Method for enhancing killing effect of universal NK cell cryopreservation preparation

Abstract

The invention relates to the technical field of cell therapy, and discloses a method for enhancing the killing effect of a universal NK cell cryopreserved preparation. The method comprises the following steps: recovering a cryopreserved NK cell preparation, centrifuging, discarding a supernatant, resuspending NK cells by using a basic culture medium, adjusting the cell density to 2 * 10 <-5 > * 10 <-1 > / ml, adding fetal calf serum, IL-2 and IL-15 into the basic culture medium until the final concentrations are 5-10%, 200 IU / ml-400 IU / ml and 50 ng / ml-150 ng / ml respectively, and culturing in an incubator with the temperature of 37 DEG C, the CO content of 5% and the saturated humidity for 12-72 hours. By optimizing culture conditions and cell factor combination, the NK cell killing effect is recovered from an invalid state to a high level of 94.7%, and an effective technical scheme is provided for clinical application of a universal NK cell cryopreservation preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell therapy, and particularly relates to a method for enhancing the killing effect of a universal NK cell cryopreservation preparation. BACKGROUND

[0002] Natural killer cells are a core component of the innate immune system, and have the ability to quickly recognize and kill abnormal cells, and can non-specifically remove tumor cells and virus-infected cells without antigen pre-sensitization. In recent years, NK cell-based immunotherapy has shown great potential for clinical application in the field of tumor treatment.

[0003] Currently, NK cell treatment preparations mainly adopt a fresh preparation mode, and are produced according to the use date. The NK cell preparation used is freshly filled on the day of reinfusion. This mode has a relatively long cycle, about 15 days or so, and the user cannot arbitrarily change the use date, which has obvious limitations. In comparison, a universal NK cell cryopreservation preparation that is safe, convenient, and has good killing effect is a better choice. The prepared NK cells can be stored for a long time using the finished cryopreservation solution, and the user can use it at any time, which has better clinical application flexibility.

[0004] NK cells are extremely sensitive to cryopreservation and resuscitation, and the survival rate and cytotoxicity of NK cells after resuscitation are significantly reduced. Studies have found that the addition of cytokines such as IL-2 can activate NK cell function again. However, the universal NK cell cryopreservation preparation prepared by the prior art still has the problem of poor killing effect after resuscitation.

[0005] The prior art usually adopts the following route: umbilical cord blood is separated to obtain umbilical cord blood mononuclear cells, which are cryopreserved; when NK cells need to be prepared, the umbilical cord blood mononuclear cells are resuscitated, cultured, and finally harvested NK cells, which are centrifuged and washed, resuspended with finished cryopreservation solution, and cryopreserved. When the NK cell cryopreservation preparation is needed, the preparation can be resuscitated for the user to reinfuse. Experiments have found that the resuscitated preparation has poor killing effect, and the possible reason is that NK cells are relatively fragile and sensitive as immune cells, and the state of the cells after resuscitation is poor, so the killing ability of the cells on target cells is poor.

[0006] In addition, the culture environment used by the prior art is usually C-level plus a biological safety cabinet or B-level plus a biological safety cabinet, which has the risk of cell product contamination, affecting the safety of the product.

[0007] Therefore, it is urgent to develop a method for effectively enhancing the killing effect of a universal NK cell cryopreservation preparation to meet the needs of clinical application. SUMMARY

[0008] The application aims to provide a method for enhancing the killing effect of a universal NK cell cryopreservation preparation, so as to solve the problem of poor killing effect of an NK cell cryopreservation preparation after recovery in the prior art.

