MethodsandMaterialsWorkingWithEnzymesAlloftheenzymesWorthingtonoffersfortissuedissociationapplicationsareavailableaslyophilizedpowdersforconvenience,versatility,andstABIlity.Assuchtheymaybestoredat2-8°C,andtheycanbeshippedwithoutspecialhandling.Whilelyophilizationmakesshippingandstoringtheenzymeseasier,specialcareisrequiredwhenopeninganyofthevials. Lyophilizedproteinstendtobeveryhygroscopicsotheyshouldnotbeopenedinhumidareas.Besurethatanyvialhasbeenbroughttoroomtemperaturebeforeopening.Ideally,thevialsshouldbetakenfromtherefrigeratoratleastahalfhourbeforeopening,andtheyshouldbeleftinadessicator.Beforeopeninganyofthevials,besureitisnotatallcooltothetouch.Allofthecellisolationenzymescitedinthissectioncanberepeatedlywarmedtoroomtemperatureandthenreturnedtotherefrigeratoraslongastheseprecautionsarefollowed. Oncedilutedwithmediaorbuffer,proteolyticenzymescanundergoautolysis.Dissolveenzymesimmediatelybeforeuse. Specialcaremustbetakenwithdeoxyribonuclease(DNASE).Thisproductisverypronetosheardenaturation.Mixgently. Reconstitutedenzymesshouldnotbestoredat2-8°C.Ifnecessarytheycanbealiquotedandfrozenat-20°C.Avoidrepeatedfreeze-thawcycles. Allenzymes,uponreconstitution,canbesterilefilteredthrougha0.22µmporesizemembrane. Generallymostoftheenzymesusedincellisolationprocedures(excepttrypsin)canbedirectlydissolvedinabalancedsaltsolutionorbufferofchoice.Stocksolutionsoftrypsinshouldbemadeinitiallybyreconstitutingtheenzymein0.001NHCl.Thissolutioncanbedilutedintothedigestionmediumorbufferimmediatelypriortouse. Thecompilationofstandardbalancedsaltsolutionswiththeirreferencesfoundinthefollowingtablecanbehelpfulinselectinganappropriatedissociationsolution. StandardSolutionTableCompositionofSelectedBalancedSaltSolutionsa,b | Ringer(c) | Tyrode(de) | Gey(f) | Earle(g) | Puck(h) | Hanks(l) | Dulbecco(PBS)(jk) | NaCl | 9.00 | 8.00 | 7.00 | 6.80 | 8.00 | 8.00 | 8.00 | KCl | 0.42 | 0.20 | 0.37 | 0.40 | 0.40 | 0.40 | 0.20 | CaCl2 | 0.25 | 0.20 | 0.17 | 0.20 | 0.012 | 0.14 | 0.40 | MgCl2ø6H2O | | 0.10 | 0.21 | | | 0.10 | 0.10 | MgSO4ø7H2O | | | 0.07 | 0.10 | 0.154 | 0.10 | | Na2HPO4ø12H2O | | | 3.00 | | 0.39 | 0.12 | 2.31 | NaH2PO4øH2O | | 0.05 | | 0.125 | | | | KH2PO4 | | | 0.03 | | 0.15 | 0.06 | 0.20 | NaHCO3 | | 1.00 | 2.27 | 2.20 | | 0.35 | | Glucose | | 1.00 | 1.00 | 1.00 | 1.10 | 1.00 | | PhenolRed | | | | 0.05 | 0.005 | 0.02 | | Atmosphere | air | air | 95%air/5%CO2 | 95%air/5%CO2 | air | air | air | a | Amountsaregivenasgramsperliterofsolution | b | Insomeinstancesthevaluesgivenrepresentcalculationsfromdatapresentedbytheauthorstoaccountfortheuseofhydratedoranhydroussalts | c | S.Ringer,J.Physiol.(London)18,425(1895) | d | M.V.Tyrode,Arch.Int.Pharmacodyn.Ther.,20,2025(1910) | e | R.C.Parker,MethodsofTissueCulture,3rded.,p.57,Harper,NewYork,1961 | f | G.O.GeyandM.K.Hey,AmJ.Cancer,27,55(1936) | g | W.R.Earle,J.Natl.CancerInst,4,165(1943) | h | T.T.Puck,S.J.Cieciura,andA.Robinson,J.Exp.Med.108,945(1958) | i | J.H.HanksandR.E.Wallace,Proc.Soc.Exp.Biol.Med.,71,196(1949) | j | PBS,phosphate-bufferedsaline | k | R.DulbeccoandM.Vogt,J.Exp.Med.,99,167(1954) |
BasicPrimaryCellIsolationProtocol(Refertoreferencesforapplicationspecificparameters) - Fornon-perfusion,minceorcuttheisolatedpieceoftissueinto2-4millimeterpieceswithsterilescissorsorscalpel.
