Culture method and application of NK (Natural Killer) cells

Abstract

The invention relates to the field of cell culture, and discloses an NK cell culture method and application, and the NK cell culture method comprises the following steps: preparing a peripheral blood or umbilical cord blood sample, and extracting PBMC from the blood sample by using a Ficoll separating medium; using an NK cell sorting kit to separate CD3-CD56 + NK cells with flow cytometry detection purity greater than or equal to 95% from the PBMC; the obtained CD3-CD56 + NK cells are inoculated into an NK amplification culture medium for in-vitro culture, the period is 24 days, one or more traditional Chinese medicine polysaccharides, IL-2 and platelet lysates are added into the amplification culture medium, and thymoquinone is added on the 21st day of cell culture. The purified NK cells are long in proliferation time and high in proliferation multiple, and can be continuously cultured for 30 days; the traditional Chinese medicine polysaccharide promotes the proliferation efficiency and cell viability of NK cells and secretion of cell factors; thymoquinone can effectively inhibit T cell proliferation and activity within 72 h, and the purity of NK culture can be further maintained.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a method for culturing NK cells and applications thereof. Background Art

[0002] Natural killer (NK) cells are effective as a drug modality for cancer immunotherapy because NK cells can kill a variety of tumor cells without deliberate immunization, activation, or prior sensitization. Unlike T cells, NK cells lack surface T cell receptors (TCRs) and do not cause graft-versus-host disease (GvHD), so NK cells have the advantage of allogeneic therapy.

[0003] The key to the clinical application of NK cells in allogeneic therapy is their purity and quantity. Studies have shown that there are multiple ways to activate and proliferate NK cells, including cytokine stimulation, receptor activator stimulation, and feeder cell stimulation. Cytokine stimulation and receptor activator stimulation require a certain proportion of other lymphocytes (such as T lymphocytes, monocytes, etc.) in the early stage. Individual differences often lead to the poor control of the proportion of other lymphocytes and uneven final NK purity. Feeder cell stimulation can cause the residue of abnormal cells, which brings certain challenges to the quality control of the finished product. Using purified NK cells as the starting point for proliferation can effectively solve the problem of low purity of NK cell culture, but the proliferation of purified NK cells under general culture conditions is relatively slow and the proliferation generations are limited.

[0004] Other studies have shown that Chinese herbal polysaccharides can directly promote the growth of immune organs and immune cells, and activate multiple signaling pathways by binding to receptors on the surface of immune cells, regulating the levels of pro-inflammatory factors, anti-inflammatory factors and chemokines in the body, thereby regulating the immune system and maintaining the activity of immune cells. The principle that tumor cells can activate NK proliferation is that certain proteins or signal molecules expressed by tumor cells directly act on NK cell surface receptors, triggering NK cell immune responses.

[0005] Therefore, exploring the high-purity, high-quantity, stable and safe culture process of purified NK cells is the current challenge faced by allogeneic NK applications. Summary of the invention

[0006] In response to the technical problems raised above, a method for culturing NK cells and application thereof are provided.

[0007] The technical means adopted by the present invention are as follows:

[0008] A method for culturing NK cells, comprising the following steps:

[0009] (1) Prepare 100 ml of peripheral blood or umbilical cord blood sample and extract PBMC from the blood sample using Ficoll separation solution;

[0010] (2) Using an NK cell sorting kit, CD3-CD56+ NK cells with a purity of ≥95% as determined by flow cytometry were isolated from PBMCs by magnetic bead negative selection technology;

[0011] (3) The separated CD3-CD56+ NK cells were inoculated into NK expansion medium and cultured in vitro for 24 days.

[0012] Preferably, the method for preparing the NK expansion medium comprises the following components:

[0013] (1) basal culture medium, selected from DMEM / F12 or IMDM;

[0014] (2) the total amount of Chinese medicine polysaccharides added to the culture medium is 50 to 200 ug / ml, including but not limited to one or more of Ganoderma lucidum polysaccharides, Ginkgo biloba polysaccharides, Bletilla striata polysaccharides, and Trametes orientalis polysaccharides;

[0015] (3) adding 1000 U / ml IL-2 to promote cell proliferation;

[0016] (4) Add 1% platelet lysate to provide cell growth factors.

