A method for culturing high-purity NK cells

Abstract

The present invention belongs to the field of cell culture technology, and specifically relates to a method for culturing high-purity NK cells, which includes: separating mononuclear cells from blood by density gradient centrifugation; using culture bottles for coating, using lymphocyte serum-free medium for mononuclear cells, adding GMP-grade recombinant proteins or humanized antibodies and blood sample serum for 20 days; after the cell culture is completed, centrifugation is performed to collect cells, NK cells are washed 3 times with saline, and then the cells are resuspended with saline and fixed to 200 ml after adding 5% human serum albumin. The present invention establishes a high-purity cell culture technology that meets GMP standards, which is not only applicable to peripheral blood but also to the culture of umbilical cord blood NK. Only 50 ml of peripheral blood and umbilical cord blood samples need to be collected, and all operations are completed under GMP conditions. The number of cells cultured by the 8L system for about 20 days can also reach the clinical treatment application standard.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and particularly relates to a method for culturing high-purity NK cells. Background Art

[0002] More and more research data show that natural killer cells (NK cells) play an important role in controlling viral infections, cancer and autoimmune diseases. In recent years, NK cells have gradually become the emerging main force in tumor immunotherapy with their unique advantages, and have demonstrated impressive strength in the treatment of malignant tumors.

[0003] NK cells belong to the large granular lymphocytes of the natural lymphoid cell family. They are derived from bone marrow lymphoid stem cells. Their differentiation and development depend on the bone marrow and thymus microenvironment. They are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs and lymph nodes, accounting for 10% to 15% of the total number of circulating lymphocytes. NK cells are an important component of natural immunity and the first line of defense against viral infection and cell mutation. They can kill cancer cells, allogeneic cells and cells infected by foreign pathogens that threaten the host without prior sensitization. They can mediate powerful anti-tumor and anti-viral responses, directly killing tumors or infected cells, and indirectly enhancing antibody and T cell-mediated responses. At present, they are considered to be key effector cells for cancer immune monitoring, transplant rejection and early viral immunity. They have shown good therapeutic effects in the treatment of acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, neuroblastoma, and solid tumors such as gastric cancer, liver cancer, lung cancer, and intestinal cancer.

[0004] A major feature of NK cells is that they can kill bacteria, viruses or cancerous cells without antigen sensitization. This allows them to respond quickly in the immune system. While other immune cells are still waiting for antigen-presenting cells to activate, NK cells can already begin to play a role and quickly go into battle to attack pathogens and abnormal cells. NK cells mainly produce cytotoxic effects by releasing lytic granules perforin and granzymes, secreting cytokines such as IFN-γ to mediate immune regulation and clearance, upregulating death-related ligands such as FasL and TRAIL to induce target cell apoptosis, and killing target cells through antibody-dependent cell-mediated cytotoxicity (ADCC) effects.

[0005] NK cells have strong anti-tumor ability and are relatively safe. They have broad application prospects in the field of tumor biological immunotherapy. At present, research on NK is mainly focused on how to better use NK cells to treat tumors, such as developing more effective tumor treatment strategies by enhancing the activity of NK cells and increasing their enrichment in tumor sites. The number and cytotoxic function of NK cells are reduced in many diseases, so exogenously introduced NK cells have the potential to be used to treat these diseases.

[0006] In addition to being isolated from autologous or allogeneic peripheral blood, NK cells can also be derived from umbilical cord blood, stem cell differentiation, etc. Many researchers have also used NK-92 for further modification. Preclinical studies have achieved good results, but the cells need to be irradiated before infusion, and the availability of donor cell numbers is still an obstacle. In addition, the most commonly used method for NK cell purification is magnetic bead sorting, which removes other cells except the target cells from the mixture cells by means of magnetic bead binding and other means to achieve the purpose of purification. However, the degree of purification of this method is limited, the separation effect is greatly affected by antibodies, and magnetic beads may be introduced during the sorting process. There is also a feeder cell culture method that uses tumor cells as the culture matrix, but there is a problem of residual tumor cells, which has certain risks. The number and purity of NK cells cultured by most pure cytokine methods are very different. Using the same induction culture method, the induction culture effect of mononuclear cells from different individuals is also uneven, and there is no absolutely stable culture program. Summary of the invention

[0007] The purpose of the present invention is to provide a method for culturing high-purity NK cells. The method can stably cultivate tens of billions of high-purity NK cells that meet GMP standards and are suitable for clinical treatment applications. Only 50 ml of peripheral blood and umbilical cord blood samples need to be collected. The method is suitable for the cultivation of not only peripheral blood but also umbilical cord blood NK cells. It has the advantages of high purity, obvious amplification effect, high cell viability, high tumor cell killing rate, and high safety. It can be used for auxiliary treatment of antiviral, anti-tumor and other aspects.

