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:

1. FORRESEARCHONLY
2. NOTapprovedforhumanorveterinaryuse,forapplicationtohumansoranimals,orforinvitrodiagnosticprocedures.

MaterialsNotProvidedClonetics®NormalHumanCellSystemsdonotincludeplasticware,glasswareorotherlaboratoryequipmentusedinacellculturelaboratory.Individualcomponentsareavailableseparately.

ProductWarrantyCULTURESHAVEAFINITELifespanINVITRO.BioWhittakerwarrantsClonetics®productsonlyifClonetics®mediaandreagentsareused.

1. Cryopreservedculturesareassuredforexperimentaluseforfifteenpopulationdoublings.
2. Proliferatingculturesareassuredforexperimentalusefortenpopulationdoublings.
3. Additionalpopulationdoublingsandsubculturesarepossible,butgrowthrate,BIOLOGicalresponsivenessandfunctionmaydeterioratewithsubsequentpassage.
4. Endothelialcellscanbecomeirreversiblycontact-inhibitedifmaintainedatconfluenceformorethantwodays.Toavoidthelossofyourcellsandforfeitureofyourwarranty,werecommendthatyousubculturecellsbeforetheyreach90%confluence.

CellIsolationClonetics®endothelialcellculturesareestablishedatBioWhittaker"scellculturefacilityfromnormalhumantissue.Thecelltypeandthepassageinwhichtheyareshippedareoutlinedbelow:

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.

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:

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

SafetyPrecautionsBioWhittakerstressestheimportanceofthefollowingprecautions:

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:

1. DecontaminatetheexternalsurfacesoftheSingleQuot®cryovialsandtheEBM®bottlewithethanolorisopropanol.
2. AsepticallyopeneachcryovialandaddtheentireamounttotheEBM®withapipette.
3. Rinseeachcryovialwiththemedium.Itmaynotbepossibletorecovertheentirevolumelistedforeachcryovial.Smalllosses,evenupto10%,shouldnotaffectthecellgrowthcharacteristicsofthesupplementedmedium.
4. Transferthelabelprovidedwitheachkittothebasalmediumbottlebeingsupplemented.Useittorecordthedateandamountofeachsupplementadded.Werecommendthatyouplacethecompletedlabeloverthebasalmediumlabeltoavoidconfusionorpossibledoublesupplementation.
5. 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.

Aftertheflaskshaveequilibratedfor30minutes:

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

SeedingAftercellsarethawed:

NOTE:DonotdispensetheentirecontentsofthecryovialintooneT-25flask!!

1. Removethecap,beingcarefulnottotouchtheinteriorthreadswithyoufingers.
2. Usingamicro-pipettewitha1000µltipsetto800µl,putthetipintothecryovialandresUSPendthecells,withagentle,slowandsteadyupanddownpipettingmotionnomorethanfivetimes.DONOTresuspendquickly,andkeepthetipnearthebottomtoavoidmakingbubbles.
3. Dispenseandequalamountofcellsintotheflaskssetupearlier.IffourT-25flaskswereprepared,setmicropipetterto250µlanddispense.IfeightT-25flaskswereprepared,setmicropipetterto125µlanddispense.
4. Replacethecaporcover,andgentlyrockthevesselstoevenlydistributethecells.Loosencapsifnecessarytopermitgasexchange(see"SetUp,"stepnumber4).
5. 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:

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

InstructionsforCryopreservedCellsSubculturing

SubculturePreparationNOTE:Thefollowinginstructionsarefora25cm2flask.Adjustallvolumesaccordinglyforothersizeflasks.

Preparationforsubculturingthefirstflask:

1. Subculturethecellswhentheyare70-90%confluentandcontainmanymitoticfiguresthroughtouttheflask.
2. Foreach25cm2ofcellstobesubcultured,allow3mlofClonetics®Trypsin/EDTA(T/E)tothawandcometoroomtemperature.
3. Foreach25cm2ofcellstobesubcultured,allow5mlofClonetics®HEPESBufferedSalineSolution(HEPES-BSS)tocometoroomtemperature.
4. Foreach25cm2ofcellstobesubcultured,allow3mlofTrypsinNeutralizingSolution(TNS)tocometoroomtemperature.
5. Removegrowthmediumfrom4·Cstorageandallowtostartwarmingtoroomtemperature.
6. 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.

Inasterilefield:

