CN107083361B - Cell culture method - Google Patents
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Abstract
The invention belongs to the field of cell culture, and particularly relates to a cell culture method. The invention provides a cell culture method, which comprises the following steps: 1) isolating peripheral blood mononuclear cells; 2) adding the isolated peripheral blood mononuclear cells of step 1) to an activating medium containing plasma and activating NK cells; 3) adding a proliferation medium to the peripheral blood mononuclear cells cultured up to day 10 in step 2) promotes proliferation of NK cells. The cell culture method provided by the invention can effectively overcome the technical defect of small amount of amplified cells in the in-vitro amplification of NK cells.
Description
Technical Field
The invention belongs to the field of cell culture, and particularly relates to a cell culture method.
Background
Cellular immunotherapy is an emerging tumor treatment model and is a novel treatment method for resisting cancer by means of autoimmunity. It collects immune cells from the body of a patient, then carries out in vitro culture and amplification, and then is infused back into the body of the patient to stimulate and enhance the autoimmune function of the body, thereby achieving the purpose of treating tumors. Unlike the immune injury caused by operation, radiotherapy and chemotherapy, the cell therapy can greatly improve the anticancer immunity of patients, eliminate the cancer cells which can not be reached by the operation therapy, lymph or transferred to other tissues and even dormant cancer cells, and compared with the treatment, the treatment is more thorough.
NK cells, natural killer cells (NK), are important immune cells of the body and are not only involved in anti-tumor, anti-rheumatic, anti-viral infection and immune regulation, but also in some cases in the development of hypersensitivity reactions and autoimmune diseases. Several other cells with anti-cancer function include killer T cells, macrophages, Dendritic Cells (DCs). Because macrophages contain many inflammatory factors, they are not currently available for cell therapy. DCs are a class of immunoregulatory cells, the main role of which is antigen processing and presentation to T cells, which in turn exert an anti-cancer effect. The anti-cancer effect of the T cells needs to be stimulated again to be effective, the T cells can only act after being activated by DC and have no killing effect when meeting antigens for the first time, but only form immunological memory, and the strong killing function can be stimulated only after meeting the antigens again. Most of the CIKs used in human today are cytokine-activated T cells. In fact, the therapeutic efficacy of killer T cells activated by cytokines alone without DC antigen presentation is very limited. Compared with CIK and DC, NK can kill tumor cells through antibody-mediated cytotoxicity (ADCC) and perforin release besides releasing cytokines, and has stronger tumor killing capacity. However, compared to CIK, there is currently relatively little treatment with NK cells, the main reason for which is that NK cell production is difficult.
Currently, there are two main methods for preparing human NK cells. One is to directly separate NK cells from PBMC by magnetic bead separation. However, because the proportion of NK cells in PBMC is small, the number of the obtained NK cells is small, and the method is only suitable for small-scale preparation of NK cells and analytical research. The other is an in vitro amplification method, which uses PBMC or separated NK cells to make the NK cells amplified in vitro through the stimulation of some special cytokines. However, most of the in vitro amplification techniques to date can only amplify NK cells dozens of times, and have low purity, poor activity and high cost, so that the clinical use requirements are difficult to meet.
Therefore, the development of a culture method capable of promoting the mass proliferation of NK cells is a technical problem to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the cell culture method disclosed by the invention can effectively overcome the technical defect of small amount of expanded cells in the in vitro expansion of NK cells.
The invention discloses a cell culture method. The method comprises the following steps:
1) isolating peripheral blood mononuclear cells;
2) adding the isolated peripheral blood mononuclear cells of the step 1) into an activation medium containing plasma, and culturing the cells until the 10 th day, wherein the activation medium containing the plasma activates the activation of the peripheral blood mononuclear cells; the activation medium comprises: anti-CD 16 monoclonal antibody, IL-2, ascorbic acid, TGF-beta 1, thymosin peptide and serum-free basal medium;
3) adding a proliferation medium to the peripheral blood mononuclear cells cultured up to the 10 th day in the step 2) and the activation medium containing plasma to continue culturing the peripheral blood mononuclear cells until the cells are recovered, the proliferation medium including: IL-1 alpha, IL-2, ascorbic acid, IL-21, IL-7, 4-1BBL, and serum-free basal media.
