A large-scale culture method of NK cells and culture medium used

Abstract

The present invention proposes a large-scale culture method of NK cells and a culture medium used, belonging to the technical field of NK cell culture. Fetal bovine serum, penicillin, streptomycin, and magnetic microspheres for stimulating amplification are added to the ACTM culture medium, and the mixture is stirred and evenly mixed to obtain a culture medium. The present invention provides a large-scale culture method of NK cells, which can efficiently activate and amplify NK cells, does not use autologous serum, is safe and effective, can better promote the proliferation of NK cells, prolong their vitality, is safe and has no side effects, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of NK cell culture, and in particular to a large-scale culture method of NK cells and a culture medium used therein. Background Art

[0002] Natural killer cells (NK) are important immune cells in the body. They are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in the occurrence of hypersensitivity and autoimmune diseases in some cases. As the first line of defense of the body's defense system, NK cells can not only play their main anti-tumor effect in the innate immune system, but also can secrete different cytokines and various chemokines in the early stage of immune response to regulate the body's acquired immune response. They are indispensable effector cells for the body to exert immune effects. In recent years, NK cells have received more extensive attention and clinical applications compared with other cells used for immunotherapy such as αβT cells, γδT cells and CIK cells due to their advantages such as high cell killing activity, rapid onset and no MHC restriction. NK cells are crucial for the body's defense and anti-tumor, but the NK cells in tumor patients are usually impaired. Therefore, killing tumor cells by exogenous introduction of normal or genetically modified NK cells with enhanced functions, namely NK cell adoptive therapy, is currently the forefront and hot spot of cancer treatment. In addition, NK cell therapy also has certain therapeutic effects on viral or bacterial infections, immune regulation and other related diseases, as well as anti-aging.

[0003] However, the number of NK cells is small, accounting for only 10%-15% of lymphocytes in peripheral blood, and the content of NK in umbilical cord blood is even lower, only about 5%. The content of NK cells in normal human peripheral blood and umbilical cord blood is far from meeting the needs of clinical treatment. In the earliest clinical trials, people often used the immunomagnetic bead method to separate NK cells (CD3- / CD56+) from the blood, and then used serum-free culture medium plus autologous plasma plus IL-2 or other cytokines to amplify the purified NK cells. The NK cells obtained by this method are of high purity, but due to the lack of support from other immune cells, the NK cell amplification multiple is not high. In addition, the immunomagnetic bead method is very expensive to purify NK cells and requires special instruments and equipment, which is not conducive to the clinical promotion of NK cell therapy. At present, most studies use mononuclear cells as starting cells to culture NK cells, and use K562 cells as trophoblasts to specifically activate and amplify NK cells. K562 cells are a type of leukemia cell, which is often used as a killing target cell for activated NK cells. However, the microcapsules released by K562 cells can cause normal hematopoietic cells to become malignant. Therefore, the safety of using K562 cells as trophoblasts in clinical applications, especially their long-term safety, is a question worthy of discussion.

[0004] Therefore, how to overcome the above-mentioned deficiencies in the existing technology and develop a new in vitro large-scale culture method for human NK cells that can efficiently activate and amplify NK cells, does not require the use of autologous serum, and is safe and effective is a technical problem that urgently needs to be solved in this field. Summary of the invention

[0005] The purpose of the present invention is to propose a large-scale culture method of NK cells and the culture medium used, which can efficiently activate and amplify NK cells, does not use autologous serum, is safe and effective, can better promote NK cell proliferation, prolong its activity, is safe and has no side effects, and has broad application prospects.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The invention provides a culture medium. Fetal bovine serum, penicillin, streptomycin and amplification-stimulating magnetic microspheres are added to ACTM (full name: ALyS505NK-AC, purchased from Zhuhai Beso Biotechnology Co., Ltd.) culture medium, and the mixture is stirred and evenly mixed to prepare the culture medium.

[0008] As a further improvement of the present invention, the content of fetal bovine serum in the ACTM culture medium is 7-12wt%, the content of penicillin is 80-120U / mL, the content of streptomycin is 80-120U / mL, and the content of stimulating amplification-promoting magnetic microspheres is 100-200μg / mL.

