Culture medium and method for preparing NK cells by inducing iPSC differentiation

Abstract

The invention relates to a culture medium and a method for preparing NK cells by inducing iPSC differentiation. According to the invention, a specific induced differentiation process and culture medium are designed, the whole differentiation process is divided into three key stages, the culture medium combined by different cell factors is specifically used, efficient induction and NK phenotype maturation are realized, high-proportion NK cells are obtained through 23-day differentiation, and iPSC suspension culture is applied to large-scale induced differentiation of the NK cells, so that the large-scale induced differentiation of the NK cells is realized. The uniformity and the induction efficiency of embryoid bodies (EB) are further improved, iPSC dynamic suspension differentiation is not limited by the bottom area of a container any more and is suitable for a bioreactor system, in addition, an amplification process based on a wave type bioreactor is designed, amplification of 200 times or above in 14 days can be achieved, the yield of 1011 or above levels of a single batch is finally achieved, and the method is suitable for large-scale production. A new thought and a new method are provided for large-scale preparation and application of the NK cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a culture medium and method for inducing iPSC differentiation to prepare NK cells. Background Technology

[0002] Natural killer (NK) cells are a type of innate immune lymphocyte that can recognize and kill virus-infected cells or tumor cells even without antigen presentation. Due to their innate tumor recognition ability, lack of GVHD risk, and the fact that they do not require individualized customization, NK cells are widely used in tumor immunotherapy, viral infection control, and immunosenescence intervention.

[0003] Induced pluripotent stem cells (iPSCs) provide a standardized and scalable seed source for NK cell therapy. By mimicking embryonic hematopoietic development in a specific cytokine-induced system, iPSCs can be progressively differentiated into hematopoietic progenitor cells and NK cells. Utilizing the unlimited self-renewal and standardized expansion capabilities of iPSCs, the donor dependence and quantity limitations of primary NK cells can be completely resolved. Based on their monoclonal origin, cell phenotype and function can be precisely regulated through gene editing (such as enhancing ADCC effects or homing ability), ensuring high batch-to-batch homogeneity of cell products. Combined with the ethically uncontroversial nature of iPSCs and their efficient differentiation process, this provides a scalable, cGMP-compliant cell resource for "off-the-shelf" immunotherapy.

[0004] To prepare iPSC-derived NK cells, researchers have mainly developed two processes: 2D and 3D. Existing 2D differentiation processes have limited production scale due to the inherent limitation of their culture surface area (batch yield is typically ≤10). 8 The lack of a three-dimensional microenvironment leads to unstable differentiation efficiency due to frequent manual manipulation, which introduces the risk of contamination and disrupts the closed system. Furthermore, the multi-stage transfer process further prolongs the production cycle and increases production costs, preventing it from becoming a standardized industrial-grade application. 3D differentiation technology effectively avoids these disadvantages. For example, CN117050940A discloses a full-process 3D suspension culture technology, using commercial StemScale medium to initiate iPSC embryoid (EB) aggregation, and driving mesoderm differentiation by precisely timing the addition of CHIR99021 (combined with Rki on day 0, and alone on day 1). In a three-dimensional dynamic suspension system, the following processes are completed sequentially: ① EB to CD34 + Hematopoietic stem cell differentiation (BMP4 / FGF2 / VEGF stepwise induction, CD34) + (proportion > 50%); ② HSPC differentiates into CD45 in suspension. + CD56 +NK cells; ③ Innovative application of IL-12+IL-15+IL-18 triple stimulation to achieve efficient expansion of memory-like NK cells in a 3D environment. This protocol maintains a three-dimensional suspension state throughout the process, obtaining high-purity (>80%) and highly active NK cells within 28 days, breaking through the scale limitations of traditional 2D processes and eliminating the need for exogenous feeder layers.

[0005] However, the current 3D differentiation process still has the following problems: (1) the low differentiation efficiency and process complexity lead to increased differences in product quality attributes between batches; (2) the low yield per batch makes it difficult to meet clinical needs. Therefore, developing a 3D culture process for NK cells prepared efficiently using iPSCs is of great significance for the application of NK cells. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a culture medium and method for inducing iPSC differentiation to prepare NK cells, and designs a specific culture medium and culture process to achieve efficient preparation of NK cells using iPSCs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a culture medium for inducing iPSC differentiation to prepare NK cells, the culture medium comprising a first culture medium to a sixth culture medium; The first culture medium includes cell culture medium supplemented with ROCK inhibitors and GSK-3 inhibitors; The second culture medium includes a cell culture medium supplemented with cytokines BMP4, bFGF, and VEGF; The third culture medium includes a cell culture medium supplemented with cytokines BMP4, bFGF, VEGF and ALK5 inhibitors; The fourth culture medium includes a cell culture medium supplemented with cytokines bFGF, VEGF and SCF; The fifth culture medium includes a cell culture medium supplemented with cytokines IL-7, SCF, FLT3L, IL-15 and IL-3; The sixth culture medium includes a cell culture medium supplemented with cytokines IL-7, SCF, FLT3L and IL-15.

[0008] In this invention, an induction culture medium system is designed, and a culture medium with a specific combination of cytokines is designed to achieve efficient induction and NK phenotype maturation.

[0009] Optionally, the cell culture medium in the first culture medium includes a suspension culture medium, such as Stemscale PSC suspension culture medium.

[0010] Optionally, the concentration of ROCK inhibitor in the first culture medium is 5~100 μM, for example, it can be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95 μM, and the concentration of GSK-3 inhibitor is 2~100 μM, for example, it can be 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95 μM.

[0011] Optionally, the cell culture medium in the second culture medium includes serum-free cell culture medium.

