一人皮肤成纤维细胞培养

Ist.NazionaleNeurologicoCarloBestaLaboratoryProceduresforHumanCellCultureAugust2004_______________________________________________________________INNCB-MarinaMora–August20041/4CopyrightEurobiobank2005PRIMARYFIBROBLASTCULTUREFROMHUMANSKINBIOPSYThisprotocoldescribesthestepsforobtainingaprimaryfibroblastcelllinefromhumanskinbiopsies.Fibroblastsarederiveddirectlyfromexcisedskinasexplants;enzymedigestionbycollagenasemayhelpobtaincellsinashortertime.Thisprotocoldescribesthedifferentstepsforobtainingaprimarycelllinefromaskinbiopsy.EquipmentandmaterialsLaminarflowhoodCO2incubatorInvertedmicroscopeSterilesurgicalInstrumentsformicrodissectionBMEfibroblastmediumPBS10xwithoutCa+2orMg+2CollagenasetypeII(4mg/ml)Petridishes100mmPasteurpipettesCultureflask,25cm215mlsterileplastictubeBMEFibroblastmediumBME80mlFoetalbovineserum20mlPenicillin-streptomycinsolution100x1mlFilterandstoreat+4°C,upto1month.Theskinbiopsysampleshouldbeshapedasadiamondandabout5-10mmindiameter.CollectthetissuesampleinsterileBMEfibroblastmedium.Procedure11.RapidlywashtheskinbiopsyinPBSinaPetridish,cutintosmallfragmentsandtransferthesetoaflask.2.UsingasterilePasteurpipettewithflame-roundedtip,distributethesmalltissuefragmentsoverthebottomsurfaceofthecultureflask.3.PasstheflaskrapidlyandcarefullythroughtheBunsenflameinordertoevaporatethemediumsothatthemincedtissuepiecesadheretotheplasticsurface,butsoasnottoheat-damagethemincedtissue.Takecarenottocookthetissue!4.CarefullyaddBMEmediumforfibroblastgrowth,firmlyclosethelidoftheflaskandplaceinCO2incubator.5.Thenextday,slightlyunscrewthelidoftheflasksothatthetissuecan“breathe.”6.Replacetheculturemediumaftertwodaysand,fromthispointon,replaceitthreetimesaweek.Ist.NazionaleNeurologicoCarloBestaPrimaryfibroblastculturefromhumanskinbiopsy_______________________________________________________________________INNCB-MarinaMora–August20042/4CopyrightEurobiobank20057.Thefibroblastswillstarttogrowfromthemincedfragmentsin2-3days.Whentherearesufficientcells,theyaredetachedenzymaticallyandplatedinPetridishes,or75cm2cultureflasks,forproliferation(seenextsteps:“Maintenanceofcellculturesindishesandflasks”and“Routinesubcultureofadherentcelllines”).Themincedfragmentsintheflaskwillcontinuetoproducecellsforawhile.Procedure21.AddcollagenasetoBMEmediumcontaining20%FBS;sothattheratiomedium:collagenaseis6:1,andfilter.2.PlacetheskinbiopsyinaPetridish,minceitintoacoarseslurryusingsterilescalpelandtransfertoa15mlsterileplastictubecontainingtheBME-collagenasesolution.3.Placethetubeinincubatorat37°Cfor24hours.4.Thenextdaycentrifugeat1600gfor10min.5.Usingasterilepipette,removesupernatantandwashpellettwicewithPBS.6.Prepare1.5mlofBMEfibroblastmediumin2flasks.7.Suspendthepelletin1mlofBMEfibroblastmedium,transfer500microliterofsuspensiontoeachofthetwoflasksanddistributeitovertheirbottomsurface.8.PlaceflasksovernightinCO2incubatorat37°Ctightlyclosed.9.Thenextday,slightlyunscrewthelidoftheflasks.10.Afterafewdays,fibroblastsshouldstarttogrow;ifcellshavedeveloped,replacetheculturemediumwithfreshmediumafteroneweek.Fromthispointon,replacetheculturemediumthreetimesaweek.MAINTENANCEOFCELLCULTURESINDISHESANDFLASKSInculture,cellsgroweitherasasinglecelllayerattachedtospeciallytreatedplasticsurfacesorinsuspension.Inordertokeepadherentcellshealthyandactivelygrowingitisusuallynecessarytosubculturethematregularintervals.EquipmentandMaterialsLaminarflowhoodCO2incubatorProliferatingmedium(BMEfibroblastmedium)pre-warmedto37°CPetridishes100mmInvertedmicroscopeProcedure1.Thegeneralmorphologyandgrowthofacellpopulation,andthepresenceofanymicrobialcontaminants,shouldbecheckedregularlyunderaninvertedmicroscopeinphasecontrast.2.Dishesorflaskswithcellsatabout70%confluencearetreatedwithtrypsin;thecellsarethenharvestedandeitherfrozenordividedforfurtherproliferation(seebelow“RoutineSubcultureofadherentcelllines”).Fordisheswithnon-confluentcellsthemediumisdiscardedandreplacedwithfreshmedium:Ist.NazionaleNeurologicoCarloBestaPrimaryfibroblastculturefromhumanskinbiopsy_______________________________________________________________________INNCB-MarinaMora–August20043/4CopyrightEurobiobank20057mlfor100mmPetridishes5mlfor60mmPetridishes5mlfor25cm2flasks.3.Mediumhastobechangedthreetimesaweek,usuallyMondays,WednesdaysandFridays.NB:Whenintroducingmediumtoflasks,anewsterilepipettemustbeusedforeachflask;whenchangingmediuminPetridishes,onepipettemaybeusedfor2or3dishesifthecelllineisthesame,butthetipmustbeflamedateachpassage.LidsofflaskscontainingcellsmustbeslightlyunscrewedafterbeingplacedintheCO2incubator.ROUTINESUBCULTUREOFADHERENTCELLLINESSubculturingrequirespriorruptureofintercellularandcell-to-substrateconnectionsusingproteolyticenzymessuchastrypsin.Afterthecellshavebeendissociatedintoasuspensionofmainlysinglecells,theyaredilutedandtransferredtonewculturedishescontainingfreshmediumortocryotubescontainingfreezingmedium.Howoftenacelllineissubcultureddependsonitsgrowthpropertieswhicharedeterminedbyobservationofcellgrowthunderthemicroscope,andbycounting.EquipmentandMat