Culture medium and culture method for selectively amplifying NK cells

Abstract

The invention discloses a culture medium and a culture method for selectively amplifying NK (Natural Killer) cells. The culture medium for selectively amplifying the NK cells comprises a KBM581 basal culture medium and an additive, and the additive comprises the following components in concentration: 500IU / mL of IL-2, 25ng / mL of IL21, 20ng / mL of IL15, 1ug / mL of OK432, 10% of human platelet lysate and 0.05 ug / mL-20ug / mL of dexamethasone sodium phosphate injection. According to the culture method for selectively amplifying the NK cells, the culture medium for selectively amplifying the NK cells is adopted, coating, cell sorting and feeder layer cells are not needed, and operation is easy. After the culture medium and the culture method for selectively amplifying the NK cells are used for culturing for 2 weeks, the amplification multiple of the NK cells can reach 200 times, the proportion of the NK cells reaches 90% or above, the viability of the NK cells reaches 95% or above, and the NK cells are stable in batches and can meet clinical requirements.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to a culture medium and culture method for selectively expanding NK cells. Background Technology

[0002] NK cells (natural killer cells) are an important component of the innate immune system, capable of clearing senescent and diseased cells, and playing an increasingly important role in anti-aging and anti-tumor activity. However, NK cells account for only about 10% of total lymphocytes in peripheral blood, a relatively small proportion and number. In aging and diseased individuals, NK cell activity is further suppressed. Therefore, the body's own NK cells are insufficient to exert effective anti-aging and anti-tumor effects. To enhance the immunotherapeutic function of NK cells, it is necessary to expand NK cells in vitro and reinfuse highly active NK cells into the human body to achieve effective anti-aging and anti-tumor effects.

[0003] However, NK cells face challenges such as low proliferation efficiency and low purity when cultured in vitro. Currently, there are three main methods for obtaining high-purity NK cells in vitro: first, the coating-based pure factor method; second, the cell sorting method; and third, the feeder cell method. The coating-based pure factor method suffers from batch-to-batch quality instability. Coating requires coating the culture dish surface with coating factors (NK-activating antibodies or cytokines) a day in advance, and due to variations in individual operating techniques, the quality of the expanded NK cells is prone to instability. The cell sorting method is costly; both flow cytometry-based and immunomagnetic bead-based methods require expensive equipment and reagents. The feeder cell method poses safety risks. While feeder cells are generally tumor cells that have undergone radiation treatment to inhibit proliferation, some may slip through the filter, potentially leading to serious medical consequences.

[0004] Currently, traditional NK cell culture media typically use animal-derived components such as fetal bovine serum. However, these components pose risks such as significant batch-to-batch variations and the potential to carry pathogens, and may also introduce immunogenicity and other safety concerns for clinical applications. Existing serum-free culture media also suffer from shortcomings such as unsatisfactory culture results and low NK cell expansion efficiency and purity. The NK cell culture medium provided by existing technology CN120349965A has complex cell components, a cumbersome preparation process, and high costs.

[0005] Therefore, there is an urgent need to study a selective culture medium and culture method for expanding NK cells that improves batch-to-batch production stability, has simple components, reduces production costs, facilitates large-scale clinical translation, and has high safety. Summary of the Invention

[0006] To address the above problems, this invention proposes an innovative solution: To achieve the above objectives, the technical solution adopted by the present invention is as follows: One of the technical solutions adopted by the present invention to solve its technical problem is: providing a culture medium for selectively expanding NK cells, including KBM581 basal culture medium and additives, wherein the components and concentrations of the additives are: 500 IU / mL IL-2, 25 ng / mL IL21, 20 ng / mL IL15, 1 ug / mL OK432 and 10% human platelet lysate, and 0.05 ug / mL-20 ug / mL dexamethasone sodium phosphate injection.

[0007] The second technical solution adopted by the present invention to solve its technical problem is: to provide a culture medium for selectively expanding NK cells, including KBM581 basal culture medium and additives, wherein the components and concentrations of the additives are: 500 IU / mL IL-2, 25 ng / mL IL21, 20 ng / mL IL15, 1 ug / mL OK432 and 10% human platelet lysate, 0.05 ug / mL-20 ug / mL dexamethasone sodium phosphate injection, and 10 ng / mL-50 ng / mL IL12.

[0008] The third technical solution adopted by the present invention to solve its technical problem is: to provide a method for culturing NK cells using the above-mentioned selective amplification NK cell culture medium, comprising the following steps: S1. Mononuclear cells were isolated from umbilical cord blood, resuspended in the selective amplification NK cell culture medium described above, and adjusted to (1-3) × 10⁻⁶. 6 After the number of cells / mL is reached, it is incubated statically for 4–7 days; S2. Replace half of the medium with the above selective amplification NK cell culture medium and culture for 2-3 days to continue stimulating NK cell proliferation; S3. Use KBM581 medium containing 500 IU / mL IL2 for half-volume medium replacement. Use this medium for half-volume medium replacement every 2-3 days, and culture for 7-14 days.

