Culture medium for immune cells and use thereof

Abstract

A culture medium for immune cells and use thereof. The culture medium comprises: 200-1000 U / mL IL-2, 10-100 ng / mL IL-4, 10-100 ng / mL IL-15, 2-20 ng / mL anti-CD3 monoclonal antibody, 2-20 ng / mL anti-CD28 monoclonal antibody, 0.5-20 μM GSK-3β inhibitor, 0.1-10 μM Wnt / β-catenin signaling pathway agonist, 1-10 μg / mL transferrin, 1-10 μg / mL ethanolamine, 1-10 μg / mL water-soluble cholesterol, 1-10 μg / mL insulin, 1-10 mM L-alanyl-L-glutamine, 1-10 μg / mL L-ascorbic acid, 1-10 μg / mL taurine, 0.1-1 μg / mL putrescine, and an RPMI 1640 medium. The culture medium can effectively keep the survival rate of cells, efficiently induce the proliferation of memory T cells, and amplify the number of memory T cells. The culture medium has the advantages of low cell culture cost and good cell quality.

Description

Culture medium of immune cells and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of cell culture, in particular to a culture medium of immune cells, a culture method and application thereof. BACKGROUND

[0002] Immune cells refer to cells participating in or related to immune response, which play a crucial role in immune cell therapy. T cells belong to one kind of immune cells, and T cells are activated by specific antigen materials, proliferate and differentiate to form two types of cells that are different in function, namely T immune effector cells and T memory cells.

[0003] The formation of memory T cells is that T cells are activated under antigen stimulation, continuously proliferate to form a large number of effector T cells, and after the antigen is cleared, most of the effector T cells die, and the remaining T cells maintain a certain number in the body to become memory T cells. Among them, memory T cells have the following phenotypes: stem cell-like memory T cells (Tscm) cells, central memory T cells (Tcm), and effector memory T cells (Tem).

[0004] Memory T cells have self-renewal and replication capacity, and can mobilize the memory killing method again to kill antigens again when the next antigen invades. Memory cells can exist in the human body for several months or even decades, can exist in the blood system for a long time, and can prevent the human body from being invaded by the corresponding pathogen again.

[0005] The existing in-vitro culture of memory T cells is mainly based on AIM-V culture medium as the basic culture medium. Although AIM-V culture medium has relatively comprehensive nutrition, its price is relatively high, and the existing culture medium still cannot realize that memory T cells have a high proportion in the cultured cells.

[0006] Therefore, a culture medium capable of effectively culturing immune cells and low in cost is needed. SUMMARY

[0007] The present application provides a culture medium of immune cells and application thereof to solve the problem of high cost of the existing culture medium and realize low-cost and efficient culture of immune cells.

[0008] As a first aspect of the present application, the present application provides a medium for culturing immune cells, which comprises: 200-1000 U / mL IL-2, 10-100 ng / mL IL-4, 10-100 ng / mL IL-15, 2-20 ng / mL anti-CD3 monoclonal antibody, 2-20 ng / mL anti-CD28 monoclonal antibody, 0.5-20 μM GSK-3β inhibitor, 0.1-10 μM Wnt / β-catenin signaling pathway agonist, 1-10 μg / mL transferrin, 1-10 μg / mL ethanolamine, 1-10 μg / mL water-soluble cholesterol, 1-10 μg / mL insulin, 1-10 mM alanine-glutamine, 1-10 μg / mL L-antibacterial acid, 1-10 μg / mL taurine, 0.1-1 μg / mL putrescine, and RPMI 1640 medium.

[0009] Preferably, the medium comprises: 200 U / mL IL-2, 10 ng / mL IL-4, 20 ng / mL IL-15, 2 ng / mL IL-21, 20 ng / mL α-GalCer, 50 ng / mL GM-CSF, 0.2 μM GSK-3β inhibitor, 2 μg / mL transferrin, 4 μg / mL ethanolamine, 2 μg / mL β-cyclodextrin, 1 μg / mL insulin, 1 mM alanine-glutamine, 1 μg / mL L-antibacterial acid.

[0010] Preferably, the GSK-3β inhibitor comprises one or more of IM-12, TWS119TFA, KY19382, AZD2858, AR-A014418.

