Culture medium for in-vitro amplification of NK cells and cell in-vitro amplification method

Abstract

The invention belongs to the technical field of culture media, and particularly relates to a culture medium for promoting in-vitro amplification of NK cells, the culture medium for in-vitro amplification of the NK cells comprises a basic culture solution, a CD16 antibody, IL2, IL12 and IL21, the basic culture solution is DMEM, and the culture medium for in-vitro amplification of the NK cells is obtained by adding autologous plasma, the CD16 antibody, the IL2, the IL12 and the IL21 on the basis of the basic culture solution. Wherein the addition amount of the autologous plasma is 10%-15%, and the final concentrations of the CD16 antibody, IL2, IL12 and IL21 are 1 [mu] g / mL to 20 [mu] g / mL, 1000 IU / mL to 2000 IU / mL, 20 ng / mL to 40 ng / mL and 5 ng / mL to 15 ng / mL. According to the method, the culture medium is optimized, the single NK cell is resuspended by using the complete culture medium, the single NK cell is added into the CD16 antibody coated cell culture plate and cultured for 21 days or more, the method promotes in-vitro amplification of the NK cell, and the obtained NK cell has the advantages of being high in purity and large in number, saving the cost and improving the killing activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of culture medium, and in particular relates to a culture medium for in vitro expansion of NK cells and a method for in vitro expansion of cells. Background Art

[0002] Natural Killer Cells (NK cells) are an important part of the human immune system. They are a type of white blood cell that is primarily responsible for identifying and destroying virus-infected cells or tumor cells. Unlike T cells, NK cells do not rely on antigen presentation, but directly recognize and attack abnormal cells through specific receptors. They play a key role in immune surveillance, detecting and removing abnormal cells in the body to prevent the spread of viruses and the development of tumors.

[0003] NK cells kill target cells by releasing cytotoxic granules (such as perforins and glancinginases) and can also secrete cytokines (such as interferon-γ) to enhance immune responses. However, some viruses and tumor cells are able to evade NK cell attack by changing surface molecules or producing inhibitory signals.

[0004] In terms of clinical application, NK cells have received extensive attention in cancer immunotherapy due to their antiviral and anti-tumor properties. Researchers are working to activate NK cells or reinject them into patients through in vitro expansion technology to enhance immune response. In addition, NK cells also play an important role in antiviral effects, such as defending against herpes viruses, influenza viruses, etc.

[0005] The existing NK cell culture medium has low efficiency in in vitro expansion of NK cells, reduced NK cell function and immune activity, insufficient activation of NK cell surface receptors, imprecise control of cell density during culture, unsuitable culture environment for NK cell growth, and insufficient purity of blood-derived cells. To this end, we proposed a culture medium for in vitro expansion of NK cells and a method for in vitro cell expansion to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a culture medium for in vitro expansion of NK cells and a method for in vitro expansion of cells, which can optimize the culture medium, use complete culture medium to resuspend single NK cells, add them to a cell culture plate coated with CD16 antibody, and culture for more than 21 days. The method promotes in vitro expansion of NK cells, and the obtained NK cells have the advantages of high purity and large quantity, saving cost and improving killing activity.

[0007] The technical solution adopted by the present invention is as follows: A culture medium for in vitro expansion of NK cells, comprising a basal culture fluid, CD16 antibody, IL2, IL12 and IL21, wherein the basal culture fluid is DMEM.

[0008] In a preferred embodiment, the culture medium for in vitro expansion of NK cells is obtained by adding autologous plasma, CD16 antibody, IL2, IL12 and IL21 to the basal culture medium, wherein the amount of autologous plasma added is 10%-15%, and the final concentrations of CD16 antibody, IL2, IL12 and IL21 are 1ug-20ug / mL, 1000-2000IU / mL, 20-40ng / mL, 5-15ng / mL.

[0009] In a preferred embodiment, the CD16 antibody-coated cell culture plate and the in vitro expansion conditions of NK cells are as follows: the cell culture plate is coated with a coating solution containing CD16 antibodies, incubated at 4° C. overnight, washed with PBS buffer, and set aside.

[0010] In a preferred embodiment, the in vitro expanded NK cell culture conditions are: inoculating NK cells into the culture medium for in vitro expanded NK cells, culturing for 21 days, and replenishing the fluid once every 2-3 days during the culture period.

[0011] In a preferred embodiment, after resuspending a single NK cell using the culture medium for in vitro expansion of NK cells, the cell density is adjusted to 0.6-0.7×10 6 cells / mL.

