细胞培养(英文版)课件

ParasYadav1,AnnuYadav1,P.Kumar1,J.S.Arora1,T.K.Datta1,S.De1,S.L.Goswami1,MukeshYadav2,ShaliniJain3,RavinderNagpal4andHariomYadav3

1DepartmentofAnimalBiotechnology,3AnimalBiochemistryDivisionand4DairyMicrobiologyDivision,NationalDairyResearchInstitute,Karnal132001(Haryana),India;2SOSinChemistry,JiwajiUniversity,Gwalior-474011,M.P.,IndiaBasicsofCellCulture ParasYadav1,AnnuYadav1,P.1IntroductionCellcultureistheprocessbywhichprokaryotic,eukaryoticorplantcellsaregrownundercontrolledconditions.Butinpracticeitreferstotheculturingofcellsderivedfromanimalcells.CellculturewasfirstsuccessfullyundertakenbyRossHarrisonin1907Rouxin1885forthefirsttimemaintainedembryonicchickcellsinacellcultureIntroductionCellcultureisth2Historicaleventsinthedevelopmentofcellculture1878:ClaudeBernardproposedthatphysiologicalsystemsofanorganismcanbemaintainedinalivingsystemafterthedeathofanorganism.1885:Rouxmaintainedembryonicchickcellsinasalineculture.1897:Loebdemonstratedthesurvivalofcellsisolatedfrombloodandconnectivetissueinserumandplasma.1903:Jollyobservedcelldivisionofsalamanderleucocytesinvitro.1907:Harrisoncultivatedfrognervecellsinalymphclotheldbythe'hangingdrop'methodandobservedthegrowthofnervefibersinvitroforseveralweeks.Hewasconsideredbysomeasthefatherofcellculture.1910:Burrowssucceededinlongtermcultivationofchickenembryocellinplasmaclots.Hemadedetailedobservationofmitosis.Historicaleventsinthedevel3Contd..1911:LewisandLewismadethefirstliquidmediaconsistedofseawater,serum,embryoextract,saltsandpeptones.Theyobservedlimitedmonolayergrowth.1913:Carrelintroducedstrictaseptictechniquessothatcellscouldbeculturedforlongperiods.1916:RousandJonesintroducedproteolyticenzymetrypsinforthesubcultureofadherentcells.1923:CarrelandBakerdeveloped'Carrel'orT-flaskasthefirstspecificallydesignedcellculturevessel.Theyemployedmicroscopicevaluationofcellsinculture.1927:CarrelandRiveraproducedthefirstviralvaccine-Vaccinia.1933:GeydevelopedtherollertubetechniqueContd..1911:LewisandLewism4Contd..1940s:Theuseoftheantibioticspenicillinandstreptomycininculturemediumdecreasedtheproblemofcontaminationincellculture.1948:EarleisolatedmouseLfibroblastswhichformedclonesfromsinglecells.Fischerdevelopedachemicallydefinedmedium,CMRL1066.1952:GeyestablishedacontinuouscelllinefromahumancervicalcarcinomaknownasHeLa(HelenLane)cells.Dulbeccodevelopedplaqueassayforanimalvirusesusingconfluentmonolayersofculturedcells.1954:Abercrombieobservedcontactinhibition:motilityofdiploidcellsinmonolayercultureceaseswhencontactismadewithadjacentcells.1955:Eaglestudiedthenutrientrequirementsofselectedcellsincultureandestablishedthefirstwidelyusedchemicallydefinedmedium.1961:HayflickandMoorheadisolatedhumanfibroblasts(WI-38)andshowedthattheyhaveafinitelifespaninculture.1964:LittlefieldintroducedtheHATmediumforcellselection.1965:Hamintroducedthefirstserum-freemediumwhichwasabletosupportthegrowthofsomecells.Contd..1940s:Theuseofthea5Contd..1965:HarrisandWatkinswereabletofusehumanandmousecellsbytheuseofavirus.1975:KohlerandMilsteinproducedthefirsthybridomacapableofsecretingamonoclonalantibody.1978:Satoestablishedthebasisforthedevelopmentofserum-freemediafromcocktailsofhormonesandgrowthfactors.1982:Humaninsulinbecamethefirstrecombinantproteintobelicensedasatherapeuticagent.1985:Humangrowthhormoneproducedfromrecombinantbacteriawasacceptedfortherapeuticuse.1986:LymphoblastoidγIFNlicensed.1987:Tissue-typeplasminogenactivator(tPA)fromrecombinantanimalcellsbecamecommerciallyavailable.1989:Recombinanterythropoietinintrial.1990:Recombinantproductsinclinicaltrial(HBsAG,factorVIII,HIVgp120,CD4,GM-CSF,EGF,mAbs,IL-2).Contd..1965:HarrisandWatkin6Majordevelopment’sincellculturetechnologyFirstdevelopmentwastheuseofantibioticswhichinhibitsthegrowthofcontaminants.SecondwastheuseoftrypsintoremoveadherentcellstosubculturefurtherfromtheculturevesselThirdwastheuseofchemicallydefinedculturemedium.Majordevelopment’sincellcu7Whyiscellcultureusedfor?

