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CN112266900A - CAR-NK cell culture method - Google Patents

CAR-NK cell culture method Download PDF

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CN112266900A
CN112266900A CN202011189833.9A CN202011189833A CN112266900A CN 112266900 A CN112266900 A CN 112266900A CN 202011189833 A CN202011189833 A CN 202011189833A CN 112266900 A CN112266900 A CN 112266900A
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湛振键
齐国光
刘世豪
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Guangdong Kangdun Innovation Industry Group Co ltd
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Guangdong Kangdun Bioengineering Technology Co Ltd
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Abstract

The invention provides a CAR-NK cell culture method, which comprises the following steps: s1, collecting peripheral blood of a healthy donor, and separating to obtain peripheral blood mononuclear cells; s2, separating NK cells from the peripheral blood mononuclear cells; s3, infecting the NK cells of the step S2 with a lentiviral vector with a chimeric antigen receptor gene; s4, carrying out amplification culture on the CAR-NK cells by using a culture medium containing interleukin-2, inositol, quercetin glucoside, formononetin, sheep placenta and galactose to obtain a large amount of CAR-NK cells. The CAR-NK cell culture method is simple to operate, and the cultured NK cells are high in purity and number, and the CAR-NK cells are high in number and killing activity.

Description

CAR-NK cell culture method
Technical Field
The invention relates to the field of cell culture, and particularly relates to a CAR-NK cell culture method.
Background
Chimeric antigen receptor (abbreviated CAR) modified immune cells use genetic engineering approaches to modify immune cells to express exogenous anti-tumor genes. The CAR gene mainly includes an extracellular recognition domain and an intracellular signaling domain: the former is used for identifying tumor surface specific molecules, and the latter is used for starting immune cell response after identifying tumor surface molecules, and plays a role in cytotoxicity. This is a new type of cell therapy that has been developed for many years, but has only been improved in recent years for clinical use. Has significant efficacy in the treatment of acute leukemia and non-hodgkin's lymphoma, and is considered to be one of the most promising tumor treatment modalities.
Natural Killer (NK) cells are an important component of the non-specific immune system, cells that are critical mediators of the innate immune system response. NK cells are a broad spectrum immune cells with specific functions of rapidly discovering and destroying abnormal cells (such as cancer or virus-infected cells), and exhibit potent activity of lysing abnormal cells without the need for pre-sensitization or HLA-typing. The use of immune cells, including NK cells, for the treatment of cancer is a new trend in recent years and this new therapy is expected to provide new cure hopes for tumors that are not effective in traditional surgery, chemotherapy and radiotherapy. However, the number of NK cells is small, the NK cells only account for 10% -15% of lymphocytes in peripheral blood, the content of NK in umbilical cord blood is lower, only about 5%, and the content of NK cells in normal human peripheral blood and umbilical cord blood is far from meeting the requirement of clinical treatment. The number and purity of NK cells are important influence factors of clinical curative effect, the number of NK cells is too small to achieve the curative effect, and the low purity can influence the killing effect on tumors.
The NK cell modified by the CAR structure can efficiently identify the tumor cell, and kill the tumor cell by various means such as releasing a killing medium, inducing apoptosis of a target cell and the like. However, the CAR-NK cell prepared by the prior art has the problems of complex operation of the preparation method, insufficient activity of the prepared cell and small quantity.
Disclosure of Invention
Aiming at the problems, the invention provides a CAR-NK cell culture method, which is realized by the following steps:
s1, collecting peripheral blood of a healthy donor, and separating to obtain peripheral blood mononuclear cells;
s2, separating NK cells from the peripheral blood mononuclear cells;
s3, infecting the NK cells of the step S2 with a lentiviral vector with a chimeric antigen receptor gene;
s4, transferring the NK cells obtained in the step S3 into a serum-free DMEM medium for culturing for 24-48 h, adjusting the cell density to (1-3). times.10 cells/mL, transferring the cells into a CAR-NK cell amplification medium for amplification culture, and culturing for 21-28 d to obtain CAR-NK cells; wherein the CAR-NK cell amplification culture medium is a DMEM culture medium containing 5-12% volume ratio of autologous inactivated serum, 120-180 ng/mL interleukin-2, 0.012-0.020 g/L inositol, 0.1-0.6 g/L quercetin glucoside, 0.02-0.06 g/L formononetin, 0.3-0.8 g/L sheep placenta and 0.1-0.8 g/L galactose.
