactivate micro-'Ei$nlurn ulrcl viruses. It acts on nucleic acids, leading to strand breakage,

Fnd elorr-linkage, and the formation of pyrimidine dimers and other prod-

l$l, H,rwevcr, because of its poor penetrating power, its usefulness is very

Fftlteet ll cnrr be used to sterilize air in cell culture cabinets and rooms, andrl'ler they have been cleaned. As the power of UV lamps decreases

Ute, I lrey should be checked on a regular basis. As found with heat, bacterialglttl unconventional agents are highly resistant. Also many organismsl)NA repuir mechanisms that can overcome limited damage.

40 4l

o

EoCLEe

Peter L. Roberts

W -Ripple

Overshoot_

Warm-up Sterilization Cool{own

Time

Figure 2. Typical cycle for a hot-air oven. points in the cycle (see section 5.4) whichimportant in confirming the effective operation and validation of an oven are indicaThe ripple effect has been magnified for illustrative purposes.

6.2 Gamma raysGamma-irradiation, although not actually performed in the laboratory, isvery important method of sterilization. It mainly acts directly on nucleacids, but indirect effects by free radicals and hydrogen peroxideiormed

2: Sterilizqtion

p*ldc. 'l'heir activity is greatest at higher temperatures and humidity levels of75::1tttl'U,. However, conditions that reduce the accessibility of the micro-

€,tgenisrns to the gas, e.g. being dried in organic or inorganic material, willd€efeu*" the effectiveness of fumigation. These fumigants act as alkylatingEEgnts and act on both the nucleic acid and protein components of the micro-

€fganisrns.

1t1,1 Iithylene oxide

€thyl.t," oxide is commonly used for sterilizing items of clean equipment inlry=terrrperature autoclaves, or in combination with steam, particularly inhglpltutr. In the laboratory some items of plasticware may be purchased pre-

It€tillzect with ethylene oxide e.g. syringes and filters. However, this method

9f eterilization can leave behind toxic residues, and other methods, e.g.

lEmrna-irradiation, may be preferable (see Section 8.1.1). Some additionalp€lntt to consider include the following:

@] Ttre gas is toxic and operator exposure needs to be controlled.

[-b] Speciat equipment is needed.

Gl tt i* not suitable for decontaminating bulk discard loads.

ir1,l lrrlrmaldehyde gas

ffilf gu* is commonly used to decontaminate or sterilize larninar flow cabinets

13€ ro,rrns used for handling cell cultures, and also for small items of equip-

EEat, A'r outline of the procedure used when treating roorhs is given in

fuAatl7. It should be noted that formaldehyde gas is toxic and thus any

Eerrary precautions, including the use of breathing apparatus, should be

*Ft€n *lr.rr" necessary.

Frotocol 7. Fumigation of rooms using formaldehyde.

llernove all unnecessary items from the room, including those thatnrust not be exposed to the gas e.g. cell cultures, sensitive electricaler;tripment, etc,

(ilcan the room to minimize the level of microbial contamination andlo allow good gas penetration.

Irrrn off air-handling system and seal up the room with masking tapeas lor as possible.

itiace a hot-plate with large saucepan, connected to a timer, in the room(tlrotime requiredto boiloffthe liquid should bedetermined in advance).Altornativoly use a dedicated formaldehyde-generating kettle."

I iii tlrc container with formalin solution (20 ml per m3 of room volume),

Irrrn on ths hot-plate or kettle and loavo tho room.

water may also be important. Unlike UV light, it has very good penetratiproperties. It is commonly used with items that cannot be heat-steiilized, ahas the advantage that, because of its good penetrating power, items cancompletely sealed and packaged. various items of disposable plasticwarebought pre-sterilized in this way e.g. tissue culture flisks and dishes, filteplastic pipettes, syringes, pipette tips, and some chemicals such as antibiotisuch items will have been expose d to 2.5 Mrad, the accepted standard r

in a cobalt-60 plant. This exposure level has been in use by industry for aperiod and has been found to be acceptable as long as only low levelllmination is present which is not unusually resistant. Bactlrial sporesviruscs lend to be somewhat more resistant to irradiation and unconventilrgcnt$ nrc cxtrcmely resistant,

t,

t,

E.

!,

7, []homical sterilization7.1 FumlgationFormaldehyele gan trr er.hylcnc oxide can be used for fumigation.el'l'ective againrt ull types ol' micro-organisms including u'irur"*,bncterial endoeporen ule s'mewhat more resistant in the case of

Both a

altethy

4S

Peter L. Roberts

Pro_tocol 7. Continued7. Lock the door, tape it up fully (including the keyhole), and attach

safety warning notice.

Leave until the next day.

lf a total exhaust cabinet or air extract is present, turn this on by aremote switch. lf this is not possible enter wearing breathing appar-atus and turn on cabinet and/or air-handling system.

Leave until the level of formaldehyde reaches an acceptable level.Testing equipment should bd used.

, The room may require cleaning to rerno-ve residues of paraformal-dehyde and it may take several days to remove the gas completely,

" Other methods for generating formaldehyde gas exist e.g- heating paraformaldehyde (10 g/m3) '

and, for those situations where fumigation is carried out regularly such as in pha(rnaceqticalmanufacturing areas, equipmenl that generates a.mist of formaldehyde (e.9. Phagojet, Labor-,atoires Phagogene).

Microbiological safety cabinets (only models that can be sealed andto the outside) that are used for handling cell cultures may also betaminated with formaldehyde (Protocol S) on a regular basis, e.g. onceweek or once a month, or after it has been,found that contaminated cultuhave been handled.

7.2 Liquid disinfectantsMany types of liquld disinfcctant exist and they have a useful role in theculturc laboratory, The mnin properties of some of the principal types

Trblo 2. Effect of liquid disinfectants dn micro-organisms

Effectiveness"

Bacteria Endospores Viruses

2: Stefilizstion

8.

9.Typc Fungi

Aldehydes +Hypochlorites +Fhonolics +Aloohol

++++- +lv- +/v

++++10.

11.tEflective (+) and non-effective (-). ln the case of non-enveloped viruses, the

3lleot may be variable or partial (vl depending on the particular virus. Examples oftht dlfferent types of disinfectant are given in Section 7.2'

in Table 2. Disinfectants can either be prepared directly using

ry reagents or be purchased in the form of proprietary formulatidns.6f the factors to be considered when using or selecting a suitable

ant are listed below.

ffhe rnnge and level of antimicrobial activity is less than with other

Egthods of sterilization, e.g. heat.

Bp€re-forming bacteria and some types of viruses may be resistant.

SEme disinfectants are neutralized by organic matter.

atnbility of 'working' dilutions varies with the type of disinfectant.

ctposure time required depends on the type of disinfectant and onit is used.

ty to the user should be considered.

disinfectants can be used in the cell culture laboratory for:

€Utlne hygiene and disinfection of items of equipment and surfaces in

€:fi llii:T:?:* curture items, e.g. grassware, arter use and prior to*e:hing und re-sterilization

tfCatrnent of used or contaminated cell culture media before disposal

most suitable type of disinfectant for a particular application varies

below;, 'l'here are a number of methods available for applying the dis-

Eat, Thcse include using a cloth or hand-held sprayer, or the use oftowels applied to liquid spills and then soaked in disinfectants. For a

!tet' dirinf'ection of a room a 'knapsack' sprayer, or large volume garden

willr n long lance should be used. In this case the necessary safety pre-

rhould be taken to avoid exposure of the face, eyes, and lungs. Itemsre or plasticware are best treated by being fully immersed in the

Protocol 8. Fumigation of laminar flow cabinets

1. Clean the cabinet.

2. Fill the integral formaldehyde generator attached to the exterior, or asmall portable generator placed inside, with c. 25 ml of formalin solution.

3. Also place any items of equipment used in cell culture procedures in thecabinet e.g. pipette-aids etc.

4. Replace the door and seal with masking tape.

5. Switch on formaldehyde generator and place a warning notice on thecabinet.

6. Leave overnight before turning on air-exhaust while opening cabinetdoor.

7. Leave to vent before cleaning cabinet to remove residues ofparaformaldehyde,

44

11t ,

4l

Peter L. Roberts

7.2.1 AldehydesFormaldehyde and glutaraldehyde can both be used as liquid disinfectanalthough glutaraldehyde is more commonly used and is probably moreive. They both have the advantage that they are not influenced by tpresence of high levels of organic matter and they inactivate all typesmicro-organisms including bacterial endospores. Formaldehyde is usedabout 4o/o, conveniently prepared by diluting 40"/". formaldehyde soluti(formalin). Glutaraldehyde is used at2/". Examples of commercialtions include Cidex (Genesis Service Ltd) and Gigasept (Sterling MedicareTreatment times are typically of the order of at least 30 min, with longer tirequired to obtain full sporicidal activity. Proprietary glutaraldehyde-disinfectants need the addition of an activator before use and then haverecommended life-time of about L week. Aldehyde vapours are considerelatively toxic due to their ability to sensitize, and they also have mutaand carcinogenic properties. Steps to limit exposure should thereforetaken.

7.2.2 HypochloriteOne of the main advantages of using hypochlorite is its relatively low cost aready availability e.g. in the form of household bleach or ChlorosChemical Distribution Ltd). However, it has the disadvantage that it is noteffective in the presence of high levels of organic matter. It alsometals and thus must not be used with centrifuge rotors or cell culcabinets. It is not very stable after dilution. The concentration recommefor use when high levels of organic matter are present is 10000 p.p.m.available chlorine, although lower concentrations have been recommefor routine hygiene e.g. 2500 p.p.m. The concentrate is stable but didisinfectant should be changed after 24 h. An exposure time of at I30 min, and preferably overnight, is recommended. For treatingsodium dichloroisocyanurate powder e.g. Haz-Tab granules (GuestLtd) can be used to release high levels of chlorine rapidly.

7.2.3 PhenolicsClear phenolic disinfectants, e.g. Hycolin (William Pearson I td), alnot inactivated by organic matter, have little activity against bacterial e

rtporcs. 'Ihey are commonly used at a concentration of 2-5"/o, or as

mcnded by the manufacturer. They also may leave sticky residues whenfor cleaning surf$ccs,

7.2.{ AlsoholEthenol ls eommonly uned tbr disinfecting surfaces and hands (pregloved, but aomo peoplc treut their hands directly). Its effect is optimalconcentratlon of 7(l:8(lol' flnd surfaces must be fully saturated to ensure t

2: Sterilization

€rposure time is sufficient. The ethanol/water mixture is then left tonaturally. Although a convenient and easy disinfectant to use, and

toxicity, it is not very effective against fungi, bacterial endospores, orloped viruses. It is best used as a cleaning agent or disinfectant for

€fltical applications e.g. for treating microbiological safety cabinets be-End after routine cell culture work (see Protocol 10).

Othersother disinfectants that can be prepared in the laboratory or purchased

EBftmcrcial formulations may have a place in the cell culture laboratory.include hydrogen peroxide at 5-10"h, acids, alkalis, ethanol (70'/.)

with 4"/" formaldehyde or 2000 p.p.m 'hypochlorite, or Mikrozidnol/aldehyde mixture, Sterling Medicare). One proprietary

nt (Virkon, Antec International), that has been shown to inactivaternnge of viruses and other micro-organisms, has three components: anng agent (peroxide), acid (pH 2.6), and detergent. With this agent the

conditions are a1.."/" solution and an exposure time of at least

FlltrationFllters for bacteria and fungi€€rlicst examples of this type of filter were first developed in the late

end used such materials as unglazed porcelain, diatomaceous earth,or sintered glass. These materials acted as depth filters in which

were trapped within the filter. For sterile filtration, such filters havebecn replaced by 0.2 pm membrane filters, first developed in the

, 'l'hey act more like sieves which trap the bacteria on their surface,gh the distinction between the two types of filter is not absolute.

rne filtration is the method commonly used for solutions that cannotlterlllzett by methods such as autoclaving, because they contain heat-labile

ts. It suffers, however, from the disadvantage that, because steril.rkrcs not take place in the final sealed container as is the case for

FBmpte with autoclaving, steps must be taken to prevent recontamination

EEFfee,t liltration and bottling. Uses in the cell culture laboratory may includerrg culture media, sera and other cell culture supplements, and any'rtl products made by the cells.

tr1i1 'l'ypee of filter$ rangr ol' filter materials is available which can be used for removing

E€gterln rrrrd fungi (8). For the filtration of liquids, these include materialspp6e trv casting processes such as cellulose acetate, cellulose nitrate, or aFi*tute ol'both, or nylon or polysulphono, Other materials with low protein-

Sadtng pluperties, e,g. polyvinylidene difluoridc, are also available although

40 47

Peter L. Roberts

for most applications in the cell culture laboratory (such as filteringculture media) this property is probably not really necessary. In additifilters can be made from polycarbonate by the irradiation-etch meThese have discrete pores with a much narrower range of sizes andact exclusively by retaining micro-organisms on their surface. Full deton the various types of filters that are available, together with appliand selection guides and details on chemical compatibility and othercan be found in the extensive literature produced by the various mfacturers such as Gelman Sciences, Nucleopore (Costar), Sartorius,Millipore, etc.

