Immune cell culture medium and method for culturing immune cells

Abstract

The present invention relates to an immune cell culture medium and a method for culturing immune cells. The immune cell culture medium comprises a cell basal medium and active components added to the cell basal medium, and the active components include: arachidonic acid, β-mercaptoethanol, resveratrol, ginkgo biloba extract, cyclodextrin, alanine-glutamine, and L-ascorbic acid. The cell basal medium comprises RPMI-1640 medium. The cost of the above immune cell culture medium is only 1 / 3 of that of the AIM-V (Gibco) culture system, but the purity, cell survival rate, and killing activity of the CD3AK cells obtained by culturing are higher than those of the AIM-V culture system. That is, the cost of culturing cells with this immune cell culture medium is low, the quality of the obtained cells is good, and the killing activity against cancer cells is strong, and it can be applied to the preparation of immune cells and / or immune cell exosomes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an immune cell culture medium and a method for culturing immune cells. Background Art

[0002] Immune cells refer to cells that participate in or are related to immune responses. They include lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. Immune cells can be classified into various types, and various immune cells play important roles in the human body. Among them, CD3AK cells are a type of novel anti-tumor immune active cells, which have the advantages of strong in vitro amplification ability, long in vitro survival time, high cytotoxic activity, strong ability to secrete lymphokines, and significant anti-tumor effects in vivo and in vitro, and have become an indispensable part of multi-cell therapy. Currently, AIM-V medium is commonly used to culture CD3AK cells. However, the survival rate of immune cells such as CD3AK cells cultured using AIM-V medium is relatively low. Summary of the Invention

[0003] Based on this, the present application provides an immune cell culture medium. By culturing immune cells using this immune cell culture medium, a relatively high survival rate of immune cells can be obtained.

[0004] In addition, a method for culturing immune cells is also provided.

[0005] An immune cell culture medium, characterized in that it comprises a cell basal medium and active components added to the cell basal medium, and the active components include: arachidonic acid, β-mercaptoethanol, resveratrol, ginkgo biloba extract, cyclodextrin, alanine-glutamine, L-ascorbic acid, and the cell basal medium comprises RPMI-1640 medium.

[0006] This study found that cell basal media such as RPMI-1640 medium are insufficient to culture immune cells, especially CD3AK cells. However, the above-mentioned active components in the immune cell culture medium of the present application, in combination with cell basal media such as RPMI-1640 medium, can promote the growth of immune cells such as CD3AK cells and improve the survival rate of immune cells. Through experimental verification, the total cell amount obtained by culturing PBMC using the above-mentioned immune cell culture medium is significantly higher than that obtained using AIM-V medium.

[0007] In some embodiments, the active components further include IL-2, anti-CD3 monoclonal antibody, and anti-CD28 monoclonal antibody.

[0008] In some embodiments, the cyclodextrin is β-cyclodextrin.

[0009] In some of these embodiments, the active components, based on the final concentration in the immune cell culture medium, include IL-2 at a concentration of 500 U / mL to 1000 U / mL, anti-CD3 monoclonal antibody at 2 ng / mL to 20 ng / mL, anti-CD28 monoclonal antibody at 2 ng / mL to 20 ng / mL, arachidonic acid at 1 μM to 10 μM, β-mercaptoethanol at 1 μM to 10 μM, resveratrol at 1 μM to 10 μM, ginkgo biloba extract at 1 μM to 10 μM, cyclodextrin at 1 μg / mL to 10 μg / mL, alanine-glutamine at 1 mM to 10 mM, and L-ascorbic acid at 1 μg / mL to 10 μg / mL.

[0010] In some of these embodiments, the active components, based on the final concentration in the immune cell culture medium, include IL-2 at 700 U / mL, anti-CD3 monoclonal antibody at 20 ng / mL, anti-CD28 monoclonal antibody at 20 ng / mL, arachidonic acid at 4 μM, β-mercaptoethanol at 5 μM, resveratrol at 6 μM, ginkgo biloba extract at 3 μM, cyclodextrin at 5 μg / mL, alanine-glutamine at 2 mM, and L-ascorbic acid at 5 μg / mL.

