Abstract
Introduction
Natural Killer (NK) cells have attracted extensive attention as therapeutic agents for hematological malignancies and solid tumors. NK cell therapies carry a lower risk of Graft-Versus-Host Disease (GVHD) in allogeneic transplantation, making them ideal candidates for “off-the-shelf” allogeneic cell therapies. However, the expansion culture of NK cells typically employs a scale-out strategy using a large number of culture vessels, making it still challenging to use NK cells as 'off-the-shelf' allogeneic cell therapies. While scalable, aerated stirred bioreactor could be an ideal approach, there have been no reports on culture evaluations specifically targeting iPCS-derived NK cells.
Methods
We developed a process for expanding iPCS-derived NK cells using a stirred culture system. The NK cell stimulation process with agonist antibodies and expansion process were repeated, and the cell expansion and quality of iPCS-derived NK cells were evaluated. Scale-up factors were evaluated using an aerated stirred bioreactor, and process scale-up was performed from 1 L to 10 L bioreactors.
Results
iPCS-derived NK cells showed higher cell expansion in stirred cultures than in static cultures. By repeated stimulation and expansion processes, iPCS-derived NK cells expanded 1000-fold with comparable cell expansion and quality. iPCS-derived NK cells could be scaled up from 1 L to 10 L aerated stirred bioreactors with comparable cell expansion and quality.
Conclusions
Through systematic process evaluation and optimization, we demonstrated that iPCS-derived NK cells can be expanded in a scalable aerated stirred bioreactor.
Keywords: Natural killer cell, Scalable process, Aerated stirred bioreactor
1. Introduction
The use of NK cells as therapeutic agents targeting hematological malignancies and solid tumors has rapidly advanced. Clinical trials have utilized NK cells derived from peripheral blood and umbilical cord blood [1,2]. Further, by genetically engineering iPSCs to express chimeric antigen receptors (CARs) and differentiating them into NK cells, it is possible to produce homogeneous and more precisely targeted CAR-NK cells, and such iPSC-derived CAR-NK cells are now under clinical trial [3]. As an additional advantage, NK cell therapies carry a low risk of GVHD even in allogeneic transplantation, making them promising candidates for “off-the-shelf” allogeneic cell therapies [4].
However, the use of NK cell therapy as an “off-the-shelf” allogeneic cell therapy requires the ability to produce NK cells in quantities sufficient to meet demand. Differentiation of iPSCs into NK cells takes approximately 45 days, but the yield of NK cells is limited and insufficient for typical therapeutic doses [5]. Although various methods are known to expand NK cells while maintaining or improving their cytotoxic activity, adding these steps increases the total production time of NK cells for therapeutic purposes to approximately 2 months [6,7]. The manufacturing costs of cell therapy products are generally significantly higher than those of traditional pharmaceuticals [8]. This is due not only to the high cost of the raw materials used in production, but also to the long-term utilization of manufacturing facilities. To make “off-the-shelf” allogeneic cell therapies commercially feasible, it is therefore necessary to produce sufficient quantities of NK cells in a shorter time frame with fewer manufacturing runs.
NK cell expansion methods are broadly categorized into feeder-based and feeder-free approaches. K562 leukemia cells are commonly used as feeder cells, but other feeder cells have also been successfully employed, including Epstein-Barr virus-transformed lymphoblastoid cells and Jurkat cells [6,9,10]. However, the use of feeder cells raises several regulatory concerns, including the need for tests to confirm the absence of microorganisms and viruses derived from the feeder cells and to ensure that no residual feeder cells remain. While feeder-free conditions can mitigate many of these regulatory concerns, these carry a trade-off in significantly reduced NK cell yields. Alternative immunogenic cytokines, such as interleukin 15, 21, 12, 18, and 27, have been evaluated for stimulating NK cells, and their use, either individually or in combination, has successfully enhanced NK cell expansion [7,[11], [12], [13], [14]]. Nevertheless, further improvements in NK cell expansion are necessary to achieve therapeutic quantities of NK cells using a feeder-free process.
To date, various strategies for NK cell expansion have been reported, including scale-out strategies using T-Flasks, G-Rex, gas-permeable bags, and WAVE bioreactors, as well as scale-up strategies utilizing suspension bioreactors [[15], [16], [17], [18], [19]]. However, few reports have described processes using scale-up-capable suspension bioreactors, which are suitable for large-scale production. In particular, cell culture processes for single-use bioreactors in the 50–2000 L range, which are commonly used in antibody drug manufacturing, have not been established.
We have investigated large-scale expansion and scale-up evaluation in suspension bioreactors through a series of steps, including evaluation of culture modes and vessels, basal media selection, and seed preparation methods. Here, we report a new process capable of 1000-fold NK cell expansion which is suitable for manufacture in scalable bioreactors.
2. Methods
2.1. NK cell banking and thawing
iPSC-derived NK cells were suspended in CryoStor CS10 (STEMCELL Technologies, USA) at a density of 50 × 106 cells/mL and transferred to cryovials. The cryovials were then placed in a CoolCell (Corning, USA) and frozen at −80 °C. For thawing, the cryovials were removed from the freezer and placed in a water bath heated to 37 °C. The contents of each vial were then added to RPMI-1640 (GibcoTM, USA) supplemented with 10 % AB serum (Valley Biomedical, USA) and centrifuged at 300×g for 10 min. The supernatant was discarded, and the cell pellet was resuspended in RPMI-1640 supplemented with 10 % AB serum and 10 ng/mL of Human IL-15 (PeproTech, USA). The cells were then transferred to a T175 flask which was maintained at 37.0 °C with 5.0 % CO2 overnight to allow the thawed NK cells to rest.
The human iPSC line used for NK cell differentiation was established at the Astellas Institute for Regenerative Medicine. The differentiation of iPSCs into NK cells was carried out using an established protocol, with modifications introduced to accommodate the specific characteristics of the iPSC line used in this study [20]. Initially, iPSCs were induced to form embryoid bodies, during which they differentiated into hematopoietic progenitor cells (HPCs). These HPCs were subsequently differentiated into NK cells. At each stage of the process, including culture vessel selection, cytokine composition, and other culture parameters, conditions were optimized to suit the properties of the iPSCs employed in this study.
Use of the human iPSC was approved by the Astellas Research Ethics Committee of Astellas Pharma. Each donor signed an informed consent agreement.