Wherein the activation medium activates NK cells of peripheral blood mononuclear cells.
Wherein the proliferation medium promotes rapid proliferation of NK cells of peripheral blood mononuclear cells.
Wherein, in the step 2), the peripheral blood mononuclear cells obtained in the step 1) are cultured in an activation medium containing plasma, the activation medium is supplemented to the peripheral blood mononuclear cells on the 3 rd, 6 th and 8 th days, and the cell density is maintained at 1 × 10 when the activation medium is supplemented6 2X 10 per ml6And maintaining the volume percentage of autologous plasma in the activated medium culture to be 4%.
Wherein, the peripheral blood mononuclear cells of the step 1) are cultured in the activation culture medium containing the plasma for 10 days and then are subjected to the operation of the step 3), and the peripheral blood mononuclear cells can fully activate the NK cells of the peripheral blood mononuclear cells after being cultured in the activation culture medium containing the plasma for 8 days, so that the proliferation culture medium of the step 3) can rapidly proliferate the NK cells of the peripheral blood mononuclear cells.
Wherein the plasma is autologous plasma, and the autologous plasma is plasma in the blood obtained by separating peripheral blood mononuclear cells in the step 1).
Wherein the plasma is inactivated plasma.
Wherein, the step 3) adds the proliferation culture medium to the peripheral blood mononuclear cells cultured to the 10 th day in the step 2) and the activation culture medium containing the plasma, and culturing the peripheral blood mononuclear cells in the step 1) until the cells are recovered, the step 2) is started to calculate when the peripheral blood mononuclear cells are cultured in the activation culture medium containing the plasma, the proliferation culture medium is supplemented on the 12 th and 14 th days of culture, and the cell density is maintained to be 1 × 10 when the proliferation culture medium is supplemented7 2X 10 per ml7One per ml.
Wherein, the proliferation medium can promote the rapid proliferation of NK cells of peripheral blood mononuclear cells.
Preferably, the volume percentage of the plasma in the activated medium culture is 4%.
Preferably, the serum-free basal medium is serum-free RPMI1640 medium.
Preferably, the activation medium comprises:
more preferably, the activation medium comprises:
the invention also provides a preparation method of the activation medium, and the activation medium is prepared by dissolving the anti-CD 16 monoclonal antibody, IL-2, ascorbic acid, TGF-beta 1 and thymosin in a serum-free basal medium and filtering the solution through a 0.1 mu m filter membrane.
Preferably, the multiplication medium comprises:
more preferably, the multiplication medium comprises:
the invention also provides a preparation method of the proliferation culture medium, wherein the proliferation culture medium is prepared by dissolving IL-1 alpha, IL-2, ascorbic acid, IL-21, IL-7 and 4-1BBL in a serum-free basal culture medium and filtering through a 0.1 mu m filter membrane.
The invention also discloses application of the cell culture method in promoting NK cell proliferation of peripheral blood mononuclear cells.
