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.,IndiaBasicsofCellCultureIntroduction•Cellcultureistheprocessbywhichprokaryotic,eukaryoticorplantcellsaregrownundercontrolledconditions.Butinpracticeitreferstotheculturingofcellsderivedfromanimalcells.•CellculturewasfirstsuccessfullyundertakenbyRossHarrisonin1907•Rouxin1885forthefirsttimemaintainedembryonicchickcellsinacellcultureHistoricaleventsinthedevelopmentofcellculture•1878:ClaudeBernardproposedthatphysiologicalsystemsofanorganismcanbemaintainedina•livingsystemafterthedeathofanorganism.•1885:Rouxmaintainedembryonicchickcellsinasalineculture.•1897:Loebdemonstratedthesurvivalofcellsisolatedfrombloodandconnectivetissueinserum•andplasma.•1903:Jollyobservedcelldivisionofsalamanderleucocytesinvitro.•1907:Harrisoncultivatedfrognervecellsinalymphclotheldbythe'hangingdrop'methodand•observedthegrowthofnervefibersinvitroforseveralweeks.Hewasconsideredbysomeas•thefatherofcellculture.•1910:Burrowssucceededinlongtermcultivationofchickenembryocellinplasmaclots.Hemadedetailedobservationofmitosis.Contd..•1911:LewisandLewismadethefirstliquidmediaconsistedofseawater,serum,embryoextract,saltsandpeptones.Theyobservedlimitedmonolayergrowth.•1913:Carrelintroducedstrictaseptictechniquessothatcellscouldbeculturedforlongperiods.•1916:RousandJonesintroducedproteolyticenzymetrypsinforthesubcultureofadherentcells.•1923:CarrelandBakerdeveloped'Carrel'orT-flaskasthefirstspecificallydesignedcellculturevessel.Theyemployedmicroscopicevaluationofcellsinculture.•1927:CarrelandRiveraproducedthefirstviralvaccine-Vaccinia.•1933:GeydevelopedtherollertubetechniqueContd..•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:LymphoblastoidγIFNlicensed.•1987:Tissue-typeplasminogenactivator(tPA)fromrecombinantanimalcellsbecamecommerciallyavailable.•1989:Recombinanterythropoietinintrial.•1990:Recombinantproductsinclinicaltrial(HBsAG,factorVIII,HIVgp120,CD4,GM-CSF,EGF,mAbs,IL-2).Majordevelopment’sincellculturetechnology•Firstdevelopmentwastheuseofantibioticswhichinhibitsthegrowthofcontaminants.•Secondwastheuseoftrypsintoremoveadherentcellstosubculturefurtherfromtheculturevessel•Thirdwastheuseofchemicallydefinedculturemedium.Whyiscellcultureusedfor?Areaswherecellculturetechnologyiscurrentlyplayingamajorrole.•ModelsystemsforStudyingbasiccellbiology,interactionsbetweendiseasecausingagentsandcells,effectsofdrugsoncells,processandtriggeringofaging&nutritionalstudies•ToxicitytestingStudytheeffectsofnewdrugs•CancerresearchStudythefunctionofvariouschemicals,virus&radiationtoconvertnormalculturedcellstocancerouscellsContd….•VirologyCultivationofvirusforvaccineproduction,alsousedtostudythereinfectiouscycle.•GeneticEngineeringProductionofcommercialproteins,largescaleproductionofvirusesforuseinvaccineproductione.g.polio,rabies,chickenpox,hepatitisB&measles•GenetherapyCellshavingafunctionalgenecanbereplacedtocellswhicharehavingnon-functionalgeneTissueculture•Invitrocultivationoforgans,tissues&cellsatdefinedtemperatureusinganincubator&supplementedwithamediumcontainingcellnutrients&growthfactorsiscollectivelyknownastissueculture•Differenttypesofcellgrownincultureincludesconnectivetissueelementssuchasfibroblasts,skeletaltissue,cardiac,epithelialtissue(liver,breast,skin,kidney)andmanydifferenttypesoftumorcells.Primaryculture•Cellswhensurgicallyorenzymaticallyremovedfromanorganismandplacedinsuitablecultureenvironmentwillattachandgrowarecalledasprimaryculture•Primarycellshaveafini