APPLICATIONNOTECTSNK-XpanderMedium
Expansionandcharacterizationofhumannaturalkiller(NK)cells
OptimizeyourNKcellexpansionprotocolwithCTSNK-XpanderMedium
Introduction
Naturalkiller(NK)cellsarelymphocytesthateitherdirectlyattackoractivatetheadaptiveimmunesysteminresponsetodetectionofmalignantorvirallyinfectedcells.They
haveawell-knownsafetyprofileandtheabilitytoactasallogeneicimmunemodulators,whichmakesNKcellsanattractiveoptionforoff-the-shelfimmunotherapeutics[1].
Intheclinicalsetting,strategiesarebeingdevelopedtoimproveNKcellfunction,persistence,andtraffickingtotumorsites[2].Theseincludeinsertingchimericantigenreceptors(CARs)andusingcomplementaryantibodiestoenhancetheidentificationanddestructionoftumorcells,aswellasexvivoexpansionandactivationtechniques.
ExpandAnalyze
?CTSNK-XpanderMedium
?RecombinanthumanIL-2
?HumanABserum
?Countess3FLAutomatedCellCounter
?AttuneNxTAcousticFocusingCytometer
?Stainingsolutionsandmonoclonalantibodies
Figure1.Optimizedworkflowforfeeder-freeexpansionandcharacterizationofNKcells.
Liuetal.[2]outlinethemanyexpansionandactivation
strategiesunderexploration,includingcytokine-only
expansion,autologousandallogeneicfeederstrategies,
engineeredcelllinefeeder–basedsystems,andeven
strategiestoengineercell-freeparticlesthatmimicthe
activityofengineeredfeedercells.Feeder-basedprotocolshavehistoricallyprovidedthehighestratesofexpansion.Thetypicalexpansionrangeislessthan10-foldfor
cytokine-onlysystems,lessthan500-foldforautologous
andallogeneicfeeder–basedsystems,andwellover1,000-foldforengineeredcelllinefeeder–basedsystems[2].
Safetyconcernsaboutfeedercells,includingtheriskof
contaminatingcellsinthefinaltherapeuticproduct,makefeeder-freesystemsadesirablechoiceforclinicalresearchapplications.However,lowerexpansionratesinfeeder-
freeculturesystemspresentachallengetowidespread
adoption.Thisisakeyreasonwhyfeeder-basedexpansionsystemsarestillfrequentlyuseddespitethesafetyand
regulatoryriskstheypose.
Herewedescribetheexpansionandcharacterization
ofNKcellsgrowninafeeder-freeculturesystemusing
Gibco?CTS?NK-Xpander?Medium,whichcansupportanaverage1,500-foldexpansionofNKcellswithintwoweeks.
ThermoFisher
SCIENTIFIC
Materialsandmethods
EnrichedNKcellswereexpandedfor14daysineitherCTSNK-Xpander
Mediumwithsupplementation,classicmediumwithsupplementation,oranalternatefeeder-freemediasystem.
NKcellcultureswerefedevery
2–3daysbeginningonday5.Thelevelofexpansionwasmeasuredoneveryfeedday,andphenotypicandfunctionalcharacterizationwasperformedonday14(Figure2).
Feeder-freeNKcellexpansionCD56?NKcellsenriched
from20differentdonorsof
peripheralbloodmononuclearcells(PBMCs)wereplatedat
1.25x10?cells/mLin200μLperwellinuntreatedThermoScientific?Nunc?96-wellplates(Cat.No.268200).
Thecellswereculturedfor14days
inoneoffourmedia:(1)CTSNK-
XpanderMedium(Cat.No.A5019001)containing500U/mLrecombinant
humanIL-2(Cat.No.PHC0023)and5%hABserum(FisherScientific
Cat.No.BP2525100);(2)axeno-freeNKspecialtymediumfromsupplier1supplementedwith500U/mL
recombinanthumanIL-2and5%hABserumperproductinstructions;
(3)Gibco?RPMI1640Medium
(Cat.No.11875093)supplementedwith500U/mLrecombinant
humanIL-2and5%FBS(Cat.No.
16140063);and(4)RPMI1640
Mediumsupplementedwith500U/mLrecombinanthumanIL-2and5%
hABserum.Thecellswerestainedwithtrypanblue(Cat.No.15250061)
andcountedusingtheInvitrogen?Countess?2FLAutomatedCell
Counter(Cat.No.AMQAX1000).
