NK cell culture system and in-vitro amplification method

Abstract

The invention discloses an NK cell culture system and an in-vitro amplification method. The invention firstly discloses a culture system of the NK cells, wherein the culture system comprises a coating solution, an activation culture medium and an amplification culture medium. The invention further discloses a method for in-vitro amplification of NK cells by using the culture system. According to the method for amplifying the NK cells in vitro, the use of protein cell factors is simplified, only two factors of IL-2 and IL-15 are matched with CD16 and progesterone to directly culture mononuclear cells, and the required cost can be reduced to a great extent; sorting is not needed, all mononuclear cells can be used for culture, the operation procedure is simplified, and the culture cost is reduced; the process is simplified, and pollution is avoided to the greatest extent; meanwhile, the cultured NK cells are stable, safe, high in activity, enhanced in expansion, high in purity, short in period, strong in killing ability, high in cost performance and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of cell and tissue culture, and more specifically, to a culture system and in vitro expansion method of NK cells. Background Art

[0002] Natural killer (NK) cells are lymphocytes (CD3 - CD56 + ), plays a key role in eliminating tumors and virus-infected cells. The recognition and killing of virus-infected and transformed cells by NK cells is regulated by the signal balance of inhibitory and activating receptors. Since NK cells have a strong ability to eliminate tumors in a targeted manner, NK cells are regarded as promising cells for adoptive immunotherapy of cancer. It has been well demonstrated that NK cell alloreactivity can control the relapse of acute myeloid leukemia (AML) in the case of haploidentical stem cell transplantation (SCT) without causing graft-versus-host disease (GVHD). In addition, clinical trials of haploidentical NK cell infusions in adult and pediatric AML after lymphocyte depletion chemotherapy have also shown good killing effects. However, NK cells account for a small proportion of peripheral blood mononuclear cells (PBMCs), containing only 5-15%. Therefore, the ex vivo activation and expansion scheme of NK cells is a top priority so that clinical trials and adoptive immunotherapy can be performed with more NK cell doses.

[0003] Progesterone (P4 / PRO), also known as progesterone, is a steroid hormone that is widely present in animals and has a variety of biological activities and functions in the process of cell culture in vitro, including cell adhesion, regulation of cell growth and proliferation, and developmental processes. Progesterone is involved in maintaining tight junctions between epithelial cells, regulating cell sensitivity, and regulating the secretion of inflammatory cytokines, especially in the culture of primary cells and immortalized cell lines. Progesterone usually begins to work at doses above 1μM, inhibits cell proliferation at doses above 10μM, and at higher concentrations (≥25μM), it also inhibits cell proliferation and or reverses the EMT (epithelial-mesenchymal transition) process of stem cells. It is worth noting that high doses of progesterone can have long-term effects on the phenotype and function of enlarged amniotic cells. In addition, progesterone can inhibit the TGF-β pathway by inducing the expression and release of key anti-inflammatory cytokines IL-10, IL-4 and TGF-β, and downregulating the expression of key pro-inflammatory cytokines (IL-1β, IL-6 and IL-12), thereby achieving effective anti-cancer effects in endometrial cells.

[0004] There are many existing methods for in vitro expansion of NK cells, which are complex and have the following disadvantages: 1. The number of expanded cells is small and does not meet the infusion standard; 2. The purity of the expanded cells is low and the killing ability is weak; 3. More protein active factors are required, which are relatively expensive and difficult to store and package; 4. Magnetic bead sorting and purification steps are required, which increases the difficulty and cost of the operation; 5. Existing technologies mostly use inactivated feeder layer cells for co-culture, but the feeder layer cells themselves are modified leukemia cells, which have certain safety risks in use and may lead to regulatory issues for NK cell products.

[0005] In view of the above limitations of NK cell expansion and culture, it is urgent to develop a new method for obtaining NK cells by in vitro expansion that is simple, rapid, efficient and safe. Summary of the invention

[0006] The purpose of the present invention is to provide a NK cell culture system and in vitro expansion method, by directly culturing mononuclear cells with a combination of CD16, progesterone and IL-2, IL-15, activating and expanding NK cells in vitro, promoting NK cell proliferation, and enhancing the killing activity of tumor cells under appropriate concentration conditions.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention first provides a NK cell culture system, which comprises a coating solution, an activation culture medium and an expansion culture medium; wherein:

[0009] The coating solution comprises CD16 and PBS buffer; the final concentration of CD16 is 1-10 μM;

[0010] The activation medium comprises serum-free medium, IL-2, IL-15 and progesterone; the final concentrations of the IL-2, IL-15 and progesterone are 500-3000 IU / mL, 10-100 ng / mL and 1-10 μM, respectively.

[0011] The expansion medium comprises serum-free medium, IL-2 and IL-15; the final concentrations of the IL-2 and IL-15 are 500-3000 IU / mL and 10-100 ng / mL, respectively.

[0012] Furthermore, the culture system also includes autologous plasma, preferably heat-inactivated autologous plasma.

[0013] In a specific embodiment of the present invention, the coating solution is prepared as follows: CD16 is added to PBS buffer, and mixed by inversion to make the final concentration of CD16 1-10 μM, and then used for later use;

[0014] Preparation of activation medium: add IL-2, IL-15 and progesterone to serum-free medium, mix by inversion, so that the final concentrations of IL-2, IL-15 and progesterone are 500-3000 IU / mL, 10-100 ng / mL and 1-10 μM, respectively, and set aside;

[0015] Preparation of expansion medium: Add IL-2 and IL-15 to serum-free medium, mix by inversion to make the final concentrations of IL-2 and IL-15 500-3000 IU / mL and 10-100 ng / mL, respectively, and set aside.

[0016] Further, the serum-free medium is one or more of KBM581 immune cell medium, SCGM serum-free medium, AIM-V serum-free medium, X-VIVO 15 serum-free medium and NK MACS serum-free medium. Specifically, the serum-free medium is one or more of Corning KBM581 immune cell medium, CellGenix SCGM serum-free medium, Thermo Fisher Scientific AIM-V serum-free medium, Lonza X-VIVO 15 serum-free medium and Miltenyi NK MACS serum-free medium.

[0017] Furthermore, the final concentration of CD16 in the coating solution is 5 μM.

[0018] Furthermore, the final concentrations of IL-2, IL-15 and progesterone in the activation medium are 2000 IU / mL, 80 ng / mL and 5 μM, respectively.

[0019] Furthermore, the final concentrations of IL-2 and IL-15 in the expansion medium are 1000-1500 IU / mL and 10-100 ng / mL, respectively; preferably 1000 IU / mL and 50 ng / mL.