[0009] To achieve the above-mentioned object, the application adopts the following technical scheme:

[0010] The application provides a method for enhancing the killing effect of a universal NK cell cryopreservation preparation, comprising the following steps: recovering a frozen NK cell preparation, transferring the recovered NK cell to a centrifuge tube containing a basic culture medium, discarding the supernatant after centrifugation; resuspending the NK cell with the basic culture medium and counting, adjusting the cell density to 2×10 6 to 5×10 6 to 5% to 10%, the final concentration of the IL-2 is 200 IU / ml to 400 IU / ml, and the final concentration of the IL-15 is 50 ng / ml to 150 ng / ml; placing the adjusted NK cell suspension in a culture box for culture for 12 h to 72 h, and the culture condition of the culture box is 37℃, 5% CO2 concentration and saturated humidity.

[0011] Preferably, the centrifugation condition is 300 g to 500 g centrifugation for 5 min to 10 min.

[0012] Preferably, the cell density is adjusted to 2×10 6 to 3×10 6 .

[0013] Preferably, the culture time is 24 h.

[0014] More preferably, the final concentration of the fetal bovine serum is 10%, the final concentration of the IL-2 is 400 IU / ml, the final concentration of the IL-15 is 100 ng / ml, and the culture time is 24 h.

[0015] In an embodiment of the application, at least one cytokine selected from IL-12, IL-18 and IL-21 is further added to the basic culture medium.

[0016] In the specific embodiment of the application, the preparation of the NK cell preparation adopts an isolator operating environment, and the whole process from the separation of umbilical cord blood to the final output of the NK cell preparation is operated in a C-class isolator, so that the safety of the prepared cells is effectively improved.

[0017] The application has the following beneficial effects:

[0018] Firstly, the application adds a culture recovery step after the freezing and recovery of the NK cell preparation, and optimally adds fetal bovine serum, IL-2 and IL-15 and the like cytokines, so that the NK cells can fully recover their cell state and biological function after recovery. Experimental data show that after 24h of culture, the killing effect of NK cells is restored to a high level of more than 90%, which is significantly better than the control group directly recovered for use. Among them, the killing rate of Example 5 with the simultaneous addition of IL-2 and IL-15 is as high as 92.6%, which is the best.

[0019] Secondly, the application determines the optimal combination of culture parameters. Through systematic experimental design, it is clear that the optimal conditions are fetal bovine serum final concentration of 10%, IL-2 final concentration of 400IU / ml, IL-15 final concentration of 100ng / ml, and culture time of 24h, which can fully restore the killing activity of NK cells, and can save cost and simplify the operation process.

[0020] Thirdly, the application uses a C-class isolator operating environment, which can more effectively reduce the risk of cell product contamination compared with the traditional biological safety cabinet, improve the safety of the prepared cells, and better meet the requirements of clinical application.

[0021] Fourthly, the method of the application is simple and easy to operate, does not require complex equipment and operation, is easy to scale up production and clinically applied, and provides a feasible technical scheme for the clinical application of a universal NK cell freezing preparation.

[0022] Fifthly, the application significantly enhances the cytotoxicity of NK cells through the synergistic effect of IL-2 and IL-15. IL-15 can promote the proliferation, survival and effector function of NK cells, and has a synergistic effect when used in combination with IL-2, so that the killing activity of NK cells is maximally restored and improved. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the application clearer, the application will be described in detail below in combination with specific examples.

[0024] The base medium used in the application can be a commonly used lymphocyte culture medium in the art, such as X-VIVO 15 medium, AIM V medium, RPMI 1640 medium and the like. The complete culture medium refers to a culture medium in which cytokines and growth factors are added to the base medium.

[0025] The finished freezing solution used in the application can be a commercially available cell freezing solution, such as a dimethyl sulfoxide-containing freezing solution, such as CryoStor CS10 freezing solution, CELLBANKER series freezing solution and the like.

[0026] The commercial inducement kit used in the present application refers to a commercially available kit for NK cell inducement and expansion, which contains cytokines and growth factors required for promoting NK cell proliferation and differentiation.