[0008] The objective of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for culturing high-purity NK cells, comprising the following steps:

[0010] (1) Mononuclear cells were isolated from blood using density gradient centrifugation;

[0011] (2) Using culture bottles for coating, monocytes are cultured in lymphocyte serum-free medium, supplemented with GMP-grade recombinant proteins or humanized antibodies and blood serum for 20 days; during the culture process, cells are counted every 3 days, and corresponding GMP-grade recombinant proteins or humanized antibodies, autologous serum and lymphocyte serum-free medium are added, and microbial, mycoplasma and endotoxin safety tests are performed after the last rehydration;

[0012] (3) After the cell culture was completed, the cells were collected by centrifugation, and the NK cells were washed three times with saline. The cells were then resuspended in saline and fixed to 200 ml with 5% human serum albumin.

[0013] Furthermore, in step (1), the blood is umbilical cord blood or peripheral blood.

[0014] Furthermore, in step (1), the specific steps of separation are:

[0015] (1.1) Collect umbilical cord blood or peripheral blood and store it in a collection bag for later use;

[0016] (1.2) After the collection bag in step (1.1) is disinfected, the umbilical cord blood or peripheral blood is placed in a centrifuge tube, centrifuged and the supernatant plasma is collected, inactivated in a water bath, allowed to stand, and then centrifuged for later use;

[0017] (1.3) Add lymph separation fluid to a high-efficiency centrifuge tube, add PBS to the centrifuge tube containing cells in step (1.2), resuspend the tube, and then add it to the high-efficiency centrifuge tube for centrifugation. Then, remove the CBMC layer from the buffy coat layer and transfer it to a new centrifuge tube. Add PBS to wash and centrifuge to obtain the cells to be used.

[0018] Furthermore, in step (1.2), the centrifugation parameters for centrifugation and collection of supernatant plasma are 800g, 15min; the water bath inactivation and standing followed by centrifugation are 56°C water bath inactivation for 30min, followed by standing at 4°C for 15min, and then centrifugation at 1200g, 20min.

[0019] Furthermore, in step (1.3), the centrifugation parameters when adding to the high-efficiency centrifuge tube are 800g, 25min; and the centrifugation parameters when adding PBS for washing and centrifugation are 500g, 10min.

[0020] Furthermore, in step (2), the specific steps of culturing are:

[0021] (2.1) Adding D-PBS coating solution containing CD16 and Retronectin to the culture flask containing the monocytes, incubating in a 37°C, 5% CO2 incubator for 2 hours, discarding the coating solution, washing the cells with D-PBS, and then inoculating the cells in a T175 culture flask, adding lymphocyte serum-free culture medium containing IL-2, IL-12, IL-15, IL-18, CD16, HER2 monoclonal antibodies, and 10% inactivated plasma to make the cell concentration 1-3*10^6 / ml, and continuing to culture in a 37°C, 5% CO2 incubator for 4 days;

[0022] (2.2) On the 4th day after the culture in step (2.1), a serum-free medium containing IL-2, IL-12, IL-15, IL-18, CD16, HER2 monoclonal antibody and 10% plasma was added to the cells to continue the culture, so that the cell concentration was within the range of 1-3*10^6 / ml, and the cells were placed in an incubator and cultured for 2 days;

[0023] (2.3) On the sixth day, based on the culture in step (2.2), a serum-free medium containing IL-2, IL-15 and 10% plasma was added to the cells to continue the culture, so that the cell concentration was within the range of 1-3*10^6 / ml, and the cells were cultured in a 37°C, 5% CO2 incubator for another 2 days;

[0024] (2.4) On the 8th day after the culture in step (2.3), the NK cells are transferred from the culture flask to a cell culture bag for large-scale expansion, and the bag is replenished or expanded every 2-3 days according to the cell growth. Each time the fluid is replenished, a lymphocyte serum-free culture medium containing IL-2 and IL-15 is added to keep the cell concentration in the range of 1-3*10^ / ml.

[0025] Furthermore, in step (3), the centrifugation parameters are 1500 rpm, 5 min.

[0026] Furthermore, the method also includes the steps of performing AOPI staining counting, bacteria, fungi, mycoplasma and endotoxin and flow cytometry phenotyping analysis on the cell suspension of the NK cells.

[0027] Furthermore, the final concentrations of CD16 and Retronectin in the culture system are 1-10 μg / ml and 10-20 μg / ml respectively;

[0028] The final concentrations of the IL-2, IL-12, IL-15, IL-18, CD16 and HER2 monoclonal antibodies in the culture system are 500-1000U / ml, 10-50ng / ml, 20-100ng / ml, 100-200ng / ml, 50-100ng / ml and 1-5μg / ml, respectively.