[0012] Optionally, the concentration of BMP4 in the second culture medium is 5~200 ng / mL, for example, it can be 10, 20, 30, 50, 80, 100, 150, 160, 170, 180, 190 or 195 ng / mL, the concentration of bFGF is 5~200 ng / mL, for example, it can be 10, 20, 30, 50, 80, 100, 150, 160, 170, 180, 190 or 195 ng / mL, and the concentration of VEGF is 5~200 ng / mL, for example, it can be 10, 20, 30, 50, 80, 100, 150, 160, 170, 180, 190 or 195 ng / mL.

[0013] Optionally, the second culture medium also contains GlutaMAX, insulin-transferrin-selenium, vitamin C injection, and thioglycerol.

[0014] Optionally, the concentration of GlutaMAX in the second culture medium is 0-10×, the concentration of insulin-transferrin-selenium is 0-10×, the concentration of VC injection is 10-100 μg / mL, for example, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 μg / mL, and the concentration of thioglycerol is 0-10 mM, for example, 2, 3, 4, 5, 6, 7, 8 or 9 mM.

[0015] Optionally, the cell culture medium in the third culture medium includes serum-free cell culture medium, such as Stempro-34 SFM serum-free culture medium.

[0016] Optionally, the concentration of BMP4 in the third culture medium is 5-200 ng / mL, for example, it can be 10, 20, 30, 50, 80, 100, 150, 160, 170, 180, 190, or 195 ng / mL, etc.; the concentration of bFGF is 5-200 ng / mL, for example, it can be 10, 20, 30, 50, 80, 100, 150, 160, 170, 180, 190, or 195 ng / mL, etc.; the concentration of VEGF is 5-200 ng / mL, for example, it can be 10, 20, 30, 50, 80, 100, 150, 160, 170, 180, 190, or 195 ng / mL, etc.; and the concentration of ALK5 inhibitor is 1-20 mM, for example, it can be 2, 3, 4, 5, 10, 15, 6, 7, 18, or 19 mM. mM, etc.

[0017] Optionally, the third culture medium also contains GlutaMAX, insulin-transferrin-selenium, vitamin C injection, and thioglycerol.

[0018] Optionally, the concentration of GlutaMAX in the third culture medium is 0-10×, the concentration of insulin-transferrin-selenium is 0-10×, the concentration of VC injection is 10-100 μg / mL, for example, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 μg / mL, and the concentration of thioglycerol is 0-10 mM, for example, 2, 3, 4, 5, 6, 7, 8 or 9 mM.

[0019] Optionally, the cell culture medium in the fourth culture medium includes serum-free cell culture medium.

[0020] Optionally, the concentration of bFGF in the fourth culture medium is 5~200 ng / mL, for example, it can be 10, 20, 30, 50, 80, 100, 150, 160, 170, 180, 190 or 195 ng / mL, the concentration of VEGF is 5~200 ng / mL, for example, it can be 10, 20, 30, 50, 80, 100, 150, 160, 170, 180, 190 or 195 ng / mL, and the concentration of SCF is 5~100 ng / mL, for example, it can be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95 ng / mL.

[0021] Optionally, the fourth culture medium also contains GlutaMAX, insulin-transferrin-selenium, vitamin C injection, and thioglycerol.

[0022] Optionally, the concentration of GlutaMAX in the fourth culture medium is 0-10×, the concentration of insulin-transferrin-selenium is 0-10×, the concentration of VC injection is 10-100 μg / mL, for example, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 μg / mL, and the concentration of thioglycerol is 0-10 mM, for example, 2, 3, 4, 5, 6, 7, 8 or 9 mM.

[0023] Optionally, the cell culture medium in the fifth culture medium includes DMEM medium and / or Ham's F12 medium.

[0024] Optionally, the concentration of IL-7 in the fifth culture medium is 5~100 ng / mL, for example, it can be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95 ng / mL, the concentration of SCF is 5~100 ng / mL, the concentration of FLT3L is 5~100 ng / mL, for example, it can be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95 ng / mL, the concentration of IL-15 is 5~100 ng / mL, for example, it can be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95 ng / mL, and the concentration of IL-3 is 0~100 ng / mL, for example, it can be 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95 ng / mL.

[0025] Optionally, the fifth culture medium may also contain human AB serum, β-mercaptoethanol, sodium selenide, ethanolamine, and vitamin C injection.

[0026] Optionally, the concentration of human AB serum in the fifth culture medium is 5%~20%, for example, it can be 6%, 7%, 8%, 10%, 15%, 16%, 17%, 18% or 19%, etc.; the concentration of β-mercaptoethanol is 0~200 μM, for example, it can be 5, 10, 20, 30, 40, 50, 100, 150, 160, 170, 180, 190 or 195 μM, etc.; the concentration of VC injection is 10~100 μg / mL, for example, it can be 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 μg / mL, etc.; the concentration of sodium selenide is 10~100 μM, for example, it can be 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 μM, etc.; and the concentration of ethanolamine is 10~100 μg / mL. ng / mL, for example, it can be 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 ng / mL, etc.

[0027] Optionally, the cell culture medium in the sixth culture medium includes DMEM medium and / or Ham's F12 medium.

[0028] Optionally, the concentration of IL-7 in the sixth culture medium is 10~100 ng / mL, for example, it can be 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 ng / mL, the concentration of SCF is 10~100 ng / mL, for example, it can be 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 ng / mL, the concentration of FLT3L is 10~100 ng / mL, for example, it can be 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 ng / mL, and the concentration of IL-15 is 10~100 ng / mL, for example, it can be 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 ng / mL.

[0029] Optionally, the sixth culture medium may also contain human AB serum, β-mercaptoethanol, sodium selenide, ethanolamine, and vitamin C injection.

