Presentation Animal Tc

8/8/2019 Presentation Animal Tc

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ANIMALANIMAL TISSUETISSUECULTURECULTURE ANDAND ITSITS

APPLICATIONSAPPLICATIONS..


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Suspension cultures are usually grown either:Suspension cultures are usually grown either:

In magnetically rotated spinner flasks or shaken rlenmeyerIn magnetically rotated spinner flasks or shaken rlenmeyer

flasks where the cells are kept actively suspended in theflasks where the cells are kept actively suspended in themedium;medium;

In stationary culture vessels such as TIn stationary culture vessels such as T--flasks and bottlesflasks and bottles

where, although the cells are not kept agitated, they arewhere, although the cells are not kept agitated, they areunable to attach firmly to the substrate.unable to attach firmly to the substrate.


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ManyMany cellcell lines,lines, especiallyespecially thosethose derivedderived fromfrom normalnormal

tissues,tissues, areare consideredconsidered toto bebe AnchorageAnchorage--Dependent,Dependent, thatthat is,is,theythey cancan onlyonly growgrow whenwhen attachedattached toto aa suitablesuitable substratesubstrate..

SomeSome cellcell lineslines thatthat areare nono longerlonger consideredconsidered normalnormal

(frequently(frequently designateddesignated asas TransformedTransformed Cells)Cells) areare frequentlyfrequently

ableable toto growgrow eithereither attachedattached toto aa substratesubstrate oror floatingfloating freefree inin

suspensionsuspension;; theythey areare AnchorageAnchorage--IndependentIndependent..

InIn addition,addition, somesome normalnormal cells,cells, suchsuch asas thosethose foundfound inin thethe

blood,blood, dodo notnot normallynormally attachattach toto substratessubstrates andand alwaysalways growgrow

inin suspensionsuspension


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Types of CellsTypes of Cells

Cultured cells are usually described based on theirCultured cells are usually described based on their

morphology (shape and appearance) or their functionalmorphology (shape and appearance) or their functional

characteristics. There are three basic morphologies:characteristics. There are three basic morphologies:

1.1. EpithelialEpithelial--like: cells that are attached to a substrate andlike: cells that are attached to a substrate and

appear flattened and polygonal in shape.appear flattened and polygonal in shape.

2.2. LymphoblastLymphoblast--like: cells that do not attach normally to alike: cells that do not attach normally to a

substrate but remain in suspension with a spherical shape.substrate but remain in suspension with a spherical shape.

3.3. FibroblastFibroblast--like: cells that are attached to a substrate andlike: cells that are attached to a substrate and

appear elongated and bipolar, frequently forming swirls inappear elongated and bipolar, frequently forming swirls in

heavy culturesheavy cultures


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Functional CharacteristicsFunctional Characteristics1.1. BiochemicalBiochemical markersmarkers cancan bebe usedused toto determinedetermine ifif cellscells areare stillstill carryingcarrying onon

specializedspecialized functionsfunctions thatthat theythey performedperformed inin vivovivo (e(e..gg..,, liverliver cellscells secretingsecreting

albumin)albumin)..

2.2. MorphologicalMorphological oror ultraultra structuralstructural markersmarkers cancan alsoalso bebe examinedexamined (e(e..gg..,,

beatingbeating heartheart cells)cells)..

3.3. Frequently,Frequently, thesethese characteristicscharacteristics areare eithereither lostlost oror changedchanged asas aa resultresult ofof

beingbeing placedplaced inin anan artificialartificial environmentenvironment..

4.4. CellCell lineline maymay bebe::

a.a. FiniteFinite..

b.b. ContinuousContinuousc.c. TransformedTransformed CellsCells areare usuallyusually easiereasier andand fasterfaster growing,growing, maymay oftenoften havehave

extraextra or or abnormalabnormal chromosomeschromosomes andand frequentlyfrequently cancan bebe growngrown inin

suspensionsuspension..

d.d. IfIf thethe cellscells formform tumorstumors whenwhen theythey areare injectedinjected intointo animals,animals, theythey areare

consideredconsidered toto bebe NeoplasticallyNeoplastically TransformedTransformed..


