CulturingChickEmbryonicCardiacMyocytesandFibroblasts

1.Culturemedium:F12Kwithoutglutamine,3%FCS,100units/ml,100ug/mlstreptomycin(myocytemedium,alsoformixedculture);andF12Kwithglutamine,5-10%FCS,100units/mlpenicillin,100ug/mlstreptomycin(fibroblastmedium),onice.FCSshouldbescreenedforitsABIlitytosupportplatingandmyocytebeating.

2.Dissectiontools:1pairoflargescissors;2pairsfinescissors;2pairsofcoarseforceps;2pairsoffineforceps;soakingin95%EtOH.

3.SalineG-Ca2+andMg2+free.

4.Enzyme-0.125%trypsin+0.05%CollagenaseinsalineD.Theseconcentrationsworkwellfor6-7dayembryos.Olderembryosneed0.25%trypsinandahigherconcentrationofcollagenase.With6-dayandyoungerembryos,nocollagenaseisnecessary.Thawthesolutionandputintheincubatorat34or39^oC..Need1ml/heart(formyocytes)or0.5ml/heart(forfibroblasts).

5.F12Kwithglutamine+20%FCS+1%penicillin/streptomycin.Samevolumeastrypsin-collagenase(~5ml),ina15mlconicaltubeandkeeponice.

6.Chamberdishes.Place1.5mlappropriatemediumoneachdishandincubateat34^oC.

7.Preplateformyocyteculture.Add1mloffibroblastmediumtoa60mmdishandputinthe34or39^oCincubator.

8.2x100mmsterilepetridishes-glassorplastic-onice.

9.Chickembryos,7-dayisoptimal,but6-or8-dayembryosarealsouseable.Need1-2embryosforeachchamberdish:oneperdishifnopreplating,2perdishwithpreplating.Eachheartyieldsabout105cells(myocytesplusfibroblasts).

10.15mlconicalcentrifugetubes.

1.Sprayeggs2xwith70%EtOH.

2.Wipetopsurfacewith95%EtOHthoroughly.Letdrycompletely.

3.Put1-2mlSalineGinoneofthepetridishestopoolheartsandkeeponiceuntiluse.

4.Removeembryostothesterile100mmpetridishwithoutsalineG.Itiseasiertograbtheembryosatfeet.Dissecteachembryooniceimmediatelyifyouareslow.Ifyouarefast,youcanremovealltheembryosfirstanddissectthemlater.

5.Beheadembryos.DissectandremovethewholeheartusingacleanpairoffinescissorsandforcepsandplaceinthedishwithsalineG.Heartscanbeidentifiedbasedonthepresenceofblood,beating,andassociatedbloodvessels.

6.Cuteachheartthroughtheventricletodrainanyremainingblood.

7.PipetoffsalineG.Reapply1-2mlsalineG,rinse,andpipetoff.

8.Repeatrinseonemoretime,oruntilsalineGlooksfairly"clean"(freeofblood).Morerinsesnecessaryformyocytes.LeavesomesalineGafterthefinalrinse,about0.2mlperheart.

9.Usingacleanpairoffinescissors,chopheartstosmallbutuniformsize-aboutthatofsandparticles.Becarefulnottochoptoolong,becausecellscanbedamaged.

10.Transfercellstoa15mlconicalcentrifugetube.Useasmallamountofsalinetorinsethedish.Repeattherinseifnecessary.Thetotalvolumeshouldbecloseto0.5mlperheart.

11.Trypsinization#1:mixtheenzymesolutionwithaPasteurpipet.Add0.5to1mlenzymetothetube.Mixbyshaking.Makesuretissuesarenotstickingtothesideofthetube.Placethetubeinthe34or39^oincubatorforatotalof~5min.Shakeorvortexthetubeevery2-3min.Donotletsolutionsittoolongandbecometooturbid(willlosealotofcells).Keeptheremainingenzymesolutionwarm.

12.Removethetubefromtheincubatorandvortexat1/2speed.Lengthoftimevortexingdependsonthelengthoftrypsinization(shortvortexingforlongtrypsinization).Typically15-20sec.Shakepiecesoffthesideandletsettlefor1-2min.

13.Pipetoffsupernatantanddiscard.

14.Trypsinization#2:add0.5to1mlenzymetothesettledpieces.Vortexat1/2speedfor10-15sec.Incubatefor~5minasinstep11.

15.Removethetubefromtheincubatorandvortexatfullspeedfor10-15sec.Shakepiecesoffthesideandletsettlefor1-2min.SUSPensionneverlooksascloudyasafterthefirsttrypsinizationbecauseofthemuchsmallnumberofredbloodcells.

16.Transferthesupernatanttothetubewithcoldmediumcontaining20%FCS.Avoidclumps.

17.Repeatthetrypsinizationonthesettledmaterials.Theenzymesolutionshouldbedividedamongthenumberoftreatment.Formyocytes,repeat3-5times.Forfibroblasts,repeat1-2times.Accumulateallsupernatantinthesametubewith20%FCS.

18.Vortexhardattheendoflasttrypsinizationfor15sectotryandbreakupclumps.Pipetupanddowntobreakupclumpsevenmore.

19.Centrifugeintable-topcentrifugeonhighsetting(notquitemaxspeed)foratleast5min.

20.Removesupernatantanddiscard.Besurenottodisturbpellet.

21.Resuspendpelletin~1mlcoldmedium(for4hearts).Breakuppelletcompletelybypipeting.

22.Formyocytesonly,transferthesuspensiontothepreplatewithwarmfibroblastmedium.Incubatefor45-50min.Collectthesupernatantina15mlconicaltube.

23.Transferallsuspensiontoplates/dishes.Usuallyinnoculumsizeis~0.2mlperchamberdishor0.3mlper60mmdish.Forfibroblasts(withoutpreplating),eachchamberdishshouldcontain~6x104cells.Formyocytes,countcellsinthesupernatantafterpreplatingandplateat4x104cellsperchamberdish.

24.Changemedium2-3hrlaterforfibroblasts;24hrlaterformyoblasts.Useappropriatemedia.Feedcellsevery2-3days.

25.Cardiacmyocytesneed3-5daystospreadout.