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Mammalian Cell CultureMammalian Cell CultureIntroductionIntroduction•• Eukaryotic cells are much more difficult toEukaryotic cells are much more difficult to

culture than most prokaryotes.culture than most prokaryotes.–– They demand complex mediaThey demand complex media–– They are very susceptible to contaminationThey are very susceptible to contamination

and overgrowth by microbes such as bacteria,and overgrowth by microbes such as bacteria,yeasts and fungi.yeasts and fungi.

Cell CultureCell Culture

•• The cultivation orThe cultivation orgrowth of cellsgrowth of cellsoutside of the hostoutside of the hostorganism.organism.

•• There are three typesThere are three typesof eukaryotic cellof eukaryotic cellculture.culture.

•• BenefitBenefit–– Allows direct access toAllows direct access to

a clean population ofa clean population ofcells.cells.

•• DisadvantageDisadvantage–– The architecture of theThe architecture of the

original tissue is lost.original tissue is lost.–– Cells also tend to loseCells also tend to lose

some of theirsome of theirdifferentiation ordifferentiation orspecialization.specialization.

Primary CulturesPrimary Cultures

•• Cells are explanted directly from a donorCells are explanted directly from a donororganism, e.g. white blood cells or nasalorganism, e.g. white blood cells or nasalbrushings.brushings.

•• They may be capable of one or twoThey may be capable of one or twodivisions in culture, and given the rightdivisions in culture, and given the rightconditions can survive for some time.conditions can survive for some time.

•• They do not continue to grow andThey do not continue to grow andeventually senesce and die.eventually senesce and die.

AssessmentAssessment

•• AdvantagesAdvantages–– They are thought toThey are thought to

represent the bestrepresent the bestexperimental modelsexperimental modelsfor for in vivoin vivo situations. situations.

–– They may expressThey may expresscharacteristics whichcharacteristics whichare not seen inare not seen incultured cells.cultured cells.•• Functional CiliaFunctional Cilia•• Regulated ContractionRegulated Contraction

•• AdvantagesAdvantages–– Have the sameHave the same

karyotype karyotype as theas theparent tissue normalparent tissue normalor abnormal.or abnormal.

•• DisadvantagesDisadvantages–– Difficult to obtain.Difficult to obtain.–– Very susceptible toVery susceptible to

contaminationcontamination

Cell Line CultureCell Line Culture

•• Secondary CultureSecondary Culture–– Originally explanted from aOriginally explanted from a

donor organism, and givendonor organism, and giventhe correct culturethe correct cultureconditions, divide and growconditions, divide and growfor some time for some time in vitroin vitro..

–– They do not continue toThey do not continue todivide indefinitely anddivide indefinitely andeventually, their physicaleventually, their physicalcharacteristics may change,characteristics may change,after which the cells willafter which the cells willeventually senesce and die.eventually senesce and die.

•• The factors which controlThe factors which controlthe replication of suchthe replication of suchcells cells in vitroin vitro are related are relatedto the degree ofto the degree ofdifferentiation of the cell.differentiation of the cell.

•• Terminally differentiatedTerminally differentiatedcells are harder tocells are harder tomaintain than lessmaintain than lessspecialized cells.specialized cells.

Finite Cell LineFinite Cell Line•• A cell line that has a limited number of possibleA cell line that has a limited number of possible

subcultivationssubcultivations or passages. or passages.•• Divide and grow for some time Divide and grow for some time in vitroin vitro

–– 50-100 generations50-100 generations

•• MRC5 cellsMRC5 cells–– Secondary humanSecondary human

embryonic lungembryonic lungfibroblastsfibroblasts

–– Undergo between 60-Undergo between 60-70 doublings before70 doublings beforesenescence.senescence.

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AssessmentAssessment

•• AdvantagesAdvantages–– Can obtain a largeCan obtain a large

population of similarpopulation of similarcells.cells.

–– Most cellularMost cellularcharacteristics arecharacteristics aremaintained.maintained.

•• DisadvantagesDisadvantages–– Cells have a tendencyCells have a tendency

to differentiate overto differentiate overtime in culture.time in culture.

–– Over time the cultureOver time the culturetends to select fortends to select foraberrant cells.aberrant cells.

Infinite Cell LineInfinite Cell Line•• A cell line that hasA cell line that has

demonstrated the potentialdemonstrated the potentialto be to be subculturedsubculturedindefinitely.indefinitely.–– Continuous cell lineContinuous cell line–– Immortal cells line.Immortal cells line.

