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: 100-1000U / mL IL-2, 5-50ng / mL IL-4, 10-100ng / mL IL-15, 1-10ng / mL IL-21, 10-100ng / mL α-GalCer, 10-100ng / mL GM-CSF, 0.1-1μM TLR7 agonist, 1-10μg / mL transferrin, 1-10μg / mL ethanolamine, 1-10μg / mL β-cyclodextrin, 1-10μg / mL insulin, 1-10mM alanine-glutamine, 1-10μg / mL L-septic acid and RPMI 1640 culture medium. The culture medium uses RPMI 1640 culture medium as a basal culture medium, and is combined with TLR7 agonist, transferrin, etc., and has the advantages of low cost, fast amplification speed, and good ability to maintain cell culture survival rate.

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, including lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc., which play a vital role in immune cell therapy.

[0003] AIM-V medium is suitable for serum-free culture of lymphocytes and T cells. As a basal medium, it can ensure that the medium contains necessary nutrients to support cell growth and reproduction. Therefore, it is widely used in cell culture. However, its price is much higher than other basal culture media. Therefore, a culture medium that can effectively culture immune cells and is low-cost is needed. Summary of the invention

[0004] 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.

[0005] As a first aspect of the present invention, the present invention provides a culture medium for immune cells, the culture medium comprising: 100-1000U / mL IL-2, 5-50ng / mL IL-4, 10-100ng / mL IL-15, 1-10ng / mL IL-21, 10-100ng / mL α-GalCer, 10-100ng / mL GM-CSF, 0.1-1μM TLR7 agonist, 1-10μg / mL transferrin, 1-10μg / mL ethanolamine, 1-10μg / mL β-cyclodextrin, 1-10μg / mL insulin, 1-10mM alanine-glutamine, 1-10μg / mL L-septic acid and RPMI 1640 culture medium.

[0006] 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 TLR7 agonist, 2μg / mL transferrin, 4μg / mL ethanolamine, 2μg / mL β-cyclodextrin, 1μg / mL insulin, 1mM alanine-glutamine, and 1μg / mL L-ascorbic acid.

[0007] Preferably, the TLR7 agonist includes one or more of Imiquimod, Compound 2, compound 16b, compound 17b, compound 20, and BBIQ.

[0008] Preferably, the culture medium further comprises 1-10% autologous plasma.

[0009] As a second aspect of the present invention, the present invention further claims to protect the application of the culture medium of the above-mentioned immune cells, specifically the application in the culture of iNKT cells.

[0010] Specifically, the present invention also claims a method for culturing iNKT cells, the method comprising:

[0011] Obtaining cells to be processed;

[0012] 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;

[0013] The cells to be treated are collected after culture.

[0014] The preferred embodiment of the iNKT cell culture method is: the concentration of cells to be treated is 3×10 6 ~8×10 6 Pieces / mL.

[0015] A preferred embodiment of the iNKT cell culture method is as follows: the cells to be processed are PBMCs, and the culture duration is 13 days.

[0016] 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.

[0017] Furthermore, the kit is used for culturing iNKT cells and / or preparing exosomes.

[0018] The present invention provides a culture medium for immune cells, which includes IL-2, IL-12, IL-4, IL-15, IL-21, α-GalCer, GM-CSF, TLR7 agonist, transferrin, ethanolamine, β-cyclodextrin insulin, alanine-glutamine, L-septic acid 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 TLR7 agonist, transferrin and the like, and has the advantages of low cost, fast amplification speed, and good ability to maintain cell culture survival rate.

[0019] The culture medium is used to culture iNKT cells, which have a rapid proliferation rate and achieve efficient culture, and can meet the needs of clinical treatment of large numbers of iNKT cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 The morphology of NKT cells in Example 1i;

[0022] Figure 2 Comparative Example 1iNKT cell morphology;

[0023] Figure 3 This is the morphology of iNKT cells in comparative example 2;

[0024] Figure 4 This is the flow cytometry analysis of iNKT cell percentage in Example 1;

[0025] Figure 5 This is the flow cytometry analysis of iNKT cell percentage in Comparative Example 1;

[0026] Figure 6 This is the flow cytometry analysis of iNKT cell ratio in Comparative Example 2;

[0027] Figure 7 The flow cytometric detection diagram of IFN-γ content in the cell supernatant in Example 1, Comparative Example 1 and Comparative Example 2. DETAILED DESCRIPTION

[0028] 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.