[0011] In some of these embodiments, the immune cell culture medium consists of RPMI-1640 medium and the active components, and the active components consist of the following components: arachidonic acid, β-mercaptoethanol, resveratrol, ginkgo biloba extract, cyclodextrin, alanine-glutamine, L-ascorbic acid, IL-2, anti-CD3 monoclonal antibody, and anti-CD28 monoclonal antibody.

[0012] Use of the above immune cell culture medium in the preparation of immune cells and / or immune cell exosomes.

[0013] In some of these embodiments, the immune cells include CD3AK cells.

[0014] A method for culturing immune cells, comprising the following steps: culturing blood monocytes using the above immune cell culture medium to obtain immune cells.

[0015] In some of these embodiments, the step of culturing the blood monocytes using the immune cell culture medium includes: inoculating the blood monocytes into the immune cell culture medium for culture, and supplementing the immune cell culture medium and plasma once every 2 - 3 days during the culture process.

[0016] In some of these embodiments, the culture time is 14 days, and the amount of plasma supplemented each time during the culture from day 0 to day 5 is 10% (v / v), and the amount of plasma supplemented each time during the culture from day 5 to day 14 is 5% (v / v). Description of the Drawings

[0017] Figure 1 Schematic diagram of the morphology of CD3AK cells obtained in Example 1;

[0018] Figure 2 Schematic diagram of the morphology of CD3AK cells obtained in Comparative Example 1;

[0019] Figure 3 Schematic diagram of the morphology of CD3AK cells obtained in Comparative Example 2;

[0020] Figure 4 Flow cytometry detection chart of the proportion of CD3AK cells obtained in Example 1;

[0021] Figure 5 Flow cytometry detection chart of the proportion of CD3AK cells obtained in Comparative Example 1;

[0022] Figure 6 Detection result chart of the killing activity of CD3AK against K562 cells obtained in Example 1 and Comparative Example 1. Detailed implementation manners

[0023] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation manners of the present invention will be given in conjunction with specific embodiments and the accompanying drawings. Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific implementations disclosed below.

[0024] An embodiment of the present application provides an immune cell culture medium, including a cell basal medium and active components added to the cell basal medium. The active components include: arachidonic acid, β-mercaptoethanol, resveratrol, ginkgo biloba extract, cyclodextrin, alanine-glutamine, L-ascorbic acid, and the cell basal medium includes RPMI-1640 medium.

[0025] This study found that cell basal media such as RPMI-1640 medium are insufficient to culture immune cells, especially CD3AK cells. The combination of the above active components in the immune cell culture medium of the present application with cell basal media such as RPMI-1640 medium can promote the growth of immune cells such as CD3AK cells and improve the survival rate of immune cells. Through experimental verification, the total cell amount obtained by culturing PBMC with the above immune cell culture medium is significantly higher than that obtained with AIM-V medium.

[0026] In some of these embodiments, the above-mentioned active components further include IL-2, anti-CD3 monoclonal antibody, and anti-CD28 monoclonal antibody. Among the above-mentioned active components, IL-2, anti-CD3 monoclonal antibody, and anti-CD28 monoclonal antibody are growth factors that promote the growth of immune cells. Arachidonic acid, resveratrol, and ginkgo biloba extract can increase the activity and function of immune cells. β-mercaptoethanol can inhibit the formation of large cell clusters, avoiding the death caused by insufficient cell nutrition within the clusters due to the overly large size of the clusters during the growth of immune cells. β-cyclodextrin, alanine-glutamine, and L-ascorbic acid are used to provide nutrition for the proliferation of immune cells and facilitate their transformation, providing a necessary living environment for the growth and reproduction of immune cells.

[0027] In some of these embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component includes IL-2 at a concentration of 500 U / mL to 1000 U / mL. IL-2 at this dosage is beneficial to improving cell viability, cell purity, and the killing ability against K562 cells. In some embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component includes IL-2 at a concentration of 500 U / mL, 600 U / mL, 700 U / mL, 800 U / mL, 900 U / mL, or 1000 U / mL.

[0028] In some of these embodiments, the anti-CD3 monoclonal antibody is the OKT3 antibody. Specifically, the anti-CD3 monoclonal antibody is the OKT3 antibody from Miltenyi Biotec GmbH, Germany, with the product number 130-093-387. It should be noted that the anti-CD3 monoclonal antibody is not limited to the antibody indicated above and can also be other anti-CD3 monoclonal antibodies.

