Induced culture method of umbilical cord blood NK cells

Abstract

The invention provides an induced culture method of umbilical cord blood NK cells, and belongs to the technical field of cell culture. According to the induction culture method of the umbilical cord blood NK cells, the umbilical cord blood NK cells with the CD3 + CD56-number larger than 90% and high killing activity can be stably obtained, the limitation of the umbilical cord blood collection time can be relieved, the method can be suitable for collecting the umbilical cord blood with the preparation time interval of 20 h, and the method has important significance on full utilization of biological resources.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to an induction and culture method of umbilical cord blood NK cells. Background Art

[0002] Natural killer (NK) cells are an important part of the human innate immune system and have excellent anti-cancer and anti-viral infection capabilities. Compared with T cell immunotherapy, NK cell-based immunotherapy has many advantages: NK cells can spontaneously detect and eliminate infected and malignant cells without the need for specific activation, do not cause graft-versus-host disease, and do not induce T cell-driven inflammatory cytokine storms. These characteristics of NK cells make them highly attractive in cell-based cancer immunotherapy. As a feasible and effective treatment method, NK cell immunotherapy has shown great promise in preclinical and early clinical studies.

[0003] The main sources of NK cells are umbilical cord blood and peripheral blood. Human NK cells account for 5%-25% of peripheral blood lymphocytes, and generally only 2%-5% in umbilical cord blood. For immunotherapy, efficient in vitro expansion of high-purity and high-efficiency umbilical cord blood NK cells is crucial.

[0004] Chinese patent CN116790499A discloses a highly efficient method for culturing and amplifying umbilical cord blood NK cells, comprising the following steps: step S1, taking fresh and healthy umbilical cord blood, centrifuging it, and obtaining a mononuclear cell layer after centrifugation; step S2, taking the mononuclear cell layer obtained in step S1 and treating it; step S3, preparing a culture medium; step S4, inducing NK cell proliferation; step S5, amplifying and culturing NK cells. The invention uses IL-2, IL-15, IL-21, TNFRSF18, OK432 and a basal culture medium to prepare an NK initial culture medium, and IL-2 and IL-12 and a basal culture medium to prepare an NK amplification culture medium; during the use of the NK initial culture medium and the NK amplification culture medium, human serum substitutes are added to replace autologous plasma, and other technical means to overcome the problems of insufficient number of umbilical cord blood NK cells, insufficient cell activity, and low flow expression rate.

[0005] However, the above method fails to solve the problem of CD3 + CD56 - The problem that NK cells cannot be used due to the number or viability being less than 90% and insufficient killing ability causes serious waste of resources. In addition, the above technology is only applicable to umbilical cord blood collected within 10 hours from the time of preparation, and its application prospects are obviously limited by the time of umbilical cord blood collection. Summary of the invention

[0006] The purpose of the present invention is to provide a method for inducing and culturing umbilical cord blood NK cells, which can not only stably obtain CD3 + CD56 - Umbilical cord blood NK cells with a number greater than 90% and high killing activity also alleviate the limitations of umbilical cord blood collection time to a certain extent.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for inducing and culturing umbilical cord blood NK cells, comprising the following steps: S1. Mix the umbilical cord blood and NK cell enrichment reagent and let it stand, then add the lymphocyte sample density separation solution, and centrifuge to obtain umbilical cord blood mononuclear cells; S2, inoculating the umbilical cord blood mononuclear cells into NK activation medium, culturing for 3 to 8 days, and obtaining intermediate cells; S3, inoculating the intermediate cells into NK proliferation medium and culturing for more than 7 days to obtain umbilical cord blood NK cells; The dosage of the NK cell enrichment reagent is 0~5μL / mL; The NK activation medium contains autologous plasma; The NK proliferation culture medium contains autologous plasma.

[0008] Preferably, the dosage of the NK cell enrichment reagent is 1~2 μL / mL.