2.2. Culture vessel evaluation study
2.2.1. NK cell stimulation
iPSC-derived NK cells were suspended in RPMI-1640 with GlutaMAX (GibcoTM, USA) supplemented with 10 % AB serum and 10 ng/mL of Human IL-15, IL-18 (R&D Systems, USA), and IL-21 (PeproTech, USA) at a density of 0.50 × 106 cells/mL to initiate the stimulation culture. The suspended iPSC-derived NK cells were then seeded onto commercially available Antibody A, Antibody B, and RetroNectin (Takara, Japan)-coated T175 flasks and cultured for 3 days in an incubator at 37.0 °C, 5.0 % CO2 for stimulation. Antibody A and Antibody B are agonist antibodies targeting NK cell activating receptors [21]. After stimulation, the NK cells were harvested by centrifugation at 300×g for 10 min.
2.2.2. NK cell expansion
Following stimulation culture, iPSC-derived NK cells were resuspended at a density of 0.20 × 106 cells/mL in RPMI-1640 with GlutaMAX, supplemented with 10 % AB serum and 10 ng/mL of human IL-15 and IL-18, to initiate the expansion culture. The stimulated iPSC-derived NK cells were expanded in G-Rex100 (Wilson Wolf, USA), 125 mL spinner flask (Corning, USA). Culture in G-Rex100 (1 L working volume) was maintained at 37.0 °C in 5.0 % CO2. Culture in 125 mL spinner flask (60 mL working volume) was maintained at 37.0 °C in 5.0 % CO2 and agitation rate of 45 rpm.
2.3. Basal media evaluation study
2.3.1. NK cell stimulation
Frozen iPSC-derived NK cells were used in this study. NK cell stimulation culture conditions were identical to those described in Section 2.2.1.
2.3.2. NK cell expansion
Following stimulation culture, iPSC-derived NK cells were resuspended at a density of 0.20 × 106 cells/mL in one of the following media: RPMI-1640, NK Xpander, X–VIVO 15 (Lonza, USA), or LGM-3 (Lonza, USA), each supplemented with 10 % AB serum and 10 ng/mL of human IL-15 and IL-18. Expansion culture was initiated using these media. The stimulated NK cells were cultured in 24-deep-well plates (Thomson, USA) and 125 mL spinner flasks. The 24-deep-well plate cultures (working volume: 3.5 mL) were maintained at 37.0 °C in a 5.0 % CO2, with shaking at 265 rpm and an orbital diameter of 19 mm. Culture conditions for the 125 mL spinner flasks were identical to those described in Section 2.2.2.
2.4. Design of experiment study for optimizing Re-stimulation culture condition
2.4.1. Design of experiment study
The iPSC-derived NK cells were suspended in NK Xpander medium supplemented with 10 % AB serum and human IL-15, IL-18, and IL-21. The suspended NK cells were then seeded onto agonist antibody A, agonist antibody B, and RetroNectin-coated 6-well plates and cultured in an incubator at 37.0 °C, 5.0 % CO2 for stimulation. Coating time, coating solution concentration (Antibody A, Antibody B and RetroNectin concentration), initial cell density, culture time and cytokine concentration (IL-15, IL-18 and IL-21 concentration) were evaluated with three levels (Table 1). The study was conducted based on a Design of Experiments (DOE) approach, which was developed using JMP software (SAS, USA) (Supplementary Table 1).
Table 1.
Process parameters and ranges tested by design of experiment.
| Factors | Unit | Abbreviation | Testing levels | ||
|---|---|---|---|---|---|
| Coating time | hours | Coating time | 18 | 23 | 28 |
| Coating solution concentration | fold | Coating conc. | 0.5 | 1.0 | 1.5 |
| Initial cell density | 106 cells/mL | ICD | 0.50 | 0.68 | 0.86 |
| Culture time | hours | Culture time | 65.5 | 70.0 | 74.5 |
| Cytokine concentration | fold | Cyt conc. | 0.75 | 1.00 | 1.25 |
Abbreviation: ICD, initial cell density. Cyt conc., cytokine concentration.
Following stimulation culture, iPSC-derived NK cells were resuspended at a density of 0.20 × 106 cells/mL in NK Xpander medium, supplemented with 10 % AB serum and 10 ng/mL of human IL-15 and IL-18, to initiate the expansion culture. The resuspended NK cells were transferred into 24-deep-well plates, and cultured for 3 days. The culture conditions in the 24-deep-well plates followed the same procedure as described in Section 2.3.2. Statistical models for fold expansion and lactate concentration were built by prediction profiler by JMP software.
2.4.2. Re-stimulation culture
Frozen iPSC-derived NK cells were used in this study. The cells were initially resuspended in NK Xpander medium supplemented with 10 % AB serum and 10 ng/mL of human IL-15, IL-18, and IL-21 at a density of 0.50 × 106 cells/mL, and transferred to T175 flasks for stimulation culture.
Following stimulation, the harvested cells were resuspended in NK Xpander medium supplemented with 10 % AB serum and 10 ng/mL of human IL-15 and IL-18 at a density of 0.20 × 106 cells/mL, and transferred to 125 mL spinner flasks for expansion culture.
Expanded iPSC-derived NK cells were collected from the 125 mL spinner flasks by centrifugation at 300×g for 10 min, and subsequently re-stimulated at target cell densities of 0.35 × 106, 0.50 × 106, and 0.68 × 106 cells/mL. Re-stimulation cultures were carried out in T175 flasks, while subsequent expansion cultures were performed in 125 mL spinner flasks.
All other culture conditions for the T175 flasks and 125 mL spinner flasks were identical to those described in Sections 2.2, 2.2.1.2, respectively.
2.5. Scalable bioreactor evaluation study
2.5.1. Scalable bioreactor evaluation
Frozen iPSC-derived NK cells were used in this study. The cells were resuspended in NK Xpander medium supplemented with 10 % AB serum and 10 ng/mL of human IL-15, IL-18, and IL-21 at a density of 0.50 × 106 cells/mL, and transferred to T175 flasks for stimulation culture.
The harvested cells were resuspended in NK Xpander supplemented with 10 % AB serum, and 10 ng/mL of human IL-15 and IL-18 at a density of 0.20 × 106 cells/mL to initiate the expansion culture. The resuspended iPSC-derived NK cells were transferred into a 125 mL spinner flask, 1 L glass bioreactor, and cultured for 3 days. Culture in 1 L glass bioreactor (600 mL working volume) was maintained at 37.0 °C in 5.0 % CO2, an agitation rate 70 rpm and a dissolved oxygen (DO) of 80 %.