1. Aspiratethemediumfromoneculturevessel.
2. Rinsethecellswith2-3mlofroomtemperatureHEPES-BSS.DON"Tforgetthisstep.Themediumcontainscomplexproteinsthatneutralizethetrypsin,makingitineffective.
3. AspiratetheHEPES-BSSfromtheflask.
4. Coverthecellswith3mlofClonetics®T/Esolution.
5. Rocktheflasktomakesureallcellscomeintocontactwiththetrypsin.
6. Tightenthecapandbeginmonitoringtheflaskunderthemicroscope.
7. Continuetoexaminethecelllayermicroscopically.a.Allowthetrypsinizationtocontinueuntil390%ofthecellsareroundedup.NOTE:Roundedupcellsarespherical,havesmoothedgesandarerefractileorshiny.Ifthecellsstillhaveprotrudingnubswhicharestillattachedtotheflask,theyneedmoretimetotrypsinize.Thisentireprocesstakesabout5-6minutes.b.Atthispoint,raptheflaskagainstthepalmofyourhandtoreleasethemajorityofcellsfromtheculturesurface.Ifonlyafewcellsdetach,youmaynothaveletthemtrypsinizelongenough.Wait30secondsandrapagain.Ifcellsstilldonotdetach,waitandrapevery30secondsthereafter.NOTE:Don"ttrytogetallcellstodetachbyrappingthemseverely.Thisactionmaydamagethecells.
8. Aftercellsarereleased,neutralizethetrypsinintheflaskwith3mlofroomtemperatureTNS.Ifthemajorityofcellsdonotdetachwithinsevenminutes,thetrypsiniseithernotwarmenoughornotactiveenoughtoreleasethecells.Harvesttheculturevesselasdescribedabove,andeitherre-trypsinizewithfresh,warmClonetics®Trypsin/EDTASolution(or)rinsewithClonetics®TrypsinNeutralizingSolutionandthenaddfresh,warmmediumtotheculturevesselandreturntoanincubatoruntilfreshtrypsinizationreagentsareavailable.
9. Quicklytransferthedetachedcellstoasterile15mlcentrifugetube.
10. Rinsetheflaskwithafinal2mlofHEPES-BSStocollectresidualcells,andaddthisrinsetothecentrifugetube.
11. Examinetheharvestedflaskunderthemicroscopetomakesuretheharvestwassuccessfulbylookingatthenumberofcellsleftbehind.Thisshouldbelessthan5%.
12. Centrifugetheharvestedcellsat220xgfor5minutestopelletthecells.a.Aspiratemostofthesupernatant,exceptfor100-200µl.b.Flickthecryovialwithyourfingertoloosenthepellet.
13. Dilutethecellpelletin4-5mlofgrowthmediumandnotethetotalvolumeofthedilutedcellsuspension.Toobtainthebestresultsfromyourcells,youwillassesscellyieldandviabilitywithTrypanBlue.TrypanBlueisadyeusedtohighlightdeadcells.Deadcellstakeupthedyeandappearblue,insteadofrefractileandcolorless.Followthesesteps:
14. Countthecellswithahemacytometerorcellcounterandcalculatethetotalnumberofcells.(seeAppendixB).Makeanoteofyourcellyieldforlateruse.Thecellsuspensionshouldcontainbetween250,000to1,000,000cells/mlforgreatestaccuracy.
15. Ifnecessary,dilutethesuspensionwithHEPESBufferedSalineSolution(HEPES-BSS)toachievethedesired"cells/ml"andre-countthecells.
16. AssesscellviabilityusingTrypanBlue(seeAppendixC).
17. Usethefollowingequationtodeterminethetotalnumberofviablecells:Total#ofViableCells=Totalcellcountxpercentviability/100Example:1,000,000cellsx60/100=600,000viablecells
18. 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)
19. Usethefollowingequationtocalculatethevolumeofcellsuspensiontoseedintoyourflasks.Seedingvolume=Totalvolumeofdilutedcellsuspension/#offlasksasdeterminedinstep18Example:4.3mlofdilutedcellsuspension/3T-75flasks=1.43mlperT-75flask
20. Prepareflasksbylabelingeachflaskwiththepassagenumber,strainnumber,celltypeanddate.
21. Carefullyopenthemediumbottleandtransfergrowthmediumtonewculturevesselstobyadding1mlgrowthmediumforevery5cm2surfaceareaoftheflask(1ml/5cm2).Example:15mlgrowthmediumfora75cm2flask.
22. Aftermixingthedilutedcellswitha5mlpipettoensureauniformsuspension,dispensethevolumeofsuspensioncalculatedaboveintothepreparedsubcultureflasks.
23. Afterdispensingthecells,gentlyrockflasktopromoteevendistribution.
24. 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:

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

InstructionsforProliferatingCells

CellPreparation:ProliferatingCellsWiththeproliferatingcultureofepidermalcellsyoureceived,dothefollowing:

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

SubculturingExamineyourculturesmicroscopicallyeveryday.

1. Subculturethecellswhentheyreach70-90%confluence.Endothelialculturesshouldhavenumerousmitoticfiguresthroughouttheflask.Cellsshouldbereadytosubculturewithin24to48hours,however,shippingconditionssuchastemperaturefluctuationsmayaffecttheactualtimeatwhichthecellsarereadyforsubculture.
2. 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)

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

APPENDIXBCellCountingUsingaHemacytometer

CELLCOUNTINGUSINGAHEMACYTOMETER

Background

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

Procedure

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

HemacytometerReferenceFigure1

5. 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).

Onceyouhavedeterminedhowtoachievehighyieldandhighviability,subculturetheremainingflasks.

APPENDIXEGrowthAreaofCommonPlasticware

APPENDIXFSeedingInto96-WellPlates

Overview

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

RequiredMaterials:

1. T-25flaskofproliferatingnormalhumancellsbetween70%and90%confluence.
2. 96-well,flat-bottom,tissuecultureplates
3. 37·Chumidifiedincubatorwith5%CO2/95%air
4. Laminarflowhoodorothersterileenvironment
5. Adjustablemultichannelpipetter(8-or12-channel)orrepeatingpipetter
6. Sterilereservoir(s)forusewithmultichannelpipetter

Procedure

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