Regulation of spontaneous activity and growth of embryonic chick heart cells in tissue culture

[摘要] 1.1.Thepercentageofembryonicheartcellsthatexhibitspontaneousactivity(%BC)intissuecultureisdependentuponawidevarietyofenvironmentalparameters,includingmodeoftissuedisaggregation,compositionoftheculturemedium,densityofcellinoculum,andothercultureconditions,aswellastheageoftheembryofromwhichthecellsaretaken.2.2.Dissociating7-dayheartswiththree8-minuteincubationcyclesin0.05%trypsinliberatesamaximalnumberofviablecells;bestresultsareobtainedwhenenoughtissueisusedfordissociationtobringtheresultantcellsuspensionto2to3×106cells/mlormore.3.3.Tobeabletodetermine%BC,cllsmustbeplatedatdensitieslowenoughtopreventextensiveintercellularcontacts.Atdensitiesaboveabout100cells/mm2manycellsmakecontactwithneighbors,makingitimpossibletodistinguishwhetheracellisbeatingbecauseofitsownelectrogenicactivityorasaresponsetoanimpulseconductedfromapacemakingneighbor.4.4.Theserumusedinaculturemediuminfluences,toamarkeddegree,thepercentageofcellswhichbeatspontaneously.Atotalof56serumsamplesweretested,yieldingarangeofeffects:fromthoseserawhichwereclearlytoxictothosewhichgave“optimal”results.Asampleofserumwasconsideredoptimalifcellsinstandardlow-potassiummedium,madewiththatserum,werehealthyinappearance,andconsistentlyyielded40–50%BC.5.5.AtadultserumlevelsofK+(4–5meq/l)approximately20%ofthecellsinaculturefrom7-dayheartbeatspontaneously.IncreasingextracellularK+causessomecellstoceaseactivity.DecreasingtheK+to1meq/lpermitsamaximalnumberoflatentpacemakerstobecomeactive.Dose-responsecurvesrelatingK+concentrationto%BChavebeendeterminedwithcellsfromembryonicheartsaged4,7,12,and18days.6.6.Thegreatestnumberofspontaneouslyactivecellsisobtainedfrom7-dayhearts.Underoptimalconditions,with1meq/lK+medium,activityrangedbetween40and50%BC,withameanof44%BC.Thegreatestnumberofactivecellseverrecordedinasingleplatewas64%BC.Olderheartsyieldedprogressivelyfewerpacemakers,toaminimummeanofabout10%BCat18days.Heartsearlierthan7daysalsoyieldedfewerspontaneouslyactivecells.7.7.Four-dayheartculturesarerelativelyinsensitivetoelevatedK+intheculturemedium.FromthisitissuggestedthatthehighintracellularNa+-contentandrapidNa+-fluxcharacteristicofearlyembryonicheartsrepresentdeviceswherebyearlypacemakercellscanmaintainaratioofsodium:potassiumconductancesufficientlylargetopermittheonsetofspontaneousactivityinthehigh-potassiumenvironmentprovidedbytheeggbeforeamnionformation.8.8.Twotypesofheartcellsmaybedistinguishedmorphologicallyincultures:Fcells,whichhavetheappearanceoftypicalfibroblasts;andMcells,whichresemblemyoblastsofskeletalmuscle.Fcellscomprise40–50%ofthecells;Mcells50–60%.MostovertpacemakersareMcells.However,asmallproportion(lessthan1%)ofthesewhichmeetallthemorphologicalcriteriaofFcells,arealsospontaneouslyactive.9.9.Mitoticactivityinheartcellculturescanbeinducedorpreventedmerelybythepresenceorabsenceofchick-embryoextract.Culturescanbeobtainedwhichexhibitbothrapidmitosisandhigh%BC,bydialyzingCEEtoremoveK+.Itisconcludedthatnonecessaryantagonismexistsbetweengrowthandthemaintenanceofadifferentiatedfunctionalstateinembryonicheartcellsculturedundertheconditionsdescribed.

[学科分类] 生物科学（综合）