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Table 9 Protocol features for stirred systems

From: Lymphocyte expansion in bioreactors: upgrading adoptive cell therapy

Author Protocol Stirred system Expansion Static control Purity Functionality
Stimulation strategy Medium Key Supplements Vessel Stir. speed
[rpm]
Aerat. rate DO Seed density [cells/mL] Volume [ml] Fold Days Fold Days   
T cells
 Ou −2019 [90] IL-2;anti CD3/CD28 beads or mABs OpTmizer CTS Serum-free 2 L Stirred vessel 70 0.01 vvm 70% 5 × 105 800 132–1011 4 > 99% CD4+ and CD8+ T cells High CD3 expression (91.7–99.4%);
T cell co-stimulatory signaling receptor (ICOS/CD278) elevated; upregulated expression of PD-1/CD279;
production of IFNγ decreased by ~ 50%
 Costariol – 2019 [89] IL-2; anti CD3/CD28 beads RPMI 1640 10% FBS; Glutamine Spinner 35 5 × 105 100 No growth CD4:CD8 ratio decreased from 4:1 to 1:1
ambr 250 100–200 14.25 mL/min 21% O2 60% 5 × 105 250 23.2 ± 1.3 7 15.2 ± 3.1 7   Effector memory cells increased from 35.69 ± 10.98% to ~ 80%;
no significant difference in T cell subpopulation profiles between static and ambr® 250
 Ramsborg – 2004 [142] IL-2; anti CD3/CD28 mABs AIM V 2% AP Spinner 60 1.5 × 105–3.0 × 105 100 5.4 (0.5–87) 15    CD3 cells > 90%;
CD8 cells preferentially expanded over CD4 cells
 Foster – 2004 [143] PHA or OKT3; 50 IL2; RPMI 1640 10% AB; Glutamine, HEPES, pyruvate Spinner 50 5 × 105 100 > 10 7 > 10 7 NLV–tetramer + CD8+ phenotype > 95% CD25 decreased exponentially. This behavior did not vary significantly between suspension and static cultures;CTL maintained their specificity toward CMVpp65
 Bohnenkamp – 2002 [140] IL-2; pre anti CD3/CD28 mABs aMEM 10% FBS Spinner 394 9 > 94% CD3+ cells;
CD4:CD8 from 2.4:1 to 1:5 (stirred vessel), 1:2.7 (suspension bioreactor) and 1:4.8 (T-flask)
1 L stirred vessel 1.35 × 105 44 10 Not different IL-2R was downregulated earlier than in the static T-flask culture;
IFNγ secretion assay against a hCMV protein maintained
Stirred 50–550 mL 5 × 105 60 10 Not different
 Hilbert −2001 [144] IL-2;anti CD3/CD28 mABs RPMI 1640 OR AIMV 10% FBS; Glutamine 18 L stirred vessel 18,000 11 10 No difference   > 90% T-cells
550 mL cell retention reactor   20–50% 3.8 × 105 550 67 5
 Carswell – 2000 [145] IL-2, pre PHA or antiCD3 RPMI 1640 10% FBS; Glutamine; pyruvate, neAAs Spinner 60 5% 100 1214 ± 374 16
NK cells
 Pierson – 1996 [81] IL-2;
pre anti-CD5/CD8 mABs
(DMEM)/Ham’s F12 10% FBS; ascorbic acid; selenite Reactor 60 40% 106 530 352 33 > 90% NK-specific cytolytic function maintained
Spinner 60 106 250 107 ± 17 28 43 ± 11 28 86 ± 9,5% CD56+/CD3-; similar to static 75% specific lysis of K562; similar to static