[0020] The present invention further provides a method for in vitro expansion of NK cells using the above culture system, comprising the following steps:

[0021] Antibody-coated cell culture flask: adding the coating solution to the cell culture flask, making the coating solution evenly dispersed on the bottom of the flask, coating, and obtaining a coated cell culture flask;

[0022] Mononuclear cells and heat-inactivated autologous plasma were prepared from human blood;

[0023] Cell inoculation: discard the coating solution in the cell culture flask after coating, wash with PBS, resuspend the mononuclear cells in the activation medium and inoculate them in the cell culture flask that has been coated and washed with PBS, add heat-inactivated autologous plasma, and culture. The day of inoculation is recorded as day 0;

[0024] Fluid replenishment culture: On the third day of culture, activation medium and heat-inactivated autologous plasma are added and cultured;

[0025] Bottle expansion culture: on the 5th day of culture, observe the cell status. When it is observed that there are many and large cell clones, add expansion medium and heat-inactivated autologous plasma, take samples and count, adjust the cell density, and transfer the cells to a new cell culture bottle or cell culture bag according to the volume of the cell suspension for culture;

[0026] Expanded culture: Starting from the 7th day of culture, add expansion medium and heat-inactivated autologous plasma, take samples and count, adjust the cell density, and transfer the cells to new cell culture bottles or cell culture bags according to the volume of the cell suspension for culture;

[0027] Continue culturing: add expansion medium and heat-inactivated autologous plasma every one or two days, take samples and count, adjust cell density, transfer to new cell culture bottles or cell culture bags according to the volume of cell suspension, and culture;

[0028] Harvesting cells: Harvest NK cells on day 12-14 of culture.

[0029] Furthermore, in the step of coating the culture flask with antibodies, the coating is incubated at 4° C. overnight or incubated in a 37° C. incubator for 2 hours.

[0030] Furthermore, the cell inoculation step has an inoculation density of 2.0-3.0×10 6 cells / mL; preferably, 2.0×10 6 cells / mL.

[0031] Furthermore, in the cell inoculation step, the heat-inactivated autologous plasma accounts for 7-10% of the total volume of the activation medium and the heat-inactivated autologous plasma; preferably, 10%.

[0032] Furthermore, in the fluid replacement culture step, the heat-inactivated autologous plasma accounts for 7-10% of the total volume of the activation culture medium and the heat-inactivated autologous plasma; preferably, it is 10%.

[0033] Furthermore, in the step of expanding the flask and culturing, when more and larger clones of cells are observed, the diameter of the cell clusters observed under a 10X microscope is about 1 cm.

[0034] Furthermore, in the bottle expansion culture step, the heat-inactivated autologous plasma accounts for 4-7% of the total volume of the expansion medium and the heat-inactivated autologous plasma; preferably, it is 5%.

[0035] Furthermore, in the expansion culture step, the heat-inactivated autologous plasma accounts for 1-3% of the total volume of the expansion medium and the heat-inactivated autologous plasma; preferably, it is 1%.

[0036] Furthermore, the culture is carried out in a 37°C, 5% CO2 incubator.

[0037] Furthermore, the cell density is adjusted to maintain the cell concentration at 0.5-1.0×10 6 cells / mL; preferably, 1.0×10 6 cells / mL.

[0038] Furthermore, the new cell culture flask or cell culture bag refers to a cell culture flask or cell culture bag that has never been coated.

[0039] Furthermore, the heat-inactivated autologous plasma is obtained by inactivating the plasma separated from human blood at 56° C. for 30 minutes and then centrifuging it.

[0040] Furthermore, the mononuclear cells are obtained by separating human blood using Ficoll.

[0041] In a specific embodiment of the present invention, the method for preparing mononuclear cells and heat-inactivated autologous plasma is:

[0042] Preparation of heat-inactivated autologous plasma: anticoagulated blood (from human blood) was centrifuged at room temperature at 650×g for 15 minutes, and the upper yellow plasma portion was inactivated in a 56°C water bath for 30 minutes; after inactivation, it was centrifuged at 900×g for 10 minutes; the supernatant was taken and frozen at -20°C for 15 minutes; it was centrifuged again at 900×g for 10 minutes, and the supernatant was taken to obtain heat-inactivated autologous plasma;

[0043] Preparation of mononuclear cells: centrifuge anticoagulated blood (from human blood) at room temperature at 650×g for 15 minutes, remove the red precipitate, add PBS of the same volume as the original blood and mix thoroughly to obtain diluted blood; slowly add diluted blood to the lymphocyte separation solution, and pay attention to moderate acceleration. Centrifuge at room temperature at 800×g for 20 minutes (speed increases and decreases slowly); after centrifugation, carefully aspirate the white film layer into a new centrifuge tube, and add PBS to mix. At this time, the amount of PBS used is 5-10 times the volume of the white film layer solution, and then centrifuge at 300×g for 10 minutes; discard the supernatant, resuspend the precipitate with 5-10 times the volume of PBS again, count the cells, and centrifuge at 300×g for 10 minutes; discard the supernatant to obtain mononuclear cells.

[0044] In the present invention, if there is no heat-inactivated autologous plasma, human AB serum can be used instead.

[0045] The present invention relies on a simple, rapid, efficient and safe method for preparing NK cells in vitro by the pure factor method. By using a combination of CD16, progesterone, IL-2 and IL-15, mononuclear cells are directly cultured, NK cells are activated and amplified in vitro, NK cell proliferation can be promoted, and tumor cell killing activity can be enhanced under appropriate concentration conditions. The NK cells prepared by this method are stable and safe (no xenogeneic components, clear chemical composition; no serum, only a small amount of heat-inactivated autologous plasma is required; pure factor reagents, no feeder cells are required; phenol red is contained, no antibiotics are contained, and they are used to generate fully active NK cells in vitro), with high cell viability (cell viability throughout the process is above 90%), strong amplification ability (400-550 times amplification in 14 days, 850-950 times amplification in 21 days), and high cell purity (CD3 - CD56 + Purity ≥ 90%, CD3 - CD16 + Purity ≥ 90%, CD56 + CD16 + Purity ≥ 90%; 21 days CD3 - CD56 + Purity ≥94%, CD3 - CD16 + Purity ≥94%, CD56 + CD16 + Purity ≥ 94%), strong killing ability (high killing ability for tumor cells), convenient operation, high cost performance, suitable for industrial production (produced and managed in accordance with GMP standards).

[0046] The human blood source of the present invention includes human peripheral blood or umbilical cord blood, and the mononuclear cells are human umbilical cord blood mononuclear cells or peripheral blood mononuclear cells (PBMC).

