细胞培养PPT(英文版)

ParasYadav1,AnnuYadav1,P.Kumar1,J.S.Arora1,T.K.Datta1,S.De1,S.L.Goswami1,MukeshYadav2,ShaliniJain3,RavinderNagpal4andHariomYadav31DepartmentofAnimalBiotechnology,3AnimalBiochemistryDivisionand4DairyMicrobiologyDivision,NationalDairyResearchInstitute,Karnal132001(Haryana),India;2SOSinChemistry,JiwajiUniversity,Gwalior-474011,M.P.,India,BasicsofCellCulture,Introduction,Cellcultureistheprocessbywhichprokaryotic,eukaryoticorplantcellsaregrownundercontrolledconditions.Butinpracticeitreferstotheculturingofcellsderivedfromanimalcells.CellculturewasfirstsuccessfullyundertakenbyRossHarrisonin1907Rouxin1885forthefirsttimemaintainedembryonicchickcellsinacellculture,Historicaleventsinthedevelopmentofcellculture,1878:ClaudeBernardproposedthatphysiologicalsystemsofanorganismcanbemaintainedinalivingsystemafterthedeathofanorganism.1885:Rouxmaintainedembryonicchickcellsinasalineculture.1897:Loebdemonstratedthesurvivalofcellsisolatedfrombloodandconnectivetissueinserumandplasma.1903:Jollyobservedcelldivisionofsalamanderleucocytesinvitro.1907:Harrisoncultivatedfrognervecellsinalymphclotheldbythehangingdropmethodandobservedthegrowthofnervefibersinvitroforseveralweeks.Hewasconsideredbysomeasthefatherofcellculture.1910:Burrowssucceededinlongtermcultivationofchickenembryocellinplasmaclots.Hemadedetailedobservationofmitosis.,Contd.,1911:LewisandLewismadethefirstliquidmediaconsistedofseawater,serum,embryoextract,saltsandpeptones.Theyobservedlimitedmonolayergrowth.1913:Carrelintroducedstrictaseptictechniquessothatcellscouldbeculturedforlongperiods.1916:RousandJonesintroducedproteolyticenzymetrypsinforthesubcultureofadherentcells.1923:CarrelandBakerdevelopedCarrelorT-flaskasthefirstspecificallydesignedcellculturevessel.Theyemployedmicroscopicevaluationofcellsinculture.1927:CarrelandRiveraproducedthefirstviralvaccine-Vaccinia.1933:Geydevelopedtherollertubetechnique,Contd.,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.,1965:HarrisandWatkinswereabletofusehumanandmousecellsbytheuseofavirus.1975:KohlerandMilsteinproducedthefirsthybridomacapableofsecretingamonoclonalantibody.1978:Satoestablishedthebasisforthedevelopmentofserum-freemediafromcocktailsofhormonesandgrowthfactors.1982:Humaninsulinbecamethefirstrecombinantproteintobelicensedasatherapeuticagent.1985:Humangrowthhormoneproducedfromrecombinantbacteriawasacceptedfortherapeuticuse.1986:LymphoblastoidIFNlicensed.1987:Tissue-typeplasminogenactivator(tPA)fromrecombinantanimalcellsbecamecommerciallyavailable.1989:Recombinanterythropoietinintrial.1990:Recombinantproductsinclinicaltrial(HBsAG,factorVIII,HIVgp120,CD4,GM-CSF,EGF,mAbs,IL-2).,Majordevelopmentsincellculturetechnology,Firstdevelopmentwastheuseofantibioticswhichinhibitsthegrowthofcontaminants.SecondwastheuseoftrypsintoremoveadherentcellstosubculturefurtherfromtheculturevesselThirdwastheuseofchemicallydefinedculturemedium.,Whyiscellcultureusedfor?,Areaswherecellculturetechnologyiscurrentlyplayingamajorrole.ModelsystemsforStudyingbasiccellbiology,interactionsbetweendiseasecausingagentsandcells,effectsofdrugsoncells,processandtriggeringofaging&nutritionalstudiesToxicitytestingStudytheeffectsofnewdrugsCancerresearchStudythefunctionofvariouschemicals,virus&radiationtoconvertnormalculturedcellstocancerouscells,Contd.,VirologyCultivationofvirusforvaccineproduction,alsousedtostudythereinfectiouscycle.GeneticEngineeringProductionofcommercialproteins,largescaleproductionofvirusesforuseinvaccineproductione.g.polio,rabies,chickenpox,hepatitisB&measlesGenetherapyCellshavingafunctionalgenecanbereplacedtocellswhicharehavingnon-functionalgene,Tissueculture,Invitrocultivationoforgans,tissues&cellsatdefinedtemperatureusinganincubator&supplementedwithamediumcontainingcellnutrients&growthfactorsiscollectivelyknownastissuecultureDifferenttypesofcellgrownincultureincludesconnectivetissueelementssuchasfibroblasts,skeletaltissue,cardiac,epithelialtissue(liver,breast,skin,kidney)andmanydifferenttypesoftumorcells.,Primaryculture,CellswhensurgicallyorenzymaticallyremovedfromanorganismandplacedinsuitablecultureenvironmentwillattachandgrowarecalledasprimaryculturePrimarycellshaveafinitelifespanPrimaryculturecontainsaveryheterogeneouspopulationofcellsSubculturingofprimarycellsleadstothegenerationofcelllinesCelllineshavelimitedlifespan,theypassageseveraltimesbeforetheybecomesenescentCellssuchasmacrophagesandneuronsdonotdivideinvitrosocanbeusedasprimaryculturesLineageofcellsoriginatingfromtheprimarycultureiscalledacellstrain,Continouscelllines,MostcelllinesgrowforalimitednumberofgenerationsafterwhichtheyceasesCelllineswhicheitheroccurspontaneouslyorinducedvirallyorchemicallytransformedintoContinouscelllinesCharacteristicsofcontinouscelllines-smaller,morerounded,lessadherentwithahighernucleus/cytoplasmratio-Fastgrowthandhaveaneuploidchromosomenumber-reducedserumandanchoragedependenceandgrowmoreinsuspensionconditions-abilitytogrowuptohighercelldensity-differentinphenotypesfromdonartissue-stopexpressingtissuespecificgenes,Typesofcells,Onthebasisofmorphology(shape&appearance)orontheirfunctionalcharacteristics.Theyaredividedintothree.Epitheliallike-attachedtoasubstrateandappearsflattenedandpolygonalinshapeLymphoblastlike-cellsdonotattachremaininsuspensionwithasphericalshapeFibroblastlike-cellsattachedtoansubstrateappearselongatedandbipolar,Culturemedia,ChoiceofmediadependsonthetypeofcellbeingculturedCommonlyusedMediumareGMEM,EMEM,DMEMetc.Mediaissupplementedwithantibioticsviz.penicillin,streptomycinetc.Preparedmediaisfilteredandincubatedat4C,Whysubculturing.?,Oncetheavailablesubstratesurfaceiscoveredbycells(aconfluentculture)growthslows&ceases.Cellstobekeptinhealthy&ingrowingstatehavetobesub-culturedorpassagedItsthepassageofcellswhentheyreachto80-90%confluencyinflask/dishes/platesEnzymesuchastrypsin,dipase,collagenaseincombinationwithEDTAbreaksthecellulargluethatattachedthecellstothesurface,Culturingofcells,CellsareculturedasanchoragedependentorindependentCelllinesderivedfromnormaltissuesareconsideredasanchorage-dependentgrowsonlyonasuitablesubstratee.g.tissuecellsSuspensioncellsareanchorage-independente.g.bloodcellsTransformedcelllineseithergrowsasmonolayerorassuspension,Adherentcells,CellswhichareanchoragedependentCellsarewashedwithPBS(freeofca&mg)solution.Addenoughtrypsin/EDTAtocoverthemonolayerIncubatetheplateat37Cfor1-2mtsTapthevesselfromthesidestodislodgethecellsAddcompletemediumtodissociateanddislodgethecellswiththehelpofpipettewhichareremainedtobeadherentAddcompletemediumdependsonthesubculturerequirementeitherto75cmor175cmflask,Suspensioncells,EasiertopassageasnoneedtodetachthemAsthesuspensioncellsreachtoconfluencyAscepticallyremove1/3rdofmediumReplacedwiththesameamountofpre-warmedmedium,Transfectionmethods,CalciumphosphateprecipitationDEAE-dextran(dimethylaminoethyl-dextran)LipidmediatedlipofectionElectroporationRetroviralInfectionMicroinjection,Celltoxicity,CytotoxicitycausesinhibitionofcellgrowthObservedeffectonthemorphologicalalterationinthecelllayerorcellshapeCharacteristicsofabnormalmorphologyisthegiantcells,multinucleatedcells,agranularbumpyappearance,vacuolesinthecytoplasmornucleusCytotoxicityisdeterminedbysubstitutingmaterialssuchasmedium,serum,supplementsflasksetc.atatime,Workingwithcryopreservedcells,Vialfromliquidnitrogenisplacedinto37Cwaterbath,agitatevialcontinuouslyuntilmediumisthawedCentrifugethevialfor10mtsat1000rpmatRT,wipetopofvialwith70%ethanolanddiscardthesupernatantResuspendthecellpelletin1mlofcompletemediumwith20%FBSandtransfertoproperlylabeledcultureplatecontainingtheappropriateamountofmediumChecktheculturesafter24hrstoensurethattheyareattachedtotheplateChangemediumasthecolourchanges,use20%FBSuntilthecellsareestablished,Freezingcellsforstorage,Removethegrowthmedium,washthecellsbyPBSandremovethePBSbyaspirationDislodgethecellsbytrypsin-verseneDilutethecellswithgrowthmediumTransferthecellsuspensiontoa15mlconicaltube,centrifugeat200gfor5mtsatRTandremovethegrowthmediumbyaspirationResuspendthecellsin1-2mloffreezingmediumTransferthecellstocryovials,incubatethecryovialsat-80CovernightNextdaytransferthecryovialstoLiquidnitrogen,Cellviability,CellviabilityisdeterminedbystainingthecellswithtrypanblueAstrypanbluedyeispermeabletonon-viablecellsordeathcellswhereasitisimpermeabletothisdyeStainthecellswithtrypandyeandloadtohaemocytometerandcalculate%ofviablecells-%ofviablecells=Nu.ofunstainedcellsx100totalnu.ofcells,Commoncelllines,Humancelllines-MCF-7breastcancerHL60LeukemiaHEK-293HumanembryonickidneyHeLaHenriettalacksPrimatecelllinesVeroAfricangreenmonkeykidneyepithelialcellsCos-7AfricangreenmonkeykidneycellsAndotherssuchasCHOfromhamster,sf9&sf21frominsectcells,Contaminantsofcellculture,CellculturecontaminantsoftwotypesChemical-difficulttodetectcausedbyendotoxins,plasticizers,metalionsortracesofdisinfectantsthatareinvisibleBiological-causevisibleeffectsontheculturetheyaremycoplasma,yeast,bacteriaorfungusoralsofromcross-contaminationofcellsfromothercelllines,EffectsofBiologicalContaminations,TheycompetesfornutrientswithhostcellsSecretedacidicoralkalineby-productscesesthegrowthofthehostcellsDegradedarginine&purineinhibitsthesynthesisofhistoneandnucleicacidTheyalsoproducesH2O2whichisdirectlytoxictocells,Detectionofcontaminants,Ingeneralindicatorsofcontaminationareturbidculturemedia,changeingrowthrates,abnormallyhighpH,poorattachment,multi-nucleatedcells,grainingcellularappearance,vacuolization,inclusionbodiesandcelllysisYeast,bacteria&fungiusuallyshowsvisibleeffectontheculture(changesinmediumturbidityorpH)MycoplasmadetectedbydirectDNAstainingwithintercalatingfluorescentsubstancese.g.Hoechst33258MycoplasmaalsodetectedbyenzymeimmunoassaybyspecificantiseraormonoclonalabsorbyPCRamplificationofmycoplasmalRNAThebestandtheoldestwaytoeliminatecontaminationistodiscardtheinfectedcelllinesdirectly,Basicequipmentsusedincellculture,Laminarcabinet-VerticalarepreferableIncubationfacilities-Temperatureof25-30Cforinsect&37Cformammaliancells,co22-5%&95%airat99%relativehumidity.Topreventcelldeathincubatorssettocutoutatapprox.38.5CRefrigerators-Liquidmediakeptat4C,enzymes(e.g.trypsin)&mediacomponents(e.g.glutamine&serum)at-20CMicroscope-Aninvertedmicroscopewith10 xto100 xmagnificationTissuecultureware-Cultureplasticwaretreatedbypolystyrene,Rulesforworkingwithcellculture,NeverusecontaminatedmaterialwithinasterileareaUsethecorrectsequencewhenworkingwithmorethanonecelllines.Diploidcells(Primarycultures,linesfortheproductionofvaccinesetc.)Diploidcells(Laboratorylines)C