LargeVessel

*AA-1001*AA-1001Rev.04/98

ProliferatingCells

Thisinstructionsheetcontainscultureinformationforallofthecelltypeslistedpreviouslyinthefollowingproliferatingformats:

1.Checkallcontainersforleakageorbreakage.

2.Forcryopreservedcells-Ifthereisdryiceleftinthepackage,placecryopreservedcellcryovialsimmediatelyintoliquidnitrogen.Ifnodryiceisleftinthepackage,thawandusethemimmediately.

Forproliferatingcells-Swabdowntheflaskofproliferatingcellswith70%ethanolorisopropanol,thenplacetheflaskin37·C,5%CO2,humidifiedincubatorandallowtoequilibrateforthreetofourhours.Aftercellshaveequilibrated,removeshippingmediumfromtheflaskfollowinginstructionsonpage18.

3.Storecellculturemediumina4·Crefrigerator.

4.Ifyouplantoproceedwithin3days,storeallgrowthsupplements,HEPESBufferedSalineSolution(HEPES-BSS)andTrypsinNeutralizingSolutionat4·C.Trypsin/EDTASolutionhasalimitedshelflifeoractivationat4·C.If,uponarrival,Trypsin/EDTAisthawed,immediatelyaliquotandrefreezeat-20·C.Iffrozen,storeat-20·C.Ifyoudonotplantosetupthecellculturewithin3days,storeallgrowthsupplementsandsubculturereagentsina-20·Cfreezer.

Pleasereadandfollowtheseinstructionscarefullyandcompletely.BioWhittakerisnotresponsIBLeforproductlossduetoimproperreceiptandhandlingofitsproductsbycustomers.Replacementproductwillbesentatthecustomer\"sexpense.

EndothelialCellSystems

1.NormalHumanEndothelialCells

*Researchresultsmayvarydependinguponmediumselection.ContactyourTechnicalSpecialistfordetails.

Theproliferatingcultures(HUVECsecondary2·-allothersquarternary4·)areshippedina25cm2flask,75cm2flaskor96-wellplatefilledwithmedium.Thecellsshouldbebetween30and80%confluentuponarrival.ACertificateofAnalysisisprovidedwitheachcellstrainandindicatesQCperformanceresultsanddonorinformation.

Thecryopreservedcultures(HUVECprimary1·-allotherstertiary3·)areshippedinascrewcapcryovialcontainingapproximately500,000cells.ACertificateofAnalysisisprovidedwitheachcellstrainandindicatesdateofcryopreservation.QCperformanceresults,donorinformationandthenumberofcellscontainedinthecryovial.

2.EndothelialCellGrowthMedium(EGM®),aseither:

EndothelialCellGrowthMedium(EGM®),(CC-3024),acompletemediumina500mlbottlewithattachedBovineBrainExtract(BBE)supplement.EGM®isamodifiedMCDB131formulationandissuppliedfullysupplementedwiththefollowing:(amountsindicatefinalconcentration,exceptBBE)

10ng/mlhEGF(humanrecombinantEpidermalGrowthFactor)1.0µg/mlHydrocortisone50µg/mlGentamicin,50ng/mlAmphotericinB3mg/mlBBE(BovineBrainExtract)(CC-4092)2ml2%v/vFBS(FetalBovineSerum),producingEGM®

(or)

EndothelialCellGrowthMediumBulletKit®(EGM®BulletKit®)(CC-3124),orMicrovascularEndothelialCellGrowthMedium(EGM®-MVBulletKit®)(CC-3125),whichcontainsa500mlbottleofEndothelialCellBasalMedium(EBM®),andallthesupplementslistedbelow,convenientlypackagedassingle-usealiquotscalledSingleQuots®.(amountsindicateconcentrationofeachSingleQuot®)

10µg/mlhEGF(humanrecombinantEpidermalGrowthFactor)(CC-4017),0.5ml1.0mg/mlHydrocortisone(CC-4035),0.5ml50mg/mlGentamicin,50µg/mlAmphotericin-B(CC-4081),0.5ml3mg/mlBBE(BovineBrainExtract)(CC-4092),2ml10mlFBS(FetalBovineSerum)(CC-4101)EGM®,(or)25mlFBS(CC-4102)EGM®-MV

(or)

EndothelialCellGrowthMediumBulletKit®-2(EGM-2®BulletKit®)(CC-3162),orMicrovascularEndothelialCellGrowthMedium-2(EGM2®-MVBulletKit®)(CC-3202),whichcontainsa500mlbottleofEndothelialCellBasalMedium-2(EBM-2®),andallthesupplementslistedbelow,convenientlypackagedassingle-usealiquotscalledSingleQuots®.

0.5mlhEGF(humanrecombinantEpidermalGrowthFactor)2.0mlhFGF-B(humanFibroblastGrowthFactor-Basicwithheparin)0.5mlVEGF(VascularEndothelialGrowthFactor)0.5mlAscrobicAcid(VitaminC)0.2mlHydrocortisone0.5mlLongR3-IGF-1(HumanRecombinantInsulin-likeGrowthFactor)0.5mlHeparin10mlFBS(FetalBovineSerum)2%EGM-2,25mlsinEGM-2-MV5%0.5mlGentamicin,Amphotercin

4.ReagentPack™(CC-5034)containsone100mlbottleofeachofthefollowingsubculturereagents:

HEPESBufferedSalineSolution(HEPES-BSS)(CC-5022)1x100mlbottleTrypsin/EDTASolution(CC-5012)1x100mlbottleTrypsinNeutralizingSolution(CC-5002)1x100mlbottle

NOTE:IfyouuseadifferentClonetics®medium,seeAppendixA,EndothelialCellMedia

EndothelialCellSystemsMediaOptions

BioWhittakerstrivestooptimizeit\"sClonetics®mediainordertosupplyit\"scustomerswiththebestproductavailablefortheproliferationofEndothelialcells.Eachcomponentofthebasalmediumandeachgrowthsupplementiscarefullytiteredforoptimalgrowth.BioWhittakercurrentlyoffersfourClonetics®mediachoicesforthegrowthofendothelialcellsallowingfordesiredperformanceandflexibility.WhenselectingamediumtouserefertospecificmediarecommendationsorcallyourformulationTechnicalSpecialistforassistance.

EGM®&EGMBulletKit®(EndothelialGrowthMedium)

• BasalmediumbasedonmodifiedMCDB-131,developedfornormalhumanendothelialcellinalow-serumenvironment.
• EGM®issupplementedgrowthmediumandincludesanattachedaliquotofBovineBrainExtract(BBE)
• EGM®BulletKit®includesbasalmediumwithallsupplementsandgrowthfactorsinseparate,frozenaliquots
• Finalserumconcentration2%
• EGM®canbeusedtogrowallofClonetics®endothelialcellsexceptmicrovascularandcoronaryartery.