[0047] The beneficial effects of the present invention are as follows:

[0048] The method for in vitro expansion of NK cells of the present invention simplifies the use of protein cytokines. Only IL-2 and IL-15 factors are needed to be combined with CD16 and progesterone to directly culture mononuclear cells, which can reduce the required cost to a large extent. No sorting is required, and all mononuclear cells can be used for culture, which simplifies the operation procedure and reduces the culture cost. The process is simplified and contamination is avoided to the greatest extent. At the same time, the NK cells obtained by culture are stable and safe, have high activity, strong expansion, high purity, short cycle, strong killing ability, high cost performance, and are suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0050] Figure 1A culture process for efficiently activating and expanding NK cells in vitro.

[0051] Figure 2 The cell status during the culture process of Example 1 (10×, scale bar: 200 μm).

[0052] Figure 3 The cell viability results during the culture process of Examples 1-3 and the comparative example are shown.

[0053] Figure 4 The results of cell proliferation before and after culture of Examples 1-3 and the comparative example are shown.

[0054] Figure 5 CD3 cells cultured for 14 days in Example 1 - CD56 + / CD3 - CD16 + / CD16 + CD56 + The cell population percentage results.

[0055] Figure 6 CD3 cells cultured for 21 days in Example 1 - CD56 + / CD3 - CD16 + / CD16 + CD56 + The cell population percentage results.

[0056] Figure 7 The killing rate results of NK cells at different effector-target ratios in Examples 1-3 and the comparative example.

[0057] Figure 8 CD3 cells cultured for 14 days in Example 2 - CD56 + / CD3 - CD16 + / CD16 + CD56 + The cell population percentage results.

[0058] Fig. 9 CD3 cells cultured for 21 days in Example 2 - CD56 + / CD3 - CD16 + / CD16 + CD56 + The cell population percentage results.

[0059] Fig.10 CD3 cells cultured for 14 days in Example 3 - CD56+ / CD3 - CD16 + / CD16 + CD56 + The cell population percentage results.

[0060] Fig.11 CD3 cells cultured for 21 days in Example 3 - CD56 + / CD3 - CD16 + / CD16 + CD56 + The cell population percentage results.

[0061] Fig.12 The cell status during the culture process of the comparative example (10×, scale bar: 200 μm).

[0062] Fig.13 The results of cell proliferation before and after comparative culture are shown in FIG.

[0063] Fig.14 For the control group, CD3 - CD56 + / CD3 - CD16 + / CD16 + CD56 + The cell population percentage results. DETAILED DESCRIPTION

[0064] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0065] The culture system for preparing NK cells in the following embodiments of the present invention is:

[0066] The culture system includes coating solution, activation culture medium, expansion culture medium and heat-inactivated autologous plasma, and the specific preparation method is as follows:

[0067] Preparation of coating solution: melt CD16 at room temperature, add CD16 to 5 mL PBS buffer, mix by inversion to make the final concentration of CD16 1-10 μM, and set aside;

[0068] Preparation of activation medium: Add IL-2, IL-15 and progesterone to KBM581 immune cell culture medium, mix by inversion, so that the final concentrations of IL-2, IL-15 and progesterone are 500-3000 IU / mL, 10-100 ng / mL and 1-10 μM, respectively, and set aside;

[0069] Preparation of expansion medium: Add 500-3000 IU / mL IL-2 and 10-1000 ng / mL IL-15 to KBM581 immune cell culture medium, invert and mix, and set aside;

[0070] Preparation of heat-inactivated autologous plasma: anticoagulated blood (from human peripheral blood or umbilical cord blood) is centrifuged at 650×g for 15 minutes at room temperature, and the upper yellow plasma portion is inactivated in a 56°C water bath for 30 minutes; after inactivation, centrifuge at 900×g for 10 minutes; take the supernatant and freeze it at -20°C for 15 minutes; centrifuge again at 900×g for 10 minutes, take the supernatant, and obtain heat-inactivated autologous plasma and store it at 4°C for later use.

[0071] Example 1 In vitro expansion of NK cells

[0072] In this embodiment, a medium concentration of progesterone components is added to the culture medium, specifically as follows:

[0073] Preparation of coating solution: melt CD16 at room temperature, add CD16 to 5 mL PBS buffer, mix by inversion to make the final concentration of CD16 5 μM, and set aside;

[0074] Preparation of activation medium: Add IL-2, IL-15 and progesterone to KBM581 immune cell culture medium, mix by inversion, so that the final concentrations of IL-2, IL-15 and progesterone are 2000 IU / mL, 80 ng / mL and 5 μM, respectively, and set aside;

[0075] Preparation of expansion medium: Add 1000 IU / mL IL-2 and 50 ng / mL IL-15 to the KBM581 immune cell culture medium, invert and mix to make the final concentrations of IL-2 and IL-15 1000 IU / mL and 50 ng / mL, respectively, and set aside.

[0076] The steps for in vitro expansion of NK cells are as follows:

[0077] 1. Antibody coated culture flask (-1 day)

[0078] Add the coating solution to the T25 cell culture flask, shake it back and forth to make the liquid evenly dispersed on the bottom of the flask, and coat it overnight at 4°C. Note: If the coated culture flask is not used immediately, it can be stored in a 4°C refrigerator. It is recommended to use it within 3 days.

[0079] 2. Preparation of peripheral blood mononuclear cells (PBMC) and heat-inactivated autologous plasma (Day 0)

[0080] Preparation of heat-inactivated autologous plasma: anticoagulated blood (source: human peripheral blood) was centrifuged at 650×g for 15 minutes at room temperature, and the upper yellow plasma portion (the lower layer is the blood cell component, which is used for the subsequent separation of PBMC) was taken and inactivated in a 56°C water bath for 30 minutes; after inactivation, centrifuged at 900×g for 10 minutes; the supernatant was taken and frozen at -20°C for 15 minutes; centrifuged again at 900×g for 10 minutes, the supernatant was taken, and the heat-inactivated autologous plasma was obtained and stored at 4°C for later use.

[0081] Preparation of PBMC: PBMCs were separated from human blood using Ficoll. The specific method was as follows:

[0082] Centrifuge the anticoagulated blood at room temperature at 650×g for 15 minutes, remove the red precipitate, add PBS of the same volume as the original blood and mix thoroughly to obtain diluted blood; slowly add the diluted blood to the lymphocyte separation solution, and pay attention to the moderate acceleration. Centrifuge at room temperature at 800×g for 20 minutes (speed increases and decreases slowly); after centrifugation, carefully aspirate the buffy coat layer into a new centrifuge tube, and add PBS to mix. At this time, the amount of PBS used is 5-10 times the volume of the buffy coat solution, and then centrifuge at 300×g for 10 minutes; discard the supernatant, resuspend the precipitate with 5-10 times the volume of PBS again, count the cells, and centrifuge at 300×g for 10 minutes; discard the supernatant to obtain PBMC for use.

