Table 2. Expansion of NK cells in vitro for clinical practice*.
| Starting material | Initial cell number | Medium | Stimulators* Feeder cells | Culture instrument | Culture time/acquired cell number | Fold proliferation | Purity | Cytotoxicity | Phenotype, cytokine production | References** | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Cord blood-derived NK cells | CD34+ cell from cord blood (CliniMACS) | (0.89–6.34)×106 | Glycostem Basal Growth Medium +10% HS | SCF, IL-7, IL-15, IL-2, Flt3L, TPO, G-CSF, IL-6, LMWH | Vuelife™ bags, WAVE Bioreactor System 2/10, BIOSTATH CultiBag RM system | 6 weeks(1.6–3.7)×109 | 1435–2657 | >90% | K562 (>40%, 10∶1) | CD56+, CD3−, NKG2D+, NCRs+, CD161+, CD314+, CD244+ | [155] |
| CD34+ cell from cord blood (CliniMACS) | (0.84–2.50)×106 | Glycostem Basal Growth Medium | SCF, TPO, IL-7, Flt3L, IL-15, IL-2, G-CSF, GM-CSF, IL-6, LIF, MIP-1α | 24-well tissue culture plates | 14–35 days(1.9–7.8)×109 | ∼104 (freshly UCB);∼103 (frozen UCB) | >95% | K562, Lama, Kasumi, BLM, nd FM3 (>75%) KG1a (∼30%) (18 h 1∶1) | CD56+, CD3−, NKG2D+, NCRs+, CD107+, 2B4+, CD161+, IFN-γ | [154] | |
| Stem cell/iPSC- derived NK cells | CD34+CD45+ cells (H9 hESC line) | — | RPMI 1640+15% defined fetal bovine serum; DMEM/Ham F12+20% heat-inactivated human serum AB | IL-3, IL-15, IL-7, SCF and Flt3L;Feeder cells: M210-B4; AFT024 | — | 30–35 days | ∼100 | >37.5% | K562, MCF7, PC3 (55%–80%), NTERA2, and U87 (20%–30%) | CD56+, CD45+, CD16+, CD94+, NKG2D+, NKp46+, CD158a+, CD158b+, IFN-γ | [159,162,163] |
| BM CD34+ | — | Dulbecco's medium supplemented with 12.5% fetal calf serum; 12.5% horse serum | IL-2; Feeder cells: stromal cells from irradiated BMMNC | — | — | ∼690 | 75% | K562 (80%, 6.6∶1) | CD3−, CD56+, CD2+, CD7+, CD8+, CD16+ | [157] | |
| PBMCs | CD3−CD56+ cells from PBMCs (CliniMACS) | (0.40±0.16)×108 | CellGro SCGM serum-freeMedium, 5%AB human serum | IL-2, IL-15, anti-CD3 monoclonal antibody(MAb) OKT3 | Baxter LifeCell culture bags | 19 days(85.5±17.2)×108 | 268.3±66.8 | 100% | K562 (>60%, 10∶1) | CD3−, CD56+, NKG2D+, NCRs+, DNAM-1 | [166] |
| CD3−CD56+ cells from PBMCs | 3.0×106 | SCGMMedium and 10% fetal bovine serum | IL-2;Feeder cells: K562-mb15-41BBL | VueLife bag system | 7 days | 90.5 (33–141) | 83.1% (72.9%–85.9%) | K562, HL-60, KG1, and U937 (>40%, 4∶1) | CD3−, CD56+, NKG2D+, NCRs+ | [165] | |
| CD56+ cells from PBMCs | (9.5–85.8)×106 | Alpha-MEM, 20% fetal bovine serum | IL-15, HC | — | 20–23 days | 23 (3.2–131.3) | 97.9% (82.7%–99.6%) | K562, (23.2%, 7.0–54.7%, 1∶1) | CD3−, CD56+, NKG2D+, NCRs+ | [196] | |
| CD56+ cells from PBMCs (CliniMACS) | 2.0×108 | X-VIVO 2010% heat inactivated human AB serum | IL-2;Feeder cells: EBV-TM-LCL cells | Flasks and bags | 21 days3×1010 | 490±260 | 84.3%±7.8% | RCC (27.6±9.3%, 1∶1) | CD3−, CD56+, CD244+, CD48+, NKG2D+sFasL, IFN-γ, GM-CSF, TNF-α, MIP-1α, MIP-1β | [164] | |