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TYPES OF ANIMAL CELLTYPES OF ANIMAL CELL

CULTURESCULTURES1. Anchorage dependent culture1. Anchorage dependent culture

Anchorage dependent cell lines can be grown by the followingAnchorage dependent cell lines can be grown by the followingmethodsmethods

a. conventional methodsa. conventional methods

b. new trendsb. new trends

multiple surface tissue prapagatormultiple surface tissue prapagator

capillary semi permeable membranescapillary semi permeable membranes

as aggregatesas aggregates

encapsulation cellsencapsulation cells

packed bed reactorpacked bed reactor


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Suspension cultureS

uspension culture:: BioreactorBioreactor

Hollow fiber reactorHollow fiber reactor

Air lift fermentorsAir lift fermentors

ChemostatesChemostates

Modes of suspension cultureModes of suspension culture

Batch cultureBatch culture

Fed batch cultureFed batch culture

Continuous cultureContinuous culture

Perfusion culturePerfusion culture


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Fermentation technology for the growth of animal cellsFermentation technology for the growth of animal cellsand their products:and their products:

Fermentors have been used for the cultivation of bacteria andFermentors have been used for the cultivation of bacteria andyeasts for a long time. Initially, fermentation was synonymousyeasts for a long time. Initially, fermentation was synonymouswith alcohol production. Later, bacteriologists learnt to use thewith alcohol production. Later, bacteriologists learnt to use thesame principles for the preparation of vitamins, organic acids,same principles for the preparation of vitamins, organic acids,antibiotics etc. This led to the rapid development of a varietyantibiotics etc. This led to the rapid development of a varietyof fermentors and methodologies.of fermentors and methodologies.


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WORK AREA AND EQUIPMENTWORK AREA AND EQUIPMENT

LaminarLaminar flowflow hoodshoods..

VerticalVertical LFHLFH

HorizontalHorizontal LFHLFH

BB.. COCO22 IncubatorsIncubators:: cellscells areare growngrown inin anan atmosphereatmosphere ofof 55--1010%%

COCO22

MicroscopesMicroscopes

PreservationPreservation.. CellsCells areare storedstored inin liquidliquid nitrogennitrogen

EE.. VesselsVessels..


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Mediaand growthMediaand growth

requirementsrequirements1. Physiological parameters1. Physiological parameters

A. temperatureA. temperature -- 37C for cells from homeother37C for cells from homeother

B. pHB. pH -- 7.27.2--7.5 and osmolality of medium must be maintained7.5 and osmolality of medium must be maintainedC. humidity is requiredC. humidity is required

D. gas phaseD. gas phase -- bicarbonate conc. and CObicarbonate conc. and CO22 tension in equilibriumtension in equilibrium

E. visible lightE. visible light -- can have an adverse effect on cells; light inducedcan have an adverse effect on cells; light inducedproduction of toxic compounds can occur in some media; cellsproduction of toxic compounds can occur in some media; cells

should be cultured in the dark and exposed to room light as littleshould be cultured in the dark and exposed to room light as littleas possibleas possible


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2. Medium requirements: (often empirical)2. Medium requirements: (often empirical)

A. Bulk ionsA. Bulk ions -- Na, K, Ca, Mg, Cl, P, Bicarb or CONa, K, Ca, Mg, Cl, P, Bicarb or CO22B. Trace elementsB. Trace elements -- iron, zinc, seleniumiron, zinc, seleniumC. sugarsC. sugars -- glucose is the most commonglucose is the most common

D. amino acidsD. amino acids -- 13 essential13 essential

E. vitaminsE. vitamins -- B, etc.B, etc.

F. choline, inositolF. choline, inositol

G. serumG. serum -- contains a large number of growth promoting activities suchcontains a large number of growth promoting activities suchas buffering toxic nutrients by binding them, neutralizes trypsin andas buffering toxic nutrients by binding them, neutralizes trypsin and

other proteases, has undefined effects on the interaction between cellsother proteases, has undefined effects on the interaction between cells

and substrate, and contains peptide hormones or hormoneand substrate, and contains peptide hormones or hormone--like growthlike growth

factors that promote healthy growth.factors that promote healthy growth.

H. antibioticsH. antibiotics -- although not required for cell growth, antibiotics arealthough not required for cell growth, antibiotics are

often used to control the growth of bacterial and fungal contaminants.often used to control the growth of bacterial and fungal contaminants.

3. Feeding3. Feeding -- 22--3 times/week3 times/week


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Measurement of growth andMeasurement of growth and

viabilityviability The viability of cells can be observed visually using anThe viability of cells can be observed visually using an

inverted phase contrast microscope. Live cells are phaseinverted phase contrast microscope. Live cells are phase

bright; suspension cells are typically rounded and somewhatbright; suspension cells are typically rounded and somewhat

symmetrical; adherent cells will form projections when theysymmetrical; adherent cells will form projections when theyattach to the growth surface.attach to the growth surface.

Viability can also be assessed using the vital dye, trypan blue,Viability can also be assessed using the vital dye, trypan blue,

which is excluded by live cells but accumulates in dead cells .which is excluded by live cells but accumulates in dead cells .