[0029] Furthermore, the final concentrations of CD16 and Retronectin in the culture system were 2.5 μg / ml and 20 μg / ml, respectively;

[0030] The final concentrations of the IL-2, IL-12, IL-15, IL-18, CD16 and HER2 monoclonal antibodies in the culture system are 1000U / ml, 10ng / ml, 80ng / ml, 100ng / ml, 100ng / ml and 5μg / ml, respectively.

[0031] Beneficial effects:

[0032] In the present invention, serum-free culture medium is used throughout the NK cell culture process, antibiotics are not added, and animal-derived components such as fetal bovine serum are not added. Inactivated serum from blood samples is used as a culture medium additive, which is safer.

[0033] The present invention has established a high-purity cell culture technology that meets the GMP standard, which is not only applicable to the culture of peripheral blood but also to the culture of umbilical cord blood NK cells, and all operations are completed under GMP conditions. The number of cells cultured in the 8L system in about 20 days can also meet the clinical treatment application standard;

[0034] The NK cells cultured by the present invention have high purity, high viability and good tumor killing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 The NK cell growth curve diagram in Example 1-2 of the present invention, wherein the umbilical cord blood NK is the NK cell growth curve in Example 1, and the peripheral blood NK is the NK cell growth curve in Example 2;

[0037] Figure 2 The NK cell activity graph in Example 1-2 of the present invention, wherein the cord blood NK is the NK cell activity curve in Example 1, and the peripheral blood NK is the NK cell activity curve in Example 2;

[0038] Figure 3The figure is a diagram of the killing effect of NK cells on tumor cells prepared by the present invention, wherein A is the killing effect of NK cells with different effector targets on human breast cancer cells MCF-7, B is the killing effect of NK cells with different effector targets on human liver cancer cells HepG2, C is the killing effect of NK cells with different effector targets on human colorectal adenocarcinoma cells HCT1 16, and D is the killing effect of NK cells with different effector targets on human acute T lymphocytic leukemia cells Jurkat;

[0039] Figure 4 These are flow cytometry phenotype detection diagrams of NK cells after 20 days of culture in Example 1-2 of the present invention, A is the flow cytometry detection of NK cell phenotype in cord blood, and B is the flow cytometry detection of NK cell phenotype in peripheral blood. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art generally understood in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Some commonly used terms in the present invention are described as follows:

[0046] CD16: Recombinant human CD16 monoclonal antibody is an IgG Fc fragment receptor, widely expressed on the surface of NK cells, neutrophils, mononuclear cells, macrophages and other cells, and is one of the main surface markers of NK cells. CD16 participates in antibody-dependent cell-mediated cytotoxicity by binding to the IgG part, and then activates NK cells. CD16 can also increase the toxicity of NK cells, upregulate the expression of genes such as IL-2R, IFN-γ, TNF-α, and increase the secretion of corresponding factors. It has the effect of activating NK cells in vitro and promoting proliferation;

[0047] HER2: Recombinant HER-2 human monoclonal antibody, an induction factor that induces the differentiation and proliferation of NK cells;

[0048] RetroNectin: A recombinant human fibronectin fragment that has physiological activities involved in cell attachment, extension, differentiation and proliferation, and can promote the adhesion and proliferation of lymphocytes;

[0049] IL-2: Interleukin 2, promotes the growth and proliferation of NK cells, promotes the survival, activation and toxicity of NK cells. IL-2 binds to receptors on the surface of NK cells, induces NK cells to express high-affinity receptors, thereby promoting cell proliferation and activation;

[0050] IL-12: Interleukin 12, by activating the phosphorylation of STAT3 and STAT1, promotes the expression of IFN-γ, IL-10 and perforin, thereby enhancing the activity and function of NK cells. It can also drive the expansion and maturation of a large number of NK cells and participate in the development of memory NK cells.

[0051] IL-15: Interleukin 15, which has the function of activating NK cells and mediating NK cell proliferation and survival, can promote the activation, proliferation and functional expression of NK cells, thereby enhancing their cytotoxicity and anti-tumor activity;

[0052] IL-18: Interleukin 18 can activate NK cells and promote the ability of NK cells to kill virus-infected and cancer cells. When used in combination with IL-12, it has a strong synergistic effect on the activation of NK cells, can induce greater IFN-γ production, and further enhance the activity of NK cells.

[0053] The experimental instruments and experimental consumables used in the present invention are shown in Table 1 and Table 2.