The invention aims to overcome the technical defects that the number of amplified cells is small when NK cells are amplified in vitro and the activity of NK cells obtained by in vitro culture is poor in the prior art. Therefore, the invention adopts a brand-new combination of cytokines, wherein IL-2 and TGF-beta 1 of the activation medium in the cell culture method can stimulate the proliferation of NK cells; the anti-CD 16 monoclonal antibody is combined with an NK cell surface molecule CD16, activates NK cells and promotes cytokine synthesis; ascorbic acid inhibits regulatory T cell proliferation; the thymosin promotes the expression of an IL-2 receptor and enhances the effect of IL-2; IL-21 in the propagation medium of the cell culture method of the invention increases the expression of the NK cell surface active receptor NKG 2D; 4-1BBL increases the expression of NK cell surface activation receptor mutual NKp46, enhances the killing function of NK cells, and stimulates the high-power amplification of the NK cells under the synergistic action with IL-21; IL-7 activates NK cells and enhances the killing capacity of the NK cells on tumor cells. The invention has developed a new cell culture method, the cell culture method disclosed in the invention has very great improvement in NK cell proliferation rate, NK cell purity and NK cell in vitro killing activity to tumor cell, PBMC cultures in vitro to the 16 th day, NK cell amplification quantity increases 600 times than quantity while inoculating, NK cell purity of in vitro culture reaches more than 70%, and can also strengthen NK cell in vitro killing activity to tumor cell, when the effective target ratio of NK cell to tumor cell is 10:1, make its in vitro killing rate to tumor cell reach more than 90%; in addition, the cell culture method disclosed by the invention does not need to use inactivated tumor cells as feeder cells of NK cells, and does not need to adopt a magnetic bead separation method to separate the NK cells from PBMC, so that the culture operation can be greatly simplified, and the culture cost is reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows NK cell proliferation curves;
FIG. 2 shows the day 16 NK cell flow phenotype;
FIG. 3 shows the inhibition rate of NK cells on K562 cells at day 16.
Detailed Description
The invention provides a cell culture method, which is used for solving the technical defect of small amplified cell amount in the existing in-vitro amplification of NK cells.
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The reagents used in the following examples are all commercially available.
Example 1
Preparing an activation culture medium and a proliferation culture medium, and comprising the following steps:
firstly, preparing an activation medium
The serum-free RPMI1640 culture medium is used as a basic culture medium, an anti-CD 16 monoclonal antibody, IL-2, ascorbic acid, TGF-beta 1 and thymosin are added into the basic culture medium and uniformly mixed, so that the final concentrations of the anti-CD 16 monoclonal antibody, the IL-2, the ascorbic acid, the TGF-beta 1 and the thymosin in the activation culture medium are respectively 60ng/ml, 250U/ml, 2ng/ml, 60ng/ml and 2 ng/ml. Filtering with 0.1 μm filter membrane for sterilization, subpackaging, and storing at 4-8 deg.C in dark.
Secondly, preparing a proliferation culture medium
Taking serum-free RPMI1640 culture medium as a basic culture medium, and adding IL-1 alpha, IL-2, ascorbic acid, IL-21, IL-7 and 4-1BBL into the basic culture medium. Mixing to make final concentration of IL-1 alpha, IL-2, ascorbic acid, IL-21, IL-7 and 4-1BBL in serum-free RPMI1640 medium 50ng/ml, 500U/ml, 2ng/ml, 60ng/ml, 2ng/ml and 10ng/ml respectively. Filtering with 0.1 μm filter membrane for sterilization, subpackaging, and storing at 4-8 deg.C in dark.
The basic medium and cytokines are commercially available, wherein the basic medium is GIBCO serum-free RPMI1640, and other cytokines are purchased from Sigma.
Example 2
Culturing the cells, comprising the following steps:
step one, separating Peripheral Blood Mononuclear Cells (PBMC), comprising the following steps:
50ml of healthy human peripheral blood was collected, PBMC were isolated using lymphocyte separation (purchased from the tertiary sea of Tianjin) according to the method reported in the literature, and the supernatant plasma (autologous plasma) was collected and extinguished at 56 ℃ for 30 min. The PBMC of peripheral blood mononuclear cells were separated by carefully aspirating the intermediate haze cells and washed 2 times with physiological saline. Activation medium was added to resuspend PBMCs and perform cell counts.
Step two, culturing the NK cells, comprising the following steps:
PBMC density was adjusted to 2X 10 using the activation medium prepared in example 16And 56 ℃ inactivated autologous plasma (plasma collected in step one of example 2) was added to make the volume percentage of autologous plasma in the activation medium 4%. Transferring NK cells of PBMC and its culture medium into T175 flask, and placing at 37 deg.C and 5% CO2Culturing in a cell culture box with the concentration.
