MethodsandMaterials

WorkingWithEnzymes

AlloftheenzymesWorthingtonoffersfortissuedissociationapplicationsareavailableaslyophilizedpowdersforconvenience,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.

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%CO₂

InmanycellisolationproceduresitisimportanttothesurvivalofthetissueduringdissociationthattheincubationmediumbebothwelloxygenatedandbufferedatphysiologicalpH.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.

EnzymaticCellHarvesting

Mostnon-malignantcellsgrowinginvitromoveaboutanddivideuntiltheyformamonolayeronecellthickcompletelycoveringthesurfacesoftheculturevessel.Movementandproliferationnormallyceasewhenconfluenceisreached.Harvestingcellsforstudy,processingorsubculturerequiresdissociationanddetachmentofthemonolayer.Limitedtreatmentofthecelllayerwiththeenzymetrypsinisthemethodmostfrequentlyapplied.

ItwasformerlythoughtthattrypsinpreparationssimplyhydrolyzedaproteinaceousadhesivebondingsubstanceresponsIBLeforthetenaciousattachmentofcellstotheirsubstratumwiththeresultantdetachmentofthecellsfromtheculturevessel.Itisnowfeltthatthemechanismofactionoftrypsinincellharvestingismorecomplex.Thissectionsummarizesrecentinformationonthissubject.

CellAdhesionandHarvesting

Duringinterphase,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.

TrypsinforCellHarvesting

In1916,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.

CellReleaseProcedure

Inordertotransferorpasscellsinmonolayerculturefromoneculturevesseltoanotheritisnecessarytoreleasecellsfromthemonolayerintosuspensionsothattheycanbeeasilyhandledbypipettinganddiluting.

Releasingcellsfromthemonolayerisalmostalwaysaccomplishedwithpurifiedtrypsinbyaprocedureknownastrypsinization.Ausualtrypsinizationprocedureisdetailedintheinsetbelow.

TrypsinizationProcedure

1. Removeculturemediumfromcells.
2. Addsteriletrypsinsolution(inBSS-balanced-saltsolution,normallycalcium-freeHanks.
3. Allowtrypsinsolutiontoactonmonolayerforseveralminutesatroomtemperatureor37°C(orlongerat4°C).
4. Removetrypsinsolutiongentlysoasnottodisturbcells.
5. AddBSSormedia(oftenwithserumortrypsininhibitortoinactivateresidualtrypsin)andagitatevesseltodisruptmonolayerandsuspendcells.

Someresearchershavefoundthatproceduresusingcrystallinetrypsincanprovideincreasedviabilityincellsaftertheyarereleased.Viabilityisusuallydeterminedbymeasuringcloningefficiency,i.e.,theabilityofasinglecelltoattachtothewallofaculturevesselanddividetoproduceacolonyofcellswhichisvisibletothenakedeyeafterstaining.

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OptimizationTechniques

GeneralGuidelines

Althoughoptimizationofacellisolationprocedureforaparticularcelltypeisdependentupontheadequaterecoveryofcellshavingvariousrequiredcharacteristics,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.

OptimizationStrategy

ReviewtheReferencesoftheWorthingtonTissueDissociationGuidefortheparticulartissueandcelltypeofinterest,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.

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CellQuantitation

Itisimportanttoquantitatetheresultsofeachdissociationstepinordertoeffectivelyevaluateeachprocedure.Theuseofacellcountingchamber(hemocytometer)foryieldquantitationandtheuseoftrypanblueforviabilityquantitationarerecommended.Theuseofahemocytometerforcellyieldquantitationisoutlined;however,newcomerstothisprocedurecanrefertomoredetaileddiscussions(seeFreshney,CultureofAnimalCells,page227).

