Culture medium for NKT cells and use method of culture medium

Abstract

The invention relates to the technical field of cell culture, in particular to a culture medium for NKT cells and a use method of the culture medium. The invention provides a culture medium for NKT cells and a use method of the culture medium. The NKT cells are prepared from PBMC cells separated from peripheral blood; the separated PBMC cells are resuspended through a culture medium and then added into the culture medium to be subjected to primary culture and then subculture, and growth active components are added at set intervals in the subculture process, so that the induction efficiency of the NKT cells can be greatly improved, amplification of the NKT cells is facilitated, and the killing efficiency of the NKT cells on cancer cells can be remarkably improved.

Description

Technical Field

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

[0002] NKT cells (natural killer-like T cells) are a new type of immune regulatory cells. The main characteristics of NKT cells are the constancy of T cell receptor gene expression, CDld restriction, and rapid and high-level cytokine production. They can both enhance and inhibit immune responses, thus playing an important role in anti-tumor, anti-infection, and inhibition of autoimmune diseases. NKT cells belong to T lymphocytes, but they have the surface markers and functional characteristics of traditional T cells and NK cells. Most NKT cells express a semi-invariant T cell receptor, which interacts with glycolipid antigens presented by MHC-I-related protein CD1d on the surface of antigen-presenting cells. NKT cells are activated in a variety of different infections and inflammatory responses, and rapidly produce a large number of immunoregulatory cytokines, which can affect the activation state and functional characteristics of many other cell types in the immune system. Therefore, NKT cells can be used to treat tumors and autoimmune diseases.

[0003] NKT cells can be expanded by induction in vitro, so the use of NKT cell therapy is a widely used tumor immunotherapy method. The current method for preparing NKT cells is to induce the isolated peripheral blood mononuclear cells (PBMC) with α-Galcer, then add induction factors for stimulation, and finally obtain a certain number of NKT cells. However, the proliferation multiples and cell phenotypes of the NKT cells finally obtained by this method are average, resulting in low killing activity against tumor cells.

[0004] Therefore, how to provide a culture medium for NKT cells and a method for using the same to solve the technical problems in the prior art of low NKT cell expansion multiples, inability to expand on a large scale, and low killing activity is an issue that technical personnel in this field urgently need to solve. Summary of the invention

[0005] The object of the present invention is to provide a culture medium for NKT cells and a method for using the same. The culture method provided by the present invention does not use a serum culture medium, and the proliferation multiple of NKT cells can reach 517.2 times, and the cultured NKT cells also have a high killing rate for tumor cells.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a culture medium for NKT cells, including a primary culture medium, a secondary culture medium and a supplementary solution;

[0008] The primary culture medium is based on a serum-free culture medium, and includes: IFN-γ 500-2000 IU / mL, G-CSF 20-40 ng / L, honeysuckle leaf extract 12-16 mg / mL, and ginsenoside 5-9 mg / mL;

[0009] The secondary culture medium is based on a serum-free culture medium, and includes: 100-200 ng / mL of humanized CD3 monoclonal antibody, 100-200 ng / mL of humanized CD28 monoclonal antibody, 3000-4000 IU / mL of IL-2, 12-16 mg / mL of honeysuckle leaf extract, and 5-9 mg / mL of ginsenoside;

[0010] The supplement solution is based on serum-free culture medium and includes: IL-15 30-40 ng / mL, IL-7 10-50 ng / mL, IL-21 10-50 ng / mL, honeysuckle leaf extract 3-5 mg / mL, and ginsenoside 2-3 mg / mL.

[0011] Preferably, the serum-free culture medium is LONZAX-VIVO 15 hematopoietic stem cell serum-free culture medium or AIM V culture medium; the mass ratio of luteolin to honeysuckle glycoside in the honeysuckle leaf extract is 1-3:3-1.

[0012] The present invention also provides a method for using the culture medium for NKT cells, comprising the following steps:

[0013] (1) inoculating peripheral blood mononuclear cells into primary culture medium for primary culture to obtain primary cells;

[0014] (2) The primary cells obtained in step (1) are inoculated into a subculture medium for subculture, during which time a supplement solution is used for fluid replenishment to obtain NKT cells.

