利用NK-Xpander进行NK细胞扩增与鉴定

APPLICATIONNOTE

CTSNK-XpanderMedium

Expansionandcharacterizationof

humannaturalkiller(NK)cells

OptimizeyourNKcellexpansionprotocolwith

CTSNK-XpanderMedium

Introduction

Naturalkiller(NK)cellsarelymphocytesthateitherdirectly

attackoractivatetheadaptiveimmunesysteminresponse

todetectionofmalignantorvirallyinfectedcells.They

haveawell-knownsafetyprofileandtheabilitytoactas

allogeneicimmunemodulators,whichmakesNKcellsan

attractiveoptionforoff-the-shelfimmunotherapeutics[1].

Liuetal.[2]outlinethemanyexpansionandactivation

strategiesunderexploration,includingcytokine-only

expansion,autologousandallogeneicfeederstrategies,

engineeredcelllinefeeder–basedsystems,andeven

strategiestoengineercell-freeparticlesthatmimicthe

activityofengineeredfeedercells.Feeder-basedprotocols

havehistoricallyprovidedthehighestratesofexpansion.

Thetypicalexpansionrangeislessthan10-foldfor

cytokine-onlysystems,lessthan500-foldforautologous

andallogeneicfeeder–basedsystems,andwellover1,000-

foldforengineeredcelllinefeeder–basedsystems[2].

Intheclinicalsetting,strategiesarebeingdevelopedto

improveNKcellfunction,persistence,andtraffickingto

tumorsites[2].Theseincludeinsertingchimericantigen

receptors(CARs)andusingcomplementaryantibodiesto

enhancetheidentificationanddestructionoftumorcells,

aswellasexvivoexpansionandactivationtechniques.

Safetyconcernsaboutfeedercells,includingtheriskof

contaminatingcellsinthefinaltherapeuticproduct,make

feeder-freesystemsadesirablechoiceforclinicalresearch

applications.However,lowerexpansionratesinfeeder-

freeculturesystemspresentachallengetowidespread

adoption.Thisisakeyreasonwhyfeeder-basedexpansion

systemsarestillfrequentlyuseddespitethesafetyand

regulatoryriskstheypose.

Expand

Analyze

•CTSNK-XpanderMedium

•RecombinanthumanIL-2

•HumanABserum

•Countess3FLAutomated

CellCounter

•AttuneNxTAcoustic

FocusingCytometer

Herewedescribetheexpansionandcharacterization

ofNKcellsgrowninafeeder-freeculturesystemusing

Gibco™CTS™NK-Xpander™Medium,whichcansupportan

average1,500-foldexpansionofNKcellswithintwoweeks.

•Stainingsolutionsand

monoclonalantibodies

Figure1.Optimizedworkflowforfeeder-freeexpansionand

characterizationofNKcells.

Materialsandmethods

EnrichedNKcellswereexpandedfor

14daysineitherCTSNK-Xpander

Mediumwithsupplementation,classic

mediumwithsupplementation,oran

alternatefeeder-freemediasystem.

NKcellcultureswerefedevery

2–3daysbeginningonday5.The

levelofexpansionwasmeasured

oneveryfeedday,andphenotypic

andfunctionalcharacterizationwas

performedonday14(Figure2).

25,000cellsplatedCTSNK-XpanderMedium

•Totalnucleatedcells

•Percentviability

in96-wellplate

+5%hABserum

+500U/mLIL-2

•Flowphenotyping

•Cytotoxicityassessment

EnrichedNKcells

fromPBMCs

14daysculturefeedingand

splittingevery2–3days

CD56+NKcells

Figure2.PrimaryNKcellexpansionandcharacterizationmethodology.

NKcellphenotypiccharacterization

NKcellshavetraditionallybeenidentifiedbythepresenceofCD56andthe

absenceofCD3receptorsontheirsurfacesviaflowcytometry.NKcellscanbe

furthercategorizedintoavarietyofsubsetsbasedonmanydifferentcellsurface

markers,includingCD16andtheKIRfamilyofreceptors.Fortheseexperiments,

expandedNKcellsweregatedforlivecellsusingtheInvitrogen™LIVE/DEAD™

FixableDeadCellStainKit(Cat.No.L34964).ThelevelsofCD56,CD3,and

CD16weremeasuredusinglabeledantibodiesspecifictothesemarkers,and

theInvitrogen™Attune™NxTAcousticFocusingCytometer(Cat.No.A42858).