[0029] In some of these embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component includes the anti-CD3 monoclonal antibody at a concentration of 2 ng / mL to 20 ng / mL. The anti-CD3 monoclonal antibody at this dosage is beneficial to improving cell viability, cell purity, and the killing ability against K562 cells. In some embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component includes the anti-CD3 monoclonal antibody at a concentration of 2 ng / mL, 6 ng / mL, 10 ng / mL, 14 ng / mL, 18 ng / mL, or 20 ng / mL.

[0030] In some of these embodiments, the anti-CD28 monoclonal antibody is the 15E8 antibody. Specifically, the anti-CD28 monoclonal antibody is the 15E8 antibody from Miltenyi Biotec GmbH, Germany, with the product number 130-093-375. It should be noted that the anti-CD28 monoclonal antibody is not limited to the antibody indicated above and can also be other anti-CD28 monoclonal antibodies.

[0031] In some of these embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises an anti-CD28 monoclonal antibody at a concentration of 2 ng / mL to 20 ng / mL. The anti-CD28 monoclonal antibody at this dosage is beneficial to improving cell survival rate, cell purity, and the killing ability against K562 cells. In some embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises an anti-CD28 monoclonal antibody at a concentration of 2 ng / mL, 6 ng / mL, 10 ng / mL, 14 ng / mL, 18 ng / mL, or 20 ng / mL.

[0032] In some of these embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises arachidonic acid at a concentration of 1 μM to 10 μM. The arachidonic acid at this dosage is beneficial to improving cell survival rate, cell purity, and the killing ability against K562 cells. In some embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises arachidonic acid at a concentration of 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, or 10 μM.

[0033] In some of these embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises β-mercaptoethanol at a concentration of 1 μM to 10 μM. The β-mercaptoethanol at this dosage is beneficial to improving cell survival rate, cell purity, and the killing ability against K562 cells. In some embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises β-mercaptoethanol at a concentration of 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, or 10 μM.

[0034] In some of these embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises resveratrol at a concentration of 1 μM to 10 μM. The resveratrol at this dosage is beneficial to improving cell survival rate, cell purity, and the killing ability against K562 cells. In some embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises resveratrol at a concentration of 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, or 10 μM.

[0035] In some of these embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises ginkgo biloba extract at a concentration of 1 μM to 10 μM. The ginkgo biloba extract at this dosage is beneficial to improving cell survival rate, cell purity, and the killing ability against K562 cells. In some embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises ginkgo biloba extract at a concentration of 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, or 10 μM.

[0036] In a specific example, the cyclodextrin is β-cyclodextrin.

[0037] In some of these embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises 1 μg / mL to 10 μg / mL of cyclodextrin. The cyclodextrin at this dosage is beneficial to improving cell viability, cell purity, and the killing ability against K562 cells. In some embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises 1 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, or 10 μg / mL of cyclodextrin.

[0038] In some of these embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises 1 mM to 10 mM of alanine - glutamine. The alanine - glutamine at this dosage is beneficial to improving cell viability, cell purity, and the killing ability against K562 cells. In some embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, or 10 mM of alanine - glutamine.

[0039] In some of these embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises 1 μg / mL to 10 μg / mL of L - ascorbic acid. The L - ascorbic acid at this dosage is beneficial to improving cell viability, cell purity, and the killing ability against K562 cells. In some embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises 1 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, or 10 μg / mL of L - ascorbic acid.

[0040] In some of these embodiments, based on the final concentration of the active component in the immune cell culture medium, the active component comprises 500 U / mL to 1000 U / mL of IL - 2, 2 ng / mL to 20 ng / mL of anti - CD3 monoclonal antibody, 2 ng / mL to 20 ng / mL of anti - CD28 monoclonal antibody, 1 μM to 10 μM of arachidonic acid, 1 μM to 10 μM of β - mercaptoethanol, 1 μM to 10 μM of resveratrol, 1 μM to 10 μM of ginkgo biloba extract, 1 μg / mL to 10 μg / mL of cyclodextrin, 1 mM to 10 mM of alanine - glutamine, 1 μg / mL to 10 μg / mL of L - ascorbic acid. The CD3AK cells cultured with the immune cell culture medium containing the above - mentioned active components have relatively high purity, cell viability, and killing activity against K562 cells.

