Culture medium combination for co-culturing immune cells and its application and culture method

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a culture medium combination for co-culturing immune cells, and its application and culture method. The present invention provides a composite culture medium for co-culturing immune cells, the composite culture medium is composed of β-mercaptoethanol, IL-2, glutamine, GM-CSF, IL-1α, IL-4, IFN-γ, bFGF and RPMI1640, and the immune cells include CD8+T lymphocytes, CD4+T lymphocytes, NK cells, B lymphocytes, DC cells and macrophages. The culture method provided by the present invention can increase the expansion multiple of CD8+T lymphocytes, and has a strong killing activity against intestinal cancer cells and liver cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a culture medium combination for co-culturing immune cells, and an application and a culture method thereof. Background Art

[0002] The co-culture technology of multiple different immune cells is an advanced method to simulate the interaction of immune cells in vivo in vitro. This method places different types of immune cells in the same culture environment and observes the interaction and regulatory mechanism between them, providing important technical support for immunology research, disease model construction, and drug screening. The co-culture technology of multiple different immune cells is based on the basic principle of cell-cell interaction. By controlling the culture conditions and environment, different types of immune cells can interact with each other in the same system. These cells can communicate with each other through direct contact, secretion of signal molecules, etc., thereby simulating the complex immune environment in the body.

[0003] Immune cell co-culture can enhance the overall function of the immune system by simulating the in vivo immune environment and promoting the interaction and synergy between immune cells. For example, in tumor treatment, by co-culturing immune cells such as NK cells and CIK cells, their anti-tumor activity can be stimulated and their ability to recognize and kill tumor cells can be enhanced. Different immune cells have different anti-tumor mechanisms, and co-culture can make them work synergistically to improve the killing effect on tumor cells. For example, NK cells and CIK cells can act on tumor cells together, directly killing or inducing tumor cell apoptosis by releasing cytotoxins and secreting cytokines. Immune cell co-culture can also regulate the body's immune function and improve the body's resistance to diseases such as tumors. For example, DC cells, as antigen-presenting cells, can activate immune cells such as T cells, while CIK cells have strong anti-tumor activity. Through DC-CIK co-culture technology, the immune system can be more effectively activated to produce efficient anti-tumor effects.

[0004] However, when co-culturing multiple different immune cells, multiple bottlenecks need to be overcome to ensure the success of the experiment and the accuracy of the results. (1) Differences in cell growth conditions. Different immune cell types have different requirements for the growth environment (such as temperature, pH value, nutrients, etc.). It is difficult to meet the growth conditions of all cells at the same time during co-culture. (2) Cell-cell interactions. There are complex interactions between immune cells, including positive and negative regulation. These interactions may be exacerbated during co-culture, affecting cell growth and function. (3) Cell contamination. Cross-contamination is prone to occur during co-culture, especially microbial contamination and intercellular contamination, resulting in inaccurate experimental results. (4) Uneven cell density and distribution. During co-culture, different cells have different growth rates and migration abilities, which may lead to uneven cell density and distribution, affecting experimental results. (5) Experimental operation and data analysis. Co-culture experiments involve multiple cell types and complex experimental steps. Experimental operations are cumbersome and data analysis is complex. In summary, the bottlenecks that need to be overcome in the co-culture of multiple different immune cells mainly include differences in cell growth conditions, cell-cell interactions, cell contamination, uneven cell density and distribution, and experimental operations and data analysis.

[0005] Therefore, there is an urgent need to provide a culture medium and a culture method that can not only effectively ensure the proliferation rate of immune cells and the consistency of the life cycles of various immune cells, but also improve cell proliferation ability, cell activity and the ability to kill cancer cells. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a composite culture medium and a culture method for co-culturing multiple immune cells, which not only improves the expansion multiple of CD8+T lymphocytes but also improves the biological activity of immune cells and the killing ability to cancer cells.

[0007] On the one hand, the present invention provides a composite culture medium for co-culturing immune cells, wherein the composite culture medium consists of β-mercaptoethanol, IL-2, glutamine, GM-CSF, IL-1α, IL-4, IFN-γ, bFGF and a basal culture medium.