The calculation is started when NK cells of peripheral blood mononuclear cells are cultured in an activation medium containing plasma, and the cells are supplemented with activation on days 3, 6 and 8Culture medium, maintaining cell density of 1 × 10 when supplemented with activating medium62X 10 per ml6And maintaining the volume percentage of autologous plasma in the activated medium culture to be 4%.
Starting calculation when NK cells of peripheral blood mononuclear cells are cultured in an activation medium containing plasma, transferring the NK cells and the activation medium to a new cell culture bag for continuous culture after the activation medium is supplemented on the 8 th day, supplementing proliferation medium to the NK cells and the activation medium in the new cell culture bag on the 10 th, 12 th and 14 th days of culture, increasing the cell density in a rapid proliferation stage, and maintaining the cell density to be 1 x 10 when the proliferation medium is supplemented7 2X 10 per ml7One per ml. At 37 deg.C, 5% CO2The culture was continued in the cell culture chamber at the concentration until day 16, and the cells were collected. As shown in FIG. 1, the number of expanded NK cells increased 600-fold as compared to the number at the time of inoculation by the time of culture to day 16.
Example 3
The mononuclear cells of the peripheral blood cultured to the 16 th day are collected, and the proportion of CD3-CD56+ CD16+ of the cells is detected by a flow cytometer. FIG. 2 shows the flow assay results of NK cells on day 16 obtained by culturing according to the method of the present invention. As shown in the results, the proportion of CD3-CD56+ CD16+ of the peripheral blood mononuclear cells obtained by the invention reaches 75%, which indicates that the purity of the NK cells cultured in vitro (percentage of the NK cells in the total cells) reaches 75% after 16 days of culture.
Example 4
The log phase K562 cells were plated in 96 well cell culture plates, 5000 cells per well. NK cells (obtained by culturing in example 2) were added to each well on the following day, and the NK cells and K562 cells were co-cultured at concentrations of 1:1, 5:1 and 10:1 in the effective target ratio for 4h, 3 parallel wells per concentration. Adding the application solution into a WST1 cell counting kit to continue culturing for 2 hours, putting a 96-hole cell culture plate into an enzyme linked immunosorbent assay to detect the absorbance OD value at 450nm, and calculating the cell killing rate according to the formula: the killing rate (%) [1- (experimental OD value-NK control OD value)/target cell control OD value ] × 100%. As shown in FIG. 3, the NK cells obtained by culturing the NK cells of the day 16 by the method of the invention kill the K562 cells by 90% when the effective target ratio of the NK cells to the K562 cells is 10: 1.
In conclusion, the invention solves the technical defect of small amount of amplified cells when NK cells are amplified in vitro in the prior art. The purpose of the present invention is to develop a cell culture method that can promote the proliferation of NK cells in large quantities without affecting the activity of NK cells.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method of cell culture comprising the steps of:
1) isolating peripheral blood mononuclear cells;
2) adding the isolated peripheral blood mononuclear cells of step 1) to an activation medium containing plasma for culture to day 10; the activation medium comprises: anti-CD 16 monoclonal antibody, IL-2, ascorbic acid, TGF-beta 1, thymosin peptide and serum-free basal medium;
the activation medium comprises:
3) adding a proliferation medium to the peripheral blood mononuclear cells cultured to the 10 th day in the step 2) and the activation medium containing plasma, until the cells are recovered, the proliferation medium comprising: IL-1 alpha, IL-2, ascorbic acid, IL-21, IL-7, 4-1BBL and serum-free basal medium;
the multiplication medium comprises:
2. the cell culture method according to claim 1, wherein the volume percentage of the plasma in the activated medium culture is 4%.