[0015] Preferably, the inoculum size in step (1) is (1-2)×10 6 Pieces / mL.

[0016] Preferably, the primary culture time in step (1) is 40 to 50 hours.

[0017] Preferably, the inoculum size in step (2) is (3-4)×10 7 Pieces / mL.

[0018] Preferably, the subculture time in step (2) is 13 to 16 days.

[0019] Preferably, the method of fluid replenishment in step (2) is: fluid replenishment is performed on the 4th, 7th, and 11th day of subculture.

[0020] Preferably, after the rehydration in step (2), the cell density is always maintained at (3-4)×10 7 Pieces / mL.

[0021] The present invention also provides the use of the NKT cells prepared by the method for using the NKT cell culture medium in the preparation of anti-tumor drugs.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The culture medium provided by the present invention has the technical effect of promoting the proliferation of NKT cells and improving the killing activity of NKT cells. Among them, various types of interleukins are added to the culture medium, so that the killing activity of NKT cells obtained by culture in the culture medium is significantly improved. The addition of IFN-γ affects the growth and differentiation of cells and has an activation effect on NKT cells. The role of G-CSF (granulocyte colony stimulating factor) in the culture medium is mainly to promote the proliferation, differentiation and activation of NKT cells, and has a significant stimulating effect on the proliferation of cells.

[0024] Ginsenoside is the main active ingredient in the traditional Chinese medicine ginseng, which belongs to the sterol compound. It has the effects of promoting cell proliferation, reducing the production of oxygen free radicals, and inhibiting inflammatory factors. The applicant found through preliminary experiments that honeysuckle leaf extract also has similar effects, and honeysuckle leaf extract and ginsenoside are used together. The ability of the two to promote cell proliferation and improve cell killing activity will be greatly enhanced. This may be related to the anti-apoptosis, anti-inflammatory and antioxidant activities of honeysuckle.

[0025] The present invention provides a culture medium for NKT cells and a method for using the same. The present invention uses PBMC cells separated from peripheral blood to prepare NKT cells. The present invention resuspends the separated PBMC cells in a culture medium, adds the cells to a culture medium, performs primary culture, and then performs subculture. During the subculture process, growth active ingredients are added at regular intervals, which can greatly improve the induction efficiency of NKT cells, facilitate the expansion of NKT cells, and significantly improve the killing efficiency of NKT cells for cancer cells. DETAILED DESCRIPTION

[0026] The present invention provides a culture medium for NKT cells, including a primary culture medium, a secondary culture medium and a supplementary solution;

[0027] In the present invention, the primary culture medium is based on a serum-free culture medium and includes: IFN-γ500-2000IU / mL, G-CSF 20-40ng / L, honeysuckle leaf extract 12-16mg / mL, and ginsenoside 5-9mg / mL; preferably IFN-γ600-1900IU / mL, G-CSF 24-36ng / L, honeysuckle leaf extract 13-15mg / mL, and ginsenoside 6-8mg / mL; further preferably IFN-γ700-1800IU / mL, G-CSF 28-32ng / L, honeysuckle leaf extract 14mg / mL, and ginsenoside 7mg / mL; more preferably IFN-γ1200IU / mL, G-CSF 30ng / L, honeysuckle leaf extract 14mg / mL, and ginsenoside 7mg / mL.

[0028] In the present invention, the secondary culture medium is based on a serum-free medium and includes: humanized CD3 monoclonal antibody 100-200 ng / mL, humanized CD28 monoclonal antibody 100-200 ng / mL, IL-23000-4000 IU / mL, honeysuckle leaf extract 12-16 mg / mL, and ginsenoside 5-9 mg / mL; preferably humanized CD3 monoclonal antibody 120-180 ng / mL, humanized CD28 monoclonal antibody 120-180 ng / mL, IL-23200-3800 IU / mL, honeysuckle leaf extract 13 ~15mg / mL, ginsenoside 6~8mg / mL; further preferably humanized CD3 monoclonal antibody 140~160ng / mL, humanized CD28 monoclonal antibody 140~160ng / mL, IL-23400~3600IU / mL, honeysuckle leaf extract 14mg / mL, ginsenoside 7mg / mL; more preferably humanized CD3 monoclonal antibody 150ng / mL, humanized CD28 monoclonal antibody 150ng / mL, IL-23500IU / mL, honeysuckle leaf extract 14mg / mL, ginsenoside 7mg / mL.