[0009] As a further improvement of the present invention, the preparation method of the stimulation amplification promoting magnetic microspheres is as follows:

[0010] S1. dissolving ferric chloride, ferrous chloride and a hydrophilic emulsifier in water to obtain an aqueous phase; dissolving an alkyl orthosilicate and a lipophilic emulsifier in ethyl acetate to obtain an oil phase; dropping the aqueous phase into the oil phase, emulsifying, dropping ammonia water, heating and stirring to react, centrifuging, washing, drying, and calcining to obtain silica balls coated with magnetic ferrosoferric oxide;

[0011] S2. The silica balls coated with magnetic ferroferric oxide are added to water, tannic acid and a catalyst are added, the reaction is heated and stirred, separated by a magnet, washed, and dried to obtain modified silica balls coated with magnetic ferroferric oxide;

[0012] S3. Add the modified magnetic ferroferric oxide-coated silica spheres into water, add NHS (N-Hydroxysuccinimide, N-hydroxysuccinimide) and EDC (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide), stir to activate, add phycocyanin and silk protein peptide, stir to react, separate with a magnet, wash, freeze-dry, and obtain protein-coupled modified magnetic ferroferric oxide-coated silica spheres;

[0013] S4. Add protein-coupled modified magnetic ferroferric oxide-coated silica spheres into water, add NHS and EDC, stir to activate, add cell complex factors, stir to react, separate with a magnet, wash, and freeze-dry to obtain stimulation amplification-promoting magnetic microspheres.

[0014] As a further improvement of the present invention, the mass ratio of ferric chloride, ferrous chloride, hydrophilic emulsifier, alkyl orthosilicate and lipophilic emulsifier in step S1 is 3.24:1.26:0.2-0.5:3-5:0.1-0.2, the hydrophilic emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80 and Tween-85; the lipophilic emulsifier is selected from at least one of Span-20, Span-40, Span-60, Span-80 and Span-85; the pH value of the solution is adjusted to 9-10 by dripping ammonia water, the temperature of the heating and stirring reaction is 70-80°C, the time is 4-7h, the emulsification speed is 5000-8000r / min, the time is 15-25min, the drying temperature is 60-70°C, the time is 2-4h, the calcination temperature is 500-700°C, and the time is 1-2h.

[0015] As a further improvement of the present invention, the mass ratio of the silica balls coated with magnetic ferrosoferric oxide, tannic acid and catalyst in step S2 is 10:3-4:0.5-1, the catalyst is a Tris-HCl solution with a pH value of 8.5-9.5, the temperature of the heating and stirring reaction is 40-50°C, and the time is 3-5h.

[0016] As a further improvement of the present invention, the mass ratio of the modified silica spheres coated with magnetic ferroferric oxide, NHS, EDC, phycocyanin and silk fibroin peptide in step S3 is 10:2-3:1-3:2-3:4-5, the stirring activation time is 20-30 minutes, and the stirring reaction time is 10-12 hours.

[0017] As a further improvement of the present invention, the mass ratio of the protein-coupled modified magnetic ferroferric oxide-coated silica spheres, NHS, EDC and cell complex factors in step S4 is 10-12:2-3:1-3:4-5, and the cell complex factors are selected from at least two of IL-2 (interleukin-2), IL-15 (interleukin-15), IL-12 (interleukin-12), IL-18 (interleukin-18), and IL-21 (interleukin-21), preferably, IL-15 and IL-12, with a mass ratio of 10:3-7; the stirring activation time is 20-30 min, and the stirring reaction time is 8-10 h.

[0018] The present invention further protects a method for large-scale culture of NK cells, which comprises inoculating NK cells in the above-mentioned culture medium, culturing, stirring with an external magnetic field, centrifuging, washing the NK cell precipitate with DPBS (Dulbecco's Phosphate-Buffered Saline), resuspending, and freezing to obtain large-scale cultured NK cells.

[0019] As a further improvement of the present invention, the culture condition is culturing in an incubator at 37° C. and 5% CO 2 saturated humidity for 7-10 days.

[0020] As a further improvement of the present invention, the inoculation density is 3-5×10 4 cell / mL; the centrifugation condition is 550-600g, and the time is 5-10min.

[0021] The present invention has the following beneficial effects:

[0022] During the NK cell culture process, the usual stirring method is stirring with the stirring paddle that comes with the bioreactor. During operation, fluid shear force will be generated, and the magnitude of the shear force will vary with the difference in the propeller type. However, the fluid shear force generated when the propeller is in operation is relatively large, which will cause damage to cells that are sensitive to shear force. Therefore, the present invention prepares a stimulation and proliferation-promoting magnetic microsphere. Through emulsification and sol-gel reaction, a silica-coated magnetic ferroferric oxide microsphere is prepared. The microsphere has magnetism and can control the movement of the stirring device through changes in the magnetic field to achieve mixing of the culture system, forming spherical stirring. This spherical stirring can significantly reduce the generation of fluid shear force, avoid the decrease in cell proliferation ability due to shear force damage, and thus can be well adapted to large-scale culture, and effectively avoid the influence of stirring on NK cell proliferation.

