A new NK cell culture medium

Abstract

The present invention provides a new NK cell culture medium, which belongs to the field of cell culture technology. It includes basal culture medium, serum, cytokines, antibiotics and auxiliary factors, wherein the basal culture medium is selected from RPMI-1640, DMEM or IMDM, the serum is fetal bovine serum or human serum, the cytokines include IL-2, IL-7, IL-15, IL-3, VEGF, GDNF, and the antibiotics are penicillin or streptomycin. The present invention introduces auxiliary factors containing paclitaxel, cocin, ethanolamine, hyaluronic acid, sodium selenite, L-ascorbic acid, pyrroloquinoline quinone, dihydroxypropylphylline, reduced glutathione, madecassoside, D-pantothenate calcium, and estradiol into the basal culture medium, which can significantly increase the proliferation multiples and cell viability of NK cells, and at the same time, promote the expression of CD56 and CD16 by progeny NK cells. Experimental results show that after a 16-day culture period, the present invention causes NK cells to proliferate 377.6 times, the cell viability reaches 98.2%, and the expression of CD16 + CD56 + The percentage of cell number reached 83.28%.

Description

Technical Field

[0001] The present invention relates to the field of cell culture technology, in particular to a new NK cell culture medium. Background Art

[0002] Natural killer (NK) cells are the third largest type of lymphocytes besides T cells and B cells. They can spontaneously identify abnormal cells in the body and quickly eliminate them through cytotoxicity. At the same time, they produce various pro-inflammatory factors and chemokines, recruit and activate other immune cells to initiate adaptive immune responses.

[0003] NK cells originate from bone marrow lymphoid stem cells. Their differentiation and development depend on the bone marrow microenvironment. They are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs and lymph nodes, accounting for about 5-20% of peripheral lymphocytes. In the blood, bone marrow, spleen and lungs, NK cells are mainly mature (CD56dimCD16 + ) and terminal differentiation (CD56dimCD16 + CD57 + ) subsets with higher effector function. In lymph nodes, tonsils and intestines, the frequency of NK cells is low and mainly consists of immature (CD56brightCD16 - ) subpopulations, which have low effector capabilities. Therefore, the source of NK cells cultured in vitro can be solid tissues (bone marrow, spleen, liver, lymph nodes, etc.), blood, or directly purchased commercial primary NK cells or NK cell lines (NK-92, etc.).

[0004] There are many challenges in culturing NK cells in vitro. NK cells account for a very low proportion in peripheral blood, the initial NK cell amplification ratio of PBMC is low, NK cells are difficult to amplify and easily exhausted, and serum or feeder cells need to be added to the culture medium. However, the current efficiency of NK cell amplification is still relatively low. In addition, it is difficult to maintain the cytotoxic effect of NK cells after amplification. Although different cytokines can be used to improve this problem (for example, IL-2, IL-12, and IL-15 can promote NK cell differentiation and amplification; IL-12 and IL-18 can enhance NK cell antigen sensitivity; IL-21 can enhance the cytotoxic effect of NK cells), the decline in activity after amplification is still a technical defect that has not been overcome in NK cell culture technology. Summary of the Invention

[0005] The present invention aims to provide a new NK cell culture medium to address the technical defects of the existing technology, so as to solve the technical problems such as low culture efficiency and low cell activity of NK cells in conventional culture media.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] A new NK cell culture medium includes basal culture medium, serum, cytokines, antibiotics, as well as paclitaxel, cocin, ethanolamine, hyaluronic acid, sodium selenite, L-ascorbic acid, pyrroloquinoline quinone, dihydroxypropylphylline, reduced glutathione, madecassoside, D-pantothenate calcium, and estradiol.