Preferably, in step S2, the cell concentration of the peripheral blood mononuclear cells is diluted to (1-2). times.107Sucking 3-5 mL of the diluted cells, inoculating the cells into a culture bottle coated with 10-15 mu g/mL of anti-CD 16 monoclonal antibody for differential culture, supplementing 100-150 ng/mL of interleukin-12, 50-70 ng/mL of interleukin 3, 0.3-0.7 g/L of lecithin and 0.01-0.02 g/L of glutathione after culturing for 1-3 days, continuously culturing for 4-6 days, and sorting out NK cells; and performing NK cell amplification culture on the sorted NK cells.
Preferably, the differentiation culture is carried out by placing a culture bottle at the temperature of 30-35 ℃ and CO2Culturing in an incubator with the volume concentration of 4-5%.
Preferably, 120-130 ng/mL interleukin-12, 55-60 ng/mL interleukin-3, 0.4-0.5 g/L lecithin and 0.01g/L glutathione are supplemented after differentiation culture for 1-3 days.
Preferably, the NK cell amplification culture is performed by serum-free DMEM culture medium for 24-48 h, the cell density of the NK cells is adjusted to (1-3). times.10 cells/mL, and then the NK cells are transferred to the NK cell amplification culture medium for amplification culture for 7-10 d; the NK cell amplification culture medium is a DMEM culture medium containing 5-12% volume ratio of autologous inactivated serum, 140-200 ng/mL interleukin-2, 0.03-0.05 g/L crotonoepoxide, 0.12-0.16 g/L sheep placenta, 0.02-0.03 g/L formononetin and 0.4-0.8 g/L galactose.
Preferably, the amplification culture is carried out by placing the culture medium at the temperature of 36-37 ℃ and CO2Culturing in an incubator with the volume concentration of 4-5%.
Preferably, the expansion culture of the step S4 is to culture to the 6 th to 8 th days, adjust the cell density to (1-3). times.10/mL, transfer the cells into a new CAR-NK cell expansion culture medium to continue the culture, and continue the culture to the interval of 3-4 days, adjust the cell density, and transfer the cells into the new CAR-NK cell expansion culture medium to culture.
Preferably, the CAR-NK cell amplification culture medium is a DMEM culture medium containing 8-10% volume ratio of autologous inactivated serum, 120-150 ng/mL interleukin-2, 0.014g/L inositol, 0.2-0.6 g/L quercetin glucoside, 0.03g/L formononetin, 0.6g/L sheep placenta and 0.5g/L galactose.
Compared with the prior art, the invention has the following beneficial effects:
the invention separates the mononuclear cells of the peripheral blood from the peripheral blood, then selects the high-purity NK cells and carries out the NK cell amplification culture; infecting NK cells with a lentiviral vector with a chimeric antigen receptor gene to obtain CAR-NK cells, and inoculating the CAR-NK cells into a culture medium containing interleukin-2, inositol, quercetin glucoside, formononetin, sheep placenta and galactose for amplification culture to obtain a large amount of CAR-NK cells. The invention obtains CAR-NK cells with high killing activity by improving the purity and the number of NK cells and the number of CAR-NK cells in the culture process.
The lecithin and the glutathione which are supplemented in the culture bottle have the synergistic promotion effect on the interleukin-12 and the interleukin-3 for promoting the differentiation of the peripheral blood cells into the NK cells, and the purity of the NK cells can be improved by combining the condition of differentiation culture; crotonoepoxy essence and formononetin added into the NK cell amplification culture medium can promote the proliferation of NK cells and increase the number of the NK cells; sheep placenta, galactose and autologous inactivated serum in the CAR-NK cell amplification culture medium provide substances required for cell proliferation for cell amplification, and interleukin-2, inositol, quercetin glucoside and formononetin are used together to enhance the proliferation effect of the CAR-NK cells, promote the rapid proliferation of the CAR-NK cells and improve the cell killing activity.