[0012] In a preferred embodiment, the autologous plasma is obtained by the following method: centrifuge the sample at 1000g for 15min, take the upper layer of plasma and inactivate it at 56°C for 20min, centrifuge it at 2000g for 15min after the inactivation, take the upper layer of inactivated plasma and place it in a 4°C refrigerator for later use, and the sample is umbilical cord blood or peripheral blood.

[0013] In a preferred embodiment, the in vitro amplification method further comprises using a carbon dioxide incubator, wherein the culture conditions in the carbon dioxide incubator are: 37° C., 5% CO 2 , and saturated humidity.

[0014] In a preferred embodiment, the single NK cell is isolated by the following method: the whole blood cells of the umbilical cord blood or peripheral blood obtained by centrifugation are supplemented with PBS and fully mixed, and then slowly added to the upper layer of the lymphocyte separation fluid, and the white film layer is aspirated along the wall of the tube by density gradient centrifugation, and the white film layer cells are washed with PBS, and centrifuged and washed three times to obtain a single NK cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of a culture medium for in vitro expansion of NK cells and a method for in vitro expansion of cells of the present invention. DETAILED DESCRIPTION

[0016] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0017] Example 1

[0018] See also Figure 1 As shown, the present invention provides a culture medium for in vitro expansion of NK cells, the in vitro expansion of NK cells culture medium comprises a basic culture fluid, CD16 antibody, IL2, IL12 and IL21, the basic culture fluid is DMEM; The culture medium for in vitro expansion of NK cells was obtained by adding autologous plasma, CD16 antibody, IL2, IL12 and IL21 to the basic culture medium, wherein the amount of autologous plasma added was 10%, and the final concentrations of CD16 antibody, IL2, IL12 and IL21 were 1ug / mL, 1000IU / mL, 20ng / mL and 5ng / mL; The conditions for in vitro expansion of NK cells by coating the cell culture plate with CD16 antibody are as follows: the cell culture plate is coated with the coating solution containing CD16 antibody, incubated at 4°C overnight, washed with PBS buffer, and set aside; The culture conditions for in vitro expanded NK cells are as follows: NK cells are inoculated into the culture medium for in vitro expanded NK cells and cultured for 21 days, during which time the cells are replenished every 2 days; After resuspending single NK cells in the culture medium for in vitro expansion of NK cells, the cell density was adjusted to 0.6 × 106 cells / mL; Autologous plasma is obtained by the following method: centrifuge the sample at 1000 g for 15 min, take the upper layer of plasma and inactivate it at 56°C for 20 min, centrifuge at 2000 g for 15 min after inactivation, take the upper layer of inactivated plasma and place it in a refrigerator at 4°C for later use. The sample is umbilical cord blood or peripheral blood; The in vitro expansion method also includes using a carbon dioxide incubator, and the culture conditions in the carbon dioxide incubator are: 37°C, 5% CO2, and saturated humidity; Single NK cells are isolated by the following method: whole blood cells of umbilical cord blood or peripheral blood obtained by centrifugation are supplemented with PBS and fully mixed, and then slowly added to the upper layer of lymphocyte separation fluid, and then through density gradient centrifugation, the white film layer is aspirated along the wall of the tube, and the white film layer cells are washed with PBS, and then centrifuged and washed three times to obtain single NK cells.

[0019] Example 2

[0020] See also Figure 1 As shown, the present invention provides a culture medium for in vitro expansion of NK cells, the in vitro expansion of NK cells culture medium comprises a basic culture fluid, CD16 antibody, IL2, IL12 and IL21, the basic culture fluid is DMEM; The culture medium for in vitro expansion of NK cells is obtained by adding autologous plasma, CD16 antibody, IL2, IL12 and IL21 to the basic culture medium, wherein the amount of autologous plasma added is 12%, and the final concentrations of CD16 antibody, IL2, IL12 and IL21 are 5ug / mL, 1200IU / mL, 25ng / mL and 8ng / mL, respectively. The expansion of NK cells also includes a carbon dioxide incubator; The conditions for in vitro expansion of NK cells by coating the cell culture plate with CD16 antibody are as follows: the cell culture plate is coated with the coating solution containing CD16 antibody, incubated at 4°C overnight, washed with PBS buffer, and set aside; The culture conditions for in vitro expanded NK cells are as follows: NK cells are inoculated into the culture medium for in vitro expanded NK cells and cultured for 21 days, during which time the cells are replenished every 2 days; After resuspending single NK cells in the culture medium for in vitro expansion of NK cells, the cell density was adjusted to 0.65 × 106 cells / mL; Autologous plasma is obtained by the following method: centrifuge the sample at 1000 g for 15 min, take the upper layer of plasma and inactivate it at 56°C for 20 min, centrifuge at 2000 g for 15 min after inactivation, take the upper layer of inactivated plasma and place it in a refrigerator at 4°C for later use. The sample is umbilical cord blood or peripheral blood; The in vitro expansion method also includes using a carbon dioxide incubator, and the culture conditions in the carbon dioxide incubator are: 37°C, 5% CO2, and saturated humidity; Single NK cells are isolated by the following method: whole blood cells of umbilical cord blood or peripheral blood obtained by centrifugation are supplemented with PBS and fully mixed, and then slowly added to the upper layer of lymphocyte separation fluid, and then through density gradient centrifugation, the white film layer is aspirated along the wall of the tube, and the white film layer cells are washed with PBS, and then centrifuged and washed three times to obtain single NK cells.

