NK cell culture solution and culture method thereof

Abstract

The invention discloses an NK cell culture solution and a culture method thereof. The culture solution comprises an amplification culture solution and an induction activation culture solution, wherein the amplification culture solution is based on a serum-free culture medium and is added with cell factors such as IL-2, IL-12, IL-15 and IL-18, and the induction activation culture solution is based on the amplification culture solution and is added with induction activation factors such as OK432, CD16-alpha, a pseudomonas aeruginosa injection, alpha-galactosylceramide and CD137L. The induction activation factor CD16-alpha added in the NK cell culture fluid can activate NK cells independently or in combination with OK432, a pseudomonas aeruginosa injection, alpha-galactosylceramide and the like, so that the NK cells grow quickly, and the CD3-C56 + ratio of the NK cells can be increased; after the CD16-alpha is combined with the CD137L, the activated NK cells can be further activated, so that the activity of the NK cells is higher, and the killing effect is stronger.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a highly efficient full-factor induced NK cell culture fluid, and a method for culturing NK cells using the culture fluid. Background Art

[0002] Natural killer cells (NK cells) are one of the important components of the human immune system. They are effector cells of the innate immune system and are derived from bone marrow lymphoid stem cells. They differentiate and mature in the bone marrow and thymus and are distributed in the blood, lymph nodes, bone marrow, etc. NK cells are different from T and B cells. They can non-specifically recognize and kill tumor cells and virus-infected cells without antigen sensitization and without MHC restriction. Due to this important function, the research and application of NK cells in the field of tumor immunotherapy have gradually been valued.

[0003] NK cells can inhibit the invasion of viruses and bacteria, help clear cancerous, diseased and aging cells in the body, and have the function of delaying the aging of the body and reducing the risk of cancer. When NK cells find abnormal cells such as cancer cells, viruses, and infected cells, they will start to attack alone. Immune cells such as T cells and B cells also attack abnormal cells such as tumor cells and cancer cells; however, due to the antigen-antibody reaction, their attack behavior will be limited. In contrast, NK cells do not need antigen-antibody reaction, can go directly to the destination, and can attack freely and flexibly.

[0004] In recent years, a large number of clinical studies on NK cell anti-tumor have been conducted at home and abroad, showing significant effects. The main sources of NK cells in clinical studies are: peripheral blood NK cells, umbilical cord blood NK cells and allogeneic universal NK cells.

[0005] According to relevant reports, there are currently three main in vitro NK cell expansion and culture technologies: the first is the magnetic bead sorting method, which sorts NK cells from mononuclear cells to obtain Nk cells with higher purity and expands them in vitro; the second is to use feeder layer cells to induce and activate NK cells; the third is to directly use factor activation methods to induce and expand NK cells from mononuclear cells. Among them, the first magnetic bead sorting method has a higher cost and a relatively low NK cell expansion multiple; the second feeder layer cell method uses tumor cells for feeding, and its safety in clinical applications has not been fully verified and there are certain risks. Therefore, these two methods are currently mostly used in scientific research. The third factor activation method is a method of culturing NK cells using factors to activate NK cells, making NK cells dominant activated bodies to obtain highly expanded and high-purity NK cells, with high safety and high clinical application value.

[0006] There are many factors that can activate NK cells, but many factors activate T cells at the same time, so it is impossible to obtain NK cells with high purity. At the same time, due to the different drugs used by people in the past, the effects of NK cell activation are different, and there will be large differences between batches caused by different blood sources, making it impossible to achieve effective stable culture of NK cells. Therefore, it is necessary to find an in vitro culture method for NK cells with high stability, low cost, no exogenous serum, no feeder cells, simple operation and high expansion multiples. Summary of the invention

[0007] Based on the above problems, the problem to be solved by the present invention is to provide a culture medium for culturing NK cells with high purity, good stability, safety and reliability, and a culture method for culturing NK cells using the culture medium.

[0008] The technical solution of the present invention is as follows: A NK cell culture medium, comprising an expansion culture medium and an induction activation culture medium; wherein: The expansion culture medium includes a serum-free culture medium, wherein the culture medium contains IL-2 with a final concentration of 0.01µg / mL-5mg / mL, IL-12 with a final concentration of 0.01µg / mL-5mg / mL, IL-15 with a final concentration of 0.01µg / mL-5mg / mL, and IL-18 with a final concentration of 0.01µg / mL-5mg / mL; The induced activation culture medium is based on the expansion culture medium, and the expansion culture medium also contains one or more of OK432 with a final concentration of 0.1µg / mL-20mg / mL, CD16-α with a final concentration of 0.1µg / mL-5mg / mL, Pseudomonas aeruginosa injection with a final concentration of 0.1µg / mL-100mg / mL, α-galactosylceramide with a final concentration of 0.1µg / mL-5mg / mL, and CD137L with a final concentration of 0.1µg / mL-5mg / mL.

[0009] In one embodiment, in the NK cell culture medium, the expansion culture medium includes a serum-free culture medium, in which one or more of IL-2 with a final concentration of 1 mg / mL, IL-12 with a final concentration of 0.1 mg / mL, IL-15 with a final concentration of 2 mg / mL, and IL-18 with a final concentration of 1 mg / mL are contained; the induced activation culture medium uses the expansion culture medium as a base liquid, and in the expansion culture medium, one or more of OK432 with a final concentration of 8 mg / mL, CD16-α with a final concentration of 2 mg / mL, Pseudomonas aeruginosa injection with a final concentration of 55 mg / mL, α-galactosylceramide with a final concentration of 3 mg / mL, and CD137L with a final concentration of 1.5 mg / mL are contained.

[0010] In one embodiment, in the NK cell culture medium, the serum-free culture medium is any one of DMEM, IMEM, RPMI-1640, KPM-581, KPM-551 and OPTMIZER TCELL EXPANSFMCTS.

[0011] The present invention also provides a method for culturing NK cells using the above culture medium, comprising the following steps: On day 0, resuspend 1.0-9.0×10 7 The resuspended cell suspension was transferred into a culture bottle, and inactivated plasma was added into the culture bottle. After being blown and mixed evenly, the culture was cultured in an incubator at 37°C and 5v / v% CO2 until the end of the culture on the 15th day. On the third day, the above-mentioned expansion culture medium is added to the culture bottle; On the 5th day, cells and culture medium were transferred into culture bags and cultured at a cell density of 0.5×10 6 / mL, supplemented with the above-mentioned amplification culture medium; On day 7, the cell density was 1.0×10 6 / mL, add the above expansion culture solution into the culture bag; On day 9, the cell density was 1.0×10 6 / mL, add the above expansion culture solution into the culture bag; On day 11, the cell density was 1.5×10 6 / mL, add the above expansion culture solution into the culture bag; On day 13, the cell density was 2.0×10 6 / mL, add the above expansion culture solution into the culture bag; On the 15th day, the culture was stopped, the cells were collected into a centrifuge tube, and centrifuged at 500 g for 10 min. After removing the supernatant, the cell pellet was washed with PBS to obtain high-purity NK cells.