•• Immortalized cell linesImmortalized cell linesare also known asare also known astransformed cells:transformed cells:–– Cells whose growthCells whose growth

properties have beenproperties have beenaltered.altered.

•• This does not necessarilyThis does not necessarilymean that these aremean that these are"cancer" or "tumor" cells,"cancer" or "tumor" cells,that are able to form athat are able to form atumor if introduced intotumor if introduced intoan experimental animal.an experimental animal.

•• In some cases these cellsIn some cases these cellsmay establish tumors inmay establish tumors inanimal models.animal models.

HeLaHeLa Cells Cells

•• Classic example of anClassic example of animmortalized cell line.immortalized cell line.–– These are humanThese are human

epithelial cells from aepithelial cells from afatal cervical carcinomafatal cervical carcinomatransformed by transformed by humanhumanpapillomaviruspapillomavirus 18 18(HPV18)(HPV18)..

HeLaHeLa Cells Cells•• Adherent cells which maintain contact inhibitionAdherent cells which maintain contact inhibition

in vitro:in vitro:–– As they spread out across the culture flask, when twoAs they spread out across the culture flask, when two

adjacent cells touch, this signals them to stopadjacent cells touch, this signals them to stopgrowing.growing.

•• Loss of contact inhibition is a classic sign ofLoss of contact inhibition is a classic sign ofoncogeniconcogenic cells: cells:–– Cells which form tumors in experimental animals.Cells which form tumors in experimental animals.–– Such cells not only form a monolayer in culture butSuch cells not only form a monolayer in culture but

also pile up on top of one another.also pile up on top of one another.•• HeLaHeLa cells are not cells are not oncogeniconcogenic in animals, but in animals, but

they may become so if further transformed by athey may become so if further transformed by avirus virus oncogeneoncogene..

AssessmentAssessment

•• AdvantagesAdvantages–– Easy to maintain inEasy to maintain in

culture.culture.–– Easy to obtain largeEasy to obtain large

population of cells.population of cells.–– Typically easy toTypically easy to

manipulate genemanipulate geneexpression.expression.

•• DisadvantagesDisadvantages–– The more aggressiveThe more aggressive

the cell line the morethe cell line the moreit changes over time init changes over time inculture.culture.

–– Not clear how theNot clear how thefunction of these cellsfunction of these cellsrelates to that of otherrelates to that of othercells, healthy orcells, healthy ordiseased.diseased.

Infinite Cell LinesInfinite Cell LinesTypes of Cell GrowthTypes of Cell Growth

•• Attachment CulturesAttachment Cultures–– To survive and grow,To survive and grow,

most cells require amost cells require asurface to which theysurface to which theycan attachcan attach

–– That is they areThat is they areanchorage dependent.anchorage dependent.

•• Without the surfaceWithout the surfaceattachment theseattachment thesecells cannot survive.cells cannot survive.

•• Suspension CulturesSuspension Cultures–– Some cells can survive andSome cells can survive and

divide while beingdivide while beingsuspended in a fluid mediasuspended in a fluid mediaand stirred or shaken.and stirred or shaken.•• Shaker CulturesShaker Cultures•• Spinner CulturesSpinner Cultures

•• A limited number of cellA limited number of celltypes can be maintainedtypes can be maintainedand grown in eitherand grown in eitherformat.format.–– Can be switched betweenCan be switched between

formats to meetformats to meetexperimental needs.experimental needs.

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Number of Cell DivisionsNumber of Cell Divisions

•• Growing cells in culture.Growing cells in culture.–– Place cells in a culture dish.Place cells in a culture dish.–– Give them nutrients,Give them nutrients,

growth factors, keep themgrowth factors, keep themfree from bacterial.free from bacterial.

–– Cells will grow to cover theCells will grow to cover thesurface of the dish.surface of the dish.

–– Can take cells out of thisCan take cells out of thisculture and start a newculture and start a newculture.culture.

•• Splitting cells from oneSplitting cells from onedish to another is adish to another is apassage.passage.

Number of Cell DivisionsNumber of Cell Divisions

•• This ability to split cells andThis ability to split cells andhave them continue tohave them continue todivide is not without limitsdivide is not without limitshowever.however.

•• Normal cells have a limit toNormal cells have a limit tothe number of times whichthe number of times whichthey can be passed inthey can be passed inculture.culture.