[0008] Preferably, in the NK cell culture medium, the final concentrations of paclitaxel, cocin, ethanolamine, hyaluronic acid, sodium selenite, L-ascorbic acid, pyrroloquinoline quinone, dihydroxypropylphylline, reduced glutathione, madecassoside, D-calcium pantothenate, and estradiol are: paclitaxel 1-1.5 mg / mL, cocin 1.5-2 mg / mL, ethanolamine 0.8-1 mg / mL, hyaluronic acid 0.5-0.8 mg / mL, Sodium selenite 0.2-0.5 mg / mL, L-ascorbic acid 0.08-0.1 mg / mL, pyrroloquinoline quinone 0.1-0.2 mg / mL, dyphylline 0.1-0.2 mg / mL, reduced glutathione 0.1-0.3 mg / mL, madecassoside 0.2-0.4 mg / mL, D-calcium pantothenate 0.08-0.1 mg / mL, and estradiol 0.05-0.1 mg / mL.

[0009] Preferably, in the NK cell culture medium, the final concentrations of paclitaxel, cocin, ethanolamine, hyaluronic acid, sodium selenite, L-ascorbic acid, pyrroloquinoline quinone, dihydroxypropylphylline, reduced glutathione, madecassoside, D-pantothenate calcium, and estradiol are: paclitaxel 1.2-1.3 mg / mL, cocin 1.6-1.8 mg / mL, ethanolamine 0.8-0.9 mg / mL, hyaluronic acid 0.6-0.7 mg / mL, selenite 0. Sodium sulfate 0.3-0.4 mg / mL, L-ascorbic acid 0.09-0.1 mg / mL, pyrroloquinoline quinone 0.15-0.18 mg / mL, dihydroxypropylphylline 0.12-0.18 mg / mL, reduced glutathione 0.15-0.25 mg / mL, madecassoside 0.25-0.35 mg / mL, D-pantothenate calcium 0.08-0.09 mg / mL, and estradiol 0.06-0.08 mg / mL.

[0010] Preferably, in the NK cell culture medium, the final concentrations of paclitaxel, cocin, ethanolamine, hyaluronic acid, sodium selenite, L-ascorbic acid, pyrroloquinoline quinone, dihydroxypropylphylline, reduced glutathione, madecassoside, D-calcium pantothenate, and estradiol are: paclitaxel 1.5 mg / mL, cocin 1.7 mg / mL, ethanolamine 0.85 mg / mL, hyaluronic acid 0.65 mg / mL, sodium selenite 0.35 mg / mL, L-ascorbic acid 0.09 mg / mL, pyrroloquinoline quinone 0.16 mg / mL, dihydroxypropylphylline 0.15 mg / mL, reduced glutathione 0.2 mg / mL, madecassoside 0.3 mg / mL, D-calcium pantothenate 0.08 mg / mL, and estradiol 0.07 mg / mL.

[0011] Preferably, the basal culture medium is RPMI-1640 culture medium, DMEM culture medium or IMDM culture medium.

[0012] Preferably, the serum is fetal bovine serum or human serum; the final concentration of the serum is 5% to 10%.

[0013] Preferably, the cytokines include IL-2, IL-7, IL-15, IL-3, VEGF, and GDNF.

[0014] Preferably, in the NK cell culture medium, the final concentrations of IL-2, IL-7, IL-15, IL-3, VEGF, and GDNF are: IL-2 25-40 pg / mL, IL-7 10-30 pg / mL, IL-15 50-70 pg / mL, IL-3 30-50 pg / mL, VEGF 5-15 ng / mL, and GDNF 20-30 ng / mL, respectively.

[0015] Preferably, the cytokines further include PDGF and bFGF.

[0016] Preferably, in the NK cell culture medium, the final concentrations of PDGF and bFGF are: PDGF 10-25 ng / ml, bFGF 5-10 ng / mL, respectively.

[0017] The present invention provides a new NK cell culture medium. It includes basal culture medium, serum, cytokines, antibiotics and auxiliary factors, wherein the basal culture medium is selected from RPMI-1640, DMEM or IMDM, the serum is fetal bovine serum or human serum, the cytokines include IL-2, IL-7, IL-15, IL-3, VEGF, GDNF, and the antibiotics are penicillin or streptomycin. The present invention introduces auxiliary factors containing paclitaxel, cocin, ethanolamine, hyaluronic acid, sodium selenite, L-ascorbic acid, pyrroloquinoline quinone, dihydroxypropylphylline, reduced glutathione, madecassoside, D-pantothenic acid calcium, and estradiol into the basal culture medium, which can significantly increase the proliferation multiples and cell viability of NK cells, and at the same time, promote the expression of CD56 and CD16 by progeny NK cells. Experimental results show that after a culture period of 16 days, the present invention causes NK cells to proliferate 377.6 times, the cell viability reaches 98.2%, and the expression of CD16 + CD56 + The percentage of cell number reached 83.28%. DETAILED DESCRIPTION