[0029] In the present invention, the supplement solution is based on serum-free culture medium and includes: IL-1530-40ng / mL, IL-710-50ng / mL, IL-2110-50ng / mL, honeysuckle leaf extract 3-5mg / mL, and ginsenoside 2-3mg / mL; preferably IL-1532-38ng / mL, IL-720-40ng / mL, IL-2120-40ng / mL, honeysuckle leaf extract 4mg / mL, and ginsenoside 3mg / mL; further preferably IL-1534-36ng / mL, IL-730ng / mL, IL-2130ng / mL, honeysuckle leaf extract 4mg / mL, and ginsenoside 3mg / mL; more preferably IL-1535ng / mL, IL-730ng / mL, IL-2130ng / mL, honeysuckle leaf extract 4mg / mL, and ginsenoside 3mg / mL.

[0030] In the present invention, the serum-free culture medium is LONZAX-VIVO 15 hematopoietic stem cell serum-free culture medium or AIM V culture medium; preferably LONZAX-VIVO 15 hematopoietic stem cell serum-free culture medium.

[0031] The present invention also provides a method for using the culture medium for NKT cells, comprising the following steps:

[0032] (1) inoculating peripheral blood mononuclear cells into primary culture medium for primary culture to obtain primary cells;

[0033] (2) The primary cells obtained in step (1) are inoculated into a subculture medium for subculture, during which time a supplement solution is used for fluid replenishment to obtain NKT cells.

[0034] In the present invention, the inoculation amount in step (1) is (1-2)×10 6 / mL; preferably 1.5×10 6 Pieces / mL.

[0035] In the present invention, the primary culture time in step (1) is 40 to 50 hours, preferably 42 to 48 hours, more preferably 44 to 46 hours, and more preferably 45 hours.

[0036] In the present invention, the inoculation amount in step (2) is 3 to 4×10 7 / mL; preferably 3.5×10 7 Pieces / mL.

[0037] In the present invention, the time of subculture in step (2) is 13 to 16 days, preferably 14 to 15 days, and more preferably 14 days.

[0038] In the present invention, the method of fluid replenishment in step (2) is: fluid replenishment is performed on the 4th, 7th, and 11th day of subculture.

[0039] In the present invention, after the rehydration in step (2), the cell density is always maintained at 3 to 4×10 7 / mL; preferably 3.5×10 7 Pieces / mL.

[0040] The present invention also provides the use of the NKT cells prepared by the method for using the NKT cell culture medium in the preparation of anti-tumor drugs.

[0041] In the present invention, the preparation method of honeysuckle leaf extract refers to patent CN202310976982.7, which is an extraction, separation and purification system and method for extracting oleuroside and honeysuckle glycoside from honeysuckle leaves.

[0042] In the present invention, the honeysuckle leaf extract is composed of honeysuckle glycoside and luteolin, wherein the mass ratio of luteolin to honeysuckle glycoside in the honeysuckle leaf extract is 1-3:3-1; preferably 2:3-1; more preferably 1:1.

[0043] In the present invention, the ginsenoside was purchased from Merck, specifically ginsenoside Rh2.

[0044] 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.

[0045] Example 1

[0046] A culture medium for NKT cells, comprising a primary culture medium, a secondary culture medium and a supplementary solution;

[0047] The primary culture medium is based on serum-free culture medium, including: IFN-γ 500 IU / mL, G-CSF 20 ng / L, honeysuckle leaf extract 12 mg / mL, ginsenoside Rh 25 mg / mL;

[0048] The secondary culture medium is based on a serum-free culture medium, and includes: humanized CD3 monoclonal antibody 100 ng / mL, humanized CD28 monoclonal antibody 100 ng / mL, IL-2 3000 IU / mL, honeysuckle leaf extract 12 mg / mL, and ginsenoside Rh 25 mg / mL;

[0049] The supplement solution is based on serum-free culture medium and includes: IL-15 30 ng / mL, IL-7 10 ng / mL, IL-2 110 ng / mL, honeysuckle leaf extract 3 mg / mL, and ginsenoside Rh 22 mg / mL.