[0008] Specifically, the basic culture medium can be one or more of RPMI1640, DMEM, MEM, IMDM or DMEM / F12.

[0009] Preferably, the basic culture medium is RPMI1640.

[0010] Further preferably, the complex culture medium is 2-5 μM β-mercaptoethanol, 8-15 ng / mL IL-2, 1-5 mM glutamine, 10-20 ng / mL GM-CSF, 1-10 ng / mL IL-1α, 5-15 ng / mL IL-4, 1-10 ng / mL IFN-γ, 1-100 ng / mL bFGF and RPMI1640.

[0011] In another aspect, the present invention provides a method for co-culturing immune cells, comprising the following steps:

[0012] S1. Preparation of immune cells: separation and extraction of CD8+T lymphocytes, CD4+T lymphocytes, NK cells, B lymphocytes, DC cells and macrophages;

[0013] S2. Cultivation of immune cells: using different induction reagents to culture the immune cells in step S1 respectively;

[0014] S3, co-culture of immune cells: mixing the immune cells from step S2 and culturing them using the above-mentioned composite culture medium;

[0015] Specifically, the mixture in step S3 may be: the number ratio of CD8+T lymphocytes, CD4+T lymphocytes, NK cells, DC cells, B lymphocytes and macrophages is 40-60:30-40:25-35:10-20:10-20:10-20.

[0016] Specifically, the immune cells are derived from peripheral blood, umbilical cord blood, bone marrow, lymphoid tissue, thymus, liver, lung or spleen.

[0017] Preferably, the immune cells may be derived from peripheral blood.

[0018] Specifically, the induction reagent in step S2 can be 5-25 μg / mL anti-CD3 antibody, 0.01-0.1M PBS buffer, 5-10 ng / mL IL-12 and Treg induction medium; the induction reagent is used for the culture of CD8+T lymphocytes, CD4+T lymphocytes, NK cells or B lymphocytes.

[0019] Further specifically, in some embodiments, the induction reagent can be: 5 μg / mL anti-CD3 antibody, 0.1 M PBS buffer and 5 ng / mL IL-12 and Treg induction medium;

[0020] In other embodiments, the induction reagent may be: 25 μg / mL anti-CD3 antibody, 0.01 M PBS buffer and 10 ng / mL IL-12 and Treg induction medium.

[0021] Specifically, the induction reagent in step S2 can be 5-15 ng / mL GM-CSF, 5-15 ng / mL IL-4, 5-15 ng / mL IL-1β and DMEM / F12 culture medium; the induction reagent is used for culturing DC cells.

[0022] More specifically, in some embodiments, the induction agent can be 5 ng / mL GM-CSF, 15 ng / mL IL-4, 5 ng / mL IL-1β and DMEM / F12 medium.

[0023] In other embodiments, the induction agent may be: 15 ng / mL GM-CSF, 5 ng / mL IL-4, 15 ng / mL IL-1β and DMEM / F12 medium.

[0024] Specifically, the induction reagent in step S2 can be 10-20% v / v fetal bovine serum, 1-100 ng / mL lipopolysaccharide, 5-15 ng / mL GM-CSF, 5-15 ng / mL IL-4 and DMEM / F12 culture medium; the induction reagent is used for the culture of macrophages.

[0025] Further specifically, in some embodiments, the induction agent can be 10% v / v fetal bovine serum, 100 ng / mL lipopolysaccharide, 5 ng / mL GM-CSF and 15 ng / mL IL-4 and DMEM / F12 culture medium.

[0026] In other embodiments, the induction agent can be: 20% v / v fetal bovine serum, 1 ng / mL lipopolysaccharide, 15 ng / mL GM-CSF and 5 ng / mL IL-4 and DMEM / F12 culture medium.

[0027] Specifically, the culturing time in step S2 is 5-10 days.

[0028] Preferably, the culturing time in step S2 is 6-8 days.

[0029] More specifically, the culturing time in step S2 is 7 days.

