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CN-122012393-A - Culture medium and method for preparing NK cells by inducing iPSC differentiation

CN122012393ACN 122012393 ACN122012393 ACN 122012393ACN-122012393-A

Abstract

The invention relates to a culture medium and a method for preparing NK cells by inducing iPSC differentiation. The invention designs a specific induction differentiation process and a culture medium, divides the whole differentiation process into three key stages, uses the culture medium combined by different cytokines pertinently, realizes efficient induction and NK phenotype maturation, obtains high-proportion NK cells by 23 days differentiation, applies iPSC suspension culture to large-scale induction differentiation of NK cells, further improves uniformity and induction efficiency of Embryoid Body (EB), and enables the iPSC dynamic suspension differentiation not to be limited by the bottom area of a container, is suitable for a bioreactor system, and in addition, designs an amplification process based on a wave bioreactor, can realize amplification for more than 200 times in 14 days, finally realizes single batch 10 11 or more grade yield, and provides a new idea and a new method for large-scale preparation and application of NK cells.

Inventors

  • XU JING
  • JI WEI
  • ZHANG YONGCHAO
  • Zhou Shuiguan
  • REN KEYONG

Assignees

  • 杭州济元基因科技有限公司

Dates

Publication Date
20260512
Application Date
20260409

Claims (10)

  1. 1. The culture medium for preparing NK cells by inducing iPSC differentiation is characterized by comprising a first culture medium, a second culture medium, a third culture medium and a fourth culture medium; Wherein the first medium comprises a cell culture medium supplemented with a ROCK inhibitor and a GSK-3 inhibitor; Wherein the second medium comprises a cell culture medium supplemented with cytokines BMP4, bFGF, and VEGF; Wherein the third medium comprises a cell culture medium supplemented with cytokines such as BMP4, bFGF, VEGF, and ALK5 inhibitors; wherein the fourth medium comprises a cell culture medium supplemented with cytokines bFGF, VEGF, and SCF; wherein the fifth medium comprises a cell culture medium supplemented with cytokines IL-7, SCF, FLT3L, IL-15, and IL-3; Wherein the sixth medium comprises a cell culture medium supplemented with cytokines IL-7, SCF, FLT3L and IL-15.
  2. 2. The medium for inducing differentiation of ipscs to make NK cells according to claim 1, wherein the cell culture medium in the first medium comprises a suspension medium; the concentration of the ROCK inhibitor in the first culture medium is 5-100 mu M, and the concentration of the GSK-3 inhibitor is 2-100 mu M; the cell culture medium in the second culture medium comprises a serum-free cell culture medium; The concentration of BMP4 in the second culture medium is 5-200 ng/mL, the concentration of bFGF is 5-200 ng/mL, and the concentration of VEGF is 5-200 ng/mL; the second culture medium is also added with Glutamax, insulin-transferrin-selenium, VC injection and thioglycerol; 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 0-100 μg/mL, and the concentration of thioglycerol is 0-10 mM.
  3. 3. The medium for inducing differentiation of ipscs to make NK cells according to claim 1, wherein the cell culture medium in the third medium comprises 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 is also added with Glutamax, insulin-transferrin-selenium, VC 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 cell culture medium in the fourth culture medium comprises 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 is also added with Glutamax, insulin-transferrin-selenium, VC injection and thioglycerol; 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 0-100 μg/mL, and the concentration of thioglycerol is 0-10 mM.
  4. 4. The culture medium for inducing iPSC differentiation to make NK cells according to claim 1, wherein the cell culture medium in the fifth culture medium comprises DMEM medium and/or Ham's F medium; the concentration of IL-7 in the fifth culture medium is 5-100 ng/mL, the concentration of SCF is 5-100 ng/mL, the concentration of FLT3L is 5-100 ng/mL, the concentration of IL-15 is 5-100 ng/mL, and the concentration of IL-3 is 0-100 ng/mL; human AB serum, beta-mercaptoethanol, sodium selenide, ethanolamine and VC injection are also added into the fifth culture medium; The concentration of human AB serum in the fifth culture medium is 5% -20%, the concentration of beta-mercaptoethanol is 0-200 mu M, the concentration of VC injection is 10-100 mu g/mL, the concentration of sodium selenide is 10-100 mu M, and the concentration of ethanolamine is 10-100 ng/mL; The cell culture medium in the sixth culture medium comprises a DMEM culture medium and/or a Ham's F culture medium; The concentration of IL-7 in the sixth culture medium is 10-100 ng/mL, the concentration of SCF is 10-100 ng/mL, the concentration of FLT3L is 10-100 ng/mL, and the concentration of IL-15 is 10-100 ng/mL; human AB serum, beta-mercaptoethanol, sodium selenide, ethanolamine and VC injection are also added into the sixth culture medium; The concentration of human AB serum in the sixth culture medium is 5% -20%, the concentration of beta-mercaptoethanol is 0-200 mu M, the concentration of VC injection is 10-100 mu g/mL, the concentration of sodium selenide is 10-100 mu M, and the concentration of ethanolamine is 10-100 ng/mL.
  5. 5. Use of the medium for inducing iPSC differentiation to prepare NK cells according to any one of claims 1-4 for preparing NK cells.
  6. 6. A method for inducing differentiation of ipscs to prepare NK cells, comprising culturing iPSC cells using the medium for inducing differentiation of ipscs to prepare NK cells according to any one of claims 1 to 4.
  7. 7. The method of inducing differentiation of ipscs to make NK cells according to claim 6, wherein the method comprises: 3D suspension culturing iPSC cells by using the first culture medium, and sequentially replacing the iPSC cells with a second culture medium, a third culture medium and a fourth culture medium to perform 3D suspension culturing to obtain hematopoietic progenitor cells; and 3D suspension culturing the hematopoietic progenitor cells by using the fifth culture medium and the sixth culture medium to obtain NK cells.
  8. 8. The method for inducing differentiation of ipscs to produce NK cells according to claim 7, further comprising a step of amplifying NK cells, comprising mixing NK cells with feeder cells for amplification culture; IL-2 is added into the culture medium of the amplification culture.
  9. 9. The method for inducing differentiation of ipscs to make NK cells according to claim 8, wherein the expansion culture is performed in a wave bioreactor.
  10. 10. The method for inducing differentiation of ipscs to prepare NK cells according to claim 6, wherein the method comprises the steps of: on day 0, 3D suspension culturing iPSC cells using the first medium; on day 1, the second culture medium is replaced to continue 3D suspension culture; 3D suspension culture is continued by replacing the culture medium with the third culture medium every 22-26 h in 2-3 days; 4-6 days, replacing the culture medium with a fourth culture medium every 22-26 hours, and continuing 3D suspension culture to obtain hematopoietic progenitor cells; replacing the culture medium with a fifth culture medium every 46-50 h in 7-13 days to continue 3D suspension culture; The culture medium is replaced by a sixth culture medium every 46-50 h in 14-22 days to continue 3D suspension culture, so as to obtain NK cells; mixing NK cells and trophoblasts on day 23-30, performing amplification culture, and adding IL-2 into a culture medium every 46-50 h; and on the 30 th day, mixing the amplified and cultured NK cells with the trophoblast cells again, performing amplification culture, adding IL-2 into the culture medium every 46-50 h, and harvesting the amplified NK cells.