[0041] In a specific example, based on the final concentration of the active components in the immunocyte culture medium, the active components include 700 U / mL of IL-2, 20 ng / mL of anti-CD3 monoclonal antibody, 20 ng / mL of anti-CD28 monoclonal antibody, 4 μM of arachidonic acid, 5 μM of β-mercaptoethanol, 6 μM of resveratrol, 3 μM of ginkgo biloba extract, 5 μg / mL of cyclodextrin, 2 mM of alanine-glutamine, and 5 μg / mL of L-ascorbic acid. The CD3AK cells cultured with the immunocyte culture medium containing the above active components have high purity, cell viability, and killing activity against K562 cells.

[0042] In one of the embodiments, the immunocytes include CD3AK cells. The immunocytes are not limited to the cells indicated above and can also be other immunocytes, such as memory T cells, NKT cells, CIK, etc.

[0043] In some of the embodiments, the active components include, based on the final concentration: 500 U / mL to 1000 U / mL of IL-2, 2 ng / mL to 20 ng / mL of anti-CD3 monoclonal antibody, 2 ng / mL to 20 ng / mL of anti-CD28 monoclonal antibody, 1 μM to 10 μM of arachidonic acid, 1 μM to 10 μM of β-mercaptoethanol, 1 μM to 10 μM of resveratrol, 1 μM to 10 μM of ginkgo biloba extract, 1 μg / mL to 10 μg / mL of cyclodextrin, 1 mM to 10 mM of alanine-glutamine, and 1 μg / mL to 10 μg / mL of L-ascorbic acid. The CD3AK cells cultured with the above immunocyte culture medium have high purity, cell viability, and killing activity against K562 cells.

[0044] In a specific example, the active components include, based on the final concentration: 700 U / mL of IL-2, 20 ng / mL of anti-CD3 monoclonal antibody, 20 ng / mL of anti-CD28 monoclonal antibody, 4 μM of arachidonic acid, 5 μM of β-mercaptoethanol, 6 μM of resveratrol, 3 μM of ginkgo biloba extract, 5 μg / mL of cyclodextrin, 2 mM of alanine-glutamine, and 5 μg / mL of L-ascorbic acid. The CD3AK cells cultured with the above immunocyte culture medium have high purity, cell viability, and killing activity against K562 cells.

[0045] In a specific example, the cell basal medium is RPMI-1640 medium. It should be noted that the cell basal medium is not limited to the medium indicated above and can also be other cell basal media.

[0046] The composition of the conventional AIM-V (Gibco) culture system is relatively complex, while the cost of the above-mentioned immune cell culture medium is only 1 / 3 of that of the AIM-V (Gibco) culture system. However, the purity of the CD3AK cells obtained by culture, the cell survival rate, and the killing activity against K562 cells are all higher than those of the AIM-V culture system. That is, the culture cost of this immune cell culture medium is low, the quality of the obtained cells is good, and the killing activity against cancer cells is strong, and it can be applied to the preparation of immune cells and / or immune cell exosomes.

[0047] An embodiment of this research also provides a method for culturing immune cells, including the following steps: culturing blood monocytes with the above-mentioned immune cell culture medium to obtain immune cells.

[0048] In the above-mentioned culture method, when culturing blood monocytes with the immune cell culture medium of the above-mentioned embodiment, the obtained cells have good quality and strong killing activity against cancer cells.

[0049] In some embodiments, the step of culturing blood monocytes with the immune cell culture medium includes: inoculating blood monocytes into the immune cell culture medium for culture, and supplementing the immune cell culture medium and plasma once every 2-3 days during the culture process.

[0050] Among them, the plasma is autologous plasma.

[0051] Among them, the culture time is 14 days. During the culture process from day 0 to day 5, the amount of plasma supplemented each time is 10% (v / v), and during the culture process from day 5 to day 14, the amount of plasma supplemented each time is 5% (v / v).

[0052] In some embodiments, the blood monocytes are peripheral blood monocytes. It should be noted that the blood monocytes are not limited to peripheral blood monocytes, and can also be blood monocytes from species sources such as recombinant factors IL-2 and IL-15.

