一人皮肤成纤维细胞培养

PRIMARYFIBROBLASTCULTUREFROMHUMANSKINBIOPSYThisprotocoldescribesthestepsforobtainingaprimaryfibroblastcelllinefromhumanskinbiopsies.Fibroblastsarederiveddirectlyfromexcisedskinasexplants;enzymedigestionbycollagenasemayhelpobtaincellsinashortertime.Thisprotocoldescribesthedifferentstepsforobtainingaprimarycelllinefromaskinbiopsy.EquipmentandmaterialsLaminarflowhoodCO2incubatorInvertedmicroscopeSterilesurgicalInstrumentsformicrodissectionBMEfibroblastmediumPBS10xwithoutCa+2orMg+2CollagenasetypeII(4mg/ml)Petridishes100mmPasteurpipettesCultureflask,25cm215mlsterileplastictubeBMEFibroblastmediumBME80mlFoetalbovineserum20mlPenicillin-streptomycinsolution100x1mlFilterandstoreat+4°C,upto1month.Theskinbiopsysampleshouldbeshapedasadiamondandabout5-10mmindiameter.CollectthetissuesampleinsterileBMEfibroblastmedium.Procedure1RapidlywashtheskinbiopsyinPBSinaPetridish,cutintosmallfragmentsandtransferthesetoaflask.UsingasterilePasteurpipettewithflame-roundedtip,distributethesmalltissuefragmentsoverthebottomsurfaceofthecultureflask.PasstheflaskrapidlyandcarefullythroughtheBunsenflameinordertoevaporatethemediumsothatthemincedtissuepiecesadheretotheplasticsurface,butsoasnottoheat-damagethemincedtissue.Takecarenottocookthetissue!CarefullyaddBMEmediumforfibroblastgrowth,firmlyclosethelidoftheflaskandplaceinCO2incubator.Thenextday,slightlyunscrewthelidoftheflasksothatthetissuecan“breathe.”Replacetheculturemediumaftertwodaysand,fromthispointon,replaceitthreetimesaweek.Thefibroblastswillstarttogrowfromthemincedfragmentsin2-3days.Whentherearesufficientcells,theyaredetachedenzymaticallyandplatedinPetridishes,or75cm2cultureflasks,forproliferation(seenextsteps:“Maintenanceofcellculturesindishesandflasks”and“Routinesubcultureofadherentcelllines”).Themincedfragmentsintheflaskwillcontinuetoproducecellsforawhile.Procedure2AddcollagenasetoBMEmediumcontaining20%FBS;sothattheratiomedium:collagenaseis6:1,andfilter.PlacetheskinbiopsyinaPetridish,minceitintoacoarseslurryusingsterilescalpelandtransfertoa15mlsterileplastictubecontainingtheBME-collagenasesolution.Placethetubeinincubatorat37°Cfor24hours.Thenextdaycentrifugeat1600gfor10min.Usingasterilepipette,removesupernatantandwashpellettwicewithPBS.Prepare1.5mlofBMEfibroblastmediumin2flasks.Suspendthepelletin1mlofBMEfibroblastmedium,transfer500microliterofsuspensiontoeachofthetwoflasksanddistributeitovertheirbottomsurface.PlaceflasksovernightinCO2incubatorat37°Ctightlyclosed.Thenextday,slightlyunscrewthelidoftheflasks.Afterafewdays,fibroblastsshouldstarttogrow;ifcellshavedeveloped,replacetheculturemediumwithfreshmediumafteroneweek.Fromthispointon,replacetheculturemediumthreetimesaweek.MAINTENANCEOFCELLCULTURESINDISHESANDFLASKSInculture,cellsgroweitherasasinglecelllayerattachedtospeciallytreatedplasticsurfacesorinsuspension.Inordertokeepadherentcellshealthyandactivelygrowingitisusuallynecessarytosubculturethematregularintervals.EquipmentandMaterialsLaminarflowhoodCO2incubatorProliferatingmedium(BMEfibroblastmedium)pre-warmedto37°CPetridishes100mmInvertedmicroscopeProcedureThegeneralmorphologyandgrowthofacellpopulation,andthepresenceofanymicrobialcontaminants,shouldbecheckedregularlyunderaninvertedmicroscopeinphasecontrast.Dishesorflaskswithcellsatabout70%confluencearetreatedwithtrypsin;thecellsarethenharvestedandeitherfrozenordividedforfurtherproliferation(seebelow“RoutineSubcultureofadherentcelllines”).Fordisheswithnon-confluentcellsthemediumisdiscardedandreplacedwithfreshmedium:7mlfor100mmPetridishes5mlfor60mmPetridishes5mlfor25cm2flasks.Mediumhastobechangedthreetimesaweek,usuallyMondays,WednesdaysandFridays.NB:Whenintroducingmediumtoflasks,anewsterilepipettemustbeusedforeachflask;whenchangingmediuminPetridishes,onepipettemaybeusedfor2or3dishesifthecelllineisthesame,butthetipmustbeflamedateachpassage.LidsofflaskscontainingcellsmustbeslightlyunscrewedafterbeingplacedintheCO2incubator.ROUTINESUBCULTUREOFADHERENTCELLLINESSubculturingrequirespriorruptureofintercellularandcell-to-substrateconnectionsusingproteolyticenzymessuchastrypsin.Afterthecellshavebeendissociatedintoasuspensionofmainlysinglecells,theyaredilutedandtransferredtonewculturedishescontainingfreshmediumortocryotubescontainingfreezingmedium.Howoftenacelllineissubcultureddependsonitsgrowthpropertieswhicharedeterminedbyobservationofcellgrowthunderthemicroscope,andbycounting.EquipmentandMaterialsLaminarflowhoodCO2incubatorProliferatingmedium(BMEfibroblastmedium)PBSTrypsin-EDTAsolution1XPetridishes100mmInvertedmicroscopeProcedureThawtrypsinat37°CandallowPBSandproliferatingmediumtoreachroomtemperature.Flaskculturescontainingtissuefragments:Usingasterilepipetteremovemediumfromflaskandreplacewith5mlPBSinordertoeliminateserumresiduethatcouldinactivatethetrypsin.AfterafewminutesremovePBS(pipette)andadd1mltrypsinPlaceflaskinincubatorat37°Cfor3-5minutesObservethecellsunderthemicroscope:iftheyareseentoberounded,theyaredetached,ifmostarenotrounded,leavethesuspensionintheincubatorforafurtherminuteortwo(untilrounded).Add5mlormoreofproliferatingmedium(addvolumeequaltoormorethanthatofvolumeoftrypsin)toinhibitenzymeactivity.Usethetipofthepipette(orgentlypipetteupanddown)todetachcellsfromthebottomoftheflask,butbecarefulnottotouch,ordetach,thetissuefragments.Transferthecells(pipette)toa100mmPetridishcontaining7mlofproliferatingmedium.Writethedate,numberofpassages,andcelllinecodeonthePetridishlidandplaceinincubator.Theinitialflask(treatedwithtrypsin)isrefilledwithproliferatingmediumandplacedinincubatorwithcapslightlyunscrewed.Petridishcultures:RemovemediumfromPetridishandaddPBS(7mlto100mmPetridish,4mlto60mmPetridish)Afterafewminutes,removePBSandaddtrypsin(1mlto100mmdishes,0.5mlto60mmdishes)Placeinincubatorat37°Cfor3-5minutes.Checkunderthemicroscopethatcellshavedetachedandproceedastrypsinizationofflaskcultures(above)Addproliferatingmedium(volumeequaltoorgreaterthanvolumeoftrypsinadded)inordertoinhibittrypsinactivity.Mechanicallydetachcellsfromdishsurfacewiththehelpofapipettetip,thengentlypipettethecellsuspensionupanddownsoastoobtainasuspensionofindividualcells.Dispenseappropriatealiquotsofthecellsuspensiontonew100mmPetridishesandadd6mlofgrowthmedium:Labeleachnewdishwithcelllinecode,dateandpassagenumber.PlacethedishesintheCO2incubatorat37°C.Thefollowingday,checkunderthemicroscopethatthecellshavereattachedandaregrowing.