[0023] At the same time, after the surface of the silica sphere coated with magnetic ferroferric oxide prepared by the present invention is modified with tannic acid, it can be coupled with phycocyanin and silk protein peptides. Silk protein peptides are a type of protein peptides with good biocompatibility, which greatly improves the biocompatibility of the stimulated amplification-promoting magnetic microspheres prepared. Phycocyanin has multiple biological activities, including anti-tumor, anti-oxidation, free radical scavenging, immunity regulation and other physiological effects, which can promote the proliferation activity of NK cells, activate the secretion of NK cell factors, and improve the anti-tumor effect of NK cells.

[0024] In addition, the surface of the prepared protein-coupled modified magnetic ferroferric oxide-coated silica spheres is further coupled with cell complex factors, including IL-15 and IL-12. IL-15 can directly stimulate the proliferation and activation of NK cells without activating Treg cells, thereby enhancing the function of NK cells; IL-2 can promote the recruitment and effector function of NK cells, and indirectly promote NK cells to produce more IFNγ, perforin, and granzyme B. The two have a synergistic effect and have a good promoting effect on the proliferation and long-term activity of NK cells.

[0025] The present invention provides a large-scale culture method for NK cells, which can efficiently activate and amplify NK cells, does not require the use of autologous serum, is safe and effective, can better promote the proliferation of NK cells, prolong their activity, is safe and has no side effects, and has broad application prospects. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0027] Preparation of NK cells: Anticoagulated peripheral blood was diluted with normal saline at a ratio of 1:1, and the dilution was spread on the upper layer of lymphocyte separation medium (Tianjin Haoyang Biological Products Technology Co., Ltd.).

[0028] The cells were centrifuged at 2000 r / min for 20 min, the buffy coat layer was aspirated, and the cells were washed three times with saline. The supernatant was discarded to obtain NK cells.

[0029] Preparation Example 1 Preparation of Stimulated Amplification-Promoting Magnetic Microspheres

[0030] Here’s how:

[0031] S1. Dissolve 3.24g ferric chloride, 1.26g ferrous chloride and 0.2g Tween-20 in 200mL water to obtain an aqueous phase; dissolve 3g ethyl orthosilicate and 0.1g Span-20 in 300mL ethyl acetate to obtain an oil phase; drop the aqueous phase into the oil phase, emulsify at 5000r / min for 15min, add ammonia water to adjust the pH value of the solution to 9, heat to 70°C, stir and react for 4h, centrifuge, wash, dry at 60°C for 2h, and calcine at 500°C for 1h to obtain silica spheres coated with magnetic ferrosoferric oxide;

[0032] S2. 10 g of silica balls coated with magnetic ferroferric oxide were added to 200 mL of water, 3 g of tannic acid and 0.5 g of catalyst were added, heated to 40 ° C, stirred for 3 h, separated by magnet, washed, and dried to obtain modified silica balls coated with magnetic ferroferric oxide;

[0033] The catalyst is a Tris-HCl solution with a pH value of 8.5;

[0034] S3. 10 g of modified silica spheres coated with magnetic ferroferric oxide were added to 200 mL of water, 2 g of NHS and 1 g of EDC were added, and the mixture was stirred for activation for 20 min, 2 g of phycocyanin and 4 g of silk protein peptide were added, and the mixture was stirred for reaction for 10 h, separated by a magnet, washed, and freeze-dried to obtain protein-coupled modified silica spheres coated with magnetic ferroferric oxide;

[0035] S4. 10 g of protein-coupled modified magnetic ferroferric oxide-coated silica spheres were added to water, 2 g of NHS and 1 g of EDC were added, and the mixture was stirred and activated for 20 min, 4 g of cell complex factors were added, and the mixture was stirred and reacted for 8 h, separated by a magnet, washed, and freeze-dried to obtain stimulation-amplification-promoting magnetic microspheres;

[0036] The cell complex factors are IL-15 and IL-12, with a mass ratio of 10:3.