[0009] The beneficial effects of this invention are as follows: To address the problems of uncontrollable batch-to-batch quality, high cost, and safety hazards in existing technologies, this invention creatively proposes a selective NK cell culture medium and method. Dexamethasone sodium phosphate injection, as an adrenocortical hormone, has anti-inflammatory and immunosuppressive effects and is clinically used for allergic and autoimmune inflammatory diseases. It may inhibit the proliferation of immune cells such as T cells, B cells, and NK cells, and is rarely used to stimulate NK cell proliferation. During research, the inventors unexpectedly discovered that a suitable concentration of dexamethasone sodium phosphate injection, combined with appropriate concentrations of cytokines such as IL2, IL21, IL15, IL12, OK432, and human platelet lysate, dissolved in KBM581 basal medium, can promote NK cell proliferation and improve NK cell purity and viability. Dexamethasone sodium phosphate injection, IL2, IL21, IL15, IL12, OK432, and human platelet lysate are important components of selective NK cell culture medium. These components work synergistically to produce a selective NK cell culture medium that promotes NK cell proliferation, increases NK cell purity and viability, improves batch-to-batch production stability, has a simple composition, reduces production costs, facilitates large-scale clinical translation, and has high safety. IL2 is an important cytokine stimulating the proliferation of immune cells (such as T cells and NK cells) and is generally used as a basic additive for in vitro expansion of immune cells. IL21 can further enhance the proliferative activity of immune cells (such as T cells and NK cells). IL15 can activate immune cells (such as T cells and NK cells), further enhancing NK cell proliferative activity. IL12 can enhance the antigen sensitivity of NK cells and increase their proliferative activity. OK432 can activate innate immune cells (such as NK cells, neutrophils, and macrophages), enhancing NK cell activity. Human platelet lysate, as a supplementary additive, provides the nutrients required for NK cell proliferation and promotes NK cell maturation. Dexamethasone sodium phosphate injection inhibits the proliferation of immune cells (such as T cells, B cells, and NK cells) in PBMCs, but its inhibitory effect on T cell proliferation is stronger than that on NK cell proliferation. Under the inhibitory effect of dexamethasone sodium phosphate injection, less cytokine is consumed due to T cell proliferation, resulting in more cytokines acting on NK cell proliferation. Through the synergistic effect of the above components, the selective amplification of NK cells is achieved.

[0010] 2. The culture method of the present invention is simple to operate, requiring no coating, no cell sorting, and no feeder cells, thus avoiding the problems caused by the three NK cell culture methods mentioned in the background art.

[0011] 3. Using the culture medium and culture method of this invention, after 2 weeks of culture, the NK cell expansion can reach 200 times and the NK cell ratio can reach more than 90%, and the results are stable between batches, which can meet clinical needs. Detailed Implementation

[0012] To better understand the present invention, the following detailed description is provided in conjunction with embodiments. However, those skilled in the art will understand that the following embodiments are not intended to limit the scope of protection of the present invention, and any changes and modifications made based on the present invention are within the scope of protection of the present invention.

[0013] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0014] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0015] KBM581 basal culture medium was purchased from Corning Incorporated, USA. OK432 was purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd. HPL was purchased from Zhejiang Baidi Biotechnology Co., Ltd.

[0016] Example 1 Preparation of culture medium for selective expansion of NK cells: Formula 1: Add 500 IU / mL IL2, 25 ng / mL IL21, 20 ng / mL IL15, 1 ug / mL OK432 and 10% HPL (human platelet lysate) to KBM581 basal medium.

[0017] Formula 2-1: Based on Formula 1, add 0.05 ug / mL dexamethasone sodium phosphate injection.

[0018] The specific steps for selectively expanding NK cell culture are as follows: S1. Mononuclear cells were isolated from umbilical cord blood, resuspended in Formula 2-1, and adjusted to (1-3) × 10⁻⁶. 6 After inoculating 1 mL of each sample into a 24-well plate, place it in a carbon dioxide incubator (37℃, 5% CO2) and incubate for 4–7 days. S2. Use formula 2-1 to replace half the medium and culture for 2-3 days to continue stimulating NK cell proliferation; S3. Monitor cell count and detect NK cell ratio using flow cytometry. Use KBM581 medium containing 500 IU / mL IL2 for half-volume medium replacement. Use this medium for half-volume medium replacement every 2-3 days. Culture for 7-14 days, collect cells by centrifugation, monitor cell count, and detect NK cell ratio using flow cytometry.