Areaswherecellculturetechnologyiscurrentlyplayingamajorrole.ModelsystemsforStudyingbasiccellbiology,interactionsbetweendiseasecausingagentsandcells,effectsofdrugsoncells,processandtriggeringofaging&nutritionalstudiesToxicitytestingStudytheeffectsofnewdrugsCancerresearchStudythefunctionofvariouschemicals,virus&radiationtoconvertnormalculturedcellstocancerouscells

Whyiscellcultureusedfor?8Contd….Virology

Cultivationofvirusforvaccineproduction,alsousedtostudythereinfectiouscycle.

GeneticEngineering

Productionofcommercialproteins,largescaleproductionofvirusesforuseinvaccineproductione.g.polio,rabies,chickenpox,hepatitisB&measles

Genetherapy

Cellshavingafunctionalgenecanbereplacedtocellswhicharehavingnon-functionalgeneContd….Virology9TissuecultureInvitrocultivationoforgans,tissues&cellsatdefinedtemperatureusinganincubator&supplementedwithamediumcontainingcellnutrients&growthfactorsiscollectivelyknownastissuecultureDifferenttypesofcellgrownincultureincludesconnectivetissueelementssuchasfibroblasts,skeletaltissue,cardiac,epithelialtissue(liver,breast,skin,kidney)andmanydifferenttypesoftumorcells.

TissuecultureInvitrocultiva10PrimarycultureCellswhensurgicallyorenzymaticallyremovedfromanorganismandplacedinsuitablecultureenvironmentwillattachandgrowarecalledasprimaryculturePrimarycellshaveafinitelifespanPrimaryculturecontainsaveryheterogeneouspopulationofcellsSubculturingofprimarycellsleadstothegenerationofcelllinesCelllineshavelimitedlifespan,theypassageseveraltimesbeforetheybecomesenescentCellssuchasmacrophagesandneuronsdonotdivideinvitrosocanbeusedasprimaryculturesLineageofcellsoriginatingfromtheprimarycultureiscalledacellstrainPrimarycultureCellswhensurg11ContinouscelllinesMostcelllinesgrowforalimitednumberofgenerationsafterwhichtheyceasesCelllineswhicheitheroccurspontaneouslyorinducedvirallyorchemicallytransformedintoContinouscelllinesCharacteristicsofcontinouscelllines-smaller,morerounded,lessadherentwithahighernucleus/cytoplasmratio-Fastgrowthandhaveaneuploidchromosomenumber-reducedserumandanchoragedependenceandgrowmoreinsuspensionconditions-abilitytogrowuptohighercelldensity-differentinphenotypesfromdonartissue-stopexpressingtissuespecificgenesContinouscelllinesMostcell12Typesofcells

