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CellIsolationTechniques

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.

**StandardSolutionTableCompositionofSelectedBalancedSaltSolutions^a,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%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

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

Someresearchershavefoundthatproceduresusingcrystallinetrypsincanprovideincreasedviabilityincellsaftertheyarereleased.Viabilityisusuallydeterminedbymeasuringcloningefficiency,i.e.,theabilityofasinglecelltoattachtothewallofaculturevesselanddividetoproduceacolonyofcellswhichisvisibletothenakedeyeafterstaining.

OptimizationTechniques

GeneralGuidelines

Althoughoptimizationofacellisolationprocedureforaparticularcelltypeisdependentupontheadequaterecoveryofcellshavingvariousrequiredcharacteristics,someguidelinescanbeestablished.Theinformationinthisguideregardingcellisolationandtheenzymesused,whencombinedwithlogicandsuitableexperimentaldesign,shouldleadtothedevelopmentofasatisfactorycellisolationmethod.(SeeFreshney1987foradetaileddiscussion.)

Cell Visibility / Cell Yield Relationship illustration

Thecomplexrelationshipbetweencellyieldandviabilitycanberepresentedbythesimplifiedillustrationsshownontheleft.Ingeneralthereisanareaofoptimizedrecoverybalancedbetweenyieldandviability;workingnearthemiddleofthisrangewillreducevariabilityintheresultsofthecellisolationprocedure.Understandingthisrelationshipandhowitcanvarywithaparticularcelltypeandapplication,canmaketheoptimizationprocesseasier.

Working Range Graph

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.

CellQuantitation

Itisimportanttoquantitatetheresultsofeachdissociationstepinordertoeffectivelyevaluateeachprocedure.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=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.

MeasureofViability

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

Thisprocedurecanbeperformedalongwiththecellcountingprocedurebutcelldensitymayrequireadjustmentinordertoobtainapproximately10⁶cellspermilliliter.

Procedure

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

Calculation

PercentViability=
	TotalCellsCounted-StainedCells	x100
	TotalCellsCounted	x100

Example

TotalCells/1mm²=182StainedCells=24

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

Note:Dyeexclusionviabilityprocedurestendtogivehighestimatesofcellviabilitywhencomparedtocellattachmentormetabolicassays,butforoptimizationofcellisolationprocedurestrypanbluedoesprovidearapidestimateofdissociationperformanceinconjunctionwithyieldquantitation.

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Cell&nbsp;&nbsp;Isolation&nbsp;&nbsp;Techniques