Cell numbers are determinedCell numbers are determined using a hemocytometerusing a hemocytometer


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TISSUE CULTURE PROCEDURESTISSUE CULTURE PROCEDURES

1. TRYPSINIZING AND SUBCULTURING CELLS FROM1. TRYPSINIZING AND SUBCULTURING CELLS FROM

A MONOLAYERA MONOLAYER

A primary culture is grown to confluency in a 60A primary culture is grown to confluency in a 60--mm petrimm petriplate or 25plate or 25--cm2 tissue culturecm2 tissue culture

flask containing 5 ml tissue culture medium. Cells areflask containing 5 ml tissue culture medium. Cells are

dispersed by trypsin treatment anddispersed by trypsin treatment and

then reseeded into secondary cultures. The process ofthen reseeded into secondary cultures. The process ofremoving cells from the primaryremoving cells from the primary

culture and transferring them to secondary cultures constitutesculture and transferring them to secondary cultures constitutes

a passage, or subculturea passage, or subculture..


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2.2.MaterialsMaterials

Primary cultures of cellsPrimary cultures of cells

HBSS without Ca2+ and MgHBSS without Ca2+ and Mg, 37, 37CC Trypsin/EDTA solution 37Trypsin/EDTA solution 37CC

Complete medium with serum: e.g., supplemented DMEM (APPENDIXComplete medium with serum: e.g., supplemented DMEM (APPENDIX2A) with 10%2A) with 10%

to 15% (v/v) FBS (complete DMEMto 15% (v/v) FBS (complete DMEM--10 or10 or --15), 3715), 37CC

Sterile Pasteur pipetsSterile Pasteur pipets

3737C warming tray or incubatorC warming tray or incubator Tissue culture plasticware or glassware including pipets and 25Tissue culture plasticware or glassware including pipets and 25--cm2 flaskscm2 flasks

or 60or 60--mm petri plates, sterilemm petri plates, sterile

NOTE: All culture incubations should be performed in a humidified 37NOTE: All culture incubations should be performed in a humidified 37C,C,5% CO25% CO2

incubator unless otherwise specified Some media (e.g., DMEM) mayincubator unless otherwise specified Some media (e.g., DMEM) may

require alteredrequire altered levels of CO2 to maintain pH 7.4.levels of CO2 to maintain pH 7.4.


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Procedure:Procedure:

Remove all medium from primary culture with a sterile Pasteur pipet. WashRemove all medium from primary culture with a sterile Pasteur pipet. Wash

adheringadhering

cell monolayer once or twice with a small volume of 37cell monolayer once or twice with a small volume of 37C HBSS withoutC HBSS without

Ca2+ andCa2+ and

Mg2+ to remove any residual FBS that may inhibit the action of trypsin.Mg2+ to remove any residual FBS that may inhibit the action of trypsin.

.Use a buffered salt solution that is Ca2+ and Mg2+ free to wash cells..Use a buffered salt solution that is Ca2+ and Mg2+ free to wash cells.

Ca2+ and Mg2+ in theCa2+ and Mg2+ in the

salt solution can cause cells to stick together.salt solution can cause cells to stick together.

.If this is the first medium change, rather than discarding medium that is.If this is the first medium change, rather than discarding medium that is

removed fromremoved from

primary culture, put it into a fresh dish or flask. The medium containsprimary culture, put it into a fresh dish or flask. The medium contains

unattached cells thatunattached cells that

may attach and grow, thereby providing a backup culture.may attach and grow, thereby providing a backup culture.


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Add 2 ml 37Add 2 ml 37C complete medium. Draw cell suspension into aC complete medium. Draw cell suspension into aPasteur pipetPasteur pipet

rinse cell layer two or three times to dissociate cells and torinse cell layer two or three times to dissociate cells and to

dislodge any remaining adherent cells.dislodge any remaining adherent cells.

As soon as cells are detached, add serum or mediumAs soon as cells are detached, add serum or mediumcontaining serum to inhibit further trypsin activity that mightcontaining serum to inhibit further trypsin activity that might

damage cells.damage cells.

If cultures are to be split 1/3 or 1/4 rather than 1/2, addIf cultures are to be split 1/3 or 1/4 rather than 1/2, add

sufficient medium such that 1 ml of cell suspension can besufficient medium such that 1 ml of cell suspension can be

transferred into each fresh culture vessel.transferred into each fresh culture vessel.


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Add an equal volume of cell suspension to fresh plates orAdd an equal volume of cell suspension to fresh plates or

flasks that have been appropriately labeled.flasks that have been appropriately labeled. Alternatively, cells can be counted using a hemacytometer orAlternatively, cells can be counted using a hemacytometer or

Coulter counter and diluted to the desired density so a specificCoulter counter and diluted to the desired density so a specificnumber of cells can be added to each culture vessel.number of cells can be added to each culture vessel.