EGM®-MVBulletKit®

• Developedformicrovascularandcoronaryarteryendothelialcells
• SamebasalmediumasinEGM®
• Finalserumconcentration5%
• EGM®-MVcanbeusedtogrowallClonetics®endothelialcellsexceptHMVEC-L

EGM®-2BulletKit®

• Refinementstobasalmedium(CCMD™130)andthegrowthfactors
• DoesnotcontainBBE
• Finalserumconcentration2%
• ImprovedcellproliferationoverEGM®
• EGM®-2canbeusedtogrowallofClonetic®endothelialcellsexceptmicrovascularandcoronaryartery

EGM®-2-MVBulletKit®

• Developedfortheenhancedgrowthoflungmicrovascularendothelialcells
• DoesnotcontainBBE
• Finalserumconcentrationincreasedto5%
• EGM®-2-MVcanbeusedtogrowallendothelialcells
• SamebasalmediaasEGM®-2

EGM®(CC-3024)EGM®BulletKit®(CC-3124)BBE(BovineBrainExtract),w/heparinhEGF(HumanEpidermalGrowthFactor)HydrocortisoneGA-1000(Gentamicin,AmphotericinB)FBS(FetalBovineSerum)10ml

EGM®-2BulletKit®(CC-3162)NoBBE(BovineBrainExtract)hEGFHydrocortisoneVEGF(VascularEndothelialGrowthFactor)hFGF-B(w/heparin)(HumanFibroblastGrowthFactor)LongR3-IGF-1(HumanRecombinantInsulin-likeGrowthFactor)AscorbicAcidHeparinGA-1000(Gentamicin,Amphotericin-B)FBS(FetalBovineSerum)10ml*CCMDstandsforCloneticsCellMediaDevelopment

EGM®-MVBulletKit®(CC-3125)BBE(BovineBrainExtract),w/heparinhEGFHydrocortisoneGA-1000(Gentamicin,AmphotericinB)FBS(FetalBovineSerum)25ml

EGM®-2-MVBulletKit®(CC-3202)NoBBE(BovineBrainExtract)hEGFHydrocortisoneVEGFhFGF-B(w/heparin)LongR3-IGF-1AscorbicAcidGA-1000(Gentamicin,Amphotericin-B)FBS(FetalBovineSerum)25ml

GeneralInformation

ProductApplicationsClonetics®NormalHumanEndothelialCellsare:

• FORRESEARCHONLY
• NOTapprovedforhumanorveterinaryuse,forapplicationtohumansoranimals,orforinvitrodiagnosticprocedures.

MaterialsNotProvidedClonetics®NormalHumanCellSystemsdonotincludeplasticware,glasswareorotherlaboratoryequipmentusedinacellculturelaboratory.Individualcomponentsareavailableseparately.

ProductWarrantyCULTURESHAVEAFINITELifespanINVITRO.BioWhittakerwarrantsClonetics®productsonlyifClonetics®mediaandreagentsareused.

• Cryopreservedculturesareassuredforexperimentaluseforfifteenpopulationdoublings.
• Proliferatingculturesareassuredforexperimentalusefortenpopulationdoublings.
• Additionalpopulationdoublingsandsubculturesarepossible,butgrowthrate,BIOLOGicalresponsivenessandfunctionmaydeterioratewithsubsequentpassage.
• Endothelialcellscanbecomeirreversiblycontact-inhibitedifmaintainedatconfluenceformorethantwodays.Toavoidthelossofyourcellsandforfeitureofyourwarranty,werecommendthatyousubculturecellsbeforetheyreach90%confluence.

CellIsolationClonetics®endothelialcellculturesareestablishedatBioWhittaker\"scellculturefacilityfromnormalhumantissue.Thecelltypeandthepassageinwhichtheyareshippedareoutlinedbelow:

ProductName	PassageForShipment
	Cryopreserved	Proliferating
AllMicrovascularEndothelialCells	3rdor4th	4thor5thpassage
HUVEC	primary	2ndpassage
AllLargeVesselEndothelialCells	3rd	4thpassage

EndothelialcellsarecryopreservedinEGM®orEGM®-2supplementedwith10%v/vfetalbovineserumand10%v/vdimethylsulfoxideasacryopreservationsolutiontoimprovecellviABIlityandseedingefficiencyuponthawing.

MediumInformationPreparation,storage,andshelflifediffersforthefollowingfiveproducts:1)FullySupplementedEGM®(CC-3024),and2)EGM®BulletKit®(CC-3124),and3)EGM®-MVBulletKit®(CC-3125)and4)EGM®-2BulletKit®(CC-3162)and5)EGM®-2-MVBulletKit®(CC-3202).Seethetablebelow.

FULLYSUPPLEMENTEDEGM®(CC-3024)
HowPrepared	StorageRequirements	ShelfLife
ThepHisapproximately7.8andosmolalityapproximately294mOsm/kg. Priortoshipping,basalmediumissupplementedwithepidermalgrowthfactor,hydrocortisone,insulin,gentamicin,amphotericin-BandFBS. Afterreceiving,youwilladdBBEimmediatelybeforeusetocompletetheEGM®formulation.	EGM®isstoredat4·Cuntilshipped.TheattachedBBEwillthawduringshipment. FullysupplementedEGM®shouldbestoredat4·C. Avoidrepeatedwarmingandcooling.Iftheentirecontentsarenotneededforasingleprocedure,transferonlytherequiredvolumetoasterilesecondarycontainer.Donotfreeze.	EGM®hasanoptimumshelflifeof5monthsfromdateofmanufacture.
	EGM®BulletKit®(CC-3124)andEGM®-MVBulletKit®(CC-3125)and EGM®-2BulletKit®(CC-3162)andEGM®-2-MVBulletKit®(CC-3202)
HowPrepared	StorageRequirements	ShelfLife
AllEGM®,EGM®-MVBulletKit®,EGM®-2andEGM®-2-MVBulletKit®componentshavebeentestedagainstClonetics®NormalHumanCells.Allsolutionsaresterile-filteredbypassagethrougha0.2µmfilter.Basalmediumisstoredat4-8·C,andgrowthfactorsarestoredat-20·Cuntilshipment.	Ifthaweduponarrival,growthfactorscanbestoredat4·CandaddedtoEBM®orEBM®-2within72hoursofreceipt. IfthawedandwillNOTbeusedwithin72hours,growthfactorsmustberefrozen.Theymayberefrozenonlyonceandthenstoredat-20·Cforuptooneyear. StoreEBM®orEBM®-2at4·C.StorefullysupplementedEGM®andEGM®-MVandEGM®-2andEGM®-2-MVat4·C. Avoidrepeatedwarmingandcooling.Iftheentirecontentsarenotneededforasingleprocedure,transferonlytherequiredvolumetoasterilesecondarycontainer.Donotfreeze.	EGM®andEGM®-MVandEGM®-2andEGM®-2-MVBulletKit®shelflifeislimitedbytheshelflifeoftheEBM®andEBM®-2,respectively,whichis1yearfromthedateofmanufacture.Whengrowthfactorsareaddedatanytimewithinthistimeperiodwerecommendusewithin1month.

QualityControlEndothelialcellsareculturedwithoutantimicrobialagentsandassayedtoensuretheabsenceofmicrobialcontaminationaftercryopreservation.

1.AllcellstrainstestnegativebyPCR6forHIV-1,hepatitisBandhepatitisC.

2.Afterrecoveryfromliquidnitrogen,cellsaretestedforviability,growthrate,morphology,seedingefficiency,proliferativecapacity,mycoplasma,yeast,fungusandbacteria.Eachculturemeetsin-housespecificationsforproliferativecapacity,(i.e.,15cumulativepopulationdoublingsafterthaw).

3.HUVECarecharacterizedbymorphologicalobservationthroughoutserialpassage.AllotherClonetics®endothelialcellstestPositiveforvonWillebrandFactorVIIIandAcetylatedLDLandtestNegativeforAlphaSmoothMuscleActin.

4.Inadditiontotheabovestaining,HMVEC-Lalsotestpositivelyforplateletendothelialcelladhesionmolecule(PECAM).

5.Beforeshipping,allbasalmediaandcellculturereagentsaretestedforproperpH,osmolality,sterility,andcellcultureperformance.Growthfactorsaretestedforsterilityandcellcultureperformance.EBM®,EBM®-2,EGM®,andBBEarealsotestedforendotoxinlevels.

SubcultureReagentStorage1.Subculturereagentsaresterile-filteredandthenstoredat-20·CuntilshippedfromBioWhittaker\"sDistributionCenters.

2.Subculturereagentsmaythawduringtransport.Theymayberefrozenonce.

