A method for culturing nk cells

Abstract

The present application relates to the technical field of cell culture, in particular to a culture method of NK cells; the culture method of NK cells of the present application, whether in the early activation / induction culture stage or in the middle and late expansion culture stage, is cultured in a culture bag throughout, without the need to transfer culture solution, easy to operate, reduces production cost, can more reduce the risk of infection, is suitable for industrial production, and at the same time ensures the total amount of NK cells after final expansion, the purity of NK cells and the activity of NK cells.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a method for culturing NK cells. Background Technology

[0002] NK cells (natural killer cells) are important immune cells involved in the body's fight against tumors and viral infections, as well as immune regulation. They are not MHC-restricted and do not depend on antibodies in killing target cells, effectively killing various tumor cells through direct and indirect pathways. Therefore, they have unique application prospects in adoptive immunotherapy. However, the inability to obtain large quantities and high purity human NK cells from the human body limits their application in immunotherapy.

[0003] Obtaining human NK cells through in vitro expansion is an important step in recent years in the study of NK cell function, especially in exploring adoptive immunotherapy.

[0004] However, existing methods for expanding NK cells in vitro mainly use cell culture flasks for culture. For example, the culture flask is first coated with an antibody, and then the isolated nucleated cells are placed into the coated culture flask for activation / induction culture. The capacity of the coated culture flask is limited, so in the later stages of large-capacity expansion culture, it is necessary to transfer the cells into larger and more numerous cell culture flasks.

[0005] Considering the limited capacity of culture flasks, those skilled in the art also use a combination of culture flasks and culture bags. For example, in the early stage of culture, the isolated nucleated cells are placed into a coated culture flask for activation / induction culture. In the middle and late stages of culture, such as around day 6-8 or day 11, the cells are transferred from the culture flask to a culture bag, culture medium is added, and large-capacity expansion culture is carried out.

[0006] However, the amplification culture method using transfer culture medium is cumbersome to operate and exposes the cells to the external environment (external reagents and contaminants), resulting in a high risk of infection. In addition, existing methods for amplifying NK cells in vitro have various adverse factors that affect the activity (survival rate), quantity, and purity of the obtained NK cells, reducing the possibility of NK cell drug development. Summary of the Invention

[0007] To address the above problems, the present invention provides a method for culturing NK cells, which includes the following steps (1) to (5).

[0008] Step (1): Collect mononuclear cells In one specific embodiment of the invention, the mononuclear cells are derived from blood, umbilical cord blood, or bone marrow; in a preferred embodiment of the invention, the blood is peripheral blood.

[0009] Specifically, in this embodiment, the inventors used density gradient centrifugation to freshly separate peripheral blood mononuclear cells from approximately 50 ml of healthy human blood.

[0010] Step (2): Initial culture In one specific embodiment of the present invention, the collected mononuclear cells are processed at a concentration of 0.6-2.4 × 10⁻⁶. 6 Cells / mL were seeded into the initial culture medium and transferred to a culture bag; the initial culture medium was serum-free cell culture medium as the basal medium, which contained 700~1200 IU / L IL-2, 0.1~0.3 ug / ml anti-CD16 antibody and 3~7 ug / ml IL-8; and the culture volume of the culture bag was controlled so that the ratio of the initial culture medium volume to the culture bag volume was 1:6~1:10.

[0011] In a preferred embodiment of the invention, the seeding density of mononuclear cells in the initial culture medium is 0.6 × 10⁶. 6 0.7×10 6 0.8×10 6 0.9×10 6 1.0×10 6 1.1×10 6 1.2×10 6 1.3×10 6 1.4×10 6 1.5×10 6 1.6×10 6 1.7×10 6 1.8×10 6 1.9×10 6 2.0×10 6 2.1×10 6 2.2×10 6 2.3×10 6 2.4×10 6 Cells / mL.

[0012] Preferably, the seeding density of mononuclear cells in the initial culture medium is 1.0 × 10⁻⁶. 6 Cells / mL.

[0013] Regarding the formulation of the initial culture medium, the basal culture medium used is serum-free culture medium for cell culture.

[0014] The term "serum-free culture medium for cell culture" refers to a serum-free culture medium used for the proliferation and culture of immune cells such as human lymphocytes, CIK cells, DC cells, and NK cells, but it does not have the function of selecting, inducing, or differentiating immune cells.

[0015] In a preferred embodiment of the present invention, the basal culture medium is a conventional, especially commercially available, serum-free culture medium for human immune cell and human lymphocyte culture (which does not contain antibodies and cell growth factors that select, induce, or differentiate immune cells and lymphocytes); more preferably, the basal culture medium is a conventional, especially commercially available, serum-free culture medium for NK cell culture (which does not contain antibodies and cell growth factors that select, induce, or differentiate NK cells).