Membrane filters come in a range of pore sizes, but 0.2 pm is consithe standard for removing bacteria and fungi. Larger pore sizes, andpre-filters e.g. made of glass-fibre, are useful in serial filtration systemsincrease the filtration capacity where significant levels of insolublemay exist, e.g. in serum, complete medium containing serum, or cell culsupernatant harvests. For effectively removing mycoplasmas, filters withpore size of 0.1 pm are required. Filters with pores of this size are nowby most commercial processors of serum products. In addition, filtrationthis level may also be useful for removing some of the larger viruses thatbe present in such products, e.g. infectious bovine rhinotracheitis virusparainfluenza-3.

Low levels of various extractable materials may be found in mefilters, e.g. surfactant used during manufacture or residues of the eoxide gas that is used by some manufacturers as a sterilizing agent (9,10).critical cell culture applications, where the complete absence of suchis necessary, filters with very low levels of extractable material whichbeen sterilized with gamma-radiation, can be used. Alternatively,practical, simply discard the first sample of filtrate. Apart from filteriliquid, membrane filters are also used. for filtering gases e.g. CO2 orsupplied to cell cultures growing in mass culture systems or via a filter insein the cap of cell culture flasks (e.g. Bibby Sterilin, Life Technologies Ltd,Costar). Various types of filter, ranging from membrane filters to sicotton wool plugs, are also used to protect liquid handling equipment suchpipettes, automatic pipette handling devices, and micropipette tips, fromor liquid-borne contamination.

tt.1.2 "l'ypes of filtration unitIrr ucklilion to thc wide range of filter types that exist, filter housingscrtttte itt ll lnng,c ol' shapes, sizes, and materials. The filter manufactunlruultl hc colrsrrllcrl lilr dctails of the physical and chemical propertieslroth lillclr nnrl lrorrsings. Sizes and formats range from the simple syrilllteL ol' ll ot 25 rtrrrr tlirnrctcr which will filter small volumes, to caol'vnrioulr xizes lrrtrl rltrpes, ol'lcn pleated to maximize filter area, for

2: Sterilization

Egille (i.c. bottle-top filters), some with integral receiving vessel, are also

Blallnlrlc.Flltcr units comprising the filter housing and the filter itself can be pur-

*aletl cither as pre-assembled, sterilized, and disposable units or as a re-

l*€Rble lu,using which, after fitting the required filter(s), can be sterilized by

il$teeluuittg. Some points to consider with regard to the different systems are

Etetl bcl.rw.

[El dlnlt,tsaule units:t ltrtr convenientO ltttvc high unit cost

I lrnvc quitity control of filter batches, including integrity, carried out by

lltc tnanufacturer

lbl re"rrsrrbte systems:t=' I t,ctpire assembly and sterilization before use, followed by disassembly

rntl cleaning after use

t utny a greater risk of failure due to improper assembly

I t nl'lcr initial purchase of the housing, have low unit costs

&19 l'rnr:tical asPects

l{emhr,,,," filtration can be carried out under positive pressure e.g. using a

SFngn lirf small volumes up to c. 50 ml, air pressure from a pump or a

fulitrc-tirr", or a peristaltic pump. Negative pressure can also be used with

Ftb=t,rl liltcrs and filter units with integral receiving vessels. However, this

*-thrxt irls the disadvantage that, with tissue culture medium containing

€F€fh'rrrrrc,, the pH rise caused by filtration is greater than that caused by thegi*ttlVn lrrcssure methods. In addition, when protein is present, frothing and

*leltt ilcrrilturation can occur. After filtration, the liquid can be collected

ff*et lrrr.t ir single final sterile container or, where smaller aliquots are

*gttn,l, tlircctly into vessels of a convenient size e.g. 500 ml bottles for basal

EEll utrlt,,'.' tncdium. The filter, cell culture, and plasticware manufacturers

$n ;truvittc various complete filtration systems designed for use with particu-

let lirprt,t volumes.

llt'l\tiltxtil 9 the typical steps involved in liquid filtration are given.

irrg lurga volunror, l)iupottblc units designed for filtration clirect into

Peter L. Roberts

Protocol 9. Continued3. Assemble the unit taking care to install all supports and o-rings in their

correct positions.

4. Attach tubing, if necessary, to inlet/outlet ports.5. wrap the items and sterilize by autoclaving at 12r.c for r5 min using a

porous-load cycre. Higher temperatures, or dry-heat sterilization, mustnot be used unless recommended by the filter manufacturer.

B. Filtration

1. Assemble any accessories that are required in a raminar frow cabinet.2. Remove the sterirized firter from its packaging using aseptic tech-

niques, and assemble the complete filtration system.3. Tighten all connections and attach to pressure system.4. Start filtering into sterile container(s), after having bled off any air in the

system via the air vent (if present).

5. Test the integrity of the filter unit and/or carry out sterility tests on thefiltered liquid as necessary.

8.1.4 Testing of filters

rpecific lilter is provided by the manufacturer.

To ensure the correct functioning of the filtration system, the assembled ri.e. filter and. housing, needs to be tested for intigrity. The manufactu:ayl- out various physical tests that are related to ttre actual pore sizebubble-point or air-flow/diffusion tests. These are then correlated withactual performance of the filter-or filter unit in tests designed to challengefilters with high levers of a smail test bacteriurl, e.g. psiudomoni dimiru,for a 0.2 pm filter. A number of quantitatiue pro"edures can be carried outthe user to confirm the integrity of the filter unit. The tests shouid be caout after and, where considered necessary, before using the filter. ihe simmethod is to confirm that significant resistance (i.e. b-ubble-poi"rpr".r"see below) is felt when trying to force air forwards or backwards throuwet filter- If air passes through unhindered then the filter is damaged.quantitative methods are:

(a) 'l'he .bubble'point method, which is based on determining the pre

requirccl to force air through the wet filter. An expected nul"lu" ,urrg"

2: Sterilization

'l'hc pressure-hold test, which is based on measuring pressure decay afterpressurizing the upstream of the filter. The test measures not only thelntcgrity of the filter but also that of the filter housing. It is usually carriedout using test kits provided by the manufacturers, which can often alsoporform the diffusion air flow/forward flow method.

In atldition to these physical tests, sterility testing of the final product, bylating a range of microbiological growth media, can be carried out. Both

y testing and sterility testing are routinely carried out in the pharma-industry.

Fllters for viruses0.1 pm membrane filters can be useful for removing large viruses,

rly when several are used in series, membrane filters of a muchpore size are required for the removal of most other viruses. Recently

llltcr manufacturers have introduced filters which fulfil this require-I Millipore (L7,12\, Asahi Chemicals (13), and Pall Corporation (14).

now filters are available in a range of formats, e.g. tangential flow units70k or 180k, Millipore), hollow-fibre cartridges for tangential or

filtration (Planoray 15, 35, 40, or 75 nm, Asahi Chemicals) or dead-

flhcr units (0.04 pm Nylon 66, Pall Corporation).All thcse systems use membrane filter technology except for the hollow-

iy$tcm which acts as a high-performance depth filter. The Viresolvel'rom Millipore and the Planoray system from Asahi have been the

fully validated of this new technology. The Viresolve system has beento remove viruses exclusively in a size-dependent fashion. Some filter

eolnc in a range of pore sizes and the most appropriate type that willffilze virus removal without removing significant levels of any important

nrnrponent or product should be selected. For instance the pore size oflerpcst Millipore Viresolve membrane has a nominal molecular weight

lrrting of L80,000 Da which is close to the molecular weight of im-nughrbulin G (IgG). It is thus essential to test the suitability of such filtersnny spccific application.

Fttme rrspccts to be considered with regard to virus-removing filters are

lbt:tl hckrw.

{C} 'ft,t* is a new technology and further developments are likely.

{bl 'ttt,' cl'l'cct of filtration on the levels of important proteins (e.g. in thenterliurn or product), must be tested.

[€] flent,tuul of large (>80 nm) viruses, e.g. infectious bovine rhinotracheitisvhrrn (lllRV), Epstein-Barr virus, and retroviruses, is most effective.

'- (ltlrer mcdium-sized viruses (c. 50-60 nm), e.g. parainfluenza-3, and:fltrrll viruses, e.g. picornaviruses, may also be removed to a lesser extent.

t€l E{fttei"rrey can be increased by using multiple units.

€I

iI

(b) 'l'ho difl'ueion air flow or forward flow method, which is based on deterrlng the uir llow

'cross the wet firter at u ,p""ifi"d pressure 1". soz ot

ttutrble-polnt presnurc). I[ is a more sopiristicatei method than bublpolnting, requlrlng n special test kit (available from manufactur.rry. anpected valuo rarrge, or built in pass/fail, is providcd by the ,unui""tu

60 [1

-.virus-removing filters have found application, in the cell culture field,:]llg1l* viruses such;s_bovin_e viraf diarrhoea virus (BVDVj u'a ottlarger viruses such as IBRV and parainfluenza-3 r.o- doui*'J"rrr-,manufacturer of bovine serum has used six filter units of 0.04 pm'to rePYDV and other possibre virar contaminants (15). virus ntte^ ar" curren

L:,:li ":llyl"d i" rhe pharmaceutical indusrry for use with a range of cculture-derived or plasma-derived biological products and further advancesthis new technology are likely.

Peter L. Roberts

(e) Costs are high relative to standard filters.(f) Units are designed for single use.(g) Methods for testing the integrity of filters, that can be carried out by t

user, have been developed.

8.3 HEPA filters

Table 3, Types of cabinet used for handling cell cultures

Protection "Typc

lamlner flow cabinet (vertical)Lamlnar llow cabinet (horizontal)Mlr;roblologlcal aafety cabinot (Class l)Mlcroblologktol rafety cobinet (Class ll).Mlerolrlologloal rdtoty cablnot (Class lll)

Operator

+++

H"Efl (high. efficiency particulate air) filters are used to filter large voluof air in sterile or clean rooms, and in laminar flow or microbiol'ogical sacabinets (L6,r7). Such filters act- as depth-type filters and are capableremoving >99.97"/" of particres of 0.3 pm or-larger. They are thus effectiagainst not only bacteria but also viruses which exist in tne atmosphrattached to dust particles and riquid droprets. In the pharmaceuticar indusstandards exist for the quality of air i.e. the maximurn level of farticles arviable micro-organisms permitted for a particular crass of room or cabinet. F,asepticfilling, an environmentwithno mbre than l00particlesof >0.5 pm/foot (3530/m3) is required to meet class M3-5 of ub rederal Standard 2other clean room classification standards, e.g. BS5295, exist. The varitypes of cabinet that use HEPA firters are lirt"d in Tabte 3. celr curtshould be considered as a potential source of viruses (see crrapters 4 andand it is thus best to handre them in a crass II microbiorogicaisalty cabi

2: Sterilization

(ME(') which provides protection to both the operator and the cells. LaminarfiB* cnbinets should only be used for preparing media and horizontal models

FU:t rrol be used for handling cell cultures (see Chapter 4). Class I MSCsBffer tro protection to the work but are recommended because they give a

hlgh arrl consistent level of operator protection, when handling pathogens inhrccr.l group 3 (18). If it is considered essential that cell cultures and hazard

33€up 3 agents are handled together in a Class II MSC, in order to providepf6teetion to the cells, then a case must be made to the Health and Safety

E*ecrrtivc or other relevant agency. Additional safety testing of the MSC isllkely t,r be required. Alternatively, a fully enclosed Class III cabinet wouldpFevlrlc lull operator protection and some level of protection to the workBE€arr*c the air, although not laminar, is filtered. A Class II MSC is in fact

l€eqrrrrlc lbr work with most common microbiological agents, i.e. hazard

fFau;r ,2 pathogens (18). Guidance on the routine use and maintenance of€Ablncts is given in Protocol 10.

i,5. I |torformance testingllE|'n liltcrs and cabinets should be tested at least once a year, or every 6

fitaurllrs in the case of a room or cabinet used for the sterile filling of pharma-c€ttlilrrl ;rroducts or for handling dangerous pathogens, to ensure they areperfurrrring correctly. Cabinet manufncturers and clean-room environment

Air I

H

XV,XE,X

Product

+

l++

dPtottrrlhlr I | ), rro prolpolkrrr ( ),

" vartlilel hilrrner 6rr rrow {V}, horrronrar raminar air frow (H}, air firtered on €xhaust (X), or airon snlry and axheurt (E, X),uAlro oomnt'nry rlhttarr r. ec a crara I rominar frow hood or cabinet.