[0053] In the above-mentioned culture method, when culturing immune cells with the immune cell culture medium of the above-mentioned embodiment, the obtained cells have good quality and strong killing activity against cancer cells, and the culture method is simple and conducive to large-scale promotion.

[0054] The following are specific examples.

[0055] Unless otherwise specified, the drugs and instruments used in the examples are all conventional selections in the art. For the experimental methods without specific conditions indicated in the examples, they are usually carried out under conventional conditions, such as the conditions described in the literature, books, or the methods recommended by the kit manufacturers.

[0056] Unless otherwise specified, in the following examples, the anti-CD3 monoclonal antibody is derived from the OKT3 antibody of Miltenyi Biotec GmbH, with the product number 130-093-387, and the anti-CD28 monoclonal antibody is derived from the 15E8 antibody of Miltenyi Biotec GmbH, with the product number 130-093-375.

[0057] Example 1

[0058] The immune cell culture medium of this example includes the following components: The basal medium is RPMI-1640 medium, and the rest includes: 700 U / mL IL-2, 20 ng / mL anti-CD3 monoclonal antibody, 20 ng / mL anti-CD28 monoclonal antibody, 4 μM arachidonic acid, 5 μM β-mercaptoethanol, 6 μM resveratrol, 3 μM ginkgo biloba extract, 5 μg / mL β-cyclodextrin, 2 mM alanine-glutamine, 5 μg / mL L-ascorbic acid.

[0059] The culture steps of this example include:

[0060] The first step: Isolate peripheral blood mononuclear cells (PBMC) from human peripheral venous blood by density gradient centrifugation;

[0061] The second step: Inoculate PBMC at an inoculation cell concentration of 3 - 8×10 6 cells / mL into a T25 culture flask containing fresh CD3AK cell culture medium, 5 mL per flask. Then place it in a carbon dioxide incubator and continue to culture for 14 days. From day 0 to day 5, supplement fresh CD3AK cell culture medium and 10% (v / v) autologous plasma every 2 - 3 days; from day 5 to day 14, supplement fresh CD3AK cell culture medium and 5% (v / v) autologous plasma every 2 - 3 days. Then, according to the cell proliferation situation, transfer the cells to a T75, T175, T225 or 1 L cell culture bag for continued culture;

[0062] The third step: After the culture is completed, collect all the cells for detection of cell viability, CD3AK cell purity, cell phenotype, killing activity, etc.

[0063] Example 2

[0064] The immune cell culture medium of this example includes the following components: The basal medium is RPMI-1640 medium, and the rest includes: 500 U / mL IL-2, 2 ng / mL anti-CD3 monoclonal antibody, 10 ng / mL anti-CD28 monoclonal antibody, 1 μM arachidonic acid, 10 μM β-mercaptoethanol, 1 μM resveratrol, 10 μM ginkgo biloba extract, 1 μg / mL β-cyclodextrin, 10 mM alanine-glutamine, 10 μg / mL L-ascorbic acid.

[0065] The culture steps in this example are the same as those in Example 1.

[0066] Example 3

[0067] The immune cell culture medium in this example comprises the following components: the basal medium is RPMI-1640 medium, and the rest includes: 1000 U / mL IL-2, 10 ng / mL anti-CD3 monoclonal antibody, 2 ng / mL anti-CD28 monoclonal antibody, 10 μM arachidonic acid, 1 μM β-mercaptoethanol, 10 μM resveratrol, 1 μM ginkgo biloba extract, 10 μg / mL β-cyclodextrin, 1 mM alanine-glutamine, 1 μg / mL L-ascorbic acid.

[0068] The culture steps in this example are the same as those in Example 1.

[0069] Control Example 1

[0070] The immune cell culture medium in this control example comprises the following components: the basal medium is AIM-V medium (Gibco, 0870112DK), and the rest includes 700 U / mL IL-2, 20 ng / mL anti-CD3 monoclonal antibody, 20 ng / mL anti-CD28 monoclonal antibody.

[0071] The culture steps in this control example are the same as those in Example 1.

[0072] Control Example 2

[0073] The immune cell culture medium in this control example comprises the following components: the basal medium is RPMI-1640 medium, and the rest includes 700 U / mL IL-2, 20 ng / mL anti-CD3 monoclonal antibody, 20 ng / mL anti-CD28 monoclonal antibody.

[0074] The culture steps in this control example are the same as those in Example 1.