[0054] Table 1 Experimental instruments used in the present invention

[0055]

[0056]

[0057] Table 2 Experimental consumables used in the present invention

[0058] Serum-free lymphocyte culture medium Bio-Tech (Beijing) Co., Ltd. Lymphatic fluid Tianjin Haoyang Huake Biotechnology Co., Ltd. Buffer D-PBS Tianjin Haoyang Huake Biotechnology Co., Ltd. High efficiency centrifuge tube Shanghai Lirui Biotechnology Co., Ltd. IL-2 Beijing Sihuan Biopharmaceutical Co., Ltd. Cell Culture Bags Shenzhen Jiake Biotechnology Co., Ltd. 225ml centrifuge tube Coming Company 0.9% Sodium Chloride Injection Sichuan Kelun Pharmaceutical Co., Ltd. Immune cell serum-free cryopreservation medium Shenzhen Jiake Biotechnology Co., Ltd. 50ml centrifuge tube Wuxi Nice Life Sciences Co., Ltd. Disposable sterile syringe (with needle) 50ml Jumin Biotechnology Co., Ltd. Disposable sterile syringe (with needle) 10ml Jumin Biotechnology Co., Ltd. T175 cell culture flask Coming Company 5ml, 10ml, 25ml pipettes Wuxi Nice Life Sciences Co., Ltd. 1.8ml inner screw cap cryotube Thermo Fisher Scientific (China) Co., Ltd. 200ml gun tip Eppendorf, USA AOPI fluorescent dye Shanghai Ruiyu Biotechnology Co., Ltd. Cell counting chamber Shanghai Ruiyu Biotechnology Co., Ltd. Type I aerobic culture bottle Zhuhai Meihua Medical Technology Co., Ltd. Type II anaerobic culture bottle Zhuhai Meihua Medical Technology Co., Ltd.

[0059] The NK cell culture method of the present invention comprises the following steps:

[0060] (1) Collecting umbilical cord blood or peripheral blood and placing it in a collection bag for storage and standby use;

[0061] (2) After the collection bag in step (1) is disinfected, the umbilical cord blood or peripheral blood is placed in a centrifuge tube, centrifuged and the supernatant plasma is collected, inactivated in a water bath, allowed to stand, and then centrifuged for later use;

[0062] (3) adding lymph separation fluid to a high-efficiency centrifuge tube, adding PBS to the centrifuge tube containing cells in step (2) and resuspending the cells, and then adding the cells to the high-efficiency centrifuge tube for centrifugation, then aspirating the CBMC layer of the buffy coat layer and transferring it to a new centrifuge tube, adding PBS for washing and centrifuging to obtain cells to be used;

[0063] (4) adding D-PBS coating solution containing CD16 and Retronectin to the cell culture bottle containing the cells to be used, discarding the coating solution after incubation, washing with D-PBS, and then inoculating the cells in a T175 culture bottle, adding lymphocyte serum-free medium containing IL-2, IL-12, IL-15, IL-18, CD16, HER2 monoclonal antibodies, and 10% inactivated plasma to make the cell concentration 1-3*10^6 / ml, and placing in an incubator for further culturing for 4 days;

[0064] (5) On the 4th day after the culture in step (4), a serum-free medium containing IL-2, IL-12, IL-15, IL-18, CD16, HER2 monoclonal antibody and 10% plasma was added to the cells to continue the culture, so that the cell concentration was within the range of 1-3*10^6 / ml, and the cells were placed in an incubator and cultured for 2 more days;

[0065] (6) On the sixth day, based on the culture in step (5), a serum-free medium containing IL-2, IL-15 and 10% plasma was added to the cells to continue culturing until the cell concentration was within the range of 1-3*10^6 / ml, and the cells were placed in an incubator and cultured for 2 more days;

[0066] (7) On the 8th day after the culture in step (6), the NK cells are transferred from the culture flask to a cell culture bag for large-scale expansion, and the fluid is replenished or the bag is expanded every 2-3 days according to the cell growth situation. Each time the fluid is replenished, a lymphocyte serum-free culture medium containing IL-2 and IL-15 is added to make the cell concentration within the range of 1-3*10^6 / ml;

[0067] (8) On the 20th day, the cell culture bag was transferred to a biosafety cabinet, the cells were collected and centrifuged to discard the supernatant, the cells were resuspended with 0.9% sodium chloride injection, and 5% human serum albumin was added to the fixed volume to obtain the NK cells.

[0068] The final concentrations of CD16 and Retronectin in the culture system are 1-10 μg / ml and 10-20 μg / ml, respectively, preferably 2.5 μg / ml and 20 μg / ml;

[0069] When adding IL-2, IL-12, IL-15, IL-18, CD16 and HER2 monoclonal antibodies, the final concentrations in the culture system are 500-1000U / ml, 10-50ng / ml, 20-100ng / ml, 100-200ng / ml, 50-100ng / ml, 1-5μg / ml, preferably 1000U / ml, 10ng / ml, 80ng / ml, 100ng / ml, 100ng / ml, 5μg / ml.