Onthebasisofmorphology(shape&appearance)orontheirfunctionalcharacteristics.Theyaredividedintothree.Epitheliallike-attachedtoasubstrateandappearsflattenedandpolygonalinshapeLymphoblastlike-cellsdonotattachremaininsuspensionwithasphericalshapeFibroblastlike-cellsattachedtoansubstrateappearselongatedandbipolarTypesofcellsOnthebasis13CulturemediaChoiceofmediadependsonthetypeofcellbeingculturedCommonlyusedMediumareGMEM,EMEM,DMEMetc.Mediaissupplementedwithantibioticsviz.penicillin,streptomycinetc.Preparedmediaisfilteredandincubatedat4CCulturemedia14Whysubculturing.?Oncetheavailablesubstratesurfaceiscoveredbycells(aconfluentculture)growthslows&ceases.Cellstobekeptinhealthy&ingrowingstatehavetobesub-culturedorpassagedIt’sthepassageofcellswhentheyreachto80-90%confluencyinflask/dishes/platesEnzymesuchastrypsin,dipase,collagenaseincombinationwithEDTAbreaksthecellulargluethatattachedthecellstothesurfaceWhysubculturing.?Oncetheav15CulturingofcellsCellsareculturedasanchoragedependentorindependentCelllinesderivedfromnormaltissuesareconsideredasanchorage-dependentgrowsonlyonasuitablesubstratee.g.tissuecellsSuspensioncellsareanchorage-independente.g.bloodcellsTransformedcelllineseithergrowsasmonolayerorassuspensionCulturingofcellsCellsarecu16Adherentcells

CellswhichareanchoragedependentCellsarewashedwithPBS(freeofca&mg)solution.Addenoughtrypsin/EDTAtocoverthemonolayerIncubatetheplateat37Cfor1-2mtsTapthevesselfromthesidestodislodgethecellsAddcompletemediumtodissociateanddislodgethecellswiththehelpofpipettewhichareremainedtobeadherentAddcompletemediumdependsonthesubculturerequirementeitherto75cmor175cmflaskAdherentcells

Cellswhichare17SuspensioncellsEasiertopassageasnoneedtodetachthemAsthesuspensioncellsreachtoconfluencyAscepticallyremove1/3rdofmediumReplacedwiththesameamountofpre-warmedmediumSuspensioncellsEasiertopass18Transfectionmethods

CalciumphosphateprecipitationDEAE-dextran(dimethylaminoethyl-dextran)LipidmediatedlipofectionElectroporationRetroviralInfectionMicroinjectionTransfectionmethods

Calciump19CelltoxicityCytotoxicitycausesinhibitionofcellgrowthObservedeffectonthemorphologicalalterationinthecelllayerorcellshapeCharacteristicsofabnormalmorphologyisthegiantcells,multinucleatedcells,agranularbumpyappearance,vacuolesinthecytoplasmornucleusCytotoxicityisdeterminedbysubstitutingmaterialssuchasmedium,serum,supplementsflasksetc.atatimeCelltoxicityCytotoxicitycaus20WorkingwithcryopreservedcellsVialfromliquidnitrogenisplacedinto37Cwaterbath,agitatevialcontinuouslyuntilmediumisthawedCentrifugethevialfor10mtsat1000rpmatRT,wipetopofvialwith70%ethanolanddiscardthesupernatantResuspendthecellpelletin1mlofcompletemediumwith20%FBSandtransfertoproperlylabeledcultureplatecontainingtheappropriateamountofmediumChecktheculturesafter24hrstoensurethattheyareattachedtotheplateChangemediumasthecolourchanges,use20%FBSuntilthecellsareestablishedWorkingwithcryopreservedcel21FreezingcellsforstorageRemovethegrowthmedium,washthecellsbyPBSandremovethePBSbyaspirationDislodgethecellsbytrypsin-verseneDilutethecellswithgrowthmediumTransferthecellsuspensiontoa15mlconicaltube,centrifugeat200gfor5mtsatRTandremovethegrowthmediumbyaspirationResuspendthecellsin1-2mloffreezingmediumTransferthecellstocryovials,incubatethecryovialsat-80CovernightNextdaytransferthecryovialstoLiquidnitrogenFreezingcellsforstorageRemo22CellviabilityCellviabilityisdeterminedbystainingthecellswithtrypanblueAstrypanbluedyeispermeabletonon-viablecellsordeathcellswhereasitisimpermeabletothisdyeStainthecellswithtrypandyeandloadtohaemocytometerandcalculate%ofviablecells-%ofviablecells=Nu.ofunstainedcellsx100totalnu.ofcellsCellviabilityCellviabilityi23CommoncelllinesHumancelllines-MCF-7breastcancerHL60LeukemiaHEK-293HumanembryonickidneyHeLaHenriettalacksPrimatecelllinesVeroAfricangreenmonkeykidneyepithelialcellsCos-7AfricangreenmonkeykidneycellsAndotherssuchasCHOfromhamster,sf9&sf21frominsectcells