52 !t:t

Peter L. Robeds

specialists are able to carry out the necessary tests. For MSC, these ichallenging the HEPA filter with dioctyl-phthalate smoke particles of0.3 pm diameter; and measuring the air in-flow and downflow velocity,necessary for the particular type of cabinet, to confirm they meet the requistandard, e.g. 855726 in the UK. An operator protection test, usingiodide generated within the cabinet (Kl-Discus test), should also be carriout when the cabinet is first installed. This test is not strictly requiredunless the cabinet is moved or relatively high risk pathogens in hazard group(18) are handled. The level of particles within a sterile room can bemonitored by the use of a particle counter. Additional biological testsproduct protection in rooms and cabinets used for sterile filling can be carriout by exposing bacteriological agar plates.

AcknowledgementsI am grateful to the engineering and quality control staffat BPL for inon the routine maintenance, testing, and validation of sterilizers. I wouldlike to thank Paul Harrison for useful discussions and Christine Thompsonhelp in the preparation of the manuscript.

ReferencesL. Block, S. S. (ed.) (1983). Disinfection, sterilization and preservation, 3rd edn.

and Febiger, Philadelphia.2. Russell, A. D., Hugo, W. B., and Ayliffe, G. A. J. (ed.) (1992). Principles

practice of disinfection, presewation and sterilization.2ndedn. Blackwell SciPublications, Oxford.

3. Threlfall, G. and Garland, S. G. (1985). In Animal cell biotechnology (ed. R.Spier and J. B. Griffiths), Vol. l, pp. 1?j-40. Academic Press, London.

4. Department of Health and Social Security (1983). Guide to goodmanufacturing practice. HMSO, I-ondon.Prusiner, S. B. (1991). Dev. Biol. Standard.,75,55.Department of Health and Social Security (1980). Sterilisers. Healthmemorandum, no. 10. HMSO, London.Bowie, J. H., Kelsey, J. C., and Thompson, G. R. (1963). Lancet,i,586Brock, T. D. (1983). Membrane filtration. Springer-Verlag, Berlin.Gelman Sciences (1993). Laboratory Solutions,l, L

Knight, D. E. (1990). Nature,343,2l8.Dileo, A. J., Allegrezza, A. E., Jr and Builder, S. E. (199). Biotechnology,182,Dileo, A, J. and Allegrezza, A. 8., Jr (1991). Nature35l,420.Manabe, S, (1992). ln Animal cell technology: basic and applied aspec* (ed.Murakami, S, Shlrahata, and H. Tachibana), pp. 15-30. Kluwer Academicllshoro, Dordrecht.

14. Pall Prococa Filtratlon Ltd, Rctcntion of Viral Contaminants by 0.04 pm Nylon

54

5.6.

7.8.9.

10.

11.

12.13.

2: Sterilization

Ftltors, Pall Scientific and Technical Report STR1358. Pall Process Filtration Ltd,

Pottarnouth.*{Jci;;" Laboratories (1987). Art to science in tissue culture, 5, 1.

*iltprr, c. J. (1985). in I'itmat cell biotechnolga @d' R' E' Spier and J' B'

blfntftg, Vol. 1, pp. l4l-f/'. Academic Press, London'

E;iii;r, t. H. (;il (1993). Laboratory-acquired infectiow, 3rd edn. Butter-

?0fthfi, London.Atlvinory committee on Dangerous Pathogens. (1990). categoriz.atio.n of Patho'

ffflns uccording to hazard ind catego'iei of containrnent' 2nd edn' HMSO'

6[

Culture mediaT. CARTWRIGHT and G. P. SHAH

Introductiongrow cells in vitro, culture conditions must mimic in vivo conditions with

llltpect to temperature, oxygen and CO2 conceffiotr, pH, osmolality, andil[trition. Most basal cell culture media cannot, by themselves, support the

of cells and it is common practice to supplement cell culture mediaanimal sera. Growing cells in serum-free media has many advantages but

ihe ideal general purpose serum-free medium has not yet been developed(fnd is almost certainly an unattainable goal). The main functions of cell,fllture media are to maintain the pH and osmolality essential for cell viability|nd to provide the nutrients and energy needed for cell growth and multipli-fftlon. The temperature and oxygen and CO2 content of the cell cultureslllUtlt also be controlled. A complete cell culture medium can be considered to

composed of two distinct parts:

(E) a basal medium that satisfies all cellular requirements for nutrients

(b) a set of components that satisfy other types of cellular requirements andpermit growth of cells in the basal medium

A nutrient is defined as a chemical substance that enters a cell and is used as

Slthor a structural component, as a substrate for biosynthesis or energyltetabolism, or in a catalytic role in such metabolism (1). Anything else

heecled for cellular proliferation is normally classified as a supplement, in-Eluding all undefined additives such as serum and other biological fluids.

U, Basal media*l'he culture medium is by far the most important single factor in culturingtnirnal cells. The function of this medium is to provide an environment forturvival and also to provide substances required by the cells which theygannot synthesize directly. The composition of early tissue culture media was

bansd on biological fluids such as plasma, lymph, and serum, and tissuegxtrects especially of embryonic origin, Basal tissue culture media wcre

T. Cortwright qnd G. P. Shoh

developed to include only the minimal components which were essentialgrowth.

2.1 Types of basal mediumThere are four main categories of basal media for mammalian cellsseveral categories for insect cells. These are

o Eagle's medium and derivatives, e.g. BME (basal medium Eagle'sEMEM (minimum essential medium with Earl's salts), AMEM (minimuessential medium with alpha modification), DMEM (Dulbecco's modiEagle's medium), GMEM (Glasgow modification of Eagle's medium),JMEM (minimum essential medium with Joklik's modification)

o Media designed at Roswell Park Memorial Institute (RPMI), e.g. RL629, RPMI 1630, and RPMI 1640

o basal medium designed for use after serum supplementation, e.g. FischerLiebovitz, Trowell, and Williams'

o basal medium designed for serum-free formulations, e.g. CMRL 1

Ham's FLO and derivatives, TCl99 and derivatives, MCDB and derivaNCTC and Waymouth

For insect cell culture, the basal media (designed empirically) are

o Grace's medium

o Schneider's medium

o Mitsuhashi and Maramorosch medium

o IPL-41mediumo Chiu and Black medium

o D-22 medium

Most cell lines derived from cold-blooded vertebrates can be culturedone or more of the above basal media when supplemented with a biologifluid. However, an allowance is usually made for differences in salt cotion to obtain the optimum osmolality. Different incubation temperatumay also require adjustments to be made to the composition ofcomponents, since pH may change with temperature due to alterations insolubility of CO2 and in ionization and pKu of buffers.

2.2 Constituents of basal media'l'hc common constitution of basal media may be considered as follows.

2.2.1 llalnnced salt solutionBulanced salt solutions (BSSs) have been used since the earliest attemptscell crrlture in vilnt. A llSS is composed of a combination of inorganic sal

thnt rnuintain plrysiological pl{ ancl osnrotic pressurc. ln acldition to

3: Culture medio

Efreets, the inorganic ions used have other important physiological roles

iaeiUOing the maintenance of membrane potential and as cofactors in enzyme

Eaett,roi and in cell attachment. The inorganic ions employed are chiefly

lie*, K', Mgt*, C**,Cl-, S@-, tcl31, and HCo3. When necessary, osmo-

lglity rn,,y be adjusted by modifying the concentration of NaCl.=:'M1rrt

RSsr oo not contain the nutrients required by cells for long'term

FElntenilnce or growth although glucose may be included' The four main

€€legrrrics of BSS are

! Ecrle's balanced salt solution (EBSS)

! Bulhccco's phosphate-buffered saline (DPBS)

! Henk's balanced salt solution (HBSS)

! Eagle's spinner salt solution (ESSS)

$Egli nrrct DPBS are intended for use equilibrated with air while EBSS and

ibBS r.quite equilibration with a gas phase containing 5"/" COz in order to

EBlntni,t the correct PH.

lrl,g lluffering sYstems

Eni*or. ,u"oia .reeo to be buffered to compensate for evolution of co2 and

EE pt,nto"tion of lactic acid from the metabolism of glucose. Media have

Hditl,,nutty been buffered with a bicarbonate buffer, often at a final con-

*?lraf i.r,r tt Z+ -tr,t. Bicarbonate forms a buffering system with dissolved

E, pr.r.tu.*d by growing cells. Horvever, when cells are growing at a low cell

*Eift' or are i" a tag piur", insufficient CO2 may be produced to maintain

H€ r=q,,ir"a optimal lU. fo. this reason, these cultures need to be grown in

S=etrri,,rptrere of s-ioy. Co2. Bicarbonate is both cheap and non-toxic to

E'==,'=tt* liut its pK" (6.1) resulis in sub-optimal buffering in the physiological

ieig=, S,,r" rn"Oiu are designed to coniain low HCO3 but high PO3o con-

Fa?uti,,,.,', and therefor" do not require incubation in a CO2-enriched

iiao*t,t,"r". sodium p_glycerophosphate is also used as a buffer in some

EFmuinri.rns. Each bisil *editr- has a recommended bicarbonate

$-He.errtr.lfion and CO2 tension to achieve and maintain correct pH and

gssrrlrrlity (Table I).=-F,,, ,,,,ir" effective buffering, without the need for elevated CO2 levels' a

faaEe .,1 organic buffers "un

b" employed. The most widely used of these

b_uiF;,- is frepes (N-2-hydroxyethyipiperazine-N'-2-ethanesulphonic acid)'

F:epes i* ,, u"ry effective buffer in the pH rangeT.2-7.6 and is more resistant

F-iepirl pl{ ciranges than bicarbonate. Some media are buffered with both

E-i=*ri,,,,,u," and tlepes' However, Hepes is both expensive and toxic to the

ettr u, rrrncentrations above 1fi) mM. Impurities in Hepes preparations have

ilac 1r."" rcported to cause cytotoxicity at lower effective Hepes concen-

Fjiftu,,*, Other organic buffers, related to Hepes, which have been used are-E:i

1,V rr'(hyclroiymethytmethyl)-2-aminoethanesulphonic acidl and Bes

5g58

T. Caftwright ond G. P. Shoh

Table 1. Recommended CO2 concentration (gas phase) to use with com-mon basal media

Basal medium

Eagle's MEM (Hank's saltslGrace's (Hank's salts)IPL-41 (Hank's salts)TC 100 (Hank's salts)Schneider's (Hank's salts)IMDMTC 199DMEM/Ham's F12RPMI 1640Ham's Fl2DMEM

NaHGO3concentration (mM)

44444

362629241444

% CO2 ingas phase

AtmosphericAtmosphericAtmosphericAtmosphericAtmospheric5\5555

10

[N,N-bis-(2-hydroxyethyl)-2-aminoethanesulphonic acid]. Althoughorganic buffers function without COr, it should be remembered that bicarbonis essential to cells as a nutrient independently of its buffering role and sufficibicarbonate for this requirement must always be present in the mediActively growing cells will usually themselves produce sufficient CO2 for

2.2.3 Energy sourcesCarbohydrates are a major energy source for cultured cells. Glucose is tmost frequently used sugar. Other sugars, e.g. maltose, sucrose,galactose, and mannose, may also be included. Glutamine can also supplymajor proportion of the required energy in some cells.

2,2,4 Amino acidsMost animal cells have a requirement for the essential amino acids, i.e.which are not synthesized in the body. In the human, these are arginicystine, histidine, isoleucine, leucine, lysine, methionine, phenylalanithreonine, tryptophan, and valine. Cysteine and tyrosine are also includedtltis group to compensate for inadequate synthesis. Most animal cellshuve a high requirement for glutamine. Glutamine acts both as an ene$ourcc nnd as a carbon source in the synthesis of nucleic acids. Otherluci(1il ntc ol'ten rclded to compensate either for a particular cell type's incaIty lo rnrke thenr or bccause they are made but lost into the medium.