[0050] The serum-free medium is LONZAX-VIVO 15 hematopoietic stem cell serum-free medium;

[0051] The mass ratio of luteolin to honeysuckle in the honeysuckle leaf extract is 1:3.

[0052] Example 2

[0053] A culture medium for NKT cells, comprising a primary culture medium, a secondary culture medium and a supplementary solution;

[0054] The primary culture medium is based on serum-free culture medium, including: IFN-γ 2000IU / mL, G-CSF 40ng / L, honeysuckle leaf extract 16mg / mL, ginsenoside Rh 29mg / mL;

[0055] The secondary culture medium is based on a serum-free culture medium, and includes: 200 ng / mL of humanized CD3 monoclonal antibody, 200 ng / mL of humanized CD28 monoclonal antibody, 4000 IU / mL of IL-2, 16 mg / mL of honeysuckle leaf extract, and 29 mg / mL of ginsenoside Rh;

[0056] The supplement solution is based on serum-free culture medium and includes: IL-15 40ng / mL, IL-7 50ng / mL, IL-2 150ng / mL, honeysuckle leaf extract 5mg / mL, and ginsenoside Rh 23mg / mL.

[0057] The serum-free medium is AIM V medium;

[0058] The mass ratio of luteolin to honeysuckle glycoside in the honeysuckle leaf extract is 3:1.

[0059] Example 3

[0060] A culture medium for NKT cells, comprising a primary culture medium, a secondary culture medium and a supplementary solution;

[0061] The primary culture medium is based on serum-free culture medium, including: IFN-γ 1200 IU / mL, G-CSF 30 ng / L, honeysuckle leaf extract 14 mg / mL, ginsenoside Rh 27 mg / mL;

[0062] The secondary culture medium is based on a serum-free culture medium, including: 150 ng / mL of humanized CD3 monoclonal antibody, 150 ng / mL of humanized CD28 monoclonal antibody, 500 IU / mL of IL-23, 14 mg / mL of honeysuckle leaf extract, and 27 mg / mL of ginsenoside Rh;

[0063] The supplement solution is based on serum-free culture medium and includes: IL-15 35 ng / mL, IL-7 30 ng / mL, IL-2 1 30 ng / mL, honeysuckle leaf extract 4 mg / mL, and ginsenoside Rh 23 mg / mL.

[0064] The serum-free medium is LONZAX-VIVO 15 hematopoietic stem cell serum-free medium;

[0065] The mass ratio of luteolin to honeysuckle glycoside in the honeysuckle leaf extract is 1:1.

[0066] Example 4

[0067] A method for using a culture medium for NKT cells, comprising the following steps:

[0068] (1) Based on the culture medium of Example 1, mononuclear cells isolated from healthy human peripheral blood were inoculated into primary culture medium and cultured for 40 h at 37° C. and 5% CO2 to obtain primary cells; the inoculation amount was 1×10 6 Pieces / mL.

[0069] (2) The primary cells obtained in step (1) were inoculated in a subculture medium and subcultured for 13 days (the day of inoculation was recorded as day 0 of culture). The inoculation amount was 3×10 7 / mL, and the fluid was replenished on the 4th, 7th, and 11th days of subculture. After replenishment, the cell density was always maintained at 3×10 7 / mL, and NKT cells were obtained.

[0070] Example 5

[0071] A method for using a culture medium for NKT cells, comprising the following steps:

[0072] (1) Based on the culture medium of Example 1, mononuclear cells isolated from peripheral blood of healthy people were inoculated into primary culture medium and cultured for 40 h at 37° C. and 5% CO2 to obtain primary cells; the inoculation amount was 2×10 6 Pieces / mL.