CommoncelllinesHumancellli24Contaminant’sofcellculture

CellculturecontaminantsoftwotypesChemical-difficulttodetectcausedbyendotoxins,plasticizers,metalionsortracesofdisinfectantsthatareinvisibleBiological-causevisibleeffectsontheculturetheyaremycoplasma,yeast,bacteriaorfungusoralsofromcross-contaminationofcellsfromothercelllinesContaminant’sofcellculture25EffectsofBiologicalContamination’sTheycompetesfornutrientswithhostcellsSecretedacidicoralkalineby-productscesesthegrowthofthehostcellsDegradedarginine&purineinhibitsthesynthesisofhistoneandnucleicacidTheyalsoproducesH2O2whichisdirectlytoxictocellsEffectsofBiologicalContamin26DetectionofcontaminantsIngeneralindicatorsofcontaminationareturbidculturemedia,changeingrowthrates,abnormallyhighpH,poorattachment,multi-nucleatedcells,grainingcellularappearance,vacuolization,inclusionbodiesandcelllysisYeast,bacteria&fungiusuallyshowsvisibleeffectontheculture(changesinmediumturbidityorpH)MycoplasmadetectedbydirectDNAstainingwithintercalatingfluorescentsubstancese.g.Hoechst33258MycoplasmaalsodetectedbyenzymeimmunoassaybyspecificantiseraormonoclonalabsorbyPCRamplificationofmycoplasmalRNAThebestandtheoldestwaytoeliminatecontaminationistodiscardtheinfectedcelllinesdirectlyDetectionofcontaminantsInge27BasicequipmentsusedincellcultureLaminarcabinet-VerticalarepreferableIncubationfacilities-Temperatureof25-30Cforinsect&37Cformammaliancells,co22-5%&95%airat99%relativehumidity.Topreventcelldeathincubatorssettocutoutatapprox.38.5CRefrigerators-Liquidmediakeptat4C,enzymes(e.g.trypsin)&mediacomponents(e.g.glutamine&serum)at-20CMicroscope-Aninvertedmicroscopewith10xto100xmagnificationTissuecultureware-CultureplasticwaretreatedbypolystyreneBasicequipmentsusedincell28RulesforworkingwithcellcultureNeverusecontaminatedmaterialwithinasterileareaUsethecorrectsequencewhenworkingwithmorethanonecelllines.Diploidcells(Primarycultures,linesfortheproductionofvaccinesetc.)Diploidcells(Laboratorylines)Continous,slowgrowinglineContinous,rapidlygrowinglinesLineswhichmaybecontaminatedVirusproducinglinesRulesforworkingwithcellcu29BasicasepticconditionsIfworkingonthebenchuseaBunsenflametoheattheairsurroundingtheBunsenSwaballbottletops&neckswith70%ethanolFlameallbottlenecks&pipettebypassingveryquicklythroughthehottestpartoftheflameAvoidingplacingcaps&pipettesdownonthebench;practiceholdingbottletopswiththelittlefingerWorkeitherlefttorightorviceversa,sothatallmaterialgoestooneside,oncefinishedCleanupspillsimmediately&alwaysleavetheworkplaceneat&tidyBasicasepticconditionsIfwor30SafetyaspectincellculturePossiblykeepculturesfreeofantibioticsinordertobeabletorecognizethecontaminationNeverusethesamemediabottlefordifferentcelllines.Ifcapsaredroppedorbottlestouchedunconditionallytouched,replacethemwithnewonesNecksofglassbottlespreferheatatleastfor60secsatatemperatureof200CSwitchonthelaminarflowcabinet20mtspriortostartworkingCellcultureswhicharefrequentlyusedshouldbesubcultered&storedasduplicatestrainsSafetyaspectincellcultureP31Otherkeyfacts…….?Useactivelygrowingcellsthatareintheirlogphaseofgrowth,whichare80-90%viableKeepexposuretotrypsinataminimumHandlethecellsgently.Donotcentrifugecellsathighspeedorroughlyre-suspendthecellsFeeding&subculturingthecellsatmorefrequentintervalsthenusedwithserumcontainingconditionsmaybenecessaryAlowerconcentrationof104cells/mltoinitiatesubcultureofrapidlygrowingcells&ahigherconcentrationof105cells/mlforslowinggrowingcellsOtherkeyfacts…….?Useactivel32ThanksThanks33