[0083] III. Cell Inoculation (Day 0)

[0084] Take out the coated T25 cell culture flask 20-30 minutes in advance and equilibrate it to room temperature, then discard the coating solution, wash with PBS, resuspend PBMC in 9 mL activation medium and inoculate it in a T25 cell culture flask (seeding density 2×10 6 cells / mL), and add 1 mL of heat-inactivated autologous plasma to a total volume of 10 mL in the T25 culture bottle. Shake the cells back and forth and left and right and place them in a 37°C, 5% CO2 incubator for culture. The day of inoculation is recorded as day 0.

[0085] IV. Fluid culture

[0086] On the third day of culture, 18 mL of activation medium and 2 mL of heat-inactivated autologous plasma were slowly added along the side wall of the T25 cell culture flask (i.e., the heat-inactivated autologous plasma accounted for 10% of the total volume of the activation medium and heat-inactivated autologous plasma, i.e., the content of heat-inactivated autologous plasma was 10%), and the total volume of the liquid in the T25 cell culture flask was 30 mL. The cells were continued to be cultured in a 37°C, 5% CO2 incubator. Be careful to avoid touching the bottom of the culture flask, do not blow the cells, and minimize operations such as counting and observation to avoid affecting the initial growth of the cells.

[0087] 5. Bottle expansion culture

[0088] On the fifth day of culture, observe the cell status. When there are many and large cell clones (the diameter of the cell clusters is about 1 cm under a 10X microscope), add expansion medium and heat-inactivated autologous plasma (heat-inactivated autologous plasma accounts for 5% of the total volume of expansion medium and heat-inactivated autologous plasma, that is, the content of heat-inactivated autologous plasma is 5%), take samples and count, and adjust the cell density to 1.0×10 6 cells / mL, expand the bottle according to the volume of the cell suspension (i.e. transfer to a new T175 cell culture flask or transfer to a cell culture bag for culture; neither the T175 cell culture flask nor the cell culture bag for transfer bottle culture needs to be coated with coating solution), and continue to culture in a 37°C, 5% CO2 incubator.

[0089] Note: Try to minimize cell counting, observation, pipetting and other operations during the first 5 days to avoid affecting the initial growth of cells.

[0090] VI. Expand training

[0091] On the 7th day of culture, expansion medium and heat-inactivated autologous plasma (the content of heat-inactivated autologous plasma was reduced to 1%) were added, samples were taken and counted, and the cell density was adjusted to 1.0×10 6 cells / mL, expand the bottle according to the volume of cell suspension, and continue to culture in a 37℃, 5% CO2 incubator.

[0092] 7. Continue to cultivate

[0093] Add expansion medium and heat-inactivated autologous plasma every other day or two, take samples and count, adjust the cell density to 1.0×106 cells / mL, expand the bottle according to the volume of the cell suspension, and continue culturing in a 37°C, 5% CO2 incubator.

[0094] 8. Harvesting cells

[0095] NK cells can be harvested on the 12th to 14th day of culture, or the harvesting time can be extended to 21 days, depending on the experimental situation.

[0096] NK cell culture process Figure 1 As shown, the cell status during the entire culture process was observed. Figure 2 As shown, during the culture process, the cell amount increased; the cells were uniformly transparent, with obvious semi-adherent aggregate growth, and some cell aggregates grew in suspension; in the later stage, the cell aggregates increased, the cells had an irregular shape, and some cells had tentacles extending.

[0097] Detect cell viability during culture (cell counter detects cell viability and number of living cells) Figure 3 As shown, the cell viability is high overall, and the cell viability of the whole process is maintained above 90%.

[0098] Calculate the cell proliferation before and after culture. Figure 4 As shown, the initial cell number was 2×10 7 cells, which expanded 539.40-fold in 14 days and 950.82-fold in 21 days.

[0099] The purity of cells during the culture process was tested, i.e. the proportion of cell-specific markers detected by flow cytometry, and CD3 - CD56 + / CD3 - CD16 + / CD16 + CD56 + The percentage of cell populations is shown in Figure 5 and 6 As shown, after 14 days of culture, the NK cells obtained were CD3 - CD56 + 94.79%, CD3 - CD16 + 94.31%, CD16 + CD56 + After 21 days of culture, the NK cells obtained had a CD3 - CD56 + 96.91%, CD3 - CD16 + 95.45%, CD16 + CD56 + It is 94.07%.

[0100] The killing rate of NK cells was detected after 14 days of culture (CCK8 detection killing principle), and the results were as follows Figure 7 When the effector-target ratio was 1:1, the killing rate of NK cells was 94.62%; when the effector-target ratio was 5:1, the killing rate of NK cells was 95.37%; when the effector-target ratio was 10:1, the killing rate of NK cells was 96.36%; when the effector-target ratio was 20:1, the killing rate of NK cells was 98.18%.

[0101] Example 2 In vitro expansion of NK cells

[0102] In this embodiment, a high concentration of progesterone component is added to the culture medium, specifically as follows:

[0103] Preparation of coating solution: melt CD16 at room temperature, add CD16 to 5 mL PBS buffer, mix by inversion to make the final concentration of CD16 5 μM, and set aside;

[0104] Preparation of activation medium: Add IL-2, IL-15 and progesterone to the KBM581 immune cell culture medium, mix by inversion, so that the final concentrations of IL-2, IL-15 and progesterone are 2000 IU / mL, 80 ng / mL and 10 μM, respectively, and set aside;

[0105] Preparation of expansion medium: Add 1000 IU / mL IL-2 and 50 ng / mL IL-15 to the KBM581 immune cell culture medium, invert and mix to make the final concentrations of IL-2 and IL-15 1000 IU / mL and 50 ng / mL, respectively, and set aside.

[0106] The steps for in vitro expansion of NK cells are as follows:

[0107] 1. Antibody coated culture flask (-1 day)

[0108] Add the coating solution to the T25 cell culture flask, shake it back and forth to make the liquid evenly dispersed on the bottom of the flask, and coat it overnight at 4°C. Note: If the coated culture flask is not used immediately, it can be stored in a 4°C refrigerator. It is recommended to use it within 3 days.