[0073] (2) The primary cells obtained in step (1) were inoculated in a subculture medium and subcultured for 16 days (the day of inoculation was recorded as day 0 of culture), with an inoculation size of 4×10 7 / mL, and the fluid was replenished on the 4th, 7th, and 11th days of subculture. After replenishment, the cell density was always maintained at 4×10 7 / mL, and NKT cells were obtained.

[0074] Example 6

[0075] A method for using a culture medium for NKT cells, comprising the following steps:

[0076] (1) Based on the culture medium of Example 1, mononuclear cells isolated from healthy human peripheral blood were inoculated into primary culture medium and cultured for 48 h at 37° C. and 5% CO2 to obtain primary cells; the inoculation amount was 1.5×10 6 Pieces / mL.

[0077] (2) The primary cells obtained in step (1) were inoculated in a subculture medium and subcultured for 14 days (the day of inoculation was recorded as day 0 of culture), with an inoculum size of 3.5×10 7 / mL, and the fluid was replenished on the 4th, 7th, and 11th days of subculture. After replenishment, the cell density was always maintained at 3.5×10 7 / mL, and NKT cells were obtained.

[0078] Control group 1

[0079] The other methods are the same as those in Example 6, except that the honeysuckle leaf extract and ginsenoside Rh2 are not added to the primary culture medium, secondary culture medium, and supplement solution in Example 3.

[0080] Control group 2

[0081] The other methods are the same as those in Example 6, except that the honeysuckle leaf extract is not added to the primary culture medium, the secondary culture medium, and the supplement solution in Example 3.

[0082] Control group 3

[0083] The other methods are the same as those in Example 6, except that honeysuckle is replaced by an equal amount of distilled water in the primary culture medium, secondary culture medium, and supplement solution in Example 3.

[0084] Control group 4

[0085] The other methods are the same as those in Example 6, except that the supplement solution in Example 3 does not contain honeysuckle leaf extract and ginsenoside Rh2.

[0086] Experimental Example 1

[0087] The proliferation multiples, viability and CD3+CD56+ effector cell ratio of NKT cells prepared by the methods of Example 6 and Control Groups 1 to 4 were measured. Three repeated tests were set up for each test group, and the results were averaged, as shown in Table 1.

[0088] Table 1 Proliferation status questionnaire

[0089] Group Multiplication times Cell viability Effector cell ratio Example 6 517.2 98.53％ 44.35％ Control group 1 343.7 96.35％ 36.22％ Control group 2 387.9 97.86％ 38.55％ Control group 3 487.36 98.36％ 42.72％ Control group 4 465.1 98.23％ 39.57％

[0090] From the contents recorded in Table 1, it can be seen that the proliferation multiple of NKT cells in the culture method provided by the present invention is high, which can reach 517.2 times, and the cell viability also reaches 98.53%, and the proportion of CD3+CD56+ effector cells also reaches 44.35%, all of which are significantly better than other control groups.

[0091] From the data of the control group, it can be seen that adding honeysuckle leaf extract and ginsenoside Rh2 during the cell culture process helps the proliferation of cells. If some of the components are discarded, it will have a significant impact on the effectiveness of the culture medium.

[0092] Experimental Example 2

[0093] MCF-7 (human breast cancer cells) in the logarithmic growth phase were used as target cells, and the cell density was adjusted to 8×10 5 50 μL of NKT cells prepared in each example were plated in a 96-well culture plate. The density of NKT cells prepared in each example was adjusted to 4×10 6 / mL, 8×10 6 / mL, 1.6×10 7 / mL, add to 96-well culture plates, 50μL per well, the effector-target ratio is 5:1, 10:1 and 20:1, and three replicate wells are set for each group. The inoculation method is as follows:

[0094] Each experimental group (a value): MCF-7 (50 μL) + NKT cells (50 μL)

[0095] Control group (b value): MCF-7 (50 μL) + serum-free medium (50 μL)

[0096] (c value): NKT (50 μL) + serum-free medium (50 μL)

[0097] Blank group (d value): serum-free medium (100 μL)

[0098] Tumor killing efficiency = [1-(a value-c value-d value) / (b value-d value)]×100%.