All other culture conditions for the T175 flasks and 125 mL spinner flasks were identical to those described in Sections 2.2.1 and 2.2.2, respectively.
2.5.2. Re-stimulation culture with scalable bioreactor
Frozen iPSC-derived NK cells were used in this study. The cells were resuspended in NK Xpander medium supplemented with 10 % AB serum and 10 ng/mL of human IL-15, IL-18, and IL-21 at a density of 0.50 × 106 cells/mL for Cycle 1, and 0.35 × 106 cells/mL for Cycles 2 and 3. All other conditions for NK cell stimulation culture were identical with the procedures described in Section 2.2.1.
The harvested cells were resuspended in NK Xpander medium supplemented with 10 % AB serum, and 10 ng/mL of human IL-15 and IL-18 at a density of 0.20 × 106 cells/mL to initiate the expansion culture. The resuspended iPSC-derived NK cells were transferred into 1 L glass bioreactor, and cultured for 3 days. The culture conditions in the 1 L glass bioreactor were identical with the procedures described in Section 2.5.1. Stimulation and expansion culture were repeated three cycles.
2.6. Scale up evaluation study
2.6.1. P/V and DO evaluation
Frozen iPSC-derived NK cells were used in this study. The cells were resuspended in NK Xpander medium supplemented with 10 % AB serum and 10 ng/mL of human IL-15, IL-18, and IL-21 at a density of 0.35 × 106 cells/mL, and transferred to T175 flasks for stimulation culture. All other conditions for NK cell stimulation culture were identical with the procedures described in Section 2.2.1.
The harvested cells were resuspended in NK Xpander supplemented with 10 % AB serum, and 10 ng/mL of human IL-15 and IL-18 at a density of 0.2 × 106 cells/mL, and transferred to 1 L glass bioreactor for expansion culture. For the agitation rate evaluation, cultures in 1 L glass bioreactor (600 mL working volume) were maintained at 37.0 °C in 5.0 % CO2 and a dissolved oxygen of 80 %, and agitation rates of 70 (P/V = 1.4) and 101 rpm (P/V = 4.2) were compared. For the DO evaluation, cultures in 1 L glass bioreactor (600 mL working volume) were maintained at 37.0 °C in 5.0 % CO2 and an agitation rate of 101 rpm, and DO of 40 and 80 % were compared.
2.6.2. Scale-up evaluation
Frozen iPSC-derived NK cells were used in this study. The cells were resuspended in NK Xpander medium supplemented with 10 % AB serum and 10 ng/mL of human IL-15, IL-18, and IL-21 at a density of 0.35 × 106 cells/mL, and transferred to T175 flasks for stimulation culture. All other conditions for NK cell stimulation culture were identical with the procedures described in Section 2.2.1.
The harvested cells were then resuspended in NK Xpander medium supplemented with 10 % AB serum and 10 ng/mL of human IL-15 and IL-18 at a density of 0.20 × 106 cells/mL to initiate the expansion culture. Cell expansion culture was conducted in 1 L single use bioreactor (1 L SUB) (Eppendorf, Germany) for all three cycles and 10 L single use bioreactor (10 L SUB) (Eppendorf, Germany) for only Cycle 3. Culture in 1 L SUB (600 mL working volume) was maintained at 37.0 °C in 5.0 % CO2, an agitation rate of 101 rpm (P/V = 4.2) and DO of 40 %. Culture in 10 L SUB (6000 mL working volume) was maintained at 37.0 °C in 5.0 % CO2, an agitation rate of 66 rpm (P/V = 4.2) and DO of 40 %.
2.7. Cell culture performance assay
Cell count and viability were analyzed using a NucleoCounter NC-200 (ChemoMetec, USA), and metabolites were analyzed using a Bioprofile FLEX (Nova Biomedical, USA).
2.8. NK cell cytotoxicity assay
K562 cells which had been genetically modified to express HiBiT-tagged protein were cocultured with NK cells at the indicated effector-to-target (E:T) ratio. After incubation, LgBiT protein and Nano-Glo HiBiT extracellular substrate (Promega, USA) were added in accordance with the manufacturer's protocol. Luminescence intensity was then measured using a SpectraMAX M3 (Molecular Devices, USA).
2.9. IFN-γ secretion assay
Human erythroleukemia targets (K562) were cocultured with NK cells at a 1:1 effector-to-target ratio. After incubation, anti-hIFN-γ mAb-smBiT and anti-hIFN-γ mAb-IgBiT (Promega, USA) were added according to the manufacturer's protocol. Luminescence intensity was measured using a SpectraMax M3.
2.10. Flow cytometry assay
The cells were washed and labeled with Live/Dead® Fixable Near IR Dead Cell Stain (Miltenyi Biotech, USA) and the following specific monoclonal antibodies: anti-CD3 BV421 (BD Sciences, USA), anti-CD45 APC (BD Sciences, USA), and anti-CD56 PE (BD Sciences, USA). Fluorescence was measured using a NovoCyte Advanteon flow cytometer (Agilent, USA).
2.11. Statistical analysis
JMP software was utilized for statistical analysis. Results are presented as mean ± standard deviation (SD). Statistical significance was determined using a Student's t-test. Statistical significance was considered as p < 0.05.
3. Results
3.1. Culture vessel evaluation
A study was conducted to determine the optimal culture mode and vessel for NK cell expansion. Following antibody stimulation, iPSC-derived NK cells were seeded and expanded in either G-Rex100 vessels or 125 mL spinner flasks. In the static G-Rex100 vessel, cell expansion reached 4.3-fold by day 3 and 7.4-fold by day 4. In contrast, the stirred 125 mL spinner flask achieved 8.2-fold expansion by day 3 (Table 2), indicating significantly faster cell growth under stirred conditions.
Table 2.