A final concentration ofA final concentration of55 104 cells/ml is appropriate for104 cells/ml is appropriate for

most subcultures.most subcultures. For primary cultures and early subcultures, 60For primary cultures and early subcultures, 60--mm petri platesmm petri plates

or 25or 25--cm2 flasks are generally used; larger vessels (e.g., 150cm2 flasks are generally used; larger vessels (e.g., 150--mm plates or 75mm plates or 75--cm2 flasks) may be used for later subcultures.cm2 flasks) may be used for later subcultures.

Cultures should be labeled with dateCultures should be labeled with date of subculture and passageof subculture and passagenumber.number.


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If necessary, feed sub confluent cultures after 3 or 4 days byIf necessary, feed sub confluent cultures after 3 or 4 days by

removing old medium and adding fresh 37removing old medium and adding fresh 37C medium.C medium.

Passage secondary culture when it becomes confluent byPassage secondary culture when it becomes confluent by

repeating steps 1 to 7, and continue to passage as necessaryrepeating steps 1 to 7, and continue to passage as necessary


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CELL FEEDINGCELL FEEDING

Use prewarmed media and have cells out of the incubator for asUse prewarmed media and have cells out of the incubator for aslittle time as possible. Feeding and sub culturing suspensionlittle time as possible. Feeding and sub culturing suspensioncultures are done simultaneously. About every 2cultures are done simultaneously. About every 2--3 days, dilute3 days, dilute

the cells into fresh media. The dilution you use will depend onthe cells into fresh media. The dilution you use will depend onthe density of the cells and how quickly they divide, whichthe density of the cells and how quickly they divide, whichonly you can determine. Typically 1:4 to 1:20 dilutions areonly you can determine. Typically 1:4 to 1:20 dilutions areappropriate for most cell lines. b. Adherent cells. About everyappropriate for most cell lines. b. Adherent cells. About every22--3 days, pour off old media from culture flasks and replace3 days, pour off old media from culture flasks and replace

with fresh media. Subculture cells as described below beforewith fresh media. Subculture cells as described below beforeconfluency is reached.confluency is reached.


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SUBCULTURING OF ADHERENTSUBCULTURING OF ADHERENT

CELLSCELLSWhen adherent cells become semiWhen adherent cells become semi--confluent, subculture using 2confluent, subculture using 2mM EDTA or trypsin/EDTA.mM EDTA or trypsin/EDTA.

TRYPSINEDTATRYPSINEDTA

a.a.

Remove medium from culture dish and wash cells in a balanced salt solutionRemove medium from culture dish and wash cells in a balanced salt solutionwithout Ca++ or Mg++. Remove the wash solution.without Ca++ or Mg++. Remove the wash solution.

b. Add enough trypsinb. Add enough trypsin--EDTA solution to cover the bottom of the cultureEDTA solution to cover the bottom of the culturevessel and then pour off the excess.vessel and then pour off the excess.

c. Place culture in the 37c. Place culture in the 37C incubator for 2 minutes.C incubator for 2 minutes.

d. Monitor cells under microscope. Cells are beginning to detach when theyd. Monitor cells under microscope. Cells are beginning to detach when theyappear rounded.appear rounded.

e. As soon as cells are in suspension, immediately add culture mediume. As soon as cells are in suspension, immediately add culture mediumcontaining serum. Wash cells once with serum containing medium andcontaining serum. Wash cells once with serum containing medium anddilute as appropriate (generally 4dilute as appropriate (generally 4--20 fold).20 fold).


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EDTA ALONEEDTA ALONE

a. Prepare a 2 mM EDTA solution in a balanced salt solution (i.e.,a. Prepare a 2 mM EDTA solution in a balanced salt solution (i.e.,

PBS without CaPBS without Ca++++

orM

gorM

g++++

).).b. Remove medium from culture vessel by aspiration and wash theb. Remove medium from culture vessel by aspiration and wash the

monolayer to remove all traces of serum. Remove salt solutionmonolayer to remove all traces of serum. Remove salt solutionby aspiration.by aspiration.

c. Dispense enough EDTA solution into culture vessels to completelyc. Dispense enough EDTA solution into culture vessels to completelycover the monolayer of cells.cover the monolayer of cells.

d. The coated cells are allowed to incubate until cells detach fromd. The coated cells are allowed to incubate until cells detach fromthe surface. Progress can be checked by examination with anthe surface. Progress can be checked by examination with aninverted microscope. Cells can be gently nudged by banging theinverted microscope. Cells can be gently nudged by banging theside of the flask against the palm of the hand.side of the flask against the palm of the hand.

e. Dilute cells with fresh medium and transfer to a sterile centrifugee. Dilute cells with fresh medium and transfer to a sterile centrifuge

tube.tube.f. Spin cells down, remove supernatant, and resuspend in culturef. Spin cells down, remove supernatant, and resuspend in culture

medium (or freezing medium if cells are to be frozen). Dilute asmedium (or freezing medium if cells are to be frozen). Dilute asappropriate into culture flasks.appropriate into culture flasks.