[0099] Wherein, the serum-free culture medium is LONZAX-VIVO 15 hematopoietic stem cell serum-free culture medium.

[0100] After the inoculated cells were cultured at 37°C and 5% CO2 for 24 hours, 10 μL of CCK-8 reagent was added to each well, shaken well, and cultured for another 3 hours at 37°C and 5% CO2. 450 Absorbance.

[0101] The results of the different effect-target ratios of each test group were averaged, as shown in Table 2.

[0102] Table 2 Killing rate of NKT cells against tumor cells

[0103] Group 5:1 10:1 20:1 Example 6 78.6％ 84.2％ 92.3％ Control group 1 68.2％ 73.2％ 86.4％ Control group 2 71.5％ 77.8％ 89.2％ Control group 3 74.6％ 82.4％ 91.5％ Control group 4 76.2％ 80.2％ 91.0％

[0104] The results are shown in Table 2. The NKT cells prepared by this method have a high killing activity against the MCF-7 cell line. When the effector-target ratio is 10:1, the killing rate of NKT cells can reach more than 80%, and when the effector-target ratio is 20:1, the killing rate of NKT cells can reach more than 90%.

[0105] It can also be seen from the data of the control group that whether or not to add honeysuckle leaf extract and ginsenoside also has a significant effect on the killing activity of cells. Therefore, only by adopting the complete technical solution of the present invention can the best culture effect be achieved.

[0106] 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 culture medium for NKT cells, characterized in that Includes primary culture medium, secondary culture medium and supplementary solution; The primary culture medium is based on a serum-free culture medium, and includes: IFN-γ 500-2000 IU / mL, G-CSF 20-40 ng / L, honeysuckle leaf extract 12-16 mg / mL, and ginsenoside 5-9 mg / mL; The secondary culture medium is based on a serum-free culture medium, and includes: 100-200 ng / mL of humanized CD3 monoclonal antibody, 100-200 ng / mL of humanized CD28 monoclonal antibody, 3000-4000 IU / mL of IL-2, 12-16 mg / mL of honeysuckle leaf extract, and 5-9 mg / mL of ginsenoside; The supplement solution is based on serum-free culture medium and includes: IL-15 30-40 ng / mL, IL-7 10-50 ng / mL, IL-21 10-50 ng / mL, honeysuckle leaf extract 3-5 mg / mL, and ginsenoside 2-3 mg / mL.

2. The culture medium for NKT cells according to claim 1, characterized in that The serum-free culture medium is LONZAX-VIVO 15 hematopoietic stem cell serum-free culture medium or AIM V culture medium; the mass ratio of luteolin to honeysuckle glycoside in the honeysuckle leaf extract is 1-3:3-1.

3. The method for using the culture medium for NKT cells according to claim 1 or 2, characterized in that: The steps include: (1) inoculating peripheral blood mononuclear cells into primary culture medium for primary culture to obtain primary cells; (2) The primary cells obtained in step (1) are inoculated into a subculture medium for subculture, during which time a supplement solution is used for fluid replenishment to obtain NKT cells.

4. The method for using a culture medium for NKT cells according to claim 3, characterized in that: The inoculum size in step (1) is (1-2)×10 6 Pieces / mL.

5. The method for using a culture medium for NKT cells according to claim 3, characterized in that: The primary culture time in step (1) is 40 to 50 hours.

6. The method for using a culture medium for NKT cells according to claim 3, characterized in that: The inoculum size in step (2) is (3-4)×10 7 Pieces / mL.

7. The method for using a culture medium for NKT cells according to claim 3, characterized in that: The time of subculture in step (2) is 13 to 16 days.

8. The method for using a culture medium for NKT cells according to claim 3, characterized in that: The method of fluid replenishment in step (2) is: fluid replenishment is performed on the 4th, 7th and 11th day of subculture.

9. The method for using a culture medium for NKT cells according to claim 3, characterized in that: After the rehydration in step (2), the cell density is always maintained at (3-4)×10 7 Pieces / mL.

10. Use of the NKT cells prepared by the method for using the NKT cell culture medium according to any one of claims 3 to 9 in the preparation of anti-tumor drugs.