THESEINSTRUCTIONSAPPLYTOORDERSCONTAININGTHEFOLLOWINGCELLPRODUCTS

CryopreservedCells(Singledonor)

ProliferatingCells

ProliferatingCellsinPreseeded®96-wellPlates

1. Checkallcontainersforleakageorbreakage.Fibroblastsareavailablein6,12,24and48wellplates,T-150andT-225flasks.PleasecallyourTechnicalSpecialistfordetailsandalsoprices.
2. Forcryopreservedcells-Ifthereisdryiceleftinthepackage,placecryopreservedcellcryovialsimmediatelyintoliquidnitrogen.Ifnodryiceisleftinthepackage,thawandusethemimmediately.Forproliferatingcells-Swabdowntheflaskofproliferatingcellswith70%ethanolorisopropanol,thenplacetheflaskin37·C,5%CO2,humidifiedincubatorandallowtoequilibrateforthreetofourhours.Aftercellshaveequilibrated,removeshippingmediumfromtheflaskfollowinginstructionsonpage20.
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.

*AA-1005*AA-1005-1Rev.04/98

FibroblastCellSystem

1.NormalHumanDermalFibroblastsandNormalHumanLungFibroblastsfromsingledonors,aseither:

Theproliferatingculturesareshippedinflasksorplatesfilledwithmedium.Thecellsshouldbebetween30and100%confluentuponarrival.ACertificateofAnalysisisprovidedwitheachcellstrainandindicatesQCperformanceresultsanddonorinformation.

Thecryopreservedculturesareshippedinascrewcapcryovialcontainingapproximately500,000cells.ACertificateofAnalysisisprovidedwitheachcellstrainandindicatesdateofcryopreservation,QCperformanceresults,donorinformationandthenumberofcellscontainedinthecryovial.

FibroblastGrowthMediumBulletKit®(FGM®BulletKit®)(CC-3130),whichcontainsa500mlbottleofFibroblastBasalMedium(FBM®)andallthesupplementslistedbelow,convenientlypackagedassingle-usealiquotscalledSingleQuots®(amountsindicateconcentrationofeachSingleQuot®)

1mg/mlhFGF(humanrecombinantFibroblastGrowthFactor)(CC-4065)0.5ml5mg/mlInsulin(CC-4021)0.5ml50mg/mlGentamicin,50mg/mlAmphotericin-B(CC-4081)0.5ml

FibroblastGrowthMedium-2BulletKit®(FGM®-2BulletKit®)(CC-3132),whichcontainsa500mlbottleofFibroblastBasalMedium(FBM®)andallthesupplementslistedbelow,convenientlypackagedassingle-usealiquotscalledSingleQuots®(amountsindicateconcentrationofeachSingleQuot®)

1mg/mlhFGF(humanrecombinantFibroblastGrowthFactor)(CC-4065)0.5ml5mg/mlInsulin(CC-4021)0.5ml50mg/mlGentamicin,50mg/mlAmphotericin-B(CC-4081)0.5ml10mlFBS(FetalBovineSerum)(CC-4101)

3.ReagentPackTM(CC-5034)containsone100mlbottleofeachofthefollowingsubculturereagents:

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

NOTE:IfyouuseadifferentClonetics®medium,seeAppendixA.

GeneralInformation

ProductApplicationsClonetics®NormalHumanNHDFandNHLFare:

1. FORRESEARCHUSEONLY.
2. NOTapprovedforhumanorveterinaryuse,forapplicationtohumansoranimals,orforinvitrodiagnosticprocedures.

MaterialsNotProvidedFibroblastCellSystemsdonotcontainplasticware,glasswareorotherlaboratoryequipmentusedinacellculturelaboratory.Individualcomponentsareavailableseparately.

ProductWarrantyCULTURESHAVEAFINITELifespanINVITRO.BioWhittakerwarrantsitsClonetics®cellsinthefollowingmanneronlyifClonetics®mediaandreagentsareused.

1. NHDFandNHLFcryopreservedculturesareassuredforexperimentaluseforfifteenpopulationdoublings.
2. NHDFandNHLFproliferatingculturesareassuredforexperimentalusefortenpopulationdoublings.
3. Additionalpopulationdoublingsandsubculturesarepossible,butgrowthrate,BIOLOGicalresponsivenessandfunctiondeterioratewithsubsequentpassage.
4. NHDFandNHLFcanbecomeirreversiblycontact-inhibitedifmaintainedatconfluenceformorethantwodays.Toavoidthelossofyourcellsandforfeitureofyourwarranty,werecommendthatyousubculturecellsbeforetheyreach90%confluence.