3.Subculturereagentscanbestoredat-20·Cforuptooneyearafterthawingonceandrefreezing.

4.TokeepTrypsin/EDTAfreshandactiveafterthawing,youmayaliquotitintofive20mlsterilecentrifugetubesandrefreezeat-20·C.Trypsin/EDTAmaybestoredfrozenuptooneyear.

5.WerecommendthatHEPES-BSSandtheTrypsinNeutralizationSolution,oncestoredat4·C,beusedwithinonemonth.

HandlingPrecautionsNormalhumancellsarefragile,andrequirespecialhandling:

• Uponreceipt,immediatelystorecryopreservedcellsinliquidnitrogen.Properlystoredcellsremainviableindefinitely.
• Uponreceipt,immediatelyplaceproliferatingcellsina37·C,5%CO2,humidifiedincubator.
• Donotusethemediumorreagentsbeyondtheexpirationdate.
• NormalhumancellsareverysensitivetoimpuritiesincommerciallyavailableTrypsin.UseonlyClonetics®Trypsin;everylotofourTrypsinistestedonnormalhumancellcultures.
• UseonlyClonetics®media.Keepmediarefrigeratedat4·C.Whenusingamedium,takejusttheamountyouneedandthenreturnthebottletotherefrigerator.
• Regularlywipeflasks,cryovials,bottlesandgloveswith70%isopropylalcoholor70%ethylalcohol.
• Becausecellsareanchoredtoonesideofaflask,alwaysaddallliquidsbypipettingthemdowntheoppositesidefromwherethecellsareattached.

SafetyPrecautionsBioWhittakerstressestheimportanceofthefollowingprecautions:

SafetyPrecautions

Asaprecautionagainstcontamination,followallproceduresforhandlingproductsofhumanoriginoutlinedin\"GuidelinestoAvoidPersonnelContaminationByInfectiveAgentsinResearchLaboratoriesThatUseHumanTissues,\"fromtheJ.OfTissueCultureMethods.2(SeeBibliography.Page25)

Alwayswearglovesandsafetyglasseswhenworkingwithallmaterials.Exercisecautionwhenworkingwithcryopreservedcells;rapidtemperaturechangesmaycausesplatteringofliquidnitrogen.

Washhandsthoroughlyafterperformingallprocedures.

Nevermouthpipet.

Donotsmoke,eatordrinkinareaswherereagentsorcellsarehandled.

Productsofhumanoriginarepotentiallybiohazardous.AlthougheachcellstraintestsnegativebyPCRforHIV-1,hepatitisBandhepatitisC,properprecautionsmustbetakentoavoidinadvertentexposure.

Theflowchartonthefollowingpageillustratesthecultureprocess.Itisfollowedbythestep-by-stepinstructions...



InstructionsforCryopreservedCellsMediumPreparation

BeforeYouBeginPerformthefollowingstepsbeforeyoubeginmediumorcellpreparation:

*Maynotbenecessaryforallend-userassays.

MediumPreparationPerformthestepsbelowinasterilefield.\"Sterilefield\"isdefinedabove.

ForthebottleoffullysupplementedEGM®,dothefollowing:

1.AddBBEtoa500mlbottleofEGM®.

a.DetachtheBBEsupplementfromthemediumbottle.

b.WipetheBBEcryovialandEGM®bottlewithethanolorisopropanol.

c.AddtheentirecontentsoftheBBEcryovial(approximately2ml)totheEGM®withapipette.RinsetheBBEcryovialwithEGM®andpipettethecontentsbackintothe500mlbottle.

d.Replacethecapandswirlthemediumgentlyafewtimestomix.

e.RecordthedatetheBBEwasaddedonthemediumlabel.

InstructionsforCryopreservedCellsMediumPreparation

FortheEGM®BulletKit®,EGM®-MVBulletKit®,EGM®-2BulleKit®orEGM®-2-MVBulletKit®dothefollowing:

• DecontaminatetheexternalsurfacesoftheSingleQuot®cryovialsandtheEBM®bottlewithethanolorisopropanol.
• AsepticallyopeneachcryovialandaddtheentireamounttotheEBM®withapipette.
• Rinseeachcryovialwiththemedium.Itmaynotbepossibletorecovertheentirevolumelistedforeachcryovial.Smalllosses,evenupto10%,shouldnotaffectthecellgrowthcharacteristicsofthesupplementedmedium.
• Transferthelabelprovidedwitheachkittothebasalmediumbottlebeingsupplemented.Useittorecordthedateandamountofeachsupplementadded.Werecommendthatyouplacethecompletedlabeloverthebasalmediumlabeltoavoidconfusionorpossibledoublesupplementation.
• Recordthenewexpirationdateonthelabelbasedontheshelflife(seetableonpage8).ThissupplementedmediumwillnowbereferredtoasEGM®,EGM®-MV,EGM®-2orEGM®-2-MV.

NOTE:Ifthereisconcernthatsterilitywascompromisedduringthesupplementationprocess,theentirenewlypreparedgrowthmediummayberefilteredtoassuresterility.Ifyourefilter,useasterile0.2µmfilter.Routinerefiltrationisnotrecommended.

InstructionsforCryopreservedCellsSetUp

SetUpTosetupvesselsforendothelialcellscomingoutofcryopreservation,dothefollowing:

1.Calculatethenumberofvesselstobesetup.RefertoyourCertificateofAnalysisfortheexactnumberofcellsinyourcryovial.RefertoAppendixE,GrowthAreaofCommonPlasticware,forhelpinadjustingthiscalculation.

NOTE:Flasksandmultiwellplatesaremosteffectivetosubculturethesecells.

Usethefollowingcalculationstodeterminethenumberofvesselstobesetupforthefollowingrecommendedseedingdensityof2500cells/cm2forHUVEC,HCAEC,HAEC,andHPAEC;5000cells/cm2forHMVEC-dNeonatal,HMVEC-dAdult,andHMVEC-L.

No.ofcellsavailable/RecommendedSeedingDensity=max.no.ofcm2thatcanbeplated

Max.no.ofcm2thatcanbeplated/Effectivegrowthareaofflask=max.no.offlasksthatcanbesetup

Example:AcryovialofHMVEC-Lwith520,000cells

520,000/5000=104cm2tobesetup

IfyouuseaT-25withaneffectivegrowthareaof25cm2

104cm2/25cm2=4flasks(roundeddowntonearestwholeno.offlasks)

AtypicalcryovialcanbeplatedintoatleastfourT-25flasksforHMVECandeightT-25flasksforallotherendothelialcells.TheadvantageofsettingupthisnumberofT-25flasksfromtheinitialcryovial,asopposedtolargerflasks,isthatitreducestheriskoflosinglargenumbersofcells.Thatis,ifyouexperiencedifficultytrypsinizingthefirstT-25flask,thereareotherremainingT-25flaskstouse.

2.Labeleachflaskwiththepassagenumber,celltype,strainnumber,anddate.

Example:ForaprimarycryovialofHUVECwithstrainnumber5658,thelabelmightappearasfollows:

1·HUVEC5658;12/12/97

3.Inasterilefield,carefullyopenthesupplementedbottleofgrowthmedium,andasepticallytransferthemediumtonewculturevesselsbyadding1mlgrowthmediumforevery5cm2surfaceareaoftheflask.

Example:5mlgrowthmediumfora25cm2flaskor60mmplate.

4.Placecapsonvesselslooselyifventedcapsarenotbeingused(i.e.,twistcapsuntiltight,thenloosenabout*turn).Allowtheculturevesselstowarmandequilibrateina37·C,5%CO2,humidifiedincubatorforatleast30minutes.

InstructionsforCryopreservedCellsThawing

ThawingNOTE:Ifmorethanonecryovialistobethawed,thawonecryovialatatimeandkeepothercryovialsinliquidnitrogenuntilreadyforuse.