[0021] Example 3

[0022] See also Figure 1 As shown, the present invention provides a culture medium for in vitro expansion of NK cells, the in vitro expansion of NK cells culture medium comprises a basic culture fluid, CD16 antibody, IL2, IL12 and IL21, the basic culture fluid is DMEM; The culture medium for in vitro expansion of NK cells is obtained by adding autologous plasma, CD16 antibody, IL2, IL12 and IL21 to the basic culture medium, wherein the amount of autologous plasma added is 13%, and the final concentrations of CD16 antibody, IL2, IL12 and IL21 are 10ug / mL, 1500IU / mL, 30ng / mL, and 10ng / mL, and the expansion of NK cells also includes a carbon dioxide incubator; The conditions for in vitro expansion of NK cells by coating the cell culture plate with CD16 antibody are as follows: the cell culture plate is coated with the coating solution containing CD16 antibody, incubated at 4°C overnight, washed with PBS buffer, and set aside; The culture conditions for in vitro expanded NK cells are as follows: NK cells are inoculated into the culture medium for in vitro expanded NK cells and cultured for 21 days, during which time the cells are replenished once every 3 days; After resuspending single NK cells in the culture medium for in vitro expansion of NK cells, the cell density was adjusted to 0.68 × 106 cells / mL; Autologous plasma is obtained by the following method: centrifuge the sample at 1000 g for 15 min, take the upper layer of plasma and inactivate it at 56°C for 20 min, centrifuge at 2000 g for 15 min after inactivation, take the upper layer of inactivated plasma and place it in a refrigerator at 4°C for later use. The sample is umbilical cord blood or peripheral blood; The in vitro expansion method also includes using a carbon dioxide incubator, and the culture conditions in the carbon dioxide incubator are: 37°C, 5% CO2, and saturated humidity; Single NK cells are isolated by the following method: whole blood cells of umbilical cord blood or peripheral blood obtained by centrifugation are supplemented with PBS and fully mixed, and then slowly added to the upper layer of lymphocyte separation fluid, and then through density gradient centrifugation, the white film layer is aspirated along the wall of the tube, and the white film layer cells are washed with PBS, and then centrifuged and washed three times to obtain single NK cells.

[0023] Example 4

[0024] See also Figure 1 As shown, the present invention provides a culture medium for in vitro expansion of NK cells, the in vitro expansion of NK cells culture medium comprises a basic culture fluid, CD16 antibody, IL2, IL12 and IL21, the basic culture fluid is DMEM; The culture medium for in vitro expansion of NK cells is obtained by adding autologous plasma, CD16 antibody, IL2, IL12 and IL21 to the basic culture medium, wherein the amount of autologous plasma added is 15%, and the final concentrations of CD16 antibody, IL2, IL12 and IL21 are 20ug / mL, 12000IU / mL, 40ng / mL and 15ng / mL, respectively. The expansion of NK cells also includes a carbon dioxide incubator; The conditions for in vitro expansion of NK cells by coating the cell culture plate with CD16 antibody are as follows: the cell culture plate is coated with the coating solution containing CD16 antibody, incubated at 4°C overnight, washed with PBS buffer, and set aside; The conditions for in vitro expansion of NK cells are as follows: NK cells are inoculated into the culture medium for in vitro expansion of NK cells and cultured for 21 days, during which time the cells are replenished once every 3 days; After resuspending single NK cells in the culture medium for in vitro expansion of NK cells, the cell density was adjusted to 0.7 × 106 cells / mL; Autologous plasma is obtained by the following method: centrifuge the sample at 1000 g for 15 min, take the upper layer of plasma and inactivate it at 56°C for 20 min, centrifuge at 2000 g for 15 min after inactivation, take the upper layer of inactivated plasma and place it in a refrigerator at 4°C for later use. The sample is umbilical cord blood or peripheral blood; The in vitro expansion method also includes using a carbon dioxide incubator, and the culture conditions in the carbon dioxide incubator are: 37°C, 5% CO2, and saturated humidity; Single NK cells are isolated by the following method: whole blood cells of umbilical cord blood or peripheral blood obtained by centrifugation are supplemented with PBS and fully mixed, and then slowly added to the upper layer of lymphocyte separation fluid, and then through density gradient centrifugation, the white film layer is aspirated along the wall of the tube, and the white film layer cells are washed with PBS, and then centrifuged and washed three times to obtain single NK cells.