[0009] Preferably, in S1, the standing time is 5 to 20 minutes; and / or, the volume ratio of the cord blood to the lymphocyte sample density separation fluid is 1.5-3:1; And / or, the centrifugal speed is 800~1200g; And / or, the centrifugation time is 10 to 30 minutes.

[0010] Preferably, in S2, the inoculation amount is 5×10 5 ~5×10 6 Pieces / mL.

[0011] Preferably, the NK activation medium contains autologous plasma at a final volume concentration of 5-20%; The NK activation medium is the NK-I additive and / or the NK-II additive in the NK cell culture special kit, and the NK cell culture special kit comes from Beijing Wukang Xinxing Technology Co., Ltd.

[0012] Preferably, in S2, the culturing process includes supplementing NK activation medium, and the frequency of supplementation is 1 to 2 days / time.

[0013] Preferably, the NK proliferation medium contains basal proliferation medium, autologous plasma and cell culture additives; The final volume concentration of the autologous plasma is 3-10%; The final volume concentration of the cell culture additive is 0.1-0.3%.

[0014] Preferably, the NK proliferation culture medium further contains IL-15 and / or IL-2; The final concentration of IL-15 is 5-15 ng / mL; The final concentration of IL-2 is 800-1200 IU / mL; And / or, the type of the basic proliferation medium is KBM 581 medium and / or NK-IV additive in a special kit for NK cell culture, and the special kit for NK cell culture comes from Beijing Wukang Xinxing Technology Co., Ltd.

[0015] Preferably, in S3, the culture process includes adding NK proliferation medium, and the added NK proliferation medium is used to maintain the cell density at 5×10 5 ~5×10 6 Pieces / mL.

[0016] Preferably, the autologous plasma is obtained by a preparation method comprising the following steps: Centrifuging the umbilical cord blood to obtain a supernatant; The supernatant is sequentially inactivated, quick-frozen, and centrifuged to obtain autologous plasma; The temperature of the inactivation treatment is 50-60°C, and the time of the inactivation treatment is 20-40 minutes; The temperature of the quick freezing treatment is -30 to -10°C, and the time of the quick freezing treatment is 5 to 20 minutes.

[0017] Beneficial effects of the present invention: The method for inducing and culturing umbilical cord blood NK cells provided by the present invention can not only stably obtain CD3 + CD56 - Umbilical cord blood NK cells with a number greater than 90% and high killing activity can also alleviate the limitation of umbilical cord blood collection time. This method is applicable to umbilical cord blood collected within a preparation time interval of 20 hours, which is of great significance for the full utilization of biological resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the changes in the number of CD3-CD56+ and CD3+ before and after separation of MNC from umbilical cord blood; Figure 2This is a statistical diagram of the number of CD3-CD56+ in umbilical cord blood NK cells after 13 days of culture; Figure 3 This is a graph showing the characterization of the killing activity of NK cells from umbilical cord blood after 13 days of culture; Figure 4 This is a statistical chart of the number of cells expanded after 13 days of culture of umbilical cord blood NK cells; Figure 5 The control group NK cells CD3 - CD56 + Quantity test result diagram. DETAILED DESCRIPTION

[0019] The present invention provides a method for inducing and culturing umbilical cord blood NK cells, comprising the following steps: S1. Mix the umbilical cord blood and NK cell enrichment reagent and let it stand, then add the lymphocyte sample density separation solution, and centrifuge to obtain umbilical cord blood mononuclear cells; S2, inoculating the umbilical cord blood mononuclear cells into NK activation medium, culturing for 3 to 8 days, and obtaining intermediate cells; S3, inoculating the intermediate cells into NK proliferation medium and culturing for more than 7 days to obtain umbilical cord blood NK cells; The dosage of the NK cell enrichment reagent is 0~5μL / mL; The NK activation medium contains autologous plasma; The NK proliferation culture medium contains autologous plasma.

[0020] In the present invention, preferably, the dosage of the NK cell enrichment reagent is further preferably 1-2 μL / mL, and the NK cell enrichment reagent can directly enrich NK cells from umbilical cord blood by negative selection.

