Immune cell culture medium and application thereof

Abstract

The present invention provides a culture medium for immune cells and its application. The culture medium includes: 200-1000U / mL IL-2, 10-100ng / mL IL-4, 10-100ng / mL IL-15, 2-20ng / mL anti-CD3 monoclonal antibody, 2-20ng / mL anti-CD28 monoclonal antibody, 0.5-20μM GSK-3β inhibitor, 0.1-10μM Wnt / β-catenin signaling pathway agonist, 1-10μg / mL transferrin, 1-10μg / mL ethanolamine, 1-10μg / mL water-soluble cholesterol, 1-10μg / mL insulin, 1-10mM alanine-glutamine, 1-10μg / mL L-ascorbic acid, 1-10μg / mL taurine, 0.1-1μg / mL putrescine and RPMI 1640 culture medium. The culture medium of the present invention can effectively maintain the survival rate of cells, and can achieve efficient induction of the proliferation of memory T cells and amplification of the number of memory T cells. The culture medium has the advantages of low cell culture cost and good cell quality.

Description

Technical Field

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

[0002] Immune cells refer to cells that participate in or are related to immune responses, and they play a vital role in immune cell therapy. T cells are a type of immune cell. After being activated by specific antigens, T cells proliferate and differentiate to form two types of cells with different functions, namely T immune effector cells and T memory cells.

[0003] Memory T cells are formed when T cells are activated under antigen stimulation and continue to proliferate to form a large number of effector T cells. After antigen clearance, most effector T cells die, while the remaining T cells maintain a certain number in the body and become memory T cells. Among them, memory T cells have the following phenotypes: stem cell-like memory T cells (Tscm), central memory T cells (Tcm), and effector memory T cells (Tem).

[0004] Memory T cells have the ability to self-renew and replicate, and will re-activate the virus-killing methods in memory the next time an antigen invades, killing the antigen again. Memory cells can exist in the human body for months or even decades, can exist in the blood system for a long time, and can protect the human body from secondary invasion by the corresponding pathogens.

[0005] The existing in vitro culture of memory T cells mainly uses AIM-V medium as the basic culture medium. Although AIM-V medium has more comprehensive nutrition, its price is relatively high, and the existing culture medium still cannot achieve a high proportion of memory T cells in the cells after culture.

[0006] Therefore, there is a need for a culture medium that can effectively culture immune cells at a low cost. Summary of the invention

[0007] The present invention provides an immune cell culture medium and application thereof, which are used to solve the problem of high cost of existing culture medium and realize low-cost and high-efficiency culture of immune cells.

[0008] As a first aspect of the present invention, the present invention provides a culture medium for immune cells, the culture medium comprising: 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-ascorbic acid, 1-10 μg / mL taurine, 0.1-1 μg / mL putrescine and RPMI 1640 culture medium.

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

[0010] Preferably, the GSK-3β inhibitor includes one or more of IM-12, TWS119 TFA, KY19382, AZD2858, and AR-A014418.

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

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

[0013] As a second aspect of the present invention, the present invention also seeks to protect the application of the culture medium of the above-mentioned immune cells, specifically the application in the culture of memory T cells.

[0014] Specifically, the present invention also claims protection for a method for culturing memory T cells, the method comprising:

[0015] Obtaining cells to be processed;

[0016] Based on any one of the above culture media, culturing the cells to be treated, replacing or supplementing the culture media and 1-10% autologous plasma based on the culture progress, until a predetermined culture time is reached;

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

[0018] The preferred embodiment of the method for culturing memory T cells is as follows: the concentration of cells to be treated is 3×10 6 ~8×10 6 Pieces / mL.

[0019] A preferred embodiment of the method for culturing memory T cells is as follows: the cells to be processed are PBMCs, and the culturing time is 14 days.

[0020] As the third aspect of the present invention, the present invention requests protection for the specific application of the culture medium of the immune cells. Specifically, the present invention requests protection for a kit, which includes the culture medium of the immune cells as described in any one of the above schemes, and the kit is used for the culture of immune cells and / or the preparation of exosomes.

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

[0022] The present invention provides a culture medium for immune cells, which includes 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-ascorbic acid, taurine, putrescine and RPMI 1640 culture medium within a limited range. The culture medium uses RPMI 1640 culture medium as a 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 ability to maintain cell culture survival rate.