[0025] Comparative Example 1 The culture medium was prepared according to the method of Example 2, except that IL21 was not added to the culture medium.

[0026] Comparative Example 2 The culture medium was prepared according to the method of Example 2, except that no CD16 antibody was added to the culture medium.

[0027] Comparative Example 3 The culture medium was prepared according to the method of Example 2, except that IL2 was not added to the culture medium.

[0028] Comparison of cell biological indicators between Examples 1-4 of the present invention and Comparative Examples 1-3

[0029] In the present invention, the culture medium is optimized, a single NK cell is resuspended in complete culture medium, added to a cell culture plate coated with a CD16 antibody, and cultured for more than 21 days. The method promotes the in vitro expansion of NK cells, and the obtained NK cells have the advantages of high purity and large quantity, saving costs, and improving killing activity.

[0030] The above is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention. The structures, devices and operating methods not specifically described and explained in the present invention shall be implemented according to the conventional means in the art unless otherwise specified and limited.

Claims

1. A culture medium for in vitro expansion of NK cells, characterized in that: The in vitro expanded NK cell culture medium comprises a basic culture fluid, CD16 antibody, IL2, IL12 and IL21, and the basic culture fluid is DMEM.

2. A culture medium for in vitro expansion of NK cells according to claim 1, characterized in that: The culture medium for in vitro expansion of NK cells is obtained by adding autologous plasma, CD16 antibody, IL2, IL12 and IL21 to the basic culture medium, wherein the amount of autologous plasma added is 10%-15%, and the final concentrations of CD16 antibody, IL2, IL12 and IL21 are 1ug-20ug / mL, 1000-2000IU / mL, 20-40ng / mL, and 5-15ng / mL.

3. A method for in vitro expansion of NK cells based on the culture medium for in vitro expansion of NK cells according to claim 2, characterized in that: The CD16 antibody coated cell culture plate and the in vitro amplification conditions of NK cells are as follows: the cell culture plate is coated with a coating solution containing CD16 antibodies, incubated at 4° C. overnight, washed with PBS buffer, and set aside.

4. A method for in vitro expansion of NK cells based on the culture medium for in vitro expansion of NK cells according to claim 2, characterized in that: The in vitro expanded NK cell culture conditions are: inoculating NK cells into the culture medium for in vitro expanded NK cells, culturing for 21 days, and performing fluid replenishment once every 2-3 days during the period.

5. A method for in vitro expansion of NK cells based on the culture medium for in vitro expansion of NK cells according to claim 2, characterized in that: After resuspending a single NK cell using the culture medium for in vitro expansion of NK cells, the cell density was adjusted to 0.6-0.7×10 6 cells / mL.

6. A method for in vitro expansion of NK cells based on the culture medium for in vitro expansion of NK cells according to claim 2, characterized in that: The autologous plasma is obtained by the following method: centrifuging the sample at 1000g for 15 minutes, taking the upper layer of plasma and inactivating it at 56°C for 20 minutes, centrifuging it at 2000g for 15 minutes after the inactivation is completed, taking the upper layer of inactivated plasma and placing it in a 4°C refrigerator for later use, and the sample is umbilical cord blood or peripheral blood.

7. A method for in vitro expansion of NK cells based on the culture medium for in vitro expansion of NK cells according to claim 2, characterized in that: The in vitro amplification method also includes using a carbon dioxide incubator, and the culture conditions in the carbon dioxide incubator are: 37° C., 5% CO2, and saturated humidity.

8. A method for in vitro expansion of NK cells based on the culture medium for in vitro expansion of NK cells according to claim 2, characterized in that: The single NK cell is isolated by the following method: adding PBS to the whole blood cells of the umbilical cord blood or peripheral blood obtained by centrifugation and fully mixing, slowly adding to the upper layer of the lymphocyte separation solution, through density gradient centrifugation, aspirating the white film layer along the tube wall, washing the white film layer cells with PBS, centrifuging and washing three times, and obtaining a single NK cell.

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