Results of cell fold expansion, consumed glucose from day 0 to day 3 and 4 (ΔGluc), produced lactate from day 0 to day 3 and 4 (ΔLac) and glucose to lactate ratio on day 3 and 4 (ΔLac/ΔGluc).
| Fold Expansion |
ΔGluc |
ΔLac |
ΔLac/ΔGluc |
|||||
|---|---|---|---|---|---|---|---|---|
| Day 3 | Day 4 | Day 3 | Day 4 | Day 3 | Day 4 | Day 3 | Day 4 | |
| G-Rex100 (n = 3) | 4.3 | 7.4 | 7.4 | 11.0 | 12.6 | 19.4 | 1.7 | 1.8 |
| 125 mL Spinner Flask (n = 3) | 8.2 | 11.4 | 6.5 | 9.4 | 12.6 | 18.9 | 2.0 | 2.0 |
Abbreviation: Lac, lactate. Gluc, glucose. n/a, not applicable. The fold expansion data for Day 3 and Day 4 in G-Rex100 were generated from different culture vessels due to sampling difficulties caused by static culture. Results represent means of multiple experiments.
Despite the difference in expansion rates, the ΔLac/ΔGluc values were comparable between the G-Rex100 and the 125 mL spinner flask, suggesting that intracellular metabolism remained similar. These findings demonstrate that stirred culture using the 125 mL spinner flask promoted more rapid NK cell expansion without altering intracellular metabolic profiles.
However, a marked reduction in the expansion rate was observed between day 3 and day 4. The average cell doubling time in the 125 mL spinner flask was 25 h by day 3 but increased to 69 h between day 3 and day 4. Notably, this decline in proliferation could not be improved by medium exchange or nutrient supplementation (data not shown). Additional expansion studies using PBS 0.1 (PBS Biotech, USA) and the WAVE bioreactor (Cytiva, USA), both employing distinct dynamic culture systems compared to the 125 mL spinner flask, did not result in enhanced cell proliferation (Supplementary Table 2).
Considering that most commercially available single-use bioreactors utilize impeller-based stirring mechanisms, the 125 ml spinner flask, which employs a similar agitation method, was selected for subsequent studies.
3.2. Basal media evaluation
An evaluation was conducted to assess the performance of different basal media for NK cell expansion. Frozen iPSC-derived NK cells were thawed, stimulated with agonist antibodies, and then seeded in a 24-deep-well plate for cell expansion. The study compared RPMI-1640, which is commonly used for NK cells, with other commercial media designed for lymphocyte culture, including NK Xpander, X–VIVO15, and LGM-3. On day 4 of culture, RPMI-1640 achieved 3.9-fold expansion, NK Xpander 6.2-fold, X–VIVO15 4.6-fold, and LGM-3 3.8-fold. To verify whether similar performance could be achieved in stirred cultures, RPMI-1640 and NK Xpander were tested using the 125 mL spinner flask. NK Xpander showed faster cell expansion than RPMI-1640, achieving similar expansion to that observed in the 24-deep-well plate. No significant differences were observed in ΔLac/ΔGluc between the media (Table 3). These results suggest that NK Xpander has higher cell expansion performance than RPMI-1640 and is also suitable for use in stirred cultures.
Table 3.
Results of cell fold expansion, consumed glucose (ΔGluc), produced lactate (ΔLac) and glucose to lactate ratio (ΔLac/ΔGluc).
| Fold Expansion |
ΔGluc |
ΔLac |
ΔGluc/ΔLac |
||
|---|---|---|---|---|---|
| Day 3 | Day 4 | Day 4 | Day 4 | Day 4 | |
| RPMI-1640 (n = 2) | 3.3 | 4.3 | 6.1 | 10.8 | 1.8 |
| NK Xpander (n = 2) | 4.0 | 6.5 | 7.6 | 13.8 | 1.8 |
Abbreviation: Lac, lactate. Gluc, glucose. Results represent means of multiple experiments.
3.3. NK cell Re-stimulation evaluation
The results obtained using the 125 mL spinner flask indicated that NK cells could undergo expansion under stirred conditions, in turn suggesting the feasibility of large-scale manufacture in stirred bioreactors. However, we observed that NK cell expansion slowed after approximately four days of culture following stimulation with agonist antibodies (data not shown). Neither medium exchange nor the addition of feed produced a recovery in cell expansion. Starting expansion culture at low cell densities and expanding cells to high densities is challenging, indicating that significant stimulated cell quantities are required to conduct large-scale expansion culture. On the other hand, differentiation from iPSCs to NK cells is time-consuming, and scaling up this differentiation process is not cost-effective. Therefore, a process is needed to produce the required number of stimulated NK cells to initiate large-scale expansion culture.
NK cell cytotoxicity and cell expansion are supported by stimulation with cytokine and agonist antibody targeting activation receptor in feeder-free conditions [22,23]. In the present study, the effects of five process parameters in the stimulation step on NK cell expansion were evaluated, namely coating time, coating solution concentration, initial cell density, incubation time, and cytokine concentration. Since agonist antibodies are adsorbed onto flasks via physical adsorption, the concentration of the coating solution and the coating time are thought to influence the amount of agonist antibody adsorbed onto the flask. Additionally, NK cell expansion is dependent on cytokine concentration [24,25]. Because initial cell density and culture duration were directly related to cell expansion, we created an experimental design to evaluate both the individual and combined effects of the process parameters, with assessment conducted on a three-level scale (Table 1). Of the factors affecting NK cell expansion, initial cell density emerged as the most important main effect (Fig. 1A). In contrast, coating time and cytokine concentration showed no statistically significant impact on NK cell expansion in the range tested.
Fig. 1.
Impact of process parameters of the stimulation step on NK cell expansion. NK cells were stimulated with various conditions set by DoE, and harvested NK cells were conducted expansion culture in 24-deep-well plate. The results of NK cell expansion were statistically analyzed. Prediction profiler of (A) NK cell fold expansion and (B) lactate concentration in expansion culture. (C) NK cells stimulated and expanded for one cycle were harvested on day 6 and seeded into antibody-coated flasks at 0.35 × 106 cells/mL, 0.50 × 106 cells/mL, or 0.68 × 106 cells/mL. The stimulated NK cells under different conditions were then seeded into 125 mL spinner flasks on day 9 of culture and expanded until day 12. The negative control shows the fold expansion trend of NK cells seeded at a density of 0.50 × 106 cells/mL in an antibody-uncoated flask. ICD indicates initial cell density.