Step	Explanation
Prepareasterilefield.	AsterilefieldconsistsofaClassIIbiologicalsafetycabinetwithafrontaccessopeningandfilteredlaminarairflow,orothersuchequivalentdevice.
Determinetheamountofmediumrequired.	ReviewtheGrowthAreaofCommonPlasticwareChart(AppendixE)todeterminetheamountofmediumtobeused.
Collectsterileinstrumentsandvessels.	SteriledisposableSEROlogicalPipettes Micropipettersandsterilepipettetips Adjustablemultichannelpipetterorrepeatingpipetter* Sterilereservoirsforusewithmultichannelpipetter* Sterile15mlcentrifugetubes Cellcultureflasks Multi-well,flat-bottomtissuecultureplates* Hemacytometerorcellcounter
Collectothersupplies.	70%alcohol(ethanolorisopropanol) Growthmedium(cell-typespecific) Protectiveglovesandgarments TrypanBlue
Planandprepareforinitialsetup.	BaseyoursetuponthenumberofcellsindicatedontheaccompanyingCertificateofAnalysis.(SeeAppendicesBandC).
Checkthecalibrationonhumidifiedincubator.	Incubatorshouldbea5%CO2/95%air,humidifiedincubator,setto37·C.

Cryopreservedcellsareverydelicate.Thawandreturnthemtocultureasquicklyaspossiblewithminimalhandling! Weareyeprotectionwhenhandlingfrozencells.Rapidtemperaturechangesmaycausesplatteringofliquidnitrogen. Centrifugationshouldnotbeperformedtoremovecellsfromthecryoprotectantcocktail.ThisactionismoredamagingthantheeffectsofDMSOresidueintheculture.

Aftertheflaskshaveequilibratedfor30minutes:

• Priortothawing,locateamicropipetter.
• Removethecryovialofcellsfromstorage.Wipecryovialwithethanolorisopropanolbeforeopening.Inasterilefield,brieflytwistthecapaquarterturntorelievetheinternalpressure,thenretighten.Donotopenthecropvialcompletely.
• Holdingthecryovial,dipthebottom3/4ofthecryovialina37·Cwaterbath,andswirlgentlyfor1-2minutesuntilcontentsarethawed.Watchyourcryovialclosely;whenthelastsliveroficemeltsremoveit.DON\"Tsubmergeitcompletely.Thawingthecellsforlongerthan3minutesresultsinlessthanoptimalresults.
• Removethecryovialimmediately,wipeitdry,andtransfertoasterilefieldwheretheequilibratedflasksshouldbewaiting,readytoseed.Rinsethecryovialwith70%alcohol,thenwipetoremoveexcess.
• Notethecolorofthethawedcryovial.Ideally,thecolorofthethawedcryovialshouldbepink.Ifthecolorisnotpink,stillseedthecells,notethecolorandmentionthisfacttoyourTechnicalSpecialistifseedingisnotsuccessful.

SeedingAftercellsarethawed:

NOTE:DonotdispensetheentirecontentsofthecryovialintooneT-25flask!!

• Removethecap,beingcarefulnottotouchtheinteriorthreadswithyoufingers.
• Usingamicro-pipettewitha1000µltipsetto800µl,putthetipintothecryovialandresUSPendthecells,withagentle,slowandsteadyupanddownpipettingmotionnomorethanfivetimes.DONOTresuspendquickly,andkeepthetipnearthebottomtoavoidmakingbubbles.
• Dispenseandequalamountofcellsintotheflaskssetupearlier.IffourT-25flaskswereprepared,setmicropipetterto250µlanddispense.IfeightT-25flaskswereprepared,setmicropipetterto125µlanddispense.
• Replacethecaporcover,andgentlyrockthevesselstoevenlydistributethecells.Loosencapsifnecessarytopermitgasexchange(see\"SetUp,\"stepnumber4).
• Returntheculturevesselsto37·C,5%CO2incubator.Laythemflatontheshelf,providingthelargestsurfaceforcellstoattach.Thecellswillanchortothebottomsurfaceoftheflask.

MaintenanceAfterSeedingNormalHumanEndothelialCellsarenottolerantorrapidtemperaturefluctuationsornutrient-deficientmedium.Feedingthemwithfreshgrowthmediumthathasbeenwarmedwillavertpotentialproblems.(Remembertowarmonlytheamountneeded.)Checkandfeedthecellsontheschedulebelow,evenonweekendsandholidays.

1.Changethegrowthmediumthedayafterseeding(toremoveresidualDMSOandunattachedcells),theneveryotherdaythereafterwhileexaminingthemdaily.

NOTE:Achangeofmediumrequiresremovalofthemediumbyaspiratingwithasterilepipetteontheoppositesideoftheflaskfromwherethecellsareattached.Thenwarm,freshmediumisaddeddownthesameside.

2.Successfullyrecoveredcultureswillexhibitthefollowing:

a.Cellswithclearnon-granularcytoplasm.

b.Numerousmitoticfiguresafterday2.

3.Feedthecellsalargervolumeofmediumastheybecomemoreconfluent.Usethistableasaguideline:

IFCELLSARE:	THENFEEDTHEM:
Under25%confluent...	1mlper5cm2
From25-45%confluent...	1.5mlper5cm2
Exceeding45%confluence...	2mlper5cm2

4.Continuefeedingthecellsuntil70-90%confluence.Ifthecellsareallowedtobecomeover-confluentandstayatconfluenceformorethan2days,theycansufferirreversiblecontactinhibitionandmaypopofftheflaskand/orbedifficulttotrypsinize.

InstructionsforCryopreservedCellsSubculturing



SubculturePreparationNOTE:Thefollowinginstructionsarefora25cm2flask.Adjustallvolumesaccordinglyforothersizeflasks.

Preparationforsubculturingthefirstflask:

• Subculturethecellswhentheyare70-90%confluentandcontainmanymitoticfiguresthroughtouttheflask.
• Foreach25cm2ofcellstobesubcultured,allow3mlofClonetics®Trypsin/EDTA(T/E)tothawandcometoroomtemperature.
• Foreach25cm2ofcellstobesubcultured,allow5mlofClonetics®HEPESBufferedSalineSolution(HEPES-BSS)tocometoroomtemperature.
• Foreach25cm2ofcellstobesubcultured,allow3mlofTrypsinNeutralizingSolution(TNS)tocometoroomtemperature.
• Removegrowthmediumfrom4·Cstorageandallowtostartwarmingtoroomtemperature.
• Preparenewculturevessels:a.PreparefivetotenT-75flasks.Thenumberofflasksneededdependsuponconfluenceandtotalyield.Largerflasksmaybeusedtosaveplasticwareandtimespentonsubsequentsubcultures.Smallerflasksreducetheriskoflosingasubstantialpartofyourculture.b.Asbefore,labeleachflaskwiththepassagenumber,strainnumber,celltype,anddate.c.Inasterilefield,carefullyopenthebottleandtransfergrowthmediumtonewculturevesselsbyadding1mlgrowthmediumforevery5cm2surfaceareaoftheflask.Example:15mlgrowthmediumfora75cm2flask.d.Ifnotusingventedcaps,loosencapsofflasks.Placethenewculturevesselsintoa37·Chumidifiedincubatorwith5%CO2andequilibratetheflaskforatleast30minutes.

SubculturingSubcultureoneflaskatatime.Allflasksfollowingthefirstflaskwillbesubculturedfollowinganoptimizationofthisprotocol(explainedlaterinthisprocedure),basedoncalculatedcellcount,cellviability,andseedingdensity.