[0011] Preferably, the Wnt / β-catenin signaling pathway agonist comprises one or more of Wnt / β-catenin agonist 1, Wnt / β-catenin agonist 2, Wnt / β-catenin agonist 3, Wnt / β-catenin agonist 4, SKL2001.

[0012] Preferably, the medium further comprises 1-10% autologous plasma.

[0013] As a second aspect of the present application, the present application also claims the use of the above-mentioned medium for culturing immune cells, specifically for culturing memory T cells.

[0014] Specifically, the present application also claims a method for culturing memory T cells, which comprises:

[0015] Obtaining cells to be treated;

[0016] Based on any one of the above-mentioned culture media, the cells to be treated are cultured, the culture medium is replaced or supplemented based on the culture progress and 1-10% autologous plasma until a predetermined culture time is reached.

[0017] The cells to be treated after culture are collected.

[0018] In a preferred embodiment of the method for culturing memory T cells, the concentration of the cells to be treated is 3x10 6 ~ 8x10 6

[0019] In a preferred embodiment of the method for culturing memory T cells, the cells to be treated are PBMCs and the culture time is 14 days.

[0020] As a third aspect of the present application, the present application claims the specific application of the culture medium for immune cells, specifically, a kit comprising the culture medium for immune cells according to any one of the above-mentioned embodiments is claimed, and the kit is used for the culture of immune cells or / and the preparation of exosomes.

[0021] Further, the kit is used for the culture of memory T cells or / and the preparation of exosomes.

[0022] The present application provides a culture medium for immune cells, which comprises IL-2, IL-4, IL-15, anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody, GSK-3β inhibitor, Wnt / β-catenin signaling pathway agonist, transferrin, ethanolamine, water-soluble cholesterol, insulin, amino acid-glutamine, L-antibacterial acid, taurine, putrescine and RPMI 1640 medium within a defined range. The culture medium takes RPMI 1640 medium as the basic culture medium, and is combined with transferrin, amino acids, insulin and putrescine, etc., and has the advantages of low cost, fast expansion speed and good cell culture survival rate.

[0023] The culture medium can be used to culture memory T cells, which can realize in vitro mass expansion, effectively increase the proportion of memory T cells, realize efficient culture of memory T cells, and meet the clinical needs. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0025] ​Figure 1 is a memory T cell culture morphology diagram of Example 1;

[0026] Figure 2 is a memory T cell culture morphology diagram of Comparative Example 1;

[0027] Figure 3 is a memory T cell culture morphology diagram of Comparative Example 2;

[0028] Figure 4 is a flow cytometry diagram of the proportion of memory T cells in Example 1;

[0029] Figure 5 is a flow cytometry diagram of the proportion of memory T cells in Comparative Example 1. DETAILED DESCRIPTION

[0030] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in conjunction with the drawings in the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0031] Through research, the present application provides a culture medium for immune cells, comprising: the culture medium comprises 200-1000 U / mL IL-2, 10-100 ng / mL IL-4, 10-100 ng / mL IL-15, 2-20 ng / mL anti-CD3 monoclonal antibody, 2-20 ng / mL anti-CD28 monoclonal antibody, 0.5-20 μM GSK-3β inhibitor, 0.1-10 μM Wnt / β-catenin signal pathway agonist, 1-10 μg / mL transferrin, 1-10 μg / mL ethanolamine, 1-10 μg / mL water-soluble cholesterol, 1-10 μg / mL insulin, 1-10 mM alanine-glutamine, 1-10 μg / mL L-anti-septic acid, 1-10 μg / mL taurine, 0.1-1 μg / mL putrescine and RPMI 1640 medium.

[0032] Among the components, RPMI 1640 is a basic culture medium containing various cell culture essential nutrients, including sugars, amino acids, vitamins, minerals, etc. Compared with using AIM-V (Gibco) basic culture medium, using RPMI 1640 basic culture medium is more cost-effective, which can reduce the corresponding culture cost. At the same time, memory T cells are suspension cells, which are more suitable for culture with RPMI 1640.

[0033] The combination of IL-2, IL-4 and IL-15 can increase the proliferation speed of memory T cells and promote the proliferation of memory T cells. Anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody are used for expanding T cells. GSK-3beta inhibitor and Wnt / beta-catenin signal pathway agonist are used for maintaining the homeostasis of cells and preventing the further differentiation of memory T cells. Transferrin can be combined with iron ions to participate in the life activities of cell proliferation. Insulin can stimulate cell growth, and alanine-glutamine can provide an important energy source for culturing cells. Taurine can enhance the proliferation of T cells. Putrescine is used for maintaining the osmotic pressure balance of cells. Transferrin, ethanolamine, insulin, alanine-glutamine, L-antibacterial acid and water-soluble cholesterol are used for providing nutrition for immune cell proliferation and facilitating the transformation of immune cells, and providing a necessary survival environment for the growth and reproduction of immune cells.

[0034] The serum-free culture medium of the present application is based on RPMI 1640 combined with other components, and each component cooperates with each other to effectively promote the proliferation of immune cells, ensure the survival rate of cells, especially effectively culture memory T cells. The survival rate of the obtained cells is high, the proportion of memory T cells is high, and the cost of the culture medium is effectively reduced.

[0035] The present application also claims the application of the culture medium of the present application, which can be used for immune cell culture, such as a corresponding kit for culturing immune cells and / or preparing exosomes, preferably for culturing memory T cells.

[0036] As the application of the cell culture medium of the present application, the present application also claims a memory T cell culture method based on the culture, and the memory T cells cultured by using the culture medium have fast proliferation speed and obvious proliferation, and the survival rate of the cultured cells is high.

[0037] In order to better illustrate the technical effects of the technical scheme of the present application, the present application constructs the following examples and comparative examples for experiments.

[0038] Example 1

[0039] The culture medium of the immune cells in this embodiment 1 comprises the following components: the basic culture medium is RPMI-1640 medium, and the remaining components include: 700 U / mL IL-2, 10 ng / mL IL-4, 5 ng / mL IL-15, 20 ng / mL anti-CD3 monoclonal antibody, 15 ng / mL anti-CD28 monoclonal antibody, 5 μM GSK-3β inhibitor, 3 μM Wnt / β-catenin signaling pathway agonist, 2 μg / mL transferrin, 4 μg / mL ethanolamine, 5 μg / mL water-soluble cholesterol, 1 μg / mL insulin, 1 mM alanine-glutamine, 1 μg / mL L-antibacterial acid, 4 μg / mL taurine, 0.8 μg / mL putrescine.

[0040] In this scheme, the selected GSK-3β inhibitor is TWS119TFA, and the Wnt / β-catenin signaling pathway agonist is SKL2001. The culture medium of this scheme of the application is tested by culturing memory T cells using PBMC cells. The culture steps of culturing memory T cells using the culture medium are as follows:

[0041] S1: PBMC is separated from human peripheral venous blood by density gradient centrifugation;

[0042] In this scheme, the selected GSK-3β inhibitor is TWS119TFA, and the Wnt / β-catenin signaling pathway agonist is SKL2001. The culture medium of this scheme of the application is tested by culturing memory T cells using PBMC cells. The culture steps of culturing memory T cells using the culture medium are as follows:

[0043] After the PBMC is separated, the survival rate of the PBMC is detected and recorded.

[0044] S2: The PBMC is inoculated into a T25 culture flask, wherein the inoculation cell concentration is 6×10 6 The inoculated culture flask is placed in a carbon dioxide incubator for culture;

[0045] S3: During the culture process, the above-mentioned culture medium is supplemented once every 2-3 days, and 10% autologous plasma is added. During the culture process, the cells are transferred to a T75, T175, T225 or 1L cell culture flask or cell culture bag for continuous culture according to the cell proliferation, and the total culture time is 14 days;

[0046] S4: After the culture is completed, the treated cells after culture are collected;

[0047] In the present scheme, suitable culture bottles or bags are replaced based on the progress of the culture to ensure sufficient culture space. At the same time, during the culture process, the proliferation of cells will cause the nutrients in the original culture medium to be consumed, so the culture medium needs to be supplemented every 2-3 days during the culture process. The supplemented is a freshly prepared culture medium, and at the same time of supplementing the culture medium, a certain amount of autologous plasma can be added to simulate an in vivo environment, which is conducive to the proliferation and growth of cells.

[0048] After the culture is completed, all cells are collected.

[0049] Comparative Example 1

[0050] The culture medium used in Comparative Example 1 includes the following components: the basic medium is AIM-V medium (Gibco, 0870112DK), and the remaining components include 700 U / mL IL-2, 10 ng / mL IL-4, 5 ng / mL IL-15, 20 ng / mL anti-CD3 monoclonal antibody, 15 ng / mL anti-CD28 monoclonal antibody, 5 μM GSK-3β inhibitor, and 3 μM Wnt / β-catenin signaling pathway agonist.