RequiredSupplies:

• ImprovedNeubauerHemocytometer
• CellCompatibleMediaorBSS
• PasteurPipetorMicropipettor
• Microscope(10X)
• Counter

Procedure:

1. Carefullycleanthecountingchambersurfaceandthecoverslipofthehemocytometerwith70%isopropanolandallowtoairdry.Becarefulnottoscratchthesesurfaces.
2. Wetthesidesofthecoverslipwithreagentgradewaterandalignthecoverslipoverthecountingchamber.
3. Takeawellmixed20-50µlaliquotofthedissociatedcellsuspensionusingeitheraPasteurpipetoramicropipettoronlydrawingthecellsintothetip.Immediatelytransferthecellsuspensiontothecountingchamberbyplacingthetipofthepipetattheedgeofthechamberandallowingthechambertofillcompletelyviacapillaryaction.Donotover-orunderfillthechamber.
4. Repeatthisprocedureusinganotheraliquotsampleforthesecondchamberontheoppositesideofthehemocytometer.
5. Placethehemocytometeronthemicroscopestageand,usingthe10Xobjective,focusonthecountingchambergridlines.Adjustthecontrastasneededtoclearlyseeboththegridandthedispersedcells.
6. Adjustthefieldareabyslowlymovingtheslidetoobtainacentralgridboundedbythreelinesonallsides(seefigurebelow).Countthetotalnumberofcellspresentinthis1mm²areaincludingthosecellswhichareonthetopandleftbordersandexcludingthoseontherightandbottomborders.
7. Foraccuracycountatleast100-500cells.Dependinguponyieldanddensitymoreorfewerareasmaybecounted.
8. Repeatthecountforthesecondchamber.Ifnosecondchamberexists,theslideshouldbecleanedandtheprocessrepeated.

Calculation:

C=Ñx10⁴

whereC=cellspermilliliterÑ=averageofcellscounted10⁴=volumeconversionfactorfor1mm²

TotalYield=CxV

whereV=totalvlaueofcells(ml)

Example:

Count₁=183cells/mm²Count₂=175cells/mm²VolumeofCells=55ml

Averagecellscounted=	Count1+Count2
	2
=	185+175
	2
=	178.5

C=178.5x10⁴=1,785,000cells/mlTotalyield=CxV=1785,000x55=98,175,000cells

Note:Forbestresultsthecelldensityshouldbeatleast10⁵cellspermilliliter.Commonerrorsoccurbyimpropermixingofthecellsuspensionpriortosamplingand/orbyallowingthecellstosettleinthepipetpriortoloadingthehemocytometercountingchamber.Avoidthecountingofmultiplecellaggregates;thepresenceofaggregatesindicatesincompletedissociationwhichmayrequirefurtheroptimizationoftheisolationparameters.Asinglecellsuspensionprovidesthebestresults.

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MeasureofViability

Oneofthesimplestmethodstoapproximatecellviabilityisthedyeexclusiontechnique.Thismethodutilizesanindicatordyetodemonstratecellmembranedamage.Cellswhichabsorbthedyebecomestainedandareconsiderednon-viable.Dyessuchastrypanblue,erythrosin,andnigrosinarecommonlyusedwithtrypanbluebeingthemostcommoninpreliminarycellisolationprocedures.

Thisprocedurecanbeperformedalongwiththecellcountingprocedurebutcelldensitymayrequireadjustmentinordertoobtainapproximately10⁶cellspermilliliter.

Procedure

1. Mix1dropoftrypanbluewithonedropofthecellsuspensionandallow1-2minutesforabsorption
2. Preparehemocytometerandloadchambersasdescribedin"CellQuantitation".
3. Countboththetotalnumberofcellsandthenumberofstained(dark)cells.

Calculation

PercentViability=
	TotalCellsCounted-StainedCells	x100
	TotalCellsCounted

Example

TotalCells/1mm²=182StainedCells=24

%Viability=	182-24	=	158	x100
	182		182
		=	86.8%Viability

Note:Dyeexclusionviabilityprocedurestendtogivehighestimatesofcellviabilitywhencomparedtocellattachmentormetabolicassays,butforoptimizationofcellisolationprocedurestrypanbluedoesprovidearapidestimateofdissociationperformanceinconjunctionwithyieldquantitation.