[0012] In one embodiment, in the NK cell culture method, on day 0, 36 mL of the induced activation culture medium is used to resuspend the mononuclear cells, and preferably the number of mononuclear cells is 7.0×10 7 indivual.

[0013] In one embodiment, in the NK cell culture method, on day 0, in the culture bottle, the volume ratio of the induced activation culture medium to the inactivated plasma is 9:1, and the final concentration of the inactivated plasma in the inducing activation culture medium is 10 v / v%.

[0014] In one embodiment, in the NK cell culture method, on the third day, inactivated plasma should be added to the culture bottle when the expansion culture medium is added, the total volume of the expansion culture medium and the inactivated plasma added is 50 mL, and the final concentration of the inactivated plasma in the expansion culture medium is 10 v / v%.

[0015] In one embodiment, in the NK cell culture method, on the 5th day and the 7th day, inactivated plasma should be added each time the expansion culture medium is added, and the final concentration of the inactivated plasma in the expansion culture medium is 10 v / v%.

[0016] In one embodiment, in the NK cell culture method, on day 9, inactivated plasma should be added when adding the expansion culture medium, and the final concentration of the inactivated plasma in the expansion culture medium is 5 v / v%.

[0017] Compared with the prior art, the present invention has the following advantages: 1) The induced activation factor CD16-α in the induced activation culture medium can activate NK cells and promote the rapid expansion and growth of NK cells; at the same time, CD16-α can also combine with the synergistic effect of induced activation factors such as OK432, Pseudomonas aeruginosa injection, and α-galactosylceramide to activate NK cells into a dominant cell population, more deeply activate NK cells to grow rapidly, and further increase the CD3-C56+ ratio (purity ratio) of NK cells; 2) After the induction activation factor CD16-α binds to CD137L, it can further activate the activated NK cells, making the NK cells more active and having a stronger killing effect; 3) The present invention adopts a method of pure factor-induced activation and culture of NK cells, which does not require coating and does not add any feeder cells (i.e., trophoblasts) and animal serum. It can eliminate medical ethics and GVHD risks, is simpler to operate, and has higher stability. It can be applied to NK cell clinical experimental research and large-scale industrial preparation, and is easier to realize clinical trial application and industrial promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The phenotype flow cytometric diagram of NK cells CD3-CD56+ was obtained for Example 1; Figure 2 The phenotype flow cytometric diagram of NK cells CD3-CD56+ was obtained for Example 2; Figure 3 The phenotype flow cytometric diagram of NK cells CD3-CD56+ was obtained for Example 3; Figure 4 The phenotype flow cytometric diagram of NK cells CD3-CD56+ was obtained for Example 4; Figure 5The phenotype flow cytometric diagram of NK cells CD3-CD56+ was obtained for Example 5; Figure 6 The phenotype flow cytometric diagram of NK cells CD3-CD56+ was obtained for Example 6; Figure 7 The phenotype flow cytometric diagram of NK cells CD3-CD56+ was obtained for Example 7; Figure 8 NK cell expansion multiple bar graph of Examples 1 to 7 Fig. 9 It is a bar graph of NK cell killing rate of Examples 1 to 7. DETAILED DESCRIPTION

[0019] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0020] 1. Obtaining Mononuclear Cells In the present invention, NK cells are cultured using mononuclear cells (PBMC) separated from umbilical cord blood.

[0021] The process technology for separating mononuclear cells from umbilical cord blood is existing technology; the specific operation is as follows: 1.1) Rough separation In the present invention, the blood is divided into two centrifuge tubes (marked as No. 1 and No. 2 centrifuge tubes) with a specification of 50 mL, each centrifuge tube is divided into 30 mL, and the blood volume is calculated.

[0022] Use a 3mL Pasteur pipette to draw up the blood sample, drop 2 drops into the EP tube, and use a blood cell analyzer to detect the cell count.

[0023] The blood in the centrifuge tube was centrifuged; during the centrifugation process, the centrifugal force was 650 g, the centrifugal time was 10 min, and the speed was first increased by 8 gears and then decreased by 8 gears.

[0024] Prepare two centrifuge tubes of the same specifications (marked as No. 3 and No. 4) and add 15 mL of lymphocyte separation solution to each centrifuge tube.

[0025] 1.2) Plasma preparation After the two centrifuge tubes containing whole blood are centrifuged, transfer the centrifuge tubes to the safety cabinet surface, and use a 25mL pipette to draw the upper plasma into a new centrifuge tube (marked as No. 5); take 1mL of plasma as a reserve sample, mark it and store it in a -20 degree refrigerator; seal the No. 5 centrifuge tube containing the remaining plasma with sealing film, place it in a 56℃ water bath, and inactivate it for 30 minutes.

[0026] After the inactivation is completed, the No. 5 centrifuge tube containing the plasma is immediately placed in a -20°C refrigerator for 15 minutes. Subsequently, the No. 5 centrifuge tube is taken out for centrifugation. At this time, the centrifugal force is 3000g and the centrifugation time is 10 minutes.

[0027] Finally, pour the plasma supernatant in centrifuge tube No. 5 into another new centrifuge tube (marked as No. 6), seal it, and store it in a refrigerator at 2-8°C for later use.

[0028] 1.3) Preparation of mononuclear cells The remaining lower layer blood cells in the above No. 1 and No. 2 centrifuge tubes were diluted with physiological saline in a ratio of 1:1 and mixed; and the corresponding blood cell dilutions were added to the No. 3 and No. 4 centrifuge tubes containing lymphocyte separation solution, respectively, and the following treatment was performed.

[0029] That is, tilt the No. 3 and No. 4 centrifuge tubes filled with lymph separation fluid at 45 degrees, and use an electric pipette to add a small amount of blood cell suspension to 1 cm above the separation fluid to spread the blood cell suspension; the purpose of tilting the centrifuge tube is to make the suspension contact with the separation fluid.

[0030] Then continue to slowly add the remaining cell suspension along the wall of No. 3 and No. 4 centrifuge tubes. During the operation, do not disrupt the liquid surface, and add up to 45mL per tube. After completion, keep No. 3 and No. 4 centrifuge tubes vertically placed, carefully transfer them to the centrifuge, and centrifuge them at a centrifugal force of 650g, a centrifugal time of 30 minutes, and increase the gear by 1 and decrease the gear by 0 (i.e. the slowest gear).