2.2.S VltnmlnnSeverttl vltunlinB ol'llre I] group are necessary for cell growth and multiplitlon, Mntty vlturnlrrx llre precursors for cofactors. The vitamins most comudtled lrt bassl me(lln nre /ral(r-itmino benzoic acid, biotin, choline, folicnieotinlc ncid, panlotltenic ncitl, pyridoxal, riboflavin, thiamine, and i

3: Culture medio

The importance of other water-soluble vitamins is less clear. Vitamin B12 has

becn reported to be essential for some cells and is included inFl2 medium.

Few data are available on the role of fat-soluble vitamins in medium. Medium109 contains both vitamin A and vitamin E.

8,2.6 Hormones and growth factorsHormones and growth factors exhibit a variety of different effects on cells.

These are included in some media (especially serum-free media) at relativelylow concentrations. Insulin and hydrocortisone are main examples but growth

faetors like NGF (nerve growth factor) and EGF (epidermal growth factor)havc also been used as well as certain interleukins, colony stimulating factors,

Bnd fibroblast growth factors (FGFs) (for examples, see Chapter 6).

t,2.7 Proteins and peptides

Although an absolute requirement for proteins and/or peptides by cells inCglture has not been established, relatively few media have been formulated

ln wtrictr cells grow rapidly in the total absence of proteins or polypeptides.

€ommon examples of protein supplements used are fetuin, a-globulin, fibro-ncctin, albumin, and transferrin.

t,2,8 Fatty acids and lipidsAn with the proteins and peptides, there is no consensus regarding an essen-

tlsl role for lipids in cell culture. However, fatty acids and lipids are important€omponents of several serum-free media.

t,2.9 Accessory factors$nrongst these are the 'trace' elements, especially iron, zinc, copper,

Glenium. A variety of other compounds, including nucleosides and

Earboxylic acid cycle intermediates, may also be added to the medium.

1,2.10 AntibioticsAlthough antibiotics are routinely used in laboratory-scale tissue culture, they

lhould ideally be avoided since resistant micro-organisms may develop and

Eell growth and function may also be adversely affected. Whenever possible,gnlibiotics are not used in industrial-scale cell culture where reliance is placed

on ;rlant which is correctly designed and of appropriate quality to maintainpullure sterility. The use of antibiotics in biopharmaceutical production is notrendily acceptable from a regulatory vigwpoint.

When antibiotics are to be used in cell culture, the key factors governing

llrcil choice are

I ubsence of cytotoxicity

t broad anti-microbial spectrum

I ucccptable cost

t nrinimum tendency to induce formatitln tlf resistant micro-organisms

01

andtri-

T. Cartwfight ond G. P. Shoh

Mixtures of penicillin (100 IU/ml) and streptomycin (50 pglml) are thefrequently used anti-bacterial agents. Gentamycin (50 p,g/ml) is more expesive but is widely used to treat persistent contaminations. AmphotericiB (2.5 pg/ml) is the most commonly used anti-fungal agent, but is cyfor some insect cells. Nystatin (25 pglml) is also an effective anti-fungalin tissue culture medium. For further information on antibiotics, see Chapter

2.3 Choice of basal mediumThe choice of medium is not always obvious and frequently remains empiin spite of many years of exhaustive research into matching particular medito specific cell types and culture conditions. Useful information is usuavailable in the literature or from the source of the cells. As a general guiBME will usually support the growth of continuous cell lines, e.g. HeLa,cells, BHK-21, and. primary cultures of human, rodent, and avian fibroblRPMI medium is intended mainly for cultures of human haemopoieticwhile Fischer's medium isintendedprimarilyformouse leukaemiccells. Iscovemodified Dulbecco's medium (IMDM) is widely considered best forcells of haemopoietic origin and supports the growth and differentiationboth human and murine primary bone marrow cultures. Most insect cells, e.Sf9, Sf21, Bombyx mori, Trichoplusia ni, and Drosophilawill grow in Gracemedium supplemented with fetal calf serum.

2.4 Preparation of basal mediumContamination of medium with micro-organisms, e.g. bacteria, 1leaSt,fungi, and with noxious chemical substances, e.g. traces of heavy metals,the greatest hazard in media preparation. For this reason, particularmust be taken in the selection and preparation of materials. High purityshould be used (see Chapter 1). Biochemicals should be of analyticalItems of glassware used in dispensing and storage of reagents and media mbe cleaned very carefully to prevent traces of toxic materials from contamiating the inner surfaces of vessels and thence becoming incorporated intomedium. Basal media are frequently prepared by diluting a series ofsolutions, e.g. amino acid and vitamin concentrates, in water. These s

solutions are stored separately in conditions appropriate to the indivicomponents. Incompatible substances are kept separate until they are mtogother to make the complete medium.

'l'hc complete medium is usually sterilized by filtration (0.1*0.2 pm).mcdir can ulso be sterilized by autoclaving, e.g. EMEM, but care musttuken to sltbilizc thc B vitamins, and glutamine should be substitutedglutunrute or udded al'tcr autoclaving. Powdered media are preparedclissolving the powtler in the rccommended amount of water and ensuringnll the conntiluentr ule completely dissolved. Unstable constituents (e

3: Culture medio

JUat before use. Glutamine is a key metabolite for growth of animal cells but itlg ttlso relatively unstable and decomposes to produce ammonia which is toxicto colls. The dipeptide glycyl glutamine has been shown to be an adequateiubstitute for glutamine for some cell lines, and is more stable than glutaminedurirrg both autoclaving and storage (2).

1,0.1 Equipment for preparation of mediaWhcther preparing medium from powder, concentrates or the constituentehemicals, the following equipment is required:

I ltigh purity chemicals and biologicals

I gttod analytical balance

I hot plate with magnetic stirrer

I vrtlumetric flasks of various volumes

I pll meter

| 0ilnlometer

a Bterilizing equipment: autoclave and membrane filtration

Whcn preparing larger volumes of medium, it may be more practical forlEme purposes to measure weights rather than volumes. A large capacitybElarrce may also be appropriate in such cases.

1,4.2 Preparation of media from powderFEr lirrge-scale tissue culture applications, it is often economical and practicaltg nrrrke up single strength media from powder as outlined below.

Protocol 1. Preparation of media from powder

Using a graduated container of appropriate capacity, dispense 90o/" althe required volume of tissue culture grade water, The temperature ofthe water should be 15-20'C.

Add the appropriate amount of powdered medium whilst gently stir-ring the water. Stir until all the powder has dissolved. Do not heat thewater,

Rinse the powdered medium container with a small amount of tissueculture grade water and add to the bulk volume.

4, Add the required amount of buffer solution and any other additives.

E. For bicarbonate-buffered media, adjust the pH to 0.2-0.3 pH units belowthe desired pH using 1 M NaOH or 1 M HCl. Stir gently while adjustingthe pH. The pH will normally rise by 0.1-0.3 pH units during filtration.

6" Make up to the final volume with tissue culture grade water and mix bygontle stirring.sodiunr lricartrorrale or u*corbntc) are usually added as a sterilc conce

T. Cartwright ond G. P. Shoh

Protocof 1. Continued7. Sterilize by filtration using a membrane with a pore size of 0.22 y,m or

less. A positive pressure (3-15 p.s.i.) is recommended to minimize theloss of CO2.

8. Store at 2-8'C in the dark.

2.4.3 Preparation of single strength media from 10xconcentrate

To prepare single strength media from a 10x concentrate, the followingneed to be taken.

2.4.4 Precautions to be taken during preparation of mediao avoid using partial quantities of prepackaged powder media because of

hygroscopic nature of many medium componentso prefilter water (0.1 pm) prior to use

o avoid excess acid/base additions. ensure that mixing vessels are properly cleaned (depyrogenation may

be appropriate for some applications)o perform filtration as soon as preparation of the medium is complete

2.4.5 Quality control of basal mediumComplete basal medium should satisfy the following criteria:

(a) it should be a clear solution

(b) it should have the correct pH at room temperature: this is specificindividual media

(c) osnrolulity should be correct-this is specific to individual mediausuully in tlre rangc 280-300 mOsmol/kg

(d) rts juclgcd by 11l'1,() analysis, amino acids should be present attiulrs cunninlent with tlro l<lrmulation

{a)

(0(:t

3: Culture medio

lhc concentrations of key elements should be confirmed by chemicalrunalysis as defined in the formulationit should comply with standard protocols for sterilitylhc endotoxin level should be less than 1 nglml

Tlto basal medium must also be able to support the growth of appropriate@lh through at least two sub-culture generations (serum supplementation is

Hfttttlly required to achieve this).

l,0,ll Storage of mediumIt tr general rule, medium is best kept at 4'C and the shelf-life at thist€lttpcrature does not usually exceed 3 months unless otherwise specified

by tt," manufacturer. Once glutamine has been added, the shelflife is, in

lettcrirl, reduced to 2-3 weeks although individual media can last muchIttttgcr than this at 4'C or even at room temperature. Media which containhhllc constituents should either be used within 2-3 weeks of preparation orlftttctl at -20'C.

!, Serum

F,l Why use serum?Hlrlorically, the first tissue culture experiments were performed using animalbttly lluids such as lymph to support cell growth. When Eagle and others, inthe lnte 1950s (3), produced basal media containing amino acids, carbo-hytlrrrtc, vitamins, and minerals, it became apparent that supplementation offfictliurn with body fluids was still needed to provide unidentified, but essen-

liul, l'actors needed for efficient cell growth. Supplementation of basalffi€rlirrrn with up to 20"/" of animal serum became widely used. Because of itsflt,lt trrntent of growth factors and its low gamma-globulin content, fetalhrvirrc serum (FBS) has been adopted as the standard supplement. FBS isnow nrost frequently used at 107" concentration although this may beClttrrrgcd for specific applications. Advantages of serum use include thefitllowing:

(rt I

lh)

Strrum represents a cocktail of most of the factors required for cellploliferation and maintenance.

Scrum is an almost universal growth supplement which is effective withrrrost cells. Using serum-supplemented medium therefore reduces therrcccl to spend time developing a specific, optimized medium formulationlirl cvery cell type under investigation.

Sclum buffers the cell culture system against a variety of perturbationsrrrrtl toxic effects such as pH change, or presence of heavy metal ions,prrltoolytic activity, or endotoxin,

'l'ltrst' points are discusscd in morc dctail in Scction 3.3.

05

Ir' I

Protocol 2. Preparation of media from 10x concentrate

1. Using a graduated container of appropriate capacity, add the appropri-ate volume of the concentrate to 8O/o ol the required volume of tissueculture grade water and mix gently.

2. Add the required amount of buffer.

3. Add the appropriate volume of 200 mM l-glutamine and any otheradditives.

4. Follow steps 5-8 of Protocol 1.

f

T. Coftwright ond G. P. Shoh

The use of serum also imposes a number of difficulties (discussed in Secti3.4) which impact on the safety, reproducibility, and cost of biopharmacecals produced in animal cells. These difficulties can be minimized by ca

selection and validation of serum sources. Although almost all new manufturing processes using animal cells are designed for serum-free medium iorder to avoid these difficulties, many existing processes still use FBsupplemented medium. This situation is unlikely to change fundamentally ithe near future since regulatory constraints generally make it impractical auneconomic to alter existing processes.

3.2 Types of serumDespite its high cost, FBS remains the most frequently used serum for medisupplementation. Several different types of serum have been proposedcheaper alternatives to FBS. Calf serum is quite widely used industriallyis available either as newborn calf serum (which has high levels of biotin)as mature calf serum. Newborn calf ^y-globulin levels are high as a result ofingestion of colostrum immediately after birth. Adult bovine serum isoccasionally but is not usually as effective as FBS or calf serum. Horse se

is also used, particularly with some human cell lines. The use of human se

has been proposed for some fastidious human cell lines, but it is not cestablished that human serum performs better in general than FBS.

Whereas FBS is usually collected at the abattoir, generally bycardiac puncture, calf serum and horse serum can be produced from 'animals. In the donor system, herds of virus-screened animals are maintaiisolated from other stock and are used exclusively for serum production.health of each individual animal is constantly monitored, with special reence to virus infection. Animals are bled at intervals by aseptic venepunctas for human blood donors. Advantages of this system over slacollection are:

o better control of animal husbandryo comprehensive knowledge of the animals' health status

full control of blood collection and processing in a fully integratedmanufacturing practice (GMP) process

improved consistency of serum quality since animals remain in theherd for several years

o full traceability from bottled serum back to the individual animal if req

Serum from donor animals is available from a number of companiesing thc Salzman Corporation, Bocknek Ltd, and TCS Biologicals Ltd.