[0016] In a preferred embodiment of the present invention, the formulation of the "serum-free culture medium for cell culture" adopts the formulation of "a serum-free culture medium for human T lymphocyte culture" disclosed in patent document CN108707579A: DMEM / F12 medium, 3-10 g / L human serum albumin, 30-100 mg / L glutathione, 5 mg / L insulin, 10 mg / L transferrin, 5 g / L sodium pyruvate, 25 μg / L sodium selenite, 50 μg / L phosphatidylcholine, 50 μg / L phosphatidylserine, 5 μg / L linoleic acid, 5 μg / L oleic acid, 0.1 g / L 2-aminoethanol hydrochloride, 5 g / L HEPES, and 5 g / L sodium bicarbonate, and water. Preferably, the serum-free culture medium for cell culture contains 5-10 g / L human serum albumin; more preferably, it contains 7.5-10 g / L human serum albumin. Preferably, the serum-free culture medium for cell culture contains 50-100 mg / L glutathione; preferably, the pH of the serum-free culture medium for cell culture is 6.8-7.4; more preferably, the pH is 7.1-7.3.

[0017] Preferably, the formulation of the "serum-free culture medium for cell culture" adopts the serum-free culture medium formulation for human T lymphocyte culture provided in Example 1 (Table 1 of the specification) of patent document CN108707579A.

[0018] Regarding the formulation of the initial culture medium, in addition to the aforementioned basal culture medium (serum-free culture medium for cell culture), antibodies and cell growth factors for the selection, induction, and differentiation of NK cells are added. In one specific embodiment of the present invention, the aforementioned basal culture medium also contains 700-1200 IU / L of IL-2, 0.1-0.3 μg / ml of anti-CD16 antibody, and 3-7 μg / ml of IL-8.

[0019] Preferably, the initial culture medium contains 1000 IU / L of IL-2, 0.2 μg / ml of anti-CD16 antibody, and 5 μg / ml of IL-8.

[0020] In one specific embodiment of the present invention, the culture volume of the culture bag needs to be controlled during the initial culture stage so that the ratio of the initial culture medium volume to the culture bag volume is 1:6 to 1:10. In a preferred embodiment of the present invention, the ratio of the initial culture medium volume to the culture bag volume is 1:6, 1:7, 1:8, 1:9, or 1:10.

[0021] Preferably, the initial culture medium volume is 50 ml, and the culture bag volume is 400 ml, with a ratio of 1:8. Specifically, the inventors used a culture bag with a total volume of 2000 ml, and used cell bag clamps or partitions to clamp the bag at one-fifth of its length. Figure 1 As shown, the culture volume of the culture bag should be controlled at 400ml.

[0022] Step (3): First round of amplification culture In one specific embodiment of the present invention, the culture volume of the culture bag is expanded so that the ratio of the expanded culture volume to the culture volume in step (2) is 3:2 to 9:5; during the first round of amplification culture, the total volume of the culture medium is 1.8 to 6 times the initial culture medium volume.

[0023] In the first round of amplification culture stage, it is necessary to increase the culture volume of the culture bag and supplement or replenish the culture medium multiple times. At the same time, it is still necessary to control the culture volume of the culture bag to be expanded and the total volume of the culture medium.

[0024] In one specific embodiment of the present invention, the ratio of the expanded culture volume to the culture volume in step (2) is 3:2, 8:5, 5:3 or 9:5; the total volume of the culture medium (the total volume of the culture medium after each replenishment) is 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 or 6 times the initial culture medium volume.

[0025] In a preferred embodiment of the present invention, in step (3), on days 1 to 7 after the initial culture, the culture volume of the culture bag is expanded and amplification culture medium A is added, and then the serum-free culture medium for cell culture is added every 1 to 2 days.

[0026] In one specific embodiment of the present invention, the amplification medium A uses serum-free cell culture medium as the basal medium, and contains 700~1200 IU / l of IL-2, 0.1~0.3 ug / ml of anti-CD16 antibody, 3~7 ug / ml of IL-8 and 0.18~0.22 ug / ml of hydrocortisone sodium succinate.

[0027] Preferably, in the amplification medium A, the basal medium (serum-free medium for cell culture) is formulated according to the serum-free medium for human T lymphocyte culture provided in Example 1 (Table 1 of the specification) of patent document CN108707579A, and contains 1000 IU / l of IL-2, 0.2 ug / ml of anti-CD16 antibody, 5 ug / ml of IL-8 and 0.20 ug / ml of hydrocortisone sodium succinate.