[0027] Example 1

[0028] The present embodiment provides a method for enhancing the killing effect of a universal NK cell cryopreservation preparation, comprising the following steps:

[0029] Preparation of NK cell preparation: 50 ml of umbilical cord blood was transferred into an isolator in a class C clean environment, and diluted at a volume ratio of umbilical cord blood to physiological saline of 1:1. A gradient density centrifuge tube was taken, 10 ml of ficoll was added to the centrifuge tube, and 10 ml of diluted umbilical cord blood was added to the centrifuge tube for centrifugation, and the centrifugation condition was 700 g for 20 min. After centrifugation, the white membrane layer in each tube was taken to harvest the umbilical cord blood mononuclear cells. 10 ml of physiological saline was added to the collected umbilical cord blood mononuclear cells, and the cell suspension was centrifuged at 500 g for 10 min, and the supernatant was discarded after centrifugation. 10 ml of physiological saline was used to resuspend the cells, and the cell suspension was centrifuged at 500 g for 10 min, and the supernatant was discarded after centrifugation. The washed umbilical cord blood mononuclear cells were counted, and the cells were resuspended with the finished product cryopreservation solution, and packaged into cryopreservation tubes according to the specification of 1×10 7 cells / ml. A program cooling instrument was used for cryopreservation.

[0030] The cryopreserved umbilical cord blood mononuclear cells were recovered on day 0, and the recovered umbilical cord blood mononuclear cells were transferred into an isolator for experimental operation. The umbilical cord blood mononuclear cells were first transferred to a 50 ml centrifuge tube containing 10 ml of basal medium, centrifuged at 300 g for 5 min, and the supernatant was discarded after centrifugation. The cells were resuspended with complete medium and counted, and then a commercial inducement kit was added. The final concentration of umbilical cord blood mononuclear cells was adjusted to 1×10 5 cells / ml with complete medium, and the cells were placed in a honeycomb incubator for culture at 37℃, 5% CO2, and saturated humidity.

[0031] On day 4, the cells were taken out of the honeycomb incubator, mixed and counted, and the cell density was adjusted to 1×10 5 cells / ml with complete medium, and the cells were returned to the honeycomb incubator for continued culture.

[0032] On day 6, the cells were taken out of the honeycomb incubator, mixed and counted, and the cell density was adjusted to 2×10 5 cells / ml with complete medium, and the cells were returned to the honeycomb incubator for continued culture.

[0033] On day 9, the cells were taken out of the honeycomb incubator, mixed and counted, and the cell density was adjusted to 8×10 5 cells / ml with complete medium, and the cells were returned to the honeycomb incubator for continued culture.

[0034] On day 11, the cells were taken out of the incubator, mixed and counted. The cell density was adjusted to 1 x 10 6 cells / ml using complete medium, and the cells were returned to the incubator.

[0035] On day 13, the cells were taken out of the incubator, mixed and counted. The cell density was adjusted to 1.5 x 10 6 cells / ml using complete medium, and the cells were returned to the incubator.

[0036] On day 15, the cells were taken out of the incubator, mixed and counted. The cells were transferred to a 500 ml centrifuge cup and centrifuged at 300 g for 5 min. The supernatant was discarded after centrifugation. The cells were resuspended in physiological saline containing 0.1% HSA and centrifuged to wash the cells. The washing was repeated four times. A small amount of finished cryopreservation solution was added to the washed cells to resuspend and count the cells. The cell density was adjusted to 5 x 10 7 cells / ml according to the counting result. The cells were filled into a cryopreservation bag, 1 x 10 9 cells per bag, and the NK cell preparation was cryopreserved using a program cooling instrument.

[0037] After the NK cell preparation was cryopreserved for 3 days, one bag of preparation was thawed. The thawed preparation was transferred into an isolator. The preparation was first transferred to a 50 ml centrifuge tube containing 10 ml of basic medium, centrifuged at 300 g for 5 min, and the supernatant was discarded after centrifugation. The cells were resuspended in a small amount of basic medium and counted. The cell density was adjusted to 2 x 10 6 cells / ml, and 10% fetal bovine serum, 400 IU / ml IL-2 and 100 ng / ml IL-15 were added to the basic medium. The cells were cultured for 24 h. The culture conditions were 37°C, 5% CO2, and saturated humidity.