[0017] Preferably, the process management of the in vitro culture comprises the following steps:

[0018] (1) Replenish the cultured NK cells every 2 to 3 days to ensure that the cell density is maintained at 2*10 6 cells / ml;

[0019] (2) adding 5–10 μg / ml of human lung mesothelioma cell extract to the culture medium on days 0, 9, and 16;

[0020] (3) On day 21, 200 μg / ml of thymoquinone was added to the culture medium to enhance cell function.

[0021] Preferably, the method for preparing the human lung mesothelioma cell extract specifically comprises the following steps:

[0022] (1) H2052 human lung mesothelioma cell line was subcultured in RPMI-1640 medium containing 10% FBS and 30-60 μM stimulator of interferon genes (STING);

[0023] (2) The inoculated cell density is 1 million / ml, and the culture medium is replenished every 2 to 3 days to maintain the cell density at 1 million / ml. After the cell number is expanded to 50 to 1000 times, the cells and culture medium are collected by centrifugation;

[0024] (3) Wash the cell pellet twice with PBS, centrifuge and discard the supernatant to obtain a pure cell pellet;

[0025] (4) Use RIPA lysis and extraction buffer containing 1x protease inhibitor and 1x phosphatase inhibitor to disrupt and extract the cell pellet in an ice bath.

[0026] Preferably, the specific operation of crushing and extracting human lung mesothelioma cells comprises the following steps:

[0027] (1) Resuspend the cell pellet in extraction buffer to a concentration of 10 million cells / ml, with no more than 10 ml per tube;

[0028] (2) Place in an ice bath for 10 minutes and then gently shake to mix well. Repeat this operation twice for a total of 30 minutes.

[0029] (3) The treated liquid was dispensed into 1.5 ml EP tubes, centrifuged at 12000 g for 20 min at 4 °C, and the supernatant was transferred to an ultrafiltration centrifuge tube for centrifugation and washing;

[0030] (4) The protein concentration was detected by BCA method, and the extract was diluted to 1 mg / ml with PBS for later use.

[0031] Preferably, the control of the culture environment is specifically as follows:

[0032] (1) culturing NK cells in an incubator at 37°C and 5% CO2;

[0033] (2) Ensure that the environmental conditions remain stable and pollution-free throughout the cultivation process.

[0034] The present invention also provides an application of NK cells collected by the above NK cell culture method in killing solid tumor cells.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] 1. After purification, NK cells proliferate for a long time and at a high multiple, and can be cultured continuously for up to 30 days;

[0037] 2. Chinese medicine polysaccharides can promote the proliferation efficiency and cell activity of NK cells and promote the secretion of cytokines;

[0038] 3. High concentrations of thymoquinone can effectively inhibit T cell proliferation and activity within 72 hours, but are non-toxic to NK cells and can further maintain the purity of NK culture;

[0039] 4. The extracts of human lung mesothelioma cells cultured with STING stimulation have strong NK cell tumor killing activity and promotion ability, especially in killing solid tumor cells, and also have strong ability to promote NK cell proliferation.

[0040] Based on the above reasons, the present invention can be widely promoted in the field of cell culture technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0042] Figure 1 It is a schematic diagram of NK cell expansion;

[0043] Figure 2 It is the flow cytometry ratio of CD3-CD56+ cells;

[0044] Figure 3 This is a graph showing the ratio of killing efficiency of lung cancer A549 cells;

[0045] Figure 4 HepG2 liver cancer cell killing efficiency ratio diagram;

[0046] Figure 5 Colon cancer HCT-8 cell killing efficiency ratio diagram;

[0047] Figure 6 The killing efficiency ratio of gastric cancer SGC-7901 cells. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Example 1

[0050] 1. Purify NK cells

[0051] 100 ml of peripheral blood was collected using a sodium heparin anticoagulant blood collection tube. PBMCs were separated according to the blood separation procedure. CD3-CD56+ cells with a flow cytometry ≥ 95% were separated according to the instructions of the NK cell sorting kit. The cells were washed once with PBS, centrifuged at 600 g for 8 min, and the cell pellet was used for later use.

[0052] 2. NK cell culture

[0053] (1) Preparation of NK expansion medium:

[0054] Take 1L DMEM / F12, add 50ug / ml Ganoderma lucidum polysaccharide, 1000U / ml IL-2 and 1% platelet lysate.

