I.Feederfreecultureconditions

Preparationoffeeder-freemedium

1. ThawmTeSR15XSupplement(Cat.no.05850,STEMCELLTechnologies)atroomtemperatureorovernightat4°C.
2. Addthe100mLofthawed5XSupplementto400mLBasalMediumforatotalvolumeof500mLaseptically.Mixwellandfilterthrougha0.2μm,low-proteinbindingfilter,ifdesired.
3. Aliquotintoappropriateamountaccordingtousageandstorethealiquotsat4°C.

CoatingplateswithMatrigel

Matrigel(Cat.no.354277,BD)shouldbealiquotedandstoredat-80°Cforlong-termuse.

1. Thawmatrigeloniceuntilliquid.Dilutematrigel1:30to1:50withpre-chilledKODMEM/F12.
2. Immediatelyusethedilutedmatrigelsolutiontocoattissueculture-treatedplates.Fora6-wellplate,use1mLofdilutedmatrigelsolutionperwell,andswirltheplatetospreadthematrigelsolutionevenlyacrossthesurface.
3. Letthecoatedplatestandfor1hat37°Corovernightat4°C.Ifplatehasbeenstoredat4°C,allowtheplatetoincubateat37°Cforatleast30minutesbeforeremovingthematrigelsolution.

ThawingcryopreservedhumaniPScells

1. QuicklythawthehumaniPScellsina37°Cwaterbathbygentlyshakingthecryovialcontinuouslyuntilhalfthawed.Removethecryovialfromthewaterbathandspraywith70%ethanoltosterilize.
2. Transferthecontentsofthecryovialtoa15mLconicaltube.Add5mLwarmmTeSR1dropwisetothetube,gentlymixingasthemediumisadded.
3. Centrifugecellsat200xgfor5minutesatroomtemperature.
4. Aftercentrifugation,aspiratethemediumfrom15mLtube.GentlyresUSPendthecellpelletin2mLmTeSR1with5µMROCKinhibitor,takingcaretomaintainthecellsassmallcellclumps.
5. Removethematrigelsolutionfromacoatedtissueculture6-wellplate.Transferthemediumcontainingthecellclumpstothematrigelcoated6-wellplate.
6. Placetheplateintothe37°Cincubatorandmovetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributetheclumpswithinthewells.Culturethecellsat37°C,with5%CO₂and95%humidity.
7. Changemediumdaily.Checkforundifferentiatedcoloniesthatarereadytopassagewhencoloniesarebigenough(approximately7-10daysafterthawing).

PassaginghumaniPScellsgrownunderfeeder-freeconditions

1. Useamicroscopetoidentifyregionsofdifferentiation.Markthedifferentiatedcoloniesusinglensmarkeronthebottomoftheplate.
2. RemoveregionsofdifferentiationbyscrapingwithaPipettetiporbyaspiration.
3. AspiratemediumfromthehumaniPScellcultureandrinsewithDPBS(2mL/well).
4. Add1mLperwellofEZStemEnzyme-FreeStemCellDissociationSolution(cat.no.M100,ALSTEM),andincubateat37°Cfor2-3minutes.Oradd0.5mLperwellofaccutase(Cat.no.SCR005,Millipore,diluted1:1withDPBSbeforeuse),andincubateat37°Cfor1minute.
5. RemoveEZStemEnzyme-FreeStemCellDissociationSolutionoraccutase,gentlyrinseeachwell2-3timeswith2mLofDMEM/F-12perwellandtransferthedetachedcellaggregatestoa15mLconicaltube.
6. Add2mL/wellmTeSR1andscrapecoloniesoffwithacelllifter.Transferthedetachedcellaggregatestoa15mLconicaltube.
7. Rinsethewellwithanadditional2mLmTeSR1tocollectanyremainingaggregates.Addtherinsetothe15mLtube.
8. Centrifugethe15mLtubecontainingtheaggregatesat200xgfor5minutesatroomtemperature.
9. Aspiratethesupernatant.ResuspendpelletinmTeSR1containing5µMROCKinhibitorbygentlypipettingandensurethatcellsaremaintainedasaggregates.
10. PlatethehumaniPScellaggregateswithmTeSR1inanewplatecoatedwithmatrigel.(Removematrigelsolutionbeforeplating).
    Note:Ifthecoloniesareatanoptimaldensity,thecellscanbesplitevery5-7daysusing1:3to1:6ratios.
11. Placetheplateintothe37°Cincubatorandmovetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributetheclumpswithinthewells.Culturethecellsat37°C,with5%CO₂and95%humidity.
12. Changemediumdaily.

CryopreservinghumaniPScells

1. PrepareEZStemfreezingmedium(Cat.no.M050,ALSTEM)onice.
2. Performsteps1-8fromPassaginghumaniPScellsgrownunderfeeder-freeconditions.
3. Gentlyaspiratethesupernatantandloosenthecellpelletbytappingthebottomofthetube.
4. Gentlyresuspendthepelletinfreezingmedium,takingcaretoleavetheclumpslargerthanthatwouldnormallybedoneforpassaging.
5. Transfer1mLofclumpsinfreezingmediumintoeachlabeledcryogenicvial.
6. Placevialsintoafreezingcontainerandplacethecontainerat-80°Covernight.
7. Transfertoaliquidnitrogentanknextday.

