应用指南 - 封闭自动化系统Rotea洗涤浓缩NK细胞 Closed,automated wash and concentration of expanded human natural killer (NK) cells

APPLICATIONNOTECTSRoteaCounterflowCentrifugationSystem

Closed,automatedwashandconcentrationofexpandedhumannaturalkiller(NK)cells

ImprovetheefficiencyofNKcellprocessingwiththeCTSRoteaCounterflowCentrifugationSystemandCTSNK-XpanderMedium

Introduction

Oneofthekeychallengesfacedbythecellandgenetherapyindustryispoorefficiencyincellprocessing.Akeyimprovementbeingmadeistheshifttowardclosedandautomatedsystems,whichcanhelpreducethe

riskofcontaminationanderror,aswellasprovidetheabilitytoprocessmultipleproductsinparallelinlesscontrolledspaces.

TheGibco™CTS™Rotea™CounterflowCentrifugation

Systemisaclosedcellprocessingsystemdeveloped

specificallyforsmall-batchcelltherapymanufacturing.

Itprovidesoutputvolumesaslowas5mLwithhigh

cellrecoveryandviability,andiscontrolledviauser-

programmablesoftwaretoenablecreationofprotocolsformanydifferentprocesses.TheCTSRoteasystemhasbeensuccessfullyusedinvariousstepsoftheCART

cellprocessingworkflow,includingisolationofperipheralbloodmononuclearcells(PBMCs),cellwashing,and

concentrationofengineeredCARTcells.

HerewedemonstrateuseoftheCTSRoteaCounterflowCentrifugationSystemforautomatedwashingand

concentrationofhumanNKcellsexpandedinGibco™CTS™NK-Xpander™Medium(Figure1).

Materialsandmethods

EnrichedNKcellswereexpandedinCTSNK-Xpander

Medium,harvestedonday17,washed,andconcentrated

usingtheCTSRoteaCounterflowCentrifugation

System.Subsequenttothis,phenotypicandfunctionalcharacterizationwasperformed.

ABCDEFG

H

2

2

1

FORWARD

Figure1.CTSRoteaCounterflowCentrifugationSystemandCTSNK-XpanderMedium.

Expand

Washandconcentrate

Analyze

•CTSNK-XpanderMedium

•IL-2recombinanthumanprotein

•HumanABserum

•CTSRotea

Counterflow

CentrifugationSystem

(instrument,

software,andsingle-usekits)

•CTSDPBS,bagformat

•Countess

AutomatedCellCounter

•Stainingsolutions

•AttuneNxT

AcousticFocusingCytometer

•Monoclonalantibodies

ThermoFisher

SCIENTIFIC

Feeder-freeNKcellexpansionandactivation

EnrichedCD56⁺NKcellsfromPBMCswereculturedpertheCTSNK-XpanderMedium

protocol

andscaledup

toafinalvolumeof1.5L.Briefly,NKcellswereplatedat

1.25x10⁵cells/mLat200µLperwellinThermoScientific™

Nunc™non-treated96-wellplatesandculturedfor

17daysinCTSNK-XpanderMedium(Cat.No.A5019001)containing500U/mLrecombinanthumanIL-2(Cat.No.

PHC0023)and5%humanABserum(FisherScientific

Cat.No.BP2525100).Thecellswerefedevery2–3daysbeginningonday5tomaintainanoptimalcelldensityof4–5x10⁵cells/mL.Ascellsgrew,theyweretransferred

frominitial48-wellplatesandsplitintomultiple6-well

plates,thenT-75flasks,andfinallytomultipleT-175

non–tissueculturetreatedflaskstoafinalvolumeof1.5L.CellswerestainedwithtrypanblueandcountedusingtheInvitrogen™Countess™IIFLAutomatedCellCounter.

Closed,automatedwashingandconcentrationofexpandedNKcells

Followingexpansion,NKcellwashingandconcentrationwereperformedusingtheCTSRoteaCounterflow

CentrifugationSystem.Priortoloadingontothe

CTSRoteasystem,asingle-usekitwasconstructed

usingbagconnectionsmadeviastandardwelding

techniques(Figure2).TheCTSRoteaSystemwasprimedbyreplacingairinthesystemwithbuffer.Thecellswereloadedintothechambertoformafluidizedbed.Fresh

washbuffer,consistingofGibco™CTS™DPBS(withoutCaCl2andMgCl2)and2%humanserumalbumin(NovaBiologics,Cat.No.68982-0643-02),wasallowedtoflow

throughthebedtowashthecells.Thecellswerethenconcentratedandharvestedfromthesystemforfurtherdownstreamprocessing.