[0055] (2) Preparation of human lung mesothelioma cell extracts:

[0056] A. Tumor cell culture: Tumor cells were obtained from H2052 human lung mesothelioma cell line and subcultured in RPMI-1640 medium containing 10% FBS and 30μM ADU-S100. 10 million cells were inoculated with a cell density of 1 million / ml. The cells were replenished every 2 to 3 days to maintain a cell density of 1 million / ml. After the cell number was expanded 50 times, the cells and culture medium were collected into a 50 ml centrifuge tube, centrifuged at 2000g for 8 min, and the supernatant was discarded. The cells were washed twice with PBS, centrifuged at 2000g for 8 min, and the supernatant was discarded. The cell pellet was used for later use.

[0057] B. Extraction solution preparation: Prepare RIPA lysis and extraction buffer (Thermo) containing 1x protease inhibitor (Roche) and 1x phosphatase inhibitor cocktail A (beyotime), and cool in an ice bath for later use;

[0058] C. Cell disruption and extraction: resuspend the cell pellet with the above buffer, 10 million / ml, no more than 10ml per tube, operate on ice bath throughout the process, let stand for 10 minutes, shake gently to evenly, let stand again, repeat the above operation twice, the whole process is 30 minutes in total;

[0059] D. Collection of cell extracts: Evenly distribute the treatment solution in step C into 1.5 ml EP tubes, centrifuge at 12000g for 20 min at 4°C, transfer the supernatant to a 10 kDa MWCO 15 ml ultrafiltration centrifuge tube, centrifuge at 3000g for 20 min, wash twice with an equal volume of PBS, centrifuge at 3000g for 20 min, resuspend with a small amount of PBS, detect the protein concentration using the BCA method, and make up to 1 mg / ml with PBS for later use.

[0060] (3) Bottle filling and rehydration

[0061] Resuspend the cell pellet from step 1 in NK expansion medium and maintain the cell density at 2*10 6 cells / ml, cultured for 24 days, refilled every 2-3 days, and the cell density was maintained at 2*10 6 cells / ml;

[0062] Add 5ug / ml of step (2) human lung mesothelioma cell extract on days 0, 9, and 16;

[0063] On day 21, 200 μg / ml thymoquinone was added.

[0064] 3. NK cell collection

[0065] After collection in the centrifuge tube, the cells were washed 2-3 times with physiological saline, centrifuged at 800 g for 10 min, and finally resuspended with physiological saline containing 1% human albumin.

[0066] Example 2

[0067] 1. Purify NK cells

[0068] 100 ml of peripheral blood was collected using a sodium heparin anticoagulant blood collection tube. PBMCs were separated according to the blood separation procedure. CD3-CD56+ cells with a flow cytometry ≥ 95% were separated according to the instructions of the NK cell sorting kit. The cells were washed once with PBS, centrifuged at 600 g for 8 min, and the cell pellet was used for later use.

[0069] 2. NK cell culture

[0070] (1) Preparation of NK expansion medium:

[0071] Take 1L IMDM, add 20ug / ml Ganoderma lucidum polysaccharide, 60ug / ml Ginkgo biloba polysaccharide, 80ug / ml Bletilla striata polysaccharide, 40ug / ml Trametes orientalis polysaccharide, 1000U / ml IL-2 and 1% platelet lysate.

[0072] (2) Preparation of human lung mesothelioma cell extracts:

[0073] A. Tumor cell culture: Tumor cells were obtained from H2052 human lung mesothelioma cell line and subcultured in RPMI-1640 medium containing 10% FBS and 60 μM 2',3'-cGAMP. 10 million cells were inoculated with a cell density of 1 million / ml. The cells were replenished every 2 to 3 days to maintain a cell density of 1 million / ml. After the cell number was expanded 1000 times, the cells and culture medium were collected into a 50 ml centrifuge tube, centrifuged at 2000 g for 8 min, and the supernatant was discarded. The cells were washed twice with PBS, centrifuged at 2000 g for 8 min, and the supernatant was discarded. The cell pellet was used for later use.

[0074] B. Extraction solution preparation: Prepare RIPA lysis and extraction buffer (Thermo) containing 1x protease inhibitor (Roche) and 1x phosphatase inhibitor cocktail A (beyotime), and cool in an ice bath for later use;

[0075] C. Cell disruption and extraction: resuspend the cell pellet with the above buffer, 10 million / ml, no more than 10ml per tube, operate on ice bath throughout the process, let stand for 10 minutes, shake gently to evenly, let stand again, repeat the above operation twice, the whole process is 30 minutes in total;

[0076] D. Collection of cell extracts: Evenly distribute the treatment solution in step C into 1.5 ml EP tubes, centrifuge at 12000g for 20 min at 4°C, transfer the supernatant to a 10 kDa MWCO 15 ml ultrafiltration centrifuge tube, centrifuge at 3000g for 20 min, wash twice with an equal volume of PBS, centrifuge at 3000g for 20 min, resuspend with a small amount of PBS, detect the protein concentration using the BCA method, and make up to 1 mg / ml with PBS for later use.