Description

Culture medium and method for preparing NK cells by inducing iPSC differentiation Technical Field The invention belongs to the technical field of biological medicines, and relates to a culture medium and a method for preparing NK cells by inducing iPSC differentiation. Background Natural Killer (NK) cells are a class of innate immune lymphocytes that are capable of recognizing and killing virus-infected or tumor cells without antigen presentation. Due to its natural tumor recognition capability, no GVHD risk, and no need for personalized customization, NK cells are widely used in tumor immunotherapy, viral infection control, and immune aging intervention. Induced pluripotent stem cells (ipscs) provide a standardized, scalable source of seed for NK cell therapy. By simulating the embryonic hematopoietic development process in a specific cytokine induction system, stepwise differentiation of ipscs into hematopoietic progenitor cells and NK cells can be achieved. The method can thoroughly solve the problems of donor dependence and quantity limitation of primary NK cells by utilizing infinite self-renewal and standardized expansion capacity of iPSC, can precisely regulate cell phenotype and functions (such as enhancing ADCC effect or homing capacity) by gene editing based on monoclonal origin characteristics, ensures high uniformity among cell product batches, and provides large-scale mass-produced cell resources conforming to cGMP specifications for 'spot' immunotherapy by combining the ethical disputed property and efficient differentiation process of iPSC. To prepare iPSC-derived NK cells, researchers have invented mainly both 2D and 3D processes. The existing 2D differentiation process has limited production scale (the single batch yield is usually less than or equal to 10 8 cells) due to the inherent limitation of the culture surface area, frequent manual operation introduces pollution risks and breaks the sealing property, the differentiation efficiency is unstable due to the lack of three-dimensional microenvironment support, and meanwhile, the multi-stage transfer operation further prolongs the production period and increases the production cost, so that the method cannot be used as a standardized industrial grade application. The 3D separation process can well avoid the disadvantages, for example, CN117050940A discloses that a whole-process 3D suspension culture technology is adopted, embryoid (EB) aggregation initiation of iPSC is realized by a commercial STEMSCALE culture medium, mesoderm differentiation is driven by adding CHIR 99021 (combination Rki on day 0 and single on day 1) in precise time sequence, ① EB differentiation to CD34 + hematopoietic stem cells (BMP 4/FGF2/VEGF stepwise induction, CD34 + proportion is more than 50%) is sequentially completed in a three-dimensional dynamic suspension system, ② HSPC is differentiated into CD45 +CD56+ NK cells in a suspension state, and IL-12+IL-15+IL-18 triple stimulation is creatively applied to ③, so that memory-like NK efficient expansion is realized in a 3D environment. The scheme keeps a whole-course three-dimensional suspension state, high purity (> 80%) and high activity NK cells are obtained within 28 days, the scale limit of the traditional 2D process is broken through, and an exogenous feeding layer is not needed. However, the current 3D differentiation process still has the problems that (1) the difference of product quality attributes among batches is increased due to lower differentiation efficiency and complexity of the process, and (2) clinical requirements cannot be well met due to the fact that the single batch yield is too low, so that the development of the 3D culture process of NK cells efficiently prepared by utilizing the iPSC has important significance for the application field of the NK cells. Disclosure of Invention Aiming at the defects and actual demands of the prior art, the invention provides a culture medium and a method for preparing NK cells by inducing iPSC differentiation, and designs a specific culture medium and a specific culture flow so as to realize the efficient preparation of NK cells by using the iPSC. In order to achieve the above purpose, the invention adopts the following technical scheme: In a first aspect, the invention provides a culture medium for inducing iPSC to differentiate and prepare NK cells, wherein the culture medium comprises a first culture medium, a sixth culture medium and a third culture medium; Wherein the first medium comprises a cell culture medium supplemented with a ROCK inhibitor and a GSK-3 inhibitor; Wherein the second medium comprises a cell culture medium supplemented with cytokines BMP4, bFGF, and VEGF; Wherein the third medium comprises a cell culture medium supplemented with cytokines such as BMP4, bFGF, VEGF, and ALK5 inhibitors; wherein the fourth medium comprises a cell culture medium supplemented with cytokines bFGF, VEGF, and SCF; wherein the fifth medium comprises a cell