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1、PROTOCOLCryopreservationandbankingofmammaliancelllinesGlynNStacey1&JohnRMasters21DivisionofCellBiologyandImaging,NationalInstituteforBiologicalStandardsandControl,BlancheLane,SouthMimms,PottersBar,HertfordshireEN63QG,UK.2DepartmentofSurgery,ProstateCancerResearchCentre,RoyalFreeandUniversityCol
2、legeLondonMedicalSchool,67RidingHouseStreet,LondonW1W7EJ,UK.CorrespondenceshouldbeaddressedtoJ.R.M.(j.masters@ucl.ac.uk).Publishedonline4December2008;doi:10.1038/nprot.2008.190sThisprotocoldescribestheprinciplesandmethodsusedforthepreparationofcryopreservedcellstocks.Followingtheseprocedureswil
3、lensuretheavailabilityofreproducibleculturesforusewithinasinglelaboratoryatdifferenttimesandfordifferentcollaboratinglaboratories.Althoughthebasicprincipleissimple,eachcelllinehascharacteristicsthatmusttobeborneinmindwhenestablishingandtestingsuchstocks.Thekeyrequirementsarereliablemethodsforcu
4、lture,cryopreservation,characterizationandqualitycontrol.natureprotocol/moc.INTRODUCTIONerutTheneedforcryopreservedcellbanksandcellbankingisaddedasacryoprotectanttotheculturemedium,followedbyna.principlescoolingatarateofapproximately11Cmin1.UnderstandingwMaintainingaconstantlygrowingcultureofc
5、ellsovermanywhichelementsofthecryopreservationprocessarecriticaltotheww/monthsisbadpractice.Anyinvitroculture,whetherprokaryoticsuccessfulpreservationandrecoveryofviableculturesisessentialto/:ptoreukaryotic,issusceptibletogeneticandphenotypicchangeprovidereliablefrozenstocksofcellsanddealwithpr
6、oblematict2hduringpropagation.Inaddition,duringconstantculture,thereiscultures.Keyconsiderationsforthesuccessfulpreservationofcellpalwaystheriskofanaccidentresultinginlossofthecells,linesincludethefollowing:uorcross-contaminationwithanothercelllineorcontaminationwithSelectionofculturesforcryop
7、reservationintheexponentialGmicroorganisms.Thegenerationofastable,cryopreservedstockofphaseofgrowth.gnicellsthatcanberegeneratedatwillhasmanyadvantagesoverMinimizationofexposureofcellstocryoprotectantduringhsiconstantculture,prim