[0021] In the present invention, preferably, in S1, the standing time is 5 to 20 min; and / or, the volume ratio of the cord blood to the lymphocyte sample density separation fluid is 1.5 to 3:1; and / or, the centrifugal speed is 800 to 1200 g; and / or, the centrifugal time is 10 to 30 min; preferably, in S2, the inoculation amount is 5×10 5 ~5×10 6 Pieces / mL.

[0022] In the present invention, preferably, the NK activation medium contains autologous plasma with a final volume concentration of 5-20%; the NK activation medium is NK-I additive and / or NK-II additive in a special kit for NK cell culture, and the special kit for NK cell culture is from Beijing Wukang Xinxing Technology Co., Ltd.; preferably, in S2, the culture process includes supplementing NK activation medium, and the frequency of supplementation is 1-2 days / time. When the NK activation medium adopts NK-I additive and / or NK-II additive in a special kit for NK cell culture, it is preferred to first supplement NK-I additive and then supplement the mixed reagent, and the mixed reagent is obtained by mixing working solution I and working solution II in equal proportions.

[0023] In the present invention, preferably, the NK proliferation medium contains basal proliferation medium, autologous plasma and cell culture additives; the final volume concentration of the autologous plasma is 3-8%; the final volume concentration of the cell culture additive is 0.1-0.3%. Preferably, the NK proliferation medium also contains IL-15 and / or IL-2; the final concentration of IL-15 is 5-15 ng / mL; the final concentration of IL-2 is 800-1200 IU / mL; and / or, the type of the basal proliferation medium is KBM581 medium and / or NK-IV additive in the NK cell culture special kit, and the NK cell culture special kit is from Beijing Wukang Xinxing Technology Co., Ltd.; preferably, in S3, the culture process includes adding NK proliferation medium, and the added NK proliferation medium is used to maintain the cell density at 5×10 5 ~5×10 6 The basal proliferation medium used for supplementation is further preferably the NK-IV additive in the NK cell culture special kit, followed by the KBM581 medium; the cell density is maintained at 5×10 5 ~5×10 6 It is more preferred to maintain the cell density at 5×10 5 The cell density was maintained at 7×10 5 The final cell density was maintained at 1×10 6 Pieces / mL.

[0024] In the present invention, preferably, the autologous plasma is obtained by a preparation method comprising the following steps: centrifuging the umbilical cord blood to obtain a supernatant; sequentially performing inactivation treatment, quick freezing treatment, and centrifugation treatment on the supernatant to obtain the autologous plasma; the inactivation treatment temperature is 50-60°C, and the inactivation treatment time is 20-40 min; the quick freezing treatment temperature is -30--10°C, and the quick freezing treatment time is 5-20 min.

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] Sources of reagents used in the present invention: Enrichment reagent (Stem cell): NK cells are directly enriched from umbilical cord blood through negative selection. The target non-NK cell elimination antibody of this kit recognizes specific cell surface markers. The present invention uses RosetteSep™ antibody cocktail, which can cross-link non-target cells in umbilical cord blood to a variety of red blood cells (RBC) to form an immune group, thereby increasing the density of non-target (rosette-shaped) cells.

[0027] The sample density separation solution was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.; NK cell culture medium: The culture of NK cells mainly requires two types of activation culture medium and proliferation culture medium. The present invention uses a special NK cell culture kit purchased from Beijing Wukang Xinxing Technology Co., Ltd. The kit includes activation culture medium (liquid No. Ⅰ, liquid No. Ⅱ) and proliferation culture medium (liquid No. Ⅳ). During the culture, the volume of the amplification culture medium in the special NK cell culture kit is only 2L, and an additional amplification culture medium-KBM581 culture medium (Corning) is supplemented later. KBM581 culture medium contains 10 ng / mL IL-15 and 1000 IU / mL IL-2; Cell culture supplements (brand: Dakoway); IL-2, brand: Beijing Sihuan Biotechnology; IL-15, Brand: Beijing Tongli Haiyuan Biological.

