MosttrADItionalmethodspublishedforisolatinghepatocytesusecrudeandpartiallypurifiedenzymepreparationsincludingvarioustypesofCollagenaseandotherproteases.MorerecentlytheuseofbettercharacterizedpreparationsofcollagenasesuchasWorthingtonTypes1and4(CLS-1,4)haveprovidedbetterresults.Allcrudecollagenasepreparationscancontainlot-variablecontaminatingproteases,esterasesandotherenzymesrequiringresearcherstopre-screenseverallotsofenzymeand/orcontinuallymodifyisolationparametersandprotocols.
TheWorthingtonHepatocyteIsolationSystemhasbeendevelopedtoprovideresearcherswithareliable,convenient,andconsistenthepatocytecellisolationsystem.Byusingthepre-optimizedcombinationofenzymescontainedinthiskit,itispossIBLetominimizethelot-to-lotvariationandimprovethequalityoftheisolatedhepatocytes.Inaddition,Worthingtonuse-testseachlotbyisolatinghepatocytesfromadultrattoassureperformance,reliABIlity,andconsistentyieldofviablecells.
ThemethodisbasedonthatdescribedbyBerry,M.N.,modifiedbySeglen,P.O.(MethodsinCellBIOLOGy,volXIII,DavidM.Prescotted.,AcademicPress,1976;Chapter4,"PreparationofIsolatedRatLiverCells",pp29-83),andfurtheroptimizedinconjunctionwithseveralresearchers.
Stability/Storage:Thereagentsarestableatambienttemperaturesfortheperiodsoftimeexpectedinnormalshippingprocedures,butthepackageshouldberefrigerateduponarrival.Contentsmaybestoredat2-8°Cfor4-6monthsbeforeuse.Storeat2-8°C.
PackageContentsThepackagecontainssufficientmaterialsforfiveseparateadultratliverperfusions.Forlargerorsmallertissueapplications,prepareproportionatevolumesofreagentsateachstepandcombinetheminthesameratioasdescribedintheprotocol.
Vial#1:10XCMF-HBSSConcentrate,1bottle,500mlSterilecalcium-andmagnesium-freeHank"sBalancedSaltSolution(CMF-HBSS).Thesolutionisusedforwashingandperfusingtheliverpriortotheadditionofthedissociatingenzymesolution.
Vial#2:22,500UnitsCollagenaseand30UnitsElastase,5VialsWorthingtoncollagenase(Code:CLS-1)andelastase(Code:ESL),filteredthrough0.22µmporesizemembrane,andlyophilized.Beforeuse,reconstitutewiththeL-15/MOPSsolutionandswirlgentlytodissolvecontentsasdirectedinthefollowingprocedure.Storeunreconstitutedvialsat2–8°C.
Vial#3:1,000UnitsDNaseIeach,5VialsWorthingtonDNaseI(Code:D),filteredthrough0.22µmporesizemembrane,andlyophilized.Beforeuse,reconstitutewithL-15/MOPSsolutionandswirlgentlytodissolvecontentsasdirectedinthefollowingprocedure.Storeunreconstitutedvialsat2–8°C.
Vial#4:0.15MMOPS,pH7.5,1bottle,75ml0.15MMOPS,pH7.5bufferconcentrate,usedtobufferthereconstitutedLeibovitzL-15media.Vial#5:7.5%SodiumBicarbonate(NaHCO3),1bottle,100ml7.5%Sodiumbicarbonateconcentrate,usedtobufferthedilutedCMF-HBSS.
Pouch,containingLeibovitzL-15MediaPowder,1x1LReconstituteentirecontentsofpouchbycuttingopentopofenvelopeandpouringcontentsintobeakercontainingapproximately800mlofcellculturegradewater.Rinsepouch2-3timeswithanadditional100mlwater.Bringtotalvolumeto1000mlandfilterthrougha0.22micronporesizemembrane.
References
Alpinietal.,"RecentAdvancesintheIsolationofLiverCells",Hepatology(1994)20:494-514.
Berry,M.N.,Edwards,A.M.,andBarritt,G.J.;IsolatedHepatocytes:Preparation,PropertiesandApplications,RHBurdonandPHVanKnippenberg,eds.,Elsevier,Amsterdam,NewYork,Oxford,Chapt.2,1991.
ChenH-L,WuH-l,FonC-C,ChenP-J,Lai,M-Y,ChenD-S(1998)Long-termcultureofhepatocytesfromhumanadults.BiomedicalScience5:435-440
DeRobertis,E.D.P.,Saez,F.A.andDeRobertis,E.M.F.:CellBiology,6thed.,W.B.SaundersCo.,Philadelphia,PA,1975.
Freshney,R.Ian:CultureofAnimalCells,AlanR.Liss,Inc.,NewYork,2000.
Hanks,J.H.,andWallace,R.E.:Proc.Soc.Exp.Biol.Med.,71,1961949.
Jakoby,W.B.,andPastan,I.H.:MethodsinEnzymologyVol.LVIIIp.121,AcademicPress,1979.
LeCluyseEL(2001)HumanhepatocytesystemsfortheinvitroevaluationofcytochromeP450enzymeexpressionandregulation.Eur.JPharmSci13:343-368.
LeCluyseEL,PLBullock,AParkinson(1996)Strategiesforrestorationandmaintenanceofnormalhepaticstructureandfunctioninlong-termculturesofrathepatocytes.Adv.DrugDelRev22:133-186.
Leibovitz,A.:TheGrowthandMaintenanceofTissue/CellCulturesinFreeGasExchangewiththeAtmosphere,Am.J.Hyg.,78,173,1963.
Seglen,P.O.,MethodsinCellBiology,Vol.XIII,DavidM.Prescotted.,Ch.4,pp.29-83,AcademicPress,1976.
WorthingtonEnzymeManual,WorthingtonBiochemicalCorp.,Freehold,NJ,1993.
WorthingtonTissueDissociationGuide,WorthingtonBiochemicalCorp.,Lakewood,NJ,2003.
RelatedProducts:
Collagenase(CLS1-4/CLSPA)
DeoxyribonucleaseI(DP/D/DCLS/D2/DPFF/DPRF)
Elastase(ES/ESL)
Hyaluronidase(HSE/HSEP)
NeutralProtease(Dispase,NPRO)
Papain(PAP/PAPL/PAP2)
ProteinaseK(PROK)
Trypsin(TL/TRL/TRL3/TRLS/TRTPCK)
TrypsinInhibitors(LBI/OI/SI/SIC)
CellIsolationOptimizimgSystem(CIT)
NeonatalCardiomyocyteIsolationSystem(NCIS)
PapainDissociiationSystem(PDS/PDS2)