ParasYadav1,AnnuYadav1,P.Kumar1,J.S.Arora1,T.K.Datta1,S.De1,S.L.Goswami1,MukeshYadav2,ShaliniJain3,RavinderNagpal4andHariomYadav3

1DepartmentofAnimalBiotechnology,3AnimalBiochemistryDivisionand4DairyMicrobiologyDivision,NationalDairyResearchInstitute,Karnal132001(Haryana),India;2SOSinChemistry,JiwajiUniversity,Gwalior-474011,M.P.,IndiaBasicsofCellCulture ParasYadav1,AnnuYadav1,P.34IntroductionCellcultureistheprocessbywhichprokaryotic,eukaryoticorplantcellsaregrownundercontrolledconditions.Butinpracticeitreferstotheculturingofcellsderivedfromanimalcells.CellculturewasfirstsuccessfullyundertakenbyRossHarrisonin1907Rouxin1885forthefirsttimemaintainedembryonicchickcellsinacellcultureIntroductionCellcultureisth35Historicaleventsinthedevelopmentofcellculture1878:ClaudeBernardproposedthatphysiologicalsystemsofanorganismcanbemaintainedinalivingsystemafterthedeathofanorganism.1885:Rouxmaintainedembryonicchickcellsinasalineculture.1897:Loebdemonstratedthesurvivalofcellsisolatedfrombloodandconnectivetissueinserumandplasma.1903:Jollyobservedcelldivisionofsalamanderleucocytesinvitro.1907:Harrisoncultivatedfrognervecellsinalymphclotheldbythe'hangingdrop'methodandobservedthegrowthofnervefibersinvitroforseveralweeks.Hewasconsideredbysomeasthefatherofcellculture.1910:Burrowssucceededinlongtermcultivationofchickenembryocellinplasmaclots.Hemadedetailedobservationofmitosis.Historicaleventsinthedevel36Contd..1911:LewisandLewismadethefirstliquidmediaconsistedofseawater,serum,embryoextract,saltsandpeptones.Theyobservedlimitedmonolayergrowth.1913:Carrelintroducedstrictaseptictechniquessothatcellscouldbeculturedforlongperiods.1916:RousandJonesintroducedproteolyticenzymetrypsinforthesubcultureofadherentcells.1923:CarrelandBakerdeveloped'Carrel'orT-flaskasthefirstspecificallydesignedcellculturevessel.Theyemployedmicroscopicevaluationofcellsinculture.1927:CarrelandRiveraproducedthefirstviralvaccine-Vaccinia.1933:GeydevelopedtherollertubetechniqueContd..1911:LewisandLewism37Contd..1940s:Theuseoftheantibioticspenicillinandstreptomycininculturemediumdecreasedtheproblemofcontaminationincellculture.1948:EarleisolatedmouseLfibroblastswhichformedclonesfromsinglecells.Fischerdevelopedachemicallydefinedmedium,CMRL1066.1952:GeyestablishedacontinuouscelllinefromahumancervicalcarcinomaknownasHeLa(HelenLane)cells.Dulbeccodevelopedplaqueassayforanimalvirusesusingconfluentmonolayersofculturedcells.1954:Abercrombieobservedcontactinhibition:motilityofdiploidcellsinmonolayercultureceaseswhencontactismadewithadjacentcells.1955:Eaglestudiedthenutrientrequirementsofselectedcellsincultureandestablishedthefirstwidelyusedchemicallydefinedmedium.1961:HayflickandMoorheadisolatedhumanfibroblasts(WI-38)andshowedthattheyhaveafinitelifespaninculture.1964:LittlefieldintroducedtheHATmediumforcellselection.1965:Hamintroducedthefirstserum-freemediumwhichwasabletosupportthegrowthofsomecells.Contd..1940s:Theuseofthea38Contd..1965:HarrisandWatkinswereabletofusehumanandmousecellsbytheuseofavirus.1975:KohlerandMilsteinproducedthefirsthybridomacapableofsecretingamonoclonalantibody.1978:Satoestablishedthebasisforthedevelopmentofserum-freemediafromcocktailsofhormonesandgrowthfactors.1982:Humaninsulinbecamethefirstrecombinantproteintobelicensedasatherapeuticagent.1985:Humangrowthhormoneproducedfromrecombinantbacteriawasacceptedfortherapeuticuse.1986:LymphoblastoidγIFNlicensed.1987:Tissue-typeplasminogenactivator(tPA)fromrecombinantanimalcellsbecamecommerciallyavailable.1989:Recombinanterythropoietinintrial.1990:Recombinantproductsinclinicaltrial(HBsAG,factorVIII,HIVgp120,CD4,GM-CSF,EGF,mAbs,IL-2).Contd..1965:HarrisandWatkin39Majordevelopment’sincellculturetechnologyFirstdevelopmentwastheuseofantibioticswhichinhibitsthegrowthofcontaminants.SecondwastheuseoftrypsintoremoveadherentcellstosubculturefurtherfromtheculturevesselThirdwastheuseofchemicallydefinedculturemedium.Majordevelopment’sincellcu40Whyiscellcultureusedfor?