[0051] The culture method and steps of Comparative Example 1 are the same as those of Example 1.

[0052] Comparative Example 2

[0053] Comparative Example 2 and Example 1 are compared, and the culture medium composition only contains the following components: the basic medium is RPMI-1640 medium, and the remaining components include 700 U / mL IL-2, 10 ng / mL IL-4, 5 ng / mL IL-15, 20 ng / mL anti-CD3 monoclonal antibody, 15 ng / mL anti-CD28 monoclonal antibody, 5 μM GSK-3β inhibitor, and 3 μM Wnt / β-catenin signaling pathway agonist.

[0054] The culture method and steps of Comparative Example 2 are the same as those of Example 1.

[0055] The following experiments are performed to test the above-mentioned Example 1, Comparative Example 1 and Comparative Example 2.

[0056] Experiment 1

[0057] Peripheral blood mononuclear cells (PBMCs) are extracted from peripheral blood using the Ficoll method (density gradient centrifugation method), and the survival rate of PBMCs is detected.

[0058] The culture of memory T cells is carried out, and after 14 days of culture, the morphology and purity of memory T cells and the survival rate of total cells are detected. During the culture process, the cell morphology at 0 days, 5 days, 10 days and 14 days is photographed and recorded.

[0059] The total cell survival rate after 14 days of culture was detected as follows:

[0060] The collected PBMC or total cells were subjected to AO / PI staining, and the cell count was performed using a cell counter to calculate the cell survival rate, as shown in Table 1 below.

[0061] Table 1: Survival rate of PBMC and total cells after 14 days of culture (%)

[0062] In combination with Figures 1-3, Figure 1 is the cell morphology during the culture process of Example 1, Figure 2 is the cell morphology of Comparative Example 1, and Figure 3 is the cell morphology of Comparative Example 2.

[0063] For Comparative Example 2, the cells appeared to be vacuolated and dead after 5 days of culture in its culture medium system, so no further culture and detection were performed.

[0064] As shown in Figure 1, it can be seen from Figure 1 that a small amount of suspended cells appeared from the 5th day, a more obvious expansion effect appeared on the 10th day, and a large amount of proliferation occurred on the 14th day.

[0065] In Comparative Example 1, small pieces of aggregation appeared on the 5th day, and the proliferation rate was faster than that of Example 1. On the 10th day, obvious adhesion appeared between the cells, and on the 14th day, obvious aggregation and adhesion between the cells occurred, the suspension of the cells decreased, and the survival rate of the cells decreased due to the lack of oxygen in the internal cells after the proliferation of the culture medium of Comparative Example 1.

[0066] In combination with Table 1 and Figures 1-3, it can be seen that, compared with Comparative Examples 1 and 2, the culture medium of Example 1 can achieve a total cell survival rate of 90% after 14 days of culture, has a high cell survival rate, and the cost of the culture medium is only 1 / 3 of that of the AIM-V culture system.

[0067] Compared with Comparative Example 2, it cannot meet the growth requirements of the cells without adding transferrin and other components.

[0068] The purity of memory T cells was detected as follows:

[0069] After 14 days of culture, the total cells were collected for memory phenotype detection. Specifically, the collected cells were detected by flow cytometry.

[0070] The proportion of stem cell-like memory T cells (Tscm), central memory T cells (Tcm), effector memory T cells (Tem), and effector T cells (Teff) in the collected cells is detected by a flow cytometry detection method, and the used antibodies are: PerCP-Cy5.5 anti-human CD3 antibody, Brilliant Violet 510 TM anti-human CD95 (Fas) antibody, Brilliant Violet 421 anti-human CD45RO antibody, and PE anti-human CD62L antibody.

[0071] The Tscm phenotype is CD3+CD95+CD62L+CD45RO-.

[0072] The Tcm phenotype is CD3+CD95+CD62L+CD45RO+.

[0073] The Tem phenotype is CD3+CD95+CD62L-CD45RO+.

[0074] The Teff phenotype is CD3+CD95+CD62L-CD45RO-.

[0075] The detection results are shown in Figures 4-5 and Figure 2.