As a further evaluation to improve NK cell expansion, a process involving repeated cycles of stimulation and expansion was evaluated. Frozen iPSC-derived NK cells were thawed, and the cycles of agonist antibody stimulation and expansion were repeated. A single cycle was defined as one round of stimulation followed by expansion, and two cycles were evaluated. In the stimulation step of cycle 2, the initial cell density that proved most effective in improving NK cell expansion was evaluated. Initial cell densities were set at 0.35 × 106, 0.50 × 106, and 0.68 × 106 cells/mL. It was observed that the lower the initial cell density at the stimulation step, the higher the NK cell expansion, similar to the results of the DoE study (Fig. 1B). By setting the initial cell density at 0.35 × 106 cells/mL, comparable expansion rates were achieved in both Cycle 1 (10.0-fold) and Cycle 2 (10.5-fold) (Fig. 1C).
3.4. Scalable bioreactor evaluation
The repeated cycles of stimulation and expansion successfully produced the required number of stimulated iPSC-derived NK cells for the initiation of large-scale expansion cultures. For large-scale expansion, a scale-up strategy is preferred over scale-out. Since the maximum size of spinner flasks is limited, an aerated stirred bioreactor was evaluated as a scalable bioreactor option. Frozen iPSC-derived NK cells were thawed, stimulated with agonist antibodies, and then seeded in both a 125 mL spinner flask and a 1 L glass bioreactor for expansion. The 1 L glass bioreactor demonstrated higher cell expansion, indicating that NK cells can be cultured in an aerated stirred bioreactor (Fig. 2A).
Fig. 2.
Cell expansion trends and qualities of iPSC-derived NK cells. (A) NK cell expansion trends. Stimulated NK cells were seeded in a 125 mL spinner flask (n = 4) and a 1 L glass bioreactor (n = 3). (B) NK cell fold expansion trend, (C) NK cell cytotoxicity and (D) INF-r secretion: Three cycles of stimulation and expansion culture were performed. Samples for NK cell cytotoxicity and INF-r secretion analyses were taken from end of each cycle shown in (B). Stimulation was conducted in agonist antibody-coated flasks, and expansion was carried out in a 1 L bioreactor, with each cycle repeated every 3 days. GBR indicates glass bioreactor. Error bars representing the standard deviations from multiple runs. ∗Denotes statistical significance (p < 0.05).
Subsequently, the aerated stirred bioreactor was used for repeated cycles of stimulation and expansion. Frozen iPSC-derived NK cells were thawed and then subjected to three cycles of stimulation and expansion. Cell expansion of 10.0-fold, 10.3-fold, and 12.7-fold was observed in Cycles 1, 2, and 3, respectively, demonstrating consistent cell expansion across cycles in the aerated stirred bioreactor (Fig. 2B). Furthermore, the quality of NK cells at the end of each cycle was evaluated. Results from the NK cell cytotoxicity assay (Fig. 2C), IFN-γ secretion assay (Fig. 2D), and flow cytometry assay (Table 4) confirmed that the quality remained consistent across all cycles.
Table 4.
Results of flow assay with analytical samples taken at the end of expansion culture in Cycle 1, 2 and 3.
| Flow assay (%) |
|||
|---|---|---|---|
| CD3+ | CD45+ | CD56+ | |
| 1 L glass bioreactor (Cycle-1) | 0.8 | 100.0 | 99.8 |
| 1 L glass bioreactor (Cycle-2) | 1.8 | 100.0 | 99.9 |
| 1 L glass bioreactor (Cycle-3) | 1.0 | 100.0 | 100.0 |
3.5. Scale-up evaluation
The study demonstrated that iPSC-derived NK cells could be cultured in a scalable aerated stirred bioreactor. Successful scale-up of the bioreactor requires appropriate setting of scale-up parameters. For aerated stirred bioreactors, scale-up strategies commonly involve parameters such as volumetric gas flow rate, mass transfer coefficient, and power per unit volume (P/V), as established in studies using CHO cells for antibody production [26,27]. To prepare for iPSC-derived NK cell scale-up, the effects of shear stress from agitation and the DO set point were evaluated. Using the 1 L glass bioreactor, agitation rates of 70 rpm (P/V = 1.4) and 101 rpm (P/V = 4.2) were evaluated. iPSC-derived NK cells expanded without a decrease in cell viability at both agitation rates (Fig. 3A). Additionally, DO set points of 40 % and 80 % were evaluated in the 1 L glass bioreactor. IPSC-derived NK cells expanded without a decrease in viability at both DO set points (Fig. 3B).
Fig. 3.
Expansion trend of iPSC-derived NK cells. (A) Stimulated NK cells were seeded in a 1 L glass bioreactor and cultured at agitation speeds of 70 rpm (P/V = 1.4) (n = 3) and 101 rpm (P/V = 4.2) (n = 3). (B) Stimulated NK cells were seeded in a 1 L glass bioreactor and cultured at dissolved oxygen set points of 80 % and 40 %. DO indicates dissolved oxygen. Error bars representing the standard deviations from multiple runs.
Based on the P/V metric, a common indicator for setting agitation speeds during scale-up, agitation speed was set at 66 rpm (P/V = 4.2) for the 10 L single use bioreactor and agitation rate was set at 79 rpm (P/V = 4.2) for the 1 L single use bioreactor. After two cycles of stimulation and expansion, the stimulated NK cells were seeded into both the 1 L single use bioreactor and the 10 L single use bioreactor to the Cycle 3 expansion culture. Cell expansion of 8.8-fold (1 L SUB), 12.9-fold (1 L SUB), and 29.6-fold (10 L SUB) was observed in Cycles 1, 2, and 3, respectively, demonstrating scale-up in the aerated stirred bioreactor with P/V metric (Fig. 4A). At the end of the expansion culture, samples were taken, and NK cell cytotoxicity assay (Fig. 4C), IFN-γ secretion assay (Fig. 4D), and flow cytometry assay (Table 5) were performed. These confirmed that quality in the 1 L single use bioreactor and 10 L single use bioreactor was comparable.
Fig. 4.
Expansion trends and qualities of iPSC-derived NK cells. (A) NK cell fold expansion trend. Three cycles of stimulation and expansion culture were performed. Cell expansion results of Cycle 1 and Cycle 2 represent cell culture in T175 flask and 1 L single use bioreactor, and cell expansion results of Cycle 3 represent cell culture in T175 flask and 10 L single use bioreactor. (B) NK cell expansion trends in Cycle 3. (C) NK cell cytotoxicity in Cycle 3. (D) INF-r secretion in Cycle 3. Stimulated NK cells were seeded in a 1 L single use bioreactor and 10 L single use bioreactor. Agitation of the 1 L single use bioreactor and 10 L single use bioreactor were set to the same P/V (79 rpm and 66 rpm, respectively) and dissolved oxygen was set to 40 %. SUB indicates single use bioreactor.