CellIsolationFibroblastculturesareestablishedatBioWhittaker"scellculturefacilityfromnormalhumantissue.

1. NHDFareisolatedaccordingtoproprietaryprocedures,incubateduntiltheyapproach90%confluence,harvestedandcryopreservedasfrozenprimaries.
2. NHLFareisolatedaccordingtoproprietaryprocedures,incubateduntiltheyreach90%confluence,subculturedonceandcryopreservedasfrozensecondarycultures.
3. NHDFandNHLFarecryopreservedinFGM®(or)FGM®-2supplementedwith10%v/vfetalbovineserumand10%v/vdimethylsulfoxideasacryopreservationsolutiontoimprovecellviABIlityandseedingefficiencyuponthawing.

QualityControlNHDFandNHLFareculturedwithoutantimicrobialagentsandassayedtoensuretheabsenceofmicrobialcontaminationaftercryopreservation.

1. AllcellstrainstestnegativebyPCR(1)forHIV-1,hepatitis-Bandhepatitis-C.
2. Afterrecoveryfromliquidnitrogen,cellsaretestedforviability,growthrate,morphology,seedingefficiency,proliferativecapacity,mycoplasma,yeast,fungusandbacteria.EachculturemeetsClonetics®productspecificationsforproliferativecapacity,(i.e.,15cumulativepopulationdoublingsafterthaw).
3. AdditionalTesting:

   CellType	VonWillebrandFactor	SmoothMuscleAlphaActin	Cytokeratins18&19
   NHLF	Negative	Negative	Negative

4. Beforeshipping,allbasalmedia,growthfactorsandcellculturereagentsaretestedforsterilityandcellcultureperformance.

SubcultureReagentStorage1.Subculturereagentsarestoredat-20·CuntilshippedfromBioWhittaker"sDistributionCenters.

2.Subculturereagentsmaythawduringtransport.Theymayberefrozenonce.

3.Subculturereagentscanbestoredat-20·Cforuptooneyearafterthawingonceandrefreezing.

4.TokeepTrypsin/EDTAfreshandactiveafterthawing,youshouldaliquotitintofive20mlsterilecentrifugetubesandrefreezeat-20·C.Trypsin/EDTAmaybestoredfrozenuptooneyear.

5.WerecommendthatHEPES-BSSandtheTrypsinNeutralizingSolution,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,alwaysaddallliquidsbypipetingthemdowntheoppositesidefromwherethecellsareattached.

SafetyPrecautionsBioWhittakerstressestheimportanceofthefollowingprecautions:

Theflowchartonthefollowingpageillustratesthecultureprocess.Itisfollowedbythestep-by-stepinstructions...

InstructionforCryopreservedCells

BeforeYouBeginPerformthefollowingstepsbeforeyoubeginmediumorcellpreparation:

*Maynotbenecessaryforallend-userassays.

MediumPreparationPerformthestepsbelowinasterilefield."Sterilefield"isdefinedabove.

FortheFGM®andFGM®-2BulletKits®,dothefollowing:

1. DecontaminatetheexternalsurfacesoftheSingleQuot®cryovialsandthebasalmediumbottlewithethanolorisopropanol.
2. Asepticallyopeneachcryovialandaddtheentireamounttothebasalmediumwithapipette.
3. Rinseeachcryovialwiththemedium.Itmaynotbepossibletorecovertheentirevolumelistedforeachcryovial.Smalllosses,evenupto10%,shouldnotaffectthecellgrowthcharacteristicsofthesupplementedmedium.
4. Transferthelabelprovidedwitheachkittothebasalmediumbottlebeingsupplemented.UseittorecordthedateandamountofeachSingleQuot®added.(Werecommendthatyouplacethecompletedlabeloverthebasalmediumlabeltoavoidconfusionorpossibledoublesupplementation.)
5. Recordthenewexpirationdateonthelabelbasedontheshelflife(seetableonpage6).ThissupplementedmediumwillnowbereferredtoaseitherFGM®orFGM®-2.

