EMBRYONICSTEMCELLSPROTOCOLIfyouareembarkingingrowingEScells,bepreparedtorefeedthemDAILY.Allproceduresshouldbecarriedoutusingsteriletechniques.ThegrowthandmaintenancemediaforEScellsisM15:DMEM(nopyruvate,highglucose)15%FBS,1XGPS,1XBME.HandlingEScells:growth,maintenance,passing,freezingandthawingisconductedinamannertoprotectandmaintainthequalityofthecellsandkeeptheminapluripotentialstate.SerumqualityiscriticalforsuccessfulgrowthofEScellsandespeciallytrueforblastocysts.Thequalityofthefeedersisveryinstrumental.Rememberalsothatinpassing,freezing,andelectroporatingEScells;itisbestthatthecellsarestillatexponentialgrowth(80%confluence)foroptimalresults.THAWING(QUICKTHAW)1.Removecellsfromthefreezerandquicklythawina37oCwaterbath.2.TransferthecellsUSPensiontoasterile15mltube.Add10-12mlsofM15mediato1mlofcellsuspension.3.Gentlymixandpelletthecellsbycentrifuging@1000rpmfor7minutes.4.Aspirateoffsupernatantandresuspendcellsinto6mlsofM15,andplateoutcellsina6-cmfeederplate.5.RefeedcellsdailywithfreshM15.Upon80-85%confluence,cellsneedtobepassageorfreeze.(M15media:DMEM,15%FBS,1XGPS,1XBME)PASSAGEOFESCELLSEScellstypicallyshouldbepassagedevery2-4days(apartfromcoloniesunderselection).Ifpassagingisneglectedthecellswilldifferentiateandyouwillselectforvariantsthatmighthavelosttotipotency.Cellsmustbefedwhenmediabeginstoturnorange.Yellowmedia(acidpH)isverybadforEScellsandshouldbeavoidedatallcosts.Ifyouareplanningtopassageandbelievethatthecellsmightturnyellowovernightfeedlastthingintheeveningandagainthenextmorningbeforepassaging.DONOTPASSAGECELLSWHENMEDIAISYELLOW.1.Checkcellsunderthemicroscopefor80-85%confluence.2.Refeedcells3-4hoursbeforepassingthem.(VERYIMPORTANT)3.Aspiratemediaoff.WashonetimewithPBS.Add500µloftrypsintoa6-cmplate,or1-1.5mloftrypsintoone10-cmplate.4.Incubate@37oCfor15minutes.5.Addmedia,M15toinactivatethetrypsin.About2mlsto1x6-cmdishor4-5mlsto1x10-cmdish.6.WithatransferPipette,pipetupanddownseveraltimestoseparatethecellsandbreakanycolonies.7.Determinethenumberoffeederplatesyouneed,dependinguponthepassageyouaredoing.Addfreshmedia,M15tothefeederplates(to1x6-cmfeederdish:6mlsofmedia;1x10-cmfeeder:12mlsofmedia).SplitratiosforEScellscanvaryfrom1:1to1:10.Donotexceed1:10.Thearearelationshipsforthevariousdishesareasfollows:DishMediaTrypsinArea(cm2)Diameter(actual)(6mm)96well200µl/well30-50µl0.30.6cm(10mm)24well1.0ml200µl1.81.5cm(30mm)6-wellplate3-4mls400µl9.63.5cm(6-cm)dish6mls0.6ml21.25.2cm(10-cm)dish12mls1.5ml608.7cm(15-cm)dish30mls2.5ml15414cmSometypicalpassagingratios:1:6=1x60mmto2x90mm1:6=1x30mmto1x90mm1:4=1x30mmto2x60mm1:5=1x24wellto1x30mm(6-wellplate)1:6=1x96wellto1x24well8.Aliquotthecellsuspensionintoplatesinthevolumespecifiedforeachplate.RemembertouseFeederplates.Alwayscheckthefeedersbeforeusingthem.Theyshouldbeconfluent,nogaps,notcontaminatedandnotdividing.Usefeedersthatareolder,(1-2weeksold),theadvantagesaremany:anycontaminationisassessed,alsoanydividingrun-awaycellscanbedetected,andthepassagewillbeearlier.Also,olderfeedershavesettlednicelyandflattened.9.Mixtohaveauniformcelldistribution.ReturnplatestotheTC37oCincubator.FREEZINGESCELLS(SLOWFREEZE)1.Checkcellsunderthemicroscopefor80-85%confluence.2.Refeedcells3-4hoursbeforepassingthem.3.Aspiratemediaoff.WashonetimewithPBS.Add500µloftrypsintoa6-cmplate,or1-1.5mloftrypsintoone10-cmplate.4.Incubate@37oCfor15minutes.5.Addmedia,M15(M15media:DMEM,15%FBS,1XGPS,1XBME)toinactivatethetrypsin.About2mlsto1x6-cmdishor4-5mlsto1x10-cmdish.6.Withatransferpipette,pipetupanddownseveraltimestoseparatethecellsandbreakanycolonies.Collectcellsuspensioninacentrifugetubeandaddmoremediatocount.7.Counta200µlaliquotandcalculatethetotalcellnumber.Fromthis,calculatethevolumeofmediarequiredtogiveafinaldensityof3.0x107cells/ml.Thisdensityisveryimportant,donotdeviatefromit.8.Pelletcellsbycentrifuging@1000rpmfor7minutes.9.Aspirateoffsupernatantandresuspendthepelletin1/2thevolumecalculatedinStep7above,withM15media.10.Add1/2thevolumewith2XFreezingMedia(60%DMEM,20%FBS,20%DMSO,freshlyprepared);thecellsuspensionisdilutedasaresult:10%DMSOisthefinalconc.Addthefreezingmediadropwise,mixingwellaftereachaddition.11.Aliquotthesuspensionintosterilefreezingvials,pre-labeledwiththecelltype(AB2.2,AB1,etc.),clonenumber,passagenumberanddate.Atypicalaliquotwouldhave0.3ml-0.4mlofEScells(@adensity=3.0x107cells/ml)thisisabout9x106cells-12x106cellstotal/vial.12.Placevialsintoafreezingcontainer.Itiscriticalthatthefreezingrateisnotfasterthan1¡C/minute.Donotuseanyuntestedstyrofoamcontainersincefreezingratesvarygreatlyandthiswillmostlikelyresultindeathofmostofyourcells.Freezecellsovernightat-70oC,(24hours).13.Nextday,transfercellstotheLiquidNitrogenfreezer(or-135oCfreezer).