Table 5.
Results of flow assay with analytical samples taken at end of expansion culture in Cycle 3.
| Flow assay (%) |
|||
|---|---|---|---|
| CD3+ | CD45+ | CD56+ | |
| 1 L single use bioreactor | 1.7 | 99.8 | 99.8 |
| 10 L single use bioreactor | 1.6 | 100.0 | 99.9 |
4. Discussion
NK cell therapy has rapidly developed as a cancer treatment, particularly for hematological malignancies. Recently, strategies have been explored to use NK cells differentiated from allogeneic iPSCs as off-the-shelf therapeutics. However, therapeutic doses typically require between 105 to 108 NK cells per kilogram of body weight, necessitating methods for the large scale production of high-quality NK cells [2,7].
Various methods for NK cell cultivation have been proposed. Feeder-based methods have been reported to achieve up to 15,000-fold expansion [28], while feeder-free methods have achieved 1000- to 3000-fold expansion [29]. However, NK cell culture densities typically range between 0.5 and 1.0 × 106 cells/mL at the harvest step, requiring large culture volumes to obtain sufficient NK cells for therapeutic doses [15,16].
In this study, we demonstrated that NK cells could be cultured in a scalable bioreactor. The scale up strategy based on P/V, which is commonly used in CHO cell culture for antibody production, was shown to be applicable to NK cells. In CHO cells, maintaining a constant P/V enabled successful scale up from 3 L to 2000 L single-use bioreactors [27]. This finding suggests that NK cells could also be scaled up to large bioreactors using a similar strategy.
The present study also demonstrated a method for producing the required number of stimulated iPSC-derived NK cells for initiating large-scale expansion culture. IPSC-derived NK cells can undergo repeated cycles of stimulation and expansion, through which they maintain both expansion and cytotoxic activity. Our present results showed that the optimization of initial cell density in the stimulation step is necessary to repeat the stimulation-expansion cycle while maintaining cell proliferation. Lactate has been reported to induce impaired energy metabolism and apoptosis [30,31]. In T cells, lactate has been reported to inhibit cell proliferation [32]. This study confirmed that among the five process parameters evaluated, initial cell density had the greatest influence on lactate accumulation, which in turn suggests that lactate needs to be maintained at a low concentration in order to perform stimulation-expansion cycles while maintaining cell expansion.
Theoretically, the repeated cycles of stimulation and expansion will allow for the generation of sufficient stimulated iPSC-derived NK cells to initiate expansion in bioreactors with capacities of several thousand liters. However, the stimulation step for iPSC-derived NK cells in this study still relies on scale-out methods, and challenges for large-scale manufacture accordingly remain. It is reported that NK cells can be stimulated by magnetic beads or polymer-based microspheres conjugated with antibodies and that NK cells can be expanded [[33], [34], [35]]. In T cells, magnetic beads have been used to activate cells under agitation conditions [36]. Developing a process that combines beads or microspheres with bioreactors for NK cell stimulation could enable scalable NK cell stimulation and expansion in bioreactors. In addition, the selection of antibody combinations for conjugation to beads or microspheres warrants further investigation. Beyond the antibodies used in this study, previous reports have shown that combinations such as anti-NKp46 antibody with anti-CD16 antibody, and anti-CD2 antibody with anti-NKp46 antibody, can also promote NK cell expansion [33,37]. However, the identification of antibody combinations that enable the repeated cycles of stimulation and expansion remains an important subject for future research.
In this study, stimulation and expansion cultures were performed using iPSC-derived NK cells that had been cryopreserved after differentiation. The differentiation of iPSCs into NK cells typically requires approximately 45 days [5]. Therefore, the use of cryopreserved, post-differentiation NK cells as the starting material offers potential advantages, including a shortened manufacturing timeline and reduced variability in NK cell quality. However, cryopreservation is known to impair NK cell viability and cytotoxic function [38]. As shown in Table 1, Table 2, a substantial reduction in expansion fold was observed when using frozen iPSC-derived NK cells, suggesting that the freeze–thaw process adversely affected their proliferative capacity. Nevertheless, as shown in Fig. 2B, proliferation tended to improve across successive stimulation and expansion cycles (Cycle 1: 10.0-fold; Cycle 2: 10.3-fold; Cycle 3: 12.7-fold). Moreover, as shown in Fig. 4A, further optimization of key process parameters—such as initial cell density at the time of stimulation, agitation rate, and DO concentration—led to enhanced NK cell expansion (Cycle 1: 8.8-fold; Cycle 2: 12.9-fold; Cycle 3: 29.6-fold). These improvements in overall cell growth were primarily attributed to increased proliferation during the expansion phase. Collectively, these findings highlight the critical importance of optimizing both stimulation and expansion culture conditions when working with cryopreserved iPSC-derived NK cells.
5. Conclusion
An upstream process has been developed to produce sufficient quantities of iPSC-derived NK cells to supply multiple patients in a single manufacturing run. Through a series of evaluations, including the selection of culture modes and vessels, media selection, development of seed preparation methods for large-scale culture, and scale-up based on P/V, we established a process that enables 1000-fold cell expansion. The iPSC-derived NK cells obtained through repeated cycles of stimulation and expansion were confirmed to be of comparable quality to iPSC-derived NK cells before the repeated cycles. The developed process also allows for expansion from frozen iPSC-derived NK cell banks, allowing a significant shortening of manufacture time through the banking of NK cells differentiated from iPSCs.
To the best of our knowledge, there have been no prior reports of iPSC-derived NK cells being expanded in a scalable, aerated, stirred bioreactor. The use of such bioreactors offers several advantages for pharmaceutical manufacturing, including a reduced number of production batches and shorter occupancy times in manufacturing suites. Moreover, the repeated stimulation and expansion strategy developed in this study enables large-scale NK cell culture starting from a small quantity of cryopreserved iPSC-derived NK cells. This approach eliminates the need to perform the labor-intensive differentiation process from iPSCs to NK cells for each manufacturing batch, thereby supporting the production of NK cells with consistent quality.
Author contributions
Conceptualization, T.K.; original draft preparation, T.K.; review and editing, T.K., I.T. and H.Y. All authors have read and agreed to the published version of the manuscript.
Institutional review board statement
Not applicable.
Informed consent statement
Not applicable.