Areaswherecellculturetechnologyiscurrentlyplayingamajorrole.ModelsystemsforStudyingbasiccellbiology,interactionsbetweendiseasecausingagentsandcells,effectsofdrugsoncells,processandtriggeringofaging&nutritionalstudiesToxicitytestingStudytheeffectsofnewdrugsCancerresearchStudythefunctionofvariouschemicals,virus&radiationtoconvertnormalculturedcellstocancerouscells

Whyiscellcultureusedfor?41Contd….Virology

Cultivationofvirusforvaccineproduction,alsousedtostudythereinfectiouscycle.

GeneticEngineering

Productionofcommercialproteins,largescaleproductionofvirusesforuseinvaccineproductione.g.polio,rabies,chickenpox,hepatitisB&measles

Genetherapy

Cellshavingafunctionalgenecanbereplacedtocellswhicharehavingnon-functionalgeneContd….Virology42TissuecultureInvitrocultivationoforgans,tissues&cellsatdefinedtemperatureusinganincubator&supplementedwithamediumcontainingcellnutrients&growthfactorsiscollectivelyknownastissuecultureDifferenttypesofcellgrownincultureincludesconnectivetissueelementssuchasfibroblasts,skeletaltissue,cardiac,epithelialtissue(liver,breast,skin,kidney)andmanydifferenttypesoftumorcells.

TissuecultureInvitrocultiva43PrimarycultureCellswhensurgicallyorenzymaticallyremovedfromanorganismandplacedinsuitablecultureenvironmentwillattachandgrowarecalledasprimaryculturePrimarycellshaveafinitelifespanPrimaryculturecontainsaveryheterogeneouspopulationofcellsSubculturingofprimarycellsleadstothegenerationofcelllinesCelllineshavelimitedlifespan,theypassageseveraltimesbeforetheybecomesenescentCellssuchasmacrophagesandneuronsdonotdivideinvitrosocanbeusedasprimaryculturesLineageofcellsoriginatingfromtheprimarycultureiscalledacellstrainPrimarycultureCellswhensurg44ContinouscelllinesMostcelllinesgrowforalimitednumberofgenerationsafterwhichtheyceasesCelllineswhicheitheroccurspontaneouslyorinducedvirallyorchemicallytransformedintoContinouscelllinesCharacteristicsofcontinouscelllines-smaller,morerounded,lessadherentwithahighernucleus/cytoplasmratio-Fastgrowthandhaveaneuploidchromosomenumber-reducedserumandanchoragedependenceandgrowmoreinsuspensionconditions-abilitytogrowuptohighercelldensity-differentinphenotypesfromdonartissue-stopexpressingtissuespecificgenesContinouscelllinesMostcell45Typesofcells