[0038] Example 2

[0039] The difference between this example and Example 1 is that the umbilical cord blood was used in an amount of 200 ml, the dilution ratio was 1:2, the ficoll was used in an amount of 20 ml, the dilution of the umbilical cord blood was used in an amount of 20 ml, the centrifugation condition was 1500 g for 40 min, the physiological saline was used in an amount of 200 ml, the cryopreservation specification of the umbilical cord blood mononuclear cells was 5 x 10 7 cells / ml, the basic medium was used in an amount of 40 ml, the centrifugation condition was 500 g for 10 min, the final concentration of the umbilical cord blood mononuclear cells was 10 x 10 5 cells / ml, the cell density was adjusted to 5 x 10 5 cells / ml on day 4, the cell density was adjusted to 6 x 10 5 cells / ml on day 6, and the cell density was adjusted to 12 x 105 Cells / ml, the cell density was adjusted to 1.5 × 10⁶ on day 11. 6 Cells / ml, the cell density was adjusted to 2.0 × 10⁶ on day 13. 6 Cells / ml, centrifuged at 500g for 10 min, washing buffer containing 0.5% HSA, NK cell density adjusted to 10 × 10⁶ cells / ml. 7 Cells / ml, after resuscitation, the cell density was adjusted to 5×10⁶ / ml. 6 The concentration of fetal bovine serum was 5%, the concentration of IL-2 was 200 IU / ml, the concentration of IL-15 was 50 ng / ml, and the culture time was 12 h.

[0040] Example 3

[0041] The difference between this embodiment and Embodiment 1 is that the cell density is adjusted to 3×10⁻⁶. 6 The culture time was 48 hours, with each cell per ml being incubated.

[0042] Example 4

[0043] The difference between this embodiment and Embodiment 1 is that the culture time is 72 hours.

[0044] Example 5

[0045] The difference between this embodiment and Example 1 is that IL-21 was added to the basal culture medium to make its final concentration 100 ng / ml.

[0046] Comparative Example 1

[0047] The difference between this comparative example and Example 1 is that after reviving the cryopreserved NK cell preparation, samples were taken directly for testing without performing a culture recovery step.

[0048] Comparative Example 2

[0049] The difference between this comparative example and Example 1 is that no fetal bovine serum was added during the post-resuscitation culture; only IL-2 and IL-15 were added.

[0050] Comparative Example 3

[0051] The difference between this comparative example and Example 1 is that IL-2 and IL-15 were not added during the post-resuscitation culture; only fetal bovine serum was added.

[0052] Comparative Example 4

[0053] The difference between this comparative example and Example 1 is that no fetal bovine serum, IL-2, and IL-15 were added during the post-resuscitation culture; only basal culture medium was used.

[0054] The following performance tests were performed on the NK cells prepared in the above embodiments and comparative examples:

[0055] 1. Cell viability detection

[0056] Cell viability was detected by trypan blue staining. 10 μl of cell suspension was mixed with 10 μl of trypan blue staining solution, and the number of live cells and dead cells was counted under a microscope to calculate the viability.

[0057] 2. Surface marker detection

[0058] Flow cytometry was used to detect the expression of NK cell surface markers CD3 and CD56. 1 × 10 6 cells were taken, and fluorescently labeled anti-CD3 and anti-CD56 antibodies were added, and incubated at 4°C in the dark for 30 min. After washing, flow cytometry was used to detect the proportion of CD3⁻CD56⁺ cells.