The CAR-NK cell culture method is simple to operate, the purity of NK cells obtained by culture is up to 99%, the number of CAR-NK cells is large, and the killing activity is up to 92.58%.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
Example 1
A method of culturing CAR-NK cells, comprising the steps of:
s1, collecting peripheral blood of a healthy donor, and separating to obtain peripheral blood mononuclear cells;
s2, diluting the cell concentration of the peripheral blood mononuclear cells to 1 × 107Pipetting 3mL of the diluted cells into a culture flask coated with 10 μ g/mL of anti-CD 16 monoclonal antibody, and placing the culture flask at 32 ℃ under CO2After culturing for 3 days in an incubator with the volume concentration of 5%, supplementing 120ng/mL interleukin-12, 55ng/mL interleukin 3, 0.5g/L lecithin and 0.01g/L glutathione, continuously culturing for 5 days, sorting NK cells, and detecting the purity of the NK cells;
culturing the selected NK cells with serum-free DMEM medium for 24h, adjusting cell density of NK cells to 3 × 10/mL, transferring to NK cell amplification medium for amplification culture, and culturing at 37 deg.C in CO2Culturing in an incubator with the volume concentration of 5% for 7d, and detecting the amplification multiple of NK cells; the NK cell amplification culture medium is a DMEM culture medium containing 8% volume ratio autologous inactivated serum, 160ng/mL interleukin-2, 0.03g/L croton epoxy essence, 0.14g/L sheep placenta essence, 0.02g/L formononetin and 0.5g/L galactose;
s3, infecting the NK cells of the step S2 with a lentiviral vector with a chimeric antigen receptor gene;
s4, transferring the NK cells obtained in the step S3 into serum-free DMEM medium for 24h, adjusting the cell density to 1.8 x 10 cells/mL, transferring the cells into CAR-NK cell amplification medium for amplification culture, and placing the medium at 37 ℃ and CO2Culturing in 5% culture box to 6d, adjusting cell density to 1.8 × 10/mL, transferring cells to new CAR-NK cell amplification culture medium, culturing, adjusting cell density every 4d, transferring cells to new CAR-NK cell amplification culture mediumCulturing in nutrient medium until reaching 21d to obtain CAR-NK cells, and detecting amplification multiple of the CAR-NK cells;
wherein the CAR-NK cell amplification culture medium is a DMEM culture medium containing 8% volume ratio autologous inactivated serum, 130ng/mL interleukin-2, 0.014g/L inositol, 0.5g/L quercetin glucoside, 0.013g/L formononetin, 0.6g/L sheep placenta and 0.5g/L galactose.
Example 2
Example 2 differs from example 1 in that:
step S2, the differentiation culture is to place the culture bottle at 37 ℃ and CO2Culturing in an incubator with the volume concentration of 4 percent.
Example 3
Example 3 differs from example 1 in that:
step S2, the expansion culture is to culture the sorted NK cells for 36h by serum-free DMEM medium, adjust the cell density of the NK cells to 1.5 multiplied by 10/mL, transfer the NK cells to the NK cell expansion medium for expansion culture, and place the medium at 37 ℃ and CO2Culturing in an incubator with the volume concentration of 5% for 7d, and detecting the amplification multiple of NK cells; the NK cell amplification culture medium is a DMEM culture medium containing 10% volume ratio autologous inactivated serum, 140ng/mL interleukin-2 and 0.4g/L galactose.
Example 4
Example 4 differs from example 1 in that:
the amplification culture of the step S4 is that the NK cells of the step S3 are transferred into serum-free DMEM medium for 48h, the cell density is adjusted to 1 x 10/mL and the cells are transferred into CAR-NK cell amplification medium for amplification culture, and the medium is placed at the temperature of 37 ℃ and CO for amplification culture2Culturing in an incubator with the volume concentration of 5%, culturing to the 9 th day, adjusting the cell density to 1 × 10/mL, transferring the cells into a new CAR-NK cell amplification culture medium for continuous culture, adjusting the cell density every 5 days, transferring the cells into the new CAR-NK cell amplification culture medium for culture, and culturing to the 28 th day to obtain the CAR-NK cells.
Example 5
Example 5 differs from example 1 in that:
step S4, the CAR-NK cell amplification culture medium is a DMEM culture medium containing 5% volume ratio autologous inactivated serum, 120ng/mL interleukin-2, 0.020g/L inositol, 0.6g/L quercetin glucoside, 0.02g/L formononetin, 0.8g/L sheep placenta and 0.1g/L galactose.