[0028] Regarding the timing of "expanding the culture volume of the culture bag and supplementing amplification medium A", preferably, it is on the 1st, 3rd, 5th or 7th day after the initial culture; more preferably, it is on the 3rd or 5th day.

[0029] Regarding the timing of "supplementing serum-free culture medium for cell culture," the interval should be 1-2 days after supplementing amplification medium A, preferably 1 day. The interval, number of supplementations, and volume of serum-free culture medium supplemented can be adjusted by those skilled in the art based on the cell proliferation and density after supplementing amplification medium A.

[0030] Preferably, on the 3rd day after the initial culture, the culture volume of the culture bag is expanded so that the ratio of the expanded culture volume to the culture volume in step (2) is 5:3, and amplification culture medium A is added, the volume of amplification culture medium A being 1 times the volume of the initial culture medium; on the 5th day after the initial culture, serum-free culture medium for cell culture is added until the total volume of the culture medium reaches 5 times the volume of the initial culture medium.

[0031] Specifically, on the 3rd day after the initial culture, the culture volume of the culture bag was increased to 667ml (using a cell bag clamp or septum to clamp one-third of the culture bag), and 50ml of amplification medium A was added; on the 5th day after the initial culture, 150ml of serum-free cell culture medium was added, bringing the total culture volume to 250ml.

[0032] Step (4): Second round of amplification culture In one specific embodiment of the present invention, the culture volume of the culture bag is expanded so that the ratio of the expanded culture volume to the culture volume in step (2) is 4:1 to 6:1; in the second round of amplification culture, the total volume of the culture medium is 18 to 32 times the volume of the initial culture medium.

[0033] In the second round of amplification culture, it is necessary to increase the culture volume of the culture bag and supplement or replenish the culture medium multiple times. At the same time, it is still necessary to control the culture volume of the culture bag to be expanded and the total volume of the culture medium.

[0034] In one specific embodiment of the present invention, the ratio of the expanded culture volume to the culture volume in step (2) is 4:1, 5:1 or 6:1; the total volume of the culture medium (the total volume of the culture medium after each replenishment) is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 times the initial culture medium volume.

[0035] In a preferred embodiment of the present invention, in step (4), on days 7 to 15 after the initial culture, the culture volume of the culture bag is expanded and amplification medium B is added, and serum-free culture medium for cell culture is added every 1 to 2 days.

[0036] In a preferred embodiment of the present invention, the amplification medium B uses serum-free cell culture medium as the basal medium, and contains 700-1200 IU / L of IL-2 and 3-7 μg / ml of IL-8. The volume of the amplification medium B is 14-16 times the volume of the initial medium.

[0037] Preferably, in the amplification medium B, the basal medium (serum-free medium for cell culture) is formulated according to the serum-free medium for human T lymphocyte culture provided in Example 1 (Table 1 of the specification) of patent document CN108707579A, and contains 1000 IU / l of IL-2 and 5 ug / ml of IL-8.

[0038] Regarding the timing of "expanding the culture volume of the culture bag and supplementing the amplification medium B", preferably, it is on the 8th, 10th, 12th or 14th day after the initial culture; more preferably, it is on the 8th or 10th day.

[0039] Regarding the timing of "supplementing serum-free culture medium for cell culture," the interval should be 1-2 days after supplementing amplification medium B, preferably 1 day. The interval, number of supplementations, and volume of serum-free culture medium supplemented can be adjusted by those skilled in the art based on the cell proliferation and density after supplementing amplification medium B.

[0040] Preferably, on the 8th day after the initial culture, the culture volume of the culture bag is expanded so that the ratio of the expanded culture volume to the culture volume in step (2) is 5:1, and amplification culture medium B is added, the volume of amplification culture medium B being 15 times the volume of the initial culture medium; on the 10th day after the initial culture, serum-free culture medium for cell culture is added until the total volume of the culture medium reaches 30 times the volume of the initial culture medium.

[0041] Specifically, on day 8 after initial culture, the culture volume of the culture bag was expanded to the full volume of the culture bag (i.e., the cell bag clamp or septum was removed, and the culture volume was 2000ml), and 750ml of amplification medium B was added; on day 10 after initial culture, 500ml of serum-free cell culture medium was added, bringing the total culture volume to 1500ml.

[0042] Step (5): Harvest cultured NK cells In one specific embodiment of the present invention, cultured NK cells are harvested on days 10 to 20 after initial culture; preferably, NK cells are harvested on days 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; more preferably, NK cells are harvested on days 13, 14, 15, and 16.