[0031] After the centrifugation of No. 3 and No. 4 centrifuge tubes is completed, carefully remove the centrifuge tubes, and the cell stratification can be clearly seen. Be careful to handle with care and keep the centrifuge tubes vertically to avoid damaging the cell stratification. Slowly transfer to the safety cabinet, use a 10mL pipette to remove the upper layer of residual plasma and the remaining 2mL, then pipette about 10mL of the second layer of white film cells in the No. 3 and No. 4 centrifuge tubes respectively and add them to the corresponding new centrifuge tubes (marked as No. 7 and No. 8).

[0032] Add physiological saline to the 50mL mark in the No. 7 and No. 8 centrifuge tubes respectively and mix well. Centrifuge No. 7 and No. 8 centrifuge tubes at a centrifugal force of 650g and a centrifugal time of 10min, and increase the speed to 8 and 8 during centrifugation. After the centrifugation, add 5-10mL of physiological saline to the No. 7 and No. 8 centrifuge tubes respectively to resuspend the cells. After the cells in each centrifuge tube are precipitated, they are combined and collected in the No. 7 centrifuge tube, and physiological saline is added to make up to the 50mL mark. After blowing evenly with a 10mL pipette, 2 drops are dropped into the EP tube for counting.

[0033] After counting, 50 million mononuclear cells are counted as a portion from the cell fluid, which are individually loaded into multiple centrifuge tubes and resuspended to 50 mL. These centrifuge tubes are then centrifuged at a centrifugal force of 300g and a centrifugal time of 10 minutes, with 8 up and 8 down gears during centrifugation. After centrifugation, the supernatant is discarded, and the precipitate in each centrifuge tube is the required mononuclear cell (PBMC); collect and set aside.

[0034] 2. Preparation of NK cell culture medium In the present invention, the NK cell culture medium includes an expansion culture medium and an induction activation culture medium, and each is prepared as follows.

[0035] Preparation of expansion culture medium: using serum-free culture medium as basal culture medium, the culture medium contains one or more of IL-2 with a final concentration of 1 mg / mL, IL-12 with a final concentration of 0.1 mg / mL, IL-15 with a final concentration of 2 mg / mL, and IL-18 with a final concentration of 1 mg / mL; Preparation of induced activation culture medium: The prepared expansion culture medium is used as the base culture medium, and the expansion culture medium also contains one or more of OK432 with a final concentration of 0.1µg / mL-20mg / mL, CD16-α with a final concentration of 0.1µg / mL-5mg / mL, Pseudomonas aeruginosa injection with a final concentration of 0.1µg / mL-100mg / mL, α-galactosylceramide with a final concentration of 0.1µg / mL-5mg / mL, and CD137L with a final concentration of 0.1µg / mL-5mg / mL.

[0036] Preferably, the serum-free culture medium of the expansion culture medium contains one or more of IL-2 with a final concentration of 0.1µg / mL-3mg / mL, IL-12 with a final concentration of 0.05µg / mL-3mg / mL, IL-15 with a final concentration of 0.1µg / mL-3mg / mL, and IL-18 with a final concentration of 0.05µg / mL-3mg / mL; and the expansion culture medium of the induced activation culture medium also contains one or more of OK432 with a final concentration of 1µg / mL-10mg / mL, CD16-α with a final concentration of 0.5µg / mL-3mg / mL, Pseudomonas aeruginosa injection with a final concentration of 10µg / mL-50mg / mL, α-galactosylceramide with a final concentration of 0.5µg / mL-3mg / mL, and CD137L with a final concentration of 0.5µg / mL-3mg / mL.

[0037] Preferably, in one embodiment, the serum-free culture medium of the expansion culture medium contains IL-2 with a final concentration of 4 mg / mL, IL-12 with a final concentration of 0.5 mg / mL, IL-15 with a final concentration of 3 mg / mL, and IL-18 with a final concentration of 1 mg / mL; and the expansion culture medium of the induced activation culture medium contains OK432 with a final concentration of 8 mg / mL, CD16-α with a final concentration of 2 mg / mL, Pseudomonas aeruginosa injection with a final concentration of 55 mg / mL, α-galactosylceramide with a final concentration of 3 mg / mL, and CD137L with a final concentration of 1.5 mg / mL.

[0038] Preferably, in one embodiment, the serum-free culture medium of the expansion culture medium contains IL-2 with a final concentration of 3 mg / mL and IL-18 with a final concentration of 0.1 mg / mL; and the expansion culture medium of the induced activation culture medium contains OK432 with a final concentration of 8 mg / mL, CD16-α with a final concentration of 2 mg / mL, Pseudomonas aeruginosa injection with a final concentration of 55 mg / mL, α-galactosylceramide with a final concentration of 3 mg / mL, and CD137L with a final concentration of 1.5 mg / mL.

[0039] Preferably, in one embodiment, the serum-free culture medium of the expansion culture medium contains IL-2 with a final concentration of 3 mg / mL and IL-15 with a final concentration of 5 mg / mL; and the expansion culture medium of the induced activation culture medium contains OK432 with a final concentration of 8 mg / mL, CD16-α with a final concentration of 2 mg / mL, Pseudomonas aeruginosa injection with a final concentration of 55 mg / mL, α-galactosylceramide with a final concentration of 3 mg / mL, and CD137L with a final concentration of 1.5 mg / mL.

[0040] Preferably, in one embodiment, the serum-free culture medium of the expansion culture medium contains IL-15 with a final concentration of 0.1µg / mL-3mg / mL and IL-18 with a final concentration of 0.05µg / mL-3mg / mL; and the expansion culture medium of the induced activation culture medium contains CD16-α with a final concentration of 4mg / mL and CD137L with a final concentration of 0.5mg / mL.

[0041] Preferably, in one embodiment, the serum-free culture medium of the expansion culture medium contains IL-12 with a final concentration of 0.05µg / mL-3mg / mL, IL-15 with a final concentration of 0.1µg / mL-3mg / mL, and IL-18 with a final concentration of 0.05µg / mL-3mg / mL; and the final concentration of CD16-α in the expansion culture medium of the induced activation culture medium is 1.5mg / mL.

[0042] The above-mentioned induction and activation factors induce the induction and activation factor CD16-α in the activation culture medium, which can activate NK cells and promote the rapid expansion and growth of NK cells; at the same time, CD16-α can also combine with induction and activation factors such as OK432, Pseudomonas aeruginosa injection, α-galactosylceramide, etc. to activate NK cells into a dominant cell population, more deeply activate the rapid growth of NK cells, and further improve the purity of NK cells; and after the induction and activation factor CD16-α binds to CD137L, it can further activate the activated NK cells, making the NK cells more active and having a stronger killing effect.