[0037] Preparation Example 2 Preparation of Stimulated Amplification-Promoting Magnetic Microspheres

[0038] Here’s how:

[0039] S1. Dissolve 3.24g ferric chloride, 1.26g ferrous chloride and 0.5g Tween-40 in 200mL water to obtain an aqueous phase; dissolve 5g ethyl orthosilicate and 0.2g Span-40 in 300mL ethyl acetate to obtain an oil phase; drop the aqueous phase into the oil phase, emulsify at 8000r / min for 25min, drop ammonia water to adjust the pH value of the solution to 10, heat to 80°C, stir and react for 7h, centrifuge, wash, dry at 70°C for 4h, and calcine at 700°C for 2h to obtain silica spheres coated with magnetic ferrosoferric oxide;

[0040] S2. Add 10 g of silica balls coated with magnetic ferroferric oxide to 200 mL of water, add 4 g of tannic acid and 1 g of catalyst, heat to 50 ° C, stir and react for 5 h, separate with a magnet, wash, and dry to obtain modified silica balls coated with magnetic ferroferric oxide;

[0041] The catalyst is a Tris-HCl solution with a pH value of 9.5;

[0042] S3. 10 g of modified silica spheres coated with magnetic ferroferric oxide were added to 200 mL of water, 3 g of NHS and 3 g of EDC were added, and the mixture was stirred and activated for 30 min, 3 g of phycocyanin and 5 g of silk protein peptide were added, and the mixture was stirred and reacted for 12 h, separated by a magnet, washed, and freeze-dried to obtain protein-coupled modified silica spheres coated with magnetic ferroferric oxide;

[0043] S4. 12g of protein-coupled modified magnetic ferroferric oxide-coated silica spheres were added to water, 3g of NHS and 3g of EDC were added, and the mixture was stirred and activated for 30min, 5g of cell complex factors were added, and the mixture was stirred and reacted for 10h, separated by a magnet, washed, and freeze-dried to obtain stimulation-amplification-promoting magnetic microspheres;

[0044] The cell complex factors are IL-15 and IL-12, with a mass ratio of 10:7.

[0045] Preparation Example 3 Preparation of Stimulated Amplification-Promoting Magnetic Microspheres

[0046] Here’s how:

[0047] S1. Dissolve 3.24g of ferric chloride, 1.26g of ferrous chloride and 0.35g of Tween-85 in 200mL of water to obtain an aqueous phase; dissolve 4g of tetraethyl orthosilicate and 0.15g of Span-85 in 300mL of ethyl acetate to obtain an oil phase; drop the aqueous phase into the oil phase, emulsify at 7000r / min for 20min, drop ammonia water to adjust the pH value of the solution to 9.5, heat to 75°C, stir and react for 5.5h, centrifuge, wash, dry at 65°C for 3h, and calcine at 600°C for 1.5h to obtain silica spheres coated with magnetic ferrosoferric oxide;

[0048] S2. 10 g of silica balls coated with magnetic ferroferric oxide were added to 200 mL of water, 3.5 g of tannic acid and 0.7 g of catalyst were added, heated to 45 ° C, stirred for 4 h, separated by magnet, washed, and dried to obtain modified silica balls coated with magnetic ferroferric oxide;

[0049] The catalyst is a Tris-HCl solution with a pH value of 9;

[0050] S3. 10 g of modified silica spheres coated with magnetic ferroferric oxide were added to 200 mL of water, 2.5 g of NHS and 2 g of EDC were added, and the mixture was stirred and activated for 25 min. 2.5 g of phycocyanin and 4.5 g of silk protein peptide were added, and the mixture was stirred and reacted for 11 h. The mixture was separated by a magnet, washed, and freeze-dried to obtain protein-coupled modified silica spheres coated with magnetic ferroferric oxide.

[0051] S4. 11 g of protein-coupled modified magnetic ferroferric oxide-coated silica spheres were added to water, 2.5 g of NHS and 2 g of EDC were added, and the mixture was stirred and activated for 25 min. 4.5 g of cell complex factors were added, and the mixture was stirred and reacted for 9 h. The mixture was separated by a magnet, washed, and freeze-dried to obtain stimulation-amplification-promoting magnetic microspheres.

[0052] The cell complex factors are IL-15 and IL-12, with a mass ratio of 10:5.

[0053] Preparation Example 4

[0054] Compared with Preparation Example 3, the difference is that the cell complex factor is a single IL-15.