[0109] 2. Preparation of peripheral blood mononuclear cells (PBMC) and heat-inactivated autologous plasma (Day 0)

[0110] Preparation of heat-inactivated autologous plasma: anticoagulated blood (source: human peripheral blood) was centrifuged at 650×g for 15 minutes at room temperature, and the upper yellow plasma portion (the lower layer is the blood cell component, which is used for the subsequent separation of PBMC) was taken and inactivated in a 56°C water bath for 30 minutes; after inactivation, centrifuged at 900×g for 10 minutes; the supernatant was taken and frozen at -20°C for 15 minutes; centrifuged again at 900×g for 10 minutes, the supernatant was taken, and the heat-inactivated autologous plasma was obtained and stored at 4°C for later use.

[0111] Preparation of PBMC: PBMCs were separated from human blood using Ficoll. The specific method was as follows:

[0112] Centrifuge the anticoagulated blood at room temperature at 650×g for 15 minutes, remove the red precipitate, add PBS of the same volume as the original blood and mix thoroughly to obtain diluted blood; slowly add the diluted blood to the lymphocyte separation solution, and pay attention to the moderate acceleration. Centrifuge at room temperature at 800×g for 20 minutes (speed increases and decreases slowly); after centrifugation, carefully aspirate the buffy coat layer into a new centrifuge tube, and add PBS to mix. At this time, the amount of PBS used is 5-10 times the volume of the buffy coat solution, and then centrifuge at 300×g for 10 minutes; discard the supernatant, resuspend the precipitate with 5-10 times the volume of PBS again, count the cells, and centrifuge at 300×g for 10 minutes; discard the supernatant to obtain PBMC for use.

[0113] III. Cell Inoculation (Day 0)

[0114] Take out the coated T25 cell culture flask 20-30 minutes in advance and equilibrate it to room temperature, then discard the coating solution, wash with PBS, resuspend PBMC in 9 mL activation medium and inoculate it in a T25 cell culture flask (seeding density 2×10 6 cells / mL), and add 1 mL of heat-inactivated autologous plasma to a total volume of 10 mL in the T25 culture bottle. Shake the cells back and forth and left and right and place them in a 37°C, 5% CO2 incubator for culture. The day of inoculation is recorded as day 0.

[0115] IV. Fluid culture

[0116] On the third day of culture, 18 mL of activation medium and 2 mL of heat-inactivated autologous plasma were slowly added along the side wall of the T25 cell culture flask (i.e., the heat-inactivated autologous plasma accounted for 10% of the total volume of the activation medium and heat-inactivated autologous plasma, i.e., the content of heat-inactivated autologous plasma was 10%), and the total volume of the liquid in the T25 cell culture flask was 30 mL. The cells were continued to be cultured in a 37°C, 5% CO2 incubator. Be careful to avoid touching the bottom of the culture flask, do not blow the cells, and minimize operations such as counting and observation to avoid affecting the initial growth of the cells.

[0117] 5. Bottle expansion culture

[0118] On the fifth day of culture, observe the cell status. When there are many and large cell clones (the diameter of the cell clusters is about 1 cm under a 10X microscope), add expansion medium and heat-inactivated autologous plasma (heat-inactivated autologous plasma accounts for 5% of the total volume of expansion medium and heat-inactivated autologous plasma, that is, the content of heat-inactivated autologous plasma is 5%), take samples and count, and adjust the cell density to 1.0×10 6 cells / mL, expand the bottle according to the volume of the cell suspension (i.e. transfer to a new T175 cell culture flask or transfer to a cell culture bag for culture; neither the T175 cell culture flask nor the cell culture bag for transfer bottle culture needs to be coated with coating solution), and continue to culture in a 37°C, 5% CO2 incubator.

[0119] Note: Try to minimize cell counting, observation, pipetting and other operations during the first 5 days to avoid affecting the initial growth of cells.

[0120] VI. Expand training

[0121] On the 7th day of culture, expansion medium and heat-inactivated autologous plasma (the content of heat-inactivated autologous plasma was reduced to 1%) were added, samples were taken and counted, and the cell density was adjusted to 1.0×10 6 cells / mL, expand the bottle according to the volume of cell suspension, and continue to culture in a 37℃, 5% CO2 incubator.

[0122] 7. Continue to cultivate

[0123] Add expansion medium and heat-inactivated autologous plasma every other day or two, take samples and count, and adjust the cell density to 1.0×10 6 cells / mL, expand the bottle according to the volume of cell suspension, and continue to culture in a 37℃, 5% CO2 incubator.

[0124] 8. Harvesting cells

[0125] NK cells can be harvested on the 12th to 14th day of culture, or the harvesting time can be extended to 21 days, depending on the experimental situation.

[0126] Detect cell viability during culture (cell counter detects cell viability and number of living cells) Figure 3 As shown, the cell viability is high overall, and the cell viability of the whole process is maintained above 90%.

[0127] Calculate the cell proliferation before and after culture. Figure 4 As shown, the initial cell number was 2×10 7 cells, which expanded 491.58-fold in 14 days and 934.78-fold in 21 days.

[0128] Detect cell purity during culture, i.e. (the proportion of cell-specific markers detected by flow cytometry) and detect CD3 - CD56 + / CD3 - CD16 + / CD16 + CD56 + The percentage of cell populations is shown in Figure 8 and 9 As shown, after 14 days of culture, the NK cells obtained were CD3 - CD56 + 92.64%, CD3 - CD16 + 93.20%, CD16 + CD56 + After 21 days of culture, the NK cells obtained had a CD3 - CD56 + 95.38%, CD3 - CD16 + 96.08%, CD16 + CD56 + It is 94.56%.

[0129] The killing rate of NK cells was detected after 14 days of culture (CCK8 detection killing principle), and the results were as follows Figure 7When the effector-target ratio was 1:1, the killing rate of NK cells was 90.25%; when the effector-target ratio was 5:1, the killing rate of NK cells was 91.78%; when the effector-target ratio was 10:1, the killing rate of NK cells was 93.11%; when the effector-target ratio was 20:1, the killing rate of NK cells was 95.34%.

[0130] Example 3 In vitro expansion of NK cells

[0131] In this embodiment, a low concentration of progesterone is added to the culture medium

[0132] Preparation of coating solution: melt CD16 at room temperature, add CD16 to 5 mL PBS buffer, mix by inversion to make the final concentration of CD16 5 μM, and set aside;

[0133] Preparation of activation medium: Add IL-2, IL-15 and progesterone to the KBM581 immune cell culture medium, mix by inversion, so that the final concentrations of IL-2, IL-15 and progesterone are 2000 IU / mL, 80 ng / mL and 2 μM, respectively, and set aside;

[0134] Preparation of expansion medium: Add 1000 IU / mL IL-2 and 50 ng / mL IL-15 to the KBM581 immune cell culture medium, invert and mix to make the final concentrations of IL-2 and IL-15 1000 IU / mL and 50 ng / mL, respectively, and set aside.