NOTE:Ifthereisconcernthatsterilitywascompromisedduringthesupplementationprocess,theentirenewlypreparedgrowthmediummayberefilteredtoassuresterility.Ifyourefilter,useasterile0.2mm,lowproteinbindingfilter.Routinerefiltrationisnotrecommended.

SetUpTosetupvesselsforNHDFandNHLFcomingoutofcryopreservation,dothefollowing:

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

NOTE:Flasksandmultiwellplatesaremosteffectivetosubculturethesecells.

Usethefollowingcalculationstodeterminethenumberofvesselstobesetupfortherecommendedseedingdensityof3500cells/cm2forNHDFand2500cells/cm2forNHLF.

No.ofcellsavailable/3500cells/cm2=max.surfaceareathatcanbeplated

Max.surfaceareathatcanbeplated/Effectivegrowthareaofflask=max.no.offlasksthatcanbesetup

Example:Acryovialwith520,000cells

520,000/3500=148cm2

IfyouuseaT-25withaneffectivegrowthareaof25cm2

148cm2/25cm2=5flasks(roundeddowntonearestwholenumberofflasks)

AtypicalcryovialcanbeplatedintoatleastfiveT-25flasks.TheadvantageofsettingupfiveT-25flasksfromtheinitialcryovial,asopposedtolargerflasks,isthatitreducestheriskoflosinglargenumbersofcells.Thatis,ifyouexperiencedifficultytrypsinizingthefirstT-25flask,therearemoreT-25flaskstouse.

2.Labeleachflaskwiththepassagenumber,celltype,strainnumber,anddate.

Example:Forfirstpassageoutofcryopreservationforlungfibroblastswithstrainnumber5099,thelabelmightappearasfollows:

3·NHLF5099;12/30/96

3.Inasterilefield,carefullyopenthesupplementedbottleofgrowthmedium,andasepticallytransferthemediumtonewculturevesselsbyadding1mlgrowthmediumforevery5cm2surfaceareaoftheflask.

Example:5mlgrowthmediumfora25cm2flaskor60mmplate.

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

ThawingNOTE:Ifmorethanonecryovialistobethawed,thawonecryovialatatimeandkeepothercryovialsinliquidnitrogenuntilreadyforuse.

Aftertheflaskshaveequilibratedfor30minutes:

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

SeedingAftercellsarethawed:

NOTE:DonotdispensetheentirecontentsofthecryovialintooneT-25flask!!

1. Removethecap,beingcarefulnottotouchtheinteriorthreadswithyourfingers.
2. Usingamicro-pipettewitha1000mltipsetto800ml,putthetipintothecryovialandresUSPendthecells,withagentle,slowandsteadyupanddownpipettingmotionnomorethanfivetimes.DONOTresuspendquickly,andkeepthetipnearthebottomtoavoidmakingbubbles.
3. Dispenseanequalamountofcellsintotheflasks.IffiveT-25flaskswereprepared,setmicropipetterto200mlanddispense.
4. Replacethecaporcover,andgentlyrockthevesselstoevenlydistributethecells.Loosencapsifnecessarytopermitgasexchange(see"SetUp,"stepnumber4,pg.11).
5. Returntheculturevesselsto37·C,5%CO2incubator.Laythemflatontheshelf,providingthelargestsurfaceforcellstoattach.Thecellswillanchortothebottomsurfaceoftheflask.

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

1.Changethegrowthmediumthedayafterseeding(toremoveresidualDMSOandunattachedcells),theneveryotherdaythereafterwhileexaminingthemdaily.

NOTE:Achangeofmediumrequiresremovalofthemediumbyaspiratingwithasterilepipetteontheoppositesideoftheflaskfromwherethecellsareattached.Thenwarm,freshmediumisaddeddownthatsameside.

2.Successfullyrecoveredcultureswillexhibitthefollowing:

a.Cellswithclearnon-granularcytoplasm.

b.Numerousmitoticfiguresafterday2.

3.Feedthecellsalargervolumeofmediumastheybecomemoreconfluent.Usethistableasaguideline:

4.Continuefeedingthecellsuntil70-90%confluence.Ifthecellsareallowedtobecomeover-confluenttheywillsuffercontactinhibitionandwillpopofftheflaskand/orbedifficulttotrypsinize.