[0030] Optionally, the concentration of human AB serum in the sixth culture medium is 5%~20%, for example, it can be 6%, 7%, 8%, 10%, 15%, 16%, 17%, 18% or 19%, etc.; the concentration of β-mercaptoethanol is 0~200 μM, for example, it can be 5, 10, 20, 30, 40, 50, 100, 150, 160, 170, 180, 190 or 195 μM, etc.; the concentration of VC injection is 10~100 μg / mL, for example, it can be 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 μg / mL, etc.; the concentration of sodium selenide is 10~100 μM, for example, it can be 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 μM, etc.; and the concentration of ethanolamine is 10~100 μg / mL. ng / mL, for example, it can be 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 ng / mL, etc.

[0031] Secondly, the present invention provides the application of the culture medium for inducing iPSC differentiation to prepare NK cells as described in the first aspect in the preparation of NK cells.

[0032] Thirdly, the present invention provides a method for inducing iPSC differentiation to prepare NK cells, the method comprising culturing iPSC cells using the culture medium for inducing iPSC differentiation to prepare NK cells as described in the first aspect.

[0033] Optionally, the method for inducing iPSC differentiation to prepare NK cells includes: iPSC cells were cultured in 3D suspension using the first culture medium, and then successively replaced with the second, third, and fourth culture media for 3D suspension culture to obtain hematopoietic progenitor cells (HSPCs); the hematopoietic progenitor cells were cultured in 3D suspension using the fifth and sixth culture media to obtain NK cells.

[0034] Optionally, the 3D suspension culture is carried out by continuous rotation at a speed of 50-80 rpm.

[0035] Optionally, the method further includes a step of expanding NK cells, including mixing NK cells with feeder cells for expansion culture.

[0036] Optionally, IL-2 is added to the culture medium for the amplification culture.

[0037] Optionally, the working concentration of IL-2 is 10~1000 IU / mL, for example, it can be 20, 30, 50, 100, 300, 500, 600, 700, 800, 900, 950 or 990 IU / mL.

[0038] Optionally, the ratio of NK cells to trophoblast cells can be 1:2.

[0039] Optionally, the amplification culture is carried out in a wave-type bioreactor.

[0040] Optionally, the method for inducing iPSC differentiation to prepare NK cells further includes the steps of reviving and expanding iPSCs.

[0041] Optionally, the resuscitation and expansion of iPSCs includes culturing iPSC cells under 3D suspension culture conditions, with the culture medium including Y27632; passaged every 3-5 days. The passaged iPSC cells are then used for induced differentiation culture.

[0042] In this invention, the induction of differentiation by using iPSCs that have been revived and expanded through 3D suspension culture can significantly improve the uniformity and induction efficiency of embryoid bodies (EB).

[0043] Optionally, the method for inducing iPSC differentiation to prepare NK cells includes the following steps: On day 0, iPSC cells were cultured in 3D suspension using the first culture medium; On day 1, the culture medium was replaced with the second medium to continue 3D suspension culture; On days 2-3, the medium was replaced with the third medium every 22-26 hours to continue 3D suspension culture; From day 4 to day 6, the culture medium was replaced with the fourth medium every 22 to 26 hours to continue 3D suspension culture and obtain hematopoietic progenitor cells. From day 7 to day 13, replace the medium with the fifth medium every 46 to 50 hours to continue 3D suspension culture; From day 14 to 22, the medium was replaced with the sixth medium every 46 to 50 hours to continue 3D suspension culture and obtain NK cells; On days 23-30, NK cells were mixed with feeder cells for expansion culture, and IL-2 was added to the culture medium every 46-50 hours. On day 30, the expanded NK cells were mixed with feeder cells again for expansion culture. IL-2 was added to the culture medium every 46-50 hours, and the expanded NK cells were harvested.

[0044] Compared with the prior art, the present invention has at least the following beneficial effects: This invention designs a specific induction differentiation process and culture medium, dividing the entire differentiation process into three key stages (CD34). + (HSPC induction, NK cell induction, NK cell maturation), with targeted addition of different cytokine combinations to achieve efficient induction and NK phenotype maturation, resulting in a high proportion and quantity of NK cells differentiated within 23 days. A 3D dynamic suspension culture and NK cell differentiation system was constructed, and iPSC suspension culture was applied to the large-scale induction and differentiation of NK cells, further improving cell cluster uniformity and induction efficiency. Further dynamic suspension differentiation is no longer limited by the container bottom area, making it suitable for bioreactor systems. Furthermore, an amplification process based on a wave-type bioreactor was designed, enabling over 200-fold amplification in 14 days, ultimately achieving a single batch size of 10... 11 The above-mentioned production levels provide new ideas and methods for the large-scale preparation and application of NK cells. Attached Figure Description

[0045] Figure 1 The flow cytometry results of HSPC cells prepared for this example; Figure 2 The flow cytometry results of HSPC cells prepared in the comparative preparation example are shown in the figure. Figure 3 This is a schematic diagram of the differentiation protocol for NK cells using iPSCs in Example 1; Figure 4 A schematic diagram of the differentiation protocol for NK cells using iPSCs, used in Comparative Example 1. Figure 5 This is a graph showing the CD56 expression results identified by flow cytometry after 23 days of differentiation in Example 1. Figure 6 Figure showing the CD56 expression results identified by flow cytometry after 28 days of differentiation in Comparative Example 1. Figure 7 Figure 1 shows the continuous killing effect of expanded NK cells on K562 cells at a 1:1 effector-to-target ratio, as shown in Example 1 and Comparative Example 1. Figure 8 Figure 1 shows the continuous killing effect of expanded NK cells on K562 cells at an effector-to-target ratio of 1:3, as shown in Example 1 and Comparative Example 1. Figure 9 The diagram shows the changes in cell morphology during the differentiation of iPSCs into HSPCs in Example 1 and Comparative Example 2. Figure 10 A graph showing the fold expansion of NK cells in a WAVE reactor; Figure 11 This is a flow cytometry result of amplified NK cells in a WAVE reactor. Detailed Implementation