[0028] Example 1 Step 1: The obtained umbilical cord blood (umbilical cord blood collected within 20 hours of preparation) was restored to room temperature, and centrifuged at 1300g for 10 minutes, with an increase speed of 7 and a decrease speed of 7 to separate the autologous plasma. The cell precipitate after the plasma was removed was restored to its original volume with physiological saline, and the plasma was inactivated at 56°C for 30 minutes, quickly frozen at -20°C for 15 minutes, and stored at 4°C for use after centrifugation.

[0029] Step 2: According to the flow cytometry results of the cord blood, add the cord blood sample to the lymphocyte sample density separation solution at a volume ratio of 2:1, centrifuge at 1000g, 20min, increase speed 3, decrease speed 1 to separate the cord blood mononuclear cells, and wash twice. The centrifugation conditions during washing are 500g, 5min, increase speed 8, decrease speed 8.

[0030] Step 3: The final separated cell suspension was divided into 2.0*106 / mL for inoculation, the volume is 30mL, containing 10% autologous plasma, this time is recorded as day 0. This culture uses the NK cell culture special kit of Beijing Wukang Xinxing Technology Co., Ltd. The reagent is prepared on the spot, and the culture medium used for inoculation is the No. Ⅰ working solution in the kit (with NK-Ⅰ additive added).

[0031] Step 4: On the second day of culture, add 30 mL of working solution No. 1 containing 5% autologous plasma.

[0032] Step 5: On the third day of culture, add 80 mL of working solution III (working solution I and working solution II mixed in equal proportions) containing 5% autologous plasma.

[0033] Step 6: On the 5th day of culture, the activation medium (working solution I and working solution II) added earlier was converted into proliferation medium (working solution IV, total volume 2L). After collecting the cell suspension, centrifuge at 526g for 5min, increase speed 8, decrease speed 8, discard the supernatant, first use a small amount of working solution IV (containing 1 tube of NK-IV additive and 1000 IU / mL IL-2) to resuspend the cells, take samples for counting, and continue to add working solution IV according to the counting results to make the cell density 5*10 5 / mL, containing 5% autologous plasma. If the plasma volume is insufficient, add cell culture supplements at a concentration of 0.20%.

[0034] Step 7: On the sixth day of culture, according to the counting results, add working solution IV to make the cell density 7*105 cells / mL, containing 0.20% cell culture additives.

[0035] Step 8: On the 7th day of culture, according to the counting results, add the remaining IV working solution in the kit. After adding 2L IV working solution, introduce new proliferation medium: KBM 581 medium (containing 10 ng / mL IL-15, 1000 IU / mL IL-2 and 0.20% cell culture supplement) to make the cell density 1*10 6 Pieces / mL.

[0036] Step 9: On the 8th, 9th and 11th days of culture, KBM581 culture medium (containing 0.20% cell culture supplement) was added to keep the cell density at 1.0*10 6 Pieces / mL.

[0037] Step 10: On the 13th day of culture, perform tests such as endotoxin, mycoplasma, fungi, flow cytometry, and killing ability, and harvest the cells after passing the tests.

[0038] Example 2 Step 1: The obtained umbilical cord blood (umbilical cord blood collected within 10 hours from the time of preparation) was restored to room temperature, and centrifuged at 1300g for 10 minutes, with an increase speed of 7 and a decrease speed of 7 to separate the autologous plasma. The cell precipitate after the plasma was removed was restored to its original volume with physiological saline, and the plasma was inactivated at 56°C for 30 minutes, quickly frozen at -20°C for 15 minutes, and stored at 4°C for use after centrifugation.

[0039] Step 2: According to the flow cytometry results of umbilical cord blood, add NK cell enrichment reagent at a concentration of 1.5 μL / mL, mix and let stand for 10 minutes, then add saline to terminate the reaction, then add the umbilical cord blood sample to the lymphocyte sample density separation solution at a volume ratio of 2:1, centrifuge at 1000g, 20min, increase speed 3, decrease speed 1 to separate the umbilical cord blood mononuclear cells, and wash twice. The centrifugation conditions during washing are 500g, 5min, increase speed 8, decrease speed 8.