| PBMCs | 2×106 NK cells | SCGMMedium and 10% fetal bovine serum | IL-2;Feeder cells: K562-mb15-41BBL | G-Rex100 flasks | 8–10 days | 209 (38–338) | 61% (54%–70%) | K562, U266 and Raji (>40%, 5∶1) | CD3−, CD56+ | [197] | |
| PBMCs | — | Serum-free medium and 10% heat-inactivated human plasma | rhIL-2; OK432;anti-CD16 | Cell-culture bag | 21 days | 637–5712 | 78.9%±11.6% | K562, Raji and Daudi (>20%, 3∶1) | CD3−, CD56+, CD158a+, CD158b1/b2+, CD159a+, CD69+, NKp30+, NKp44+, NKp46+, IFN-γ, TNF-α | [198] | |
| PBMC | 1.5×106 | cRPMI | Il-2;Feeder cells: K562-mbIL15-41BBL cells | T-25 or T-75 culture flasks | 14 days | 165 (4–567) | 45.6% (7.4%–76.4%) | K562, MCF-7, LNCaP, DU145, PC-3 | CD3−, CD56+, NKG2D+, NCRs+ | [199] | |
| PBMCs | (4.6–9.7)×108 | CellGro SCGM serum-free medium5% human serum | IL-2 | Wave Bioreactor System 2/10 | 21 days(9.8×109) | Mean 77-fold | Mean 37.5% | K562 (>25%, 10∶1) | CD3−, CD56+, CD244+, CD11a+, CD69+, NKG2D+, NCR+ | [200] | |
| CD3-depleted PBMCs | 107 CD3−depleted cells | AIMV media 10% hu AB serum | IL-2;Feeder cells: OKT3-loaded autologous PBMC | Cell-culture bags | 21 days(4.70±2.10)×1010 | — | ≥93% | 888 (82±12%, 10∶1) | CD3−, CD56+, CD16+, NKG2D+ | 176 | |
| NK cell lines | NK-92 | (2.5×105/mL)×25 mL/bag | X-Vivo 10 serum-free mediaamino acids and 2.5% human AB plasma | IL-2 (500 IU/ml) | 1 l Vuelife culture bag | 15–17 days>1×109/bag | >200 | ≥80% (viability) | K562 (72%); Raji (58%) (10∶1) | CD3−, CD56+, IL-6, IL-8, IL-10 | [170,171] |
| 1×107/bioreactor | Optimized clinical-grade media | IL-2 (100∼500 IU/ml) | Controlled stirred bioreactor | 11–16 days>1010/bioreactor | >1000 | >95% (viability) | Highly lytic to leukemia, lymphoma, malignant melanoma, prostate cancer, squamous cell carcinoma, breast cancer | Positive: CD56, CD2, CD7, C11a, CD28, CD45, CD54Negative: CD1, CD3, CD4, CD8, CD14, CD16, CD20, CD23, CD34, HLA-DR | [131] | ||
| NKG | (1×105/ml)×200 ml/bag | α-MEM medium10% fetal bovine serum +10% horse serum | IL-2 (100 IU/ml) | WAVE Bioreactor | 12–14 days>1010/bag | >1000 | >90% (viability) | K562 (>50%), Ho-8910 (>60%), Daudi (>70%), LoVo (>35%) (10∶1) | CD56+, CD16−, CD27−, CD3−, αβTCR−, γδTCR−, CD4−, CD8−, CD19−, CD161−, CD45+,CXCR4+, CCR7+, CXCR1−, CX3CR1−;IFN-γ, TNF-α, IL-6, IL-10 | [127] |
Abbreviations: GM-CSF, granulocyte monocyte-colony stimulating factor; hESCs, human embryonic stem cells; IFN, interferon; iPSCs, induced pluripotent stem cells; NCR, natural cytotoxicity receptor; NK, natural killer; PBMC, peripheral blood mononuclear cell; SCF, stem cell factor; UCB, umbilical cord blood.
*Ex vivo expansion of NK cells for clinical immunotherapy under GMP conditions.
**Only references in the most recent 3 years were cited for the ex vivo expansion of NK cells from PBMCs.