Beginningonday5,cultureswerefedevery2–3daystomaintainanoptimalcelldensityof4–5x10?cells/mL.
25,000cellsplatedin96-wellplate
EnrichedNKcellsCD56+NKcells
fromPBMCs
CTSNK-XpanderMedium+5%hABserum
+500U/mLIL-2
14daysculturefeedingandsplittingevery2–3days
?Totalnucleatedcells
?Percentviability
?Flowphenotyping
?Cytotoxicityassessment
Figure2.PrimaryNKcellexpansionandcharacterizationmethodology.
NKcellphenotypiccharacterization
NKcellshavetraditionallybeenidentifiedbythepresenceofCD56andthe
absenceofCD3receptorsontheirsurfacesviaflowcytometry.NKcellscanbefurthercategorizedintoavarietyofsubsetsbasedonmanydifferentcellsurfacemarkers,includingCD16andtheKIRfamilyofreceptors.Fortheseexperiments,expandedNKcellsweregatedforlivecellsusingtheInvitrogen?LIVE/DEAD?
FixableDeadCellStainKit(Cat.No.L34964).ThelevelsofCD56,CD3,andCD16weremeasuredusinglabeledantibodiesspecifictothesemarkers,andtheInvitrogen?Attune?NxTAcousticFocusingCytometer(Cat.No.A42858).
NKcellfunctionality
Figure3illustratesthefunctionalcharacterizationstrategy.NKcells(effector
cells)expandedinCTSNK-XpanderMediumwereco-incubatedwithK562
targetcellslabeledwiththeInvitrogen?CellTrace?CFSECellProliferationKit
(Cat.No.C34570)for2hours.TheratiosofNKcellstoK562cellswere0.625:1,1.25:1,2.5:1,and5:1.Followingincubation,degranulationwasassessedbytheexpressionofCD107aonCD56?NKcells,measuredusingalabeledantibodyspecifictothismarker,andtheAttuneNxTAcousticFocusingCytometer.NK
cellcytotoxicitywasassessedbymeasuringK562celldeathontheAttune
NxTFlowCytometer;thegatewassetforCFSE-labeledK562cells,and
percentagesofliveanddeadcellsweredeterminedusingtheLIVE/DEADstainkit(Cat.No.L34964).
ExpandedNKcells
CSFE-labeledK562cells
+
Target
IncubateexpandedNKcellswithlabeledK562cellsfor2hr
?NKcelldegranulation
?MeasureK562celldeath
Efector
Figure3.NKcellfunctionalitywasassessedbymeasurementofdegranulationandtheabilityoftheNKcellstokillK562targetcells.
Results
NKcellsthatwereexpandedusingCTSNK-XpanderMediummaintainedtheirphenotypeandfunctionality.
Feeder-freeNKcellexpansion
PBMC-derivedNKcellsfrom20differentdonorswere
expandedfor14daysineitherCTSNK-XpanderMedium,classicmedia,oranNKspecialtymediumfromsupplier1.Asexpected,expansionlevelsvariedfromdonor
todonorandbetweenthedifferentmediasystems
tested.Cellexpansioninthedifferentmediaover14daysiscomparedinFigure4A,andFigure4Bshowsadonor-to-donorcomparison.ExpansioninCTSNK-Xpander
Mediumwassignificantlyhigherthanitwasinanyothermediumsystemtested(p<0.0001).InCTSNK-XpanderMedium,expansionwassignificantlyhigherthanitwasinthemediumfromsupplier1for80%ofthedonorstested.
Expansionofcellsfromthe20donorsinsupplier1mediumwas732-foldonaverageafter14days,whereasexpansionofcellsfromthesamedonorsinCTSNK-XpanderMediumwas1,508-foldonaverageafter14days.
A
Foldexpansion
1,800
1,600
1,400
1,200
1,000
800
600
400
200
0
Day5Day7Day10Day12Day14
Medium
NK-XpanderRPMI-FBS RPMI-hABSupplier1
B
Medium
NK-XpanderSupplier1
3,500
Day14foldexpansion
3,000
2,500
2,000
1,500
1,000
500
0
Donor1
Donor2
Donor3
Donor4
Donor5
Donor6
Donor7
Donor8
Donor9
Donor10
Donor11
Donor12
Donor13
Donor14
Donor15
Donor16
Donor17
Donor18
Donor19
Donor20
Donorblinded
Figure4.ExpansionofNKcellcultureinCTSNK-XpanderMediumcomparedtoexpansioninothermediasystems.(A)Average
expansionofcellsfrom20differentdonorsover14days.Errorbars
represent1standarderrorfromthemean.(B)Donor-to-donorcomparisonafter14daysofexpansion.Errorbarsrepresent1standarddeviationfromthemean.