•• This number does varyThis number does varyfrom cell type to cell type,from cell type to cell type,but commonly the limit isbut commonly the limit isbetween 50 and 100between 50 and 100passages.passages.

HayflickHayflick’’ss Phenomenon Phenomenon

•• Cells will continue toCells will continue togrow and divide normallygrow and divide normallyfor a limited number offor a limited number ofpassagespassages

•• When they get to aWhen they get to acertain point even if theycertain point even if theyare given the appropriateare given the appropriatenutrients, they simplynutrients, they simplystop dividing and willstop dividing and willeventually die.eventually die.

Cel

l Num

ber

Passage Number

HayflickHayflick’’ss Phenomenon and Aging Phenomenon and Aging

•• There appears to be aThere appears to be acorrelation between thecorrelation between themaximal number ofmaximal number ofpassages and aging.passages and aging.

•• The number of passagesThe number of passagesdecreases when cells aredecreases when cells areharvested from olderharvested from olderindividuals.individuals.

Age (years)Pa

sses

70

0-0.5 100

ProgeriaProgeria

•• A collection of defects whichA collection of defects whichcauses premature aging.causes premature aging.

•• Genetic disorder which causesGenetic disorder which causesphysical symptoms like grayphysical symptoms like grayhair, wrinkled skin, hair loss,hair, wrinkled skin, hair loss,muscle degeneration.muscle degeneration.

•• Child of age 4 or 5 appears likeChild of age 4 or 5 appears likethey are 80.they are 80.

•• Cells from these individualsCells from these individualsshow dramatically decreasedshow dramatically decreasedpassage number.passage number.

Passage Number and CancerPassage Number and Cancer

•• Cancer cells appear to be immortal.Cancer cells appear to be immortal.•• In the early 1900In the early 1900’’s cervical cancer cells weres cervical cancer cells were

removed from a woman.removed from a woman.–– HeLaHeLa cells. cells.–– Helen Lang, Henrietta Lack.Helen Lang, Henrietta Lack.

•• These cells have been grown in culture and usedThese cells have been grown in culture and usedextensively in science.extensively in science.–– HeLaHeLa cells have been passed well over 1,000 times cells have been passed well over 1,000 times

and show no sign of slowing their grow rate.and show no sign of slowing their grow rate.

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Contact InhibitionContact Inhibition•• The phenomenon observedThe phenomenon observed

in normal animal cells thatin normal animal cells thatcauses them to stopcauses them to stopdividing when they comedividing when they comeinto contact with oneinto contact with oneanother.another.

•• Cells in a culture flask withCells in a culture flask withthe appropriate nutrientsthe appropriate nutrientsand the cells grow andand the cells grow anddivide.divide.

•• Continues until the cells areContinues until the cells arecovering the entire surface.covering the entire surface.

•• At that point they stopAt that point they stopdividing.dividing.

Contact InhibitionContact Inhibition

•• These cells can beThese cells can betriggered to begintriggered to begindividing again by givingdividing again by givingthem more room.them more room.

•• The cells now being in anThe cells now being in anenvironment where theyenvironment where theyare not in contact withare not in contact withone another begin toone another begin todivide again.divide again.

Contact InhibitionContact Inhibition

•• Cancer cells do not display contact inhibition.Cancer cells do not display contact inhibition.•• Put them in a culture dish, they will grow to create aPut them in a culture dish, they will grow to create a

single layer of cellssingle layer of cells

•• Then they will continue to grow multiple layers andThen they will continue to grow multiple layers andcreate piles of cells.create piles of cells.

GROWTH CYCLE IN ATTACHEMENTGROWTH CYCLE IN ATTACHEMENTCULTURECULTURE

•• Eukaryotic cells inEukaryotic cells inattachment cultureattachment culturehave a characteristichave a characteristicgrowth cycle similargrowth cycle similarto bacteria.to bacteria.

•• The growth cycle isThe growth cycle istypically divided intotypically divided intothree phases.three phases.–– Lag PhaseLag Phase–– Log PhaseLog Phase–– Plateau PhasePlateau Phase

GROWTH CYCLE IN ATTACHEMENTGROWTH CYCLE IN ATTACHEMENTCULTURECULTURE

92105760000

675000710000885000770000

Day 6

75674603125

642500532500547500690000

Day 5

63656303750

380000227500287500320000

Day 4

38588186875

190000237500145000175000

Day 3

17722139375

117500155000152500132500

Day 2

5774110000

115000115000105000105000

Day 1

StandardDeviation

Average #of Cells

Cells/WellDays (in culture)

Lag PhaseLag Phase

•• This is the time following subculture andThis is the time following subculture andreseeding during which there is little evidence ofreseeding during which there is little evidence ofan increase in cell number.an increase in cell number.