Example 6
Example 6 differs from example 1 in that:
the amplification culture is carried out by placing the culture medium at 35 deg.C and CO2Culturing in an incubator with 3% volume concentration.
Example 7
Example 7 differs from example 1 in that:
step S2 is to dilute the cell concentration of the peripheral blood mononuclear cells to 1X 107Pipetting 3mL of the diluted cells into a culture flask coated with 16 μ g/mL of anti-CD 16 monoclonal antibody, and placing the culture flask at 32 ℃ under CO2After culturing for 3 days in an incubator with the volume concentration of 5%, supplementing 120ng/mL interleukin-12, 55ng/mL interleukin 3, 0.6g/L soybean phospholipid and 0.01g/L glutamine, continuously culturing for 4-6 days, sorting NK cells, detecting the purity of the NK cells, and performing amplification culture on the sorted NK cells.
Comparative example 1
A conventional culture method of CAR-NK cells, comprising the steps of:
s1, collecting peripheral blood of a healthy donor, and separating to obtain peripheral blood mononuclear cells;
s2, separating NK cells from the peripheral blood mononuclear cells;
s3, infecting the NK cells sorted in the step S2 with a lentiviral vector with a chimeric antigen receptor gene;
s4, adjusting the cell density of the NK cells of the step S3 to 1 × 10/mL, transferring the cells to CAR-NK cell amplification culture medium for amplification culture, and placing the culture medium at 37 ℃ and CO2Culturing in 5% volume incubator, supplementing solution every 4 days at cell density of 1.0 × 10/mL,culturing to 21d to obtain CAR-NK cells, and detecting the amplification multiple of the CAR-NK cells; the CAR-NK cell amplification culture medium is a DMEM culture medium containing 8% volume-ratio autologous inactivated serum and 130ng/mL interleukin-2.
Comparative example 2
Comparative example 2 differs from example 1 in that:
step S4, the CAR-NK cell amplification culture medium is a DMEM culture medium containing 8% volume ratio of autologous inactivated serum, 130ng/mL interleukin-2, 0.02g/L mannitol, 0.6g/L sheep placenta and 0.5g/L galactose.
Comparative example 3
Comparative example 3 differs from example 1 in that:
step S4, the CAR-NK cell amplification culture medium is a DMEM culture medium containing 13% volume ratio of autologous inactivated serum, 110ng/mL interleukin-2, 0.010g/L inositol, 0.1-0.6 g/L quercetin glucoside, 0.08g/L formononetin, 0.2g/L sheep placenta and 0.9g/L galactose.
Detecting the purity of the cells by adopting flow cytometry;
detecting the cell amplification multiple by adopting a cell counting method;
the cell killing activity is detected by MTT method, and the cell concentration is adjusted to 105Per ml, per effector cell: target cells were placed in a ratio of 20:1 into a 96-well plate, with separate target and effector cell groups, each group having 3 duplicate wells. And (3) incubating for 24h, adding MTT solution into each hole, continuing to incubate for 4h, removing the suspension by using a plate centrifuge, adding 150mL of dimethyl sulfoxide solution into each hole, oscillating, measuring an absorption value (the result is expressed by a mean value) A on an enzyme-linked immunosorbent assay, and calculating the killing activity according to a killing activity calculation formula, wherein the result is shown in the following table. Wherein, the effector cell is a mononuclear cell separated from peripheral blood, and the target cell is an in vitro passage cell strain K562.
Killing activity (%) [ target cell a value- (effector cell a value-experimental group a value) ]/target cell a value × 100%
Figure BDA0002752460670000081
From the above experimental results, it can be seen that the NK cells cultured by the culturing method of the present invention have high purity, large cell number, large amplification factor of CAR-NK cells, and high killing activity, the lecithin, glutathione and differentiation culture conditions added to the culture flask have a promoting effect on the differentiation of peripheral blood cells into NK cells, the crotonoepoxy and formononetin in the NK cell amplification medium can promote the proliferation of NK cells, and the inositol, quercetin glucoside and formononetin in the CAR-NK cell amplification medium have a promoting effect on the proliferation of CAR-NK cells.