[0046] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0047] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0048] In a specific embodiment of this invention, the feeder cells (K562 cells expressing IL21-CD137L irradiated by X-rays), Stemscale PSC suspension medium were purchased from Thermo Fisher (Catalog No. A4965001); Y27632 was purchased from Stemcell (Catalog No. 72304); CHIR-99021 was purchased from Wako (Catalog No. 038-23101); Stempro-34... SFM serum-free culture medium was purchased from Thermo Fisher (catalog number 10639011); GlutaMAX was purchased from Thermo Fisher (catalog number 35050061); BMP4 was purchased from R&D (catalog number 314-BP-010); bFGF was purchased from R&D (catalog number 3718-FB-025); VEGF was purchased from R&D (catalog number 293-VE-010); SB-431542 was purchased from MedChemExpress (catalog number HY-10431); SCF (human stem cell factor) was purchased from R&D. The following products were purchased: IL-15 (catalog number 255-SC-050), IL-3 (catalog number 300-18), IL-7 (catalog number 200-07-500), and IL-2 (catalog number 200-02). The TPO (thrombopoietin) was purchased from PeproTech. FLT3L (FMS-like tyrosine kinase 3 ligand) was purchased from R&D.

[0049] In a specific embodiment of the present invention, the culture medium includes: M1 medium (first medium): Stemscale PSC suspension medium with 10 μM Y27632 (ROCK inhibitor) and 10 μM CHIR-99021 (GSK-3 inhibitor) added. M2 medium (second medium): Stempro-34 SFM serum-free medium supplemented with 1×GlutaMAX, 1×Insulin-Transferrin-Selenium, 50 μg / mL VC injection, 0.4 mM 1-thioglycerol, 10 ng / mL BMP4, 10 ng / mL bFGF and 10 ng / mL VEGF; M3 medium (third medium): Stempro-34 SFM serum-free medium supplemented with 1×GlutaMAX, 1×Insulin-Transferrin-Selenium, 50 μg / mL VC injection, 0.4 mM 1-thioglycerol, 10 ng / mL BMP4, 10 ng / mL bFGF, 10 ng / mL VEGF and 6 μM SB-431542 (ALK5 inhibitor); M4 medium (fourth medium): Stempro-34 SFM serum-free medium supplemented with 1×GlutaMAX, 1×Insulin-Transferrin-Selenium, 50 μg / mL VC injection, 0.4 mM 1-thioglycerol, 10 ng / mL bFGF, 10 ng / mL VEGF and 20 ng / mL SCF; M5 medium: Stempro-34 SFM serum-free medium supplemented with 1×GlutaMAX, 1×Insulin-Transferrin-Selenium, 50 μg / mL VC injection, 0.4 mM 1-thioglycerol, 10 ng / mL bFGF, 10 ng / mL VEGF, 20 ng / mL SCF, 50 ng / mL TPO and 50 ng / mL FLT3L; M6 medium (fifth medium): 56.7% DMEM medium supplemented with 28.3% Ham's F12 medium, 15% human AB serum, 25 μM β-mercaptoethanol, 5 ng / mL sodium selenide, 50 μM ethanolamine, 20 μg / mL VC injection, 20 ng / mL SCF, 20 ng / mL IL-7, 10 ng / mL FLT3L, 10 ng / mL IL-15 and 5 ng / mL IL-3; M7 medium (sixth medium): 56.7% DMEM medium supplemented with 28.3% Ham's F12 medium, 15% human AB serum, 25 μM β-mercaptoethanol, 5 ng / mL sodium selenide, 50 μM ethanolamine, 20 μg / mL VC injection, 20 ng / mL SCF, 20 ng / mL IL-7, 10 ng / mL FLT3L and 10 ng / mL IL-15.

[0050] Preparation Example This preparation example induces iPSC differentiation into HSPC. Using commercially available Vitronectin to coat six-well culture plates, iPSCs were revived, resuspended in Stemfit Basic 03 complete medium, and then added to the coated culture plates. 10 μM Y-27632 was added, and the plates were incubated in a tri-gas incubator (5% O2, 5% CO2, 37℃). The initial revival was recorded as P+0 generation. Cells were passaged every 4 days, and each passage was recorded as p+1, p+2, p+3, and so on.

[0051] When iPSCs reached 70% confluence at passage P+2, the cells were examined and found to have healthy morphology and no spontaneous differentiation. The iPSCs were then dissociated into single cells using an accutase. The collected single cells were counted and seeded in Stemfit03 complete medium (day 0) containing 10 μM Y-27632 and 10 μM CHIR-99021.

[0052] Cells were continuously incubated in a three-gas incubator (5% O2, 5% CO2, 37°C) on a CO2-resistant orbital oscillator at 70 rpm.

[0053] 24 hours later (day 1), replace the culture medium with fresh M2 differentiation medium.

[0054] Replace the M3 differentiation medium in the culture system every 24 hours (on days 2-3).

[0055] Replace the culture medium with fresh M4 differentiation medium every 24 hours (days 4-6).

[0056] Embryoids (EBs) were harvested on day 7, and CD34 / CD43 expression was detected by flow cytometry after the EBs were dissociated into single cells using Accutase.

[0057] Comparative preparation example This comparative example used commercially available Vitronectin-coated six-well culture plates. iPSCs were revived, resuspended in Stemfit Basic 03 complete medium, and then added to the coated culture plates. 10 μM of Y-27632 was added, and the plates were cultured in a tri-gas incubator (5% O2, 5% CO2, 37℃). The initial revival was recorded as P+0 generation. Cells were passaged every 4 days, and each passage was recorded as p+1, p+2, p+3, and so on.

[0058] When iPSCs reached 70% confluence at passage P+2, the cells were examined and found to have healthy morphology and no spontaneous differentiation. Accutase was used to dissociate the iPSCs into single cells. The collected single cells were counted and seeded in APEL2 medium supplemented with 10 μM Y27632, 40 ng / mL SCF, 20 ng / mL VEGF, and 20 ng / mL BMP4.