[0043] Preferably, NK cells are harvested on day 14.

[0044] In some embodiments, the method further includes measuring or sorting cultured cells using fluorescence activated cell sorting (FACS).

[0045] In other respects, the present invention also provides an NK cell obtained by the culture method described above.

[0046] Beneficial effects of the present invention As described in the background section, existing methods for in vitro expansion of NK cells generally involve initial culture in culture flasks, followed by later transfer of the culture medium to larger or more numerous culture flasks or larger or more numerous culture bags for large-volume expansion. This is a common practice for those skilled in the art.

[0047] Multiple transfers of culture medium are cumbersome and pose a risk of exposure to the external environment (external reagents and contaminants). Culturing within a single system throughout the entire process is not only easier to operate and reduces production costs, but also lowers the risk of infection, making it more suitable for industrial production and meeting cGMP requirements.

[0048] However, those skilled in the art still insist on using the method of culturing in culture flasks in the early stage and transferring the culture medium in the middle and later stages in the existing technology. This method mainly has the following technical problems: 1) Antibody coating and immobilization: In the initial activation / induction culture process, those skilled in the art use culture flasks coated with antibodies (e.g., T75 culture flasks). If larger culture flasks (e.g., T175 culture flasks) are used, the efficiency and uniformity of coating and immobilization are much lower than with T75 culture flasks; and if culture bags are used, the antibody cannot be immobilized on the inner wall of the culture bag at all; therefore, based on the practical problems of operation (coating and immobilization), those skilled in the art would not consider using culture bags in the initial activation / induction culture process.

[0049] 2) Amplification fold and purity: Existing research has shown that, in the early activation / induction culture stage, the average amplification fold in culture flasks is more than twice that in culture bags. Furthermore, the percentage of NK cells (average purity) in the product from culture flasks is significantly higher than the purity of the product from culture bags. Therefore, based on the amplification effect, those skilled in the art would not consider using culture bags in the early activation / induction culture process.

[0050] Therefore, the practice of culturing in culture flasks in the early stages and transferring the culture medium in the middle and later stages is not only a common practice but also a mindset of those skilled in the art.

[0051] In the process of researching and attempting to use culture bags as the entire culture system, the inventors of this application broke away from the aforementioned mindset. On the one hand, they abandoned the conventional operation of "antibody coating and fixation," and on the other hand, they tried to temporarily disregard the issue of amplification effects in the early stages.

[0052] In the early activation / induction culture stage, because the culture bag could not be used for antibody coating and fixation, the inventors often encountered cell adhesion due to "antibody-antibody" linkage during the experiment, which seriously affected the cell proliferation effect.

[0053] Through numerous experiments and studies, the inventors analyzed the possible reasons behind this situation, such as the choice of antibodies and the choice of cytokine combinations, especially the trade-off between "antibody concentration and cell inoculation concentration".

[0054] The inventors experimented with sequentially adding various antibodies to the culture medium (e.g., anti-NKp46 antibody, anti-CD16 antibody, and CD3, CD4, CD8, CD28, CD52, CD56, CD137, Her-2, etc.), and also tried various cell growth factors and their combinations (any one or more combinations of IL-2, IL-12, IL-15, IL-18, IL-21); however, the initial proliferation effect was still very poor. Therefore, the inventors continued to conduct optimization and improvement experiments based on this.

[0055] In the initial culture stage, the volume of the cell culture medium is small, while the overall volume of the culture bag is large. The inventors discovered that the balance between the "ratio control of culture medium volume to culture bag volume" and the "cell seeding density" in the initial stage is also very important.

[0056] Through experimentation and exploration, the inventors discovered that by controlling the relationship between the culture medium volume and culture volume in the initial stage, as well as the initial inoculation concentration, and by using a specific combination of antibodies and cell growth factors in the initial culture medium (700~1200 IU / l of IL-2, 0.1~0.3 ug / ml of anti-CD16 antibody, and 3~7 ug / ml of IL-8), NK cell activation / induction culture can be achieved in the culture bag without the need for antibody coating and fixation, and cell adhesion will not occur due to "antibody-antibody" interconnection.

[0057] In addition, in order to achieve culture in the same culture bag throughout the entire process, the inventors used components such as culture bag clamps or partitions to control the culture volume of the culture bag. During the two rounds of expansion culture in the middle and later stages, the culture volume was gradually increased, and the culture area on one side of the culture bag and the total volume of the culture medium were strictly controlled to ensure the final expansion multiple, NK cell purity and NK cell activity (survival rate).