[0055] Preparation Example 5

[0056] Compared with Preparation Example 3, the difference is that the cell complex factor is a single IL-12.

[0057] Comparative Preparation Example 1

[0058] Compared with Preparation Example 3, the difference is that step S2 is not performed.

[0059] Comparative Preparation Example 2

[0060] Compared with Preparation Example 3, the difference is that no phycocyanin is added in step S3.

[0061] Comparative Preparation Example 3

[0062] Compared with Preparation Example 3, the difference is that step S3 is not performed.

[0063] Comparative Preparation Example 4

[0064] Compared with Preparation Example 3, the difference is that step S4 is not performed.

[0065] Test Example 1

[0066] The specific surface areas of the stimulation-amplification-promoting magnetic microspheres prepared in Preparation Examples 1-5 and Comparative Preparation Examples 1-4 were measured using a 3-FLEX 3500 multi-station high-throughput gas adsorption instrument. The results are shown in Table 1.

[0067] Table 1

[0068] Group <![CDATA[Specific surface area (m 2 / g)]]> Preparation Example 1 452 Preparation Example 2 454 Preparation Example 3 457 Comparative Preparation Example 1 372 Comparative Preparation Example 2 433 Comparative Preparation Example 3 410 Comparative Preparation Example 4 395

[0069] It can be seen from the above table that the stimulation-amplification-promoting magnetic microspheres prepared in Preparation Examples 1-3 of the present invention have a larger specific surface area.

[0070] Example 1

[0071] The present embodiment provides a culture medium, wherein fetal bovine serum, penicillin, streptomycin, and the stimulation-amplification-promoting magnetic microspheres prepared in Preparation Example 1 are added to the ACTM culture medium, and the mixture is stirred for 15 minutes to prepare the culture medium, wherein the content of fetal bovine serum in the ACTM culture medium is 7wt%, the content of penicillin is 80U / mL, the content of streptomycin is 80U / mL, and the stimulation-amplification-promoting magnetic microspheres is 100μg / mL.

[0072] Example 2

[0073] The present embodiment provides a culture medium, wherein fetal bovine serum, penicillin, streptomycin, and the stimulation-amplification-promoting magnetic microspheres prepared in Preparation Example 2 are added to the ACTM culture medium, and the mixture is stirred for 15 minutes to prepare the culture medium, wherein the content of fetal bovine serum in the ACTM culture medium is 12wt%, the content of penicillin is 120U / mL, the content of streptomycin is 120U / mL, and the stimulation-amplification-promoting magnetic microspheres are 200μg / mL.

[0074] Example 3

[0075] The present embodiment provides a culture medium, wherein fetal bovine serum, penicillin, streptomycin, and the stimulation-amplification-promoting magnetic microspheres prepared in Preparation Example 3 are added to the ACTM culture medium, and the mixture is stirred for 15 minutes to prepare the culture medium, wherein the content of fetal bovine serum in the ACTM culture medium is 10wt%, the content of penicillin is 100U / mL, the content of streptomycin is 100U / mL, and the stimulation-amplification-promoting magnetic microspheres are 150μg / mL.

[0076] Example 4

[0077] Compared with Example 3, the difference is that the stimulation and amplification promoting magnetic microspheres are prepared by Preparation Example 4.

[0078] Example 5

[0079] Compared with Example 3, the difference is that the stimulation and amplification promoting magnetic microspheres are prepared by Preparation Example 5.

[0080] Comparative Example 1

[0081] Compared with Example 3, the difference is that the stimulation and amplification promoting magnetic microspheres are prepared by Comparative Preparation Example 1.

[0082] Comparative Example 2

[0083] Compared with Example 3, the difference is that the stimulation and amplification promoting magnetic microspheres are prepared by Comparative Preparation Example 2.

[0084] Comparative Example 3

[0085] Compared with Example 3, the difference is that the stimulation and amplification promoting magnetic microspheres are prepared by Comparative Preparation Example 3.

[0086] Comparative Example 4

[0087] Compared with Example 3, the difference is that the stimulation and amplification promoting magnetic microspheres are prepared by Comparative Preparation Example 4.

[0088] Comparative Example 5

[0089] Compared with Example 3, the difference is that no stimulation and amplification-promoting magnetic microspheres are added.

