CN115710577A - NK cell culture medium and method for in-vitro amplification of NK cells - Google Patents
NK cell culture medium and method for in-vitro amplification of NK cells Download PDFInfo
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- YDDUMTOHNYZQPO-YVUSBIGSSA-N 1,3-Dicaffeoylquinic acid Natural products O=C(O[C@@H]1[C@H](O)[C@H](O)C[C@](OC(=O)/C=C/c2cc(O)c(O)cc2)(C(=O)O)C1)/C=C/c1cc(O)c(O)cc1 YDDUMTOHNYZQPO-YVUSBIGSSA-N 0.000 claims abstract description 14
- YDDUMTOHNYZQPO-UHFFFAOYSA-N 1,3-bis{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-4,5-dihydroxycyclohexanecarboxylic acid Natural products OC1C(O)CC(C(O)=O)(OC(=O)C=CC=2C=C(O)C(O)=CC=2)CC1OC(=O)C=CC1=CC=C(O)C(O)=C1 YDDUMTOHNYZQPO-UHFFFAOYSA-N 0.000 claims abstract description 14
- JUHOZYRSRTUDPA-UHFFFAOYSA-N 1,3-di-O-caffeoyl quinic acid methyl ester Natural products C1C(C(=O)OC)(OC(=O)C=CC=2C=C(O)C(O)=CC=2)CC(O)C(O)C1OC(=O)C=CC1=CC=C(O)C(O)=C1 JUHOZYRSRTUDPA-UHFFFAOYSA-N 0.000 claims abstract description 14
- YDDUMTOHNYZQPO-BBLPPJRLSA-N 1,3-di-O-caffeoylquinic acid Natural products O[C@@H]1C[C@@](C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)(OC(=O)C=Cc1ccc(O)c(O)c1)C(O)=O YDDUMTOHNYZQPO-BBLPPJRLSA-N 0.000 claims abstract description 14
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- 239000002609 medium Substances 0.000 claims abstract description 10
- KSMRODHGGIIXDV-YFKPBYRVSA-N N-acetyl-L-glutamine Chemical compound CC(=O)N[C@H](C(O)=O)CCC(N)=O KSMRODHGGIIXDV-YFKPBYRVSA-N 0.000 claims abstract description 9
- 229960005488 aceglutamide Drugs 0.000 claims abstract description 9
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- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 abstract description 8
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Abstract
The invention discloses an NK cell culture medium and a method for amplifying NK cells in vitro, wherein the culture medium contains: 10-20% FBS, 1640 medium of 40-60. Mu.g/mL penicillin-streptomycin solution; 10-200 mg/mL human plasma transferrin; 40-90 μ M1,3-dicaffeoylquinic acid; 20-70 μ M aceglutamide; 500-1500 IU/mL human recombinant IL-2;10-200 IU/mL human recombinant IL-15;10-200 IU/mL human recombinant IL-18;10-200 IU/mL human recombinant IL-21. The invention discloses that the expression levels of Perforin, granzyme B and CD107a of NK cells can be remarkably enhanced and the killing activity of the NK cells to K562 can be remarkably enhanced under the combined stimulation condition that 1,3-dicaffeoylquinic acid and aceglutamide are added according to a specific proportion for the first time.
Description
Technical Field
The invention relates to the field of cell culture, in particular to an NK cell culture medium and a method for amplifying NK cells in vitro.
Background
Natural killer cells (NK) are important immune cells of the body, mainly distributed in the peripheral blood. NK cells, as the first line of defense of the body, can not only exert their main tumoricidal effects in the natural immune system, but also secrete different cytokines and various chemokines to regulate the body's acquired immune response in the early stage of the immune response, and are the indispensable effector cells of the body to exert immune effects.
The content of NK cells in peripheral blood is far from meeting clinical requirements, and at present, the NK cells are amplified mainly by using stimulation of cytokines, but the amplified NK cells by the method have a certain gap with the clinical requirements. How to provide a culture medium capable of amplifying a large amount of high-activity NK cells is one of the problems to be solved in the field of NK cell in-vitro amplification.
The application of PD-1 blocker in enhancing NK cell killing power is disclosed in Chinese patent application CN 113897334A. In the process that NK cells kill target cells, the NK cells are triggered by signals, and first through the degranulation process, perforin and granzyme B are released to reach the target cells, wherein Perforin is used for perforating a channel on a target cell membrane to further mediate granzyme B to enter the target cells, and after granzyme B enters the target cells, DNA breakage of the target cells is triggered to enable the target cells to die. CD107a is also an important factor involved in NK cell degranulation.