[0076] Table 2: Proportion of memory T cells in each experimental group

[0077] As can be seen from Figures 4-5 and Table 2 above, the proportion of cells expressing CD3+CD95+T in the cells cultured according to the present application is much higher than that of Comparative Example 1, and among the obtained memory T cells, the proportion of Tscm cells obtained in Example 1 is lower than that of Comparative Example 1, and the proportion of Tcm is higher, and Tscm cells can self-renew and differentiate into Tcm and Tem cells, which also indicates that the culture medium system of Example 1 of the present application can better promote the proliferation and differentiation of memory T cells.

[0078] The memory T cells cultured by the culture medium in Example 1 of the present application can account for more than 90% of the total cells, which is higher than that of the AIM-V culture system.

[0079] The culture medium of Example 1 of the present application can effectively expand memory T cells, providing a guarantee for the subsequent research and clinical use of memory T cells.

[0080] In summary, the culture medium for immune cells of the present application can effectively maintain the survival rate of cells, and can effectively induce the proliferation of memory T cells and expand the number of memory T cells, and the culture medium has the advantages of low cost for culturing cells and good cell quality.

[0081] The application also provides a kit comprising the culture medium of the immune cells as described above, which can be applied in memory T cell culture, supernatant exosome preparation, etc.

[0082] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A medium for culturing immune cells, characterized in that, The culture medium comprises 200-1000 U / mL IL-2, 10-100 ng / mL IL-4, 10-100 ng / mL IL-15, 2-20 ng / mL anti-CD3 monoclonal antibody, 2-20 ng / mL anti-CD28 monoclonal antibody, 0.5-20 μM GSK-3β inhibitor, 0.1-10 μM Wnt / β-catenin signaling pathway agonist, 1-10 μg / mL transferrin, 1-10 μg / mL ethanolamine, 1-10 μg / mL water-soluble cholesterol, 1-10 μg / mL insulin, 1-10 mM alanine-glutamine, 1-10 μg / mL L-antibacterial acid, 1-10 μg / mL taurine, 0.1-1 μg / mL cadaverine and RPMI 1640 medium.

2. The medium for immune cells according to claim 1, characterized in that, The culture medium comprises 700 U / mL IL-2, 10 ng / mL IL-4, 5 ng / mL IL-15, 20 ng / mL anti-CD3 monoclonal antibody, 15 ng / mL anti-CD28 monoclonal antibody, 5 μM GSK-3β inhibitor, 3 μM Wnt / β-catenin signaling pathway agonist, 2 μg / mL transferrin, 4 μg / mL ethanolamine, 5 μg / mL water-soluble cholesterol, 1 μg / mL insulin, 1 mM alanine-glutamine, 1 μg / mL L-antibacterial acid, 4 μg / mL taurine, 0.8 μg / mL cadaverine.

3. The medium for immune cells according to claim 1, wherein, The GSK-3β inhibitor comprises one or more of IM-12, TWS119 TFA, KY19382, AZD2858, AR-A014418.

4. The medium for culturing immune cells according to claim 1, characterized by, The Wnt / β-catenin signaling pathway agonist comprises one or more of Wnt / β-catenin agonist 1, Wnt / β-catenin agonist 2, Wnt / β-catenin agonist 3, Wnt / β-catenin agonist 4, SKL2001.

5. The medium for immune cells according to claim 1, wherein, The culture medium further comprises 1-10% autologous plasma.

6. A culture method of memory T cells, characterized by, The method comprises: obtaining cells to be treated; culturing the cells to be treated based on the culture medium of any one of claims 1-4, replacing or supplementing the culture medium and 1-10% autologous plasma based on the culturing progress until a predetermined culturing time is reached; collecting the cells to be treated after culturing.

7. The method of claim 6, wherein the T memory cells are cultured in the presence of IL-7 and IL-15. The concentration of the cells to be treated is 3 x 10 6 ~ 8 x 10 6 cells / mL.

8. The method of claim 6, wherein the T memory cells are cultured in the presence of IL-7 and IL-15. The cells to be treated are PBMCs, and the culturing time is 14 days.

9. A kit characterized in that, The kit comprises the culture medium of any one of claims 1-5, and is used for culturing immune cells or / and preparing exosomes.

10. The kit of claim 9, wherein The kit is used for culturing memory T cells or / and preparing exosomes. The kit is used for culturing memory T cells or / and preparing exosomes.

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