[0090] Example 6

[0091] This example provides a method for large-scale cultivation of NK cells. NK cells are inoculated in the culture medium prepared in Example 1 at a density of 3×10 4 cell / mL, cultured in an incubator at 37°C and 5% CO2 saturated humidity for 8 days, stirred with an external magnetic field with a magnetic field strength of 0.1 T, centrifuged at 550g for 5 minutes, washed the NK cell pellet with DPBS, and resuspended to obtain large-scale cultured NK cells.

[0092] Example 7

[0093] This example provides a method for large-scale cultivation of NK cells. NK cells are inoculated in the culture medium prepared in Example 2 at a density of 5×10 4 cell / mL, cultured in an incubator at 37°C and 5% CO2 saturated humidity for 8 days, stirred with an external magnetic field with a magnetic field strength of 0.15T, centrifuged at 600g for 10min, washed the NK cell pellet with DPBS, and resuspended to obtain large-scale cultured NK cells.

[0094] Example 8

[0095] This example provides a method for large-scale cultivation of NK cells. NK cells are inoculated in the culture medium prepared in Example 3 at a density of 4×10 4 cell / mL, cultured in an incubator at 37°C and 5% CO2 saturated humidity for 8 days, stirred with an external magnetic field with a magnetic field strength of 0.12T, centrifuged at 576g for 7min, washed the NK cell pellet with DPBS, and resuspended to obtain large-scale cultured NK cells.

[0096] Example 9

[0097] Compared with Example 8, the difference is that the culture medium is prepared by Example 4.

[0098] Example 10

[0099] Compared with Example 8, the difference is that the culture medium is prepared by Example 5.

[0100] Comparative Example 6

[0101] Compared with Example 8, the difference is that the culture medium is prepared by Comparative Example 1.

[0102] Comparative Example 7

[0103] Compared with Example 8, the difference is that the culture medium is prepared by Comparative Example 2.

[0104] Comparative Example 8

[0105] Compared with Example 8, the difference is that the culture medium is prepared by Comparative Example 3.

[0106] Comparative Example 9

[0107] Compared with Example 8, the difference is that the culture medium is prepared by Comparative Example 4.

[0108] Comparative Example 10

[0109] Compared with Example 8, the difference is that the culture medium is prepared from Comparative Example 5, no external magnetic field is added for stirring, and a traditional stirring paddle is used for stirring.

[0110] Test Example 1

[0111] 0.5 mL of cell suspension was taken from Examples 6-10 and Comparative Examples 6-10 for counting and calculation of the expansion multiples. The results are shown in Table 2.

[0112] Table 2

[0113] Group Amplification factor Example 6 45.2±3.33* Example 7 45.9±3.15* Example 8 46.4±3.74* Example 9 37.8±4.56 Example 10 38.2±3.97 Comparative Example 6 22.8±3.89 Comparative Example 7 39.7±4.05 Comparative Example 8 33.9±4.12 Comparative Example 9 25.7±4.17 Comparative Example 10 (control group) 19.4±0.79

[0114] Note: * compared with comparative example 10, P < 0.05.

[0115] As can be seen from the above table, the methods in Examples 6-8 of the present invention cultured NK cells at a greater expansion multiple.

[0116] Test Example 2

[0117] The NK cells cultured in Example 6-10 or Comparative Example 6-10 were collected as effector cells (E), K562 cells (chronic myeloid leukemia cell line) were used as target cells (T), and CCK8 was used to detect cell activity to evaluate the killing activity of NK cells against K562 cells. 5NK cells and 1×10 4 K562 cells (the ratio of effector cells to target cells (E / T) was 10:1) were added into 0.1 mL IMDM medium (Iscove's Modified Dulbecco's Medium) + 10% fetal bovine serum medium as the experimental group. 5 NK cells and 1×10 4 K562 cells were added to the above culture medium as the control group. After incubation in the incubator for 24 h, 10 μL CCK-8 was added and incubated in the incubator for another 4 h. The absorbance (OD) value at 450 nm was measured. 1×10 5 NK cells, 1×10 4 K562 cells and 1×10 5 NK cells and 1×10 4 The OD values ​​obtained by co-culture of K562 cells were OD E OD T OD E / T The killing activity was calculated according to the following formula. The results are shown in Table 3.