[0077] (3) Bottle filling and rehydration

[0078] Resuspend the cell pellet from step 1 in NK expansion medium and maintain the cell density at 2*10 6 cells / ml, cultured for 24 days, refilled every 2-3 days, and the cell density was maintained at 2*10 6 cells / ml;

[0079] On days 0, 9, and 16, 10 ug / ml of the human lung mesothelioma cell extract from step (2) was added; on day 21, 200 μg / ml of thymoquinone was added.

[0080] 3. NK cell collection

[0081] Example 3

[0082] 1. Purify NK cells

[0083] 100 ml of umbilical cord blood was collected using a sodium heparin anticoagulant blood collection tube. After separating PBMCs according to the blood separation procedure, CD3-CD56+ cells with a flow cytometry ≥ 95% were separated according to the instructions of the NK cell sorting kit. Finally, the cells were washed once with PBS, centrifuged at 600 g for 8 min, and the cell pellet was used for later use.

[0084] 2. NK cell culture

[0085] (1) Preparation of NK expansion medium:

[0086] Take 1L IMDM, add 40ug / ml selenized lily polysaccharide, 30ug / ml ginkgo polysaccharide, 30ug / ml wolfberry polysaccharide, 1000U / ml IL-2 and 1% platelet lysate.

[0087] (2) Preparation of human lung mesothelioma cell extracts:

[0088] A. Tumor cell culture: Tumor cells were obtained from H2052 human lung mesothelioma cell line and subcultured in RPMI-1640 medium containing 10% FBS and 40μM ADU-S100. 10 million cells were inoculated with a cell density of 1 million / ml. The cells were replenished every 2 to 3 days to maintain a cell density of 1 million / ml. After the cell number was expanded 500 times, the cells and culture medium were collected into a 50 ml centrifuge tube, centrifuged at 2000g for 8 min, and the supernatant was discarded. The cells were washed twice with PBS, centrifuged at 2000g for 8 min, and the supernatant was discarded. The cell pellet was used for later use.

[0089] B. Extraction solution preparation: Prepare RIPA lysis and extraction buffer (Thermo) containing 1x protease inhibitor (Roche) and 1x phosphatase inhibitor cocktail A (beyotime), and cool in an ice bath for later use;

[0090] C. Cell disruption and extraction: resuspend the cell pellet with the above buffer, 10 million / ml, no more than 10ml per tube, operate on ice bath throughout the process, let stand for 10 minutes, shake gently to evenly, let stand again, repeat the above operation twice, the whole process is 30 minutes in total;

[0091] D. Collection of cell extracts: Evenly distribute the treatment solution in step C into 1.5 ml EP tubes, centrifuge at 12000g for 20 min at 4°C, transfer the supernatant to a 10 kDa MWCO 15 ml ultrafiltration centrifuge tube, centrifuge at 3000g for 20 min, wash twice with an equal volume of PBS, centrifuge at 3000g for 20 min, resuspend with a small amount of PBS, detect the protein concentration using the BCA method, and make up to 1 mg / ml with PBS for later use.

[0092] (3) Bottle filling and rehydration

[0093] Resuspend the cell pellet from step 1 in NK expansion medium and maintain the cell density at 2*10 6 cells / ml, cultured for 24 days, refilled every 2-3 days, and the cell density was maintained at 2*10 6 cells / ml;

[0094] On days 0, 9, and 16, 7 ug / ml of step (2) human lung mesothelioma cell extract was added;

[0095] On day 21, 200 μg / ml thymoquinone was added.

[0096] 3. NK cell collection

[0097] Comparative Example 1

[0098] 1. Purify NK cells

[0099] 100 ml of peripheral blood was collected using a sodium heparin anticoagulant blood collection tube. PBMCs were separated according to the blood separation procedure. CD3-CD56+ cells with a flow cytometry ≥ 95% were separated according to the instructions of the NK cell sorting kit. The cells were washed once with PBS, centrifuged at 600 g for 8 min, and the cell pellet was used for later use.