[0043] In the preparation process of the above-mentioned NK cell culture medium, the serum-free culture medium used is one of DMEM, IMEM, RPMI-1640, KPM-581, KPM-551 or OPTMIZER TCELL EXPANSFMCTS; preferably, the serum-free culture medium is OPTMIZER TCELL EXPANSFMCTS.

[0044] In the above-mentioned NK cell culture medium, cytokines IL-2, IL-12, IL-15, IL-18, etc. are basic culture factors commonly used for cell culture. At present, NK culture medium basically contains all the latter parts of these cytokines.

[0045] 3. In vitro expansion and culture of NK cells In the present invention, the NK seed cells used in the in vitro expansion culture of NK cells are derived from the mononuclear cells (PBMC) obtained in step 1.3) of the first item of the present invention; the NK cell culture medium is derived from the expansion culture medium and the induced activation culture medium prepared separately in the second item.

[0046] 3.1) NK cell culture On day 0, resuspend 1.0-9.0×10 7 The resuspended cell suspension was transferred into a T75 culture flask, and the fire-extinguished plasma was added into the culture flask. After being blown and mixed evenly, the culture was cultured in an incubator at 37°C and 5v / v% CO2 for 15 days until the culture was completed on the 15th day. On the third day, the expansion medium was added to the culture flask; On the fifth day, the cells and culture medium in the culture flask were transferred to the culture bag and cultured at a cell density of 0.5×10 6 / mL, add the corresponding volume of amplification culture medium; On day 7, the cell density was 1.0×10 6 / mL, add the corresponding volume of amplification culture medium to the culture bag; On day 9, the cell density was 1.0×106 / mL, add the corresponding volume of amplification culture medium to the culture bag; On day 11, the cell density was 1.5×10 6 / mL, add the corresponding volume of amplification culture medium to the culture bag; On day 13, the cell density was 2.0×10 6 / mL, add the corresponding volume of expansion culture medium to the culture bag, continue to culture until the 15th day, and then stop the culture.

[0047] 3.2) Cell collection and washing On the 15th day, after stopping the cell culture, the cells were collected and transferred to a centrifuge tube, centrifuged at 500g for 10 minutes, and the supernatant was removed and the cell pellet was repeatedly washed 2 to 3 times with PBS to obtain high-purity NK cells.

[0048] In the above NK cell culture process, preferably, on day 0, the volume of the mononuclear cells resuspended in the induction activation culture medium is 36 mL. Of course, the number of mononuclear cells can also be 1.0×10 7 pcs, 2.0×10 7 pcs, 3.0×10 7 pcs, 4.5.0×10 7 pcs, 5.0×10 7 pcs, 6.0×10 7 pcs, 7.0×10 7 8.0×10 7 pcs, 9.0×10 7 In the present invention, the number of mononuclear cells is preferably 7.0×10 7 In the culture bottle, the volume ratio of the induction activation culture medium to the inactivated plasma is 9:1, preferably the volumes of the induction activation culture medium and the inactivated plasma added are 36 mL and 4 mL, respectively, and the final concentration of the inactivated plasma in the induction activation culture medium is 10 v / v%.

[0049] In the above NK cell culture process, preferably, on the third day, when the expansion culture medium is added to the culture bottle, inactivated plasma should also be added, the total volume of the inactivated plasma and the expansion culture medium is 50 mL, and the final concentration of the inactivated plasma in the expansion culture medium is 10 v / v%.

[0050] In the above NK cell culture process, preferably, on the 5th day and the 7th day, inactivated plasma should be added each time the expansion culture medium is added, and the final concentration of the inactivated plasma in the expansion culture medium is 10 v / v%.

[0051] In the above NK cell culture process, preferably, on day 9, when the expansion culture medium is added, inactivated plasma should also be added, and the final concentration of the inactivated plasma in the expansion culture medium should be 5 v / v%.

[0052] The following describes in detail the process of using PBMCs separated from umbilical cord blood as seed cells to expand and culture NK cells through some specific examples.

[0053] In the following examples, the NK cells used were all derived from step 1.3) of item 1 above. In the following examples, in order to facilitate the detection test, the initial number of mononuclear cells was 7.0×10 7 indivual.

[0054] (I) NK cell culture

[0055] Example 1 1.1. Preparation of NK cell culture medium Prepare 1000 mL expansion culture medium: use OPTMIZER TCELL EXPANSFMCTS serum-free medium as the basal medium, which contains IL-2 with a final concentration of 4 mg / mL, IL-12 with a final concentration of 0.5 mg / mL, IL-15 with a final concentration of 3 mg / mL, and IL-18 with a final concentration of 1 mg / mL; Prepare 50 mL of induced activation culture medium: take the prepared amplification culture medium and use it as the base culture medium, add OK432, CD16-α, Pseudomonas aeruginosa injection, α-galactosylceramide and CD137L to the amplification culture medium, and make the amplification culture medium contain OK432 with a final concentration of 8 mg / mL, CD16-α with a final concentration of 2 mg / mL, Pseudomonas aeruginosa injection with a final concentration of 55 mg / mL, α-galactosylceramide with a final concentration of 3 mg / mL and CD137L with a final concentration of 1.5 mg / mL.

[0056] 1.2 NK cell culture On day 0, 36 mL of the induced activation culture medium prepared in step 1.1 of this example was resuspended in 7.0×10 7 The resuspended cell suspension was transferred into a T75 culture flask, and 4 mL of inactivated plasma was added to the culture flask, and the final concentration of the inactivated plasma in the induction activation medium was 10 v / v%, and after being pipetted and mixed evenly, the culture was cultured in an incubator at 37°C and 5 v / v% CO2 until the end of the culture on the 15th day; On the third day, the culture flask was supplemented with the expansion culture medium prepared in step 1.1 of this example and 50 mL of inactivated plasma was added, and the final concentration of the inactivated plasma in the expansion culture medium was 10 v / v; On the fifth day, the cells and culture medium in the culture flask were transferred to the culture bag and cultured at a cell density of 0.5×10 6 / mL, add the expansion culture medium prepared in step 1.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 7, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 1.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 9, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 1.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 11, the cell density was 1.5×10 6 / mL, add the expansion culture medium prepared in step 1.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 13, the cell density was 2.0×10 6 / mL, add the expansion culture medium prepared in step 1.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On the 15th day, after stopping the cell culture, the cells were collected and transferred to a 50 mL centrifuge tube and centrifuged at 500 g for 10 min. After removing the supernatant, the cell pellet was repeatedly washed 2 to 3 times with PBS to obtain high-purity NK cells.