3.3 Constituents of serumSerum is urr cl'l'cclivc growth-promoting supplement for practically all typescell (for $ontc cxr:el'rliolrs, scc Chaptcr 6) because <tf its complcxity and

00g7

3: Culture medio

ffiUlligrlicity of growth-promoting, cell protection, and nutritional factors that

It Crtrrltins. These can be divided into specific polypeptides which stimulate

Fll growth (growth factors), carrier proteins, cell protective agents, cell

IttAehnrent factors, and nutrients (some of which may be small molecules

Thlclr rrre attached to carrier proteins). Some serum macromolecules can fillFttrc tlran one of these roles.

}fl,| Growth factorsFolypr,ptide growth factors are of particular importance in serum. These

l=t(l kl)a proteins act via specific cell surface receptors as signals which

lllttttrlrrtc cell proliferation or differentiation. In some cases, the presence

Ef eer.tain growth factors may not be stimulatory as such but may still be

lllpttlirrl since deprivation of the factor initiates a pre-programmed auto-

d€rlructive sequence of events (apoptosis) which results in the death of cells

EVetr lhough they may be fully provided with nutrients and be maintained

Efldet' optimal culture conditions (4).

llll'l'crcnt cell types have different growth factor requirements and the same

llttwtlr l'actors may stimulate or inhibit depending on the cell type and the

ifnwttr lactor concentration (5). Different types of serum (and different

bllClr.* of the same serum type) may contain different absolute and relative

bVelx ol different growth factors. This is one of the main reasons why growth

lertlrrg of serum batches is necessary to ensure satisfactory performance withlhe rpccilic cell line of interest.

1S,2 AlbuminAllrrrrnirr is the major protein component of serum and exerts several effects

lltlr,lr contribute to the growth and maintenance of cells in culture. It functionspt il r,rrlricr protein for a range of small molecules, particularly lipids. Transport

ttf lirtty acids is an important function of albumin since these are essential for€€llr lrrrt are toxic in the unbound form and are also very poorly soluble in water.

Elcr uitls and fat-soluble vitamins may also bind to albumin. (Other lipids such as

€holcstcrol, cholesterol esters, triglycerides, and phospholipids are transported

ilt rcrrrrn in micellar form complexed with specific lipoproteins.)Allrrrrrrin also has specific binding sites for thyroxine and for metal ions such

Bt Nir I and Cuz *. There is evidence that albumin may also bind other metals

Fltrl rrlso carry other, unidentified components which support cells in culture.'l'ltt. rrlrsorptive capacity of albumin also enables it to act in a detoxifying rolehy lrirrtling toxic metal ions and other inhibitory factors.

Allrrrrrtin also functions as a pH buffer and protects cells against damage by

therrr lorccs that may occur in stirred or pumped culture systems. This latternl'|elt irppcars to be entirely mechanical and related to the hydrodynamic

Fl'npc!li(js of the medium since cells become protected immediately after

€rilltiorr ol'albumin, before the albumin prcparation has had time to exert

aty possiblc mctabolic cffccts (6).

T. Cartwright ond G. P. Shoh

3.3.3 Transferrinlron is an essential trace element for cultured animal cells but can be toxic iprgs,ented in an inappropriate form in the medium. Iron salts are also sparinglsoluble in medium. Transferrin (siderophilin) is the major iron transpor

3; Culture medio

Table 2. .Nutritional and protective factors which may be sup'plied by serum

protein in vertebrates, representing 3-6"/" of total ."iur protein. ;

transferrin/Fe3+ complex is taken up via specific cell receptors and, arelease ofthe iron, apotransferrin is liberated from the cell and recycled. Itnot clear if iron transport is the only role of transferrin; some reports sugg(that it may also transport vanadium, and others that it might have a widerin heavy metal detoxification.

copper may also be transported by a carrier protein (ceruloplasmin) and r

chelated form by small peptides such as Gry-His-iys (GHL, liveigrowth factor

Factor

Specific growth factors: EGF, PDGF, lGF,

FGF, lL-1, lL-6. insulinTrace elements

lronTincSelenium(also Co, Cu, l, Mn, Mo. Cr, Ni, V. As,

Si, F, SN)Lipids

CholesterolLinoleic acidSteroids

PolyaminesPutrescineOrnithineSpermidine

Attachment factorsFibronectinLamininFetuin

Mechanical protectionAlbumin

Buffering capacityAlbumin

Neutralization of toxic factorsAlbumin

Transport of metalsTransferri n/Fe3*Ceru loplasmin/Cu 2+

Protease inhibitorsct, antitrypsinct2 macroglobulin

Goncentration

1-100 ng/ml

1-10 pM0.1-1 pM0.01 p.M

c. 10 pM0.01-0.1 pM

0.01-1 pM0.01-1 pM0.01-1 pM

1-10 pg/ml

2-4 mglml

1.5-2.5 mg/ml0.7-2.0 mg/ml

Serum contains two major classes of wide spectrum protease inhibitors,antitrypsin and a2 macroglobulin, each representing around 2/o of the tcserum proteins. Proteases are secreted naturally by many cell types (toextent which depends on culture conditions) and are ,n"d in the sub-cultof anchorage-dependent cells. The powerful anti-protease activity of seruprevents proteolytic damage to cells and to products.

3.3.5 Attachment factorsserum also provides attachment factors which facilitate the bindinganchorage-dependent cells to the substratum. The major serum proteinvolved in attachment is fibronectin. Fetuin and laminin also play a role.A summary of the main components of serum that are known toimportant in culture medium is given in Table 2.

3.4 Potential problems with the use of serumThere are a number of serious disadvantages incurred when serum is usedsupplement culture medium. These have different impacts depending ondifferent uses to which the cultured cels are put; in the proiuction ofpharmaceuticals, compliance with rigorous rigulatory controls concepolcntial contamination by viruses and other adventitious agents is the priconcenl, whilc this may be of limited relevance in researclistudies. Theelil'licultics e ncountcred when using serum are detailed below.

3.3.4 Anti-proteases

ll.4.l l,ur:k ol' reproducibilityselrln bnlclrcs viu'y considerably depending on the characteristics ofHolrrce rrrrirnulr rnctl, orr.thc lbcd stuffs employed, on the time of year,llil'l'erenl bulclrr:s trorrruirr dil'l'crent absoluie and relative levels of grrl'ucloru, ('ertrrlrr lircrom nrrry bc cleficient in somc batchcs whilc others mayprcsclrl rrl ereernivc, ilrlrihitory levcls lirr somc cell typcs.

Vuri:rtions in performance of this sort are not tolerable in manufacturing

fft'r!('..sscs and are countered by the batch reservation system where batches

Ale lrr.ltl on reserve by the serum producer while the would-be user completes

lerlirrg ol'samples for efficacy in his own specific system. The situation is also

llrprovcd if the reserved batches are large enough to permit production over

a t'orrsitlcrable period.trr r.xpcrimental cell biology, it is also important to be aware of the inherent

Vntillrility ol serum which renders it very difficult to study the specific effects

rtf lrrolcculcs such as growth factors, cytokines, adhesion molecules, or matrix

Fuurlx)ncnl"s, all ol'which arc prosent itt unclcfined levels in serum.

68 00

T. Cartwright ond G. P. Shah

The presence of specific antibodies in serum may also profoundly affectresults obtained. This is especially true in the culture of viruses. Antibodiesserum may result from a natural infection with the virus in question orrelated species (which may be transmitted transplacentally in some cases),from prior vaccination of the animals used. It should be rememberedsome antibodies traverse the placenta and that even FBS may contain si

ficant inhibitory activity to infectious agents to which the mother hadexposed.

Serum may also vary depending on the quality and the reproducibilitythe procedures used for its collection. For instance, the length of timetween collection of the blood and removal of the cells is critical, and mbe minimized if lysis of cells and the release of cellular contents (possiincluding viruses) is to be kept low. Sterility of the operation and seveother process parameters are also critical. Reputable serum suppliersinvested heavily in the quality of their operation and serum productiGMP standards is a more complex process than many users realize. Relitissue culture quality serum is accordingly an expensive product.

3.4.2 Risk of contaminationSerum can represent a major route for the introduction into cell culturesadventitious agents including bacteria, fungi, mycoplasma, and viruses.could be disruptive in research projects and dangerous in pharmaceutimanufacture. In order to minimize risks of contamination, suppliersapply rigorous health checks to the animals used, use GMP facilitiescollection and processing of serum, employ thorough quality controland ensure rigorous batch documentation to permit verification of theprocess. The process employs aseptic collection by cardiac puncture orvenepuncture, aseptic clotting and clot removal, clarification, and sterilition by filtration terminating in double 0.1 pm filters followed byfilling. Several companies are now producing 40 nm pore size filtersshould provide additional safeguards by achieving better clearance of viHowever, it should be recognized that some of the higher molecularproteins (e.g. IgM) may also be removed by such filters. Serum is thenfor microbial sterility, for contamination by certain viruses, and forcapacity to support the growth of test cells. An example of the qualitytests routinely performed on serum batches is shown in Figure 1.

As the recent outbreak of bovine spongiform encephalopathy (BSE)the UK illustrates all too clearly, this approach to testing cannot eliminaterisks of contamination. As a further line of defence, regulatory a

now specify thut tlnly serum from specified countries of origin, wherelar agentr uf coneern nre thought not to occur, can be used in theof pharmaceulicnl, veterinary, and sometimes diagnostic products.restrictionn arb unlikely to nffect the research user, but are mandatory

3: Culture medio

FOETAL CALF SER(]M - CERTIIICATE OF ANALYS$

Bovine Viral DianhoeaPsrainfluenza 3

lnfectious Bovine Rhinohacheitis

Frimn Epittt"tiel Cell Growth Capacity

fiyel,tmritlyttidona Growth capacity

a't37"emOsmolftgmg/mlrng/mlng/mlPgl^l-g/nl

EU/ml

% of control% of control% of control% of control% of control% of control

Signed .'...............""""'Date

Signed .'..'...'..........."""Date

ldrtivo Cloning EfficiencY

lehllvo Plating EfficiencY

€yftrtoxicity check

Ereuttmntttkrn APProval

idrl'l l{olcase

F$lr t, An example of the type of quality control testing regime which should be

lppl I er I t o batc h es ot "" -'-ro I El* ::^t: ::^ :'": ::i*::l::' :: : ::n""'4

"ff

;';t;BHil,J'i#:'J;r;" ,;;;;'Jrpprlirn" originar documentation used to senerate this

l,ffinrary .hoet Bhould o" rr"if"[i" for examinition on request. The precise specification

lllgft l|n tho analysis certificate depend on the specific type of serum in question' All sera

h$Ed rhould, of course, rnot" no d"t""trbl" conteminaiion in the sterility tests' (NB: for

5lrsnr ol non-bovine .tis;;:;1;;;;;;tiil wlll be based on other virus tvpes')

7071

T. Cortwright ond G. p. Shoh 3: Culture medio

) is such an expensive commodity there is a temptation for unscrupulouss to misrepresent the origin and the quality of the material (7). It is

pgrtrnt, therefore, foruserswho require highquality serum of definedoriginUgc u reputable supplier who has direct sources in the required production

. Such suppliers will be pleased to provide comprehensive and verifiable,documentation for every serum batch and to allow audits of all stages

the production process. This approach is important, both for validation ofqunlity of individual batches and for continuity of supply.

€ontinuity of supply and consistency of quality are, of course, also import-trt the research user who may not be operating under strict regulatoryrnints. Again this is best achieved by dealing with reputable suppliersItuvo the physical and logistical resources to provide this service.

It nlrould be noted that, independently of the regulatory needs of thetology industry, movement of serum between countries is also subject

f€rtriction because of concerns by governments over animal health.

I Selection ofserumion of serum type and of serum batch is essentially based on empiricalion by the user. When a given cell type is first grown, it may be

hwhile to test its capacity for growth on cheaper serum types, possiblyrrg mixtures of different types of serum.

Altllrugh serum offered commercially will have been subjected to thorough, irrcluding tests for cell growth performance (Figure /), it is still routine

marry users to 'batch test' serum in their own system before buying. Forllelum suppliers will generally provide samples free of charge from

tlilTerent batches for the user to test in his own laboratory to select thernost suitable for his specific requirements. The quantity of serum

nliully required by the user is held on reserve until testing is completed.lry the user is primarily to check performance (Protocol3) but may

$16 lrrvolve verification of the absence of adventitious agents or analysis offfiy ltirrirrneter that is of particular importance for the user.