Data availability statement
All data generated or analyzed during this study, which support the findings of this study, are included within this article and its supplementary information files. Researchers interested in further analysis not present in the manuscript may contact the corresponding author.
Funding
This research was funded by Astellas.
Declaration of competing of interest
The authors declare no conflicts of interest.
Acknowledgments
We gratefully acknowledge the feedback and support of colleagues at Astellas Institute for Regenerative Medicine.
Footnotes
Peer review under responsibility of the Japanese Society for Regenerative Medicine.
Supplementary data to this article can be found online at https://doi.org/10.1016/j.reth.2025.07.014.
Appendix A. Supplementary data
The following is the supplementary data to this article:
References
- 1.Choi I., Yoon S.R., Park S.Y., Kim H., Jung S.J., Jang Y.J., et al. Donor-derived natural killer cells infused after human leukocyte antigen-haploidentical hematopoietic cell transplantation: a dose-escalation study. Biol Blood Marrow Transplant. 2014;20(5):696–704. doi: 10.1016/j.bbmt.2014.01.031. [DOI] [PubMed] [Google Scholar]
- 2.Marin D., Li Y., Basar R., Rafei H., Daher M., Dou J., et al. Safety, efficacy and determinants of response of allogeneic CD19-specific CAR-NK cells in CD19+ B cell tumors: a phase 1/2 trial. Nat Med. 2024;30(3):772–784. doi: 10.1038/s41591-023-02785-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Li Y., Hermanson D.L., Moriarity B.S., Kaufman D.S. Human iPSC-Derived natural killer cells engineered with chimeric antigen receptors enhance anti-tumor activity. Cell Stem Cell. 2018;23(2):181–192. doi: 10.1016/j.stem.2018.06.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Liu E., Marin D., Banerjee P., Macapinlac H.A., Thompson P., Basar R., et al. Use of CAR-transduced natural killer cells in CD19-Positive lymphoid tumors. N Engl J Med. 2020;382(6):545–553. doi: 10.1056/NEJMoa1910607. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Lupo K.B., Moon J.I., Chambers A.M., Matosevic S. Differentiation of natural killer cells from induced pluripotent stem cells under defined, serum- and feeder-free conditions. Cytotherapy. 2021;23(10):939–952. doi: 10.1016/j.jcyt.2021.05.001. [DOI] [PubMed] [Google Scholar]
- 6.Lim S.A., Kim T.J., Lee J.E., Sonn C.H., Kim K., Kim J., et al. Ex vivo expansion of highly cytotoxic human NK cells by cocultivation with irradiated tumor cells for adoptive immunotherapy. Cancer Res. 2013;73(8):2598–2607. doi: 10.1158/0008-5472.CAN-12-2893. [DOI] [PubMed] [Google Scholar]
- 7.Choi Y.H., Lim E.J., Kim S.W., Moon Y.W., Park K.S., An H.J. IL-27 enhances IL-15/IL-18-mediated activation of human natural killer cells. J Immunother Cancer. 2019;7(1):168. doi: 10.1186/s40425-019-0652-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Ten Ham R.M.T., Nievaart J.C., Hoekman J., Cooper R.S., Frederix G.W.J., Leufkens H.G.M., et al. Estimation of manufacturing development costs of cell-based therapies: a feasibility study. Cytotherapy. 2021;23(8):730–739. doi: 10.1016/j.jcyt.2020.12.014. [DOI] [PubMed] [Google Scholar]
- 9.Granzin M., Stojanovic A., Miller M., Childs R., Huppert V., Cerwenka A. Highly efficient IL-21 and feeder cell-driven ex vivo expansion of human NK cells with therapeutic activity in a xenograft mouse model of melanoma. OncoImmunology. 2016;5(9) doi: 10.1080/2162402X.2016.1219007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Picard L.K., Claus M., Fasbender F., Watzl C. Human NK cells responses are enhanced by CD56 engagement. Eur J Immunol. 2022;52(9):1441–1451. doi: 10.1002/eji.202249868. [DOI] [PubMed] [Google Scholar]
- 11.Marks-Konczalik J., Dubois S., Losi J.M., Sabzevari H., Yamada N., Feigenbaum L., et al. IL-2-induced activation-induced cell death is inhibited in IL-15 transgenic mice. Proc Natl Acad Sci U S A. 2000;97(21):11445–11450. doi: 10.1073/pnas.200363097. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.D'Andrea A., Rengaraju M., Valiante N.M., Chehimi J., Kubin M., Aste M., et al. Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells. J Exp Med. 1992;176(5):1387–1398. doi: 10.1084/jem.176.5.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Heinze A., Grebe B., Bremm M., Huenecke S., Munir T.A., Graafen L., et al. The synergistic use of IL-15 and IL-21 for the generation of NK cells from CD3/CD19-Depleted grafts improves their ex vivo expansion and cytotoxic potential against neuroblastoma: perspective for optimized immunotherapy post haploidentical stem cell transplantation. Front Immunol. 2019;10:2816. doi: 10.3389/fimmu.2019.02816. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Felices M., Lenvik A., Chu S., McElmurry R., Cooley S., Tolar J., et al. Continuous IL-15 signaling leads to functional exhaustion of human natural killer cells through metabolic changes that alters their in vivo anti-tumor activity. Blood. 2016;128(22):551. [Google Scholar]
- 15.Huang H., Zhang S., Zhao Y., Xu R., Tan W.S., Cai H. Suspension culture promoted the expansion of NK-92 cells ex vivo by enhancing the expression of IL-2 receptor. Biotechnol J. 2024;19(3) doi: 10.1002/biot.202300654. 2024. [DOI] [PubMed] [Google Scholar]
- 16.Lapteva N., Durett A.G., Sun J., Rollins L.A., Huye L.L., Fang J., et al. Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications. Cytotherapy. 2012;14(9):1131–1143. doi: 10.3109/14653249.2012.700767. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Lapteva N., Szmania S.M., van Rhee F., Rooney C.M. Clinical grade purification and expansion of natural killer cells. Crit Rev Oncog. 2014;19(1–2):121–132. doi: 10.1615/critrevoncog.2014010931. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Bröker K., Sinelnikov E., Gustavus D., Schumacher U., Pörtner R., Hoffmeister H., et al. Mass production of highly active NK cells for cancer immunotherapy in a GMP conform perfusion bioreactor. Front Bioeng Biotechnol. 2019;7:194. doi: 10.3389/fbioe.2019.00194. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Pierson B.A., Europa A.F., Hu W.S., Miller J.S. Production of human natural killer cells for adoptive immunotherapy using a computer-controlled stirred-tank bioreactor. J Hematother. 1996;5:475–483. doi: 10.1089/scd.1.1996.5.475. [DOI] [PubMed] [Google Scholar]