- Addthetissuepiecestotheappropriatebufferorbalancedsaltsolutiononiceandwash2-3times.
- Addappropriateamountofenzyme(s)andincubateatoptimumtemperature(usually37°C)forappropriatetime,mixingintermittently.
- Gentlydispersethecellsbypipeting(trituration).
- FilterthecellsUSPensionthroughfinemesh.
- Allowthecellstosettleanddecantexcessliquidcontainingenzymes.Washandrepeat2-3times.
- Resuspendcellsinappropriatemediumorbuffer.
- Quantitatecellyieldandviability.
- Seedcellsforculture,ifrequired.
Perfusionproceduresrequirespecialequipmentandtechniquesforrecirculatingthebuffers,mediaandenzymes.Pleaserefertoreferencedtextsforadditionalinformationandguidance. Equilibrationwith95%O2:5%CO2InmanycellisolationproceduresitisimportanttothesurvivalofthetissueduringdissociationthattheincubationmediumbebothwelloxygenatedandbufferedatphysiologicalpH.Bothrequirementsaresatisfiedwhenthemediumisequilibratedwith95%O2:5%CO2.SeveralbalancedsaltsolutionscontainthepHsensitiveindicatordye,phenolred.Whenitisredorpurpleincolor,themediumistooalkaline.Thissometimesoccurswhenthetissueisplacedinthedissociationenzymesolution.ReequilibrationwithO2:CO2isusuallynecessarypriortoincubation. Gasshouldnotbebubbleddirectlyintoanysolutioncontainingprotein.ThiscanresultinfrothinganddenaturationoftheproteinwithlossofBIOLOGicalactivity.Gascanbesterilizedbypassagethrougha0.22micronmembranefilterorthroughasterilefiberplugsuchasthecottonpluginasterilePasteurorvolumetricPipette.Whilemixingthesolution,passO2:CO2continuouslythroughthespaceabovetheliquiduntilcolorindicatespH7.2-7.4.Thebalancedsaltsolutionisoftenpre-gassedbutshouldbeequilibratedwithsterileO2:CO2eachtimethebottleisopened. BufferedbalancedsaltsolutionswillusuallymaintainconstantpHregardlessofthedegreeofoxygenation/carbonationandasaresultcanbeeasiertoworkwith.Certaincelltypesmaybesensitivetoparticularbuffersalts.Thereferencetablescanbeusefulinselectinganappropriatebalancedsaltsolution,buffer,ordissociationmediaforaspecificapplication. Trituration(Celldispersionthroughmildpumpingaction) Thiscanbeacrucialprocedure.Itservestobreakupthetissuefragmentsfollowingincubationinthedissociationmix.Ifdonetoovigorously,cellswillbedestroyedloweringviability;tooweaklyandtissuefragmentswillbeleftintactloweringyield.Gentletrituration,usinga10mlpipette,constitutesfillingandemptyingthebarrelatarateofabout3.0mlpersec.Youcanbestdetermineasuitablerateforyourtissuethroughtrialanderror.Avoidbubblingthecellsuspension. EnzymaticCellHarvestingMostnon-malignantcellsgrowinginvitromoveaboutanddivideuntiltheyformamonolayeronecellthickcompletelycoveringthesurfacesoftheculturevessel.Movementandproliferationnormallyceasewhenconfluenceisreached.Harvestingcellsforstudy,processingorsubculturerequiresdissociationanddetachmentofthemonolayer.Limitedtreatmentofthecelllayerwiththeenzymetrypsinisthemethodmostfrequentlyapplied. ItwasformerlythoughtthattrypsinpreparationssimplyhydrolyzedaproteinaceousadhesivebondingsubstanceresponsIBLeforthetenaciousattachmentofcellstotheirsubstratumwiththeresultantdetachmentofthecellsfromtheculturevessel.Itisnowfeltthatthemechanismofactionoftrypsinincellharvestingismorecomplex.Thissectionsummarizesrecentinformationonthissubject. CellAdhesionandHarvestingDuringinterphase,fibroblast-likecellsinculturearespreadoutonthesubstratuminacharacteristic,spindle-shapedconfiguration.Therearedifferencesofopinionastowhethertheactualareasofcelladhesionaredistributedovermostoftheundersurfaceofthecellorarelocalizedinrelativelynarrowpatchesnearthecellmargins,principallyinthevicinityofrufflingactivity.Ineithercase,theseareasofadhesionappeartobecomposedofclustersofattachmentpoints,eachabout1µmindiameter.TheindividualattachmentpointsareapparentlythedistalportionsofacellCytoskeletonstructureboundtothesubstratum. Withinminutesaftersubjectingculturedcellstocoldtemperatures,chelatingagentsortrypsinsolutions,theychangeshapedrasticallybyroundingupandblebbing.Electronmicrographsshowmanylongretractionfiberswithadiameterof0.25-0.5µmrunningfromthesurfaceoftheroundedcellbodytoenlarged,terminalbulbattachmentpointspreviouslylocatedontheflattenedcell\"sundersurface. Thecellsremainattachedtothesubstratumuntilthefibersarebroken,eithermechanicallybytappingorshakingtheculturevessel,orchemicallybythecontinuedactionofchelatorsand/ortrypsin.(Coldtemperaturesalonearesufficientforroundingupbutnotfordetachment.Theseconditionsalsogreatlydiminishtheentryoftrypsinintothecell.)Soonaftercelldetachmentfromthesurfaceoftheculturevessel,andsubcultureintonewvesselscontainingtrypsin-freemedium,cytoplasmflowsintothebrokenretractionfibersandrefillsthem.Withinanhourtheroundedcellsbegintotakeontheircharacteristicshape. TrypsinforCellHarvestingIn1916,RousandJonesused\"thetrypsinpowdersofMerck,BrublerandKahlbaum\"todigesttheplasmaclotsinwhichlivingcellsweregrowinginordertoobtainacellsuspensionforsubculturing.VogelaarandErlichmanin1934werethenextresearcherstoutilizethedigestiveenzymesinacrudetrypsinpreparationtoliquifythecoagulatedplasmainwhichhumanfibroblastsweregrowingpriortosubculturing.TechniquesusingtrypsinsimilartothoseusedtodaywereintroducedbyScherer,SyvertonandGeyin1953toharvestthethennewlycultivatedHeLacellstrainforsubculturingandbiochemicalanalysis.TheseworkerstestedbothrecrystallizedtrypsinandNF1:250trypsinforcellharvestingandfoundthatthepurifiedtrypsinwasmorepotentandlesstoxictocells.NeverthelesstheNF1:250preparationwasemployedforroutineharvestingsimplybecauseitwaslessexpensive. RelativelycrudepancreaticpreparationslikeNF1:250trypsinarestillusedtodayforcellharvestinginspiteofthefactthattheyexhibitconsiderablelot-to-lotvariabilityandcontainextraneoussubstancesandotherenzymaticactivities.Impuritiesincrudetrypsincancauseunnecessarydamagetocellsandareductionofcloningefficiency.Useofhigherpuritycrystallinetrypsincaneliminatemanyofthesedifficulties. NoneofthecontaminantspresentintheNF1:250materialsappearstobeessentialforcellharvestingactivitysincepurifiedtrypsinisveryeffectiveformonolayerdissociation,andsincecrudeNF1:250trypsinplussoybeantrypsininhibitorisineffective. McKeehanandHamreportmarkedlyimprovedviabilityandmultiplicationpotentialtosinglecellsinlowserummediumwhenharvestingwithcrystallinetrypsinatreducedtemperatures,i.e.,at4°C. CellReleaseProcedureInordertotransferorpasscellsinmonolayerculturefromoneculturevesseltoanotheritisnecessarytoreleasecellsfromthemonolayerintosuspensionsothattheycanbeeasilyhandledbypipettinganddiluting. Releasingcellsfromthemonolayerisalmostalwaysaccomplishedwithpurifiedtrypsinbyaprocedureknownastrypsinization.Ausualtrypsinizationprocedureisdetailedintheinsetbelow. TrypsinizationProcedure