[0059] Cells were continuously incubated in a three-gas incubator (5% O2, 5% CO2, 37°C) on a CO2-resistant orbital oscillator at 70 rpm.

[0060] Every 24 hours, the culture system was replenished with fresh APEL2 medium containing 10 μM Y27632, 40 ng / mL SCF, 20 ng / mL VEGF, and 20 ng / mL BMP4.

[0061] EB was harvested on day 7, and CD34 / CD43 expression was detected by flow cytometry after the EB spheres were dissociated into single cells using Accutase.

[0062] The flow cytometry results of the prepared HSPC cells are as follows: Figure 1 As shown, the flow cytometry results of the HSPC cells prepared in the comparative preparation example are as follows: Figure 2 As shown, the results indicate that the optimized combination of cytokines and small molecule compounds in this invention enhances CD34. + HSPC induction efficiency was assessed by optimizing the combination of factors and small molecules used to induce HSPC, as well as determining the appropriate dosage of each factor and small molecule and the induction differentiation medium. This resulted in a CD34 induction rate of >80% after 7 days. + HSPC differentiation efficiency.

[0063] Example 1 This embodiment utilizes iPSCs in suspension culture to prepare and expand NK cells. Resuscitate iPSCs, resuspend cells in StemScale medium, and add to a 125 mL shake flask. The cell seeding density is 1.5e5 / mL, and the culture medium volume is 25 mL. Add 10 μM Y-27632 and incubate in a tri-gas incubator (5% O2, 5% CO2, 37℃) on a CO2-resistant orbital shaker at 70 rpm. The initial resuscitation is recorded as P+0 generation. Subculture every 4 days, and each subculture is recorded as P+1, P+2, P+3, etc. When P+2 generation iPSCs were cultured to day 4, the cells were examined and found to have healthy morphology and no spontaneous differentiation. The iPSC cells were then dissociated into single cells using Accutase. The collected single cells were counted and seeded in M1 differentiation medium (day 0), and the cells were examined and found to have healthy morphology and no spontaneous differentiation. The embryoid (EB) spheres formed by the suspended iPSC cells were then dissociated into single cells using Accutase. The collected single cells were counted and seeded in differentiation medium supplemented with 10 μM Y27632 and 10 μM CHIR 99021 (day 0).

[0064] Cells were incubated in a tri-gas incubator (5% O2, 5% CO2, 37°C) by continuous rotation at 70 rpm on a CO2-resistant orbital oscillator.

[0065] 24 hours later (day 1), replace the culture medium with fresh M2 differentiation medium.

[0066] Replace the M3 differentiation medium in the culture system every 24 hours (on days 2-3).

[0067] Replace the culture medium with fresh M4 differentiation medium every 24 hours (days 4-6).

[0068] Every 48 hours (days 7-13), fresh M6 differentiation medium was added to the culture system. Cells were continuously incubated at 37°C and 5% CO2 in a CO2-resistant orbital oscillator at 70 rpm.

[0069] Replace the culture medium with fresh M7 differentiation medium every 48 hours (days 14-22).

[0070] On day 23, differentiated single cells were harvested and CD45 / CD56 expression was detected by flow cytometry. The harvested NK cells and feeder cells were seeded into a G-REX cell culture system at a ratio of 1:2, using NK Macs medium containing 5% human serum and 200 IU / mL IL-2.

[0071] On day 26, IL-2 with a final concentration of 200 IU / mL was added to the NK amplification system.

[0072] On day 28, IL-2 with a final concentration of 200 IU / mL was added to the NK amplification system.

[0073] On day 30, NK cells and IL-2 at a final concentration of 200 IU / mL were added to the NK expansion system at a ratio of 1:2.

[0074] On day 33, IL-2 with a final concentration of 200 IU / mL was added to the NK amplification system.

[0075] On day 35, IL-2 with a final concentration of 200 IU / mL was added to the NK amplification system.

[0076] On day 37, the expanded NK cells were harvested and their continuous killing ability against K562 target cells was tested.

[0077] Example 2 This embodiment expands NK cells in a WAVE bioreactor.

[0078] Resuscitate iPSCs, resuspend cells in StemScale medium, and add to a 125 mL shake flask. The cell seeding density is 1.5e5 / mL, and the culture medium volume is 25 mL. Add 10 μM Y-27632 and incubate in a tri-gas incubator (5% O2, 5% CO2, 37℃) on a CO2-resistant orbital shaker at 70 rpm. The initial resuscitation is recorded as P+0 generation. Subculture every 4 days, and each subculture is recorded as P+1, P+2, P+3, etc. When P+2 generation iPSCs were cultured to day 4, the cells were examined and found to have healthy morphology and no spontaneous differentiation. The iPSC cells were then dissociated into single cells using an accutase. The collected single cells were counted and seeded in M1 differentiation medium (day 0), and the cells were examined and found to have healthy morphology and no spontaneous differentiation. The embryoid (EB) spheres formed by the suspended iPSC cells were then dissociated into single cells using an accutase. The collected single cells were counted and seeded in differentiation medium supplemented with 10 μM MY27632 and 10 μM CHIR 99021 (day 0).

[0079] Cells were incubated in a tri-gas incubator (5% O2, 5% CO2, 37°C) by continuous rotation at 70 rpm on a CO2-resistant orbital oscillator.

[0080] 24 hours later (day 1), replace the culture medium with fresh M2 differentiation medium.

[0081] Replace the M3 differentiation medium in the culture system every 24 hours (on days 2-3).

[0082] Replace the culture medium with fresh M4 differentiation medium every 24 hours (days 4-6).