[0018] The following is a detailed description of specific embodiments of the present invention. To avoid excessive unnecessary detail, well-known structures or functions will not be described in detail in the following examples. Approximate language used in the following examples can be used for quantitative expression to indicate that a certain amount of variation is allowed without changing the basic function. Unless otherwise defined, technical and scientific terms used in the following examples have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0019] Example 1

[0020] Penicillin, streptomycin, IL-2, IL-7, IL-15, IL-3, VEGF, GDNF, paclitaxel, cocin, ethanolamine, hyaluronic acid, sodium selenite, L-ascorbic acid, pyrroloquinoline quinone, dihydroxypropylphylline, reduced glutathione, madecassoside, calcium D-pantothenate, and estradiol were added to RPMI-1640 culture medium containing 5% fetal bovine serum to the following concentrations: penicillin 100 U / mL, streptomycin 0.1 mg / mL, IL-2 30 pg / mL, IL-7 20 pg / mL, IL-15 60 pg / mL, IL-3 40 pg / mL, VEGF 10 ng / mL, GDNF NK cell culture medium was obtained by adding 1% paraformaldehyde, 25 ng / mL of dapoxetine, 1.5 mg / mL of paclitaxel, 1.7 mg / mL of cocin, 0.85 mg / mL of ethanolamine, 0.65 mg / mL of hyaluronic acid, 0.35 mg / mL of sodium selenite, 0.09 mg / mL of L-ascorbic acid, 0.16 mg / mL of pyrroloquinoline quinone, 0.15 mg / mL of dyphylline, 0.2 mg / mL of reduced glutathione, 0.3 mg / mL of madecassoside, 0.08 mg / mL of calcium D-pantothenate, and 0.07 mg / mL of estradiol.

[0021] NK cell culture medium was added to a T75 suspension culture flask at a volume of 2.8 × 10 6 Peripheral blood-derived NK cells were inoculated at a concentration of 100 cells / mL and placed in an incubator for culture at 37°C, 5% CO2 concentration, and 95% humidity.

[0022] During the first few days of culture, avoid pipetting the cell pellet to reduce mechanical damage. On day 3, gently rehydrate the cells and avoid shaking the culture flask. On day 5, spin the cells. After day 10, replenish the culture medium at a 1.5x volume every two days. Harvest the cells after day 16 of culture.

[0023] Flow cytometry was used to detect the phenotype of collected NK cells and analyze the expression of CD16 + CD56 + The results are recorded in Table 1.

[0024] Trypan blue staining was used to examine the initial cell inoculation amount and the total number of cells harvested after culture, the proliferation times were calculated, and the viability of the harvested NK cells was statistically analyzed. The results are recorded in Table 2.

[0025] Example 2

[0026] Based on the technical solution of Example 1, the estradiol component was omitted from the NK cell culture medium, and other conditions remained consistent with those of Example 1.

[0027] Example 3

[0028] On the basis of the technical solution of Example 1, the D-calcium pantothenate component was omitted from the NK cell culture medium, and other conditions remained the same as those of Example 1.

[0029] Example 4

[0030] On the basis of the technical solution of Example 1, the NK cell culture medium omitted the madecassoside component, and other conditions remained the same as those of Example 1.

[0031] Example 5

[0032] Based on the technical solution of Example 1, the reduced glutathione component was omitted from the NK cell culture medium, and other conditions remained consistent with those of Example 1.

[0033] Example 6

[0034] Based on the technical solution of Example 1, the NK cell culture medium omitted the dihydroxypropylphylline component, and other conditions remained consistent with Example 1.

[0035] Example 7

[0036] On the basis of the technical solution of Example 1, the pyrroloquinoline quinone component was omitted from the NK cell culture medium, and other conditions remained the same as those of Example 1.