OverviewofSubculturePreparation

SubculturePreparationNOTE:Thefollowinginstructionsarefora25cm2flask.AdjustallvolumesaccordinglyPreparationforothersizeflasks.

Preparationforsubculturingthefirstflask:

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

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

Inasterilefield:

1. Aspiratethemediumfromoneculturevessel.
2. ForNHLFandNHDFgrowninFGM®-2,rinsethecellswith2-3mlofroomtemperatureHEPES-BSS.DON"Tforgetthisstep.Themediumcontainscomplexproteinsthatneutralizethetrypsin,makingitineffective.ForNHDFgrowninFGM®,skipsteps2and3.
3. AspiratetheHEPES-BSSfromtheflask.
4. Coverthecellswith3mlofClonetics®T/Esolution.Usecold(4·C)T/EsolutionforNHDFgrowninFGM®.
5. Rocktheflasktomakesureallcellscomeintocontactwiththetrypsin.
6. Tightenthecapandbeginmonitoringtheflaskunderthemicroscope.
7. Continuetoexaminethecelllayermicroscopically.a.Allowthetrypsinizationtocontinueuntil390%ofthecellsareroundedup.NOTE:Roundedupcellsarespherical,havesmoothedgesandarerefractileorshiny.Ifthecellsstillhaveprotrudingnubswhicharestillattachedtotheflask,theyneedmoretimetotrypsinize.Thisentireprocesstakesabout1to2minutes,underoptimalconditions.b.Atthispoint,raptheflaskagainstthepalmofyourhandtoreleasethemajorityofcellsfromtheculturesurface.Ifonlyafewcellsdetach,youmaynothaveletthemtrypsinizelongenough.Wait30secondsandrapagain.Ifcellsstilldonotdetach,waitandrapevery30secondsthereafter.NOTE:Don"ttrytogetallcellstodetachbyrappingthemseverely.Thisactionmaydamagethecells.
8. Aftercellsarereleased,neutralizethetrypsinintheflaskwith3mlofroomtemperatureTNS.Ifthemajorityofcellsdonotdetachwithinfourminutes,thetrypsiniseithernotwarmenoughornotactiveenoughtoreleasethecells.Harvesttheculturevesselasdescribedabove,andeitherre-trypsinizewithfresh,warmClonetics®Trypsin/EDTASolution(or)rinsewithClonetics®TrypsinNeutralizingSolutionandthenaddfresh,warmgrowthmediumtotheculturevesselandreturntoanincubatoruntilfreshtrypsinizationreagentsareavailable.
9. Quicklytransferthedetachedcellstoasterile15mlcentrifugetube.
10. Rinsetheflaskwithafinal2mlofHEPES-BSStocollectresidualcells,andaddthisrinsetothecentrifugetube.
11. Examinetheharvestedflaskunderthemicroscopetomakesuretheharvestwassuccessfulbylookingatthenumberofcellsleftbehind.Thisshouldbelessthan5%.
12. Centrifugetheharvestedcellsat220xgfor5minutestopelletthecells.a.Aspiratemostofthesupernatant,exceptfor100-200ml.b.Flickthecryovialwithyourfingertoloosenthepellet.
13. Dilutethecellsin4-5mlofgrowthmediumandnotethetotalvolumeofthedilutedcellsuspension.
14. Duringtheseprocedureskeepthecellsiniceuntiltheyareplated.Toobtainthebestresultsfromyourcells,youwillassesscellyieldandviabilitywithTrypanBlue.TrypanBlueisadyeusedtohighlightdeadcells.Deadcellstakeupthedyeandappearblue,insteadofrefractileandcolorless.Evaluateonbright-fieldmicroscope.Followthesesteps:
15. Countthecellswithahemacytometerorcellcounterandcalculatethetotalnumberofcells.(SeeAppendixB.)Makeanoteofyourcellyieldforlateruse.Thecellsuspensionshouldcontainbetween250,000to1,000,000cells/mlforgreatestaccuracy.
16. Ifnecessary,dilutethesuspensionwithHEPESBufferedSalineSolution(HEPES-BSS)toachievethedesired"cells/ml"andre-countthecells.
17. AssesscellviabilityusingTrypanBlue(seeAppendixC).
18. Usethefollowingequationtodeterminethetotalnumberofviablecells:Total#ofViableCells=Totalcellcountxpercentviability/100Example:1,000,000cellsx60/100=600,000viablecells
19. Determinethetotalnumberofflaskstoinoculatebyusingthefollowingequation.Thenumberofflasksneededdependsuponcellyieldandseedingdensity.Largerflasksmaybeusedtosaveplasticwareandtimespentonsubsequentsubcultures.Smallerflasksreducetheriskoflosingasubstantialpartofyourcultureifcontaminationoccurs.NOTE:Recommendedseedingdensityis3500cells/cm2forNHDFand2500cells/cm2forNHLF.Total#offlaskstoinoculate=Total#ofviablecells/GrowthAreaofFlaskxRecommendedSeedingDensityExample:600,000viablecells/75cm2x3500cells/cm2=2T-75flasks(roundeddowntonearestwholenumber)
20. Usethefollowingequationtocalculatethevolumeofcellsuspensiontoseedintoyourflasks.Seedingvolume=Totalvolumeofdilutedcellsuspension/#offlasksasdeterminedinstep18Example:4.3mlofdilutedcellsuspension/2T-75flasks=2.15mlperT-75flask
21. Prepareflasksbylabelingeachflaskwiththepassagenumber,strainnumber,celltype,anddate.
22. Carefullyopenthemediumbottleandtransfergrowthmediumtonewculturevesselsbyadding1mlgrowthmediumforevery5cm2surfaceareaoftheflask(1ml/5cm2).Example:15mlgrowthmediumfora75cm2flask.
23. Aftermixingthedilutedcellswitha5mlpipettoensureauniformsuspension,dispensethevolumeofsuspensioncalculatedaboveintothepreparedsubcultureflasks.
24. Afterdispensingthecells,gentlyrockflasktopromoteevendistribution.
25. Ifnotusingventedcaps,loosencapsofflasks.Placethenewculturevesselsintoa37·Chumidifiedincubatorwith5%CO2..