[0040] Step 3: The final separated cell suspension was divided into 2.0*10 6 / mL for inoculation, the volume is 30mL, containing 10% autologous plasma, this time is recorded as day 0. This culture uses the NK cell culture special kit of Beijing Wukang Xinxing Technology Co., Ltd. The reagent is prepared on the spot, and the culture medium used for inoculation is the No. Ⅰ working solution in the kit (with NK-Ⅰ additive added).

[0041] Step 4: On the second day of culture, add 30 mL of working solution No. 1 containing 5% autologous plasma.

[0042] Step 5: On the third day of culture, add 80 mL of working solution III (working solution I and working solution II mixed in equal proportions) containing 5% autologous plasma.

[0043] Step 6: On the 5th day of culture, the activation medium (working solution I and working solution II) added earlier was converted into proliferation medium (working solution IV, total volume 2L). After collecting the cell suspension, centrifuge at 526g for 5min, increase speed 8, decrease speed 8, discard the supernatant, first use a small amount of working solution IV (containing 1 tube of NK-IV additive and 1000 IU / mL IL-2) to resuspend the cells, take samples for counting, and continue to add working solution IV according to the counting results to make the cell density 5*10 5 / mL, containing 5% autologous plasma. If the plasma volume is insufficient, add cell culture supplements at a concentration of 0.20%.

[0044] Step 7: On the sixth day of culture, according to the counting results, add working solution IV to make the cell density 7*105 cells / mL, containing 0.20% cell culture additives.

[0045] Step 8: On the 7th day of culture, according to the counting results, add the remaining IV working solution in the kit. After adding 2L IV working solution, introduce new proliferation medium: KBM 581 medium (containing 10 ng / mL IL-15, 1000 IU / mL IL-2 and 0.20% cell culture supplement) to make the cell density 1*10 6 Pieces / mL.

[0046] Step 9: On the 8th, 9th and 11th days of culture, KBM581 culture medium (containing 0.20% cell culture supplement) was added to keep the cell density at 1.0*10 6 Pieces / mL.

[0047] Step 10: On the 13th day of culture, perform tests such as endotoxin, mycoplasma, fungi, flow cytometry, and killing ability, and harvest the cells after passing the tests.

[0048] Example 3 Step 1: The obtained umbilical cord blood (umbilical cord blood collected within 20 hours of preparation) was restored to room temperature, and centrifuged at 1300g for 10 minutes, with an increase speed of 7 and a decrease speed of 7 to separate the autologous plasma. The cell precipitate after the plasma was removed was restored to its original volume with physiological saline, and the plasma was inactivated at 56°C for 30 minutes, quickly frozen at -20°C for 15 minutes, and stored at 4°C for use after centrifugation.

[0049] Step 2: According to the flow cytometry results of umbilical cord blood, add NK cell enrichment reagent at a concentration of 1.5 μL / mL, mix and let stand for 10 minutes, then add saline to terminate the reaction, then add the umbilical cord blood sample to the lymphocyte sample density separation solution at a volume ratio of 2:1, centrifuge at 1000g, 20min, increase speed 3, decrease speed 1 to separate the umbilical cord blood mononuclear cells, and wash twice. The centrifugation conditions during washing are 500g, 5min, increase speed 8, decrease speed 8.

[0050] Step 3: The final separated cell suspension was divided into 2.0*10 6 / mL for inoculation, the volume is 30mL, containing 10% autologous plasma, this time is recorded as day 0. This culture uses the NK cell culture special kit of Beijing Wukang Xinxing Technology Co., Ltd. The reagent is prepared on the spot, and the culture medium used for inoculation is the No. Ⅰ working solution in the kit (with NK-Ⅰ additive added).

[0051] Step 4: On the second day of culture, add 30 mL of working solution No. 1 containing 5% autologous plasma.