•• It is a period of adaptation during which the cellIt is a period of adaptation during which the cellreplaces elements of the replaces elements of the glycocalyxglycocalyx lost during lost duringtrypsinizationtrypsinization, attaches to the substrate, and, attaches to the substrate, andspreads out.spreads out.

•• During spreading the cytoskeleton reappearsDuring spreading the cytoskeleton reappearsand its reappearance is probably an integral partand its reappearance is probably an integral partof the spreading process.of the spreading process.

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Log PhaseLog Phase

•• This is the period of exponential increase in cellThis is the period of exponential increase in cellnumber following the lag period and terminatingnumber following the lag period and terminatingone or two doublings after confluence isone or two doublings after confluence isreached.reached.

•• The length of the log phase depends on theThe length of the log phase depends on theseeding density, the growth rate of the cells,seeding density, the growth rate of the cells,and the density at which cell proliferation isand the density at which cell proliferation isinhibited by density.inhibited by density.

•• In the log phase the growth fraction is highIn the log phase the growth fraction is high(usually 90%-100%) and the culture is in its(usually 90%-100%) and the culture is in itsmost reproducible form.most reproducible form.

Log PhaseLog Phase

•• It is the optimal time for sampling sinceIt is the optimal time for sampling sincethe population is at its most uniform andthe population is at its most uniform andviability is high.viability is high.

•• The cells are, however, randomlyThe cells are, however, randomlydistributed in the cell cycle and, for somedistributed in the cell cycle and, for somepurposes, may need to be synchronized.purposes, may need to be synchronized.

Plateau PhasePlateau Phase

•• Toward the end of the log phase, theToward the end of the log phase, theculture becomes confluentculture becomes confluent–– All the available growth surface is occupiedAll the available growth surface is occupied

and all the cells are in contact withand all the cells are in contact withsurrounding cells.surrounding cells.

•• Following confluence the growth rate ofFollowing confluence the growth rate ofthe culture is reduced, and in some cases,the culture is reduced, and in some cases,cell proliferation ceases almost completelycell proliferation ceases almost completelyafter one or two further populationafter one or two further populationdoublings.doublings.

Plateau PhasePlateau Phase

•• At this stage, the culture enters theAt this stage, the culture enters theplateau (or stationary) phase, and theplateau (or stationary) phase, and thegrowth fraction falls to between 0 andgrowth fraction falls to between 0 and10%.10%.

•• There may be a relative increase in theThere may be a relative increase in thesynthesis of specialized versus structuralsynthesis of specialized versus structuralproteins and the constitution and chargeproteins and the constitution and chargeof the cell surface may be changed.of the cell surface may be changed.

Know Your CellsKnow Your Cells•• When you start a series of experiments,When you start a series of experiments,

someone in the lab may provide you with a tubesomeone in the lab may provide you with a tubeor flask of cells, with a brief description aboutor flask of cells, with a brief description aboutthe care of the cells. The cells may be easy tothe care of the cells. The cells may be easy tomaintain, but the more you know about themaintain, but the more you know about thecells, the more finely attuned you are to thecells, the more finely attuned you are to thecellcell’’s quirks, the quicker and more clear thes quirks, the quicker and more clear theinterpretation of results will be. Look upinterpretation of results will be. Look upreferences for the cells. Speak to people whoreferences for the cells. Speak to people whohave used the cells and ask for advice. Mosthave used the cells and ask for advice. Mostimportantly, monitor the cells constantly, untilimportantly, monitor the cells constantly, untilyou are the expert on their growth.you are the expert on their growth.

At the Bench At the Bench –– Chapter 10 Chapter 10

Aseptic TechniqueAseptic Technique

•• Aseptic technique is the execution ofAseptic technique is the execution oftissue culture procedures withouttissue culture procedures withoutintroducing contamination microorganismsintroducing contamination microorganismsfrom the environment.from the environment.

•• Most of the problems related to cellMost of the problems related to cellculture are associated with the lack ofculture are associated with the lack ofgood aseptic technique.good aseptic technique.