[0070] When IL-2 and IL-15 are added, the final concentration in the culture system is 200-1000U / ml, 10-100ng / ml, more preferably 1000U / ml, 100ng / ml.

[0071] The technical solution of the present invention will be described in detail below by way of embodiments.

[0072] Example 1

[0073] In this embodiment, NK cells are cultured using cord blood, which specifically includes the following steps:

[0074] 1. Cord Blood Collection

[0075] Conduct a survey on the health status of the mothers and fill out the "Health Status Questionnaire" to exclude the risks of infectious diseases such as hepatitis A, hepatitis B, hepatitis C, and syphilis. After the mother and her family sign the "Informed Consent for Cord Blood Disposal", the doctor will collect the cord blood of the full-term newborn, place it in a cord blood collection bag, store it in a refrigerator at 2-8℃, and transport it to the laboratory for processing within 24 hours. The maternal blood and cord blood will be handed over to a third party for infectious disease testing.

[0076] 2. Isolation of Cord Blood Mononuclear Cells (CBMNCs)

[0077] 2.1 Plasma preparation

[0078] Disinfect the outer surface of the cord blood collection bag with 75% alcohol and place it in a biosafety cabinet; wipe the entire cord blood collection bag with an alcohol cotton ball and place it on a test tube rack. Use a 50ml syringe to transfer the cord blood to a 50ml centrifuge tube; centrifuge at 800g for 15min. After centrifugation, collect the supernatant plasma on the upper layer, keep 5ml of the sample for infectious source microorganism testing, and inactivate the remaining plasma in a 56℃ water bath for 30min; then let it stand at 4℃ for 15min; after standing, centrifuge at 1200g for 20min. Take the supernatant plasma and place it at 4℃ for later use.

[0079] 2.2 Extraction and separation of cord blood mononuclear cells (CBMNCs)

[0080] Add 13 ml of lymph separation fluid to the high-efficiency centrifuge tube, add PBS to the centrifuge tube containing cells to 35 ml, fully resuspend, and slowly add to the high-efficiency centrifuge tube; centrifuge at 800g for 25 minutes. After centrifugation, aspirate the CBMC layer of the buffy coat layer and transfer it to a new 50 ml centrifuge tube, add 30 ml of PBS to wash, and centrifuge twice, 500g for 10 minutes.

[0081] 3. Cultivation of NK cells from cord blood

[0082] 3.1 Counting of cord blood mononuclear cells (CBMNCs)

[0083] After the above cell washing and centrifugation, 40 ml of culture medium was added to resuspend the cells, and the cell suspension was placed in a 1.8 ml cryotube for counting; each group was counted twice.

[0084] 3.2 Induction, differentiation and expansion of NK cells from cord blood

[0085] Day 0: Place the T175 cell culture flask in a biosafety cabinet, wipe the entire flask with an alcohol cotton ball, add D-PBS coating solution containing CD16 and Retronectin, seal the flask with a sealing film, shake gently to cover the entire surface, and incubate in a 37°C, 5% CO2 incubator for 2 hours. Then discard the coating solution, wash once with 15ml D-PBS, inoculate the cells in a T175 culture flask, add lymphocyte serum-free medium containing IL-2, IL-12, IL-15, IL-18, CD16, HER2 monoclonal antibodies, and 10% inactivated plasma, so that the cell concentration is 1-3*10^ / ml, and continue to culture in a 37°C, 5% CO2 incubator.

[0086] Day 4: Add lymphocyte serum-free culture medium containing IL-2, IL-12, IL-15, IL-18, CD16, HER2 monoclonal antibody and 10% plasma and continue culturing until the cell concentration is in the range of 1-3*10^6 / ml, and place in a 37°C, 5% CO2 incubator for continued cultivation.

[0087] Day 6: Add lymphocyte serum-free medium containing IL-2, IL-15 and 10% plasma to continue culturing, so that the cell concentration is within the range of 1-3*10^6 / ml, and continue culturing in a 37°C, 5% CO2 incubator. The subsequent culture system is lymphocyte serum-free medium supplemented with IL-2 and IL-15.

[0088] Day 8: Transfer NK cells from the culture flask to a cell culture bag for large-scale expansion, and replenish or expand the bag every 2-3 days according to the cell growth. Add 500-1000ml of lymphocyte serum-free culture medium each time to make the cell concentration within the range of 1-3*10^6 / ml.