[0052] Step 5: On the third day of culture, add 80 mL of working solution III (working solution I and working solution II mixed in equal proportions) containing 5% autologous plasma.

[0053] Step 6: On the 5th day of culture, the activation medium (working solution I and working solution II) added earlier was converted into proliferation medium (working solution IV, total volume 2L). After collecting the cell suspension, centrifuge at 526g for 5min, increase speed 8, decrease speed 8, discard the supernatant, first use a small amount of working solution IV (containing 1 tube of NK-IV additive and 1000 IU / mL IL-2) to resuspend the cells, take samples for counting, and continue to add working solution IV according to the counting results to make the cell density 5*10 5 / mL, containing 5% autologous plasma. If the plasma volume is insufficient, add cell culture supplements at a concentration of 0.20%.

[0054] Step 7: On the sixth day of culture, according to the counting results, add working solution IV to make the cell density 7*105 cells / mL, containing 0.20% cell culture additives.

[0055] Step 8: On the 7th day of culture, according to the counting results, add the remaining IV working solution in the kit. After adding 2L IV working solution, introduce new proliferation medium: KBM 581 medium (containing 10 ng / mL IL-15, 1000 IU / mL IL-2 and 0.20% cell culture supplement) to make the cell density 1*10 6 Pieces / mL.

[0056] Step 9: On the 8th, 9th and 11th days of culture, KBM581 culture medium (containing 0.20% cell culture supplement) was added to keep the cell density at 1.0*10 6 Pieces / mL.

[0057] Step 10: On the 13th day of culture, perform tests such as endotoxin, mycoplasma, fungi, flow cytometry, and killing ability, and harvest the cells after passing the tests.

[0058] Experimental example CD3 of NK cells - CD56 + Detection Flow cytometry was used to detect the surface markers of NK cells, including CD3 / CD16+CD56 / CD45 / CD4 / CD19 / CD8. - CD56 + It is a representative marker of NK cells and can directly reflect the proportion of NK cells. Therefore, we only focus on CD3 - CD56 + Quantity analysis.

[0059] The MNC cells before and after separation in step 2 of each scheme of Examples 1 to 3 were tested, and the CD3 -CD56 + With CD3 + The number of detection conditions is as follows Figure 1 shown.

[0060] The results show that CD3 in Example 2 and Example 3 - CD56 + The number of CD3 + The number of samples was reduced, and there was no obvious difference before and after separation in Example 1.

[0061] After 13 days of culture, the NK cells CD3 - CD56 + The number of tests was tested and the results Figure 2 Shown: CD3 corresponding to Examples 1-3 - CD56 + The proportion of NK cells in Example 1 was significantly different from that in other examples. This indicates that the separation and culture scheme provided by the present invention can reduce the number of CD3 + Quantity, increase CD3 - CD56 + The number is more conducive to the cultivation of umbilical cord blood NK cells.

[0062] Determination of NK cell cytotoxic activity The lactate dehydrogenase release method was used to determine the killing effect of NK cells on K562 tumor cells. Figure 3 The killing activity of NK cells after culture was measured. The killing activity of NK cells was evaluated by the effector-target ratio (NK:K562) corresponding to a killing rate of 50%.

[0063] As can be seen from the figure, the effector-target ratio corresponding to a killing rate of 50% in Example 1 is around 5:1, and the effector-target ratio corresponding to a killing rate of 50% in Example 2 and Example 3 is around 1:10, indicating that the NK cultured in the present invention has a strong killing ability.

[0064] NK cell proliferation Figure 4 The cell expansion results of umbilical cord blood NK cells cultured for 13 days. As can be seen from the figure, after 13 days of culture, the number of cells in Example 1 can reach 7.86*10 9 The number of cells corresponding to Example 2 and Example 3 can be increased to 2.59*10 10 pcs, 2.17*10 10 This indicates that the culture scheme provided by the present invention can obtain a large number of NK cells in a short time.