Further, in the prior application CN114606187a of the present application, there is also disclosed an NK cell culture medium, characterized in that the culture medium contains: 10-20% FBS, 1640 medium of 50. Mu.g/mL penicillin-streptomycin solution; 10-200 mg/mL human plasma transferrin; 100uM 1, 3-dicaffeoylquinic acid; 1000 IU/mL human recombinant IL-2;10-200 IU/mL human recombinant IL-15;10-200 IU/mL human recombinant IL-18;10-200 IU/mL human recombinant IL-21. By adding 1,3-dicaffeoylquinic acid into the culture medium, the killing activity of NK cells on K562 can be obviously enhanced, and the promotion effect is enhanced along with the increase of the effective target ratio.
However, in the subsequent studies of the applicant, it was found that the increase of the killing activity of the NK cells against the tumor cells is limited only by adding 1,3-dicaffeoylquinic acid, and in order to solve the technical problem, the applicant has conducted further studies and completed the present invention.
Disclosure of Invention
The invention mainly solves the technical problem of providing an NK cell culture medium and a method for amplifying NK cells in vitro, and the NK cells with high activity and stronger killing property can be amplified.
In order to solve the above-mentioned problems, according to a first aspect of the present invention, there is provided an NK cell culture medium comprising:
10-20% FBS, 1640 medium of 40-60. Mu.g/mL penicillin-streptomycin solution;
10-200 mg/mL human plasma transferrin;
40-90 μ M1,3-dicaffeoylquinic acid;
20-70 μ M aceglutamide;
500-1500 IU/mL human recombinant IL-2;
10-200 IU/mL human recombinant IL-15;
10-200 IU/mL human recombinant IL-18
10-200 IU/mL human recombinant IL-21.
In a preferred embodiment, the amount of 1,3-dicaffeoylquinic acid in the medium is 50-80 μ M; preferably 50-60. Mu.M.
In a preferred embodiment, the amount of acetylglutamide in the medium is 30 to 70 μ M; preferably 30-40. Mu.M.
In a second aspect of the present invention, there is provided a method for in vitro expansion of NK cells, the method comprising: the NK cells were suspended in the above NK cell culture medium, and the NK cell-suspended medium was transferred to a cell culture flask, and the cell culture flask was placed in an incubator for culture.
In one embodiment, the cell density is adjusted to 1-5X 10 when transferring NK cells to a cell culture flask 5 Each/ml.
In one embodiment, the incubator parameters are humidity saturation, 5% CO 2 、37℃。
Compared with the prior art, the invention achieves the following remarkable improvements:
the invention discloses that the expression levels of Perforin, granzyme B and CD107a of NK cells can be remarkably enhanced and the killing activity of the NK cells on K562 can be remarkably enhanced under the combined stimulation condition of adding 1,3-dicaffeoylquinic acid and aceglutamide in a specific proportion for the first time.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.
Example 1: preparation and in-vitro amplification method of NK cell culture medium
The NK cell culture medium comprises the following components:
1640 medium containing 10% FBS, 50. Mu.g/mL penicillin-streptomycin solution;
20mg/mL human plasma transferrin;
50 μ M1,3-dicaffeoylquinic acid;
30 μ M aceglutamide;
1000 IU/mL human recombinant IL-2;
50 IU/mL human recombinant IL-15;
50 IU/mL human recombinant IL-21.
The medium was depleted of 100uM 1, 3-dicaffeoylquinic acid fraction and used as a control.
The method for in vitro expansion of NK cells can be performed as follows:
1. separating human peripheral blood mononuclear cell (PB-MC) by anticoagulation of venous heparin, diluting Hank's solution in equal volume, performing density gradient centrifugation with Fi-coll to obtain PBMC, washing, and adjusting density of PBMC to 2 × 10 9 Purifying NK cells by an immunomagnetic bead method, and operating according to the kit instructions (flow detection shows that the purity of the NK cells>95%)。
2. Suspending the prepared NK cell strain in the above NK cell culture medium and control group NK cell culture medium, and adjusting the density of NK cells to 1-5 × 10 5 Transferring to T75 cell culture flask for culture under humidity saturation, 5% CO2, and 37 deg.C incubator.
3. NK cells were collected for further 48h, washed with PBS and resuspended to a cell density of 1X 10 6 one/mL of the cells were inoculated into 0.1mL of flow tubes, and the cells were incubated with PE-labeled Perforin (Perforin), PE-labeled Granzyme B (Granzyme B), and APC-labeled CD107a for flow cytometry detection, and the detection results are shown in Table 1.
Example 2:
same as in example 1, except that the amount of acetylglutamide was 50. Mu.M.
Example 3:
the same as example 1 except that the amount of 1,3-dicaffeoylquinic acid was 80. Mu.M.
Comparative example 1:
the same as example 1 except that without aceglutamide, 1,3-dicaffeoylquinic acid was present in an amount of 100. Mu.M.
Comparative example 2:
the same as example 1 except that no 1,3-dicaffeoylquinic acid was added, the amount of acetylglutamide was 80. Mu.M.