[0089] Day 20: Transfer the cell culture bag to the biosafety cabinet, collect the cells into a 225ml centrifuge tube and centrifuge at 1500rpm for 5min. After centrifugation, discard the supernatant, resuspend the cells with an appropriate amount of 0.9% sodium chloride injection, and add 5% human albumin to make the volume 200ml. Keep a sample of the cell suspension for AOPI staining count, bacteria and fungi, mycoplasma and endotoxin, and flow cytometry phenotyping.

[0090] Example 2

[0091] In this embodiment, NK cells are cultured using peripheral blood, specifically comprising the following steps:

[0092] 1. Peripheral Blood Collection

[0093] Conduct a health status survey and fill out the "Health Status Questionnaire" to exclude the risks of infectious diseases such as hepatitis A, hepatitis B, hepatitis C, and syphilis. After the volunteer signs the "Informed Consent for Peripheral Blood Disposal", the doctor will collect 50 ml of peripheral blood, place it in an anticoagulant tube, store it in a 2-8℃ refrigerator, and transport it to the laboratory for processing within 24 hours.

[0094] 2. Isolation of Peripheral Blood Mononuclear Cells (PBMNC)

[0095] 2.1 Plasma preparation

[0096] Disinfect the outer surface of the peripheral blood collection tube with 75% alcohol and place it in a biosafety cabinet; wipe the entire peripheral blood collection tube with an alcohol cotton ball and place it on a test tube rack. Use a 50ml syringe to transfer peripheral blood to a 50ml centrifuge tube; centrifuge at 800g for 15min. After centrifugation, collect the supernatant plasma on the upper layer, keep 5ml of the sample for infectious source microorganism testing, and inactivate the remaining plasma in a 56℃ water bath for 30min; then let it stand at 4℃ for 15min; after standing, centrifuge at 1200g for 20min. Take the supernatant plasma and put it at 4℃ for later use.

[0097] 2.2 Extraction and separation of peripheral blood mononuclear cells (PBMNC)

[0098] Add 13 ml of lymph separation fluid to the high-efficiency centrifuge tube, add PBS to the centrifuge tube containing cells to 35 ml, fully resuspend, and slowly add to the high-efficiency centrifuge tube; centrifuge at 800g for 25 minutes. After centrifugation, aspirate the PBMC layer of the buffy coat layer and transfer it to a new 50 ml centrifuge tube, add 30 ml of PBS to wash, and centrifuge twice, 500g for 10 minutes.

[0099] 3. Cultivation of Peripheral Blood NK Cells

[0100] 3.1 Counting of peripheral blood mononuclear cells (PBMNCs)

[0101] After the above cell washing and centrifugation, 40 ml of culture medium was added to resuspend the cells, and the cell suspension was placed in a 1.8 ml cryotube for counting; each group was counted twice.

[0102] 3.2 Induction, differentiation and expansion of peripheral blood NK cells

[0103] Day 0: Place the T175 cell culture flask in a biosafety cabinet, wipe the entire flask with an alcohol cotton ball, add D-PBS coating solution containing CD 16 and Retronectin, seal the flask with a sealing film, shake gently to cover the entire surface, and incubate in a 37°C, 5% CO2 incubator for 2 hours. Then discard the coating solution, wash once with 15ml D-PBS, inoculate the cells in a T175 culture flask, add lymphocyte serum-free medium containing IL-2, IL-12, IL-15, IL-18, CD16, HER2 monoclonal antibodies, and 10% inactivated plasma, so that the cell concentration is 1-3*10^6 / ml, and continue to culture in a 37°C, 5% CO2 incubator.

[0104] Day 4: Add lymphocyte serum-free culture medium containing IL-2, IL-12, IL-15, IL-18, CD16, HER2 monoclonal antibody and 10% plasma and continue culturing until the cell concentration is in the range of 1-3*10^6 / ml, and place in a 37°C, 5% CO2 incubator for continued cultivation.

[0105] Day 6: Add lymphocyte serum-free medium containing IL-2, IL-15 and 10% plasma to continue culturing, so that the cell concentration is within the range of 1-3*10^6 / ml, and continue culturing in a 37°C, 5% CO2 incubator. The subsequent culture system is lymphocyte serum-free medium supplemented with IL-2 and IL-15.

[0106] Day 8: Transfer NK cells from the culture flask to a cell culture bag for large-scale expansion, and replenish or expand the bag every 2-3 days according to the cell growth. Add 500-1000ml of lymphocyte serum-free culture medium each time to make the cell concentration within the range of 1-3*10^6 / ml.