[0100] 2. NK cell culture

[0101] (1) Preparation of NK expansion medium:

[0102] Take 1 L of IMDM and add 1000 U / ml of IL-2 and 1% platelet lysate.

[0103] (2) Bottle and fluid replenishment

[0104] Resuspend the cell pellet from step 1 in NK expansion medium and maintain the cell density at 2*10 6 cells / ml, cultured for 24 days, refilled every 2-3 days, and the cell density was maintained at 2*10 6 cells / ml.

[0105] 3. NK cell collection

[0106] Comparative Example 2

[0107] 1. Isolation of PBMC and NK Culture

[0108] 100 ml of peripheral blood was collected using a heparin sodium anticoagulant blood collection tube. After separating PBMCs according to the blood separation procedure, NK cells were cultured using the NK cell culture kit available on the market according to the instruction manual (the culture time of the kit is 15 days). After 15 days, the fluid was replenished every 2 to 3 days until the 24th day. After the fluid was replenished, the cell density was maintained at 2*10 6 cells / ml;

[0109] 2. NK cell collection

[0110] Detection

[0111] 1. NK cell expansion

[0112] like Figure 1 The figure shows a schematic diagram of NK cell expansion, and the results are as follows:

[0113] (1) Examples 1 to 3 and Comparative Example 1 show that human lung mesothelioma cell extracts and Chinese herbal polysaccharides can significantly enhance the proliferation of purified NK cells;

[0114] (2) Examples 1 to 3 and Comparative Example 2 show that the effects of human lung mesothelioma cell extracts and Chinese herbal polysaccharides on NK proliferation after purification can achieve the culture effect of PBMC using the NK kit, and can continue to maintain a high proliferation multiple after 15 days (the culture effect of Comparative Example 2 began to weaken after exceeding the 15-day culture time of the kit, and the NK proliferation rate slowed down).

[0115] 2. Flow cytometry

[0116] like Figure 2 As shown, samples were taken on days 0, 15, and 24 to detect the CD3 / CD56 flow cytometry phenotype of cells. The results show that:

[0117] (1) The results of Examples 1 to 3 and Comparative Example 1 on days 0 and 15 showed that the purity of the purified NK cells would decrease after culture, but the comparison of the results on day 24 showed that the thymoquinone added on day 21 of Examples 1 to 3 had a significant inhibitory effect on T cells. The NK flow cytometry of Examples 1 to 3 on day 24 reached more than 99%, while the flow cytometry of Comparative Example 1 on day 24 was less than 95%;

[0118] (2) From the results of comparative example 2, the NK flow cytometry ratio decreased after the 15-day culture time of the kit, and the kit was not sufficient to maintain the long-term stability of the NK flow cytometry.

[0119] 3. Tumor killing

[0120] like Figures 3 to 6 As shown, cells on the 24th day were taken, and 100 million cells were collected from comparative examples 1 and 2, and the NK cell sorting kit was used to further purify the NK flow cytometry ratio by magnetic bead negative selection, so that the purified CD3-CD56+ flow cytometry ratio was ≥99% (this step was to control the consistency of NK flow cytometry variables), and they were co-cultured with A549 (lung cancer cell line), HepG2 (liver cancer cell line), HCT-8 (colon cancer cell line), and SGC-7901 (gastric cancer cell line) at a 5:1 NK cell: tumor cell ratio, and the tumor killing efficiency was detected using a real-time label-free cell analysis system (Real time xCELLigence analysis system, RTCA).

[0121] The following are the test data of different cancer cell killing efficiency (respectively Figures 3 to 6 correspond):

[0122] Lung Cancer A549 Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Oh 0.00％ 0.00％ 0.00％ 0.00％ 0.00％ 6h 15.24％ 13.12％ 13.66％ 5.12％ 7.55％ 12h 16.12％ 14.26％ 15.72％ 6.15％ 7.86％ 18h 18.12％ 15.26％ 19.20％ 7.61％ 9.73％ 24h 20.20％ 18.16％ 21.17％ 9.11％ 12.26％ 30h 23.54％ 19.46％ 25.26％ 12.16％ 15.62％ 36h 27.16％ 22.85％ 29.42％ 13.64％ 19.42％ 42h 33.06％ 27.16％ 35.14％ 16.74％ 22.19％ 48h 42.15％ 35.10％ 41.26％ 17.66％ 26.02％ 54h 54.18％ 45.14％ 49.78％ 18.23％ 33.14％ 60h 63.28％ 52.67％ 55.16％ 18.66％ 36.59％ 66h 69.41％ 57.74％ 62.43％ 19.15％ 38.92％ 72h 75.43％ 65.30％ 68.63％ 19.17％ 43.80％ 78h 77.42％ 68.80％ 73.43％ 19.58％ 46.87％ 84h 79.11％ 72.40％ 76.54％ 18.26％ 48.79％ 90h 78.16％ 73.06％ 74.16％ 17.79％ 48.04％ 96h 80.20％ 71.46％ 72.19％ 17.46％ 46.46％