[0058] The NK cell culture method of the present invention, whether in the early activation / induction culture stage or the mid-to-late expansion culture stage, is cultured entirely in a culture bag without the need to transfer culture medium. It is easy to operate, reduces production costs, and can further reduce the risk of infection. It is suitable for industrial production, while also ensuring the total amount, purity, and activity of NK cells after final expansion. Attached Figure Description

[0059] Figure 1 A schematic image showing the culture volume (culture area) of the culture bag controlled by partitions. Detailed Implementation

[0060] The present invention will be further illustrated below with reference to specific embodiments, but these embodiments do not limit the invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. All culture procedures were performed at 37°C and 5% CO2.

[0061] Example 1

[0062] This embodiment provides a method for culturing NK cells, which includes the following steps.

[0063] Step (1): Collect mononuclear cells.

[0064] Peripheral blood mononuclear cells were freshly isolated from approximately 50 ml of healthy human blood using density gradient centrifugation.

[0065] Step (2) Initial culture.

[0066] The collected mononuclear cells were measured at 1.0 × 10⁻⁶. 6 Cells / mL were seeded into the initial culture medium and transferred to a culture bag; the initial culture medium used was serum-free culture medium for cell culture (the formulation of which was based on the serum-free culture medium for human T lymphocyte culture provided in Example 1 (Table 1) of patent document CN108707579A) as the basal medium, and contained 1000 IU / L IL-2, 0.2 μg / ml anti-CD16 antibody and 5 μg / ml IL-8; and the culture volume of the culture bag was controlled so that the ratio of the initial culture medium volume to the culture bag volume was 1:8. Specifically, the inventors used a culture bag with a total volume of 2000 ml, and used a cell bag clamp or partition to clamp the bag at one-fifth of its capacity. Figure 1 As shown, the culture volume of the culture bag is controlled at 400ml, and the initial culture medium volume is 50ml.

[0067] Step (3): First round of amplification culture.

[0068] On the 3rd day after the initial culture, the culture volume of the culture bag is increased so that the ratio of the increased culture volume to the culture volume in step (2) is 5:3, and amplification culture medium A is added, the volume of amplification culture medium A being 1 times the volume of the initial culture medium; on the 5th day after the initial culture, serum-free culture medium for cell culture is added until the total volume of the culture medium reaches 5 times the volume of the initial culture medium.

[0069] Specifically, on the 3rd day after the initial culture, the culture volume of the culture bag was increased to 667ml (using a cell bag clamp or septum to clamp one-third of the culture bag), and 50ml of amplification medium A was added; on the 5th day after the initial culture, 150ml of serum-free cell culture medium was added, bringing the total culture volume to 250ml.

[0070] Step (4): Second round of amplification culture.

[0071] On the 8th day after the initial culture, the culture volume of the culture bag was increased so that the ratio of the increased culture volume to the culture volume in step (2) was 5:1, and amplification medium B was added, the volume of amplification medium B being 15 times the volume of the initial culture medium; on the 10th day after the initial culture, serum-free culture medium for cell culture was added until the total volume of the culture medium reached 30 times the volume of the initial culture medium.

[0072] Specifically, on day 8 after initial culture, the culture volume of the culture bag was expanded to the full volume of the culture bag (i.e., the cell bag clamp or septum was removed, and the culture volume was 2000ml), and 750ml of amplification medium B was added; on day 10 after initial culture, 500ml of serum-free cell culture medium was added, bringing the total culture volume to 1500ml.

[0073] (5) Harvest cultured NK cells.

[0074] Cultured NK cells were harvested on day 14 after initial culture. Example 2

[0075] The only difference between the culture method in Example 2 and Example 1 is that the serum-free culture medium (basal culture medium) used for cell culture is commercially available serum-free cell culture medium purchased from Auside Biotechnology.

[0076] Other operations and parameters are the same as in Example 1, and will not be repeated here.

[0077] Example 3

[0078] The cultivation method in Example 3 differs from that in Example 1 as follows: The initial inoculation concentration was 0.6 × 10⁶. 6 Cells / mL.

[0079] The initial culture medium contained 800 IU / L IL-2, 0.15 μg / ml anti-CD16 antibody, and 4 μg / ml IL-8; amplification culture medium A contained 800 IU / L IL-2, 0.15 μg / ml anti-CD16 antibody, 4 μg / ml IL-8, and 0.18 μg / ml hydrocortisone sodium succinate; amplification culture medium B contained 800 IU / L IL-2 and 4 μg / ml IL-8. The initial culture medium volume is 50 ml, and the culture volume of the culture bag is controlled at 350 ml (the ratio of the two is 1:7). First round of amplification culture: After the initial culture, on the 3rd day, the culture volume of the culture bag was increased to 560ml so that the ratio of the expanded culture volume to the culture volume in step (2) was 8:5.