[0029] After research, the present invention provides a culture medium for immune cells, including: 100-1000U / mL IL-2, 5-50ng / mL IL-4, 10-100ng / mL IL-15, 1-10ng / mL IL-21, 10-100ng / mL α-GalCer, 10-100ng / mL GM-CSF, 0.1-1μM TLR7 agonist, 1-10μg / mL transferrin, 1-10μg / mL ethanolamine, 1-10μg / mL β-cyclodextrin, 1-10μg / mL insulin, 1-10mM alanine-glutamine, 1-10μg / mL L-septic acid and RPMI 1640 culture medium.

[0030] 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 basic culture medium, RPMI 1640 basic culture medium is cheaper and can reduce the corresponding culture cost.

[0031] IL-2 and IL-15 can promote the proliferation and activation of iNKT cells; GM-CSF and IL-4 can induce the proliferation of DC cells; IL-21 can promote the proliferation of participating cells; TLR7 agonists have a strong ability to induce the secretion of cytokines by DC antigen-presenting cells in vivo, and can promote the secretion of type I interferon and IL-12 more significantly, thereby further promoting the activation and proliferation of immune cells. Transferrin, ethanolamine, β-cyclodextrin, insulin, alanine-glutamine and L-ascorbic acid 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.

[0032] The serum-free culture medium of the present invention uses RPMI 1640 as a basic culture medium combined with other components, can effectively promote the proliferation of immune cells, ensure the viability of cells, and effectively reduce the cost of the culture medium.

[0033] 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 for the culture of iNKT cells, and the immune cells may also be NK cells, T cells, etc.

[0034] As an application of the cell culture medium of the present invention, the present invention also claims protection for an iNKT cell culture method based on the culture medium. The iNKT cells cultured using the culture medium have a fast and obvious proliferation rate, and the corresponding cultured cells have a high survival rate.

[0035] 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.

[0036] Example 1

[0037] The culture medium of immune cells in Example 1 includes the following components: the basal culture medium is RPMI-1640 culture medium, and the rest includes 200U / mL IL-2, 10ng / mL IL-4, 20ng / mL IL-15, 2ng / mL IL-21, 20ng / mLα-GalCer, 50ng / mL GM-CSF, 0.2μMTLR7 agonist, 2μg / mL transferrin, 4μg / mL ethanolamine, 2μg / mLβ-cyclodextrin, 1μg / mL insulin, 1mM alanine-glutamine, 1μg / mL L-ascorbic acid. Among them, the TLR7 agonist selected in this scheme is Imiquimod. For the culture medium of the scheme of the present invention, PBMC cells are used to culture iNKT cells for testing. The culture steps of iNKT cells cultured with this culture medium are as follows:

[0038] S1: PBMCs were isolated from human peripheral venous blood using density gradient centrifugation;

[0039] S2: PBMCs were inoculated into T25 culture flasks at a cell density of 5×10 6 / mL, 5mL / bottle. The inoculated culture bottle is placed in a carbon dioxide incubator for culture;

[0040] 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 13 days;

[0041] S4: After the culture is completed, the cells to be treated are collected after the culture;

[0042] Specifically, after the culture is completed, all cells are collected.

[0043] Comparative Example 1

[0044] The culture medium used in Comparative Example 1 includes the following components: the basal culture medium is AIM-V culture medium, and the remaining components include 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, and 50 ng / mL GM-CSF.

[0045] Among them, the cultivation method and steps of Comparative Example 1 are the same as those of Example 1.

[0046] Comparative Example 2

[0047] Compared with Example 1, the culture medium composition of Comparative Example 2 only contains the following components: the basic culture medium is RPMI-1640 culture medium, and the rest includes 200U / mL IL-2, 10ng / mL IL-4, 20ng / mL IL-15, 2ng / mL IL-21, 20ng / mL α-GalCer, and 50ng / mL GM-CSF.

[0048] Among them, the cultivation method and steps of Comparative Example 2 are the same as those of Example 1.

[0049] The following experiments were performed on the above-mentioned Example 1, Comparative Example 1 and Comparative Example 2 for testing.

[0050] Experiment 1

[0051] Peripheral blood mononuclear cells (PBMC) were extracted from peripheral blood using the Ficoll method (density gradient centrifugation), and the survival rate of PBMC was detected. Then iNKT cells were cultured, and the morphology and purity of iNKT cells and the survival rate of total cells were detected after 13 days of culture.