[0123] Hepatocellular Carcinoma HepG2 Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Oh 0.00％ 0.00％ 0.00％ 0.00％ 0.00％ 6h 10.56％ 8.27％ 6.71％ 3.09％ 7.42％ 12h 13.54％ 12.12％ 10.42％ 6.10％ 11.62％ 18h 15.67％ 14.86％ 12.52％ 7.61％ 13.21％ 24h 18.46％ 18.12％ 15.88％ 9.02％ 16.13％ 30h 22.72％ 23.68％ 19.20％ 11.65％ 19.O4％ 36h 27.86％ 28.16％ 24.O3％ 12.62％％ 23.16％ 42h 32.89％ 34.62％ 29.40％ 12.82％ 26.47％ 48h 39.88％ 41.73％ 34.76％ 14.88％ 3O.62％ 54h 49.12％ 51.19％ 41.76％ 15.O6％ 34.88％ 6Oh 58.65％ 62.70％ 49.93％ 15.12％ 39.06％ 66h 65.43％ 68.12％ 54.62％ 16.44％ 43.18％ 72h 71.04％ 70.16％ 57.62％ 17.20％ 45.26％ 78h 74.78％ 71.64％ 61.O6％ 16.68％ 46.O6％ 84h 77.1O％ 72.O1％ 64.16％ 16.32％ 46.76％ 90h 76.98％ 72.62％ 66.76％ 14.82％ 46.15％ 96h 76.76％ 72.04％ 66.62％ 12.16％ 45.30％

[0124] Colon Cancer HCT-8 Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 oh 0.00％ 0.00％ 0.00％ 0.00％ 0.00％ 6h 8.26％ 9.71％ 9.05％ 5.16％ 6.08％ 12h 13.30％ 15.62％ 14.62％ 5.16％ 9.88％ 18h 15.12％ 17.12％ 16.75％ 7.08％ 12.14％ 24h 19.13％ 19.62％ 18.26％ 8.76％ 14.68％ 30h 21.76％ 21.68％ 20.26％ 9.43％ 17.53％ 36h 25.16％ 25.16％ 24.03％ 11.64％ 20.13％ 42h 28.65％ 29.46％ 28.66％ 13.44％ 23.42％ 48h 32.16％ 34.26％ 34.43％ 15.68％ 25.61％ 54h 38.76％ 38.12％ 39.15％ 15.45％ 28.15％ 60h 47.16％ 46.15％ 44.60％ 16.09％ 31.62％ 66h 55.22％ 53.41％ 49.18％ 16.68％ 34.16％ 72h 62.16％ 58.16％ 55.98％ 17.22％ 37.26％ 78h 65.76％ 61.14％ 63.75％ 17.76％ 37.11％ 84h 67.44％ 64.12％ 71.50％ 17.51％ 35.65％ 90h 68.11％ 64.66％ 73.04％ 15.77％ 33.12％ 96h 67.12％ 64.19％ 72.16％ 16.25％ 30.07％

[0125] Gastric CancerSGC-7901 Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Oh 0.00％ 0.00％ 0.00％ 0.00％ 0.00％ 6h 6.55％ 8.61％ 6.46％ 2.12％ 4.62％ 12h 5.47％ 10.85％ 7.15％ 3.62％ 6.24％ 18h 8.13％ 10.22％ 9.11％ 3.99％ 6.16％ 24h 11.62％ 11.03％ 10.62％ 5.14％ 6.84％ 30h 15.42％ 13.46％ 12.28％ 6.49％ 8.25％ 36h 19.65％ 17.73％ 16.19％ 7.85％ 10.46％ 42h 23.15％ 21.67％ 19.49％ 9.06％ 13.56％ 48h 27.12％ 25.14％ 23.18％ 11.26％ 16.27％ 54h 33.62％ 28.64％ 26.84％ 13.24％ 18.16％ 60h 39.74％ 33.06％ 33.16％ 13.76％ 23.08％ 66h 47.58％ 39.40％ 39.78％ 14.15％ 27.78％ 72h 55.14％ 45.55％ 46.25％ 13.67％ 32.44％ 78h 59.62％ 51.46％ 54.16％ 13.08％ 35.65％ 84h 63.12％ 56.16％ 59.46％ 12.43％ 35.23％ 90h 64.12％ 59.42％ 62.16％ 11.75％ 33.59％ 96h 64.06％ 62.13％ 63.44％ 10.46％ 33.22％