[0080] Second round of amplification culture: After the initial culture, on the 8th day, the culture volume of the culture bag was increased to 2000ml so that the ratio of the expanded culture volume to the culture volume in step (3) was 40:7.

[0081] Other operations and parameters are the same as in Example 1, and will not be repeated here.

[0082] Example 4

[0083] The cultivation method in Example 4 differs from that in Example 1 as follows: The initial inoculation concentration was 2.4 × 10⁻⁶. 6 Cells / mL.

[0084] The initial culture medium contained 1200 IU / L IL-2, 0.3 μg / ml anti-CD16 antibody, and 7 μg / ml IL-8; amplification culture medium A contained 1200 IU / L IL-2, 0.3 μg / ml anti-CD16 antibody, 7 μg / ml IL-8, and 0.22 μg / ml hydrocortisone sodium succinate; amplification culture medium B contained 1200 IU / L IL-2 and 7 μg / ml IL-8. The initial culture medium volume is 50 ml, and the culture volume of the culture bag is controlled at 500 ml (the ratio of the two is 1:10). First round of amplification culture: After the initial culture, on the 3rd day, the culture volume of the culture bag was increased to 900ml so that the ratio of the expanded culture volume to the culture volume in step (2) was 9:5.

[0085] Second round of amplification culture: After the initial culture, on the 8th day, the culture volume of the culture bag was increased to 2000ml so that the ratio of the expanded culture volume to the culture volume in step (2) was 4:1.

[0086] Other operations and parameters are the same as in Example 1, and will not be repeated here.

[0087] Comparative Example 1a The difference between Comparative Example 1a and Example 1 is that Comparative Example 1a uses a conventional culture method, first culturing in a culture flask coated with antibody, then transferring the first round of amplification culture to a larger culture flask, and finally introducing the second round of amplification culture into a culture bag.

[0088] Specifically For the initial culture process, T75 culture flasks coated and immobilized with anti-CD16 antibody were used. 50 ml of initial culture medium (initial inoculation density 8.0 × 10⁻⁶) was added. 7 Cells / mL, 50ml of serum-free culture medium supplemented with 1000 IU / L IL-2 and 5 μg / mL IL-8. On day 3 after initial culture, the culture medium was transferred to an empty T175 culture flask and 50 ml of amplification medium A was added. On day 5 after initial culture, 150 ml of serum-free cell culture medium was added and the medium was transferred to two empty T175 culture flasks. On day 8 after initial culture, the culture medium was transferred to a 2000ml culture bag and 750ml of amplification medium B was added; on day 10 after initial culture, 500ml of serum-free cell culture medium was added, bringing the total culture medium volume to 1500ml.

[0089] Other parameters are the same as in Example 1, and will not be repeated here.

[0090] Comparative Example 1b The difference between Comparative Example 1b and Example 1 is that Comparative Example 1b was cultured in a complete culture bag throughout the entire process, and the culture volume was not controlled (culture volume 2000ml).

[0091] Other parameters are the same as in Example 1, and will not be repeated here.

[0092] Comparative Example 1c The difference between Comparative Example 1c and Example 1 is as follows: The initial vaccination density is the standard vaccination density: 3 × 10⁻⁶ 6 cells / mL; The initial culture medium contained a standard combination of cytokines (IL-2, IL-15, and IL-12) at standard concentrations: 1000 IU / L of IL-2, 0.2 μg / ml of anti-CD16 antibody, 1000 IU / L of IL-15, and 10 ng / ml of IL-12.

[0093] Other parameters are the same as in Example 1, and will not be repeated here.

[0094] Results data For Examples 1-4 and Comparative Examples 1a-1c, 1 ml of the harvested NK cell culture medium was used for cell counting and cell viability testing. Three parallel experiments were performed in each group. The results of cell number, proliferation rate and cell survival rate are shown in Table 1 below.

[0095] Table 1

[0096] FACS detection of NK (CD56-positive) cell ratio: Cells were washed once with PBS and incubated at 4°C for 30 minutes with an appropriate amount of the antibodies listed in Table 2 below. The labeled cells were then washed with PBS and fixed in 4% PFA before data acquisition. Data acquisition was performed using FACSCalibur (BD), and data were analyzed using CellQuest or FloMax software. For the analysis, SSC / FSC gates were used. NK cells were gated as the CD3-CD56+ population. NK-like T cells and T cells were gated as the CD3+CD56+ and CD3+CD56- populations, respectively. The results of the NK (CD56-positive) cell ratio assay are shown in Table 2 below.