Removeculturemediumfromcells.Addsteriletrypsinsolution(inBSS-balanced-saltsolution,normallycalcium-freeHanks.Allowtrypsinsolutiontoactonmonolayerforseveralminutesatroomtemperatureor37°C(orlongerat4°C).Removetrypsinsolutiongentlysoasnottodisturbcells.AddBSSormedia(oftenwithserumortrypsininhibitortoinactivateresidualtrypsin)andagitatevesseltodisruptmonolayerandsuspendcells.Someresearchershavefoundthatproceduresusingcrystallinetrypsincanprovideincreasedviabilityincellsaftertheyarereleased.Viabilityisusuallydeterminedbymeasuringcloningefficiency,i.e.,theabilityofasinglecelltoattachtothewallofaculturevesselanddividetoproduceacolonyofcellswhichisvisibletothenakedeyeafterstaining.
OptimizationTechniquesGeneralGuidelinesAlthoughoptimizationofacellisolationprocedureforaparticularcelltypeisdependentupontheadequaterecoveryofcellshavingvariousrequiredcharacteristics,someguidelinescanbeestablished.Theinformationinthisguideregardingcellisolationandtheenzymesused,whencombinedwithlogicandsuitableexperimentaldesign,shouldleadtothedevelopmentofasatisfactorycellisolationmethod.(SeeFreshney1987foradetaileddiscussion.) Thecomplexrelationshipbetweencellyieldandviabilitycanberepresentedbythesimplifiedillustrationsshownontheleft.Ingeneralthereisanareaofoptimizedrecoverybalancedbetweenyieldandviability;workingnearthemiddleofthisrangewillreducevariabilityintheresultsofthecellisolationprocedure.Understandingthisrelationshipandhowitcanvarywithaparticularcelltypeandapplication,canmaketheoptimizationprocesseasier. Fortroubleshootingpurposesvariouspossibleresults,alongwithsuggestedcorrectiveactionsarelistedbelow.Keepinmindthattherearenoclearlinesbetweenthequadrantsbutratherconvergingzoneswithvariableareasofoverlap. Lowyield/LowViability | Over/underdissociation,cellulardamage.Changetolessdigestivetypeenzymeand/ordecreaseworkingconcentration.(e.g.fromtrypsintoCollagenase/fromType2collagenasetoType1). | LowYield/HighViability | Underdissociation.Increaseenzymeconcentrationand/orincubationtimeandmonitorbothyieldandviabilityresponse.Ifyieldremainspoor,evaluateamoredigestivetypeenzymeand/ortheadditionofsecondaryenzyme(s). | HighYield/LowViability | Gooddissociation,cellulardamage.Enzymeoverlydigestiveand/orattoohighaworkingconcentration.Reduceconcentrationand/orincubationtimeandmonitoryieldandviabilityresponse.Trydilutingtheproteolyticactionbyaddingbovineserumalbumin(BSA)(0.1-0.5%w/v)orsoybeantrypsininhibitor(0.01-0.1%w/v)tothedissociation.Tryusinglessproteolyticenzymealthoughyieldmaybeaffectedandshouldbemonitored. | HighYield/HighViability | Theplacetobe.Considerevaluatingtheeffectofdissociationparameterstolearntheirlimitationsforfuturereference. |
