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)
Materials:
• BG01Vhumanembryonicstemcells
• Tissueculturedishes(60mm;Fisher,Catalog#08-772B,100mm;Fisher,Catalog#08-772E,orequivalent)
• 15mLconicaltubes(CorningCostar,Catalog#430052orequivalent)
• Pipettesandpipettetips
Equipment:
• 37°Cand5%CO₂humidifiedincubator
• Centrifuge(lowspeedclinicalorequivalent)
• Hemacytometer
• Invertedmicroscope

Procedure

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

References

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

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