AssessingYieldandViabilitySeveralfactorscontributetolowcellcountandlowcellviability.Anexampleofyieldviabilityassessmentisprovidedinthechartbelow.Todeterminethereasonforlowyield/viability,followthesesteps:

1.Studythesamplechartbelow.Itisasampleofhighyield,highviability.

a.Notethe"soliddot"onthefar,leftsideofthesquare.Itindicateshighyield,oracellcountofmorethan250,000forNHDFandmorethan500,000forNHLF.

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

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

2.Now,usingtheblankdiagrambelowplotyourcellyieldandcellviability.Followthesesteps:

a.Marka(·)ontheYaxistoindicatethetotalcellcountofyourculture.

bMarka(·)ontheXaxistoindicatethecalculatedpercentviabilityofyourculture.

3.Ifyourresultfallsintoanyquadrantotherthanthe"HighYield-HighViability"quadrant,refertoAppendixD,ImprovingCellYieldandViability,beforeproceedingtoyournexttrypsinization.

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.Feedwithvolumesasoutlinedinthetableonpage13.

6.Passageagainwhenthecellsare70-90%confluent.(Ifseededattherecommendedseedingdensity,thisshouldtake5-9days.)

InstructionsforProliferatingCells

CellPreparation:ProliferatingCellsWiththeproliferatingcultureofNHDForNHLFyoureceived,dothefollowing:

1. Examinetheculturemicroscopicallyforanysignsofdistressduringshipment(i.e.,detachment,rounding-uporatypicalmorphology).Checktherelativecelldensityandestimate"%confluency."Thecultureshouldbe30-80%confluentuponreceipt.Somecellulardetachmentisnormal.PleasecallClonetics®TechnicalSpecialistimmediatelyifcellslookseverelydistressed.
2. Decontaminatetheexternalsurfaceofthecellcultureflaskbywipingwith70%ethanolorisopropanol.
3. Incubatethesealedflaskat37·C,5%CO2forthreetofourhourstoequilibratetemperature.
4. Warmanappropriateamountofgrowthmedium(seetableonpage13)to37·Cinasterilecontainer.Warmingtheentirebottlecanshortenthelifeofthemedium.Neverwarmgrowthmediumunderhotrunningwateroranyotheruncontrolledtemperaturesource.NEVERMICROWAVE!
5. Inasterilefield,carefullyopenthecellcultureflask,removethemediumandreplaceitwiththewarmed,freshgrowthmedium.Asepticallyremoveanymediuminsidetheneckorcapareabecauseitcanfacilitatemicrobialcontamination.
6. Loosenthecap,andreturntheflasktothe37·Chumidifiedincubatorwith5%CO2foratleast24hours.