[0057] Example 2

[0058] 2.1. Preparation of NK cell culture medium Prepare 1000 mL expansion culture medium: OPTMIZER TCELL EXPANSFMCTS serum-free medium is used as the basal medium, and the medium contains IL-2 with a final concentration of 0.01 mg / mL, IL-12 with a final concentration of 5 mg / mL, IL-15 with a final concentration of 0.01 mg / mL, and IL-18 with a final concentration of 5 mg / mL; Prepare 50 mL of induced activation culture medium: take the prepared amplification culture medium and use it as the base culture medium, add OK432, CD16-α, Pseudomonas aeruginosa injection, α-galactosylceramide and CD137L to the amplification culture medium, and make the amplification culture medium contain OK432 with a final concentration of 0.1 mg / mL, CD16-α with a final concentration of 5 mg / mL, Pseudomonas aeruginosa injection with a final concentration of 100 mg / mL, α-galactosylceramide with a final concentration of 0.1 mg / mL and CD137L with a final concentration of 5 mg / mL.

[0059] 2.2 NK cell culture On day 0, 36 mL of the induced activation culture medium prepared in step 2.1 of this example was resuspended in 7.0×10 7 The resuspended cell suspension was transferred into a T75 culture flask, and 4 mL of inactivated plasma was added to the culture flask, and the final concentration of the inactivated plasma in the induction activation medium was 10 v / v%, and after being pipetted and mixed evenly, the culture was cultured in an incubator at 37°C and 5 v / v% CO2 until the end of the culture on the 15th day; On the third day, the culture flask was supplemented with the expansion culture medium prepared in step 2.1 of this example and 50 mL of inactivated plasma was added, and the final concentration of the inactivated plasma in the expansion culture medium was 10 v / v; On the fifth day, the cells and culture medium in the culture flask were transferred to the culture bag and cultured at a cell density of 0.5×10 6 / mL, add the expansion culture medium prepared in step 2.1 of this example and inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 7, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 2.1 of this example and inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 9, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 2.1 of this example and inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 11, the cell density was 1.5×10 6 / mL, add the expansion culture medium prepared in step 2.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 13, the cell density was 2.0×10 6 / mL, add the expansion culture medium prepared in step 2.1 of this example and inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On the 15th day, after stopping the cell culture, the cells were collected and transferred to a 50 mL centrifuge tube and centrifuged at 500 g for 10 min. After removing the supernatant, the cell pellet was repeatedly washed 2 to 3 times with PBS to obtain high-purity NK cells.

[0060] Example 3

[0061] 3.1. Preparation of NK cell culture medium Prepare 1000 mL expansion culture medium: use OPTMIZER TCELL EXPANSFMCTS serum-free medium as the basal medium, which contains IL-2 with a final concentration of 0.05 mg / mL, IL-12 with a final concentration of 0.01 mg / mL, IL-15 with a final concentration of 5 mg / mL, and IL-18 with a final concentration of 0.01 mg / mL; Prepare 50 mL of induced activation culture medium: take the prepared amplification culture medium and use it as the base culture medium, add OK432, CD16-α, Pseudomonas aeruginosa injection, α-galactosylceramide and CD137L to the amplification culture medium, and make the amplification culture medium contain OK432 with a final concentration of 20 mg / mL, CD16-α with a final concentration of 0.1 mg / mL, Pseudomonas aeruginosa injection with a final concentration of 0.1 mg / mL, α-galactosylceramide with a final concentration of 5 mg / mL and CD137L with a final concentration of 0.1 mg / mL.

[0062] 3.2 NK cell culture On day 0, 36 mL of the induced activation culture medium prepared in step 3.1 of this example was resuspended in 7.0×10 7 The resuspended cell suspension was transferred into a T75 culture flask, and 4 mL of inactivated plasma was added to the culture flask, and the final concentration of the inactivated plasma in the induction activation medium was 10 v / v%, and after being pipetted and mixed evenly, the culture was cultured in an incubator at 37°C and 5 v / v% CO2 until the end of the culture on the 15th day; On the third day, the culture flask was supplemented with the expansion culture medium prepared in step 3.1 of this example and 50 mL of inactivated plasma was added, and the final concentration of the inactivated plasma in the expansion culture medium was 10 v / v; On the fifth day, the cells and culture medium in the culture flask were transferred to the culture bag and cultured at a cell density of 0.5×10 6 / mL, add the expansion culture medium prepared in step 3.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 7, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 3.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 9, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 3.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 11, the cell density was 1.5×10 6 / mL, add the expansion culture medium prepared in step 3.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 13, the cell density was 2.0×10 6 / mL, add the expansion culture medium prepared in step 3.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On the 15th day, after stopping the cell culture, the cells were collected and transferred to a 50 mL centrifuge tube and centrifuged at 500 g for 10 min. After removing the supernatant, the cell pellet was repeatedly washed 2 to 3 times with PBS to obtain high-purity NK cells.

[0063] Example 4 (An inducible activation factor) 4.1. Preparation of NK cell culture medium Prepare 1000 mL of expansion culture medium: use IMEM serum-free medium as the basal medium, and include IL-2 at a final concentration of 3 mg / mL in the medium; Prepare 50 mL of induced activation culture medium: take the prepared amplification culture medium and use it as the base culture medium, add CD16-α to the amplification culture medium, and make the amplification culture medium contain CD16-α at a final concentration of 1 mg / mL.

[0064] 4.2 NK cell culture On day 0, 36 mL of the induced activation culture medium prepared in step 4.1 of this example was resuspended in 7.0×10 7 The resuspended cell suspension was transferred into a T75 culture flask, and 4 mL of inactivated plasma was added to the culture flask, and the final concentration of the inactivated plasma in the induction activation medium was 10 v / v%, and after being pipetted and mixed evenly, the culture was cultured in an incubator at 37°C and 5 v / v% CO2 until the end of the culture on the 15th day; On the third day, the culture flask was supplemented with the expansion medium prepared in step 4.1 of this example and 50 mL of inactivated plasma was added, and the final concentration of the inactivated plasma in the expansion medium was 10 v / v; On the fifth day, the cells and culture medium in the culture flask were transferred to the culture bag and cultured at a cell density of 0.5×10 6 / mL, add the expansion culture medium prepared in step 4.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 7, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 4.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 9, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 4.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 11, the cell density was 1.5×10 6 / mL, add the expansion culture medium prepared in step 4.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 13, the cell density was 2.0×10 6 / mL, add the expansion culture medium prepared in step 4.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On the 15th day, after stopping the cell culture, the cells were collected and transferred to a 50 mL centrifuge tube and centrifuged at 500 g for 10 min. After removing the supernatant, the cell pellet was repeatedly washed 2 to 3 times with PBS to obtain high-purity NK cells.