Examples 1-7 and comparative example 1 show that the CAR-NK cell culture method of the invention has higher purity and amplification factor of the obtained NK cells and higher amplification factor and killing activity of the CAR-NK cells compared with the conventional culture method. Examples 1-7 and comparative example 2 show that inositol, quercetin glucoside, formononetin and interleukin-2 of the invention are used together to remarkably enhance the proliferation effect of CAR-NK cells, and enhance the killing activity of CAR-NK cells while increasing the amplification multiple of the CAR-NK cells. Examples 1-7 and comparative example 3 show that the usage amount of each component of the CAR-NK cell amplification medium of the invention is the optimal usage amount range for promoting CAR-NK cell proliferation of the invention.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (7)

1. A method of culturing CAR-NK cells, comprising the steps of:
s1, collecting peripheral blood of a healthy donor, and separating to obtain peripheral blood mononuclear cells;
s2, separating NK cells from the peripheral blood mononuclear cells;
s3, infecting the NK cells of the step S2 with a lentiviral vector with a chimeric antigen receptor gene;
s4, transferring the NK cells obtained in the step S3 into a serum-free DMEM medium for culturing for 24-48 h, adjusting the cell density to (1-3). times.10 cells/mL, transferring the cells into a CAR-NK cell amplification medium for amplification culture, and culturing for 21-28 d to obtain CAR-NK cells; wherein the CAR-NK cell amplification culture medium is a DMEM culture medium containing 5-12% volume ratio of autologous inactivated serum, 120-180 ng/mL interleukin-2, 0.012-0.020 g/L inositol, 0.1-0.6 g/L quercetin glucoside, 0.02-0.06 g/L formononetin, 0.3-0.8 g/L sheep placenta and 0.1-0.8 g/L galactose.
2. The method for culturing CAR-NK cells according to claim 1, wherein the cell concentration of peripheral blood mononuclear cells is diluted to (1-2). times.10 in step S27Sucking 3-5 mL of the diluted cells, inoculating the cells into a culture bottle coated with 10-15 mu g/mL of anti-CD 16 monoclonal antibody for differential culture, supplementing 100-150 ng/mL of interleukin-12, 50-70 ng/mL of interleukin 3, 0.3-0.7 g/L of lecithin and 0.01-0.02 g/L of glutathione after culturing for 1-3 days, continuously culturing for 4-6 days, and sorting out NK cells; and performing NK cell amplification culture on the sorted NK cells.
3. The method for culturing CAR-NK cells according to claim 2, wherein the differentiation culture is performed by placing a culture flask at a temperature of 30-35 ℃ and CO2Culturing in an incubator with the volume concentration of 4-5%.
4. The CAR-NK cell culture method according to claim 2, wherein the NK cell expansion culture is performed by serum-free DMEM culture medium for 24-48 h, the cell density of NK cells is adjusted to (1-3) x 10 cells/mL, and the cells are transferred to the NK cell expansion culture medium for expansion culture for 7-10 d; the NK cell amplification culture medium is a DMEM culture medium containing 5-12% volume ratio of autologous inactivated serum, 140-200 ng/mL interleukin-2, 0.03-0.05 g/L crotonoepoxide, 0.12-0.16 g/L sheep placenta, 0.02-0.03 g/L formononetin and 0.4-0.8 g/L galactose.
5. A method of culturing CAR-NK cells according to claim 1 or 4The method is characterized in that the amplification culture is carried out by placing the culture medium at the temperature of 36-37 ℃ and CO2Culturing in an incubator with the volume concentration of 4-5%.
6. The method for culturing CAR-NK cells according to claim 1, wherein the amplification culture of step S4 is to culture the cells to 6-8 days, the cell density is adjusted to (1-3). times.10/mL, and the cells are transferred to a new CAR-NK cell amplification culture medium for further culture, and the culture is continued to be every 3-4 days, the cell density is adjusted, and the cells are transferred to a new CAR-NK cell amplification culture medium for culture.
7. The method for culturing CAR-NK cells according to claim 1 or 6, wherein the CAR-NK cell expansion medium is a DMEM medium containing 8-10% by volume of autologous inactivated serum, 120-150 ng/mL interleukin-2, 0.014g/L inositol, 0.2-0.6 g/L quercetin glucoside, 0.03g/L formononetin, 0.6g/L sheep placenta, and 0.5g/L galactose.
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