Ascaleshowingtherelativedigestivepoweroftheenzymescommonlyusedfollowsforreference.Refertothisscalewhentroubleshootingadissociationandplanningisolationstrategy. OptimizationStrategyReviewtheReferencesoftheWorthingtonTissueDissociationGuidefortheparticulartissueandcelltypeofinterest,andthenapplythisinformationtothepracticalapplicationoftissuedissociation.Anexampleofabasicoptimizationstrategyfollows: Basedupontheenzyme(s)cited,workingconcentrationsandthebufferormediasystemused,setupproposedpreliminarydissociationconditionssimilartotheclosestavailablereference(s)listedinthetables. Ifamajorityofthemostsimilarreferencedprocedurescitetheuseofmorethanoneenzyme,optimizetheconcentrationoftheprimaryenzyme(theoneatthehighestrelativeconcentration)beforeaddingthesecondaryenzyme(s).Forexample,ifthetwomostsimilarreferencescitecollagenase0.1%withDNase0.01%andcollagenase0.075%withhyaluronidase0.025%,optimizethecollagenaseconcentrationempiricallybeforeevaluatingtheeffectsofeitherthehyaluronidaseorthedeoxyribonuclease. Afteroptimizingtheprimaryenzyme\"sconcentrationandincubationconditionsevaluateanysecondaryenzyme(s). Initiallyvarytheconcentrationoftheprimaryenzymeapproximately50%relativetothereferencedprocedure(s).Theaboveexampleofcollagenaseconcentrations0.1%and0.075%suggestsanevaluationofenzymeconcentrationsbetween0.025%and0.15%.Theconcentrationincrementsshouldbeevenlydistributedtocoverthisentirerange.Asaresultincrementalconcentrationsof0.025%,0.05%,0.075%,0.10%,0.125%and0.15%wouldbeindicated.Tosimplifytheinitialscreeningthemiddleoftherangecanbeselectedand,afterevaluationofyieldandviabilityresults,adecisioncanbemaderegardingtheneedforfurtherstudies.Inthiscaseinitialcollagenaseconcentrationsevaluatedmaybe0.05%,0.075%,0.10%and0.125%. Historically,mosttissuedissociationandcellisolationprotocolshavecitedtheenzymeconcentrationusedintermsofweightperunitvolume(w/v).Morerecently,however,someresearchershavebeguntousetheenzymesonanactivitybasis,thatis,unitspermilliliter(u/ml).Useeithermethodbutconsidertheadvantagesanddisadvantagesofeach: a)ThetrADItionalweightperunitvolumemethodmostlikelyresultedfromtheuseofcruder,partiallypurifiedmixturesofenzymesandisusedindependentlyofanyspecificorcontaminatingactivitieswhichmaybepresent.Withsomeofthesecrudepreparationsthelot-to-lotvariationcanbesignificantresultinginuptoatwo-folddifferenceintheamountofenzymaticactivityaddedonaweightbasis. b)Addingbyactivitycanresultinapossibletwo-folddifferenceintheamountofweightaddedtoadissociation;however,normalizesthepotencyusedbasedupontheprimaryactivityforeachlot. Bothmethodsignoretherelativecontaminantactivitylevels.Uponestablishingabasicmethod,considerpre-samplingdifferentlotsofenzyme(s)toevaluatethesefactorsandtoselectalotofenzymewhichhasminimaleffectuponthecriticalparametersofaspecificapplication. Important:Foraccurateevaluationofaparticularprocedure\"sperformance,cellyieldandviabilityshouldbequantitatedandcompared.Afteroptimizingbasicdissociationandisolationconditions,thespecificapplicationparameterssuchasmetabolicfunction(s)orreceptorbindingcapabilityshouldalsobeevaluated.Basedupontheseresultsthemethodmaybejudgedsuitableforuseorre-optimizedforhigherretentionofnativecellularcharactaristics.