3. The cell culture method according to claim 1, wherein the serum-free basal medium is serum-free RPMI1640 medium.
4. The cell culture method of claim 1, wherein the activation medium comprises:
5. a preparation method of an activation medium is characterized in that the activation medium is prepared by dissolving an anti-CD 16 monoclonal antibody, IL-2, ascorbic acid, TGF-beta 1 and thymosin in a serum-free basal medium and filtering the solution through a 0.1 mu m filter membrane;
the activation medium comprises:
6. the cell culture method according to claim 1, wherein the proliferation medium comprises:
7. a method for preparing a proliferation culture medium is characterized in that the proliferation culture medium is prepared by dissolving IL-1 alpha, IL-2, ascorbic acid, IL-21, IL-7 and 4-1BBL in a serum-free basal culture medium and filtering the solution through a 0.1 mu m filter membrane;
the multiplication medium comprises:
8. use of the cell culture method of any one of claims 1, 2, 3, 4, or 6 for promoting NK cell proliferation.
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| CN108504636A (en) * | 2018-04-13 | 2018-09-07 | 上海锦映生物科技有限公司 | A kind of efficient NK cell culture processes |
| CN109161527A (en) * | 2018-09-25 | 2019-01-08 | 深圳市五零生命科技有限公司 | A kind of efficient NK methods for cell expansion |
| CN109294988B (en) * | 2018-11-12 | 2021-05-11 | 友康恒业生物科技（北京）有限公司 | NK cell induction kit |
| CN111718899B (en) * | 2020-06-19 | 2021-11-23 | 珠海贝索细胞科学技术有限公司 | Culture method for in vitro induced NK (natural killer) cells after resuscitation of cryopreserved human PBMCs (peripheral blood mononuclear cells) |
| CN112877288B (en) * | 2021-03-05 | 2021-11-02 | 广州达博生物制品有限公司 | NK cell culture system and application |
| CN115197909B (en) * | 2021-04-12 | 2023-11-17 | 北京和斯瑞生物技术有限公司 | NK cell in-vitro culture method |
| CN113528439A (en) * | 2021-09-03 | 2021-10-22 | 东莞再立健生物科技有限公司 | NK cell culture method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011053321A1 (en) * | 2009-10-30 | 2011-05-05 | University Of Arkansas For Medical Science | Use of autologous effector cells for treatment of multiple myeloma |
| CN104357390A (en) * | 2014-10-15 | 2015-02-18 | 深圳源正细胞医疗技术有限公司 | Method for simultaneous and efficient amplification of CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells |
| CN105754941A (en) * | 2016-04-18 | 2016-07-13 | 广州复大医疗股份有限公司 | In-vitro induction and proliferation culture method for peripheral blood NK (natural killer) cells |
-
2017
- 2017-06-14 CN CN201710447706.6A patent/CN107083361B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011053321A1 (en) * | 2009-10-30 | 2011-05-05 | University Of Arkansas For Medical Science | Use of autologous effector cells for treatment of multiple myeloma |
| CN104357390A (en) * | 2014-10-15 | 2015-02-18 | 深圳源正细胞医疗技术有限公司 | Method for simultaneous and efficient amplification of CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells |
| CN105754941A (en) * | 2016-04-18 | 2016-07-13 | 广州复大医疗股份有限公司 | In-vitro induction and proliferation culture method for peripheral blood NK (natural killer) cells |
Non-Patent Citations (4)
| Title |
|---|
| Ascorbic acid promotes proliferation of natural killer cell populations in culture systems applicable for natural killer cell therapy;MIRELLE J.A.J. HUIJSKENS等;《Cytotherapy》;20151231;第17卷(第5期);第613-620页 * |
| Natural killer (NK) cell generation in bone marrow culture: role of IL-lα;Emira Ayroldi等;《IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY》;19911231;第13卷(第4期);第589-606页 * |
| 不同细胞因子组合对人自然杀伤细胞体外高纯度扩增的实验研究;刘新玲 等;《国际免疫学杂志》;20151231;第38卷(第6期);第501-506页 * |
| 最优化细胞因子诱导杀伤细胞/自然杀伤细胞少因子培养体系的探索;吴燕峰 等;《中山大学学报（医学科学版）》;20090818;第30卷(第4期);第361-366页 * |
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