- 20.Zhu H., Kaufman D.S. An improved method to produce clinical-scale natural killer cells from human pluripotent stem cells. Methods Mol Biol. 2019;2048:107–119. doi: 10.1007/978-1-4939-9728-2_12. [DOI] [PubMed] [Google Scholar]
- 21.Karmakar S., Pal P., Lal G. Key activating and inhibitory ligands involved in the mobilization of natural killer cells for cancer immunotherapies. ImmunoTargets Ther. 2021;10:387–407. doi: 10.2147/ITT.S306109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Merino A., Zhang B., Dougherty P., Luo X., Wang J., Blazar B.R., et al. Chronic stimulation drives human NK cell dysfunction and epigenetic reprograming. J Clin Investig. 2019;129(9):3770–3785. doi: 10.1172/JCI125916. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Nakazawa T., Morimoto T., Maeoka R., Matsuda R., Nakamura M., Nishimura F., et al. Establishment of an efficient ex vivo expansion strategy for human natural killer cells stimulated by defined cytokine cocktail and antibodies against natural killer cell activating receptors. Regen Ther. 2022;21:185–191. doi: 10.1016/j.reth.2022.07.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Törnroos H., Hägerstrand H., Lindqvist C. Culturing the human natural killer cell line NK-92 in Interleukin-2 and Interleukin-15 - implications for clinical trials. Anticancer Res. 2019;39(1):107–112. doi: 10.21873/anticanres.13085. [DOI] [PubMed] [Google Scholar]
- 25.Chaix J., Tessmer M.S., Hoebe K., Fuséri N., Ryffel B., Dalod M., et al. Cutting edge: priming of NK cells by IL-18. J Immunol. 2008;181(3):1627–1631. doi: 10.4049/jimmunol.181.3.1627. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Xing Z., Kenty B.M., Li Z.J., Lee S.S. Scale-up analysis for a CHO cell culture process in large-scale bioreactors. Biotechnol Bioeng. 2009;103(4):733–746. doi: 10.1002/bit.22287. [DOI] [PubMed] [Google Scholar]
- 27.Xu S., Hoshan L., Jiang R., Gupta B., Brodean E., O'Neill K., et al. A practical approach in bioreactor scale-up and process transfer using a combination of constant P/V and vvm as the criterion. Biotechnol Prog. 2017;33(4):1146–1159. doi: 10.1002/btpr.2489. [DOI] [PubMed] [Google Scholar]
- 28.Min B., Choi H., Her J.H., Jung M.Y., Kim H.J., Jung M.Y., et al. Optimization of large-scale expansion and cryopreservation of human natural killer cells for anti-tumor therapy. Immune Netw. 2018;18(4) doi: 10.4110/in.2018.18.e31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Huang R.S., Lai M.C., Shih H.A., Lin S. A robust platform for expansion and genome editing of primary human natural killer cells. J Exp Med. 2021;218(3) doi: 10.1084/jem.20201529. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Brand A., Singer K., Koehl G.E., Kolitzus M., Schoenhammer G., Thiel A., et al. LDHA-associated lactic acid production blunts tumor immunosurveillance by T and NK cells. Cell Metab. 2016;24(5):657–671. doi: 10.1016/j.cmet.2016.08.011. [DOI] [PubMed] [Google Scholar]
- 31.Harmon C., Robinson M.W., Hand F., Almuaili D., Mentor K., Houlihan D.D., et al. Lactate-mediated acidification of tumor microenvironment induces apoptosis of liver-resident NK cells in colorectal liver metastasis. Cancer Immunol Res. 2019;7(2):335–346. doi: 10.1158/2326-6066.CIR-18-0481. [DOI] [PubMed] [Google Scholar]
- 32.Fischer K., Hoffmann P., Voelkl S., Meidenbauer N., Ammer J., Edinger M., et al. Inhibitory effect of tumor cell-derived lactic acid on human T cells. Blood. 2007;109(9):3812–3819. doi: 10.1182/blood-2006-07-035972. [DOI] [PubMed] [Google Scholar]
- 33.Johnson C.D.L., Zale N.E., Frary E.D., Lomakin J.A. Feeder-cell-free and serum-free expansion of natural killer cells using cloudz microspheres, G-Rex6M, and human platelet lysate. Front Immunol. 2022;13 doi: 10.3389/fimmu.2022.803380. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Dorsch M., Urlaub D., Bönnemann V., Bröde P., Sandusky M., Watzl C. Quantitative analysis of human NK cell reactivity using latex beads coated with defined amounts of antibodies. Eur J Immunol. 2020;50(5):656–665. doi: 10.1002/eji.201948344. [DOI] [PubMed] [Google Scholar]
- 35.Kim J., Phan M.T., Hwang I., Park J., Cho D. Comparison of the different anti-CD16 antibody clones in the activation and expansion of peripheral blood NK cells. Sci Rep. 2023;13(1):9493. doi: 10.1038/s41598-023-36200-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Costariol E., Rotondi M.C., Amini A., Hewitt C.J., Nienow A.W., Heathman T.R.J., et al. Demonstrating the manufacture of human CAR-T cells in an automated stirred-tank bioreactor. Biotechnol J. 2020;15(9) doi: 10.1002/biot.202000177. [DOI] [PubMed] [Google Scholar]
- 37.Nakazawa T., Morimoto T., Maeoka R., Matsuda R., Nakamura M., Nishimura F., et al. Establishment of an efficient ex vivo expansion strategy for human natural killer cells stimulated by defined cytokine cocktail and antibodies against natural killer cell activating receptors. Regen Ther. 2022;21:185–191. doi: 10.1016/j.reth.2022.07.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Mark C., Czerwinski T., Roessner S., Mainka A., Hörsch F., Heublein L., et al. Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells. Nat Commun. 2020;11:5224. doi: 10.1038/s41467-020-19094-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
All data generated or analyzed during this study, which support the findings of this study, are included within this article and its supplementary information files. Researchers interested in further analysis not present in the manuscript may contact the corresponding author.