[0097] Table 2

[0098] As can be seen from the data in Tables 1 and 2, the NK cell number obtained by using the culture methods of Examples 1-4 of the present invention reached 3.2-5.8 billion, the proliferation multiple reached 59-116 times, the survival rate reached 97-100%, and the purity of the obtained NK cells was 69.1-75.63%.

[0099] The culture method in Example 2, using a conventional commercially available serum-free cell culture medium, also achieved good culture results; however, the data on proliferation rate, survival rate, and NK cell ratio were slightly lower than those in Example 1.

[0100] As a preferred embodiment, the serum-free culture medium used in Example 1 is more suitable for the culture method of the present invention.

[0101] Example 3 used a lower initial seeding density; generally, reducing the initial density affects cell-cell interactions, thus reducing culture efficiency; using the method of this invention, at 0.6 × 10⁻⁶... 6 Even at an initial seeding concentration of 1 cell / mL, we still achieved good proliferation fold, total NK cell count, survival rate, and NK cell ratio.

[0102] Example 4 used a higher initial seeding density; generally, while a higher cell density leads to a higher final cell count, it also increases the number of dead cells, resulting in a lower fold increase. Example 4 of this invention still achieved a moderate fold increase and a favorable total NK cell count, with the NK cell ratio slightly lower than that of Example 1.

[0103] The inventors of this invention experimented with various seeding concentrations. To obtain optimal total NK cell count, proliferation rate, cell viability, and NK cell ratio, the initial cell seeding density needed to be controlled between 0.6 and 2.4 × 10⁻⁶. 6 Cells / mL.

[0104] Comparative Example 1a used a conventional culture method: first, the culture was carried out in antibody-coated culture flasks; after the first round of amplification, the culture was transferred to larger culture flasks; and after the second round of amplification, it was transferred to culture bags. This is a commonly used culture method in the market and in scientific research. However, antibody coating requires additional time and expense, and the operation steps are more complicated and prone to contamination during the culture process.

[0105] In contrast, the method of the present invention simplifies the antibody coating procedure and the cumbersome operation steps of moving the container in passaged cells, making it suitable for large-scale culture needs. At the same time, it can still achieve excellent culture results in terms of cell number, proliferation rate and cell survival rate, and even better than conventional culture methods.

[0106] Comparative Example 1b was cultured entirely in a complete culture bag, but the volume of the culture bag was not controlled. Comparative Example 1b was significantly worse in terms of the number of cells obtained, the fold increase, and the cell viability.

[0107] Comparative Example 1c used conventional seeding density and conventional combinations and concentrations of growth factors. However, the results of Comparative Example 1c showed that although the cell number was similar to that of Examples 1-4 of this invention, the fold increase was lower, and the cell viability was significantly reduced. Clearly, the use of anti-CD16 antibody, combined with conventional combinations and concentrations of growth factors, is not suitable for the culture method of this invention, resulting in a significant increase in dead cells and a significant decrease in cell viability.

[0108] In the description of this specification, the references to terms such as "an embodiment," "some embodiments," and "example" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0109] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in the claims, they should all fall within the protection scope of the present invention.

Claims

1. A method for culturing NK cells, characterized in that, The cultivation method includes the following steps: (1) Collect mononuclear cells; (2) Initial culture: The collected mononuclear cells were cultured at a culture temperature of 0.6~2.4×10⁻⁶. 6 10 cells / mL were seeded into the initial culture medium and then transferred to a culture bag; The initial culture medium uses serum-free cell culture medium as the basal medium, which contains 700~1200 IU / l of IL-2, 0.1~0.3 ug / ml of anti-CD16 antibody and 3~7 ug / ml of IL-8; Furthermore, the culture volume of the culture bag is controlled so that the ratio of the initial culture medium volume to the culture volume of the culture bag is 1:6 to 1:10; (3) First round of amplification culture: Expand the culture volume of the culture bag so that the ratio of the expanded culture volume to the culture volume in step (2) is 3:2 to 9:5; during the first round of amplification culture, the total volume of the culture medium is 1.8 to 6 times the volume of the initial culture medium; (4) Second round of amplification culture: The culture volume of the culture bag is increased so that the ratio of the expanded culture volume to the culture volume in step (2) is 4:1 to 6:1; During the second round of amplification culture, the total volume of the culture medium is 18 to 32 times the volume of the initial culture medium; (5) Harvest cultured NK cells.