[0126] Result description:

[0127] From the results of killing efficiency of the four types of tumor cells, the killing efficiency of Examples 1 to 3 within 96 hours all reached more than 60%, indicating that the killing effect on solid tumors is stronger.

[0128] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, a person of ordinary skill in the art should understand that any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for culturing NK cells, characterized in that: The following steps are involved: (1) Prepare 100 ml of peripheral blood or umbilical cord blood sample and extract PBMC from the blood sample using Ficoll separation solution; (2) Using an NK cell sorting kit, CD3-CD56+ NK cells with a purity of ≥95% as determined by flow cytometry were isolated from PBMCs by magnetic bead negative selection technology; (3) The separated CD3-CD56+ NK cells were inoculated into NK expansion medium and cultured in vitro for 24 days.

2. The method for culturing NK cells according to claim 1, characterized in that: The preparation method of the NK expansion culture medium comprises the following components: (1) basal culture medium, selected from DMEM / F12 or IMDM; (2) the total amount of Chinese medicine polysaccharides added to the culture medium is 50 to 200 ug / ml, including but not limited to one or more of Ganoderma lucidum polysaccharides, Ginkgo biloba polysaccharides, Bletilla striata polysaccharides, and Trametes orientalis polysaccharides; (3) adding 1000 U / ml IL-2 to promote cell proliferation; (4) Add 1% platelet lysate to provide cell growth factors.

3. The method for culturing NK cells according to claim 2, characterized in that: The process management of the in vitro culture comprises the following steps: (1) Replenish the cultured NK cells every 2 to 3 days to ensure that the cell density is maintained at 2*10 6 cells / ml; (2) adding 5–10 μg / ml of human lung mesothelioma cell extract to the culture medium on days 0, 9, and 16; (3) On day 21, 200 μg / ml thymoquinone was added to the culture medium to enhance cell function.

4. The method for culturing NK cells according to claim 3, characterized in that: The method for preparing the human lung mesothelioma cell extract specifically comprises the following steps: (1) H2052 human lung mesothelioma cell line was subcultured in RPMI-1640 medium containing 10% FBS and 30-60 μM interferon gene stimulator; (2) The inoculated cell density is 1 million / ml, and the culture medium is replenished every 2 to 3 days to maintain the cell density at 1 million / ml. After the cell number is expanded to 50 to 1000 times, the cells and culture medium are collected by centrifugation; (3) Wash the cell pellet twice with PBS, centrifuge and discard the supernatant to obtain a pure cell pellet; (4) Use RIPA lysis and extraction buffer containing 1x protease inhibitor and 1x phosphatase inhibitor to disrupt and extract the cell pellet in an ice bath.

5. The method for culturing NK cells according to claim 4, characterized in that: The specific operation of the human lung mesothelioma cell crushing and extraction comprises the following steps: (1) Resuspend the cell pellet in extraction buffer to a concentration of 10 million cells / ml, with no more than 10 ml per tube; (2) Place in an ice bath for 10 minutes and then gently shake to mix well. Repeat this operation twice for a total of 30 minutes. (3) The treated liquid was dispensed into 1.5 ml EP tubes, centrifuged at 12000 g for 20 min at 4 °C, and the supernatant was transferred to an ultrafiltration centrifuge tube for centrifugation and washing; (4) The protein concentration was detected by BCA method, and the extract was diluted to 1 mg / ml with PBS for later use.

6. The method for culturing NK cells according to claim 1, characterized in that: Control of the training environment, specifically: (1) culturing NK cells in an incubator at 37°C and 5% CO2; (2) Ensure that the environmental conditions remain stable and pollution-free throughout the cultivation process.

7. Use of NK cells collected by the NK cell culture method according to any one of claims 1 to 6 in killing solid tumor cells.

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