It la irrrportant to bear in mind that tests of serum batches for growth orpforlrrctivity should always be performed in an identical system (in terms of

. tA:ut rrrcclium, cell conditions, and culture configuration) to that in which thetsfurrr will finally be used, and should always include a previously evaluated

tsfeterrcc scrum as control. It is recommended that the cells are sub-cultured

Bl lesxt llrrcc times as indicated below. At each passage, growth results shouldFE lrrrurrrrlizcd relative to the reference serum. Yields within l-20yo of the

tsfetrrrcc ricrum would normally be considered satisfactory. In addition to cell

IlElrl, r'clls should also be examined at each passage for any indication ofpftoluxicily or of abnormal morphology. l'ests of cell growth from very low

tsedirrg lcvels may bc important lbr spccific applications. Tests of cloning

Effielerrcy or of plating efficiency ntay be pcrl'ormed to assqss this.

manufacturers of biopharmaceuticals. This has resulted in the creation

lif f T":?,:1""-"^l::":l^?l]:countries*iin-N"*z"uruna,-au,iJiu,a,,duS A o ccupyin g th e his.!e1 rr ac::i N;,;,e";i;ffi i. ;.frJillllil;lLirlthe cost of serum and because of this, an extensive ,black market, has srorup supplying relarively low price serum of Joubtfur orilin ffi;,l#;fi;* ?"i::,::l:,,T* f : ":_.,rg

t g tle re quired ri gorous" staro".o,' f zl .To reduce further the risk of virar conlminatron, serum can be tIii"o n

#:::-::::*:::ii ?q:",: *:h- q ^p--p-pioru",o,," or by gamma-inadiatiHeat inactivation (usuatv at 56'i f;t-1';t;;'-"",t"1 5ir;ffiTil:::lgeneral, a' of these processes also resurt in decreased growth-promotcapacity and increased cost.

3.4.3 Availability and costSerum is a by-product of the meat industry. As such its supply (pa

::?,," _lr"".Tt *'n;T.l,^""i Tll,:,ir--R-Jh", "r in the d iffe re nt pcountries. The_ supprv of geniine N"* y'Juna eesr;u; ffi;#H;i::*:j "',tl':::,: ^":.iT:111 : :"'v rr ig_ h ;'ice. il ;;";; ;i' o,l *in,,,significant investment and.operating

"oi, -" incurred b"";t;;'ft::,;*:l':i:"::,,Tyl::T:or,ig tg cvrl f.in"ipr".. correctry couected, pcessed, and validated FBS will always th;r"fr;;';;;;,"br;;i""ffiiil:of a tissue culture process.

3.4.4 Influence on downstream processing

l*:::::,1.::-:f^':r_1T I, tissue culture medium presenrs particular difficurwhen purifving a protein secreted bv rh" ";il.;;;;#ffi;lion,.J!njt: about^4-8 mg of protein d ;r io ,r," medium wh'e recombir;uumDlnproteins are frequently expressed at levers of tens of micrograms per mr.::: :*y::,:1"^,",1r_lurificarion otthe required protein may be difficand in some cases it may even be i-por;;i;';; Iiffi'#%ff#;acceptable purification process.

Monoclonal antibodies may be secreted by hybridomas at higher

[i;-3'-*l*,':':"-::::"1'"9u"i{*.;;i#;;iffi ',"l""}u.rvserum-containing medium because of ttre -'--r e'^"vsr! rv r,ur'J rr

-. il**:t"1 * I i "

_, "our

i n- (si gn i n c"'', "li,'",li; ;i Jilg" q uantities

tn c.mmerciat production ot p,ir" prot;i":,;*rJ.ii# pro""rring:::.,1l':r:l]Ivcr 80%, of rhe totat pro""r, cost and may determine the via,f the crrtire pr.cess. In such d.;;;;;;;;;,i#:",},[I;:procdB' ,fi ile

^rm supprementation is a criticar disadvantag".

- -"'

3.tl Sourclng and solection of serumFor rosron;.alreatry trincunsctr, *rr some apprications the geographic source$crunr may bo trerermrncd rry regurut.ry agencies. Becau-"se Gnim l"speciar

72 78

T. Cortwright ond G. P. Shoh

3.6 Serum storage and useSerum is rapidly frozen by the supplier immediately after bottling and isat -20"C. Few data are available on the shelf-life of serum held in thisbut 2 years has become accepted as a rule of thumb. Measurement ofstability in real time is difficult (due to the difficulty of standardizingculture over the period required) and time-consuming, while acceleradegradation tests are not interpretable when applied to frozenHowever, the one rigorous real-time study of which we are awarethat 2 years at -20'C is a conservative estimate of shelf-life and thatcould be extended to 5 years.

When required, serum should be thawed rapidly and with gentle mixingminimize protein denaturation due to salt concentration effects. Anwuter bath at 37'C is best although serum should be removed as soon as

lhnwed and not allowed to warm up.'l'hnwed $crum should be clear and there should be no significant

tlttn, Once tlurwcd, scrum can be held at 4"C for a maximum of 2-3Scrum rhorrltl ttol be re-frozen.

4. Replruconlont of serum in mediumMuclt of the prorcrrt urrderstnnding of theculture atcms l'r'unr Llugle's work (3) on

nutritional requirements of cells

3: Culture medio

whenever possible it is desirable

A properly designed serum-free

It rcproducible

I h not reliant on the economics of the world cattle market

I rlrnplifies downstream purification

I hns rro unknown factors e'g' viruses, or growth inhibitors

A rrumber of cell types have been grown successfully in serum-free cell

iturc media, usually in a medium spLcifically developed for one cell line.

ffi= '"q"r..-L;;t;icell lines diffei greatly and success of a serum-free

et-Elt,,"i formulation with one cell line *": i"tpit"n1"11ut,t:::11^:^t::t:$,i=g6;i;t;imitu, "ett

lines. A great deal of effort has gone inro developing

EfUtr,-1r"" .edia, but until recenlly, success has been limited. However' with

FJlit",,tin"ution of essential gro*ih factors and nutrients required by differ-

Enl .*ffr, several very effect--ive serum-free media have been formulated'=frOfr

t lists the estabiished cell lines which are able to proliferate in defined

Rgtllr,,,t without adaptation; those requiring a period of-'weaning off' serum

iiE-rut, included. A-variety of tissue so^t'rces and species are represented;

gl*ri' ,fr"r" are unique combinations of growth factors and hormones that

!f'nr,,r., .ptimal proliferation of specific "ill typ". (see also Chapter 6)' The

Hu-*i .,t"ti*,ent iequirement app;ars to be for the polypeptide hormone'

iHliiir'i, ,,,rJ fo, the iron-transport prote.in, transferrin. other supplements

iHeh,,t.' polypeptide and steroii growth hormones, polypeptide growth fac-

kfr, 1,,,." clements, reducing agents, diamines, vitamins' and albumin com-

bb*u,t with unsaturated fatiy icids. An important consideration for some

Hnll,rrti'ns is that animal-derived supplcments or proteins can pose con-

Hiiiii,,,i,tt risks similar to those of sorum (see Section 3.4'2)==Buu,.r,,l

commercially procluced, reudy to usc serum-free media are now

Protocol 3. Functional testing of serum batches

The following is a test of the capacity of serum to support growth of therequired cells over several passages.

1. Prepare flasks containing an appropriate growth medium supple-mented with either 5% of the serum to be tested or 5Y" of a previouslytested reference serum (use of 5% serum gives a more sensitive indica-tion of the growth-promoting capacity of a batch than does use ofhigher percentages).

2. Seed the flasks with a number of cells appropriate to the cell line used(generally 1000-1500 cells/ml for attached cells).

3. lncubate the cells for 5-7 days, harvest the cells, and count.

4. Split the cells 1 :5 (or at other appropriate ratio); seed cells from the testserum flasks into fresh medium supplemented with 5olo test serum, andthe reference serum cells into 5olo reference serum.

5. lncubate flasks for 5-7 days as before, harvest, and count.

6. Repeat steps 4 and 5.

7. Calculate growth-promoting activity relative to the reference serum(generally accept reference serum value 1207o).

Erowins mammalian cells. Based on this information' many attempts have

6a.n *""4" i" replace serum in part or in full by serum-derived factors or by

$mpt"tety synthetic media. one approach is to reduce the serum require-

iiini Uy supitementing culture medium with processed serum products' Con-

tfoltcct process serum ieplacements (CPSR) are prepared by processes that

}iEi,f A"nn"a products with much higher batch to batch consistency than

Ginl cpSR products are derived from bovine plasma and have lower

Srntcin and endotoxin levels than serum. Natural serum can also be replacedgy ;;;pi";"nted/fortifi ed serum'

. Serum -1- b:, l"^tj*,*:t I j"?llj;

Ciu*rii'factors, hormones, proteins, other protein stabilizers, and trace

A;;;;;. S-;ch fortified ,"*- can be used at a much lower concentration

$en normal serum.

the fundamentul requirements

76

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3: Culture medio

|Yalluble which have been designed for particular cell types. These are sum-

Gltlzcd in Table 4. It should, however, be remembered that different strains

!f thc same cell type may have different medium requirements, and that 'fine

Hnlng' of these commercial media may be necessary to obtain optimum

tslUlts with a specific individual strain or construct.

Irt Design of serum-free mediadclined serum-free medium is one in which a group of components are

rlated together to optimize performance of a single cell type. Each com-included is of known purity and is present at a known concentration.important factors must be considered to achieve this goal. Amongst

rurc the origin of the cell line, i.e. species and tissue, the compatibility ofcomponents and their interactions, and the specific application for

the cell line is being cultured, e.g. production of biomass or generationpnrtluct. The two main approaches generally followed in designing a serum-

tttcdium are:

Rcduced serum: in this approach, the concentration of serum in the basal

nrctlium is progressively reduced whilst other components, e.g. growthfuctors and hormones, are added to identify the factor(s) capable oftcstoring growth to the level obtained in the presence of serum. This

Ittoccss can be very lengthy because at each change, growth assays using

tlrc scrum-supplemented control and repeat verification assays need to be

rkrnc (30).

Busal medium: a different approach is to add components (singly or incombinations) to a basal medium in a stepwise manner until a medium is

progressively 'built up' to give a similar or equivalent cell growth to thetc nr m-supplemented medium.

frf ultlt"t of these approaches, the following critical factors need to be

*inrltlcrcd in designing an efficient, defined serum-free medium.

Irl,l llusal mediumlfte rclcction of basal medium can be extremely important in terms of energy

l€Utccs, buffers, and inorganic ions. Generally the starting basal medium

kfntrrlrrlion is chosen on the basis of the known preferences of the required

tsll llrr.'.

lrl,t l,iPidsfrerc irrclude ethanolamine, phosphoethanolamine, sterols, fatty acids, and

Fhugrlrolipids. In serum-supplemented media, they are usually carried on

hgut'orrr,rlccules, principally proteins. In serum-free media, fatty acids are

BtUulty provided in a bound form (eithcr to albumin or to other serum

p1tlslrrr) rtr in the form of phospholipid'enclosed vesicles (i.e. liposomcs). If

77

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3: Culture medio

tsfum albumin is used directly as a lipid source, it should be noted that thelipid content of albumin may be dependent on the methods used

Its purification; the solvent precipitation frequently used may result instripping of lipid from the protein. It should also be noted that

ized human albumin will have been stabilized with octanoic acid orhydrophobic stabilizing agents prior to heating and that it may bent to replace these with more physiologically relevant lipids before

ln cell culture. Recent developments have permitted the use of totallyic hydrophilic carriers such as cyclodextrins for the transport of lipids

Bufferingmaintain a proper environment for the metabolism, growth, and

rning of cells. Major ions (Na+, K+, HCOI, and HPO!-) are usuallycd as the principal components in pH control, along with H* and OH-,cnter into the ion balance. Other components, including amino acids, if

in high concentrations, can contribute to the buffering power of a

um, Besides bicarbonate, zwitterionic organic buffers like Hepes, Bes,'lbs may be used in systems in which strict control of the gas phase is notrcd. However, careful consideration must be given to the concentration

lltcsc buffers which can be toxic to the cells (32). Some of these buffersbiologically important cations (33). A useful buffer for use in the

rcc of low or no bicarbonate is sodium glycerophosphate (34).