Onthebasisofmorphology(shape&appearance)orontheirfunctionalcharacteristics.Theyaredividedintothree.Epitheliallike-attachedtoasubstrateandappearsflattenedandpolygonalinshapeLymphoblastlike-cellsdonotattachremaininsuspensionwithasphericalshapeFibroblastlike-cellsattachedtoansubstrateappearselongatedandbipolarTypesofcellsOnthebasis46CulturemediaChoiceofmediadependsonthetypeofcellbeingculturedCommonlyusedMediumareGMEM,EMEM,DMEMetc.Mediaissupplementedwithantibioticsviz.penicillin,streptomycinetc.Preparedmediaisfilteredandincubatedat4CCulturemedia47Whysubculturing.?Oncetheavailablesubstratesurfaceiscoveredbycells(aconfluentculture)growthslows&ceases.Cellstobekeptinhealthy&ingrowingstatehavetobesub-culturedorpassagedIt’sthepassageofcellswhentheyreachto80-90%confluencyinflask/dishes/platesEnzymesuchastrypsin,dipase,collagenaseincombinationwithEDTAbreaksthecellulargluethatattachedthecellstothesurfaceWhysubculturing.?Oncetheav48CulturingofcellsCellsareculturedasanchoragedependentorindependentCelllinesderivedfromnormaltissuesareconsideredasanchorage-dependentgrowsonlyonasuitablesubstratee.g.tissuecellsSuspensioncellsareanchorage-independente.g.bloodcellsTransformedcelllineseithergrowsasmonolayerorassuspensionCulturingofcellsCellsarecu49Adherentcells

CellswhichareanchoragedependentCellsarewashedwithPBS(freeofca&mg)solution.Addenoughtrypsin/EDTAtocoverthemonolayerIncubatetheplateat37Cfor1-2mtsTapthevesselfromthesidestodislodgethecellsAddcompletemediumtodissociateanddislodgethecellswiththehelpofpipettewhichareremainedtobeadherentAddcompletemediumdependsonthesubculturerequirementeitherto75cmor175cmflaskAdherentcells

Cellswhichare50SuspensioncellsEasiertopassageasnoneedtodetachthemAsthesuspensioncellsreachtoconfluencyAscepticallyremove1/3rdofmediumReplacedwiththesameamountofpre-warmedmediumSuspensioncellsEasiertopass51Transfectionmethods

CalciumphosphateprecipitationDEAE-dextran(dimethylaminoethyl-dextran)LipidmediatedlipofectionElectroporationRetroviralInfectionMicroinjectionTransfectionmethods

Calciump52CelltoxicityCytotoxicitycausesinhibitionofcellgrowthObservedeffectonthemorphologicalalterationinthecelllayerorcellshapeCharacteristicsofabnormalmorphologyisthegiantcells,multinucleatedcells,agranularbumpyappearance,vacuolesinthecytoplasmornucleusCytotoxicityisdeterminedbysubstitutingmaterialssuchasmedium,serum,supplementsflasksetc.atatimeCelltoxicityCytotoxicitycaus53WorkingwithcryopreservedcellsVialfromliquidnitrogenisplacedinto37Cwaterbath,agitatevialcontinuouslyuntilmediumisthawedCentrifugethevialfor10mtsat1000rpmatRT,wipetopofvialwith70%ethanolanddiscardthesupernatantResuspendthecellpelletin1mlofcompletemediumwith20%FBSandtransfertoproperlylabeledcultureplatecontain