[0030] Specifically, in some embodiments, the ratio of the number of CD8+T lymphocytes, CD4+T lymphocytes, NK cells, DC cells, B lymphocytes and macrophages in step S3 is 60:30:35:10:20:10.

[0031] Specifically, in some other embodiments, the number ratio of CD8+T lymphocytes, CD4+T lymphocytes, NK cells, DC cells, B lymphocytes and macrophages in step S3 is 40:40:25:20:10:20.

[0032] Specifically, the co-cultivation time in step S3 is 3-5 days.

[0033] Preferably, the co-cultivation time in step S3 is 3-4 days.

[0034] In yet another aspect, the present invention provides a culture obtained by the aforementioned method.

[0035] In another aspect, the present invention provides use of the aforementioned culture in preparing a drug for preventing and / or treating liver cancer.

[0036] In yet another aspect, the present invention provides use of the aforementioned culture in the preparation of a drug for preventing and / or treating colorectal cancer.

[0037] In yet another aspect, the present invention provides a medicine comprising the aforementioned culture.

[0038] Specifically, the drug is in the form of an injection, a gel, a cell membrane, a patch, a microsphere, a microparticle or a composite material.

[0039] More specifically, the composite material comprises mesenchymal stem cells combined with other biological materials, growth factors, drugs, etc. to form a composite material with specific functions.

[0040] The technical effects achieved by the present invention are:

[0041] (1) Improve the proliferation capacity of co-cultured immune cells, that is, the expansion multiple of CD8+T lymphocytes.

[0042] (2) Improve the tumor killing ability of co-cultured immune cells, namely, the killing ability against liver cancer cells and colorectal cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is the killing rate of the co-culture on liver cancer cells.

[0044] Figure 2 is the killing rate of the co-culture against colorectal cancer cells. DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0046] The purchase sources of the main reagents of the present invention are shown in Table 1:

[0047] Table 1

[0048]

[0049] Example 1

[0050] The method for co-culturing a plurality of different immune cells proposed in the present invention specifically comprises the following steps:

[0051] S1. Take fresh peripheral blood and use flow cytometry to sort out the required multiple immune cells, wherein the multiple immune cells are CD8+T lymphocytes, CD4+T lymphocytes, NK cells, B lymphocytes, DC cells and macrophages.

[0052] S2. Use different induction reagents to culture separately in vitro for 7 days to complete the initial expansion of each immune cell.

[0053] S3. Culture the various immune cells together in a complex culture medium for 3-4 days in the following quantitative ratio: the quantitative ratio of CD8+T lymphocytes, CD4+T lymphocytes, NK cells, DC cells, B lymphocytes and macrophages is 60:30:35:10:20:10.

[0054] The inducers of CD8+T lymphocytes, CD4+T lymphocytes, NK cells and B lymphocytes are: 5 μg / mL anti-CD3 antibody, 0.1M PBS buffer and 5ng / mL IL-12 and Treg induction culture medium.

[0055] The DC cell induction reagents are: 5 ng / mL GM-CSF, 15 ng / mL IL-4, 5 ng / mL IL-1β and DMEM / F12 culture medium.

[0056] The macrophage induction reagents include: 10% v / v fetal bovine serum, 100 ng / mL lipopolysaccharide, 5 ng / mL GM-CSF and 15 ng / mL IL-4 and DMEM / F12 culture medium.

[0057] The complex culture medium described in step S3 is: 5 μM β-mercaptoethanol, 8 ng / mL IL-2, 5 mM glutamine, 10 ng / mL GM-CSF, 10 ng / mL IL-1α, 5 ng / mL IL-4, 10 ng / mL IFN-γ, 1 ng / mL bFGF and RPMI1640 culture medium.

[0058] Example 2

[0059] The difference from Example 1 is that: Step S3: the number ratio of the CD8+T lymphocytes, CD4+T lymphocytes, NK cells, DC cells, B lymphocytes and macrophages is 40:40:25:20:10:20.