[0019] Example 2 The difference from Example 1 is as follows: Steps S1 and S2 use formula 2-2. Formula 2-2: Based on Formula 1, add 1 ug / mL dexamethasone sodium phosphate injection.

[0020] Example 3 The difference from Example 1 is as follows: Steps S1 and S2 use formula 2-3: Formula 2-3: Based on Formula 1, add 20 ug / mL dexamethasone sodium phosphate injection.

[0021] Example 4 The difference from Example 1 is as follows: Steps S1 and S2 use formula 3-1: Formula 3-1: Based on Formula 2-2, add 10 ng / mL IL12.

[0022] Example 5 The difference from Example 1 is as follows: Steps S1 and S2 use formula 3-2: Formula 3-2: Based on Formula 2-2, add 25 ng / mL IL12.

[0023] Example 6 The difference from Example 1 is as follows: Steps S1 and S2 use formula 3-3: Formula 3-3: Based on Formula 2-2, add 50 ng / mL IL12.

[0024] Example 7 The difference from Example 2 is as follows: NK amplification was performed using another umbilical cord blood sample.

[0025] Example 8 The difference from Example 2 is as follows: NK amplification was performed using another umbilical cord blood sample.

[0026] Example 9 The difference from Example 5 is as follows: NK amplification was performed using another umbilical cord blood sample.

[0027] Example 10 The difference from Example 5 is as follows: NK amplification was performed using another umbilical cord blood sample.

[0028] Comparative Example The difference from Example 1 is as follows: Steps S1 and S2 use formula 1.

[0029] The number and proportion of NK cells cultured after steps S1 and S3 in Examples 1-10 and the comparative examples were detected respectively. The results are shown in Tables 1, 2 and 3.

[0030] Table 1. Results of NK cell expansion fold and NK cell proportion under different formulations.

[0031] Upon testing, 1×10⁻⁶ ppm was found in the sample during step S1. 6 As shown in Table 1, after culture in step S3, the NK cell percentage of Formula 1 was 81.48%, with a 62-fold increase in cell number. The NK cell percentage of Formula 2-1 was 91.52%, with a 215-fold increase in cell number. The NK cell percentage of Formula 2-2 was 91.68%, with a 217-fold increase in cell number. The NK cell percentage of Formula 2-3 was 92.36%, with a 213-fold increase in cell number. When using 1 μg / mL dexamethasone sodium phosphate injection, the NK cell percentage of Formula 3-1 was 93.58%, with a 218-fold increase in cell number; the NK cell percentage of Formula 3-2 was 94.30%, with a 224-fold increase in cell number; and the NK cell percentage of Formula 3-3 was 93.84%, with a 221-fold increase in cell number.

[0032] Compared with formula 1, formulas 2-1, 2-2, 2-3 and formulas 3-1, 3-2, 3-3 can significantly improve the NK cell expansion fold, NK cell purity and viability, and the quality is stable between batches.

[0033] Table 2. Results of NK cell expansion fold and NK cell ratio among different batches under Formula 2-2

[0034] Table 3. Results of NK cell expansion fold and NK cell ratio among different batches under formulation 3-2

[0035] As shown in Tables 2 and 3, under formulas 2-2 and 3-2, the NK amplification fold, purity, and viability of different batches were not significantly different, and the quality was relatively stable.

[0036] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A culture medium for selectively amplifying NK cells, characterized in that, The formulation includes KBM581 basal culture medium and additives, the components and concentrations of which are: 500 IU / mL IL-2, 25 ng / mL IL21, 20 ng / mL IL15, 1 ug / mL OK432 and 10% human platelet lysate, and 0.05 ug / mL-20 ug / mL dexamethasone sodium phosphate injection.

2. The culture medium according to claim 1, characterized in that, It also includes IL12 at concentrations of 10 ng / mL to 50 ng / mL.

3. A method for culturing NK cells using the selective amplification NK cell culture medium as described in claim 1 or claim 2, characterized in that, Includes the following steps: S1. Mononuclear cells are isolated from umbilical cord blood and resuspended in the selective NK cell amplification culture medium as described in claim 1 or claim 2, adjusted to (1-3) × 10⁻⁶. 6 After the number of cells / mL is reached, it is incubated statically for 4–7 days; S2. Use the selective amplification NK cell culture medium as described in claim 1 or claim 2 to perform a half-volume medium replacement and culture for 2-3 days to continue stimulating NK cell proliferation; S3. Use KBM581 medium containing 500 IU / mL IL2 for half-volume medium replacement. Use this medium for half-volume medium replacement every 2-3 days, and culture for 7-14 days.

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