CellQuantitationItisimportanttoquantitatetheresultsofeachdissociationstepinordertoeffectivelyevaluateeachprocedure.Theuseofacellcountingchamber(hemocytometer)foryieldquantitationandtheuseoftrypanblueforviabilityquantitationarerecommended.Theuseofahemocytometerforcellyieldquantitationisoutlined;however,newcomerstothisprocedurecanrefertomoredetaileddiscussions(seeFreshney,CultureofAnimalCells,page227). RequiredSupplies:- ImprovedNeubauerHemocytometer
- CellCompatibleMediaorBSS
- PasteurPipetorMicropipettor
- Microscope(10X)
- Counter
Procedure:Carefullycleanthecountingchambersurfaceandthecoverslipofthehemocytometerwith70%isopropanolandallowtoairdry.Becarefulnottoscratchthesesurfaces.Wetthesidesofthecoverslipwithreagentgradewaterandalignthecoverslipoverthecountingchamber.Takeawellmixed20-50µlaliquotofthedissociatedcellsuspensionusingeitheraPasteurpipetoramicropipettoronlydrawingthecellsintothetip.Immediatelytransferthecellsuspensiontothecountingchamberbyplacingthetipofthepipetattheedgeofthechamberandallowingthechambertofillcompletelyviacapillaryaction.Donotover-orunderfillthechamber.Repeatthisprocedureusinganotheraliquotsampleforthesecondchamberontheoppositesideofthehemocytometer.Placethehemocytometeronthemicroscopestageand,usingthe10Xobjective,focusonthecountingchambergridlines.Adjustthecontrastasneededtoclearlyseeboththegridandthedispersedcells.Adjustthefieldareabyslowlymovingtheslidetoobtainacentralgridboundedbythreelinesonallsides(seefigurebelow).Countthetotalnumberofcellspresentinthis1mm2areaincludingthosecellswhichareonthetopandleftbordersandexcludingthoseontherightandbottomborders.Foraccuracycountatleast100-500cells.Dependinguponyieldanddensitymoreorfewerareasmaybecounted.Repeatthecountforthesecondchamber.Ifnosecondchamberexists,theslideshouldbecleanedandtheprocessrepeated.Calculation:C=Ñx104whereC=cellspermilliliterÑ=averageofcellscounted104=volumeconversionfactorfor1mm2TotalYield=CxVwhereV=totalvlaueofcells(ml)Example:Count1=183cells/mm2Count2=175cells/mm2VolumeofCells=55mlAveragecellscounted= | Count1+Count2 | | 2 | = | 185+175 | | |
| 2 | | | | | = | 178.5 | C=178.5x104=1,785,000cells/mlTotalyield=CxV=1785,000x55=98,175,000cellsNote:Forbestresultsthecelldensityshouldbeatleast105cellspermilliliter.Commonerrorsoccurbyimpropermixingofthecellsuspensionpriortosamplingand/orbyallowingthecellstosettleinthepipetpriortoloadingthehemocytometercountingchamber.Avoidthecountingofmultiplecellaggregates;thepresenceofaggregatesindicatesincompletedissociationwhichmayrequirefurtheroptimizationoftheisolationparameters.Asinglecellsuspensionprovidesthebestresults.
MeasureofViabilityOneofthesimplestmethodstoapproximatecellviabilityisthedyeexclusiontechnique.Thismethodutilizesanindicatordyetodemonstratecellmembranedamage.Cellswhichabsorbthedyebecomestainedandareconsiderednon-viable.Dyessuchastrypanblue,erythrosin,andnigrosinarecommonlyusedwithtrypanbluebeingthemostcommoninpreliminarycellisolationprocedures. Thisprocedurecanbeperformedalongwiththecellcountingprocedurebutcelldensitymayrequireadjustmentinordertoobtainapproximately106cellspermilliliter. ProcedureMix1dropoftrypanbluewithonedropofthecellsuspensionandallow1-2minutesforabsorptionPreparehemocytometerandloadchambersasdescribedin\"CellQuantitation\".Countboththetotalnumberofcellsandthenumberofstained(dark)cells.CalculationPercentViability= | | | TotalCellsCounted-StainedCells | x100 | | TotalCellsCounted | ExampleTotalCells/1mm2=182StainedCells=24%Viability= | 182-24 | = | 158 | x100 | | 182 | | 182 | | | | = | 86.8%Viability | Note:Dyeexclusionviabilityprocedurestendtogivehighestimatesofcellviabilitywhencomparedtocellattachmentormetabolicassays,butforoptimizationofcellisolationprocedurestrypanbluedoesprovidearapidestimateofdissociationperformanceinconjunctionwithyieldquantitation. |