[0060] The inducers of CD8+T lymphocytes, CD4+T lymphocytes, NK cells and B lymphocytes are: 25 μg / mL anti-CD3 antibody, 0.01M PBS buffer and 10 ng / mL IL-12 and Treg induction culture medium.

[0061] The DC cell induction reagents are: 15 ng / mL GM-CSF, 5 ng / mL IL-4, 15 ng / mL IL-1β and DMEM / F12 culture medium.

[0062] The macrophage induction reagents include: 20% v / v fetal bovine serum, 1 ng / mL lipopolysaccharide, 15 ng / mL GM-CSF and 5 ng / mL IL-4 and DMEM / F12 culture medium.

[0063] The complex culture medium described in step S3 is RPMI1640 culture medium containing 2 μM β-mercaptoethanol, 15 ng / mL IL-2, 1 mM glutamine, 20 ng / mL GM-CSF, 1 ng / mL IL-1α, 15 ng / mL IL-4, 1 ng / mL IFN-γ, and 100 ng / mL bFGF.

[0064] The rest is the same as in Example 1.

[0065] Comparative Example 1

[0066] The difference from Example 1 is that GM-CSF is not added to the composite culture medium described in step S3, and the rest is the same as Example 1.

[0067] Comparative Example 2

[0068] The difference from Example 1 is that bFGF is not added to the composite culture medium described in step S3, and the rest is the same as Example 1.

[0069] Comparative Example 3

[0070] The difference from Example 1 is that GM-CSF and bFGF are not added to the composite culture medium described in step S3, and the rest is the same as Example 1.

[0071] Comparative Example 4

[0072] The difference from Example 1 is that the number ratio of CD8+T lymphocytes, CD4+T lymphocytes, NK cells, DC cells, B lymphocytes and macrophages in step S3 is 50:20:15:5:5:5.

[0073] Effect Example 1 Flow cytometry detection

[0074] Flow cytometry was used to detect and analyze the expansion multiples of CD8+T lymphocytes on the 4th day of mixed culture compared with that before mixed culture (repeated detection 3 times). The results are shown in Table 2:

[0075] Table 2

[0076]

[0077] As shown in Table 2, the composite culture medium provided by Examples 1-2 of the present invention can significantly improve the expansion multiple of CD8+T lymphocytes, while the composite culture medium in Comparative Examples 1-3 does not add GM-CSF and bFGF or one of them, which significantly affects the expansion efficiency of CD8+T lymphocytes. As shown in Comparative Example 4, the number ratio of CD8+T lymphocytes, CD4+T lymphocytes, NK cells, DC cells, B lymphocytes and macrophages also has a significant effect on the expansion multiple of CD8+T lymphocytes.

[0078] Effect Example 2 Cytotoxicity against cancer cells

[0079] 2.1 Cytotoxicity of co-cultures against hepatoma cells

[0080] CD8+T lymphocytes, CD4+T lymphocytes, NK cells, DC cells, B lymphocytes and macrophages cultured for 4 days were used as effector cells, and SMM C-7721 hepatoma cell line (Biyuntian, C6865) was used as target cells. SMM C-7721 hepatoma cells in logarithmic growth phase were selected and the concentration was adjusted to 1×10 5 / mL, and the cell concentration of effector cells was adjusted to: 1×10 6 / mL, 2×10 6 / mL, the effector-target ratio was 10:1 and 20:1 respectively. The experiment was divided into 3 groups, each with 3 replicates. The experimental group added 100 μL / well of effector and target cells, the target cell group added 100 μL / well of target cells and IMDM culture medium, and the effector cell group added 100 μL / well of effector cells and IMDM culture medium. The cells were placed in a 37°C, 5% CO2 incubator for 24 hours, and MTT reagent 20 μL / well was added for incubation at 37°C for 4 hours, and then detergent reagent 100 μL / well was added for incubation at 37°C for 2 hours. After the precipitate was fully dissolved, the absorbance (A) was detected by an enzyme-linked immunosorbent assay (wavelength 570 nm) to calculate the killing rate. The calculation formula is: killing rate = [1-(A experimental well-A effector well / A target cell well)] × 100%. The test results are shown in Figure 1 and Table 3:

[0081] Table 3 Killing rate of co-culture on liver cancer cells

[0082]

[0083] From the results in Table 3, it can be seen that the various immune cell cultures cultured in Examples 1-2 of the present invention have a strong killing effect on liver cancer.