[0083] Every 48 hours (days 7-13), fresh M6 differentiation medium was added to the culture system. Cells were continuously incubated at 37°C and 5% CO2 in a CO2-resistant orbital oscillator at 70 rpm.

[0084] Replace the culture medium with fresh M7 differentiation medium every 48 hours (days 14-22).

[0085] On day 23, differentiated single cells were harvested and CD45 / CD56 expression was analyzed by flow cytometry. Differentiated NK cells were expanded using a WAVE bioreactor. 2.16e8 cells were seeded from the harvested NK cells into a 5L cell culture bag. Similarly, 4.32e8 feeder cells were added to the culture bag. Expanding culture was performed using NK Macs medium containing 5% human serum and 200 IU / mL IL-2, with an initial medium volume of 432.7 mL. The WAVE bioreactor parameters were adjusted to an angle of 6° and a rotation speed of 6 rpm (day 0).

[0086] On day 3, the culture medium volume in the cell culture bag was increased to 1000 mL using NK Macs medium containing 5% human serum and 200 IU / mL IL-2.

[0087] On day 5, the culture medium volume in the cell culture bag was increased to 1500 mL using NK Macs medium containing 5% human serum and 200 IU / mL IL-2.

[0088] On day 7, based on the number of cells in the cell culture bag, feeder cells were added at a ratio of 1:2. The parameters of the WAVE reactor were adjusted to an angle of 6° and a rotation speed of 7 rpm, and perfusion operations were started on the amplification system from that day. The glucose concentration in the culture system was maintained at 1 g / L, and the perfusion rate and drainage rate were adjusted daily from day 7 to day 13 according to this standard.

[0089] On day 9, the parameters of the WAVE reactor were adjusted to an angle of 7° and a rotation speed of 8 rpm.

[0090] On day 12, the parameters of the WAVE reactor were adjusted to an angle of 8° and a rotation speed of 8 rpm.

[0091] On day 14, the expanded NK cells were harvested, the expansion fold was calculated, and the expression of cell surface markers was detected by flow cytometry.

[0092] Example 3 This embodiment utilizes adherent iPSCs to prepare NK cells.

[0093] Six-well culture plates were coated with commercially available vitronectin. iPSCs were revived, resuspended in Stemfit Basic 03 complete medium, and then added to the coated plates. 10 μM Y-27632 was added, and the plates were cultured in a tri-gas incubator (5% O2, 5% CO2, 37℃). The initial revival was recorded as P+0 generation. The cells were passaged every 4 days, and each passage was recorded as P+1, P+2, P+3, and so on.

[0094] When iPSCs reached 70% confluence at passage P+2, the cells were examined and found to have healthy morphology and no spontaneous differentiation. The iPSCs were then dissociated into single cells using an accutase. The collected single cells were counted and seeded in Stemfit03 complete medium (day 0) containing 10 μM Y-27632 and 10 μM CHIR-99021.

[0095] Cells were incubated in a tri-gas incubator (5% O2, 5% CO2, 37°C) by continuous rotation at 70 rpm on a CO2-resistant orbital oscillator.

[0096] 24 hours later (day 1), replace the culture medium with fresh M2 differentiation medium.

[0097] Replace the M3 differentiation medium in the culture system every 24 hours (on days 2-3).

[0098] Replace the culture medium with fresh M4 differentiation medium every 24 hours (days 4-6).

[0099] Every 48 hours (days 7-13), fresh M6 differentiation medium was added to the culture system. Cells were continuously incubated at 37°C and 5% CO2 in a CO2-resistant orbital oscillator at 70 rpm.

[0100] Replace the culture medium with fresh M7 differentiation medium every 48 hours (days 14-22).

[0101] On day 23, differentiated single cells were harvested and CD45 / CD56 expression was detected by flow cytometry.

[0102] Comparative Example 1 This comparative study utilized HSPCs formed from iPSCs after 14 days of differentiation to prepare NK cells and expand iNK cells.

[0103] Using commercially available Vitronectin to coat six-well culture plates, iPSCs were revived, resuspended in Stemfit Basic 03 complete medium, and then added to the coated culture plates. 10 μM Y-27632 was added, and the plates were incubated in a tri-gas incubator (5% O2, 5% CO2, 37℃). The initial revival was recorded as P+0 generation. Cells were passaged every 4 days, and each passage was recorded as P+1, P+2, P+3, etc.

[0104] When iPSCs reached 70% confluence at passage P+2, the cells were examined and found to have healthy morphology and no spontaneous differentiation. Accutase was used to dissociate the iPSCs into single cells. The collected single cells were counted and seeded in Stemfit03 complete medium containing 10 μM Y-27632 and 10 μM CHIR-99021 (day 0).

[0105] The cells were continuously incubated in a three-gas incubator (5% O2, 5% CO2, 37°C) on a CO2-resistant orbital oscillator at a speed of 70 rpm.

[0106] 24 hours later (day 1), replace the culture medium with fresh M2 differentiation medium.

[0107] Replace the M3 differentiation medium in the culture system every 24 hours (on days 2-3).

[0108] Replace the culture medium with fresh M4 differentiation medium every 24 hours (days 4-6).

[0109] Every 48 hours (days 7-13), fresh M5 differentiation medium was added to the culture system. Cells were continuously incubated at 37°C and 5% CO2 in a CO2-resistant orbital oscillator at 70 rpm.

[0110] Replace the culture medium with fresh M6 differentiation medium every 48 hours (days 14-20).

[0111] Replace the M7 differentiation medium in the culture system with fresh medium every 48 hours (days 21-27). (This step is compared with Example 1.) On day 28, differentiated single cells were harvested and CD45 / CD56 expression was detected by flow cytometry. The harvested NK cells and X-ray irradiated K562 cells expressing IL-21-CD137L (feeder cells) were seeded into the GREX culture system at a ratio of 1:2. The culture medium used was NK Macs medium containing 5% human serum and 200 IU / mL IL-2.