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THAWING FROZEN CELLSTHAWING FROZEN CELLS

a. Remove cells from frozen storage and quickly thaw in a 37a. Remove cells from frozen storage and quickly thaw in a 37CC

water bath by gently agitating vial.water bath by gently agitating vial.

b. As soon as the ice crystals melt, pipette gently into a cultureb. As soon as the ice crystals melt, pipette gently into a culture

flask containing prewarmed growth medium.flask containing prewarmed growth medium.

c. Log out cells in the "Liquid Nitrogen Freezer Log" Book.c. Log out cells in the "Liquid Nitrogen Freezer Log" Book.


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VIABLE CELL COUNTVIABLE CELL COUNT

USING A HEMOCYTOMETERTO DETERMINE TOTALUSING A HEMOCYTOMETERTO DETERMINE TOTALCELL COUNTS AND VIABLE CELL NUMBERSCELL COUNTS AND VIABLE CELL NUMBERS

Trypan blue is one of several stains recommended for use inTrypan blue is one of several stains recommended for use indye exclusion procedures for viable cell counting. This methoddye exclusion procedures for viable cell counting. This methodis based on the principle that live cells do not take up certainis based on the principle that live cells do not take up certaindyes, whereas dead cells do.dyes, whereas dead cells do.


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MAINTENANCEMAINTENANCE

Cultures should be examined daily, observing the morphology,Cultures should be examined daily, observing the morphology,

the color of the medium and the density of the cells. A tissuethe color of the medium and the density of the cells. A tissue

culture log should be maintained that is separate from yourculture log should be maintained that is separate from yourregular laboratory notebook. The log should contain: the nameregular laboratory notebook. The log should contain: the name

of the cell line, the medium components and any alterations toof the cell line, the medium components and any alterations to

the standard medium, the dates on which the cells were splitthe standard medium, the dates on which the cells were split

and/or fed, a calculation of the doubling time of the cultureand/or fed, a calculation of the doubling time of the culture

(this should be done at least once during the semester), and(this should be done at least once during the semester), andany observations relative to the morphology, etc.any observations relative to the morphology, etc.


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A. Growth pattern.A. Growth pattern. Cells will initially go through a quiescentCells will initially go through a quiescent

or lag phase that depends on the cell type, the seeding density,or lag phase that depends on the cell type, the seeding density,the media components, and previous handling. The cells willthe media components, and previous handling. The cells will

then go into exponential growth where they have the highestthen go into exponential growth where they have the highest

metabolic activity. The cells will then enter into stationarymetabolic activity. The cells will then enter into stationary

phase where the number of cells is constant, this isphase where the number of cells is constant, this is

characteristic of a confluent population (where all growthcharacteristic of a confluent population (where all growthsurfaces are covered).surfaces are covered).


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B. Harvesting.B. Harvesting. Cells are harvested when the cellsCells are harvested when the cells

have reached a population density which suppresseshave reached a population density which suppressesgrowth. Ideally, cells are harvested when they are in agrowth. Ideally, cells are harvested when they are in asemisemi--confluent state and are still in log phase. Cellsconfluent state and are still in log phase. Cellsthat are not passaged and are allowed to grow to athat are not passaged and are allowed to grow to aconfluent state can sometime lag for a long period ofconfluent state can sometime lag for a long period oftime and some may never recover. It is also essentialtime and some may never recover. It is also essentialto keep your cells as happy as possible to maximizeto keep your cells as happy as possible to maximizethe efficiency of transformation. Most cells arethe efficiency of transformation. Most cells arepassaged (or at least fed) three times a week.passaged (or at least fed) three times a week.


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MechanicalMechanical -- A rubber spatula can be used to physicallyA rubber spatula can be used to physically

remove the cells from the growth surface. This method isremove the cells from the growth surface. This method is

quick and easy but is also disruptive to the cells and may resultquick and easy but is also disruptive to the cells and may result

in significant cell death. This method is best when harvestingin significant cell death. This method is best when harvesting

many different samples of cells for preparing extracts, i.e.,many different samples of cells for preparing extracts, i.e.,

when viability is not important.when viability is not important.