Collectcells

Primesystem

Washcells

Loadcellsinchamber

AllCTSRoteaSystemprotocolswerewrittenusing

theGibco™CTS™Rotea™ProtocolBuilderdesktop

application.Table1liststhestepsoftheNKcellwashingandconcentrationprotocol,andFigure2illustratestheconfigurationoftheGibco™CTS™Rotea™Single-UseKit.

DGNKcellsinmedium

0mL

Figure2.CTSRoteaSingle-UseKitconfigurationforNKcellwashingandconcentration.

BDPBS+2%HSA

250mL

Celloutputbag

0mL

H

AWaste

0mL

Table1.SequenceofNKcellwashingandconcentrationprotocolontheRoteasystem,includinginitialprimingsteps.

StepDescriptionFlowpathSpeedFlowrateSteptypeTrigger

Primingsequence

1Pre-primeBtoA0xg100mL/minNormalInputbubblesensor

2

Lubricaterotarycoupling

BtoA

0xg

100mL/min

Normal

Volume:15mL

3

PrimechamberandlineA

BtoA

10xg

100mL/min

Normal

Volume:15mL

4

Addprimingvolume

BtoA

10xg

100mL/min

Normal

Volume:50mL

5

PrimebubbletrapandlineB

AtoB

10xg

100mL/min

Normal

Volume:15mL

6

PrimelineD

AtoD

10xg

50mL/min

Normal

Volume:5mL

7

Pressureprime

AtoEF

10xg

0mL/min

Pressureprime

8

Primepause

JtoK

10xg

25mL/min

Pause

Volume:3mL

9

Rampspeedtoinitiatebed

JtoK

2,200xg

50mL/min

Pause

Time:10sec

LoadingandwashingtheNKcells

10

Initiatebed

DtoG

2,200xg

50mL/min

Normal

Time:4min

11

Loadinputmaterial

DtoA

2,250xg

35mL/min

Normal

Volume:1xinputaliquot(mL)Inputbubblesensor,pause

12

Adjustspeedforwash

JtoK

2,400xg

25mL/min

Pause

Time:15sec

13

Wash

BtoA

2,400xg

25mL/min

Normal

Volume:30mL

14

Concentratebedforharvest

JtoK

2,500xg

15mL/min

Pause

Time:10sec

15

Harvest

BtoH

2,500xg

50mL/min

Harvest

Volume:1xharvestvolume(mL)

16

Ramptostop

KtoJ

500xg

50mL/min

Pause

Time:5sec

NKcellphenotypiccharacterization

ExpandedNKcellsweregatedforlivecellsusing

Invitrogen™LIVE/DEAD™FixableVioletDeadCellStainKit.TheirCD56,CD3,andCD16levelswerethenmeasuredusingappropriateantibodiesandtheInvitrogen™Attune™NxTAcousticFocusingCytometer.

NKcellfunctionality

NKeffectorcellsexpandedinCTSNK-XpanderMediumwerecoincubatedwithK562targetcellslabeledwith

theInvitrogen™CellTrace™CFSECellProliferationKitatNK:K562cellratiosof0.625:1,1.25:1,2.5:1,and5:1for

2hours.Followingincubation,degranulationwasassessedbasedontheexpressionofCD107abyCD56⁺NKcells,

measuredontheAttuneNxTAcousticFocusingCytometer.NKcellcytotoxicitywasassessedbymeasuringK562celldeathontheAttuneNxTsystembygatingforCFSE-labeledK562cellsandmeasuringthepercentageofdeadcells

usingtheLIVE/DEADstainkit.

Results

NKcellswereexpandedto1.83x10⁹cellsinafinalvolumeof1.62LusingCTSNK-XpanderMedium.

CellswashedandconcentratedusingtheCTSRotea

CounterflowCentrifugationSystemshowedhighrecoveryandviabilitypostwashandmaintainedtheirphenotypeandfunctionality.