[0084] 2.2 Cytotoxicity of co-cultures against colorectal cancer cells

[0085] The SMM C-7721 liver cancer cell line in 2.1 was replaced with the Caco-2 colorectal cancer cell line (ATCC, Shenzhen Haodi Huatuo Biotechnology Co., Ltd., HTX1665), and the rest was the same as 2.1. The test results are shown in Figure 2 and Table 4:

[0086] Table 4 Killing rate of co-culture on colorectal cancer cells

[0087]

[0088] From the results in Table 4, it can be seen that the various immune cell cultures cultured in Examples 1-2 of the present invention also have a strong killing effect on intestinal cancer.

Claims

1. A method for co-culturing immune cells, characterized in that: The following steps are involved: S1. Preparation of immune cells: separation and extraction of CD8+T lymphocytes, CD4+T lymphocytes, NK cells, B lymphocytes, DC cells and macrophages; S2. Cultivation of immune cells: using different induction reagents to culture the immune cells in step S1; S3, co-culture of immune cells: mixing the immune cells from step S2 and culturing them using a composite culture medium; The mixture of step S3 is as follows: the number ratio of CD8+T lymphocytes, CD4+T lymphocytes, NK cells, DC cells, B lymphocytes and macrophages is 40-60: 30-40: 25-35: 10-20: 10-20: 10-20; In step S2, the induction reagents for CD8+T lymphocytes, CD4+T lymphocytes, NK cells, and B lymphocytes are: 5-25 μg / mL anti-CD3 antibody, 0.01-0.1 M PBS buffer, 5-10 ng / mL IL-12, and Treg induction medium; In step S2, the induction reagents for DC cells are: 5-15 ng / mL GM-CSF, 5-15 ng / mL IL-4, 5-15 ng / mL IL-1β and DMEM / F12 medium; The macrophage induction reagent in step S2 is: 10-20% v / v fetal bovine serum, 1-100 ng / mL lipopolysaccharide, 5-15 ng / mL GM-CSF, 5-15 ng / mL IL-4 and DMEM / F12 medium; The complex culture medium in step S3 consists of 2-5 μM β-mercaptoethanol, 8-15 ng / mL IL-2, 1-5 mM glutamine, 10-20 ng / mL GM-CSF, 1-10 ng / mL IL-1α, 5-15 ng / mL IL-4, 1-10 ng / mL IFN-γ, 1-100 ng / mL bFGF and basal culture medium; The culturing time in step S2 is 5-10 days, and the co-culturing time in step S3 is 3-5 days.

2. The method according to claim 1, characterized in that The basic culture medium is one or more of RPMI1640, DMEM, MEM, IMDM or DMEM / F12.

3. The method according to claim 1, characterized in that The immune cells are derived from peripheral blood, umbilical cord blood, bone marrow, lymphatic tissue, thymus, liver, lung or spleen.

4. The method according to claim 1, characterized in that In step S3, the ratio of the number of CD8+T lymphocytes, CD4+T lymphocytes, NK cells, DC cells, B lymphocytes and macrophages is 60:30:35:10:20:

10.

5. The method according to claim 1, characterized in that In step S3, the number ratio of CD8+T lymphocytes, CD4+T lymphocytes, NK cells, DC cells, B lymphocytes and macrophages is 40:40:25:20:10:

20.

6. An immune cell culture, characterized in that Obtained by the method according to any one of claims 1 to 5.

7. Use of the immune cell culture according to claim 6 in the preparation of a drug for treating liver cancer.

8. Use of the immune cell culture according to claim 6 in the preparation of a drug for treating colorectal cancer.

9. A drug, characterized in that Comprising the immune cell culture according to claim 6.

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