ES細(xì)胞培養(yǎng)-試驗方法

ES細(xì)胞培養(yǎng)-實驗方法EScellculturemediaandsolutionsES細(xì)胞培養(yǎng)需要較高的實驗條件，培養(yǎng)血清要用純度較高的(ES級別)的胎牛血清，為防止細(xì)胞分化，需要在培養(yǎng)皿底部鋪種滋養(yǎng)層細(xì)胞(Feedercells)并在培養(yǎng)基中加入白細(xì)胞抑制因子(LIF)。培養(yǎng)細(xì)胞的平皿和吸管均為一次性聚乙烯材料。DMEMwithhighglucose0.1mMion-essentialaminoacids(100xstock,aliquoted,storedat4C)1mMsodiumpyruvate(100xstock,aliquoted,storedat4C)(D)10-4MB-mercaptoethanol(100xstock,aliquoted,storedat-20C)(E)2mML-glutamine(100xstock,aliquoted,storedat-20C))15%FBSg用純度較高的(ES級別)的胎牛血清)Penicillinandstreptomycin(finalconcentration50仙g/mleach))1000U/mlLIF白細(xì)胞抑制因子，抑制ES細(xì)胞分化PreparationofEMFIfeederlayersRegentsFrozenvialsofprimaryembryofibroblastsTissueculturedishesPBSwithoutCa2+andMg2+0.05%tripsininsaline/EDTADMEM+10%FBSMitomycinC(stock1mg/mlinPBSstoredindarkat4Candusedwithintwoweeks,mitomycinCistoxic;wearglovesandusecautionwhenhandling)MethodsThawafrozenvialEMFIcellsquicklyat37C.Addcellsto10mlDMEM+10%FBSandcentrifuge(270g,5min)Decantsupernatant,resuspendthecellpelletgentlyin10mlDMEM+10%FBS,andsplitontofive150mmplateseachcontainingatotalof25mlDMEM+10%FBSMixwell.注意搖動混勻，不要單純只按一個方向搖動，以使細(xì)胞較均勻地分布于平皿中，。Incubatecellsat37C,5%CO2.Whenthecellsformaconfluentmonolayer(approx.threedays)eachplateshouldeitherbe:(a)Thrypsinized,splitontofiveadditional150mmdishes,andgrownuntiltheyformaconfluentmonolayer,or(b)DirectlytreatedwithmitomycinC(絲裂霉素)toinhibitcellgrowthanddivision.因為ES細(xì)胞與滋養(yǎng)細(xì)胞共培養(yǎng)時，未經(jīng)絲裂霉素處理的滋養(yǎng)細(xì)胞增殖很快，會與ES細(xì)胞競爭養(yǎng)分，此步絲裂霉素處理可使滋養(yǎng)細(xì)胞失去增殖，但仍保持存活。Removethemediumfromtheconfluentplatesand10mlDMEM+10%FBScontaining100仙lmitomycinC(1mg/mlstock).Swirlplatestoensureanevendistributionofmedium.Incubatecellsat37C,5%COfor2-2.5h.Washthemonolayerofcellstwicewith10mlPBSperdish.Add5mltrypsin/EDTAtoeachplate.incubate37C,5%CO2untilthecellscomeofftheplate.Add10mlDMEM+10%FBStoeachplateandbreakanycellaggregatesbygentlypipetting.Centrifugecells(270g,5min)andresuspendthepelletinDMEM+10%FBS.Countthecellsanddilutetoaconcentrationof2x105cells/ml.PlatethecellsimmediatelyontotissueculturedishescontainingDMEM+10%FBSfortheappropriatecelldensitiesandvolumesofmediumfordifferentplatesizes.Allowfeederstoattachatleast2h,butpreferablyovernight,beforeaddingEScells.ChangethemediumtoEScellmediumimmediatelybeforeaddingEScells.MitomycinCtreatedEMFIfeederscanbeusedforuptosevendayswithmediumchangeseverythreetofourdays.PreparatiooonofastockofmitomycinCtreatedEMFIcellsReagents正常的滋養(yǎng)細(xì)胞貼壁生長于培養(yǎng)皿底面，呈梭形，應(yīng)當(dāng)均勻分布，并將皿底完全覆蓋。thawafrozenviaofEMFIcellsquicklyat37C.Addcellsto10mlDMEM+10%FBSandcentrifuge(270g,5min).Decantsupernatant,resuspendthecellpelletgentlyin10mlDMEM+10%FBS,andsplitontofive150mmplateseachcontainingatotalof25mlDMEM+10%FBS.Mixwell.incubatecellsat37C,5%CO2.Whenthecellsformaconfluentmonolayer(approx.threedays)eachplateshouldtrypsinized,splitontofiveadditional150mmdishes,andgrownuntiltheyformaconfluentmonolayerRemovethemediumfromtheconfluentplatesand10mlDMEM+10%FBScontaining1001mitomycinC(1mg/mlstock).Swirlplatestoensureanevendistributionofmedium.Incubatecellsat37C,5%COfor2-2.5h.Washthemonolayerofcellstwicewith10mlPBSperdish.Add5mltrypsin/EDTAtoeachplate.0.incubate37C,5%CO2untilthecellscomeofftheplate.