[0118]

[0119] Table 3

[0120] Group Killing activity (%) Example 6 95.6 Example 7 96.1 Example 8 96.8 Example 9 92.1 Example 10 91.4 Comparative Example 6 79.5 Comparative Example 7 90.8 Comparative Example 8 85.2 Comparative Example 9 83.5 Comparative Example 10 72.4

[0121] It can be seen from the above table that the NK cells cultured by the methods in Examples 6-8 of the present invention have greater killing activity against tumor cells.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, 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 culture medium, characterized in that Add fetal bovine serum, penicillin, streptomycin, and stimulation and amplification-promoting magnetic microspheres to the ACTM culture medium, stir and mix evenly to prepare the culture medium; the preparation method of the stimulation and amplification-promoting magnetic microspheres is as follows: S1. Dissolve ferric chloride, ferrous chloride and a hydrophilic emulsifier in water to obtain an aqueous phase; dissolve an alkyl orthosilicate and a lipophilic emulsifier in ethyl acetate to obtain an oil phase; drop the aqueous phase into the oil phase, emulsify, drop ammonia water, heat and stir to react, centrifuge, wash, dry, and calcine to obtain silica balls coated with magnetic ferrosoferric oxide; the mass ratio of the ferric chloride, ferrous chloride, hydrophilic emulsifier, alkyl orthosilicate, and lipophilic emulsifier is 3.24:1.2 6:0.2-0.5:3-5:0.1-0.2; the pH value of the solution is adjusted to 9-10 by dropping ammonia water, the temperature of the heating and stirring reaction is 70-80°C, the time is 4-7h, the speed of the emulsification is 5000-8000r / min, the time is 15-25min, the temperature of the drying is 60-70°C, the time is 2-4h, the temperature of the calcination is 500-700°C, the time is 1-2h; S2. Add the silica balls coated with magnetic ferroferric oxide into water, add tannic acid and a catalyst, heat and stir to react, separate with a magnet, wash, and dry to obtain modified silica balls coated with magnetic ferroferric oxide; the mass ratio of the silica balls coated with magnetic ferroferric oxide, tannic acid, and catalyst is 10:3-4:0.5-1; the catalyst is a Tris-HCl solution with a pH value of 8.5-9.5, and the temperature of the heating and stirring reaction is 40-50°C for 3-5h; S3. Add the modified magnetic ferroferric oxide coated silica spheres into water, add NHS and EDC, stir to activate, add phycocyanin and silk fibroin peptide, stir to react, separate by magnet, wash, freeze-dry, and obtain protein-coupled modified magnetic ferroferric oxide coated silica spheres; the mass ratio of the modified magnetic ferroferric oxide coated silica spheres, NHS, EDC, phycocyanin and silk fibroin peptide is 10:2-3:1-3:2-3:4-5, the stirring activation time is 20-30min, and the stirring reaction time is 10-12h; S4. Add protein-coupled modified silica balls coated with magnetic ferroferric oxide into water, add NHS and EDC, stir for activation, add cell complex factors, stir for reaction, separate with a magnet, wash, freeze-dry, and obtain stimulation amplification promoting magnetic microspheres; the mass ratio of the protein-coupled modified silica balls coated with magnetic ferroferric oxide, NHS, EDC and cell complex factors is 10-12:2-3:1-3:4-5, the cell complex factors are IL-15 and IL-12, and the mass ratio is 10:3-7; the stirring activation time is 20-30min, and the stirring reaction time is 8-10h.

2. The culture medium according to claim 1, characterized in that The ACTM culture medium contains 7-12 wt % fetal bovine serum, 80-120 U / mL penicillin, 80-120 U / mL streptomycin, and 100-200 μg / mL stimulation and amplification promoting magnetic microspheres.

3. The culture medium according to claim 1, characterized in that In step S1, the hydrophilic emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85; the lipophilic emulsifier is selected from at least one of Span-20, Span-40, Span-60, Span-80, and Span-85.

4. A method for large-scale cultivation of NK cells, characterized in that: The NK cells are inoculated into the culture medium according to any one of claims 1 to 3, cultured, stirred in an external magnetic field, centrifuged, and the NK cell precipitate is washed with DPBS, resuspended, and frozen to obtain large-scale cultured NK cells.

5. The method for large-scale cultivation of NK cells according to claim 4, characterized in that: The culture conditions are 37° C., 5% CO 2 saturated humidity in an incubator for 7-10 days, and the magnetic field strength of the external magnetic field is 0.1-0.15T.

6. The method for large-scale cultivation of NK cells according to claim 4, characterized in that: The inoculation density is 3-5×10 4 cell / mL; the centrifugation condition is 550-600g, and the time is 5-10min.

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