[0112] On day 31, IL-2 with a final concentration of 200 IU / mL was added to the NK amplification system.

[0113] On day 33, IL-2 with a final concentration of 200 IU / mL was added to the NK amplification system.

[0114] On day 35, NK cells and IL-2 at a final concentration of 200 IU / mL were added to the NK expansion system at a ratio of 1:2.

[0115] On day 38, IL-2 with a final concentration of 200 IU / mL was added to the NK amplification system.

[0116] On day 40, IL-2 with a final concentration of 200 IU / mL was added to the NK amplification system.

[0117] On day 42, the expanded NK cells were harvested and their continuous killing ability against K562 target cells was tested.

[0118] Schematic diagrams of the differentiation protocols for NK cells using iPSCs in Example 1 and Comparative Example 1 are shown below. Figure 3 and Figure 4 As shown in the figure, the CD56 expression results identified by flow cytometry after 23 days / 28 days of differentiation in Example 1 and Comparative Example 1 are as follows. Figure 5 and Figure 6 As shown, the results indicate that the present invention designs a specific differentiation-inducing strategy, dividing the entire differentiation process into three key stages (CD34). + This approach (inducing hematopoietic progenitor cells, NK cells, and NK cell maturation) involves the targeted addition of specific cytokine combinations and dosages to achieve highly efficient induction and NK phenotype maturation, requiring fewer inducing factors and significantly reducing costs. Optimizing the factor combination for inducing HSPCs shortens the differentiation time, allowing more time for NK cell induction and maturation. Ultimately, over 90% NK cell proportion is obtained within 23 days.

[0119] To test the NK cell killing effect, the experimental procedure included seeding K562-GFP cells (K562 cells expressing green fluorescent protein, which are ideal target cells for detecting NK cell killing effects) into 96-well plates at a rate of 1.5E4 cells / well. The NK cells to be tested were added according to the appropriate effector-to-target ratio, mixed thoroughly, and then the seeded 96-well plates were placed in an IncuCyte instrument to detect the intensity of green fluorescence in real time. The fluorescence intensity was used to reflect the strength of the NK cell killing ability. The amplified NK cells prepared in Example 1 and Comparative Example 1 were tested at a 1:1 ratio. Figure 7 ) and 1:3 ( Figure 8 The study investigated the continuous killing effect of NK cells on K562 cells at a target-effect ratio of 14 days. The results showed that the NK cells obtained after co-incubating and expanding with feeder cells had a significantly higher killing ability against K562 cells than NK cells differentiated and expanded by other methods.

[0120] The cell morphological changes during the differentiation of iPSCs into HSPCs in Examples 1 and 3 are as follows: Figure 9As shown in Table 1, the final number of cells differentiated from iPSCs into NK cells indicates that applying three-dimensional dynamic suspension culture of iPSCs (3D culture during the resuscitation and expansion phases) to the large-scale induction and differentiation of NK cells significantly improves cell cluster uniformity and induction efficiency. Further dynamic suspension differentiation will no longer be limited by the container bottom area, making it suitable for bioreactor systems. Furthermore, the entire culture and differentiation process is carried out at a uniform rotation speed, simplifying operation and avoiding batch-to-batch differences in product quality attributes caused by different rotation speeds.

[0121] Table 1 The fold expansion curve of NK cells in the WAVE reactor in Example 2 is shown in Figure 2. Figure 10 As shown, the flow cytometry results of the amplified NK cells are as follows: Figure 11 As shown, the results indicate that 5 L-scale NK cell expansion was achieved using feeder cells and the Wave bioreactor system, and each batch of iPSCs could stably differentiate into 10 cells at a scale of 125 mL shake flasks. 9 CD56 (Level 56 and above) + CD3 - NK cells with a purity of over 90% were cultured in a feeder cell and 5 L wave-type bioreactor system, achieving a more than 200-fold expansion in 14 days, ultimately yielding 10 cells per batch. 11 Production at the above levels.

[0122] In summary, this invention constructs a three-dimensional dynamic suspension culture and differentiation system for NK cells. Applying iPSC three-dimensional dynamic suspension culture to the large-scale induction and differentiation of NK cells significantly improves cell cluster uniformity and induction efficiency. Further dynamic suspension differentiation will no longer be limited by the container bottom area, making it suitable for bioreactor systems. A specific induction and differentiation process can be designed, dividing the entire differentiation process into three key stages (CD34...). + HSPC induction, NK cell induction, and NK cell maturation were achieved by selectively adding different combinations and doses of cytokines, resulting in highly efficient induction and NK phenotype maturation. A high proportion of NK cells differentiated within 23 days. Furthermore, an amplification process based on a Wave bioreactor was designed, enabling over 200-fold amplification in 14 days, ultimately achieving a single batch size of 10... 11 The above-mentioned production levels provide new ideas and methods for the large-scale preparation and application of NK cells.

[0123] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A culture medium for inducing iPSC differentiation to prepare NK cells, characterized in that, The culture medium includes the first to the sixth culture media; The first culture medium includes cell culture medium supplemented with ROCK inhibitors and GSK-3 inhibitors; The second culture medium includes a cell culture medium supplemented with cytokines BMP4, bFGF, and VEGF; The third culture medium includes a cell culture medium supplemented with cytokines BMP4, bFGF, VEGF and ALK5 inhibitors; The fourth culture medium includes a cell culture medium supplemented with cytokines bFGF, VEGF, and SCF; The fifth culture medium includes a cell culture medium supplemented with cytokines IL-7, SCF, FLT3L, IL-15 and IL-3; The sixth culture medium includes a cell culture medium supplemented with cytokines IL-7, SCF, FLT3L and IL-15.