[0135] The steps for in vitro expansion of NK cells are as follows:

[0136] 1. Antibody coated culture flask (-1 day)

[0137] Add the coating solution to the T25 cell culture flask, shake it back and forth to make the liquid evenly dispersed on the bottom of the flask, and coat it overnight at 4°C. Note: If the coated culture flask is not used immediately, it can be stored in a 4°C refrigerator. It is recommended to use it within 3 days.

[0138] 2. Preparation of peripheral blood mononuclear cells (PBMC) and heat-inactivated autologous plasma (Day 0)

[0139] Preparation of heat-inactivated autologous plasma: anticoagulated blood (human peripheral blood is selected as the blood source) is centrifuged at 650×g for 15 minutes at room temperature, and the upper yellow plasma portion (the lower layer is the blood cell component, which is used for the subsequent separation of PBMC) is placed in a 56°C water bath for inactivation for 30 minutes; after inactivation, centrifuge at 900×g for 10 minutes; take the supernatant and freeze it at -20°C for 15 minutes; centrifuge again at 900×g for 10 minutes, take the supernatant, and obtain the heat-inactivated autologous plasma and store it at 4°C for later use.

[0140] Preparation of PBMC: PBMCs were separated from human blood using Ficoll. The specific method was as follows:

[0141] Centrifuge the anticoagulated blood at room temperature at 650×g for 15 minutes, remove the red precipitate, add PBS of the same volume as the original blood and mix thoroughly to obtain diluted blood; slowly add the diluted blood to the lymphocyte separation solution, and pay attention to the moderate acceleration. Centrifuge at room temperature at 800×g for 20 minutes (speed increases and decreases slowly); after centrifugation, carefully aspirate the buffy coat layer into a new centrifuge tube, and add PBS to mix. At this time, the amount of PBS used is 5-10 times the volume of the buffy coat solution, and then centrifuge at 300×g for 10 minutes; discard the supernatant, resuspend the precipitate with 5-10 times the volume of PBS again, count the cells, and centrifuge at 300×g for 10 minutes; discard the supernatant to obtain PBMC for use.

[0142] III. Cell Inoculation (Day 0)

[0143] Take out the coated T25 cell culture flask 20-30 minutes in advance and equilibrate it to room temperature, then discard the coating solution, wash with PBS, resuspend PBMC in 9 mL activation medium and inoculate it in a T25 cell culture flask (seeding density 2×10 6 cells / mL), and add 1 mL of heat-inactivated autologous plasma to a total volume of 10 mL in the T25 culture bottle. Shake the cells back and forth and left and right and place them in a 37°C, 5% CO2 incubator for culture. The day of inoculation is recorded as day 0.

[0144] IV. Fluid culture

[0145] On the third day of culture, 18 mL of activation medium and 2 mL of heat-inactivated autologous plasma were slowly added along the side wall of the T25 cell culture flask (i.e., the heat-inactivated autologous plasma accounted for 10% of the total volume of the activation medium and heat-inactivated autologous plasma, i.e., the content of heat-inactivated autologous plasma was 10%), and the total volume of the liquid in the T25 cell culture flask was 30 mL. The cells were continued to be cultured in a 37°C, 5% CO2 incubator. Be careful to avoid touching the bottom of the culture flask, do not blow the cells, and minimize operations such as counting and observation to avoid affecting the initial growth of the cells.

[0146] 5. Bottle expansion culture

[0147] On the fifth day of culture, observe the cell status. When there are many and large cell clones (the diameter of the cell clusters is about 1 cm under a 10X microscope), add expansion medium and heat-inactivated autologous plasma (heat-inactivated autologous plasma accounts for 5% of the total volume of expansion medium and heat-inactivated autologous plasma, that is, the content of heat-inactivated autologous plasma is 5%), take samples and count, and adjust the cell density to 1.0×10 6 cells / mL, expand the bottle according to the volume of the cell suspension (i.e. transfer to a new T175 cell culture flask or transfer to a cell culture bag for culture; neither the T175 cell culture flask nor the cell culture bag for transfer bottle culture needs to be coated with coating solution), and continue to culture in a 37°C, 5% CO2 incubator.

[0148] Note: Try to minimize cell counting, observation, pipetting and other operations during the first 5 days to avoid affecting the initial growth of cells.

[0149] VI. Expand training

[0150] On the 7th day of culture, expansion medium and heat-inactivated autologous plasma (the content of heat-inactivated autologous plasma was reduced to 1%) were added, samples were taken and counted, and the cell density was adjusted to 1.0×10 6 cells / mL, expand the bottle according to the volume of cell suspension, and continue to culture in a 37℃, 5% CO2 incubator.

[0151] 7. Continue to cultivate

[0152] Add expansion medium and heat-inactivated autologous plasma every other day or two, take samples and count, and adjust the cell density to 1.0×10 6 cells / mL, expand the bottle according to the volume of cell suspension, and continue to culture in a 37℃, 5% CO2 incubator.

[0153] 8. Harvesting cells

[0154] NK cells can be harvested on the 12th to 14th day of culture, or the harvesting time can be extended to 21 days, depending on the experimental situation.

[0155] Detect cell viability during culture (cell counter detects cell viability and number of living cells) Figure 3 As shown, the cell viability is high overall, and the cell viability of the whole process is maintained above 90%.

[0156] Calculate the cell proliferation before and after culture. Figure 4 As shown, the initial cell number was 2×10 7 cells, which expanded 407.32-fold in 14 days and 881.93-fold in 21 days.

[0157] Detect cell purity during culture, i.e. (the proportion of cell-specific markers detected by flow cytometry) and detect CD3 - CD56 + / CD3 - CD16 + / CD16 + CD56 + The percentage of cell populations is shown in Fig.10 and 11 As shown, the NK cells obtained after 14 days of culture were CD3 - CD56 + 93.52%, CD3 - CD16 +93.11%, CD16 + CD56 + After 21 days of culture, the NK cells obtained had a CD3 - CD56 + 93.63%, CD3 - CD16 + 94.63%, CD16 + CD56 + It is 90.60%.