Proteolytic enzymesProteolytic enzymes -- Trypsin, collagenase, or pronase,Trypsin, collagenase, or pronase,usually in combination with EDTA, causes cells to detachusually in combination with EDTA, causes cells to detach

from the growth surface. This method is fast and reliable butfrom the growth surface. This method is fast and reliable but

can damage the cell surface by digesting exposed cell surfacecan damage the cell surface by digesting exposed cell surface

proteins. The proteolysis reaction can be quickly terminated byproteins. The proteolysis reaction can be quickly terminated by

the addition of complete medium containing serumthe addition of complete medium containing serum

EDTAEDTA -- EDTA alone can also be used to detach cells andEDTA alone can also be used to detach cells and

seems to be gentler on the cells than trypsin.seems to be gentler on the cells than trypsin.


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The standard procedure for detaching adherent cells is as follows:The standard procedure for detaching adherent cells is as follows:

1. Visually inspect daily1. Visually inspect daily

2. Release cells from monolayer surface by the following2. Release cells from monolayer surface by the followingprocedure.procedure.

Release cells from monolayer surfaceRelease cells from monolayer surface

wash once with a buffer solutionwash once with a buffer solution

treat with dissociating agenttreat with dissociating agent

observe cells under the microscope. Incubate until cellsobserve cells under the microscope. Incubate until cellsbecome rounded and loosen when flask is gently tapped withbecome rounded and loosen when flask is gently tapped withthe side of the hand.the side of the hand.

Transfer cells to a culture tube and dilute with mediumTransfer cells to a culture tube and dilute with mediumcontaining serum.containing serum.

Spin down cells, remove supernatant and replace with freshSpin down cells, remove supernatant and replace with freshmedium.medium.

Count the cells in a hemacytometer, and dilute asCount the cells in a hemacytometer, and dilute asappropriate into fresh medium.appropriate into fresh medium.


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PRESERVATION AND STORAGE.PRESERVATION AND STORAGE.

LiquidLiquid NN22 isis usedused toto preservepreserve tissuetissue cultureculture cells,cells, eithereither inin thetheliquidliquid phasephase ((--196196C)C) oror inin thethe vaporvapor phasephase ((--156156C)C).. FreezingFreezingcancan bebe lethallethal toto cellscells duedue toto thethe effectseffects ofof damagedamage byby iceicecrystals,crystals, alterationsalterations inin thethe concentrationconcentration ofof electrolytes,electrolytes,dehydration,dehydration, andand changeschanges inin pHpH.. ToTo minimizeminimize thethe effectseffects ofof

freezing,freezing, severalseveral precautionsprecautions areare takentaken..


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SAFETY CONSIDERATIONSSAFETY CONSIDERATIONS

Assume all cultures are hazardous since they may harbor latentAssume all cultures are hazardous since they may harbor latent

viruses or other organisms that are uncharacterized. Theviruses or other organisms that are uncharacterized. Thefollowing safety precautions should also be observed:following safety precautions should also be observed:

pipetting: use pipette aids to prevent ingestion and keeppipetting: use pipette aids to prevent ingestion and keep

aerosols down to a minimumaerosols down to a minimum

no eating, drinking, or smokingno eating, drinking, or smoking wash hands after handling cultures and before leaving the labwash hands after handling cultures and before leaving the lab

decontaminate work surfaces with disinfectant (before anddecontaminate work surfaces with disinfectant (before and

after)after)


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autoclave all wasteautoclave all waste

use biological safety cabinet (laminar flow hood) whenuse biological safety cabinet (laminar flow hood) when

working with hazardous organisms. The cabinet protectsworking with hazardous organisms. The cabinet protectsworker by preventing airborne cells and viruses releasedworker by preventing airborne cells and viruses releasedduring experimental activity from escaping the cabinet; thereduring experimental activity from escaping the cabinet; thereis an air barrier at the front opening and exhaust air is filteredis an air barrier at the front opening and exhaust air is filteredwith a HEPA filter make sure cabinet is not overloaded andwith a HEPA filter make sure cabinet is not overloaded and

leave exhaust grills in the front and the back clear (helps toleave exhaust grills in the front and the back clear (helps tomaintain a uniform airflow)maintain a uniform airflow)

use aseptic techniqueuse aseptic technique

dispose of all liquid waste after each experiment and treat withdispose of all liquid waste after each experiment and treat withbleachbleach


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Some of the Problems Faced by Cultured CellsSome of the Problems Faced by Cultured Cells

AvoidingAvoiding ContaminationContamination

TwoTwo typestypes ofof contaminationcontamination

BiologicalBiological ee..gg.. viruses,viruses, bacteria,bacteria, fungifungi etcetc

ChemicalChemical ee..gg.. endotoxinsendotoxins metalmetal ionsions andand tracestraces ofof disinfectantsdisinfectants..