USEONLYCLONETICS®TRYPSIN/EDTAWHICHHASACONCENTRATIONOF0.025%TRYPSIN/O.O1%EDTA. THECONCENTRATIONOFTRYPSIN/EDTAFROMOTHERSUPPLIERSMAYBEASHIGHAS0.25%TRYPSIN(=10xTHERECOMMENDEDCONCENTRATION)WHICHWILLDETRIMENTALLYEFFECTCLONETICS®CELLS.

Inasterilefield:

• Aspiratethemediumfromoneculturevessel.
• Rinsethecellswith2-3mlofroomtemperatureHEPES-BSS.DON\"Tforgetthisstep.Themediumcontainscomplexproteinsthatneutralizethetrypsin,makingitineffective.
• AspiratetheHEPES-BSSfromtheflask.
• Coverthecellswith3mlofClonetics®T/Esolution.
• Rocktheflasktomakesureallcellscomeintocontactwiththetrypsin.
• Tightenthecapandbeginmonitoringtheflaskunderthemicroscope.
• Continuetoexaminethecelllayermicroscopically.a.Allowthetrypsinizationtocontinueuntil390%ofthecellsareroundedup.NOTE:Roundedupcellsarespherical,havesmoothedgesandarerefractileorshiny.Ifthecellsstillhaveprotrudingnubswhicharestillattachedtotheflask,theyneedmoretimetotrypsinize.Thisentireprocesstakesabout5-6minutes.b.Atthispoint,raptheflaskagainstthepalmofyourhandtoreleasethemajorityofcellsfromtheculturesurface.Ifonlyafewcellsdetach,youmaynothaveletthemtrypsinizelongenough.Wait30secondsandrapagain.Ifcellsstilldonotdetach,waitandrapevery30secondsthereafter.NOTE:Don\"ttrytogetallcellstodetachbyrappingthemseverely.Thisactionmaydamagethecells.
• Aftercellsarereleased,neutralizethetrypsinintheflaskwith3mlofroomtemperatureTNS.Ifthemajorityofcellsdonotdetachwithinsevenminutes,thetrypsiniseithernotwarmenoughornotactiveenoughtoreleasethecells.Harvesttheculturevesselasdescribedabove,andeitherre-trypsinizewithfresh,warmClonetics®Trypsin/EDTASolution(or)rinsewithClonetics®TrypsinNeutralizingSolutionandthenaddfresh,warmmediumtotheculturevesselandreturntoanincubatoruntilfreshtrypsinizationreagentsareavailable.
• Quicklytransferthedetachedcellstoasterile15mlcentrifugetube.
• Rinsetheflaskwithafinal2mlofHEPES-BSStocollectresidualcells,andaddthisrinsetothecentrifugetube.
• Examinetheharvestedflaskunderthemicroscopetomakesuretheharvestwassuccessfulbylookingatthenumberofcellsleftbehind.Thisshouldbelessthan5%.
• Centrifugetheharvestedcellsat220xgfor5minutestopelletthecells.a.Aspiratemostofthesupernatant,exceptfor100-200µl.b.Flickthecryovialwithyourfingertoloosenthepellet.
• Dilutethecellpelletin4-5mlofgrowthmediumandnotethetotalvolumeofthedilutedcellsuspension.Toobtainthebestresultsfromyourcells,youwillassesscellyieldandviabilitywithTrypanBlue.TrypanBlueisadyeusedtohighlightdeadcells.Deadcellstakeupthedyeandappearblue,insteadofrefractileandcolorless.Followthesesteps:
• Countthecellswithahemacytometerorcellcounterandcalculatethetotalnumberofcells.(seeAppendixB).Makeanoteofyourcellyieldforlateruse.Thecellsuspensionshouldcontainbetween250,000to1,000,000cells/mlforgreatestaccuracy.
• Ifnecessary,dilutethesuspensionwithHEPESBufferedSalineSolution(HEPES-BSS)toachievethedesired\"cells/ml\"andre-countthecells.
• AssesscellviabilityusingTrypanBlue(seeAppendixC).
• Usethefollowingequationtodeterminethetotalnumberofviablecells:Total#ofViableCells=Totalcellcountxpercentviability/100Example:1,000,000cellsx60/100=600,000viablecells
• Determinethetotalnumberofflaskstoinoculatebyusingthefollowingequation:Thenumberofflasksneededdependsuponcellyieldandseedingdensity.Largerflasksmaybeusedtosaveplasticwareandtimespentonsubsequentsubcultures.Smallerflasksreducetheriskoflosingasubstantialpartofyourcultureifcontaminationoccurs.NOTE:recommendedseedingdensityof2500cells/cm2forHUVEC,HCAEC,HAEC,andHPAEC;5000cells/cm2forHMVEC-dNeonatal,HMVEC-dAdult,andHMVEC-L.Total#offlaskstoinoculate=Total#ofviablecells/GrowthareaofflaskxRecommendedSeedingDensityExample:600,000viablecells/75cm2x2500cells/cm2=3T-75flasks(roundeddowntonearestwholenumber)
• Usethefollowingequationtocalculatethevolumeofcellsuspensiontoseedintoyourflasks.Seedingvolume=Totalvolumeofdilutedcellsuspension/#offlasksasdeterminedinstep18Example:4.3mlofdilutedcellsuspension/3T-75flasks=1.43mlperT-75flask
• Prepareflasksbylabelingeachflaskwiththepassagenumber,strainnumber,celltypeanddate.
• Carefullyopenthemediumbottleandtransfergrowthmediumtonewculturevesselstobyadding1mlgrowthmediumforevery5cm2surfaceareaoftheflask(1ml/5cm2).Example:15mlgrowthmediumfora75cm2flask.
• Aftermixingthedilutedcellswitha5mlpipettoensureauniformsuspension,dispensethevolumeofsuspensioncalculatedaboveintothepreparedsubcultureflasks.
• Afterdispensingthecells,gentlyrockflasktopromoteevendistribution.
• Ifnotusingventedcaps,loosencapsofflasks.Placethenewculturevesselsintoa37·Chumidifiedincubatorwith5%CO2.

AssessingCellYieldandViabilitySeveralfactorscontributetolowcellcountandlowcellviability.Anexampleofyieldandviabilityassessmentisprovidedinthechartbelow.Todeterminethereasonforlowyield/visibility,followthesesteps:

1.Studythesamplechartbelow.Itisasampleofhighyield,highviability.

a.Notethe\"soliddot\"ontheYaxisorfar,leftsideofthesquare.Itindicateshighyield,ormorethan500,000cellcount.

b.Notethe\"soliddot\"ontheXaxisorbottomlineofthesquare.Itindicateshighviability,ormorethan50%viability.

c.Extendalinefromeachdotasshowninthechart.Thepointwherethelinesintersect(thebold\"X\")islocatedintheHighYield/HighViabilityquadrant.Thus,thesampleisoptional.

2.Now,usingtheblankdiagrambelow,plotyourcellyieldandcellviability.Followthesesteps:

a.Marka(*)ontheYaxistoindicatethetotalcellcountofyourculture.

b.Marka(*)ontheXaxistoindicatethecalculatedpercentviabilityofyourculture.



3.Ifyourresultfallsintoanyquadrantotherthanthe\"HighYield/HighViability\"quadrant,refertoAppendixD,ImprovingCellYieldandViability,beforeproceedingtoyournexttrypsinization.