(5-10min)..Add10mlDMEM+10%FBStoeachplateandbreakanycellaggregatesbygentlypipetting..Centrifugecells(270g,5min)andresuspendthepelletinfreezingmedium.Freezingallthecellsfromeachplateinonefreezingvialin1mlof1xfreezingmediumandstoreat-70Cforoneday.Transferthevialstoliquidnitrogen..tomakefeederplatesthawafrozenvialofmitomycinCtreatedEMFIcellsquicklyat37C.Addcellsto10mlDMEM+10%FBSandcenrifuge(270g,5min)..Decantsupernatant,andresuspendthecellpelletgentlyin30mlDMEM+10%FBS..Seedcellsdirectlyintotissuecultureplates.Dependingofthesizeoftheplatesrequiredput10ml/100mmplate,5ml/60mmplate,5ml/60mmplate,or1.5ml/35mmplate..Allowfeederstoattachpreferablyovernight,beforeaddingEScellsoratleast2hifusinggelatinizedplates..ChangethemediumtoEScellmediumimmediatelybeforeaddingEScells.GrowthofEScellsonfeedersplatesEquipmentandreagents100mmdishescontainingfeederlayersEScellmediumbrieflypre-warmedto37C0.05%trypsininsaline/EDTAPBSwithoutCa2+andMg2+Gelatin,0.1%solutioninwater,autoclavedMethodQuicklythawonevialoffrozenESbywarminginyourhandorat37C,andtransfercellstoa12mltubecontaining10mlofEScellmediumbeforealltheicehasdisappeared.Centrifugeat270gfor5min.Aspiratesupernatantandresuspendpelletin10mlEScellmediumandplateona100mmdishwithafeederlayer.Changethemediumthenextdaybyswirlingthemediumindishtocollectdebris,thenaspirate.Addfreshmediumgentlytothesideoftheplatesothatthefeederlayerisnotdisturbed.Onthesecondday(cellshouldbejustsubconfluent)washcellstwicewithPBSandadd2mltrypsin/EDTA.Incubateforapprox.5minat37Cuntilcellsbegintocomeofftheplate.Gentlyagitatetheplateandobserveunderamicroscope.Whentrypsinizationiscomplete,cellsshoulddetachassmallclumps,notasasinglesheetorsinglecells.Add5mlEScellmediumandgentlypipettethecellsupanddowntobreakcellsclumps,Ifthecellsaresticky,gentlypipettebeforeaddingmedium.Transfertoasterile12mltubeandpelletcellsincentrifuge(5min,270g).Aspiratesupernatantandgentlyresuspendcellpelletin5-7mlmedium.Add1mlofthecellsuspention(about2-5x106cells)toafresh100mmfeederlayerdishcontaining9mlEScellmedium.DisperseEScellsevenlybypipettinggentlyandrockingtheplatepriortoincubatingat37C,5%COChangethemediumthenextdayandpasscellseveryseconddayasdescribedabove.正常的未分化的ES細(xì)胞應(yīng)當(dāng)呈球狀細(xì)胞團(tuán)附著于滋養(yǎng)細(xì)胞層表面，細(xì)胞團(tuán)的邊緣與滋養(yǎng)細(xì)胞層因有空間距離而形成折光的亮邊，如果細(xì)胞有分化，則細(xì)胞團(tuán)的球形邊緣部分或全部平坦化，與底層滋養(yǎng)細(xì)胞之間的亮邊消失，分化了的細(xì)胞不應(yīng)再使用。LongtermfreezingofEScellstocksMethod短期保存可以放在-70C,但長期保存應(yīng)轉(zhuǎn)入液氮。Trypsinizecellsfroma100mmdish.Pelletcellsbycentrifugationandrespensionin3mlprecooledfreezingmedium.Immediatelyaliquot1mlofthecellsuspentionintofreezingvialsonice.ImmediatelytransfervialtoaStyrofoamboxpre-cooledto-70C,orslow-coolcontainer,andthenintoa-70Cfrezer.Itisimportanttoworkquickly.After24htransferthetubesondryicetoliquidnitrogen.SandardelectroporationEScellsEuipmentandreagentsElectroporationapparatus:Bio-RadGenePulserandCapacitanceExtenderEScellmediumPBSwithoutCa2+andMg2+100mmgelatinizedplatesGeneticin(G418)OntheseconddayafterpassingrecentlythawedEScells,trypsinizecells,buttrypsinizeforlongertoobtainsinglecells.Afterpipettingthecellsgentlyin3mltrypsintobreakuptheclumps,add7mlmediumandpipettegentlyupanddownagain.Pre-platethecellsbyincubatingtheminthedishesfor15-20mintoallowfeedercellstore-attachtotheplate.HarvesttheEScellsbycarefullymixingandwithdrawingmedium,andtransfercellstoa50mlFbiningthecellsfromtwotofiveplate.Pelletthecellsfor5minat270gAspiratethesupernatantandresuspendthecellsinaminimalvolumeofPBS(?1ml/100mmplatestartingculture).Keepcellsonice.CounttheESCellsusingahaemocytometerandadjustcellconcentrationto7-10x106cells/mlwithsterilePBS.Mix0.8mlofthecellsuspentionwith25-40仙goflinearizedvectorDNA,andtransferintoanelectroprationcavettewhichhasbeenpre-cooledonice.Setuptheelectroporationconditionsinadvance:240V,500FfortheBio-RadGenePulserusingtheCapacitanceExtender.Transfereachcuvetteholderwithelectrodesfacingtheoutputleads.Delivertheelectricpulse.Removeeachcuvettefromthecuvetteholderandplaceonicefor20min.RemovethecellsfromeachcuvetteanddiluteinappropriatevolumeofEScellmedium(15-20ml/cuvette).Cellsfromseveralcuvettescanbecombined.Transfer10mlresuspendedEScells.Dispersecellsevenlybyrockingtheplate.Changemediumthenextmorning.Twodaysafertheelectroporation,begindrugselectionChangethemediumeverydayifworkingwithgancyclvir,sinceitcanbreakdownandbecometoxictothecells,otherwiseeverytwodayswhenusingG418selectiononly.Widespreadcelldeathshouldbeapparentafertwotothreedaysofdrugselection.初次采用的G418的濃度為200ug/ml,雖然也能看到大批的細(xì)胞死亡，但陰性對照(不加DNA而用TE的電轉(zhuǎn))亦見大批細(xì)胞死亡。Afteraboutsixtoeightdaysofselection,individualdrug-resistantcoloniesshouldhaveappearedandbelargeenoughtopickandsubcloneforscreeningbySouthernblotanalysisorbyPCRandtofreezeforstorage.GrowingdrugresistantclonesMethodPreparetwosetsof96-wellplates,onecontaining35仙loftrypsin/EDTAandonegelatincoated.我們沒有明膠處理的培養(yǎng)皿，而是預(yù)先在皿中鋪種滋養(yǎng)細(xì)胞層，但滋養(yǎng)細(xì)胞過于稀疏，造成ES細(xì)胞發(fā)生分化，以后實驗應(yīng)當(dāng)避免，應(yīng)使滋養(yǎng)細(xì)胞稠密完全CircleallvisibleEScellcoloniesthatwillbepickedonthebottomeachplatewithamarkerbyholdingplateuptolightorusinganinvertedmicroscope.WashEScellcontainingplatestwicewithPBS.LeavecellsinPBSduringpicking.Afterpicking.replacePBSwithESmediumandreturnplatetoincubatorifadditional.smallercolonieswillbepickedonsubsequentdays.Pickthe1.5-2mmdrug—resistantEScellclonesthatformafterabouteightdaysofselectionwithadrawn-outPasteurpipetteorayellowGilsontipunderCadissectingmicroscope.Pickcoloniesofsimilarsize,ortakeanequivalentportionoflargecolonies.挑取細(xì)胞克隆時將細(xì)胞間的顯微鏡置于超凈臺中，一半鏡身(物鏡)在隔離玻璃里面，一半鏡身(目鏡)在外面，紫外照射消毒。操作時需要幾個人，以加快速度。注意ES細(xì)胞的形態(tài)應(yīng)當(dāng)保持球形細(xì)胞團(tuán)，而發(fā)生分化的細(xì)胞克隆應(yīng)當(dāng)舍棄。TransfereachcolonyinaminimalvolumeofPBSintoonewellofaVshaped96-wellplatecontaining35仙lof0.05%trypsininsaline/EDTAatroomtemperature.Usuallyeveryotherrowisusedand48coloniespickedatonetime.Afterpicking48colones.incubateplateforapprox.10minat37c,5%CO2,untilcellclumpsbreaksup.Stopthereactionbyadding100仙lofESmediumcontainingG418toeachwellusingmultichannelpipettor.G418的濃度宜提高至400-600ug/ml.Mixcellsgentlybypipettingupanddownandtransfertogelatinized96-welltissuecultureplate.WasheachV-wellfromthetrpsinplatewithanother100仙lofmediumperwellandaddtosamewellofthe96-wellgelatinizedplate.Changethemediumthenextday,andthendailythereafter.Ifthecolonieshavenotgrownto?80%confluenceintwotothreedays.tryplatethecellsusingthefollowingprocedure:AspiratethemediumandwashthecellswithPBS.Add35nloftrypsin/EDTAandincubatefor5minat37C,oruntilthecellsliftoffthedish.Add200nlEScellmediumandbreakcoloniesupbygentlypipettingupanddown.Changethemedium12-24hlater..Repeatsteps1-11onsecondandthirdpickingdays.usingoriginalplatesofelectroporatedcells..Whenthecoloniesin96-wellplateshavereachatleast80%confluenceyoushouldpassagethemandfreezeyourclonesandpreparereplicaforDNAanalysis.FreezingdrugresistantclonesandpreparationofreplicaplatesforSouthernblotanalysisMethodPrepar