ES细胞培养-实验方法

ES细胞培养-实验方法EScellculturemediaandsolutionsES细胞培养需要较高的实验条件，培养血清要用纯度较高的（ES级别）的胎牛血清，为防止细胞分化，需要在培养皿底部铺种滋养层细胞（Feedercells）并在培养基中加入白细胞抑制因子（LIF）。培养细胞的平皿和吸管均为一次性聚乙烯材料。(A)DMEMwithhighglucose(B)0.1mMnon-essentialaminoacids(100×stock,aliquoted,storedat4℃)(C)1mMsodiumpyruvate(100×stock,aliquoted,storedat4℃)(D)10-4Mβ­mercaptoethanol(100×stock,aliquoted,storedat-20℃)(E)2mML­glutamine(100×stock,aliquoted,storedat-20℃)(F)15%FBS要用纯度较高的（ES级别）的胎牛血清(G)Penicillinandstreptomycin(finalconcentration50μg/mleach)(H)1000U/mlLIF白细胞抑制因子，抑制ES细胞分化PreparationofEMFIfeederlayersRegentsFrozenvialsofprimaryembryofibroblastsTissueculturedishesPBSwithoutCa2+andMg2+0.05%tripsininsaline/EDTADMEM+10%FBSMitomycinC(stock1mg/mlinPBSstoredindarkat4℃andusedwithintwoweeks,mitomycinCistoxic;wearglovesandusecautionwhenhandling)Methods1.ThawafrozenvialEMFIcellsquicklyat37℃.2.Addcellsto10mlDMEM+10%FBSandcentrifuge(270g,5min)3.Decantsupernatant,resuspendthecellpelletgentlyin10mlDMEM+10%FBS,andsplitontofive150mmplateseachcontainingatotalof25mlDMEM+10%FBS.Mixwell.注意摇动混匀，不要单纯只按一个方向摇动，以使细胞较均匀地分布于平皿中，。4.Incubatecellsat37℃,5%CO2.5.Whenthecellsformaconfluentmonolayer(approx.threedays)eachplateshouldeitherbe:(a)Thrypsinized,splitontofiveadditional150mmdishes,andgrownuntiltheyformaconfluentmonolayer,or(b)DirectlytreatedwithmitomycinC（丝裂霉素）toinhibitcellgrowthanddivision.因为ES细胞与滋养细胞共培养时，未经丝裂霉素处理的滋养细胞增殖很快，会与ES细胞竞争养分，此步丝裂霉素处理可使滋养细胞失去增殖，但仍保持存活。6.Removethemediumfromtheconfluentplatesand10mlDMEM+10%FBScontaining100μlmitomycinC(1mg/mlstock).Swirlplatestoensureanevendistributionofmedium.7.Incubatecellsat37℃,5%CO2for2-2.5h.8.Washthemonolayerofcellstwicewith10mlPBSperdish.9.Add5mltrypsin/EDTAtoeachplate.10.incubate37℃,5%CO2untilthecellscomeofftheplate.11.Add10mlDMEM+10%FBStoeachplateandbreakanycellaggregatesbygentlypipetting.12.Centrifugecells(270g,5min)andresuspendthepelletinDMEM+10%FBS.13.Countthecellsanddilutetoaconcentrationof2×105cells/ml.14.PlatethecellsimmediatelyontotissueculturedishescontainingDMEM+10%FBSfortheappropriatecelldensitiesandvolumesofmediumfordifferentplatesizes.15.Allowfeederstoattachatleast2h,butpreferablyovernight,beforeaddingEScells.16.ChangethemediumtoEScellmediumimmediatelybeforeaddingEScells.MitomycinCtreatedEMFIfeederscanbeusedforuptosevendayswithmediumchangeseverythreetofourdays.PreparatiooonofastockofmitomycinCtreatedEMFIcellsReagents正常的滋养细胞贴壁生长于培养皿底面，呈梭形，应当均匀分布，并将皿底完全覆盖。1.thawafrozenviaofEMFIcellsquicklyat37℃.2.Addcellsto10mlDMEM+10%FBSandcentrifuge(270g,5min).3.Decantsupernatant,resuspendthecellpelletgentlyin10mlDMEM+10%FBS,andsplitontofive150mmplateseachcontainingatotalof25mlDMEM+10%FBS.Mixwell.4.incubatecellsat37℃,5%CO2.5.Whenthecellsformaconfluentmonolayer(approx.threedays)eachplateshouldtrypsinized,splitontofiveadditional150mmdishes,andgrownuntiltheyformaconfluentmonolayer6.Removethemediumfromtheconfluentplatesand10mlDMEM+10%FBScontaining100μlmitomycinC(1mg/mlstock).Swirlplatestoensureanevendistributionofmedium.7.Incubatecellsat37℃,5%CO2for2-2.5h.8.Washthemonolayerofcellstwicewith10mlPBSperdish.9.Add5mltrypsin/EDTAtoeachplate.10.incubate37℃,5%CO2untilthecellscomeofftheplate.(5-10min).11.Add10mlDMEM+10%FBStoeachplateandbreakanycellaggregatesbygentlypipetting.12.Centrifugecells(270g,5min)andresuspendthepelletinfreezingmedium.Freezingallthecellsfromeachplateinonefreezingvialin1mlof1×freezingmediumandstoreat-70℃foroneday.Transferthevialstoliquidnitrogen.13.tomakefeederplatesthawafrozenvialofmitomycinCtreatedEMFIcellsquicklyat37℃14.Addcellsto10mlDMEM+10%FBSandcenrifuge(270g,5min).15.Decantsupernatant,andresuspendthecellpelletgentlyin30mlDMEM+10%FBS.16.Seedcellsdirectlyintotissuecultureplates.Dependingofthesizeoftheplatesrequiredput10ml/100mmplate,5ml/60mmplate,5ml/60mmplate,or1.5ml/35mmplate.17.Allowfeederstoattachpreferablyovernight,beforeaddingEScellsoratleast2hifusinggelatinizedplates.18.ChangethemediumtoEScellmediumimmediatelybeforeaddingEScells.GrowthofEScellsonfeedersplatesEquipmentandreagents100mmdishescontainingfeederlayersEScellmediumbrieflypre-warmedto37℃0.05%trypsininsaline/EDTAPBSwithoutCa2+andMg2+Gelatin,0.1%solutioninwater,autoclavedMethod1.QuicklythawonevialoffrozenESbywarminginyourhandorat37℃,andtransfercellstoa12mltubecontaining10mlofEScellmediumbeforealltheicehasdisappeared.2.Centrifugeat270gfor5min.3.Aspiratesupernatantandresuspendpelletin10mlEScellmediumandplateona100mmdishwithafeederlayer.4.Changethemediumthenextdaybyswirlingthemediumindishtocollectdebris,thenaspirate.Addfreshmediumgentlytothesideoftheplatesothatthefeederlayerisnotdisturbed.5.Onthesecondday(cellshouldbejustsubconfluent)washcellstwicewithPBSandadd2mltrypsin/EDTA.6.Incubateforapprox.5minat37℃untilcellsbegintocomeofftheplate.7.Gentlyagitatetheplateandobserveunderamicroscope.Whentrypsinizationiscomplete,cellsshoulddetachassmallclumps,notasasinglesheetorsinglecells.8.Add5mlEScellmediumandgentlypipettethecellsupanddowntobreakcellsclumps,Ifthecellsaresticky,gentlypipettebeforeaddingmedium.9.Transfertoasterile12mltubeandpelletcellsincentrifuge(5min,270g).10.Aspiratesu