NKcellphenotypiccharacterization
ThepurityofNKcellsexpandedinCTSNK-Xpander
Mediumandthesupplier1mediumwascomparable,whileNKcellsexpandedinRPMImediumsupplementedwith
eitherFBSorhABserumhadlowerpurity(Figure5).CellsgrowninCTSNK-XpanderMediumhadsignificantlygreaterexpansionbutsimilarpurityrelativetocellsgrowninotherNKcell–specificexpansionsystems.
A
B
Day14cellpurity
CelltypemNKcellsmNKTcellsTcells
100
%Celltype
80
Other
60
40
20
0
NK-XpanderRPMI-FBSRPMI-hABSupplier1
Medium
C
Foldexpansion
1,800
1,600
1,400
1,200
1,000
800
600
400
200
0
NK-XpanderRPMI-FBSRPMI-hABSupplier1
Medium
Figure5.PhenotypiccharacterizationofNKcells.ExpansionofNKcellsinCTSNK-XpanderMediumwassignificantlyhigher,withnoloss
inpurity.(A)Gatingstrategy,(B)purity,and(C)expansionbyday14indifferentmediasystems.Errorbarsrepresent1standarderrorfromthemean.
NKcellfunctionalityAB
NKcellsgrowninCTSNK-XpanderMediumretainedtheirdose-dependentabilitytodegranulate(Figure6),andtheydisplayedcytolyticfunctionality(Figure7)afterexposuretoK562targetcells.ThesearekeyindicatorsofNKcell
functionalityinvitro.
AB
Degranulation(%)
C
80
70
60
50
40
30
20
10
0
0.625:11.25:12.5:15:1
Cellkilling(%)
120
100
80
60
40
20
0
C
0.625:11.25:12.5:15:1
NK:K562ratio
Figure7.NKcellsexpandedinCTSNK-XpanderMediumcankill
K562targetcellsinadose-dependentmanner.Representativegating
isshownfor(A)K562cellsonlyand(B)K562cellsexposedtoNKcells.
(C)ThepercentageofK562cellkillingatvaryingratiosofNKcellstoK562cells.EachdotrepresentsoneNKcelldonor.
NK:K562ratio
Figure6.NKcellsexpandedinCTSNK-XpanderMediumarecapableofdegranulationinadose-dependentmanner,asdemonstrated
bysurfaceCD107aexpression.ComparisonofCD107aexpression
in(A)NKcellsonlyand(B)NKcellsexposedtoK562cells.(C)The
percentageofNKcelldegranulation(CD107a?)atvaryingratiosofNKcellstoK562cells.EachdotrepresentsoneNKcelldonor.
gibco
Conclusions
Whilefeeder-freeculturesystemsaretypicallyseenas
asaferchoiceforNKcellexpansionthanfeeder-basedculturesystems,NKcellexpansioninexistingfeeder-freesystemslikeRPMIandserumissignificantlylower.TherecentdevelopmentofNKcell–specificspecialtymediahasbegunthetransitiontotrulyfeeder-freeculture
systems,buttheystillleavemuchtobedesiredintermsofexpansion.
CTSNK-XpanderMediumoffersasolutiontothis
challenge:feeder-freeNKcellculturewithCTSNK-XpanderMediumcandeliveruptoanaverage1,500-foldexpansionoffunctionalNKcells.CTSNK-XpanderMediumisaxeno-freeNKcellculturemediumformulatedforfeeder-free
culturethatcanhelpreduceregulatoryrisksforyourNKcellexpansionprotocol.
Orderinginformation
Product
Quantity
Cat.No.
Expand
CTSNK-XpanderMedium
500mLbottle
A5019001
5Lbag
A5019002
HumanIL-2RecombinantProtein
1mg
PHC0023
CTSDPBS,withoutcalciumchloride,withoutmagnesiumchloride
2Lbag
A1285602
HumanABSerum
100mL
FisherScientific,BP2525100
Nuncnon-treated96-wellplates
Caseof160
268200
Nuncnon-treated48-wellplates
Analyze
Caseof75
150787
Countess3FLAutomatedCellCounter
1instrument
AMQAF2000
TrypanBlueSolution,0.4%
100mL
15250061
CellTraceCFSECellProlife
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