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Methods:Methods:

1. proper training in and use of good aseptic technique on1. proper training in and use of good aseptic technique onthe part of the cell culturist.the part of the cell culturist.

Secondly properly designed maintained and sterilizedSecondly properly designed maintained and sterilized

equipments, plastic wares, glass wares and media .The carefulequipments, plastic wares, glass wares and media .The careful

and selective (limited) use of antibiotics designed for use inand selective (limited) use of antibiotics designed for use in

tissue culture can also help avoid culture loss due to biologicaltissue culture can also help avoid culture loss due to biological

contamination.contamination.


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Finding A Happy EnvironmentFinding A Happy Environment

AA happyhappy environmentenvironment isis thethe oneone whichwhich

AllowsAllows thethe cellcell toto survivesurvive..

AllowsAllows thethe cellcell toto dividedivide andand redivideredivide ii..ee.. increasedincreased mitosismitosis..

CarryingCarrying outout impimp inin vivovivo physiologicalphysiological oror biochemicalbiochemical

functionsfunctions suchsuch asas musclemuscle contractionscontractions oror secretionsecretion ofof

enzymesenzymes oror hormoneshormones..


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BASIC ENVOIRNMENTALBASIC ENVOIRNMENTAL

REQUIREMENTS FOR HAPPY CELLSREQUIREMENTS FOR HAPPY CELLS

Controlled temperature (depends upon host organism) coldControlled temperature (depends upon host organism) cold

blooded vertebrates temp is 18 to 25C. While mammalian itsblooded vertebrates temp is 18 to 25C. While mammalian its

36 to 37C.36 to 37C.

Good substrate for cell attachment such as collagen, gelatin,Good substrate for cell attachment such as collagen, gelatin,

fibronectin and laminin.fibronectin and laminin.

Appropriate medium and incubator that maintains the correctAppropriate medium and incubator that maintains the correct

pH and osmolality.pH and osmolality.


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How to Decide if Cultured Cells AreHow to Decide if Cultured Cells AreHappyHappy

TheThe MorphologyMorphology oror cellcell shapeshape usualyusualy stainingstaining isis donedone toto

findfind outout anyany problemproblem.. AlsoAlso aa difficultdifficult characteristiccharacteristic toto

quantifyquantify andand measuredmeasured preciselyprecisely..

GrowthGrowth raterate determinationdetermination..

PlatingPlating EfficiencyEfficiency..

ExpressionExpression ofof aa specializedspecialized functionfunction..


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Whatis Cell Culture UsedWhatis Cell Culture Used

For?For?Cell culture has become one of the major tools used in cell andCell culture has become one of the major tools used in cell and

molecular biology. Some of the important areas where cellmolecular biology. Some of the important areas where cell

culture is currently playing a major role are briefly describedculture is currently playing a major role are briefly described

below:below:

Model SystemsModel Systems

Toxicity TestingToxicity Testing

Cancer ResearchCancer Research


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VirologyVirology

CellCell--BasedManufacturingBasedManufacturing

Genetic CounselingGenetic Counseling

Genetic EngineeringGenetic Engineering

Gene TherapyGene Therapy Drug Screening and DevelopmentDrug Screening and Development


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Model SystemsModel Systems

Cell cultures provide a good model system for studyingCell cultures provide a good model system for studying

1) basic cell biology and biochemistry,1) basic cell biology and biochemistry,

2) the interactions between disease2) the interactions between disease--causing agents and cells,causing agents and cells, 3) the effects of drugs on cells,3) the effects of drugs on cells,

4) the process and triggers for aging, and 5) nutritional4) the process and triggers for aging, and 5) nutritional

studies.studies.


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Toxicity TestingToxicity Testing

Cultured cells are widely used alone or in conjunctionCultured cells are widely used alone or in conjunction

with animal tests to study the effects of new drugs,with animal tests to study the effects of new drugs,

cosmetics and chemicals on survival and growth in acosmetics and chemicals on survival and growth in awide variety of cell types. Especially important arewide variety of cell types. Especially important are

liverliver-- and kidneyand kidney--derived cell cultures.derived cell cultures.