[0052] The method for detecting PBMC and total cell viability after 13 days of culture is as follows:

[0053] 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.

[0054] Table 1. PBMC and total cell survival rate after 13 days of culture (%)

[0055] Group PBMC Total cells obtained after 13 days of culture Example 1 98.07±0.45 89.89±1.04 Comparative Example 1 98.07±0.45 80.93±1.06 Comparative Example 2 98.07±0.45 33.84±0.52

[0056] Among them, it can be seen from Table 1 that compared with Comparative Examples 1 and 2, the culture medium of Example 1 of the present invention can still reach a total cell survival rate of more than 89% after 13 days of culture, and there are still relatively high surviving cells, indicating that the culture medium of the present invention can better maintain the activity of cells. Compared with Comparative Example 2, under the condition of not adding components such as TLR7 agonists, its cell survival rate is low and cannot effectively guarantee the growth needs of cells.

[0057] During the culture process, the cell morphology was recorded on the 3rd, 6th, 9th and 13th days. Figure 1-3 , among which, 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 the comparative example. Figure 1 As shown, from Figure 1 It can be concluded that the cells began to aggregate from the 6th day of culture, and had an obvious expansion effect on the 9th day, and had large agglomeration in the cell morphology on the 13th day. In contrast, in Comparative Example 1, obvious expansion only occurred on the 9th day, and on the 13th day, there were also small agglomerations. Compared with the culture medium used in Example 1, its proliferation rate was slower and the culture efficiency was low. The culture medium of Comparative Example 2 could not maintain cell survival well during the culture process and no obvious cell proliferation occurred.

[0058] The iNKT cell purity detection method is as follows:

[0059] After culturing PBMC for 13 days, total cells were collected to detect the purity of C1 subtype iNKT cells and C2 subtype iNKT cells. Flow cytometry was used to detect the purity of C1 subtype iNKT cells and C2 subtype iNKT cells in the collected cells. The antibodies used were: PerCP-Cy5.5-CD3 antibody, FITC-CD244 antibody, APC-CXCR6 antibody. C1 subtype iNKT cells were CD3+CXCR6+CD244-, and C2 subtype iNKT cells were CD3+CXCR6+CD244+. The proportion of C1 type iNKT cells and C2 type iNKT cells in CD3+T cells is shown in Figure 2. Figure 4-6 shown.

[0060] Table 2. Proportion of iNKT cells in each experimental group

[0061]

[0062] from Figure 4-6 As can be seen from Table 2 above, the culture medium in Example 1 of the present invention can culture iNKT cells accounting for more than 70% of the total cells.

[0063] The above results indicate that the culture medium of Example 1 of the present invention can achieve effective expansion of iNKT cells, providing a guarantee for subsequent research and clinical use of iNKT cells.

[0064] Experiment 2

[0065] Flow cytometry was used to detect cytokines in the collected supernatant, and component content tests were performed on Example 1, Comparative Example 1 and Comparative Example 2, specifically, the content of cytokines IFN-γ, TNF-α, IL-8, IL-4, IL-12p70 and IL-17 in the cell supernatant. The specific test results are shown in FIG. Figure 7 shown.

[0066] Combined with Figure 7 ,from Figure 7 It can be seen that the IFN-γ level of Example 1 of the present invention is as high as 256.66 pg / mL, which is higher than the secretion levels of Comparative Examples 1 and 2. From the results, it can be concluded that the iNKT cells obtained after 13 days of culture in the culture medium of the present invention can secrete a higher level of IFN-γ without target cell stimulation.

[0067] 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 proliferation of iNKT cells and amplification of the number of iNKT cells, and the cultured iNKT cells are rich in IFN-γ factor.

[0068] 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 iNKT cell culture, supernatant exosome preparation and other aspects.

[0069] 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: 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 μMTLR7 agonist, 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-ascorbic acid and RPMI 1640 culture medium, wherein the TLR7 agonist is Imiquimod.

2. A method for culturing iNKT cells, characterized in that: The method comprises: Obtaining cells to be processed; Based on the culture medium of claim 1, 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.

3. The method for culturing iNKT cells according to claim 2, characterized in that: The concentration of cells to be treated is 3×10 6 ~8×10 6 Pieces / mL.

4. The method for culturing iNKT cells according to claim 3, characterized in that: The cells to be treated are PBMCs, and the culture time is 13 days.

5. A kit, characterized in that: The kit comprises the culture medium as claimed in claim 1, and the kit is used for culturing iNKT cells.

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