MaintenanceAfterSubculturingAfter24hours:

• Examinethecellsmicroscopically.Atleast60%ofthecellsshouldhaveattachedtothecultureflask.Somecellswillbelooselyadherent,butmostwillhavespreadoutonthecultureflasksurface.Atthisstage,mostcellswillbesingleorinsmallcolonies.
• Changetheculturemediumtoremoveresidualtrypsinandnon-attachedcells.
• Incubateforanadditional24hours,andre-examinetheculture.a.Atthisstage,thevesselshouldhaveseveralmitoticfiguresindicatingthatthecellshaveresumedactivegrowth.b.Iffewmitoticfiguresareobserved,contactyourClonetics®TechnicalSpecialistforassistance.
• Changethemediumagain48hoursaftertheday1feeding,andevery48hoursthereafterwhileexaminingtheculturedaily.
• Feedwithvolumesasoutlinedinthetableonpage16.
• Passageagainwhenthecellsreach70-90%confluence.(Ifseededatrecommendedseedingdensity,thisshouldtake5-9days).

InstructionsforProliferatingCells

CellPreparation:ProliferatingCellsWiththeproliferatingcultureofepidermalcellsyoureceived,dothefollowing:

• Examinetheculturemicroscopicallyforanysignsofdistressduringshipment(i.e.,detachment,rounding-uporatypicalmorphology).Checktherelativecelldensityandestimate\"%confluency.\"Thecultureshouldbe30-100%confluentuponreceipt.Somecellulardetachmentisnormal.PleasecallyourTechnicalSpecialistimmediatelyifcellslookseverelydistressed.
• Decontaminatetheexternalsurfaceofthecellcultureflaskbywipingwith70%ethanolorisopropanol.
• Incubatethesealedflaskat37·C,5%CO2forthreetofourhourstoequilibratetemperature.
• Warmanappropriateamountofgrowthmedium(seetableonpage16)to37·Cinasterilecontainer.Warmingtheentirebottlecanshortenthelifeofthemedium.Neverwarmmediumunderhotrunningwateroranyotheruncontrolledtemperaturesource.NEVERMICROWAVE!
• Inasterilefield,carefullyopentheendothelialcellcultureflask,removethemediumandreplaceitwiththewarmed,freshmedium.Asepticallyremoveanymediuminsidetheneckorcapareabecauseitcanfacilitatemicrobialcontamination.
• Ifyouareusinganonventedcap,loosenthecap,andreturntheflasktothe37·Chumidifiedincubatorwith5%CO2foratleast24hours.

SubculturingExamineyourculturesmicroscopicallyeveryday.

• Subculturethecellswhentheyreach70-90%confluence.Endothelialculturesshouldhavenumerousmitoticfiguresthroughouttheflask.Cellsshouldbereadytosubculturewithin24to48hours,however,shippingconditionssuchastemperaturefluctuationsmayaffecttheactualtimeatwhichthecellsarereadyforsubculture.
• Seepages18-21fordetailedsubculturinginstructions.

FurtherInformationonCultureofEndotheliaCells

BIBLIOGRAPHY

1)Gimbrone,M.A.(1976)Cultureofvascularendothelium.Prog.Hemost.Thromb.,3:1-29.

2)Grizzle,W.E.,andS.S.Polt.(1988)GuidelinestoAvoidPersonnelContaminationByInfectiveAgentsinResearchLaboratoriesThatUseHumanTissues,J.ofTissueCultureMethods,Vol.11,No.4.

3)Hoshi,H.andW.L.McKeehan.(1986)Isolation,growthrequirements,cloning,prostacyclinproductionandlifespanofhumanadultendothelialcellsinlowserumculturemedium.InVitroCellularandDevelopmentalBiology,22(1),51-56.

4)Voyta,J.C.,Netland,P.A.,Via,D.P.andB.R.Zetter.(1984)Specificlabelingofendothelialcellsusingfluorescentacetylated-lowdensitylipoprotein.J.CellBiology,81(A),99.

5)Maciag,T.,Cerundolo,J.,Ilsley,S.,Kelley,P.R.andR.Forand.(1979)AnEndothelialCellGrowthFactorfromBovineHypothalamus:IdentificationandPartialCharacterization.Proc.Natl.Acad.Sci.,U.S.A.,79:5674-5678.

6)PolymeraseChainReaction(PCR)technologyiscoveredbyU.S.Patents4,683,195,4,683,202,and4,965,188ownedbyHoffmanLa-Roche,Inc.

TECHNICALSERVICE:

BioWhittakerInc.Clonetics®Products9245BrownDeerRoadSanDiego,CA92121(800)852-5663

INTERNATIONALTECHINICALSERVICES

BioWhittaker,Inc.Clonetics®Products8830BiggsFordRoadWalkersville,MD21793(800)898-7025FAX:301-845-2924E-mail:techsup@biowhittaker.com

ORDERS:

BioWhittaker,Inc.Clonetics®Products8830BiggsFordRoadWalkersville,MD21793(800)344-6618

APPENDIXAKeratinocyteMedia

OVERVIEWOFENDOTHELIALCELLMEDIA

500mlBottles(exceptwhereindicated)

CC-3121EBM®	EndothelialCellBasalMedium,serum-free(nogrowthfactors)
CC-3129EBM®-PRF	SameformulationasCC-3121,withoutphenolred
CC-3024EGM®	Acompletemediumina500mlbottlewithattachedBovineBrainExtract(BBE)supplementandsupplementedwiththefollowing:(amountsindicatefinalconcentration,exceptBBE)10ng/mlhEGF(humanrecombinantEpidermalGrowthFactor)1µg/mlHydrocortisone50µg/mlGentamicin,50ng/mlAmphotericin-B3mg/mlBBE(BovineBrainExtract)(CC-4092),2ml2%FBS(FetalBovineSerum)
CC-3124EGM®BulletKit®	Kitwhichcontainsa500mlbottleofEndothelialCellBasalMedium(EBM®,CC-3121)andEGM®SingleQuots®(CC-4133)whichcontainsallofthesupplementslistedbelow,convenientlypackagedassingle-usealiquots.(amountsindicateconcentrationofeachSingleQuot®)10ng/mlhEGF(humanrecombinantEpidermalGrowthFactor)(CC-4017),0.5ml1.0mg/mlHydrocortisone(CC-4035),0.5ml50mg/mlGentamicin,50µg/mlAmphotericin-B(CC-4081),0.5ml3mg/mlBBE(BovineBrainExtract)(CC-4092),2mlFBS(FetalBovineSerum)(CC-4101),10ml
CC-3125EGM®-MVBulletKit®	Kitwhichcontainsa500mlbottleofEndothelialCellBasalMedium(EBM®,CC-3121)andEGM®-MVSingleQuots®(CC-4143)whichcontainsallofthesupplementslistedbelow,convenientlypackagedassingle-usealiquots.(amountsindicateconcentrationofeachSingleQuot®)10ng/mlhEGF(humanrecombinantEpidermalGrowthFactor)(CC-4017)0.5ml1.0mg/mlHydrocortisone(CC-4035)0.5ml50mg/mlGentamicin,50µg/mlAmphotericin-B(CC-4081)0.5ml3mg/mlBBE(BovineBrainExtract)(CC-4092)2mlFBS(FetalBovineSerum)(CC-4102),25ml
CC-3126EGLM™BulletKit®	EGM®LabelingMediumBulletKit®(500ml)thatconsistsofthefollowing:
CC-3159EGLM™-2BulletKit®	EGM®-2LabelingMediumBulletKit®(500ml)thatconsistsofthefollowing:
CC-3127/3128	EBLM™EndothelialCellBasalLabelingMediumorEBLM™-2EndothelialCellBasalLabelingMediumwithoutthefollowingnutrients:Myo-Inositol,Thymidine,Proline,Isoleucine,Leucine,Methionine,andCysteine.
CC-4142/4180	EGLM™SingleQuots®Kit,EGM®labelingSingleQuots®orEGLM™-2SingleQuot®Kit,EGM®-2labelingSingleQuots®consistingofthefollowing:3.5128mg/mlL-Cysteine(CC-4069)5ml16.3963mg/mlL-Isoleucine(CC-4070)2ml7.2064mg/mlMyo-Inositol(CC-4076)0.5ml13.1170mg/mlL-Leucine(CC-4077)5ml7.4605mg/mlL-Methionine(CC-4078)1ml11.5130mg/mlL-Proline(CC-4079)0.5ml0.02422mg/mlThymidine(CC-4080)0.5ml
CC-4133/4176	EGM®SingleQuots®(seeCC-3124)orEGM®-2SingleQuots®(seeCC-3162)
CC-3162EGM®-2BulletKit®	Kitwhichcontainsa500mlbottleofEndothelialCellBasalMedium-2(EBM®-2,CC-3156)andEGM®-2SingleQuots®(CC-4176)whichcontainsallofthesupplementslistedbelow,convenientlypackagedassingle-quots.0.5mlhEGF(humanrecombinantEpidermalGrowthFactor)(CC-4317)0.2mlHydrocortisone(CC-4112)2.0mlhFGF-B(humanFibroblastGrowthFactorBasicwithheparin)(CC-4113)0.5mlVEGF(VascularEndothelialGrowthFactor)(CC-4114)0.5mlR3-IGF-1(HumanRecombiantInsulin-likeGrowthFactor)(CC-4115)0.5mlAscorbicAcid(Vitamin)(CC-4116)0.5mlGentamicin,Amphotericin-B(CC-4381)0.5mlHeparin(CC-4396)10mlsFBS(FetalBovineSerum)(CC-4101)2%
CC-3202EGM®-2MVBulletKit®	Kitwhichcontainsa500mlbottleofEndothelialCellBasalMedium-2(EBM®-2,C3156)andEGM®-2-MVSingleQuots®(CC-4147)whichcontainsallofthesupplementslistedbelow,convenientlypackagedassingle-quots0.5mlhEGF(humanrecombinantEpidermalGrowthFactor)(CC-4317)0.2mlHydrocortisone(CC-4112)2.0mlhFGF-B(humanFibroblastGrowthFactorBasicwithheparin)(CC-4113)0.5mlVEGF(VascularEndothelialGrowthFactor)(CC-4114)0.5mlR3-IGF-1(HumanRecombiantInsulin-likeGrowthFactor)(CC-4115)0.5mlAscorbicAcid(Vitamin)(CC-4116)0.5mlGentamicin,Amphotericin-B(CC-4381)25mlsFBS(FetalBovineSerum)(CC-4102)5%