[0023] Using this culture medium to cultivate memory T cells can achieve large-scale in vitro expansion, effectively increase the proportion of memory T cells, and achieve efficient cultivation of memory T cells to meet clinical needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1This is a morphological diagram of memory T cell culture in Example 1;

[0026] Figure 2 This is the morphological diagram of memory T cell culture in comparative example 1;

[0027] Figure 3 This is the morphological diagram of memory T cell culture in comparative example 2;

[0028] Figure 4 This is a flow cytometry diagram of the memory T cell ratio in Example 1;

[0029] Figure 5 This is a flow cytometry graph of the memory T cell ratio in Comparative Example 1. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

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

[0033] The combination of IL-2, IL-4, and IL-15 can increase the proliferation rate of memory T cells and promote their proliferation. Anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies are used to expand T cells. GSK-3β inhibitors and Wnt / β-catenin signaling pathway agonists work together to maintain cell homeostasis and prevent further differentiation of memory T cells. Transferrin can bind to iron ions to participate in the life activities of cell reproduction. Insulin can stimulate cell growth, and alanine-glutamine, as a cell energy source, provides an important energy source for cultured cells. Taurine can enhance the proliferation of T cells. Putrescine is used to maintain the balance of cell osmotic pressure. Transferrin, ethanolamine, insulin, alanine-glutamine, L-ascorbic acid, and water-soluble cholesterol are used to provide nutrition for the proliferation of immune cells and facilitate their transformation, providing the necessary living environment for the growth and reproduction of immune cells.

[0034] The serum-free culture medium of the present invention uses RPMI 1640 as a basic culture medium combined with other components. The components cooperate with each other to effectively promote the proliferation of immune cells and ensure the viability of cells, especially to effectively culture memory T cells. By using it to culture memory T cells, the obtained cells have a high survival rate and a high proportion of memory T cells, and at the same time, the cost of the culture medium is effectively reduced.

[0035] The present invention also requests protection for the application of the culture medium of the present scheme, and the specific application may be application in immune cell culture, such as a corresponding kit, which is used for the culture of immune cells and / or the preparation of exosomes, and the corresponding kit is preferably used in the culture of memory T cells, etc.

[0036] As an application of the cell culture medium of the present invention, the present invention also claims protection for a memory T cell culture method based on the culture medium. The memory T cells cultured using the culture medium proliferate rapidly and significantly, and the corresponding cultured cells have a high survival rate.

[0037] In order to better illustrate the technical effect of the technical solution of the present invention, the present invention constructs the following embodiments and comparative examples for experiments.

[0038] Example 1

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

[0040] Among them, the GSK-3β inhibitor selected in this scheme is TWS119 TFA, and the Wnt / β-catenin signaling pathway agonist is SKL2001. For the culture medium of the scheme of the present invention, PBMC cells are used to culture memory T cells for testing. The culture steps of using the culture medium to culture memory T cells are as follows:

[0041] S1: PBMCs were isolated from human peripheral venous blood using density gradient centrifugation;

[0042] The selected subjects are healthy volunteers without blood-related diseases. Peripheral blood is drawn from healthy volunteers using the Ficoll method (density gradient centrifugation method), and PBMCs are separated from the drawn peripheral blood.

[0043] After PBMCs were isolated, the survival rate of PBMCs was detected and recorded.

[0044] S2: PBMCs were inoculated into T25 culture flasks at a cell density of 6×10 6 / mL, 5mL / bottle. The inoculated culture bottle is placed in a carbon dioxide incubator for culture;

[0045] S3: During the culture process, the above culture medium is freshly prepared and 10% autologous plasma is added every 2 to 3 days. During the culture process, the cells are transferred to T75, T175, T225 or 1L cell culture flasks or cell culture bags according to the cell proliferation situation and continue to be cultured for a total of 14 days;

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

[0047] In this scheme, appropriate culture bottles or culture 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 of the original culture medium to be consumed. Therefore, during the culture process, the culture medium needs to be supplemented every 2-3 days. The supplemented culture medium is freshly prepared. While supplementing the culture medium, a certain amount of autologous plasma can be added to simulate an in vivo environment to facilitate cell proliferation and growth.

[0048] After the culture was completed, all cells were collected.

[0049] Comparative Example 1

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

[0051] Among them, the cultivation method and steps of Comparative Example 1 are the same as those of Example 1.

[0052] Comparative Example 2

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

[0054] Among them, the cultivation method and steps of Comparative Example 2 are the same as those of Example 1.

[0055] The following experiments were performed on the above-mentioned Example 1, Comparative Example 1 and Comparative Example 2 for testing.

[0056] Experiment 1

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

[0058] The memory T cells were cultured, and the morphology and purity of the memory T cells and the survival rate of the total cells were detected after 14 days of culture. During the culture process, the cell morphology was photographed and recorded on the 0th day, the 5th day, the 10th day, and the 14th day.

[0059] The method for detecting PBMC and total cell viability after 14 days of culture is as follows:

[0060] The collected PBMC or total cells were stained with AO / PI, and the cells were counted using a cell counter to calculate the cell survival rate, as shown in Table 1.

[0061] Table 1. PBMC and total cell survival rate after 14 days of culture (%)

[0062] Group PBMC Total cells obtained after 14 days of culture Example 1 99.12±0.42 90.02±1.90 Comparative Example 1 99.12±0.42 78.29±2.64 Comparative Example 2 99.12±0.42 /

[0063] Combined with Figure 1-3 , among which, attached Figure 1 The cell morphology during the culture process of Example 1 is shown in FIG. Figure 2 The cell morphology of Comparative Example 1 is shown in FIG. Figure 3 This is the cell morphology of Comparative Example 2.