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Aseptic TechniqueAseptic Technique

•• Whenever possible, work in a cell culture hood.Whenever possible, work in a cell culture hood.–– This will prevent airborne organisms from entering yourThis will prevent airborne organisms from entering your

cultures.cultures.

•• Work areas should be wiped clean with 70% ethanolWork areas should be wiped clean with 70% ethanolbefore and after use.before and after use.

–– It may help to rinse your hands with ethanol as well or toIt may help to rinse your hands with ethanol as well or towear gloves.wear gloves.

•• Never leave sterile flasks, bottles, or Never leave sterile flasks, bottles, or petripetri dishes open dishes opento the environment.to the environment.

–– Do not remove the cover until the instant you are ready to useDo not remove the cover until the instant you are ready to useit, and replace the cap as soon as you are finished.it, and replace the cap as soon as you are finished.

Aseptic TechniqueAseptic Technique•• Never place caps removed from bottles or flasks downNever place caps removed from bottles or flasks down

on the bench surface. Instead, grasp it in the pinkyon the bench surface. Instead, grasp it in the pinkyfinger of one hand and keep it facing down.finger of one hand and keep it facing down.

–– Once a bottle is open, keep it tilted so that airborneOnce a bottle is open, keep it tilted so that airbornemicroorganisms will not fall directly into the medium.microorganisms will not fall directly into the medium.

•• Sterile pipettes should not be removed from theirSterile pipettes should not be removed from theircontainer until just before use.container until just before use.

–– They should be kept at the sterile bench or in the culture hoodThey should be kept at the sterile bench or in the culture hoodonly.only.

•• Use a separate sterile pipette for each manipulation.Use a separate sterile pipette for each manipulation.–– Do not draw from a bottle more than once with the sameDo not draw from a bottle more than once with the same

pipette.pipette.•• Do not talk, sing, chew gum, or breathe directly in yourDo not talk, sing, chew gum, or breathe directly in your

culture when performing sterile technique.culture when performing sterile technique.•• Techniques should be performed as rapidly as possibleTechniques should be performed as rapidly as possible

to minimize exposure to microorganisms.to minimize exposure to microorganisms.

Culture MediaCulture Media•• IonsIons

–– Na, K, Ca, Mg, Na, K, Ca, Mg, ClCl, P, Bicarbonate, P, Bicarbonate•• Trace elementsTrace elements

–– iron, zinc, seleniumiron, zinc, selenium•• SugarsSugars

–– glucose is the most commonglucose is the most common•• Amino acidsAmino acids

–– 13 essential13 essential•• VitaminsVitamins•• SerumSerum

–– Contains a large number of growth promoting activities such asContains a large number of growth promoting activities such asbuffering toxic nutrients by binding them, neutralizes buffering toxic nutrients by binding them, neutralizes trypsintrypsin and other and otherproteasesproteases

–– Contains peptide hormones or hormone-like growth factors thatContains peptide hormones or hormone-like growth factors thatpromote healthy growth.promote healthy growth.

•• Antibiotics - although not required for cell growth, antibiotics areAntibiotics - although not required for cell growth, antibiotics areoften used to control the growth of bacterial and fungaloften used to control the growth of bacterial and fungalcontaminants.contaminants.

Eyeballing Cell CulturesEyeballing Cell Cultures•• Check the pH of the culture medium by looking at theCheck the pH of the culture medium by looking at the

color of the indicator in the culture medium, phenol red.color of the indicator in the culture medium, phenol red.•• Using a microscope, check the attachment of the cellsUsing a microscope, check the attachment of the cells

growth surface. Most of the cells should appear wellgrowth surface. Most of the cells should appear wellattached and spread out in a healthy culture.attached and spread out in a healthy culture.

•• The growth of a culture can be estimated byThe growth of a culture can be estimated bymicroscopically following the development of a full cellmicroscopically following the development of a full cellsheet (sheet (confluencyconfluency).).

•• Cell shape is an important guide.Cell shape is an important guide.–– Round cells in an Round cells in an uncrowdeduncrowded culture are not good (unless they culture are not good (unless they

happen to be dividing cellshappen to be dividing cells——look for doublets of dividing cells).look for doublets of dividing cells).•• One of the most valuable early guides in evaluating theOne of the most valuable early guides in evaluating the

success of a subculture is the rate at which the cells in asuccess of a subculture is the rate at which the cells in anewly established culture attach and spread out.newly established culture attach and spread out.