一 人皮肤成纤维细胞培养

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1、Ist.NazionaleNeurologicoCarloBestaPrimaryfibroblastculturefromhumanskinbiopsy_______________________________________________________________________PRIMARYFIBROBLASTCULTUREFROMHUMANSKINBIOPSYThisprotocoldescribesthestepsforobtainingaprimaryfibroblastcellline

2、fromhumanskinbiopsies.Fibroblastsarederiveddirectlyfromexcisedskinasexplants;enzymedigestionbycollagenasemayhelpobtaincellsinashortertime.Thisprotocoldescribesthedifferentstepsforobtainingaprimarycelllinefromaskinbiopsy.Equipmentandmaterials·Laminarflowhood·

3、CO2incubator·Invertedmicroscope·SterilesurgicalInstrumentsformicrodissection·BMEfibroblastmedium·PBS10xwithoutCa+2orMg+2·CollagenasetypeII(4mg/ml)·Petridishes100mm·Pasteurpipettes·Cultureflask,25cm2·15mlsterileplastictubeBMEFibroblastmediumBME80mlFoetalbovin

4、eserum20mlPenicillin-streptomycinsolution100x1mlFilterandstoreat+4°C,upto1month.Theskinbiopsysampleshouldbeshapedasadiamondandabout5-10mmindiameter.CollectthetissuesampleinsterileBMEfibroblastmedium.Procedure11.RapidlywashtheskinbiopsyinPBSinaPetridish,cutin

5、tosmallfragmentsandtransferthesetoaflask.2.UsingasterilePasteurpipettewithflame-roundedtip,distributethesmalltissuefragmentsoverthebottomsurfaceofthecultureflask.3.PasstheflaskrapidlyandcarefullythroughtheBunsenflameinordertoevaporatethemediumsothattheminced

6、tissuepiecesadheretotheplasticsurface,butsoasnottoheat-damagethemincedtissue.Takecarenottocookthetissue!4.CarefullyaddBMEmediumforfibroblastgrowth,firmlyclosethelidoftheflaskandplaceinCO2incubator.5.Thenextday,slightlyunscrewthelidoftheflasksothatthetissueca

7、n“breathe.”6.Replacetheculturemediumaftertwodaysand,fromthispointon,replaceitthreetimesaweek.INNCB-MarinaMora–August20044/4CopyrightEurobiobank2005Ist.NazionaleNeurologicoCarloBestaPrimaryfibroblastculturefromhumanskinbiopsy__________________________________

8、_____________________________________1.Thefibroblastswillstarttogrowfromthemincedfragmentsin2-3days.Whentherearesufficientcells,theyaredetachedenzymaticallyandplatedinPetridishes,or75cm2culturef