[0037] Example 8

[0038] On the basis of the technical solution of Example 1, L-ascorbic acid was omitted from the NK cell culture medium, and other conditions remained the same as those of Example 1.

[0039] Example 9

[0040] On the basis of the technical solution of Example 1, the sodium selenite component was omitted from the NK cell culture medium, and other conditions remained the same as those of Example 1.

[0041] Example 10

[0042] Based on the technical solution of Example 1, the hyaluronic acid component was omitted from the NK cell culture medium, and other conditions remained consistent with those of Example 1.

[0043] Example 11

[0044] Based on the technical solution of Example 1, the NK cell culture medium omitted the ethanolamine component, and other conditions remained consistent with Example 1.

[0045] Example 12

[0046] Based on the technical solution of Example 1, the NK cell culture medium omitted the Cohen component, and other conditions remained consistent with Example 1.

[0047] Example 13

[0048] On the basis of the technical solution of Example 1, the paclitaxel component was omitted from the NK cell culture medium, and other conditions remained the same as those of Example 1.

[0049] Example 14

[0050] Based on the technical solution of Example 1, paclitaxel, cocin, ethanolamine, hyaluronic acid, sodium selenite, L-ascorbic acid, pyrroloquinoline quinone, dihydroxypropylphylline, reduced glutathione, madecassoside, D-calcium pantothenate, and estradiol were omitted from the NK cell culture medium, and other conditions remained the same as in Example 1.

[0051] Table 1. NK cells cultured and expressing CD16 + CD56 + The percentage of cells

[0052]

[0053]

[0054] The above experimental results show that the cofactors introduced by the present invention (paclitaxel, cohen, ethanolamine, hyaluronic acid, sodium selenite, L-ascorbic acid, pyrroloquinoline quinone, dyphylline, reduced glutathione, madecassoside, calcium D-pantothenate, and estradiol) only have a positive effect on the immune activity of progeny NK cells when added simultaneously, and the improvement in cellular immune activity is exceptionally significant. The technical effects of the present invention cannot be achieved when any of the cofactors are missing, and the cellular immune activity is essentially the same as when none of the cofactors are added.

[0055] Table 2 Proliferation times and cell viability of cultured NK cells

[0056]

[0057]

[0058] The above experimental results demonstrate that the cofactors introduced by the present invention (paclitaxel, cohen, ethanolamine, hyaluronic acid, sodium selenite, L-ascorbic acid, pyrroloquinoline quinone, dyphylline, reduced glutathione, madecassoside, calcium D-pantothenate, and estradiol) can significantly increase the proliferation rate and cell viability of NK cells. Furthermore, the absence of any of these components significantly impacts these effects. Thus, the present invention demonstrates a definite and significant enhancement of NK cell expansion efficiency.

[0059] The embodiments of the present invention are described in detail above, but the contents are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the scope of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A new NK cell culture medium, characterized in that: include: Penicillin, streptomycin, IL-2, IL-7, IL-15, IL-3, VEGF, GDNF, paclitaxel, calcineurin, ethanolamine, hyaluronic acid, sodium selenite, L-ascorbic acid, pyrroloquinoline quinone, diprophylline, reduced glutathione, madecassoside, calcium D-pantothenate, and estradiol were added to RPMI-1640 culture medium containing 5% fetal bovine serum to the following concentrations: penicillin 100 U / mL, streptomycin 0.1 mg / mL, IL-2 30 pg / mL, IL-7 20 pg / mL, IL-15 60 pg / mL, IL-3 40 pg / mL, VEGF 10 ng / mL, GDNF 25 ng / mL, paclitaxel 1.5 mg / mL, calcineurin 1.7 mg / mL, ethanolamine 0.85 mg / mL, hyaluronic acid 0.65 mg / mL, sodium selenite 0.35 mg / mL, L-ascorbic acid 0.09 mg / mL, pyrroloquinoline quinone 0.16 mg / mL, dyphylline 0.15 mg / mL, reduced glutathione 0.2 mg / mL, madecassoside 0.3 mg / mL, D-calcium pantothenate 0.08 mg / mL, and estradiol 0.07 mg / mL; obtain NK cell culture medium.

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