[0059] 3. Killing experiment

[0060] The lactate dehydrogenase release method was used to detect the killing activity of NK cells on K562 target cells. Different effector-to-target ratios were set, i.e. 0:1, 1:10, 1:4, 1:2, and 1:1. NK cells and K562 target cells were mixed at the set ratio, and incubated at 37°C for 4 h. The amount of lactate dehydrogenase released in the supernatant was detected, and the killing rate was calculated.

[0061] The test results are shown in Table 1.

[0062] Table 1. Performance test results of examples and comparative examples

[0063] Sample Cell viability (%) CD3- CD56+ (%) Killing rate (%) Effector:target ratio 0:1 Killing rate (%) Effector:target ratio 1:10 Killing rate (%) Effector:target ratio 1:4 Killing rate (%) Effector:target ratio 1:2 Killing rate (%) Effector:target ratio 1:1 Example 1 92 88 15.2 24.1 44.7 68.4 90.4 Example 2 85 82 14.8 20.5 43.3 65.4 84.3 Example 3 90 87 15.0 25.5 48.3 61.4 87.8 Example 4 91 86 15.1 23.3 45.5 65.5 88.1 Example 5 93 90 14.9 38.2 70.5 82.6 94.7 Comparative Example 1 88 85 15.3 12.6 14.9 15.7 16.2 Comparative Example 2 87 84 15.1 18.4 28.7 35.2 42.8 Comparative Example 3 86 83 15.4 16.8 24.3 29.6 36.5 Comparative Example 4 85 82 15.2 14.2 18.5 21.3 25.7

[0064] As shown in Table 1, the NK cells prepared in Examples 1 to 5 all have good cell viability and surface marker expression, and more importantly, exhibit excellent killing activity. At an effector-to-target ratio of 1:1, the killing rate is all above 84%, among which the killing rate of Example 1 reaches 90.4%, and the killing rate of Example 5 is as high as 94.7%, which is the best. The killing effect shows a clear gradient relationship with the increase of the effector-to-target ratio, indicating that the NK cells have a dose-dependent killing ability.

[0065] Example 5 is based on Example 1 with the additional addition of IL-21, and the killing effect is significantly improved. At the effector-to-target ratio of 1:10, the killing rate of Example 5 is 38.2%, which is significantly higher than 24.1% of Example 1; at the effector-to-target ratio of 1:4, the killing rate of Example 5 reaches 70.5%, which is much higher than 44.7% of Example 1; at the effector-to-target ratio of 1:2, the killing rate of Example 5 reaches 82.6%, which is significantly higher than 68.4% of Example 1; at the effector-to-target ratio of 1:1, the killing rate of Example 5 reaches 94.7%, which is further improved compared with 90.4% of Example 1. This fully shows that the combined use of IL-2, IL-15 and IL-21 can produce significant synergistic effect, and the killing function of NK cells can be maximally enhanced.

[0066] In contrast, the detection after the resuscitation of Comparative Example 1 shows that the cell viability and surface marker expression are normal, but the killing experiment shows that the killing rate at each effector-to-target ratio is basically consistent, and is between 12% and 16%, without obvious gradient relationship, indicating that the NK cell preparation has no in vitro killing effect. This verifies the necessity of the technical scheme of the present application, that is, the NK cell cryopreservation preparation needs to be cultured and recovered after resuscitation to exert the killing activity.

[0067] Comparative Examples 2 to 4 respectively verify the synergistic effect of fetal bovine serum, IL-2 and IL-15. Comparative Example 2 only adds IL-2 and IL-15 without adding fetal bovine serum, Comparative Example 3 only adds fetal bovine serum without adding IL-2 and IL-15, and the killing effects of the two are significantly lower than that of Example 1 which simultaneously adds the three. Comparative Example 4 does not add fetal bovine serum, IL-2 and IL-15, and the killing effect is the worst, only slightly better than Comparative Example 1 without culture. These data fully show that the synergistic addition of fetal bovine serum, IL-2 and IL-15 plays an important role in recovering the killing activity of NK cells.