Table 1: perforin, granzyme B, CD107a expression level
The experimental results show that the expression levels of Perforin, granzyme B and CD107a of NK cells can be remarkably enhanced under the stimulation condition of adding the 1,3-dicaffeoylquinic acid and aceglutamide in a specific ratio.
Example 4: killing experiment of NK cells on K562 cells
The NK cells cultured on day 15 in the cell proliferation ability test were used as effector cells and K562 cells as target cells.
First, effector cell solution is prepared: the NK cells cultured for 15 days in examples 1-3 and comparative examples 1-2 were washed 2 times with 2% FBS-containing RPMI 1640 medium, and 1X 10% FBS-containing RPMI 1640 medium 6 Cell suspension/ml. Target cells K562 cells were washed once with 2% FBS-containing RPMI 1640 medium, 1X 10% FBS-containing RPMI 1640 medium 5 Cell suspension/ml. The cells were inoculated into a 96-well plate at an effective target ratio of 10. The absorbance was measured by MTT method, i.e., 150. Mu.L of dimethyl sulfoxide was added to each well, gently shaken, and the A490 value was measured by a microplate reader to calculate the tumor killing rate, and the results are shown in Table 2.
Wherein: tumor killing rate% =1- (test well-effector cell well)/target cell well.
Table 2: experimental results on tumor killing rate
The experimental result shows that the combined stimulation condition of 1,3-dicaffeoylquinic acid and aceglutamide added in a specific proportion can obviously enhance the killing activity of NK cells to K562.
In conclusion, the NK cell culture medium and the method for amplifying NK cells in vitro can amplify NK cells with high activity and stronger killing property.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (8)
1. An NK cell culture medium, comprising:
10-20% FBS, 1640 medium of 40-60. Mu.g/mL penicillin-streptomycin solution;
10-200 mg/mL human plasma transferrin;
40-90 μ M1,3-dicaffeoylquinic acid;
20-70 μ M aceglutamide;
500-1500 IU/mL human recombinant IL-2;
10-200 IU/mL human recombinant IL-15;
10-200 IU/mL human recombinant IL-18;
10-200 IU/mL human recombinant IL-21.
2. The culture medium of claim 1, wherein the amount of 1,3-dicaffeoylquinic acid in the culture medium is 50-80 μ Μ.
3. The culture medium of claim 1, wherein the amount of 1,3-dicaffeoylquinic acid in the culture medium is 50-60 μ Μ.
4. The culture medium according to claim 1, wherein the amount of acetylglutamide in the culture medium is 30-70 μ M.
5. The culture medium according to claim 1, wherein the amount of acetylglutamide in the culture medium is 30-40 μ M.
6. A method of expanding NK cells in vitro, comprising: suspending NK cells in the NK cell culture medium of any one of claims 1 to 5, transferring the NK cell suspended medium to a cell culture flask, and culturing the cell culture flask in an incubator.
7. The method according to claim 6, wherein the cell density is adjusted to 1 to 5X 10 when transferring the NK cells to the cell culture flask 5 One per ml.
8. The method of claim 6, wherein the incubator parameter is humidity saturation, 5% CO 2 、37℃。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0653066B2 (en) * | 1987-02-18 | 1994-07-20 | 工業技術院長 | New cell line |
| CN109294985A (en) * | 2018-10-25 | 2019-02-01 | 江苏普瑞康生物医药科技有限公司 | A method of culture medium system and NK cell expansion ex vivo for NK cell expansion ex vivo |
| CN114032210A (en) * | 2021-11-06 | 2022-02-11 | 曾东升 | Culture medium and culture method for NK cell amplification |
| CN114606187A (en) * | 2022-05-13 | 2022-06-10 | 北京汉氏联合生物技术股份有限公司 | NK cell culture medium and method for in-vitro amplification of NK cells |
| WO2022255793A1 (en) * | 2021-06-01 | 2022-12-08 | 주식회사 박셀바이오 | Composition containing feeder cell for proliferating natural killer cell |
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- 2022-12-12 CN CN202211587869.1A patent/CN115710577A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0653066B2 (en) * | 1987-02-18 | 1994-07-20 | 工業技術院長 | New cell line |
| CN109294985A (en) * | 2018-10-25 | 2019-02-01 | 江苏普瑞康生物医药科技有限公司 | A method of culture medium system and NK cell expansion ex vivo for NK cell expansion ex vivo |
| WO2022255793A1 (en) * | 2021-06-01 | 2022-12-08 | 주식회사 박셀바이오 | Composition containing feeder cell for proliferating natural killer cell |
| CN114032210A (en) * | 2021-11-06 | 2022-02-11 | 曾东升 | Culture medium and culture method for NK cell amplification |
| CN114606187A (en) * | 2022-05-13 | 2022-06-10 | 北京汉氏联合生物技术股份有限公司 | NK cell culture medium and method for in-vitro amplification of NK cells |
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