JournalofVisualizedExperimentswww.jove.comCopyright©2009JournalofVisualizedExperimentsandStemgentDecember2009|34|e1427|Page1of3VideoArticleCultureandMaintenanceofHumanEmbryonicStemCellsLiaKent11ResearchandDevelopment,StemgentCorrespondenceto:LiaKentatlia.kent@stemgent.comURL:http://www.jove.com/video/1427DOI:doi:10.3791/1427Keywords:DevelopmentalBiology,Issue34,Humanembryonicstemcell,medium,hES,maintenance,thaw,EScells,stemcell,cellculture,pluripotency,differentiation,passage,freezeDatePublished:12/22/2009Citation:Kent,L.CultureandMaintenanceofHumanEmbryonicStemCells.J.Vis.Exp.(34),e1427,doi:10.3791/1427(2009).AbstractHumanembryonicstem(hES)cellsmustbemonitoredandcaredforinordertomaintainhealthy,undifferentiatedcultures.Atminimum,theculturesmustbefedeverydaybyperformingacompletemediumchangetoreplenishlostnutrientsandtokeeptheculturesfreeofunwanteddifferentiationfactors.Althoughasmallamountofdifferentiationisnormalandexpectedinstemcellcultures,thecultureshouldberoutinelycleanedupbymanuallyremoving,or"picking"differentiatedareas.IdentifyingandremovingexcessdifferentiationfromhEScellculturesareessentialtechniquesinthemaintenanceofahealthypopulationofcells.VideoLinkThevideocomponentofthisarticlecanbefoundathttp://www.jove.com/video/1427/Protocol1.Maintenance:ObservingCulturesUndertheMicroscope1.RemoveoneplateofhEScellsfromthe37°Cincubatorandbringtothemicroscope.2.Observethecoloniesatlowpower,using2Xor5Xmagnification,toassesstheoverallculturequality.Notethefollowing:normalmediumcolor,absenceofanyvisiblecontamination,overallcolonysize,qualityanddensity,andanyotherobservations.3.Observemediumcolorchanges.DuetothechangeinpHandthedepletionofnutrientsinthemediumafteranovernightincubationwiththehEScells,themediumwillchangefromaredhuetoa"straw"color.Ifthemediumappearspurple,abasicpHshifthasoccurred.Ifthemediumappearsextremelyyellow,themediumhasacidified.Veryyellowmediummaybetheresultofextremelyovercrowdedcoloniesinthewellorcontamination.Themediumshouldappearclearatalltimes...