[0068] NK cells will experience multiple stresses such as osmotic pressure change, ice crystal formation and temperature shock during freezing and resuscitation, resulting in cell membrane damage, metabolic disorder and down-regulation of functional protein expression. Although the resuscitated NK cells maintain basic survival ability and phenotypic characteristics, their intrinsic cytotoxic function is in an inhibited state.

[0069] The present application provides suitable culture conditions after resuscitation, so that the NK cells can gradually recover their physiological state. Fetal bovine serum provides rich growth factors, hormones and nutrients, supports the repair of cell membrane and the recovery of metabolic function. IL-2 as a key cytokine can bind to the IL-2 receptor on the surface of NK cells, activate the intracellular signal transduction pathway, promote cell proliferation and activation. IL-2 can also up-regulate the expression of NK cell surface activating receptors, enhance their recognition ability to target cells, and promote the synthesis and release of cytotoxic molecules such as perforin and granzyme.

[0070] IL-15 plays a crucial role in the development, maturation, and functional maintenance of NK cells. IL-15 can bind to the β and γ subunits of the IL-2 receptor, activating multiple signaling pathways such as JAK-STAT, PI3K-AKT, and MAPK, promoting the proliferation, survival, and effector function of NK cells. IL-15 can also upregulate the expression of activating receptors such as NKG2D, NKp30, NKp44, and NKp46 on the surface of NK cells, enhancing their recognition and killing ability of tumor cells. In addition, IL-15 can promote the secretion of cytokines such as IFN-γ and TNF-α by NK cells, enhancing their anti-tumor immune effects.

[0071] The synergistic effect of fetal bovine serum, IL-2, and IL-15 is reflected in multiple aspects. Fetal bovine serum provides the necessary metabolic basis and nutritional support for the signal transduction of IL-2 and IL-15, while IL-2 and IL-15 activate cells from a dormant state to a functional state. IL-2 and IL-15 exert synergistic effects through different receptor subunit combinations and signaling pathways, collectively promoting the proliferation, activation, and recovery of cytotoxic function of NK cells. The combined effect of the three enables NK cells to recover their inherent killing activity in a relatively short period of time, regaining the ability to recognize and eliminate tumor cells.

[0072] IL-21, as another important member of the γ-chain cytokine family, can further enhance the function of NK cells. IL-21 promotes the maturation and cytotoxic function of NK cells by activating the STAT3 signaling pathway. IL-21 can also enhance the sensitivity of NK cells to IL-2 and IL-15 signals, producing a synergistic effect. The data in Example 5 fully demonstrate the superiority of the combined use of IL-2, IL-15, and IL-21, which exhibit the strongest killing activity at all effector-to-target ratios.

[0073] Optimization of culture time is also crucial. Although 12h of culture can partially restore cell function, the effect is not as good as 24h. The effects of 24h, 48h, and 72h of culture are similar, and considering cost and operational convenience, 24h is the best choice. This time window corresponds exactly to the complete process of NK cells from stress recovery to functional activation.

[0074] The technical scheme of the present application provides a feasible solution for the clinical application of the general NK cell cryopreservation preparation, so that the cryopreservation preparation can quickly recover the killing activity after resuscitation, achieve the therapeutic effect even exceeding that of the fresh preparation, and meanwhile, the advantages of flexible use and easy standardized management of the cryopreservation preparation are retained. In particular, through the optimized combination of various cytokines such as IL-2, IL-15 and IL-21, the anti-tumor potential of NK cells can be maximally stimulated, and more effective cell treatment products are provided for tumor immunotherapy.