[0075] Test:

[0076] Peripheral blood mononuclear cells (PBMC) were extracted from the peripheral blood of healthy volunteers by the Ficoll method (density gradient centrifugation method), and the survival rate of PBMC was detected. The results are shown in Table 1. After detecting the survival rate of PBMC, it was used for culturing CD3AK cells in each example and control example. After 14 days of culture, the performance of CD3AK cells was detected.

[0077] (1) After culturing PBMC and Examples 1-3 and Control Examples 1-2 for 14 days, the total cell survival rate was detected. The detection method is as follows:

[0078] The collected PBMC or total cells were stained with AO / PI, and cell counting was performed using a cell counter to calculate the cell viability. The morphology of CD3AK cells in Example 1 and Comparative Examples 1-2 was detected, and the results are shown in Table 1 and Figure 1-3 as follows. Figure 1 Figure 4 shows the schematic diagram of the morphology of CD3AK cells in Example 1; Figure 2 Figure 5 shows the schematic diagram of the morphology of CD3AK cells in Comparative Example 1; Figure 3 Figure 6 shows the schematic diagram of the morphology of CD3AK cells in Comparative Example 2.

[0079] Table 1 Total cell viability of PBMC and after 14 days of culture (%)

[0080] Group PBMC Total cells obtained after 14 days of culture Example 1 98.60±0.45 94.02±3.40 Example 2 98.60±0.45 95.11±0.72 Example 3 98.60±0.45 93.04±0.86 Comparative Example 1 98.60±0.45 84.82±2.92 Comparative Example 2 98.60±0.45 ——

[0081] From Figure 1-3 it can be seen that in the culture medium system of Comparative Example 2, cell vacuolization and death occurred after 3-5 days of culture, so subsequent culture and detection were no longer carried out.

[0082] As can be seen from Table 1, the total cell viability of Examples 1-3 after 14 days of culture was significantly better than that of Comparative Examples 1-2. Thus, it can be seen that the immunocyte culture medium of the present application is more conducive to improving the cell viability of the cultured CD3AK cells than the AIM-V culture system.

[0083] (2) The purity of CD3AK cells in Example 1 and Comparative Example 1 was detected, and the detection method was as follows:

[0084] After PBMC was cultured for 14 days, total cells were collected for CD3AK purity detection. Flow cytometry was used to detect the proportion of CD3AK cells in the collected cells.

[0085] The antibodies used were: PerCP-Cy5.5 anti-human CD3 antibody, PE anti-human CD4 antibody, and FITC anti-human CD8 antibody. The detection results are shown in Table 2 and Figure 4-5 . Figure 4 Figure 7 shows the schematic diagram of flow cytometry for the proportion of CD3AK cells in Example 1; Figure 5 Figure 8 shows the schematic diagram of flow cytometry for the proportion of CD3AK cells in Comparative Example 1.

[0086] Table 2 Proportion of CD3AK cells in each experimental group

[0087]

[0088] As Figure 4 - Figure 5As can be seen from Table 2, the purity of the CD3AK cells obtained in Example 1 is significantly better than that of the cells obtained in Comparative Example 1. It can be seen that the immunocyte culture medium of the present application is more conducive to improving the purity of the cultured CD3AK cells than the AIM-V culture system.

[0089] (3) The method for detecting the killing activity of CD3AK cells against K562 is as follows:

[0090] Collect K562 cells in the logarithmic growth phase and the CD3AK cells amplified from Examples 1 to 3 and Comparative Example 1 in culture;

[0091] Using K562 cells as target cells and CD3AK cells as effector cells, prepare a single-cell suspension of K562 cells in the logarithmic growth phase, stain with AO / PI and count, and then adjust the cell density to 1×10 5 cells / mL;

[0092] Add the K562 cell suspension to a 96-well plate, 50 μL per well, and add effector cells (Cd3AK) according to different effector / target ratios (20:1, 10:1, 5:1), also 50 μL per well:

[0093] At the same time, set up natural release wells for effector cells and target cells, maximum release wells for target cells, natural release wells for the culture medium, and volume correction control wells, with a volume of 100 μL per well, and set up 3 replicates for each;

[0094] Incubate in a 37°C, 5% CO2 incubator for 12 h. Add 10 μL of lysis solution to each well of the maximum release wells for target cells 45 min before the end of the reaction;