SubculturingExamineyourculturesmicroscopicallyeveryday.

1. Subculturethecellswhentheyreach70-90%confluency.NHDFandNHLFculturesshouldhavemanymitoticfiguresthroughouttheflask.Cellsshouldbereadytosubculturewithin24to48hours,however,shippingconditionssuchastemperaturefluctuationsmayaffecttheactualtimeatwhichthecellsarereadyforsubculture.
2. Avoidthelossofyourcultureduetocontactinhibitionbysubculturingcellsatnomorethan90%confluency.Seepages15-17fordetailedsubculturinginstructions.

BIBLIOGRAPHY

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

2)Cytokeratin18&19.CallyourClonetics®TechnicalSpecialistforareferenceonthisprocedure.

3)Wagner,D.D.,Olmsted,J.B.andV.J.Marder.(1982)ImmunolocalizationofVonWillebrandProteininWeibel-PaladeBodiesofHumanEndothelialCells.JournalofCellBiology.,95:355-360.

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

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

INTERNATIONALTECHNICALSERVICE:

BioWhittakerInc.CloneticsProducts8830BiggsFordRd.Walkersville,MD21793-0127301-898-7025FAX:301-845-2924E-mail:techsup@biowhittaker.com

ORDERS:

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

APPENDIXAOVERVIEWOFFIBROBLASTMEDIA

500mlBottles(exceptwhereindicated)

NOTE:AllClonetics®Mediacanbecustomformulatedtomeetyourresearchneeds.ContactyourTechnicalSpecialistformoreinformation.

APPENDIXBCELLCOUNTINGUSINGAHEMACYTOMETER

ProperuseofahemacytometeriscriticalforobtaininganaccuratecountofcellsandisaprocedureusedbyBioWhittakertodeterminethesuspensioncountsforClonetics®cellstrains.Ahemacytometerconsistsofathic,kenedglassslideintowhichasmallchamberhasbeencuttoallowfortheintroductionofcellstobecounted.Thefloorofthechamberisdivided(etched)intoninesections;usuallyonlythefourcornersectionsareusedincellcounting(SeeFigure1below).Withacoverslipinplace,eachsquareofthehemacytometerrepresentsatotalvolumeof0.1mm3or10-4cm3.Since1cm3isapproximatelyequivalentto1ml,thecellconcentrationperml(andthetotalnumberofcells)canbedetermined.

1.Prepareacellsuspensionasinstructedinstep13onpage16.

2.Prepareahemacytometerforuse.

a.Carefullycleanallsurfacesofthehemacytometerandcoverslip.

b.Takecaretoensurethatallsurfacesarecompletelydryusingnon-lintingtissue.

c.Centerthecoversliponthehemacytometer.

3.Pipetapproximately9microliters(thisvolumewillvaryslightingwiththebrandofhemacytometer)ofthecellsuspensionintooneofthetwocountingchambers.

a.Useacleanpipettip.

b.Besurethatthesuspensionisthoroughly,butgently,mixedbeforedrawingthesamples.

c.FillthechambersslowlyandsteADIly.

d.Avoidinjectingbubblesintothechambers.

e.Donotoverfillorunderfillthechambers.

4.CounttheCells.

a.Allowthecellsuspensiontosettleforatleast10seconds.

b.Countallofthecellsineachofthefour1mm3cornersquareslabeledAthruDinFigure1onthenextpage.

1)DOcountthecellstouchingthetoporleftborders.

2)DONOTcountthecellstouchingthebottomorrightborders.

5.DeterminetheCellCount.

a.Calculatethetotalcellscountedinthefourcornersquares.

1)Ifthetotalcellcountislessthan100,orifmorethan10%ofthecellscountedappeartobeclustered,carefullyre-mixtheoriginalcellsuspensionandrepeatsteps2through4(above).

2)Ifthetotalcellcountisgreaterthan400,dilutethesuspensionsothecountwillbe100-400cells.ThenrepeatSteps2-4(above).