[0065] Example 5 (Two inducing activation factors) 5.1. Preparation of NK cell culture medium Prepare 1000 mL of expansion culture medium: use KPM-581 serum-free medium as the basal medium, and the medium contains IL-2 with a final concentration of 3 mg / mL, IL-15 with a final concentration of 3 mg / mL, and IL-18 with a final concentration of 0.1 mg / mL; Prepare 50 mL of induced activation culture medium: take the prepared expansion culture medium and use it as the base culture medium, add CD16-α and CD137L to the expansion culture medium, and make the expansion culture medium contain a final concentration of 3 mg / mL CD16-α and 2 mg / mL CD137L.

[0066] 5.2 NK cell culture On day 0, 36 mL of the induced activation culture medium prepared in step 5.1 of this example was resuspended in 7.0×10 7 The resuspended cell suspension was transferred into a T75 culture flask, and 4 mL of inactivated plasma was added to the culture flask, and the final concentration of the inactivated plasma in the induction activation medium was 10 v / v%, and after being pipetted and mixed evenly, the culture was cultured in an incubator at 37°C and 5 v / v% CO2 until the end of the culture on the 15th day; On the third day, the culture flask was supplemented with the expansion medium prepared in step 5.1 of this example and 50 mL of inactivated plasma was added, and the final concentration of the inactivated plasma in the expansion medium was 10 v / v; On the fifth day, the cells and culture medium in the culture flask were transferred to the culture bag and cultured at a cell density of 0.5×10 6 / mL, add the expansion culture medium prepared in step 5.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 7, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 5.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 9, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 5.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 11, the cell density was 1.5×10 6 / mL, add the expansion culture medium prepared in step 5.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 13, the cell density was 2.0×10 6 / mL, add the expansion culture medium prepared in step 5.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On the 15th day, after stopping the cell culture, the cells were collected and transferred to a 50 mL centrifuge tube and centrifuged at 500 g for 10 min. After removing the supernatant, the cell pellet was repeatedly washed 2 to 3 times with PBS to obtain high-purity NK cells.

[0067] Example 6 (Multiple induction activation factors) 6.1. Preparation of NK cell culture medium Prepare 1000 mL expansion culture medium: use OPTMIZER TCELL EXPANSFMCTS serum-free medium as the basal medium, with a final concentration of 0.5 mg / mL of IL-12, a final concentration of 3 mg / mL of IL-15, and a final concentration of 1 mg / mL of IL-18 in the medium; Prepare 50 mL of induced activation culture medium: take the prepared amplification culture medium and use it as the base culture medium, add OK432, CD16-α and Pseudomonas aeruginosa injection solution to the amplification culture medium, and make the amplification culture medium contain OK432 with a final concentration of 12 mg / mL, CD16-α with a final concentration of 1 mg / mL and Pseudomonas aeruginosa injection solution with a final concentration of 20 mg / mL; 6.2 NK cell culture On day 0, 36 mL of the induced activation culture medium prepared in step 6.1 of this example was resuspended in 7.0×10 7 The resuspended cell suspension was transferred into a T75 culture flask, and 4 mL of inactivated plasma was added to the culture flask, and the final concentration of the inactivated plasma in the induction activation medium was 10 v / v%, and after being pipetted and mixed evenly, the culture was cultured in an incubator at 37°C and 5 v / v% CO2 until the end of the 15-day culture; On the third day, the culture flask was supplemented with the expansion medium prepared in step 6.1 of this example and 50 mL of inactivated plasma was added, and the final concentration of the inactivated plasma in the expansion medium was 10 v / v; On the fifth day, the cells and culture medium in the culture flask were transferred to the culture bag and cultured at a cell density of 0.5×10 6 / mL, add the expansion culture medium prepared in step 6.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 7, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 6.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 9, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 6.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 11, the cell density was 1.5×10 6 / mL, add the expansion culture medium prepared in step 6.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On day 13, the cell density was 2.0×106 / mL, add the expansion culture medium prepared in step 6.1 of this example and add inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v; On the 15th day, after stopping the cell culture, the cells were collected and transferred to a 50 mL centrifuge tube and centrifuged at 500 g for 10 min. After removing the supernatant, the cell pellet was repeatedly washed 2 to 3 times with PBS to obtain high-purity NK cells.

[0068] Example 7 (Comparative Example 1) 7.1. Preparation of NK cell culture medium Prepare 1000 mL of NK cell expansion culture medium: use KPM-581 serum-free medium as the basal medium, and the medium contains IL-2 with a final concentration of 4 mg / mL, IL-12 with a final concentration of 0.5 mg / mL, IL-15 with a final concentration of 3 mg / mL, and IL-18 with a final concentration of 1 mg / mL; Prepare 50 mL of NK cell induction activation culture medium: KPM-581 serum-free medium is used as the basic medium, and the medium contains fludarabine with a final concentration of 0.1~10ug / mL, IL-2 with a final concentration of 500IU / mL, and IL-15 with a final concentration of 100μg / mL.

[0069] 1.2 NK cell culture On day 0, 36 mL of the induced activation culture medium prepared in step 7.1 of this example was resuspended in 7.0×10 7 The resuspended cell suspension was transferred into a T75 culture flask, and 4 mL of inactivated plasma was added to the culture flask, and the final concentration of the inactivated plasma in the induction activation medium was 10 v / v%, and after being pipetted and mixed evenly, the culture was cultured in an incubator at 37°C and 5 v / v% CO2 until the end of the culture on the 15th day; On the third day, the culture flask was supplemented with the expansion medium prepared in step 7.1 of this example and 50 mL of inactivated plasma was added, and the final concentration of the inactivated plasma in the expansion medium was 10 v / v; On the fifth day, the cells and culture medium in the culture flask were transferred to the culture bag and cultured at a cell density of 0.5×10 6 / mL, add the expansion culture medium prepared in step 7.1 of this example and inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 7, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 7.1 of this example and inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 9, the cell density was 1.0×10 6 / mL, add the expansion culture medium prepared in step 7.1 of this example and inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 11, the cell density was 1.5×10 6 / mL, add the expansion culture medium prepared in step 7.1 of this example and inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On day 13, the cell density was 2.0×10 6 / mL, add the expansion culture medium prepared in step 7.1 of this example and inactivated plasma to the culture bag, and make the final concentration of inactivated plasma in the expansion culture medium be 10v / v%; On the 15th day, after stopping the cell culture, the cells were collected and transferred to a 50 mL centrifuge tube and centrifuged at 500 g for 10 min. After removing the supernatant, the cell pellet was repeatedly washed 2 to 3 times with PBS to obtain high-purity NK cells.