Feeder-freeNKcellexpansion

CD56+NKcellsenriched

from20differentdonorsof

peripheralbloodmononuclear

cells(PBMCs)wereplatedat

1.25x105cells/mLin200µLperwell

inuntreatedThermoScientific™Nunc™

96-wellplates(Cat.No.268200).

Thecellswereculturedfor14days

inoneoffourmedia:(1)CTSNK-

XpanderMedium(Cat.No.A5019001)

containing500U/mLrecombinant

humanIL-2(Cat.No.PHC0023)and

5%hABserum(FisherScientific

Cat.No.BP2525100);(2)axeno-free

NKspecialtymediumfromsupplier

1supplementedwith500U/mL

recombinanthumanIL-2and5%hAB

serumperproductinstructions;

(3)Gibco™RPMI1640Medium

(Cat.No.11875093)supplemented

with500U/mLrecombinant

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,degranulationwasassessedbythe

expressionofCD107aonCD56+NKcells,measuredusingalabeledantibody

specifictothismarker,andtheAttuneNxTAcousticFocusingCytometer.NK

cellcytotoxicitywasassessedbymeasuringK562celldeathontheAttune

NxTFlowCytometer;thegatewassetforCFSE-labeledK562cells,and

percentagesofliveanddeadcellsweredeterminedusingtheLIVE/DEADstain

kit(Cat.No.L34964).

ExpandedNK

cells

CSFE-labeled

K562cells

IncubateexpandedNKcells

withlabeledK562cellsfor2hr

humanIL-2and5%FBS(Cat.No.

16140063);and(4)RPMI1640

+

•NKcelldegranulation

Mediumsupplementedwith500U/mL

recombinanthumanIL-2and5%

hABserum.Thecellswerestained

withtrypanblue(Cat.No.15250061)

andcountedusingtheInvitrogen™

Countess™2FLAutomatedCell

Counter(Cat.No.AMQAX1000).

Beginningonday5,cultureswerefed

every2–3daystomaintainanoptimal

celldensityof4–5x105cells/mL.

•MeasureK562celldeath

Eꢀector

Target

Figure3.NKcellfunctionalitywasassessedbymeasurementofdegranulationandtheability

oftheNKcellstokillK562targetcells.

Results

NKcellsthatwereexpandedusingCTSNK-Xpander

Mediummaintainedtheirphenotypeandfunctionality.

NKcellphenotypiccharacterization

ThepurityofNKcellsexpandedinCTSNK-Xpander

Mediumandthesupplier1mediumwascomparable,while

NKcellsexpandedinRPMImediumsupplementedwith

eitherFBSorhABserumhadlowerpurity(Figure5).Cells

growninCTSNK-XpanderMediumhadsignificantlygreater

expansionbutsimilarpurityrelativetocellsgrowninother

NKcell–specificexpansionsystems.

Feeder-freeNKcellexpansion

PBMC-derivedNKcellsfrom20differentdonorswere

expandedfor14daysineitherCTSNK-XpanderMedium,

classicmedia,oranNKspecialtymediumfromsupplier

1.Asexpected,expansionlevelsvariedfromdonor

todonorandbetweenthedifferentmediasystems

tested.Cellexpansioninthedifferentmediaover14days

iscomparedinFigure4A,andFigure4Bshowsadonor-

to-donorcomparison.ExpansioninCTSNK-Xpander

Mediumwassignificantlyhigherthanitwasinanyother

mediumsystemtested(p<0.0001).InCTSNK-Xpander

Medium,expansionwassignificantlyhigherthanitwasin

themediumfromsupplier1for80%ofthedonorstested.

Expansionofcellsfromthe20donorsinsupplier1medium

was732-foldonaverageafter14days,whereasexpansion

ofcellsfromthesamedonorsinCTSNK-XpanderMedium

was1,508-foldonaverageafter14days.