Trace elementsrr.jor ions, i.e. Na+, K+, Ca2+, Mgt*, Cl-, HPO?-, and HCO', are

involved in maintaining electrolyte balance and contributing toic cquilibrium of the system. Trace elements are also included in manyl'rce media because of their beneficial effects. Inter-relationships existrr Fe2+, Znz+,and Cu2* ions which are needed for many cells. Mostl'roe media also include Co2+ and SeO3-. Cells derived from heart andliltr-l'roe media also include Co'* and SeOS-. Cells derived from heart and

Itry tissue have a high requirement for K+ whilst Caz* is required forEEfllrol ol'mitosis (35) and the Ca2*/Mg2+ ratio is important in controlling cellpfttllli'rntion and transformation (36). Selenium is proving to be important forflHlty cc'll types (1). Other trace elements include Sn, V, Al, and As (37). Ironll lterpucntly added as a transferrin complex but can also be added in otherfuflttx srrch as ferric citrate, ferrous nitrate, or ferrous sulphate.

l,l,F Mechanical stabilizers and adhesion factons

fttr optirnal growth, cells grown in suspension culture require protection fromlheur .lrrc to agitation (air bubbles, stirrer, and shaker). Shear damage can be

Etlttccrl by increasing the viscosity of the medium. Carboxymethyl cellulose

titl glrlyvinylpyrrolidone have been used lbr this purpose. The most widelyBlerl nlrcnr protectant is Pluronic F-68. 'Ihis is a non-ionic block copolymer

T. Cartwright and G. P. Shah 3: Culture medio

!,!,8 Adaptation of cells to low serum/serum-free mediumfFhe following protocol describes a generalized procedure to adapt cells to low!€funr supplemented medium or serum-free mediuln. The procedure shouldFigurc that cell viability and protein synthesis are not compromised at any

Edaptation stage.

with an average molecular weight of 8400 Da, consisting of a central blockpolypropylene (20"/" by weight) and blocks of polyoxyethylene at bothPluronic F-68 has been demonstrated to have a significant effect in protectianimal cells grown in suspension in sparged or stirred bioreactors. Thetective effect is thought to be exerted through the formation of an istructure of adsorbed molecules on the cell surface. It is thought that thydrophobic portion of the molecule interacts with the cell membrane,the polyoxyethylene oxygen may form hydrogen bonds with water molecu Protocol 4. Adaptation of cells to low serum/serum-free mediumto generate a hydration sheath, which provides the protection from lamshear'stress and cell-bubble interactions (38,39).

Cell attachment and growth of anchorage-dependent cells can be iby pretreatment of the substrate in a variety of ways. The substrate cantreated with adhesive glycoproteins such as fibronectin, laminin, chondroitepibolin, or serum spreading factor (see also Chapter 4, Section 5.4).

4.2.6 Selection of componentsThe following checklist is a useful starting point for consideration ofponents for inclusion in a serum-free medium formulation:

o transport proteins, e.g. transferrin, bovine serum albumin, and

o stabilizing proteins, e.g. aprotinin, bovine serum albumin, fetuin,soyabean trypsin inhibitor

o growth regulators, e.g. insulin, hydrocortisone, and triiodothyronineo growth factors, e.g. EGF, FGF, NGF, platelet derived growth factor,

insulinlike growth factors (somatomedins)

o attachment proteins, e.g. fibronectin, collagen, laminin, fetuin, andspreading factor

o crude extracts, e.g. bovine pituitary extract, brain extract, liver extract,tissue digests

o essential nutrients, e.g. cholesterol, linoleic acid, ethanolamine, andelements

4,2,7 Practical hints on solubilizing specific componentso Riboflavin, folic acid, tyrosine, and cystine need dissolving in NaOH.o Insulin needs to be dissolved in HCl.r Fatty acids, lipids, and fat-soluble vitamins can be dissolved

solutions or attached to either protein carriers, e.g. BSA andor surfactunts, e.g. Tween 80 and Pluronic F-68.

Pluronie F-68 is nrore soluble in cold water and, when making Pluronicsolutiona, it chould be added to the water rather than the other way rou

Hypoxenthlne dicsolvec etsily on heating,

Determine the optimal seeding density in serum-supplementedmedium which allows for 5- to 15-fold growth during the experimont/culture period.

Using the above optimal starting density, replace the basal mediumwith the serum-free medium to be tested. Set up a series of cultures inthis medium with varying concentrations of serum (e.g. 1-10%). En-

sure that enough replicate cultures are set up to allow meaningfulinterpretation of results. lnclude selective agents to maintain gene copynumber if recombinant cell lines are used.

Select the culture condition that gives 60-80% (or more) of the cellgrowth of the control cultures and, using this condition, expand thecells to a larger scale (e.9. 24-well plate to 10 ml suspension to 50 mlsuspension to 100 ml suspension, or 24-well plate to 25 cm2 flask to 75

cm2 flask to 175 cm2 flask). Check for expression of the desired charac-teristic or the correctly processed product. lf none of the conditionsused achieve 60-80% of control growth, consider alternative serum-free media or adding supplements.

Make afrozen bankof cells atthisstage (e.g. 5 x 106to 1 x l0Tcellslml)from an exponentially growing culture (see Chapter 4, Section g).

Set up a second series of cultures as in step 2 above, reducing theserum supplementation further (e.9. 5% going down to 0.1%). Growcells for 3-6 days.

Repeat step 3 until serum supplementation is eliminated.

Prepare a frozen bank of the serum-free adapted cells and check for theabsence of mycoplasma (see Chapter 8, Section 4,3).

4'tr,11 Difficulties that may be encountered with serum-free mediumWlrcrr cclls are grown in serum-free conditions, they no longer benefit fromthe rrrrrltiple protective and nutritional effects that serum provides (Table 2).The robustness of the process in serum-free medium depends on attention tothe lirllowing points:

(e) ('clls appear more fastidious in the absence of serum: design of a dedi-cnted medium for each ccll typc is unually necessary for optimal results.

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T. Cartwright ond G. P. Shoh

(b) Culture conditions become more critical in serum-free medium: betcontrol of key process parameters (pH, oxygenation, etc.) is therenecessary.

(c) Serum-free medium has a reduced capacity to inactivate or absorbmaterials (e.9. heavy metals, endotoxin, etc.). Greater attention topurity of components and to depyrogenation is required. Antibioticsexhibit increased cytotoxicity in serum-free medium.

(d) Specific shear protective agents may need to be added.

(e) A significant adaptation period may be required before cells are tuweaned on to serum-free medium (30). This makes the design andof serum-free medium a long and labour-intensive process.

4,2,10 Direct influences of culture conditions and mediumcomponents on protein expression

Regardless of whether the medium used contains serum or not, manyin the culture environment affect the quantity and the authenticity of proteiproduccd by the cells. Apart from dependence on the basic capacity of tmedlum to supply cells with nutrients and oxygen and to maintainosmolnlity, etc., protein expression may be affected by other, more sufaetors. []or example extracellular matrices (ECM) can affect theregukrtion und maintenance of normal cellular functions (e.g. normal mmnry epithclial cells when cultured on collagen substrate produce 4- tomore casein than when grown on polystyrene substrate). Also, thetion ol' nutrient medium can affect the glycosylation of the expressed proteinClucose limitation often results in incomplete and/or aberrant protein glsylution; ammonium ion accumulation in cell cultures can result in glprotoins deficient in terminal sialylation; treatment of cells with differehormones, vitamins, differentiation factors, etc., may often result in alteglycosylation patterns in glycoproteins secreted by the cells (40).

4,2,71 Component interaction and factorial experimentaldeeign

ln lhc pnst, the design of serum-free media was predominantly empirical.mttnl cttm nton approach was to reduce the serum supplementation progressi('wetrtirrg ul'l'') and to determine the critical factors involved in celland pnrlcin cxpression.

Bntcrl olt lltis upproach, a database is now becoming established fornutrlllolml requlrenrents o[ the most commonly used cell types (see6) and lltln lttformulion cnn often be applied generically. In general, tlormctl ccll llnen huvc u lower requirement for growth factors than untrans.fblntod calh and irr sorrre instances, protein can be completely eliminafrttnt tho ntedlum (xee'l'uhle 4).

'l'ho ndvent of rnpid rncl sensitive nnulyticul mcthodrr (e,g, t{PLC) har

3: Culture medio

cnabled the rapid measurement of low molecular weight nutrient utilizationduring cell culture. Medium optimization can proceed based on these data bytupplementation of rapidly utilized components and reduction of under-usedeomponents. However, interpretation of such studies is complicated by thedynamic interaction between utilization of different components. This has ledtcveral groups to base optimization studies on factorial experimental design

tlmed at accommodating complex systems involving multiple interacting fac-

tors (41). Additional complications arise since cellular metabolism may alterduring the course of a fermentation and different nutrients may becomeeritical at different phases of the culture (42). T\erefore, reductionistepproaches to medium design should be applied with caution.

0. Influence of cell culture systems on choice ofmedium

'l'hc efficiency of an animal cell culture system depends on the interaction ofmnny different factors. The support that the medium provides for the cells is a

erucial element but this in turn is influenced by the type of culture system inwhich the cells are propagated.

C'ultures may be operated in a simple batch mode, or as fed batches, or as a

pcrfused system. The cells may be grown in suspension culture or attached toturl'aces, or they may be grown at very high density (in excess of 108 cells/mlln various types of plug flow reactor) or at densities around 105-106 cels/ml inFlrrrple cultuies.

Cells may be enclosed in compartments which favour the development ofkrcal micro-environments (as in hollow-fibre bioreactors or macroporous

eurrier cultures) or they may be fully exposed to the bulk medium. They maycrpcrience significant shear forces (either by pumped medium flow or byngitation) or they may be completely isolated from shear force (e.g. in thellrlcrior of macroporous carriers or when encapsulated).

ln these different situations, the environment experienced by the cells

nrudulates the support that they require from the culture medium.

d.l Batch or perfusion culturesWhcn cells are grown as batch cultures, all the nutrients required for thedrrrirtion of the culture must be present in the initial medium. The two majorenergy sources, glucose and glutamine, need to be present at unphysiologic-nlly high concentrations which may lead to the production of high concentra-llorrs of the toxic metabolites, lactate and ammonia. In the fed batchagrproach, glucose and glutamine can be added at intervals as they become

tlcpleted, thus limiting toxicity and improving efficiency of utilization.ln perfusion systems, i.e. culture systems where cells are retained in

llre f'ermenter while medium flows through, fresh medium is continuously

82 88

T. Cortwright ond G. P. Shoh

supplied and spent medium removed. Ideally, the perfusion rate and thecentration of each component in the medium should be adjusted to matchconsumption rate of each nutrient. This requires detailed knowledge ofcells' nutritional needs and of the rate of production of possible toxicproducts. Careful design of the medium is therefore needed to match mediucomposition to the cells' metabolic needs. Another point that requiressideration is that perfusion rate should not be so rapid as to flush out aulogous growth factors.

5.2 Anchorage-dependent cellsMany of the cell types which are currently the focus of intenseactivity, such as endothelial cells and epithelial cells, will only growfunction when attached to surfaces. Some of the cells used in vaccine mafacture, such as human diploid fibroblasts, Vero cells and MDCK cells, a

exhibit anchorage dependence.The need for cells to be attached to surfaces imposes at least two

requirements on the medium. Firstly, although some of these cells canthesize their own attachment factors, attachment is generally acceleratedviability improved if factors such as fibronectin and laminin, which formof the ECM, are incorporated in the medium formulation. These factorsto the surface on which the cells will attach and act as ligands for specificsurfaca receptors called integrins.

An alternative approach is to precoat the culture surface with ECMnents such as collagen or fibronectin (see Chapter 4, Section 5.4). Recentlyan engineered fibronectin substitute called Pronectin (available from ProteiPolymer Technologies) has been developed for this purpose. This has tadvantage of promoting better attachment than the natural matrix proteisince it contains multiple copies of the sequence Arg-Gly-Asp recognizedintegrins, and also it avoids the need to add animal-derived material toculture system with the attendant risks of viral contamination (43).

Polypeptide growth factors (particularly members of the FGF familyhind to elements of the ECM which may act as a slow release poolthesc lactors. The dynamic interplay between growth factors, the matriruncl thc attached cells may be an important aspect of cellular physi(44).