[0070] (II) NK cell test 1. NK cell CD3-CD56+ detection Figures 1 to 7 The CD3-CD56+ phenotype flow cytograms corresponding to the NK cell culture days 0 and 15 of Examples 1 to 7, i.e., the NK cell purity ratio flow cytograms; wherein P1 represents the flow cytogram detected on day 0; and P2 represents the flow cytogram detected on day 15.

[0071] On the 15th day, the NK cells cultured in Examples 1 to 7 were respectively taken to prepare cell suspensions and the final cell concentration was adjusted to 1×10 6 / mL, added labeled CD3-CD56+ monoclonal antibody, incubated at room temperature in the dark for 15 minutes, washed away excess antibody, and tested the purity ratio of NK cells (CD3-C56+) on flow cytometer. The test results are shown in Table 1. Figures 1 to 7 Flow cytometry plots of NK cell phenotypes are shown.

[0072] Since Examples 1 to 7 used PBMC seed cells for NK cell culture, and the initial content of NK cells in the PBMC seed cells also varied to a certain extent, the CD3-CD56+ ratio of NK cells (NK cell purity ratio) in the PBMC seed cells on day 0 was detected by flow cytometry as shown in Table 1.

[0073] Table 1 NK cell (CD3-C56+) purity statistics

[0074] From Table 1, Figures 1 to 7 It can be seen that when NK cells were cultured to the 15th day: 1) in Examples 1 to 3, the proportion of cultured NK cells (CD3-C56+) was 93%~99%; 2) in Examples 4, 5 and 6, the proportion of cultured NK cells (CD3-C56+) was 34.51%, 54.79% and 61.80%; 3) in Example 7, the proportion of cultured NK cells (CD3-C56+) was 31.1%.

[0075] Therefore, the ratio of NK cells (CD3-C56+) cultured in Examples 1 to 3 is 1 to 3 times that in Examples 4, 5, 6, and 7. This is because in the NK cell culture process of Examples 1 to 3, the induction activation factor CD16-α in the NK cell culture fluid components can activate NK cells and promote the rapid proliferation and growth of NK cells; at the same time, CD16-α can also combine with the mutual synergistic effect of induction activation factors such as OK432, Pseudomonas aeruginosa injection, and α-galactosylceramide to further induce and activate NK cells into a dominant cell population, and further promote the rapid proliferation and growth of NK cells, resulting in a significant increase in the CD3-C56+ ratio (purity ratio) of NK cells.

[0076] 2. NK cell growth count statistics During the NK cell culture process of Examples 1 to 7, samples were taken on day 0, day 3, day 5, day 7, day 9, day 11, day 13 and day 15, and the total number of cells was counted using a cell counter. The statistical results are shown in Table 2.

[0077] Table 2 Total cell count

[0078] When PBMC seed cells are cultured to expand NK cells, the following formulas ① and ② need to be used to calculate the number of NK cells and the expansion multiple of NK cells: NK cell number = total cell number * NK cell purity ratio; ① NK cell expansion multiple = number of NK cells on day n / number of NK cells on day 0; ② In the above-mentioned Examples 1 to 7, the purity ratios of NK cells in PBMC detected by flow cytometry on day 0 and day 15 are respectively shown in Table 1. In the NK cell culture, the number of NK cells on day 0 and day 15 can be calculated according to the purity ratio of NK cells (CD3-C56+) in Table 1 and the total number of cells in Table 2, and using formulas ① and ②, and the calculation results are shown in Table 3; Figure 8 The NK cell expansion multiple curve graph was obtained by using the NK cell expansion multiple data in Table 3.

[0079] Table 3 Statistical table of NK cell culture and expansion

[0080] From Table 3 and Figure 8 It can be seen that during the NK cell culture process of PBMC, the expansion multiples of NK cells in Examples 1 to 3 (i.e., 5061 to 6327 times) were about 2 to 5 times (1248 to 2291 times) of the expansion multiples of NK cells in Examples 4 to 7 during the 15th day of culture, indicating that the NK cells in the PBMC of Examples 1 to 3 grew faster. This is because: the induced activation factor CD16-α of NK cells can activate NK cells and promote the rapid expansion and growth of NK cells; at the same time, CD16-α can also activate NK cells into a dominant cell population in combination with the mutual synergistic effect of inducing activation factors such as OK432, Pseudomonas aeruginosa injection, and α-galactosylceramide, and more deeply activate the rapid growth of NK cells, and finally the number of NK cells obtained in Examples 1 to 3 is greater.

[0081] 3. NK cell killing test On the 15th day of culturing the NK cells in Examples 1 to 7, samples were collected into three groups of centrifuge tubes, each group of 7 centrifuge tubes, and the three groups of centrifuge tubes were numbered 1#, 2#, and 3#, respectively; wherein, the 7 centrifuge tubes in Group 1# corresponded to the NK cells in Examples 1 to 7, respectively; the 7 centrifuge tubes in Group 2# corresponded to the NK cells in Examples 1 to 7, respectively; and the 7 centrifuge tubes in Group 3# corresponded to the NK cells in Examples 1 to 7, respectively. The concentrations of the NK cells in the three groups of centrifuge tubes were adjusted to approximately 1.0×10 6 / mL, 2.0×10 6 / mL, 4.0×10 6 / mL of cell suspension was used as the three groups of effector cells (E).

[0082] K562 cells were used as target cells (T), and effector NK cells and target K562 cells were plated at effector-target ratios of 1:1, 5:1, 10:1, and 20:1, with 3 replicates per group. Each well of NK cells and K562 target cells was 500 μl, and 500 μl of amplification culture medium was added to each well.

[0083] Effector cell NK cell natural release group: 1.0×10 6 / mL, 2.0×10 6 / mL, 4.0×10 6 / mL NK cells, 500 μl per well, 3 replicates per group, and 500 μl of expansion culture medium was added to each well.

[0084] K562 target cell maximum release group: 1.0×10 5 / mL of target cells, 500 μl per well, 3 replicates per group, 500 μl of amplification culture medium per well, and culture in a 37°C, 5% CO2 incubator for 40 minutes.

[0085] K562 target cell natural release group: 1.0×10 5 / mL of target cells, 500 μl per well, 3 replicates per group, and 500 μl of expansion culture medium per well.