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Cancer ResearchCancer Research

SinceSince bothboth normalnormal cellscells andand cancercancer cellscells cancan bebe growngrown inin

culture,culture, thethe basicbasic differencesdifferences betweenbetween themthem cancan bebe closelyclosely

studiedstudied.. InIn addition,addition, itit isis possible,possible, byby thethe useuse ofof chemicals,chemicals,

virusesviruses andand radiation,radiation, toto convertconvert normalnormal culturedcultured cellscells toto

cancercancer causingcausing cellscells.. Thus,Thus, thethe mechanismsmechanisms thatthat causecause thethe

changechange cancan bebe studiedstudied.. CulturedCultured cancercancer cellscells alsoalso serveserve asas aa

testtest systemsystem toto determinedetermine suitablesuitable drugsdrugs andand methodsmethods forfor

selectivelyselectively destroyingdestroying typestypes ofof cancercancer..


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VirologyVirology

OneOne ofof thethe earliestearliest andand majormajor usesuses ofof cellcell cultureculture isis thethe

replicationreplication ofof virusesviruses inin cellcell culturescultures (in(in placeplace ofof

animals)animals) forfor useuse inin vaccinevaccine productionproduction.. CellCell culturescultures

areare alsoalso widelywidely usedused inin thethe clinicalclinical detectiondetection andandisolationisolation ofof viruses,viruses, asas wellwell asas basicbasic researchresearch intointo howhow

theythey growgrow andand infectinfect organismsorganisms..


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CELL BASED MANUFACTURINGCELL BASED MANUFACTURING

Use of cells as replacement tissue and organ.Use of cells as replacement tissue and organ.

large scale production of viruses for use in vaccines.large scale production of viruses for use in vaccines.

Large scale production if genetically engineered cellLarge scale production if genetically engineered cellfor the production of commercially imp protein.for the production of commercially imp protein.


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Genetic CounselingGenetic Counseling

Amniocentesis,Amniocentesis, aa diagnosticdiagnostic techniquetechnique thatthat enablesenables

doctorsdoctors toto removeremove andand cultureculture fetalfetal cellscells fromfrom pregnantpregnant

women,women, hashas givengiven doctorsdoctors anan importantimportant tooltool forfor thethe earlyearly

diagnosisdiagnosis ofof fetalfetal disordersdisorders.. TheseThese cellscells cancan thenthen bebe

examinedexamined forfor abnormalitiesabnormalities inin theirtheir chromosomeschromosomes andand

genesgenes usingusing karyotyping,karyotyping, chromosomechromosome paintingpainting andand otherother

molecularmolecular techniquestechniques..


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Genetic EngineeringGenetic Engineering

TheThe abilityability toto transfecttransfect oror reprogramreprogram culturedcultured cellscells withwith

newnew geneticgenetic materialmaterial (DNA(DNA andand genes)genes) hashas providedprovided aa

majormajor tooltool toto molecularmolecular biologistsbiologists wishingwishing toto studystudy thethe

cellularcellular effectseffects ofof thethe expressionexpression ofof thesestheses genesgenes (new(newproteins)proteins).. TheseThese techniquestechniques cancan alsoalso bebe usedused toto produceproduce

thesethese newnew proteinsproteins inin largelarge quantityquantity inin culturedcultured cellscells forfor

furtherfurther studystudy.. InsectInsect cellscells areare widelywidely usedused asas miniatureminiature

cellscells factoriesfactories toto expressexpress substantialsubstantial quantitiesquantities ofof proteinsproteins

thatthat theythey manufacturemanufacture afterafter beingbeing infectedinfected withwithgeneticallygenetically engineeredengineered baculobaculo virusesviruses..


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Drug Screening and DevelopmentDrug Screening and Development

CellCell--based assays have become increasingly important for thebased assays have become increasingly important for the

pharmaceutical industry, not just for cyto toxicity testing butpharmaceutical industry, not just for cyto toxicity testing but

also for high throughput screening of compounds that mayalso for high throughput screening of compounds that may

have potential use as drugs. Originally, these cell culture testshave potential use as drugs. Originally, these cell culture tests

were done in 96 well plates, but increasing use is now beingwere done in 96 well plates, but increasing use is now being

made of 384 and 1536 well plates.made of 384 and 1536 well plates.


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Gene TherapyGene Therapy

The ability to genetically engineer cells has also led to their useThe ability to genetically engineer cells has also led to their use

for gene therapy. Cells can be removed from a patient lacking afor gene therapy. Cells can be removed from a patient lacking a

functional gene and the missing or damaged gene can then befunctional gene and the missing or damaged gene can then be

replaced. The cells can be grown for a while in culture and thenreplaced. The cells can be grown for a while in culture and thenreplaced into the patient. An alternative approach is to place thereplaced into the patient. An alternative approach is to place the

missing gene into a viral vector and then infect the patient withmissing gene into a viral vector and then infect the patient with

the virus in the hope that the missing gene will then be expressedthe virus in the hope that the missing gene will then be expressed

in the patients cells.in the patients cells.


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