II.Feeder-dependentcultureconditions

PreparationofhumanESmedium

KnockoutDMEM/F12containing20%knockoutserumreplacement,2mMglutamine,0.1mMnonessentialaminoacids,0.1mM2-mercaptoethanol,10ng/mlbFGF,and50Uand50µg/mlpenicillinandstreptomycin.

ThawingcryopreservedhumaniPScells

Toinsurethehighestlevelofviability,besuretowarmmediumto37°Cbeforeusingonthecells.DuetothelowsurvivalrateofcryopreservedhumaniPScells,therecoveryisexpectedtotakeatleastoneweek.

1. QuicklythawthehumaniPScellsina37°Cwaterbathbygentlyshakingthecryovialcontinuouslyuntilhalfthawed.Removethecryovialfromthewaterbathandspraywith70%ethanoltosterilize.
2. Transferthecontentsofthecryovialtoa15mLconicaltube.Add5mLwarmhumanESmediumdropwisetothetube,gentlymixingasthemediumisadded.
3. Centrifugecellsat200xgfor5minutesatroomtemperature.
4. Whilecentrifuging,removeMEFmediumfromthefeedercellplates,andwashthewellstwicewithKnockoutDMEM/F12.Thenadd1mlofhumanESMediumwith5µMROCKinhibitor(Y-27632,StemRD)toonewellof6-wellplate.
5. Aftercentrifugation,aspiratethemediumfrom15mLtube.Gentlyresuspendthecellpelletin1mLfreshhumanESmediumcontaining5µMROCKinhibitor(Y-27632),takingcaretomaintainthecellsassmallcellclumps.
6. Transferthemediumcontainingthecellclumpstoonewellof6-wellplatewithMEFfeedercells.
7. Placetheplateintothe37°Cincubatorandmovetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributetheclumpswithinthewells.Culturethecellsat37°C,with5%CO₂and95%humidity.
8. Changemediumdaily.Checkforundifferentiatedcoloniesthatarereadytopassagewhencoloniesarebigenough(approximately7-10daysafterthawing).

PassaginghumaniPScellsgrownunderfeeder-dependentconditions

1. Aspiratethemediumandwashthecellstwicewith1mlofPBS.
2. RemovePBScompletely,add0.5mlofAccutase(Cat.no.SCR005,Millipore,diluted1:1withDPBSbeforeuse)andincubatefor1-2minat37°C.
3. Tapthebottomoftheplatetodislodgethecellsfromthebottomoftheplate.Thenaspiratetheaccutase.
4. Add1mlofDMEM/F12totheplateandcarefullywashthefeedercells,andaspiratethemedium.Repeat.
5. Add1mlofhumanESmediumcontaining5µMROCKinhibitortotheplateandsuspendthecellcoloniesbygentlypipettingupanddown.Itisimportantnottobreakupthecoloniesintosinglecells.
6. RemoveaplateofMEFfeedercellsfromtheincubator.AspiratetheMEFmedium.WashoncewithKODMEM/F12medium.
7. Distribute0.2-0.3mlofthehumaniPScellsuspensiontoeachwellofa6-wellplate.
8. Add1mLhumanESmediumtotheoriginalwellandscrapecoloniesoffwithacelllifter.
9. Distribute0.2-0.3mlofthehumaniPScellsuspensiontoeachwellofa6-wellplate.
10. AddhumanESmediumwithROCKinhibitortoafinalvolumeof2mlperwell.RightafterplatingtheiPScells,gentlyswirltheplateback-and-forthandside-to-sideandincubateat37°C.
Note:Ifthecoloniesareatanoptimaldensity,thecellscanbesplitevery5-7daysusing1:3to1:6ratios.
11. After24hours,removethemediaandreplacewithhumanESmedia(withoutROCKinhibitor).
12. ThehumanESmediamustbechangedeverydayandhumaniPScellssubculturedevery5-7days.Trackthepassagenumberofthecells.

CryopreservinghumaniPScells

1. PrepareEZStemfreezingmedium(Cat.no.M050,ALSTEM)onice.
2. Performsteps1-6fromPassaginghumaniPScellsgrownunderfeeder-dependentconditions.
3. Transferthedetachedcellaggregatestoa15mLconicaltube.
4. Add1mLhumanESmediumandscrapecoloniesoffwithacelllifter.
5. Transferthecellsuspensiontothe15mLconicaltube.
6. Centrifugethe15mLtubecontainingtheaggregatesat200xgfor5minutesatroomtemperature.
7. Gentlyaspiratethesupernatantandloosenthecellpelletbytappingthebottomofthetube.
8. Gentlyresuspendthepelletinfreezingmedium,takingcaretoleavetheclumpslargerthanthatwouldnormallybedoneforpassaging.
9. Transfer1mLofclumpsinfreezingmediumintoeachlabeledcryogenicvial.
10. Placevialsintoanisopropanolfreezingcontainerandplacethecontainerat-80°Covernight.
11. Transfertoaliquidnitrogentanknextday.