[0095] After the reaction ends, pipette 50 μL of LDH enzyme reaction solution and 50 μL of supernatant from each well into another new 96-well plate, react in the dark at room temperature for 30 min, and then add 50 μL of reaction termination solution, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value;

[0096] Calculate the killing activity according to the following formula: Killing activity (%) = (OD value of the measurement tube - OD value of the natural release tube of target cells - OD value of the natural release tube of effector cells) / (OD value of the maximum release tube of target cells - OD value of the natural release tube of target cells) × 100%. The calculation results are as shown in Figure 6 Table 3. Figure 6 Figure 30 is a schematic diagram of the killing activity of CD3AK against K562 cells in Example 1 and Comparative Example 1.

[0097] Table 3. Killing activity of CD3AK against K562 in each experimental group (%)

[0098]

[0099] As shown inFigure 6 As can be seen from Table 3, the killing activity of the CD3AK cells obtained in Examples 1-3 against K562 cells is superior to that of the CD3AK cells obtained in Comparative Example 1 against K562 cells. It can be seen that the immunocyte culture medium of the present application is more conducive to improving the killing activity of the cultured CD3AK cells against K562 cells than the AIM-V culture system.

[0100] In summary, the cost of the immunocyte culture medium of the present application is only 1 / 3 of that of the AIM-V (Gibco) culture system, but the purity, cell survival rate, and killing activity of the CD3AK cells obtained by culturing are all higher than those of the AIM-V culture system. That is, the cost of culturing cells with this immunocyte culture medium is low, the quality of the obtained cells is good, and the killing activity against cancer cells is strong, and it can be applied to the preparation of immunocytes and / or immunocyte exosomes.

[0101] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0102] The above-described embodiments only represent several implementation manners of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. An immune cell culture medium, characterized in that The immune cell culture medium is used to culture CD3AK cells. The immune cell culture medium is composed of RPMI-1640 culture medium and active components. The active components are composed of the following components: arachidonic acid, β-mercaptoethanol, resveratrol, ginkgo leaf extract, cyclodextrin, alanine-glutamine, L-ascorbic acid, IL-2, anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody, and the final concentration of the active components in the immune cell culture medium is 500U / mL to 1000U / mL. IL-2, 2ng / mL~20ng / mL anti-CD3 monoclonal antibody, 2ng / mL~20ng / mL anti-CD28 monoclonal antibody, 1μM~10μM arachidonic acid, 1μM~10μM β-mercaptoethanol, 1μM~10μM resveratrol, 1μM~10μM ginkgo leaf extract, 1μg / mL~10μg / mL cyclodextrin, 1mM~10mM alanine-glutamine, 1μg / mL~10μg / mL L-ascorbic acid.

2. The immune cell culture medium according to claim 1, characterized in that The active components are calculated as 700 U / mL IL-2, 20 ng / mL anti-CD3 monoclonal antibody, 20 ng / mL anti-CD28 monoclonal antibody, 4 μM arachidonic acid, 5 μM β-mercaptoethanol, 6 μM resveratrol, 3 μM ginkgo leaf extract, 5 μg / mL cyclodextrin, 2 mM alanine-glutamine, and 5 μg / mL L-ascorbic acid based on the final concentration in the immune cell culture medium.

3. The immune cell culture medium according to any one of claims 1 to 2, characterized in that The cyclodextrin is β-cyclodextrin.

4. Use of the immune cell culture medium according to any one of claims 1 to 3 in preparing CD3AK cells and / or CD3AK cell exosomes.

5. A method for culturing immune cells, characterized in that: The method comprises the following steps: using the immune cell culture medium according to any one of claims 1 to 3 to culture blood mononuclear cells to obtain CD3AK cells.

6. The culture method according to claim 5, characterized in that: The step of culturing the blood monocytes using the immune cell culture medium comprises: inoculating the blood monocytes into the immune cell culture medium for culturing, and replenishing the immune cell culture medium and plasma every 2-3 days during the culturing process.

7. The culture method according to claim 6, characterized in that: The culture time is 14 days. During the culture process from day 0 to day 5, the amount of plasma supplemented each time is 10% (v / v). During the culture process from day 5 to day 14, the amount of plasma supplemented each time is 5% (v / v).

Images