2. The culture medium for inducing iPSC differentiation to prepare NK cells according to claim 1, characterized in that, The cell culture medium in the first culture medium includes a suspension culture medium; The concentration of ROCK inhibitor in the first culture medium is 5~100 μM, and the concentration of GSK-3 inhibitor is 2~100 μM; The cell culture medium in the second culture medium includes serum-free cell culture medium; The concentrations of BMP4, bFGF, and VEGF in the second culture medium were 5-200 ng / mL, 5-200 ng / mL, and 5-200 ng / mL, respectively. The second culture medium also contains GlutaMAX, insulin-transferrin-selenium, vitamin C injection and thioglycerol; The concentrations of GlutaMAX, insulin-transferrin-selenium, VC injection, and thioglycerol in the second culture medium were 0-10×, 0-10×, 0-100 μg / mL, and 0-10 mM, respectively.

3. The culture medium for inducing iPSC differentiation to prepare NK cells according to claim 1, characterized in that, The cell culture medium in the third culture medium includes serum-free cell culture medium; The concentration of BMP4 in the third culture medium is 5~200 ng / mL, the concentration of bFGF is 5~200 ng / mL, the concentration of VEGF is 5~200 ng / mL, and the concentration of ALK5 inhibitor is 1~20 mM. The third culture medium also contains GlutaMAX, insulin-transferrin-selenium, vitamin C injection and thioglycerol; The concentration of GlutaMAX in the third culture medium is 0~10×, the concentration of insulin-transferrin-selenium is 0~10×, the concentration of VC injection is 0~100 μg / mL, and the concentration of thioglycerol is 0~10 mM. The fourth culture medium includes a serum-free cell culture medium; The concentration of bFGF in the fourth culture medium is 5~200 ng / mL, the concentration of VEGF is 5~200 ng / mL, and the concentration of SCF is 5~100 ng / mL. The fourth culture medium also contains GlutaMAX, insulin-transferrin-selenium, vitamin C injection and thioglycerol; The concentrations of GlutaMAX, insulin-transferrin-selenium, VC injection, and thioglycerol in the fourth culture medium are 0-10×, 0-10×, 0-100 μg / mL, and 0-10 mM, respectively.

4. The culture medium for inducing iPSC differentiation to prepare NK cells according to claim 1, characterized in that, The cell culture medium in the fifth culture medium includes DMEM medium and / or Ham's F12 medium; The concentrations of IL-7, SCF, FLT3L, IL-15, and IL-3 in the fifth culture medium are 5-100 ng / mL, 5-100 ng / mL, 0-100 ng / mL, and 0-100 ng / mL, respectively. The fifth culture medium also contains human AB serum, β-mercaptoethanol, sodium selenide, ethanolamine, and vitamin C injection. The concentration of human AB serum in the fifth culture medium is 5%~20%, the concentration of β-mercaptoethanol is 0~200 μM, the concentration of VC injection is 10~100 μg / mL, the concentration of sodium selenide is 10~100 μM, and the concentration of ethanolamine is 10~100 ng / mL. The cell culture medium in the sixth culture medium includes DMEM medium and / or Ham's F12 medium; The concentrations of IL-7, SCF, FLT3L, and IL-15 in the sixth culture medium were 10-100 ng / mL, 10-100 ng / mL, 10-100 ng / mL, and 10-100 ng / mL, respectively. The sixth culture medium also contains human AB serum, β-mercaptoethanol, sodium selenide, ethanolamine, and vitamin C injection. The sixth culture medium contains 5%–20% human AB serum, 0–200 μM β-mercaptoethanol, 10–100 μg / mL VC injection, 10–100 μM sodium selenide, and 10–100 ng / mL ethanolamine.

5. The application of the culture medium for inducing iPSC differentiation to prepare NK cells according to any one of claims 1-4 in the preparation of NK cells.

6. A method for inducing iPSC differentiation to prepare NK cells, characterized in that, The method includes culturing iPSC cells using the culture medium for preparing NK cells by inducing iPSC differentiation as described in any one of claims 1-4.

7. The method for inducing iPSC differentiation to prepare NK cells according to claim 6, characterized in that, The method includes: iPSC cells were cultured in 3D suspension using the first culture medium, and then the culture medium was successively replaced with the second, third and fourth culture media for 3D suspension culture to obtain hematopoietic progenitor cells. The hematopoietic progenitor cells were cultured in 3D suspension using the fifth and sixth culture media to obtain NK cells.

8. The method for inducing iPSC differentiation to prepare NK cells according to claim 7, characterized in that, The method also includes a step of expanding NK cells, including mixing NK cells with feeder cells for expansion culture; The culture medium used for the amplification culture contains IL-2.

9. The method for inducing iPSC differentiation to prepare NK cells according to claim 8, characterized in that, The amplification culture was carried out in a wave-type bioreactor.

10. The method for inducing iPSC differentiation to prepare NK cells according to claim 6, characterized in that, The method includes the following steps: On day 0, iPSC cells were cultured in 3D suspension using the first culture medium; On day 1, the culture medium was replaced with the second medium to continue 3D suspension culture; On days 2-3, the medium was replaced with the third medium every 22-26 hours to continue 3D suspension culture; From day 4 to day 6, the culture medium was replaced with the fourth medium every 22 to 26 hours to continue 3D suspension culture and obtain hematopoietic progenitor cells. From day 7 to day 13, replace the medium with the fifth medium every 46 to 50 hours to continue 3D suspension culture; From day 14 to 22, the medium was replaced with the sixth medium every 46 to 50 hours to continue 3D suspension culture and obtain NK cells; On days 23-30, NK cells were mixed with feeder cells for expansion culture, and IL-2 was added to the culture medium every 46-50 hours. On day 30, the expanded NK cells were mixed with feeder cells again for expansion culture. IL-2 was added to the culture medium every 46-50 hours, and the expanded NK cells were harvested.

Images