[0075] The above merely describes the preferred embodiments of the present application, and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. The above merely describes the preferred embodiments of the present application, and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for enhancing the killing effect of a universal NK cell cryopreservation preparation, characterized in that... This includes the following steps: Revive the frozen NK cell preparation, transfer the revived NK cells to centrifuge tubes containing basal culture medium, centrifuge, and discard the supernatant; The NK cells were resuspended in basal culture medium and counted, and the cell density was adjusted to 2 × 10⁶ cells / year. 6 5 x 10 cells / ml 6 pcs / ml; Fetal bovine serum, interleukin IL-2, and interleukin IL-15 were added to the basal culture medium to achieve a final concentration of 5%-10% for the fetal bovine serum, a final concentration of 200 IU / ml-400 IU / ml for the IL-2, and a final concentration of 50 ng / ml-150 ng / ml for the IL-15. The adjusted NK cell suspension was placed in an incubator and cultured for 12-72 hours. The incubator conditions were 37°C, 5% CO2 concentration, and saturated humidity.

2. The method according to claim 1, characterized in that... The centrifugation conditions are 300g-500g for 5min-10min.

3. The method according to claim 1, characterized in that... The cell density was adjusted to 2×10⁻⁶. 6 3 x 10 cells / ml 6 per ml.

4. The method according to claim 1, characterized in that... The culture time is 24 hours.

5. The method according to claim 1, characterized in that... The final concentration of the fetal bovine serum was 10%, the final concentration of IL-2 was 400 IU / ml, the final concentration of IL-15 was 100 ng / ml, and the culture time was 24 h.

6. The method according to claim 1, characterized in that... At least one cytokine selected from IL-12, IL-18, and IL-21 is also added to the basal culture medium.

7. The method according to claim 1, characterized in that... The preparation method of the NK cell preparation includes the following steps: Transfer 50ml-200ml of umbilical cord blood into the isolator and dilute it with saline at a volume ratio of 1:1 to 1:

2. Take a gradient density centrifuge tube, add 10ml-20ml of Ficoll, add the diluted umbilical cord blood to the centrifuge tube and centrifuge at 700g-1500g for 20min-40min. After centrifugation, collect the white membrane layer to harvest umbilical cord blood mononuclear cells. Add physiological saline to the collected umbilical cord blood mononuclear cells, centrifuge and wash, then resuspend the cells in the prepared cryopreservation solution at a ratio of 1×10⁻⁶. 7 5 x 10 cells / ml 7 Frozen storage at a specification of 1 unit / ml; The frozen umbilical cord blood mononuclear cells were revived, centrifuged, resuspended in complete culture medium, and counted. NK cell induction and expansion culture were then performed using a commercial induction kit, and the final concentration of umbilical cord blood mononuclear cells was adjusted to 1 × 10⁻⁶ cells using complete culture medium. 5 pcs / ml - 10×10 5 Cells / ml, placed in a honeycomb incubator for incubation; NK cells were harvested on day 15 of culture, centrifuged, washed, resuspended in cryopreservation buffer, and counted. The cell density was adjusted to 5 × 10⁶ cells / year. 7 pcs / ml - 10×10 7 Quantity / ml, filled into cryopreservation bags, 1×10 per bag 9 Cells were cryopreserved using a programmed cooling system.

8. The method according to claim 7, characterized in that... The complete culture medium is a culture medium for which cytokines and growth factors are added to the basic culture medium.

9. The method according to claim 7, characterized in that... The isolator is designed for use in Class C cleanroom environments.

10. The method according to claim 7, characterized in that... Cells were removed from the honeycomb incubator on days 4, 6, 9, 11, and 13, mixed, counted, and then adjusted to a cell density of 1×10⁻⁶ cells using complete culture medium. 5 5 x 10 cells / ml 5 10 cells / ml, adjusted to 2×10 on day 6. 5 6 x 10 units / ml 5 8 x 10^9 cells / ml, adjusted to 8 x 10^9 cells / ml on day 9. 5 12 x 10 cells / ml 5 10 cells / ml, adjusted to 1×10 on day 11. 6 1.5 × 10⁻¹¹ ml 6 The concentration of cells / ml was adjusted to 1.5 × 10⁻⁶ on day 13. 6 pcs / ml - 2.0 × 10 6 per ml.