[0107] Day 20: Transfer the cell culture bag to the biosafety cabinet, collect the cells into a 225ml centrifuge tube and centrifuge at 1500rpm for 5min. After centrifugation, discard the supernatant, resuspend the cells with an appropriate amount of 0.9% sodium chloride injection, and add 5% human albumin to make the volume 200ml. Keep a sample of the cell suspension for AOPI staining count, bacteria and fungi, mycoplasma and endotoxin, and flow cytometry phenotyping.

[0108] Test example

[0109] 1. Determination of NK cell growth and proliferation

[0110] Using the AO / PI dual fluorescence cell counting method, 1 ml of samples were collected for counting on the 0th, 4th, 8th, 12th, 16th, and 20th days of NK cell culture, and the counting was repeated twice. The NK cell concentration and volume were obtained based on the counting results, thereby obtaining the number of NK cells. The measurement results are shown in Figure 1 As shown by Figure 1 It can be seen that under the same culture technology system, after Examples 1-2 were cultured in the 8L system for a period of 20 days, the number of NK cells reached more than 2*10^10 after 22 days, which met the clinical application level.

[0111] 2. NK cell viability determination

[0112] Using the AO / PI dual fluorescence cell method, 1 ml of samples were taken on the 0th, 4th, 8th, 12th, 16th, and 20th days of NK cell culture to calculate the viability, and the counts were repeated twice to obtain the cell viability of NK cells at different time points. The results are shown in Figure 2 As shown by Figure 2 It can be seen that the cell viability of the NK cells prepared in Examples 1-2 of the present invention after the completion of the culture is above 90%, maintaining a high viability state, and the cells may have apoptosis of some non-target cells during the early stage of the induced culture, so that the cell viability is slightly reduced, and then after a large number of expansion, the cells do not lose cell viability during the culture and proliferation process and maintain a high viability.

[0113] 3. Determination of the killing effect of NK cells on tumor cells MCF-7, HepG2, HCT1 16, and Jurkat

[0114] (1) Subcultured human breast cancer cells MCF-7, human liver cancer cells HepG2, human colorectal adenocarcinoma cells HCT116, and human acute T lymphoblastic leukemia cells Jurkat were made into cell suspensions, and the tumor cell concentration was counted and adjusted to 5×10^4 cells / ml.

[0115] (2) The NK cells prepared in Example 1 were set as effector cells E, and the tumor cell line was set as target cell T. The NK cells with a culture period of 22 days were taken to prepare cell suspensions of different concentrations. The final concentrations of CTL cells were adjusted to 2.5×10^5 cells / ml, 5×10^5 cells / ml, and 1×10^6 cells / ml according to the effector-target ratios (5:1, 10:1, and 20:1) required by the experiment.

[0116] (3) The cells were inoculated into 96-well cell culture plates at effector-target ratios of 5:1, 10:1, and 20:1 (i.e., experimental groups), and target cell control group and effector cell control group were set up, with 3 replicate wells in each group and a total volume of 200 μl in each well.

[0117] (4) After plating, the cells were cultured in a 37°C, 5% CO2 incubator. CCK-8 reagent was added after 4 h, 24 h, and 48 h of co-culture, respectively. The cells were cultured in a cell culture incubator for another 2 h, and the OD450 value (i.e., A value, the absorbance of the test substance at a wavelength of 450 nm) was measured using an ELISA reader.

[0118] (5) Calculate the killing rate: killing rate = [1-(A value of the experimental group-A value of the effector cell control group) ÷ A value of the target cell control group] × 100%. The measurement results are as follows Figure 3 As shown by Figure 3 It can be seen that the killing effect of NK on tumor cells increases with the action time and the increase of effector cells, and the killing effect of NK gradually increases with the increase of effector-target ratio. And the killing effect of NK cells with an effector-target ratio of 10:1 on tumor cells in vitro can reach about 90% after 24 hours.

[0119] 4. Determination of NK cell phenotype by flow cytometry

[0120] The two types of NK cells cultured for 20 days were washed with sodium chloride and prepared into a 5×10^6 cell suspension. Antibodies to CD3, CD16, and CD56 were added. After incubation at room temperature in the dark for 15 minutes, the cells were tested on a flow cytometer to analyze the proportion of CD3-CD16+CD56+ cells. The final test results were as follows: Figure 4As shown, according to flow cytometry phenotyping analysis, the CD3-CD16+CD56+ of the cells in Example 1-2 were all above 95%.

[0121] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for culturing high-purity NK cells, characterized in that: The following steps are involved: (1) Isolate mononuclear cells from blood using density gradient centrifugation; (2) Using culture flasks for coating, monocytes are cultured for 20 days using lymphocyte serum-free medium, supplemented with GMP-grade recombinant proteins or humanized antibodies and blood sample serum; During the culture process, the cells were counted every 3 days, and the corresponding GMP-grade recombinant protein or humanized antibody, autologous serum and lymphocyte serum-free medium were added. After the last rehydration, the safety tests of microorganisms, mycoplasma and endotoxin were carried out; (3) After the cell culture was completed, the cells were collected by centrifugation, and the NK cells were washed three times with saline. The cells were then resuspended in saline and the volume was fixed to 200 ml after adding 5% human serum albumin.