'l'lre sccond rcquirement that anchorage dependence imposes on meditir the rrced to inhibit the proteolytic enzymes that are used for releasieellr I'ront tlrc substrate during sub-culture in order to minimize cell damage,ln the pre$ence ol'$erum, this is achieved by the endogenous protease in.hitritoru. lrr cerunr-l'ree medium, protection can sometimes be achieved byrlnaing tho cellr with mcdium containing soya bean trypsin inhibitor aftertrypalrrllntion, lt nhoulel bo noted, however, thnt thc crucle 'trypsin' prep.uration corrrmorrly ured in ccll culture contuins proteolylic uctivities otherthan trypnln End thnl r singlc, purified protcasc lnhltritor nlry not provide

3: Culture medio

adcquate protection. [It will be found that highly purified trypsin is much lesscflicient at releasing cells from surfaces than the impure preparations usuallyenrployed (45).1

8.2.1 Aggregate culturesWhen cells that are normally anchorage-dependent are grown in conditionswhcre attachment factors are limiting, they tend to form aggregates which canbe propagated in suspension. Such cultures can be useful for vaccine productionor other large-scale applications, but a limitation may be the heterogeneity oftbc cultures and a tendency for necrotic regions to develop in the centre of thelarger aggregates with subsequent cell lysis and the liberation of cell contentsend debris into the culture.

6.3 Stirred suspension culturesA primary concern in suspension cultures is the protection required againstClnmage to cells by shear forces. In serum-containing medium this protectionlr supplied by serum proteins, particularly albumin. In serum-free conditions,rcvcral synthetic polymers (usually at around 1 g/litre) have been used to fillthis role (see Section 4.2.5).

llccent studies have shown that shear damage occurs when turbulent eddiesol'similar size to the suspended cells are produced in the liquid (46). Thispltcnomenon appears to be more associated with bubble formation and col-lapsc such as is produced by cavitation or air entrainment caused by thelmpe ller, or by bubble disengagement from the surface when sparging, rathertltln by shear forces in the body of the liquid. Pluronic F-68 is particularlyefl'cctive in protecting cells from this type of damage and is now widely usedln scrum-free medium as a protective agent against shear (see Section 4.2.5).

l,imitation of oxygenation is the main factor which currently restricts the;crlc and density of many animal cell cultures, and cell damage by sparging orby high speed agitation precludes the use of these methods to increaseruxygenation within the culture, although improved physical protective agentsfor the cells may improve this situation. An alternative approach which iswidcly used is to perform oxygenation in a compartment of the bioreactorwltich is physically separated from the cells in order to avoid contact betweencells and bubbles. This implies an efficient separation system and an adequatecirculation system to ensure that oxygenated medium reaches all cells in thelriorcactor.

Irr some reactor configurations (some spin filter devices, hollow-fibre sys-le rrrs) the cells are completely separated from the vessel in which oxygenationurcurs. In others such as encapsulated cells or cells in macroporous carriers,llrc oxygenation may occur in the same vessel as the cells, but they areptrrtccted from direct contact with bubbles or liquid turbulence.

An important consideration whcn Pluronic F-68 or other protective agentsere udded to products intendecl fbr therapeutic use is the need to remove

84 $[

T. Cortwright and G. P. Shoh

these effectively during downstream processing. Anti-foaming agents (par-

ticularly silicones) are sometimes added to cultures, but their use is best

avoided when possible since such materials are notoriously difficult to elimin-ate completely from the final product.

5.4 High-density culture systemsAs already indicated, several of the culture systems currently in use provide

cells at veiy high density, often in excess of 108 cells/ml. This has advantages

in that higher concentrations of product can be generated but imposes the

need for better control of fermentation conditions to ensure that the cells are

kept within the specified environmental limits. High cell density systems can

be divided into two t5rpes, homogenous systems where the cells are continuallymixed and plug flow systems where cells are held immobile while medium

flows past them. Many configurations of these types of bioreactor exist-wewill only consider two which represent the best performing examples of theirclass.

5.4.1 Macroporous carrier systems

These systems employ porous particles which contain pores large enough topermit cells to enter the particle and to colonize the internal space. The

particles can be of neutral buoyancy and used as microcarriers or they can be

of higher density (typically 1.3-1.6 g/cm3) and employed in a fluidized bed

configuration. It is this configuration which permits very efficient mass trans'fer, a homogenous cell culture, and high cell density (47).

A particular feature of this and other high density systems is that the

are able to form their own local micro-environment while still being able

receive required nutrients and release toxic waste products to the bulkmedium. An important consequence of this is the reduced need for grow

factors and matrix components since these are secreted by the cells and

maintained in their own micro-environment. In cases where cells do notnaturally produce the required factors they may relatively easily be

gineered to do so.ln addition, since cells inside the particles are not directly exposed to the

bulk medium, protection against shear is much less of a problem. However,lhe protease levels secreted by cells in high density cultures may be significant

nrrd purticulur attention should be paid to the need to include protease

lrrlribltorx to protcct product from degradation'ln hlgh.density cultures, medium may rapidly become depleted in particu-

lur eotnlxtttentt und umino acid analysis of input and output medium streamg

xlrould bc perlirrmecl to determine whether particular amino acids becomo

llrnlting und lu perrnit ndjustment of medium concentration and/or flow

uccordlngly, lt rhould lrc notcd that amino acid deficiency may be one factor,wlrieh crtn lncluco lhe prtxluction of proteolytic enzymer by nnimal cells (48),

Another imporlnnt cunnitlcrution with high-dcnnity cultur€$ in tho provision

3: Culture media

rul'adequate instrumentation to ensure that pH, pO2, and temperature remainwithin the required limits.

6.4.2 Hollow-fibre systemsln hollow-fibre systems, the cells are held in the bioreactor separated from themcdium flow by the walls of capillary tubes (hollow fibres) through whichmcdium flows. Typically the capillary walls are impervious to macromoleculesbut allow the passage of low molecular weight nutrients including oxygen (49).

In common with other plug flow systems, hollow-fibre bioreactors mayrul'l'er from the development of gradients of nutrients, particularly of oxygen,along the length of the medium flow path. Adequate medium flow rate isfequired to limit the heterogeneity of the culture that this may produce.Unlike the situation with macroporous carriers, profein products generated inhollow-fibre systems are not released into the bulk medium, but are retainedby the capillary membrane in the cell compartment from where they can beperiodically or continuously harvested.

6.5 Very low density culturesekrning of cells requires that cellular proliferation is achieved at very low cellderrsities. Not surprisingly, here the opposite situation pertains to that dis-eusscd above and cells require maximum support from the medium. Serumsttpplementation alone is often not sufficient and'conditioned medium' har-vestcd from actively growing cells is frequently used. This contains undefinedmncromolecules including growth factors and detoxifying factors, and lowmolccular weight compounds such as tricarboxylic acid cycle intermediateswhich may help with culture establishment. In extreme cases it may be necessaryItt use 'feeder layers' of metabolically active but non-proliferating cells (usuallyptoduced by irradiation) which are co-cultured with the required cell (see

eltrrpter 5, Section 2.4.4, and Chapters 6 and 7).More recently, efficient media for clonal growth have been developed

wltich are based on standard media supplemented with recombinant growthfsclors appropriate to the required cells and with efficient pH controlse lricved by the use of buffers such as Hepes or Mops. CO2 is also an essentialrerlrrirement and since cells at low density may not produce enough to satisfythis, incubation in an atmosphere containing CO2at an appropriate concen-Ittrlion for the medium used is essential.

tl. In-house medium development and productionversus commercial supply

6.f Economic considerationsMrtlium production from the basic raw materials is a very major undertakingIrrvolving as it does the sourcing und quality control of a large number of

80 $7

T. Cartwright ond G. P. Shoh

individual components. In addition, the technology involved in milling andmixing powders and ensuring homogeneity and compatibility of the differentcomponents in the mixture is complex and not likely to exist within mostbiotechnology companies or cell culture laboratories.

For most users, the real choice to be made is between the different types ofmedium formulation and presentation that are available commercially. Theseinclude ready-to-use single strength (1x) medium, medium concentrates(10x or in some cases 50x) or pre-mixed powder. Liquid medium is alsosupplied in a variety of packaging ranging from small bottles (usually 5ffi ml)to flexible plastic containers with volumes from one to several hundred litres"

Laboratory-scale operations are likely to favour bottled single strengthmedium. For small process operations, single strength medium in 10 or20 litre bags is a particularly convenient approach. On larger scales the choicedepends on technical and economic factors which are specific to each manu-facturer. Use of large containers of single strength medium permits a mini-mum of investment in preparation, quarantine, and quality control facilitiesand equipment, and staff requirements may also be reduced. However, it isexpensive both in terms of the litre cost of medium and possibly also in thecost of delivery. Storage of large medium volumes in acceptable conditionsmay also incur significant costs (note that this is also true for the storageduring quarantine of medium prepared in-house). A compromise is the use ofmedium concentrates. In this case it is necessary to invest in plant for theproduction of tissue culture quality water and the vessels and pipeworkrequired for dilution. Continuous flow dilutors are available for on-line dilu-tion. The cheapest approach in raw material costs is to buy powdered mediumand prepare this for use on site. This requires considerable investment in anadequate medium kitchen with ancillary facilities and appropriate personnelto operate it.

When evaluating these options, it is important from an economic viewpointthat the true cost of the installation and operation of the required mediumkitchen facilities are fully evaluated. These include the installation itself, theprovision of adequate technical services, clean room facilities and disposables,truined operatives, and the necessary quality control backup.

0.2 Development of a dedicated medium(hnrnrercinl dccisions regarding in-house development or sub-contracting ton crtrnrnerciul organization may also be important when optimization ofurctliunr tirr t specilic cell type or process is required. Here the key question ishow I'recptcrrtly it is necessary to develop a new dedicated medium andwhather lho nruintcnance of in-house medium development expertise andfucllltier is curnonricully justified by this frequency. Several of the mediumproduetiun lruunes ol'l'er conlidential contract serviccs in which a team whichis orrgaged lirll'tinrc in medium development will optimizc modium lbrmula-tlonn r*peciflcally lirr lhe customer's cells.

3: Culture medio

6.3 Quality assurance and quality controlClritical elements in quality assurance for medium are the choice of supplier,evaluation of the documentation provided by the supplier and audit of thettupplier's facilities when appropriate. For basal and serum-free media, manyruppliers have registered a drug master file with the relevant regulatorynuthorities which provides the necessary guarantees concerning the manufac-turing process. For serum, confidence must be based almost entirely on thebltch documentation provided.

'fo assure consistent performance of medium, standard procedures must beret up in-house for all operations concerning the production of medium fromhrught-in components. This includes standardization of the storage con-ditions and the duration of storage of the complete medium before use, sincenany complete media have only limited stability.

I-ocal quality control testing by the user before committing a medium batchtrt a production process is still of critical importance. Testing of serum batcheshtts already been discussed in Section 3.5.1. Key tests to be applied to batchesol'liquid and powdered medium are summarized in Section 2.4.5. Simple testssuch as measurement of osmolality and of pH should be applied to everyItrcdium preparation (including those from the same batch of powder or ofconcentrate) to ensure that no error in solution or dilution has been made.

Sterility of the final medium before use is a less simple question because thetirnc required for completion of the sterility test (7-10 days) may not beeornpatible with storage of the complete medium which may be unstable overllris period. Some operators assume sterility if all preparation procedureshnve been performed without incident and then verify sterility in parallel withproduction. The speed and sensitivity of sterility testing can be improved byllrc use of filter concentration methods to detect very low levels of contamin-rul i ng micro-organisms.

l{cferencesl. llettger, W. and Ham, R. (1982). Adv. Nutr. Res., 4,249..1. ltoth,E,., Ollenschlager, G., Hamilton, A., Langer, K., Fekl, W., and Jaksez, R.

.l,1

I

(r

( 1988). In Vitro Cell Dev. Biol., ?A,96.. liagle, H. (1955). Science,122, 501., Williams, G. T., Smith, C. A., Spooncer, E., Dexter, T. M., and Taylor, D. R.

(1990). Nature, 34.3,76.. Moses, H. L., Coffrey, R. J., Leof, E. B., Lyons, R. M., and Kesi-Oja, J. (1987).

.l . Physiol., Suppl. 5, 1.

. Van der Pol, L. and Tramper,J. (1992).In Anirnalcelltechnology: developments,processes and produclr (ed. R. E. Spier, J. B. Griffiths, and C. MacDonald), pp.It)2-4. Buttcrworth-Hcinemann, Oxford.

7. llodgson, J. (1993). Biol'[echnoktgy, ll,49.It. f lrryashi, l. and Sato, A. (1976), Nature,2S9, 132.

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