[0086] Culture medium blank control test group: 500 μl per well, 3 replicate wells per group, 500 μl amplification culture medium was added to each well.

[0087] After plating, centrifuge the culture plate at 200g for 6 minutes and place it in a 37°C, 5% CO2 incubator for 4 hours. 60 minutes before the end of the culture, remove the culture plate, add thawing solution to the target cell maximum release group, centrifuge at 200g for 6 minutes, continue to culture for 60 minutes, remove it, and then gently mix the cells, centrifuge at 300g for 5 minutes, collect the supernatant, and use LDH (lactate dehydrogenase) to measure the absorbance value at a wavelength of 492nm. The killing rate is calculated according to the following formula: Kill rate = {(ABC) / (DC)}×100%; in: A- represents the corrected value of absorbance of the test group; B-indicates the effector cell natural release group; C-indicates the target cell natural release group; D-indicates the target cell maximum release group; The results of the kill rate calculation are shown in Table 4 and Fig. 9 , Fig. 9 This is a killing curve of NK cells against tumor cells K562; Fig. 9 It is drawn according to the specific data in Table 4; wherein, in the corresponding effect-target ratio, the bar graph is arranged from left to right as Example 1 to Example 7.

[0088] Table 4 NK cell killing rate statistics

[0089] From Table 4 and Fig. 9It can be seen that under the same effect-target ratio (e.g., 5:1), the NK cell killing effect of Examples 1 to 3 is significantly higher than that of Examples 4 to 7. This is because in Examples 1 to 3, the induced activation factor CD16-α activates NK cells and promotes the rapid proliferation and growth of NK cells; at the same time, CD16-α can also combine with the synergistic effect of induced activation factors such as OK432, Pseudomonas aeruginosa injection, and α-galactosylceramide to activate NK cells into a dominant cell population, more deeply activate NK cells to grow rapidly, and further increase the CD3-C56+ ratio (purity ratio) of NK cells, and finally obtain more NK cells, and the killing effect is more obvious; in addition, after the induced activation factor CD16-α binds to CD137L, it can further activate the activated NK cells, making the NK cells more active and having a stronger killing effect.

[0090] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the attached claims.

Claims

1. A NK cell culture medium, characterized in that The NK cell culture fluid comprises an expansion culture fluid and an induction activation culture fluid; wherein: The expansion culture medium includes a serum-free culture medium, wherein the culture medium contains one or more of IL-2 with a final concentration of 0.01µg / mL-5mg / mL, IL-12 with a final concentration of 0.01µg / mL-5mg / mL, IL-15 with a final concentration of 0.01µg / mL-5mg / mL, and IL-18 with a final concentration of 0.01µg / mL-5mg / mL; The induced activation culture medium is based on the expansion culture medium, and the expansion culture medium also contains one or more of OK432 with a final concentration of 0.1µg / mL-20mg / mL, CD16-α with a final concentration of 0.1µg / mL-5mg / mL, Pseudomonas aeruginosa injection with a final concentration of 0.1µg / mL-100mg / mL, α-galactosylceramide with a final concentration of 0.1µg / mL-5mg / mL, and CD137L with a final concentration of 0.1µg / mL-5mg / mL.

2. The NK cell culture medium according to claim 1, characterized in that The expansion culture medium includes a serum-free culture medium, which contains one or more of IL-2 with a final concentration of 1 mg / mL, IL-12 with a final concentration of 0.1 mg / mL, IL-15 with a final concentration of 2 mg / mL, and IL-18 with a final concentration of 1 mg / mL; the induced activation culture medium uses the expansion culture medium as a base liquid, and further contains one or more of OK432 with a final concentration of 8 mg / mL, CD16-α with a final concentration of 2 mg / mL, Pseudomonas aeruginosa injection with a final concentration of 55 mg / mL, α-galactosylceramide with a final concentration of 3 mg / mL, and CD137L with a final concentration of 1.5 mg / mL in the expansion culture medium.

3. The NK cell culture medium according to claim 1 or 2, characterized in that The serum-free culture medium is any one of DMEM, IMEM, RPMI-1640, KPM-581, KPM-551 and OPTMIZER TCELL EXPANSFMCTS.

4. A method for culturing NK cells, characterized in that: The steps include: On day 0, resuspend 1.0-9.0×10 7 The resuspended cell suspension was transferred into a culture bottle, and inactivated plasma was added into the culture bottle. After being blown and mixed evenly, the culture was cultured in an incubator at 37°C and 5v / v% CO2 until the end of the culture on the 15th day. On the third day, the expansion culture medium as claimed in claim 1 is added to the culture bottle; On the 5th day, cells and culture medium were transferred into culture bags and cultured at a cell density of 0.5×10 6 / mL, supplemented with the amplification culture medium as described in claim 1; On day 7, the cell density was 1.0×10 6 / mL, adding the expansion culture solution as claimed in claim 1 to the culture bag; On day 9, the cell density was 1.0×10 6 / mL, adding the expansion culture solution as claimed in claim 1 to the culture bag; On day 11, the cell density was 1.5×10 6 / mL, adding the expansion culture solution as claimed in claim 1 to the culture bag; On day 13, the cell density was 2.0×10 6 / mL, adding the expansion culture solution as claimed in claim 1 to the culture bag; On the 15th day, the culture was stopped, the cells were collected into a centrifuge tube, and centrifuged at 500 g for 10 min. After removing the supernatant, the cell pellet was washed with PBS to obtain high-purity NK cells.

5. The culture method according to claim 4, characterized in that On day 0, the mononuclear cells were resuspended in 36 mL of the induction and activation culture medium.

6. The culture method according to claim 4 or 5, characterized in that: On day 0, the number of mononuclear cells was 7.0 × 10 7 indivual.

7. The culture method according to claim 4, characterized in that: On day 0, in the culture bottle, the volume ratio of the induction activation culture medium to the inactivated plasma was 9:1, and the final concentration of the inactivated plasma in the induction activation culture medium was 10 v / v%.

8. The culture method according to claim 4, characterized in that: On the third day, the inactivated plasma should also be added to the culture bottle when the expansion culture medium is added. The total volume of the expansion culture medium and the inactivated plasma added is 50 mL, and the final concentration of the inactivated plasma in the expansion culture medium is 10 v / v%.

9. The culture method according to claim 4, characterized in that: On the 5th and 7th days, inactivated plasma should be added each time the expansion medium is added, and the final concentration of the inactivated plasma in the expansion medium is 10 v / v%.

10. The culture method according to claim 4, characterized in that: On day 9, inactivated plasma should be added when adding the expansion culture medium, and the final concentration of the inactivated plasma in the expansion culture medium is 5 v / v%.

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