2. The cultivation method according to claim 1, characterized in that, In step (3), on days 1 to 7 after the initial culture, the culture volume of the culture bag is increased and amplification medium A is added, and then serum-free culture medium for cell culture is added every 1 to 2 days. The amplification medium A uses serum-free cell culture medium as the basal medium, and contains 700~1200 IU / L IL-2, 0.1~0.3 ug / ml anti-CD16 antibody, 3~7 ug / ml IL-8 and 0.18~0.22 ug / ml hydrocortisone sodium succinate; the volume of the amplification medium A is 0.8~1.2 times the volume of the initial medium.

3. The cultivation method according to claim 2, characterized in that, The amplification medium A contains 1000 IU / L of IL-2, 0.2 μg / ml of anti-CD16 antibody, 5 μg / ml of IL-8, and 0.20 μg / ml of hydrocortisone sodium succinate.

4. The cultivation method according to claim 2, characterized in that, On the third day after the initial culture, the culture volume of the culture bag is increased so that the ratio of the increased culture volume to the culture volume in step (2) is 5:3, and amplification culture medium A is added, the volume of which is 1 times the volume of the initial culture medium; on the fifth day after the initial culture, serum-free culture medium for cell culture is added until the total volume of the culture medium reaches 5 times the volume of the initial culture medium.

5. The cultivation method according to claim 1, characterized in that, In step (4), on days 7 to 15 after the initial culture, the culture volume of the culture bag is expanded and amplification medium B is added, and then serum-free culture medium for cell culture is added every 1 to 2 days. The amplification medium B uses serum-free cell culture medium as the basal medium, and contains 700~1200 IU / l of IL-2 and 3~7 ug / ml of IL-8; the volume of the amplification medium B is 14~16 times the volume of the initial medium.

6. The cultivation method according to claim 5, characterized in that, The amplification medium B contains 1000 IU / L of IL-2 and 5 μg / ml of IL-8.

7. The cultivation method according to claim 5, characterized in that, On the 8th day after the initial culture, the culture volume of the culture bag is increased so that the ratio of the increased culture volume to the culture volume in step (2) is 5:1, and amplification culture medium B is added, the volume of which is 15 times the volume of the initial culture medium; on the 10th day after the initial culture, serum-free culture medium for cell culture is added until the total volume of the culture medium reaches 30 times the volume of the initial culture medium.

8. The cultivation method according to claim 1, characterized in that, In step (2), the seeding concentration of the mononuclear cells is 1 × 10⁻⁶. 6 cells / mL; The initial culture medium contained 1000 IU / L of IL-2, 0.2 μg / ml of anti-CD16 antibody, and 5 μg / ml of IL-8.

9. The cultivation method according to any one of claims 1 to 8, characterized in that, The serum-free culture medium for cell culture consists of the following components: DMEM / F12 medium, 3-10 g / L human serum albumin, 30-100 mg / L glutathione, 5 mg / L insulin, 10 mg / L transferrin, 5 g / L sodium pyruvate, 25 μg / L sodium selenite, 50 μg / L phosphatidylcholine, 50 μg / L phosphatidylserine, 5 μg / L linoleic acid, 5 μg / L oleic acid, 0.1 g / L 2-aminoethanol hydrochloride, 5 g / L HEPES, and 5 g / L sodium bicarbonate, as well as water.

10. The cultivation method according to claim 9, characterized in that, The serum-free culture medium for cell culture contains 5-10 g / L human serum albumin.

11. The cultivation method according to claim 10, characterized in that, The serum-free culture medium for cell culture contains 7.5-10 g / L human serum albumin.

12. The cultivation method according to claim 9, characterized in that, The serum-free culture medium for cell culture contains 50-100 mg / L glutathione.

13. The cultivation method according to claim 9, characterized in that, The serum-free culture medium used for cell culture has a pH of 6.8-7.

4.

14. The cultivation method according to claim 13, characterized in that, The serum-free culture medium used for cell culture has a pH of 7.1-7.

3.

15. The cultivation method according to any one of claims 1 to 8, characterized in that, In steps (2)-(4), the culture volume of the culture bag is controlled by a culture bag clamp or a partition.

16. The cultivation method according to any one of claims 1 to 8, characterized in that, The mononuclear cells are derived from blood, umbilical cord blood, or bone marrow.

17. The cultivation method according to claim 16, characterized in that, The blood in question is peripheral blood.

18. An NK cell obtained by the culture method according to any one of claims 1 to 17.

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