A

B

Day14cellpurity

Celltype

100

80

NKcells

NKTcells

Tcells

Other

60

40

20

A

1,800

1,600

1,400

1,200

1,000

800

Medium

NK-Xpander

0

RPMI-FBS

RPMI-hAB

Supplier1

NK-Xpander

RPMI-FBS

RPMI-hAB

Supplier1

Medium

C

1,800

1,600

1,400

1,200

1,000

800

600

400

200

0

Day5

Day7

Day10

Day12

Day14

B

3,500

Medium

600

NK-Xpander

Supplier1

3,000

2,500

2,000

1,500

1,000

500

400

200

0

NK-Xpander

RPMI-FBS

RPMI-hAB

Supplier1

Medium

Figure5.PhenotypiccharacterizationofNKcells.ExpansionofNK

cellsinCTSNK-XpanderMediumwassignificantlyhigher,withnoloss

inpurity.(A)Gatingstrategy,(B)purity,and(C)expansionbyday14

indifferentmediasystems.Errorbarsrepresent1standarderrorfrom

themean.

0

Donorblinded

Figure4.ExpansionofNKcellcultureinCTSNK-XpanderMedium

comparedtoexpansioninothermediasystems.(A)Average

expansionofcellsfrom20differentdonorsover14days.Errorbars

represent1standarderrorfromthemean.(B)Donor-to-donorcomparison

after14daysofexpansion.Errorbarsrepresent1standarddeviationfrom

themean.

NKcellfunctionality

A

B

NKcellsgrowninCTSNK-XpanderMediumretainedtheir

dose-dependentabilitytodegranulate(Figure6),andthey

displayedcytolyticfunctionality(Figure7)afterexposure

toK562targetcells.ThesearekeyindicatorsofNKcell

functionalityinvitro.

A

B

C

120

100

80

60

40

20

0

C

80

70

60

50

40

30

20

10

0

0.625:1

1.25:1

2.5:1

5:1

NK:K562ratio

Figure7.NKcellsexpandedinCTSNK-XpanderMediumcankill

K562targetcellsinadose-dependentmanner.Representativegating

isshownfor(A)K562cellsonlyand(B)K562cellsexposedtoNKcells.

(C)ThepercentageofK562cellkillingatvaryingratiosofNKcellstoK562

cells.EachdotrepresentsoneNKcelldonor.

0.625:1

1.25:1

2.5:1

5:1

NK:K562ratio

Figure6.NKcellsexpandedinCTSNK-XpanderMediumarecapable

ofdegranulationinadose-dependentmanner,asdemonstrated

bysurfaceCD107aexpression.ComparisonofCD107aexpression

in(A)NKcellsonlyand(B)NKcellsexposedtoK562cells.(C)The

percentageofNKcelldegranulation(CD107a+)atvaryingratiosofNKcells

toK562cells.EachdotrepresentsoneNKcelldonor.

Conclusions

Whilefeeder-freeculturesystemsaretypicallyseenas

asaferchoiceforNKcellexpansionthanfeeder-based

culturesystems,NKcellexpansioninexistingfeeder-free

systemslikeRPMIandserumissignificantlylower.The

recentdevelopmentofNKcell–specificspecialtymedia

hasbegunthetransitiontotrulyfeeder-freeculture

systems,buttheystillleavemuchtobedesiredinterms

ofexpansion.

CTSNK-XpanderMediumoffersasolutiontothis

challenge:feeder-freeNKcellculturewithCTSNK-Xpander

Mediumcandeliveruptoanaverage1,500-foldexpansion

offunctionalNKcells.CTSNK-XpanderMediumisaxeno-

freeNKcellculturemediumformulatedforfeeder-free

culturethatcanhelpreduceregulatoryrisksforyourNK

cellexpansionprotocol.

Orderinginformation

Product

Quantity

Cat.No.

Expand

500mLbottle

5Lbag

A5019001

CTSNK-XpanderMedium

A5019002

HumanIL-2RecombinantProtein

CTSDPBS,withoutcalciumchloride,withoutmagnesiumchloride

HumanABSerum

1mg

PHC0023

2Lbag

A1285602

100mL

FisherScientific,BP2525100

268200

Nuncnon-treated96-wellplates

Caseof160

Caseof75

Nuncnon-treated48-wellplates

150787

Analyze

Countess3FLAutomatedCellCounter

1instrument

100mL

AMQAF2000

15250061

C34570

TrypanBlueSolution,0.4%

CellTraceCFSECellProliferationKit

1kit

eBioscienceFlowCytometryStainingBuffer

FcRe