Activation and Expansion of Human Treg Cells

DynabeadsHumanTregExpanderoffersasimplemethodforexpansionofTregcellsthatdoesnotrequireantigen-presentingcellsorantigen.JustaddDynabeads?HumanTregExpanderandrecombinantIL-2(rIL-2)totheTregculturetoexpandtheTregcells.Cellculturesshowingsignsofexhaustioncanbere-stimulatedbyaddingfreshbeadsandrIL-2.

Ready-to-useDynabeads?HumanTregExpanderofferthefirstartificialantigen-presentingcellstoprovidesimultaneoussignalstoTCR/CD3andCD28forfullactivationandexpansionofhumanTregcells.

2mlDynabeads?HumanTregExpander

2x107beads/mlinphosphatebufferedsaline(PBS),pH7.4,w/0.1%humanserumalbumin(HSA).

Magnet:AnyDynalMPC

Buffer1:PBSw/0.1%BSA,pH7.4

CulturalMedium:OpTmizer?T-CellExpansionSFM(Gibco)serumfree1XformulationdesignedtosupportthecultureandexpansionofhumanTcells(orequivalent)

Roundbottomtissuecultureplatesortissuecultureflasksofsuitablesize

HumidifiedCO2 incubator

Dynal CD4CD25TregKit

rIL-2

1. DynabeadsWashingProcedure

Dynabeads shouldbewashedbeforeusewiththeaidofamagnet.

（1）ResUSPendtheDynabeads inthevial.

（2）TransferthedesiredvolumeofDynabeads toatube.

（3）AddthesamevolumeofBuffer1astheinitialvolumeofDynabeads,oratleast1ml,andmix.

（4）Placethetubeinamagnetfor1-2minutesuntiltheDynabeads?areseparated;discardthesupernatant.Removethetubefromthemagnet.

（5）ResuspendthewashedDynabeadsinthesamevolumeofBuffer1astheinitialvolumeofDynabeads.

2. IsolationofHumanTregCells

ForisolationofhumanTregcellsitisrecommendedtousetheDynalCD4CD25TregKit.

3. ExpansionofHumanTregCellsDay0:

Startwith1x105 Tregcellsin100μlCultureMediuminaroundbottom96welltissuecultureplate.

Add20μlDynabeadsHumanTregExpander.

Add500U/mlrIL-2.

Day1:

Add100μlCultureMediumcontaining

500U/mlrIL-2.

Day3:

Resuspendandsplitthecultureinhalfandadd100μlCultureMediumcontaining

500U/mlrIL-2 perwell.

Day5-7:

Splittheculturewhenneeded.Examineculturesdaily,notingcellsizeandshape.Cellshrinkingandreducedproliferationrateistypicallyobservedinexhaustedcellcultures.Countthecellsatleasttwiceweeklyafterthoroughresuspension.

Whenthecelldensityexceeds2x106cells/mlorwhenthemediumbecomesyellow,splitculturestoadensityof0.5-1.0x106cells/mlinCultureMediumcontaining500U/mlrIL-2.Growthecellsuntilthewellishalf-full(~500,000cells)andtransferthecellsfroma96welltoa24wellplate.

Day8:

RemovetheDynabeadsbyresuspendingthecellsandtransferringthecellstoasuitabletube.Placethetubeinamagnetfor1-2minutesuntiltheDynabeads?areseparated.CentrifugethesupernatantandresuspendthecellpelletinfreshCultureMediumcontaining100U/mlrIL-2.SplitthecultureswhenneededandrestthecellsuntilDay21-24inculturemediumwith100U/mlrIL-2.IncubateinahumidifiedCO2incubatorat37°C.

4. Re-stimulationofHumanTregCells

Thecellscanbere-stimulated15-18daysafterthefirststimulation,orwhencellshrinkingandreducedrateofproliferationisobserved.

（1）Beforere-stimulation:

i. Countthecellsandsplittheculturestoadensityof1x106 cells/mlinCultureMedium.

ii. UsethevolumesgiveninTable2andfollowtheprotocolbelow.

（2）Day0:

i. Startwith1x106Tregcellsin1mlCultureMediumina24welltissuecultureplate.

ii. Add50μlDynabeadsTregExpander.

iii. Add100U/mlrIL-2.

（3）Day1:

i. Add100μlCultureMediumcontaining100U/mlrIL-2 perwell.

（4）Day2or3:

i. Resuspendandsplitthecultureinhalfandadd100μlCultureMediumcontaining100U/mlrIL-2 perwell.

（5）Day5-7:

i. Splittheculturewhenneeded.

ii. Examineculturesdaily,notingcellsizeandshape.Cellshrinkingandreducedproliferationrateistypicallyobservedinexhaustedcellcultures.

iii. Countthecellsatleasttwiceweeklyafterthoroughresuspension.

Whenthecelldensityexceeds2x106 cells/mlorwhenthemediumbecomesyellow,splitculturestoadensityof0.5-1.0x106 cells/mlinCultureMediumcontaining100U/mlrIL-2.

（6）Day8:

i. RemovetheDynabeadsbyresuspendingthecellsandtransferringthecellstoasuitabletube.

ii. Placethetubeinamagnetfor1-2minutesuntiltheDynabeadsareseparated.

iii. CentrifugethesupernatantandresuspendthecellpelletinfreshCultureMediumcontaining100U/mlrIL-2.

iv. SplitthecultureswhenneededandrestthecellsuntilDay21-24inculturemediumwith100U/mlrIL-2.

Thisproductisforresearchuseonly.Notintendedforanyanimalorhumantherapeuticordiagnosticuseunlessotherwisestated.Followappropriatelaboratoryguidelines.

Thisproductcontains0.02%sodiumazideasapreservative,whichiscytotoxic.Avoidpipettingbymouth!

Sodiumazidemayreactwithleadandcopperplumbingtoformhighlyexplosivemetalazides.Whendisposingthroughplumbingdrains,flushwithlargevolumesofwatertopreventazidebuildup.