[0158] The killing rate of NK cells was detected after 14 days of culture (CCK8 detection killing principle), and the results were as follows Figure 7 When the effector-target ratio was 1:1, the killing rate of NK cells was 91.35%; when the effector-target ratio was 5:1, the killing rate of NK cells was 92.89%; when the effector-target ratio was 10:1, the killing rate of NK cells was 94.24%; when the effector-target ratio was 20:1, the killing rate of NK cells was 95.91%.

[0159] Comparative Example In vitro expansion of NK cells

[0160] In this comparative example, no progesterone component was added to the culture medium.

[0161] Preparation of coating solution: melt CD16 at room temperature, add CD16 to 5 mL PBS buffer, mix by inversion to make the final concentration of CD16 5 μM, and set aside;

[0162] Preparation of activation medium: Add IL-2 and IL-15 to the KBM581 immune cell culture medium, invert and mix to make the final concentrations of IL-2 and IL-15 2000 IU / mL and 80 ng / mL respectively, and set aside;

[0163] Preparation of expansion medium: Add 1000 IU / mL IL-2 and 50 ng / mL IL-15 to the KBM581 immune cell culture medium, invert and mix to make the final concentrations of IL-2 and IL-15 1000 IU / mL and 50 ng / mL, respectively, and set aside.

[0164] The steps for in vitro expansion of NK cells are as follows:

[0165] 1. Antibody coated culture flask (-1 day)

[0166] Add the coating solution to the T25 cell culture flask, shake it back and forth to make the liquid evenly dispersed on the bottom of the flask, and coat it overnight at 4°C. Note: If the coated culture flask is not used immediately, it can be stored in a 4°C refrigerator. It is recommended to use it within 3 days.

[0167] 2. Preparation of peripheral blood mononuclear cells (PBMC) and heat-inactivated autologous plasma (Day 0)

[0168] Preparation of heat-inactivated autologous plasma: anticoagulated blood (human peripheral blood is selected as the blood source) is centrifuged at 650×g for 15 minutes at room temperature, and the upper yellow plasma portion (the lower layer is the blood cell component, which is used for the subsequent separation of PBMC) is placed in a 56°C water bath for inactivation for 30 minutes; after inactivation, centrifuge at 900×g for 10 minutes; take the supernatant and freeze it at -20°C for 15 minutes; centrifuge again at 900×g for 10 minutes, take the supernatant, and obtain the heat-inactivated autologous plasma and store it at 4°C for later use.

[0169] Preparation of PBMC: PBMCs were separated from human blood using Ficoll. The specific method was as follows:

[0170] Centrifuge the anticoagulated blood at room temperature at 650×g for 15 minutes, remove the red precipitate, add PBS of the same volume as the original blood and mix thoroughly to obtain diluted blood; slowly add the diluted blood to the lymphocyte separation solution, and pay attention to the moderate acceleration. Centrifuge at room temperature at 800×g for 20 minutes (speed increases and decreases slowly); after centrifugation, carefully aspirate the buffy coat layer into a new centrifuge tube, and add PBS to mix. At this time, the amount of PBS used is 5-10 times the volume of the buffy coat solution, and then centrifuge at 300×g for 10 minutes; discard the supernatant, resuspend the precipitate with 5-10 times the volume of PBS again, count the cells, and centrifuge at 300×g for 10 minutes; discard the supernatant to obtain PBMC for use.

[0171] III. Cell Inoculation (Day 0)

[0172] Take out the coated T25 cell culture flask 20-30 minutes in advance and equilibrate it to room temperature, then discard the coating solution, wash with PBS, resuspend PBMC in 9 mL activation medium and inoculate it in a T25 cell culture flask (seeding density 2×10 6 cells / mL), and add 1 mL of heat-inactivated autologous plasma to a total volume of 10 mL in the T25 culture bottle. Shake the cells back and forth and left and right and place them in a 37°C, 5% CO2 incubator for culture. The day of inoculation is recorded as day 0.

[0173] IV. Fluid culture

[0174] On the third day of culture, 18 mL of activation medium and 2 mL of heat-inactivated autologous plasma were slowly added along the side wall of the T25 cell culture flask (i.e., the heat-inactivated autologous plasma accounted for 10% of the total volume of the activation medium and heat-inactivated autologous plasma, i.e., the content of heat-inactivated autologous plasma was 10%), and the total volume of the liquid in the T25 cell culture flask was 30 mL. The cells were continued to be cultured in a 37°C, 5% CO2 incubator. Be careful to avoid touching the bottom of the culture flask, do not blow the cells, and minimize operations such as counting and observation to avoid affecting the initial growth of the cells.

[0175] 5. Bottle expansion culture

[0176] On the fifth day of culture, observe the cell status. When there are many and large cell clones (the diameter of the cell clusters is about 1 cm under a 10X microscope), add expansion medium and heat-inactivated autologous plasma (heat-inactivated autologous plasma accounts for 5% of the total volume of expansion medium and heat-inactivated autologous plasma, that is, the content of heat-inactivated autologous plasma is 5%), take samples and count, and adjust the cell density to 1.0×10 6 cells / mL, expand the bottle according to the volume of the cell suspension (i.e. transfer to a new T175 cell culture flask or transfer to a cell culture bag for culture; neither the T175 cell culture flask nor the cell culture bag for transfer bottle culture needs to be coated with coating solution), and continue to culture in a 37°C, 5% CO2 incubator.

[0177] Note: Try to minimize cell counting, observation, pipetting and other operations during the first 5 days to avoid affecting the initial growth of cells.

[0178] VI. Expand training

[0179] On the 7th day of culture, expansion medium and heat-inactivated autologous plasma (the content of heat-inactivated autologous plasma was reduced to 1%) were added, samples were taken and counted, and the cell density was adjusted to 1.0×10 6 cells / mL, expand the bottle according to the volume of cell suspension, and continue to culture in a 37℃, 5% CO2 incubator.

[0180] 7. Continue to cultivate

[0181] Add expansion medium and heat-inactivated autologous plasma every other day or two, take samples and count, and adjust the cell density to 1.0×10 6 cells / mL, expand the bottle according to the volume of cell suspension, and continue to culture in a 37℃, 5% CO2 incubator.

[0182] 8. Harvesting cells

[0183] NK cells can be harvested on the 12th to 14th day of culture, or the harvesting time can be extended to 21 days, depending on the experimental situation.

[0184] NK cell culture process Figure 2 As shown, the cell status during the entire culture process was observed. Fig.12 As shown, during the culture process, the cell amount increases; the cells are in a uniform and transparent state, with obvious semi-adherent aggregate growth, and some cell aggregates grow in suspension; in the later stage, the cell aggregates increase, the cells have an irregular shape, and some cells have tentacles extending. Compared with the example, the cell amount is lower and the aggregates are large and loose.