NOTE:Ifsatisfactoryresultsarenotachieved,contactyourClonetics®TechnicalSpecialistbytelephoning800-852-5663.

b.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

Trypanblueisadyethatenableseasyidentificationofdeadcells.Deadcellstakeupthedyeandappearbluewithunevencellmembranes.Bycontrast,livingcellsrepelthedyeandappearrefractileandcolorless.

1.Preparethehemacytometerforuse.

a.Carefullycleanallsurfacesofthehemacytometerandcoverslip.

b.Takecaretoensurethatallsurfacesarecompletelydryusingnon-lintingtissue.

c.Centerthecoversliponthehemacytometer.

2.Transfer50mlof0.4%TrypanBlueintoacleantube.

3.Add50mlofthepreparedcellsuspensionintothetubecontainingthestain.

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.Allowthesuspensiontosettleinthechambersforatleast10seconds.

b.Countallofthestainedcellsineachofthefourcornersquaresofthehemacytometer.

c.Separatelycountalloftheunstainedcellsinthesamesquares.

d.Calculatethecellviabilityusingtheequation:

%CellViability=numberofunstained(living)cells/Totalcellscounted(stained+unstained)x100%

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

%Cellviability=200/300x100%=67%

IMPROVINGCELLYIELDANDVIABILITY

Background

Severalfactors,oracombinationoffactors,contributetolowcellcountandlowcellviability.Ifcellyieldorviabilityisunsatisfactory,usethefollowinginformationtoincreasethesuccessrateoffuturecultures.

ImprovingCellYield

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

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

Onceyouhavedeterminedhowtoachievehighyieldandhighviability,subculturetheremainingflasks.

APPENDIXEGROWTHAREAOFCOMMONPLASTICWARE

APPENDIXFSeedingIntoMulti-WellPlates

Overview

Acultureflaskofnormalhumancellsisharvestedbytrypsinizationandsubsequenttrypsininhibitortreatment.Thecellsarecentrifuged,resuspendedingrowthmediumandcounted.ThedesirednumberofcellsisthenaddedtowellsofsterileMulti-welltissuecultureplates.Theplatesareincubatedina37oC,5%CO2humidifiedincubatorforonetothreedaystoallowforcelladherenceandgrowth.Seedingdensitieswillvarysomewhatwithyourexperimentalrequirements.WerecommendadensityforDermalFibroblastsandLungFibroblastsof10,000cells/cm2forallmultiwellplates.

RequiredMaterials:

1. T-25flaskofproliferatingnormalhumancellsbetween70%and90%confluence.
2. Flat-bottom,Multi-welltissuecultureplates
3. 37oChumidifiedincubatorwith5%CO2/95%air
4. Laminarflowhoodorothersterileenvironment
5. Adjustablemultichannelpipetter(8-or12-channel)orrepeatingpipetter
6. Sterilereservoir(s)forusewithmultichannelpipetter

Procedure

1. Followthestepsonpages13-15forsubculturepreparationandsubculturing.Thenfollowsteps2-4below.
2. Sincethecells/mlcalculationcomputedonp.20is"perml",onemustincreasethecellconcentrationby4timesbeforeseeding96wellplates(toaccommodatethe1:4dilutionwhenadding250ulofsuspendedcellsperwell).Whenmakingthecellsuspension,adjustthecellconcentrationwithgrowthmedium.
3. Transferthedilutedcellsuspensiontoasterilereservoir.Usingamultichannel(8-or12-channel)pipetterequippedwithsterilepipettetips,add250mlofthedilutedcellsuspensiontoeachwellofthelabeled96-well,flatbottom,tissuecultureplate(s).RESUSPENDTHECELLSUSPENSIONOFTENDURINGTHESEEDINGPROCEDURETOENSUREAUNIFORMNUMBERANDDISTRIBUTIONOFCELLSINTOEACHWELLBYPIPETTINGUPANDDOWNAFEWTIMESBETWEENEVERYOTHERDISPENSING.
4. Coverandincubatetheplatesfor1to3daysat37oC/5%CO2.(Incubationperiodsexceeding3daysaregenerallynotrecommendedbecauseofevaporationofmediumfromtheedgewellsoftheplate.)

NOTE:BeforeusingtheMulti-wellplatecultureinabioassay,examinethemmicroscopicallyforthepresenceofmitoticfiguresasaconfirmationthatthecellshaveresumedactivegrowth.(Doesnotapplyforallend-userassays.)

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