应用指南 - 利用NK-Xpander进行NK细胞扩增与鉴定 Expansion and characterization of human natural killer (NK) cells

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

1.25x10⁵cells/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,cultureswerefedevery2–3daystomaintainanoptimalcelldensityof4–5x10⁵cells/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