[0065] Referring to the culture method of Chinese patent CN116790499A, the same materials as in Example 1 were used for separation and culture. The culture time was the same as in Example 1. After 13 days of culture, the NK cell CD3 - CD56 + The quantity is tested and the test results are shown in Figure 5 The results showed that CD3 - CD56 + The number of NK cells is 71.84%, and the number of cells is only 5.17*10^9. The proportion of NK cells and the number of cells are significantly different from those in Example 1, indicating that the embodiments provided by the present invention can achieve better results.

[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for inducing and culturing umbilical cord blood NK cells, characterized in that: The following steps are involved: S1. Mix the umbilical cord blood and NK cell enrichment reagent and let it stand, then add the lymphocyte sample density separation solution, and centrifuge to obtain umbilical cord blood mononuclear cells; S2, inoculating the umbilical cord blood mononuclear cells into NK activation medium, culturing for 3 to 8 days, and obtaining intermediate cells; S3, inoculating the intermediate cells into NK proliferation medium and culturing for more than 7 days to obtain umbilical cord blood NK cells; The dosage of the NK cell enrichment reagent is 0~5μL / mL; The NK activation medium contains autologous plasma; The NK proliferation culture medium contains autologous plasma.

2. The method for inducing and culturing umbilical cord blood NK cells according to claim 1, characterized in that: The dosage of the NK cell enrichment reagent is 1~2 μL / mL.

3. The method for inducing and culturing umbilical cord blood NK cells according to claim 1, characterized in that: In S1, the standing time is 5 to 20 minutes; and / or, the volume ratio of the cord blood to the lymphocyte sample density separation fluid is 1.5-3:1; And / or, the centrifugal speed is 800~1200g; And / or, the centrifugation time is 10 to 30 minutes.

4. The method for inducing and culturing umbilical cord blood NK cells according to claim 1, characterized in that: In S2, the inoculation volume is 5×10 5 ~5×10 6 Pieces / mL.

5. The method for inducing and culturing umbilical cord blood NK cells according to claim 1, characterized in that: The NK activation medium contains autologous plasma with a final volume concentration of 5-20%; The NK activation medium is the NK-I additive and / or the NK-II additive in the NK cell culture special kit, and the NK cell culture special kit comes from Beijing Wukang Xinxing Technology Co., Ltd.

6. The method for inducing and culturing umbilical cord blood NK cells according to claim 5, characterized in that: In S2, the culture process includes adding NK activation medium, and the frequency of the addition is 1 to 2 days / time.

7. The method for inducing and culturing umbilical cord blood NK cells according to claim 1, characterized in that: The NK proliferation culture medium contains a basic proliferation culture medium, autologous plasma and a cell culture additive; The final volume concentration of the autologous plasma is 3-10%; The final volume concentration of the cell culture additive is 0.1-0.3%.

8. The method for inducing and culturing umbilical cord blood NK cells according to claim 7, characterized in that: The NK proliferation culture medium also contains IL-15 and / or IL-2; The final concentration of IL-15 is 5-15 ng / mL; The final concentration of IL-2 is 800-1200 IU / mL; And / or, the type of the basic proliferation medium is KBM 581 medium and / or NK-IV additive in a special kit for NK cell culture, and the special kit for NK cell culture comes from Beijing Wukang Xinxing Technology Co., Ltd.

9. The method for inducing and culturing umbilical cord blood NK cells according to claim 7, characterized in that: In S3, the culture process includes adding NK proliferation medium, and the added NK proliferation medium is used to maintain the cell density at 5×10 5 ~5×10 6 Pieces / mL.

10. The method for inducing and culturing umbilical cord blood NK cells according to any one of claims 1 to 9, characterized in that: The autologous plasma is obtained by a preparation method comprising the following steps: Centrifuging the umbilical cord blood to obtain a supernatant; The supernatant is sequentially inactivated, quick-frozen, and centrifuged to obtain autologous plasma; The temperature of the inactivation treatment is 50-60°C, and the time of the inactivation treatment is 20-40 minutes; The temperature of the quick freezing treatment is -30 to -10°C, and the time of the quick freezing treatment is 5 to 20 minutes.

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