[0064] For Comparative Example 2, in its culture medium system, cells died of cavitation after 5 days of culture, so no further culture and detection were performed.

[0065] like Figure 1 As shown, from Figure 1 It can be concluded that a small amount of suspended cells began to appear on the 5th day, a more obvious expansion effect appeared on the 10th day, and a large amount of proliferation appeared on the 14th day.

[0066] In Comparative Example 1, small aggregates appeared on the 5th day, and the proliferation rate was faster than that of Example 1. On the 10th day, there was obvious adhesion between cells, and on the 14th day, obvious aggregates appeared, the cells adhered to each other, and the suspension of cells decreased. In addition, after the proliferation of the cells in the culture medium of Comparative Example 1, obvious aggregates appeared, and the internal cells were hypoxic, resulting in a decrease in cell survival rate.

[0067] Comprehensive Table 1 and Figure 1-3 It can be concluded that, compared with Comparative Examples 1 and 2, the culture medium of Example 1 used in the present invention can achieve a total cell survival rate of 90% after 14 days of culture, and the cell survival rate is high, and the culture medium cost is only 1 / 3 of that of the AIM-V culture system.

[0068] Compared with Comparative Example 2, it cannot meet the cell growth requirements without adding components such as transferrin.

[0069] The purity test method of memory T cells is as follows:

[0070] After culturing PBMC for 14 days, total cells were collected for memory phenotype detection, specifically, the collected cells were detected by flow cytometry.

[0071] Flow cytometry was used to detect 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. The antibodies used were: PerCP-Cy5.5anti-human CD3 antibody, Brilliant Violet 510 TM anti-human CD95(Fas) antibody, Brilliant Violet 421anti-human CD45RO antibody, PE anti-human CD62L antibody.

[0072] Among them, the Tscm phenotype is CD3+CD95+CD62L+CD45RO-;

[0073] The Tcm phenotype is CD3+CD95+CD62L+CD45RO+;

[0074] The phenotype of Tem is CD3+CD95+CD62L-CD45RO+;

[0075] The phenotype of Teff is CD3+CD95+CD62L-CD45RO-.

[0076] Test results such as Figure 4-5 and Figure 2 shown.

[0077] Table 2. Percentage of memory T cells in each experimental group

[0078]

[0079] from Figure 4-5 As can be seen from Table 2 above, the proportion of cells expressing CD3+CD95+T in the cells cultured by the present invention is much higher than that in Comparative Example 1, and among the memory T cells obtained, the memory T cells obtained in Example 1, among them, the proportion of Tscm cells is lower than that in the Comparative Example, while the proportion of Tcm is higher, and Tscm cells can self-renew and differentiate into Tcm and Tem cells, which also shows that the culture medium system of Example 1 of this scheme can better promote the proliferation and differentiation of memory T cells.

[0080] By using the culture medium in Example 1 of the present invention, the memory T cells cultured therein can account for more than 90% of the total cells, which is higher than the AIM-V culture system.

[0081] The culture medium of Example 1 of the present invention can achieve effective expansion of memory T cells, providing a guarantee for subsequent research and clinical use of memory T cells.

[0082] In summary, the culture medium of immune cells of the present invention can effectively maintain the survival rate of cells, and can achieve efficient induction of the proliferation of memory T cells and amplification of the number of memory T cells. The culture medium has the advantages of low cell culture cost and good cell quality.

[0083] The present invention also provides a kit, which includes the culture medium of immune cells as described above, and the kit can be applied to memory T cell culture, supernatant exosome preparation and other aspects.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for immune cells, characterized in that: The culture medium includes: 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-ascorbic acid, 4 μg / mL taurine, 0.8 μg / mL putrescine and RPMI 1640 culture medium.

2. The immune cell culture medium according to claim 1, characterized in that The GSK-3β inhibitor includes one or more of IM-12, TWS119 TFA, KY19382, AZD2858, and AR-A014418.

3. The culture medium for immune cells according to claim 1, characterized in that: The Wnt / β-catenin signaling pathway agonist includes one or more of Wnt / β-catenin agonist 1, Wnt / β-catenin agonist 2, Wnt / β-catenin agonist 3, Wnt / β-catenin agonist 4, and SKL2001.

4. The culture medium for immune cells according to claim 1, characterized in that The culture medium also includes 1-10% autologous plasma.

5. A method for culturing memory T cells, characterized in that: The method comprises: Obtaining cells to be processed; Based on the culture medium described in any one of claims 1 to 3, culturing the cells to be treated, replacing or supplementing the culture medium and 1-10% autologous plasma based on the culture progress, until a predetermined culture time is reached; The cells to be treated are collected after culture.

6. The method for culturing memory T cells according to claim 5, characterized in that: The concentration of cells to be treated is 3×10 6 ~8×10 6 Pieces / mL.

7. The method for culturing memory T cells according to claim 5, characterized in that: The cells to be treated are PBMCs, and the culture time is 14 days.

8. A kit, characterized in that: The kit comprises a culture medium as claimed in any one of claims 1 to 4, and the kit is used for culturing immune cells.

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