OurmediaformulationlaboratorycanprovidecustomformulationsofanyClonetics®medium.Minimumorderoftenliters(20bottles).CallyourTechnicalSpecialistformoreinformation.

APPENDIXBCellCountingUsingaHemacytometer

CELLCOUNTINGUSINGAHEMACYTOMETER

Background

ProperuseofahemacytometeriscriticalforobtaininganaccuratecountofcellsandisaprocedureusedbyCloneticstodeterminethesuspensioncountsforallcellstrains.Ahemacytometerconsistsofathickenedglassslideintowhichasmallchamberhasbeencuttoallowfortheintroductionofcellstobecounted.Thefloorofthechamberisdivided(etched)intoninesections;usuallyonlythefourcornersectionsareusedincellcounting(SeeFigure1below).Withacoverslipinplace,eachsquareofthehemacytometerrepresentsatotalvolumeof0.1mm3or10-4cm3.Since1cm3isapproximatelyequivalentto1ml,thecellconcentrationperml(andthetotalnumberofcells)canbedetermined.

Procedure

• Prepareacellsuspensionasinstructedinstep13onpage20.
• Prepareahemacytometerforuse.a.Carefullycleanallsurfacesofthehemacytometerandcoverslip.b.Takecaretoensurethatallsurfacesarecompletelydryusingnon-lintingtissue.c.Centerthecoversliponthehemacytometer.
• Pipetapproximately9microliters(thisvolumewillvaryslightingwiththebrandofhemacytometer)ofthecellsuspensionintooneofthetwocountingchambers.a.Useacleanpipettip.b.Besurethatthesuspensionisthoroughly,butgently,mixedbeforedrawingthesamples.c.FillthechamberslowlyandsteADIly.d.Avoidinjectingbubblesintothechambers.e.Donotoverfillorunderfillthechambers.
• CounttheCells.a.Allowthecellsuspensiontosettleforatleast10seconds.b.Countallofthecellsineachofthefour1mm3cornersquareslabeledAthruDinFigure1onthenextpage.1)DOcountthecellstouchingthetoporleftborders2)DONOTcountthecellstouchingthebottomandrightborders.

HemacytometerReferenceFigure1



• DeterminetheCellCount.a.Calculatethetotalcellscountedinthefourcornersquares.1)Ifthetotalcellcountislessthan100,orifmorethan10%ofthecellscountedappeartobeclustered,carefullyre-mixtheoriginalcellsuspensionandrepeatsteps2through4(above).2)Ifthetotalcellcountisgreaterthan400,dilutethesuspensionsothecountwillbe100-400cells.ThenrepeatSteps2-4(above).NOTE:Ifsatisfactoryresultsarenoteachieved,contactyourTechnicalSpecialistbytelephoning800-852-5663b.Calculatethecellcountusingtheequation:cells/ml=(n)x104,where:n=theaveragecellcountpersquareofthefourcornersquarescounted.Example:Ifthecalculatedaverage(n)ofcellsinthefour1mmcornersquaresofthehemacytometeris30:cells/ml=(n)x104(or)cells/ml=30x10,000=300,000cells/ml.c.Determinethetotalnumberofcellsinthetotalsuspensionvolume.1)Determinethetotalvolumeofthecellsuspension.2)Multiplythevolumeofthecellsuspensionbythe\"cells/ml\"valuecalculatedabove.Example:Iftheinitialsuspensionvolumeis2ml:cells/mlxtotalvolume=300,000cells/mlx2ml=600,000cells.

APPENDIXCAssessmentofCellViabilitywithTrypanBlue

Background

Trypanblueisadyethatenableseasyidentificationofdeadcells.Deadcellstakeupthedyeandappearbluewithunevencellmembranes.Bycontrast,livingcellsrepelthedyeandappearrefractileandcolorless.

UsingTrypanBlue

1.Preparethehemacytometerforuse.

a.Carefullycleanallsurfacesofthehemacytometerandcoverslip.

b.Takecaretoensurethatallsurfacesarecompletelydryusingnon-lintingtissue.

c.Centerthecoversliponthehemacytometer.

2.Transfer50µlof0.4%TrypanBlueintoacleantube.

3.Add50µlofthepreparedcellsuspensionintothetubecontainingthestain.

4.Mixthesolutionthoroughly,butgently.Takecaretoavoidmakingexcessivebubbles.

5.Allowthemixturetositfor2-3minutesaftermixing.(Donotletthecellssitinthedyeformorethanfiveminutesbecauseboththelivinganddeadcellswillbegintotake-upthedyeafterfiveminutes).

6.Pipetapproximately9microlitersoftheTrypanBlue/cellsuspensionmixture(thisvolumewillvarywithbrandofhemacytometer)intooneofthetwocountingchambers.

a.Useacleanpipettip.

b.Besurethatthesuspensionismixedthoroughlybutgentlybeforedrawingthesamples.

c.Fillthechambersslowlyandsteadily.

d.Avoidinjectingbubblesintothechambers.

e.Donotoverfillorunderfillthechambers.

7.DetermineCellViability.

a.Allowthesuspensiontosettleinthechamberforatleast10seconds.

b.Countallofthestainedcellsineachofthefourcornersquaresofthehemacytometer.

c.Separatelycountalloftheunstainedcellsinthesamesquares.

d.Calculatethecellviabilityusingtheequation:%CellViability=numberofunstained(living)cellsx100/Totalcellscounted(stained+unstained)

Example:Ifatotalof300cells(stained+unstained)arecountedand200areidentifiedaslivingcells(unstained),thentheviabilityiscalculatedas:

%Cellviability=200/300x100%=67%

APPENDIXDImprovingCellYieldandViability

Background

Severalfactors,oracombinationoffactors,contributetolowcellcountandlowcellviability.Ifcellyieldorviabilityifunsatisfactory,usethefollowinginformationtoincreasethesuccessrateoffuturecultures.

ImprovingCellYield

Ifyourcellyieldislow(lessthan50%),determinethecause(s)andpossiblesolution(s)usingthetablebelow.Thensubcultureonemoreflaskapplyingtheappropriatesolution(s).

ImprovingCellViability

Ifyourcellviabilityislow(lessthan50%),determinethepossiblecause(s)andsolution(s)usingthetablebelow.Thensubcultureonemoreflaskapplyingtheappropriatesolution(s).

LowYield(CellCount)
CONDITION	POSSIBLECAUSES	SOLUTIONS
Majorityofcellsdidnotdetach.	InactiveorcoldTrypsin/EDTA. ImproperstorageofTrypsin/EDTA. ExposuretimetoTrypsin/EDTAwastooshort. Trypsin/EDTAisneutralized. Vesselwasnot\"rapped\"firmlyenoughduringtrypsinization.	UseTrypsin/EDTAatroomtemperature. Storeat-20·Cuntilreadyforuse;thawandallowittocometoroomtemperaturebrieflybeforesubculturing. ExposuretimetoTrypsin/EDTAisusually5-6minutes. BesuretorinsetheculturecompletelywithHEPES-BSSbeforetrypsinization. Useamoderateamountofforcewhenrapping(seepage19).
Lowyield,95%ofthecellsdetachedbuttheyieldwaslow.	Culturewasunderconfluentattrypsinization.	Besuretotrypsinizeat70-90%confluencewithnumerousmitoticfiguresthroughouttheflask.

Onceyouhavedeterminedhowtoachievehighyieldandhighviability,subculturetheremainingflasks.

APPENDIXEGrowthAreaofCommonPlasticware

	LowViability(<50%viable)
CONDITION	POSSIBLECAUSES	SOLUTIONS
Trypsin/EDTAdamagedthecells	Trypsin/EDTAusedatthewrongconcentration. ExposuretimeofthecellstoTrypsin/EDTAwastoolong. Trypsin/EDTAwasusedaboveroomtemperature.Trypsinbecomesmoreactiveattemperaturesaboveroomtemperature. Failedtoneutralizethetrypsin.Prolongedexposuretotrypsinwilldamagecells. Vesselwas\"rapped\"toofirmly(seepage19)duringtrypsinization.Rappingtoohardtoreleasecellscausescellmembranedamage.	Cloneticsrecommendsatrypsinconcentrationof0.025%andanEDTAconcentrationof0.01% Donottrypsinizelongerthan7minutes. DONOTUSEEVENMILDLYHEATEDTrypsin/EDTA. NeutralizetheT/EwithTNStoeliminatecelldamageduetotrypsin. Usemoderateforcewhenrapping.
Culturevesselwastooconfluent;wascompletelycoveredwithcells.	Culturewastooconfluentattrypsinization.	Besuretotrypsinizeat70-90%confluencewithaboutfivemitoticfiguresperfieldofview.
Cellgrowthslowedbefore80%confluenceandcellslookdullandnonrefractile.	Themostprobablecauseisfailuretoincreasethevolumeofmediumusedasthecellconfluencyincreased.Thecellsbecomemildlystarvedandarenotabletorecoveraftertrypsinization.	Changemediumandincreasevolumeasrecommended.Pleaseobserveallguidelines.

Flasks	Effective GrowthArea	InitialNumberofCellstoSeedat5000cells/cm2 HMVEC	InitialNumberofCellstoSeedat2500cells/cm2 ALLOTHERS	ExpectedNumber ofEndothelialCellsattimeofHarvest
T-25	25cm2	125,000	62,500	700,000
T-75	75cm2	375,000	187,500	2,100,000
T-150	150cm2	750,000	375,500	4,205,600
Dishes	Effective GrowthArea	InitialNumberofCellstoSeedat5000cells/cm2 HMVEC	InitialNumberofCellstoSeedat2500cells/cm2 ALLOTHERS	ExpectedNumber ofEndothelialCellsattimeofHarvest
35mm	9.6cm2	48,000	24,000	268,800
60mm	28.0cm2	140,000	70,000	784,000
100mm	78.5cm2	392,500	196,250	2,198,000
150mm	176.6cm2	883,000	441,500	4,944,800
MultiwellPlates	EffectiveGrowthAreaPerwell	InitialNumberofCellstoSeedat10,000cells/cm2 HMVEC	InitialNumberofCellstoSeedat10,000cells/cm2 ALLOTHERS	ExpectedNumber ofEndothelialCellsattimeofHarvest
6well	9.60cm2	96,000	96,000	268,800
12well	3.80cm2	38,000	38,000	106,400
24well	2.00cm2	20,000	20,000	56,000
48well	.75cm2	7,500	7,500	21,000
96well	.32cm2	3,200	3,200	8,960

APPENDIXFSeedingInto96-WellPlates

Overview

Acultureflaskofnormalhumancellsisharvestedbytrypsinizationandsubsequenttrypsininhibitortreatment.Thecellsarecentrifuged,resuspendedinqrowthmediumandcounted.Thedesirednumberofcellsisthenaddedtowellsofsterile96-welltissuecultureplates.Theplatesareincubatedina37·C,5%CO2humidifiedincubatorforonetothreedaystoallowforcelladherenceandgrowth.Seedingdensitieswillvarysomewhatwithyourexperimentalrequirements.WerecommendadensityforEndothelialCellsof10,000cells/cm2forallmultiwellplates.

RequiredMaterials:

• T-25flaskofproliferatingnormalhumancellsbetween70%and90%confluence.
• 96-well,flat-bottom,tissuecultureplates
• 37·Chumidifiedincubatorwith5%CO2/95%air
• Laminarflowhoodorothersterileenvironment
• Adjustablemultichannelpipetter(8-or12-channel)orrepeatingpipetter
• Sterilereservoir(s)forusewithmultichannelpipetter

Procedure

• Followthestepsonpages18-21forsubculturepreparationandsubculturing.Thenfollowsteps2-4below.
• Sincethecells/mlcalculationcomputedonp.20is\"perml\",onemustincreasethecellconcentrationby4timesbeforeseeding96wellplates(toaccommodatethe1:4dilutionwhenadding250ulofsuspendedcellsperwell).Whenmakingthecellsuspension,adjustthecellconcentrationwithgrowthmedium.
• Transferthedilutedcellsuspensiontoasterilereservoir.Usingamultichannel(8-or12-channel)pipetterequippedwithsterilepipettetips,add250µlofthedilutedcellsuspensiontoeachwellofthelabeled96-well,flatbottom,tissuecultureplates(s).RESUSPENDTHECELLSUSPENSIONOFTENDURINGTHESEEDINGPROCEDURETOENSUREAUNIFORMNUMBERANDDISTRIBUTIONOFCELLSINTOEACHWELLBYPIPETTINGUPANDDOWNAFEWTIMESBETWEENEVERYOTHERDISPENSING.
• Coverandincubatetheplatesfor1to3daysat37·C/5%CO2.(Incubationperiodsexceeding3daysaregenerallynotrecommendedbecauseofevaporationofmediumfromtheedgewellsoftheplate).NOTE:Beforeusingthe96-wellplatecultureinabioassay,examinethemmicroscopicallyforthepresenceofmitoticfiguresasaconfirmationthatthecellshaveresumedactivegrowth.(Doesnotapplyforallend-userplate.)