Feeder-freeNKcellexpansionandactivation

PBMC-derivedNKcellsculturedinCTSNK-Xpander

Mediumexpandedby1,700-foldonaverageafter17days(Figure3).Theculturesstartedat1.13x10⁶cellsin9mLandincreasedto1.83x10⁹cellsin1.62L.

Foldexpansion

2,000

1,500

1,000

500

0

05710121417

Run1Run2Time(days)

Figure3.FoldexpansionofNKcellsculturedinCTSNK-XpanderMediumfor17days.

Closed,automatedwashingandconcentrationofexpandedNKcells

ExpandedcellswereloadedintotheCTSRoteasystemtoformastabilizedbedforsubsequentwashingin

CTSDPBS.Recoverywas~90%withhighviabilityandmaintenanceofcellularphenotype(Figure4).

A

120

89%

96%97%

100

Percentage

80

60

40

20

0

InputOutputRecovery

viabilityviability

Pre-wash

B

Post-wash

C

Figure4.NKcellwashingandconcentration.(A)NKcellviabilityandrecoveryaveragedoverfourwashingandconcentrationruns.(B)FlowcytometrystainingforCD56,CD16,andCD3beforeandafterwashingwiththeCTSRoteaSystemNKcellwashandconcentrationprotocol.

(C)CTSRoteaSystemsoftwareimagewithachamberofNKcellsinafluidizedbed.

NKcellfunctionality

CellswashedandconcentratedusingtheCTSRoteasystemmaintained

cytolyticfunctionandwereabletodegranulate(Figure5)andkillK562targetcells(Figure6)inadose-dependentmanner.

NKcellsonly

CCD--A::SSCACCD-A::SSC-A

Degranulation(%)

100

90

80

70

60

1.0M

800K

600K

400K

200K

0

CD107a

50

40

30

20

10

0

NKcells+K562cells1.0M

800K

600K

400K

200K

0

CD107a

Post-RoteaPre-Rotea

0.625:11.25:12.5:1

Efector:targetratio

5:1

Figure5.MaintenanceofNKcelldegranulationcapabilityafterwashingandconcentration.

K562cellsonly

100

Court

K562celldeath(%)

90

80

70

60

50

800

600

400

200

0

LIVE/DEADViolet

NKcells+K562cells

40

800

30

600

Court

20

400

10

200

0

0

LIVE/DEADViolet

Post-RoteaPre-Rotea

0.625:11.25:12.5:1

Efector:targetratio

5:1

Figure6.MaintenanceofNKcellcytotoxicityafterwashingandconcentration.

Conclusions

Criticalimprovementstocellandgenetherapymanufacturingcanbeachievedbyreducingriskandhands-ontimeusingregulation-compliantreagents

andclosedmanufacturingsystems.WehavedemonstratedefficientNKcellexpansioninafeeder-freeculturesystemaswellashighrecoveryafter

washingandconcentratingthecellsusingaclosed,automatedcounterflowcentrifugationsystem.Herewehavedemonstratedefficientexpansionof

NKcellsinafeeder-freeculturesystemusingCTSNK-XpanderMedium,andhighrecoveryofcellsduringwashandconcentrationusingtheCTSRotea

CounterflowCentrifugationSystem.

gibco

Orderinginformation

ProductQuantityCat.No.

Expansion

500mLbottleA5019001

CTSNK-XpanderMedium

5LbagA5019002

HumanIL-2RecombinantProtein

1mg

PHC0023

HumanABSerum

100mL

FisherScientific,BP2525100

Nuncnon-treated96-wellplates

Caseof160

268200

Nuncnon-treated48-wellplates

Analysis

Caseof75

150787

Countess3FLAutomatedCellCounter

1instrument

AMQAF2000

TrypanBlueSolution,0.4%

100mL

15250061

CellTraceCFSECellProliferationKit

1kit

C34570

eBioscienceFlowCytometryStainingBuffer

600mL

004222-26

FcReceptorBindingInhibitorPolyclonalAntibody

100tests

14-9161-73

UltraCompeBeadsCompensationBeads

100tests

01-2222-42

ArCAmineReactiveCompensationBeadKit

1kit

A10346

AttuneNxTAcousticFocusingCytometer

1instrument

A24858

CD56MonoclonalAntibody(CMSSB)

100tests

120567-42

CD3MonoclonalAntibody(OKT3)

100tests

11-0037-