Introduction

Humanembryonicstem(hES)cellsarepluripotentstemcellsderivedfrompre-implantationembryosthatcanbemaintainedandexpandedinanundifferentiatedstateorinducedtodifferentiatealongsomaticorgermcelllineages.Theycanbemaintainedeitheronalayerofmitoticallyinactivatedhumanormousefeedercells(1)orusingmouseorhumanfeedercellconditionedmedium(2)(R&DSystems,Catalog#AR005orAR007,respectively).TheprotocolbelowhasbeenusedwiththeBG01VlineofhEScells(3,4).PleasenotethatotherhEScelllinesmayrequiremodificationsofthisprotocol.OptimalcultureconditionsmustbedeterminedbytheinvestigatorforeachhESline.

MaterialsRequired

• RecombinanthumanFGFbasic(R&DSystems,Catalog#233-FB,4114-TC,orequivalent)
• Accutase(InnovativeCellTechnologies,Catalog#AT104orequivalent)
• Cultrex®BasementMembraneExtract(BME)(R&DSystems,Catalog#3433-005-01orequivalent)
• DMEM/F12(Invitrogen,Catalog#12500-096orequivalent)

• BG01Vhumanembryonicstemcells
• Tissueculturedishes(60mm;Fisher,Catalog#08-772B,100mm;Fisher,Catalog#08-772E,orequivalent)
• 15mLconicaltubes(CorningCostar,Catalog#430052orequivalent)
• Pipettesandpipettetips

• 37°Cand5%CO₂humidifiedincubator
• Centrifuge(lowspeedclinicalorequivalent)
• Hemacytometer
• Invertedmicroscope

Procedure

1. ThawingandExpandingCryopreservedCells:
   1. PreparetheCultrexBMEcoatedplate.
      1. ThawCultrexBMEoniceat2-8°Covernight.
      2. AliquotthawedCultrexBMEintopre-cooledtubesandstoreat=-20°C.
      3. Thawthealiquotoniceat2-8°Covernight.
      4. DiluteCultrexBME1:40inDMEM/F12.Thiscanbestoredforupto2weeksat2-8°C.
      5. CoatthedesirednumberofplateswithdilutedCultrexBME(approximately2.5mLper60mmplate)andincubatefor1-2hoursatroomtemperature.
      6. RemovetheCultrexBMEsolutionimmediatelypriortoplatingthecells.
   2. ThawingofBG01VhESCells:
      1. WarmtheHumanFeederCellConditionedMediumto37°C.
      2. ThawthevialofBG01VhEScellsbywarminguntiljustthawedandthenimmediatelytransfertoa15mLconicaltubecontainingatleast5mLofpre-warmedHumanFeederCellConditionedMedium.
      3. Spinat200xgfor4minutes.
      4. Removethesupernatantandgentlyflickthepellet.ResUSPendthepelletinanappropriateamountofHumanFeederCellConditionedMediumsupplementedwith4ng/mLofrecombinanthumanFGFbasic.
      5. AddtheBG01VhEScellsuspensiontotheCultrexBMEcoatedplate.
      6. Growinthe37°C,5%CO₂incubator.Changethemediumdailyandmonitorthecells.Passagethecellsatthedesiredconfluency.
   3. PassagingofBG01VhESCells:
      1. PreparethedesirednumberofplatesbycoatingwithCultrexBMEasdescribedabove,1-2hourspriortopassagingthecells.
      2. WarmtheHumanFeederCellConditionedMediumto37°C.
      3. RemovetheHumanFeederCellConditionedMediumfromthecells.Add1mLofAccutasesolutiontoeach60mmplate.Incubateatroomtemperaturefor5-10minutesoruntilthecellsbegintosloughofftheplate.
      4. Pipettegentlyovertheplateuntilallthecellshavebeendetached.
      5. Pipettethecellsuspensionupanddowntobreakuplargecellclumps.
      6. Removethecellsuspensiontoa15mLconicaltubecontaining5mLofHumanFeederCellConditionedMediumandspinat200xgfor4minutes.
      7. ResuspendthepelletinHumanFeederCellConditionedMediumandcountthecellsusingahemacytometer.
      8. Platethedesirednumberofcells(approximately1.0x10⁶cells/60mmplate)ontheCultrexBMEcoatedplateinHumanFeederCellConditionedMediumcontaining4ng/mLofrhFGFbasic.
      9. Changethemediumdaily.Monitorthecellsforthedesiredconfluency.

References

1. Thomson,J.A.etal.(1998)Science282:1145.
2. Xu,C.etal.(2001)NatureBiotechnology19:971.
3. Zeng,X.etal.(2004)Restor.Neuro.Neurosci.22:421.
4. Plaia,T.etal.(2006)StemCells24:531.