[0185] Detect cell viability during culture (cell counter detects cell viability and number of living cells) Figure 3As shown, the cell viability is generally high, ranging from 82% to 92% throughout the entire process, with most being below 90%.

[0186] Calculate the cell proliferation before and after culture. Figure 4 As shown, the initial cell number was 2×10 7 cells, which expanded 154.32-fold in 14 days and 361.20-fold in 21 days.

[0187] Detect cell purity during culture, i.e. (the proportion of cell-specific markers detected by flow cytometry) and detect CD3 - CD56 + / CD3 - CD16 + / CD16 + CD56 + The percentage of cell populations is shown in Fig.13 and 14 As shown, the NK cells obtained after 14 days of culture were CD3 - CD56 + 67.08%, CD3 - CD16 + 56.49%, CD16 + CD56 + After 21 days of culture, the NK cells obtained had a CD3 - CD56 + 76.17%, CD3 - CD16 + 65.85%, CD16 + CD56 + It is 70.92%.

[0188] The killing rate of NK cells was detected after 14 days of culture (CCK8 detection killing principle), and the results were as follows Figure 7 When the effector-target ratio was 1:1, the killing rate of NK cells was 72.58%; when the effector-target ratio was 5:1, the killing rate of NK cells was 80.15%; when the effector-target ratio was 10:1, the killing rate of NK cells was 85.34%; when the effector-target ratio was 20:1, the killing rate of NK cells was 89.57%.

[0189] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A NK cell culture system, characterized in that: The culture system includes coating solution, activation culture medium and expansion culture medium; wherein, The coating solution comprises CD16 and PBS buffer; the final concentration of CD16 is 1-10 μM; The activation medium comprises serum-free medium, IL-2, IL-15 and progesterone; the final concentrations of IL-2, IL-15 and progesterone are 500-3000 IU / mL, 10-100 ng / mL and 1-10 μM, respectively; The expansion medium comprises serum-free medium, IL-2 and IL-15; the final concentrations of the IL-2 and IL-15 are 500-3000 IU / mL and 10-100 ng / mL, respectively.

2. The culture system according to claim 1, characterized in that The culture system also includes autologous plasma; preferably, it is heat-inactivated autologous plasma.

3. The culture system according to claim 1, characterized in that: The final concentrations of IL-2, IL-15 and progesterone in the activation medium are 2000 IU / mL, 80 ng / mL and 5 μM, respectively; preferably, the final concentration of CD16 in the coating solution is 5 μM.

4. The culture system according to claim 1, characterized in that: The final concentrations of IL-2 and IL-15 in the expansion medium are 1000-1500 IU / mL and 10-100 ng / mL, respectively; preferably 1000 IU / mL and 50 ng / mL.

5. The culture system according to claim 1, characterized in that: The serum-free culture medium is one or more of KBM581 immune cell culture medium, SCGM serum-free culture medium, AIM-V serum-free culture medium, X-VIVO 15 serum-free culture medium and NK MACS serum-free culture medium.

6. A method for in vitro expansion of NK cells using the culture system according to any one of claims 1 to 5, characterized in that: The steps include: Antibody-coated cell culture flask: adding the coating solution to the cell culture flask, making the coating solution evenly dispersed on the bottom of the flask, coating, and obtaining a coated cell culture flask; Mononuclear cells and heat-inactivated autologous plasma were prepared from human blood; Cell inoculation: discard the coating solution in the cell culture flask after coating, wash with PBS, resuspend the mononuclear cells in the activation medium and inoculate them in the cell culture flask that has been coated and washed with PBS, add heat-inactivated autologous plasma, and culture. The day of inoculation is recorded as day 0; Fluid replenishment culture: On the third day of culture, activation medium and heat-inactivated autologous plasma are added and cultured; Bottle expansion culture: on the 5th day of culture, observe the cell status. When it is observed that there are many and large cell clones, add expansion medium and heat-inactivated autologous plasma, take samples and count, adjust the cell density, and transfer the cells to a new cell culture bottle or cell culture bag according to the volume of the cell suspension for culture; Expansion culture: On the 7th day of culture, add expansion medium and heat-inactivated autologous plasma, take samples and count, adjust the cell density, and transfer to a new cell culture bottle or cell culture bag according to the volume of the cell suspension for culture; Continue culturing: add expansion medium and heat-inactivated autologous plasma every one or two days, take samples and count, adjust cell density, transfer to new cell culture bottles or cell culture bags according to the volume of cell suspension, and culture; Harvesting cells: Harvest NK cells on day 12-14 of culture.

7. The method according to claim 6, characterized in that The cell inoculation step has an inoculation density of 2.0-3.0×10 6 cells / mL; preferably, 2.0×10 6 cells / mL; and / or, The cell density is adjusted in the steps of expanding the flask culture, expanding the culture and continuing the culture to maintain the cell concentration at 0.5-1.0×10 6 cells / mL; preferably, 1.0×10 6 cells / mL.

8. The method according to claim 6, characterized in that In the cell inoculation step, the heat-inactivated autologous plasma accounts for 7-10% of the total volume of the activation medium and the heat-inactivated autologous plasma; preferably, 10%; and / or, In the rehydration culture step, the heat-inactivated autologous plasma accounts for 7-10% of the total volume of the activation medium and the heat-inactivated autologous plasma; preferably, 10%; and / or, In the bottle expansion culture step, the heat-inactivated autologous plasma accounts for 4-7% of the total volume of the expansion medium and the heat-inactivated autologous plasma; preferably, 5%; and / or, In the expansion culture step, the heat-inactivated autologous plasma accounts for 1-3% of the total volume of the expansion medium and the heat-inactivated autologous plasma; preferably, it is 1%.

9. The method according to claim 6, characterized in that The antibody coating culture flask step comprises incubating overnight at 4°C or incubating in a 37°C incubator for 2 hours; Preferably, the culture is carried out at 37°C and 5% CO2; More preferably, the new cell culture flask or cell culture bag refers to a cell culture flask or cell culture bag that has never been coated.

10. The method according to claim 6, characterized in that The heat-inactivated autologous plasma is obtained by separating plasma from human blood and inactivating it at 56° C. for 30 minutes and then centrifuging it; Preferably, the mononuclear cells are obtained by separating human blood using Ficoll; More preferably, the human blood source includes human peripheral blood or umbilical cord blood, and the mononuclear cells are human peripheral blood mononuclear cells or umbilical cord blood mononuclear cells.

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