2. The culture method according to claim 1, characterized in that In step (1), the blood is cord blood or peripheral blood.

3. The culture method according to claim 1, characterized in that In step (1), the specific steps of separation are: (1.1) Collect umbilical cord blood or peripheral blood and store it in a collection bag for later use; (1.2) After the collection bag in step (1.1) is disinfected, the umbilical cord blood or peripheral blood is placed in a centrifuge tube, centrifuged and the supernatant plasma is collected, inactivated in a water bath, allowed to stand, and then centrifuged for later use; (1.3) Add lymph separation fluid to a high-efficiency centrifuge tube, add PBS to the centrifuge tube containing cells in step (1.2) and resuspend, then add it to the high-efficiency centrifuge tube for centrifugation, then aspirate the CBMC layer of the buffy coat layer and transfer it to a new centrifuge tube, add PBS to wash and centrifuge to obtain the cells to be used.

4. The culture method according to claim 3, characterized in that In step (1.2), the centrifugation parameters for centrifugation and collection of supernatant plasma are 800g, 15min; the water bath inactivation and standing followed by centrifugation are 56°C water bath inactivation for 30min, followed by standing at 4°C for 15min, and then centrifugation at 1200g, 20min.

5. The culture method according to claim 3, characterized in that: In step (1.3), the centrifugation parameters when adding to the high-efficiency centrifuge tube are 800g, 25min; the centrifugation parameters when adding PBS for washing and centrifugation are 500g, 10min.

6. The culture method according to claim 1, characterized in that In step (2), the specific steps of culturing are: (2.1) Add D-PBS coating solution containing CD16 and Retronectin to the culture flask containing the monocytes, incubate in a 37°C, 5% CO2 incubator for 2 hours, discard the coating solution, wash the cells with D-PBS, inoculate the cells in a T175 culture flask, add lymphocyte serum-free culture medium containing IL-2, IL-12, IL-15, IL-18, CD16, HER2 monoclonal antibodies, and 10% inactivated plasma, so that the cell concentration is 1-3*10^6 / ml, and continue to culture in a 37°C, 5% CO2 incubator for 4 days; (2.2) On the 4th day after the culture in step (2.1), add lymphocyte serum-free culture medium containing IL-2, IL-12, IL-15, IL-18, CD16, HER2 monoclonal antibody and 10% plasma to continue the culture, so that the cell concentration is in the range of 1-3*10^6 / ml, and continue the culture in an incubator for 2 days; (2.3) On the 6th day after the culture in step (2.2), add lymphocyte serum-free medium containing IL-2, IL-15 and 10% plasma to continue the culture, so that the cell concentration is in the range of 1-3*10^6 / ml, and continue the culture in a 37°C, 5% CO2 incubator for 2 days; (2.4) On the 8th day after culture in step (2.3), the NK cells are transferred from the culture flask to the cell culture bag for large-scale expansion, and the bag is replenished or expanded every 2-3 days according to the cell growth. Each time the fluid is replenished, lymphocyte serum-free culture medium containing IL-2 and IL-15 is added to keep the cell concentration in the range of 1-3*10^6 / ml.

7. The culture method according to claim 1, characterized in that In step (3), the centrifugation parameters are 1500 rpm, 5 min.

8. The culture method according to claim 1, characterized in that The method also includes the steps of performing AOPI staining counting, bacteria, fungi, mycoplasma and endotoxin and flow cytometry phenotyping analysis on the cell suspension of the NK cells.

9. The culture method according to claim 6, characterized in that: The final concentrations of CD16 and Retronectin in the culture system are 1-10 μg / ml and 10-20 μg / ml respectively; The final concentrations of the IL-2, IL-12, IL-15, IL-18, CD16 and HER2 monoclonal antibodies in the culture system are 500-1000 U / ml, 10-50 ng / ml, 20-100 ng / ml, 100-200 ng / ml, 50-100 ng / ml and 1-5 μg / ml, respectively.

10. The culture method according to claim 9, characterized in that: The final concentrations of CD16 and Retronectin in the culture system are 2.5 μg / ml and 20 μg / ml respectively; The final concentrations of the IL-2, IL-12, IL-15, IL-18, CD16 and HER2 monoclonal antibodies in the culture system are 1000 U / ml, 10 ng / ml, 80 ng / ml, 100 ng / ml, 100 ng / ml and 5 μg / ml, respectively.

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