Use of berberine in tolerogenic DC cells culturing

Abstract

Provided are a tolerogenic DC cells culture medium and the use thereof in tolerogenic DC cells culturing. The culture medium comprises: a basic culture medium, a cytokine, berberine, and autologous plasma, wherein the concentration of berberine is 0.25-5 μM, and the cytokine comprises 100-1000 U / mL GM-CSF, 10-50 ng / mL IL -4, and 100-1000 U / mL TGF-β. The expression levels of costimulatory molecules CD80, CD83, CD86 and HLA-DR of DC cells cultured by means of the culture medium are significantly reduced.

Description

Application of berberine in tolerogenic DC cell culture TECHNICAL FIELD

[0001] The present application relates to the technical field of DC cell culture, and particularly relates to application of berberine in tolerogenic DC cell culture. BACKGROUND

[0002] Immune tolerance refers to a state of self or harmless foreign antigens being static through various mechanisms, and the process does not cause immune damage to the body, which is beneficial to maintain the stability of self-immunity. In clinical practice, immune tolerance can be used for autoimmune diseases, prevention of transplant rejection, control of allergic reactions and the like.

[0003] DC cells, full name dendritic cells (Dendritic Cells, abbreviated as DCs), are the most powerful professional antigen presenting cells in the body. Tolerogenic DC cells (Tolerogenic Dendritic Cells, abbreviated as tolDCs) are a kind of dendritic cells with the ability to induce immune tolerance, which have the characteristics of low expression of costimulatory molecules such as CD80, CD86 and the like, which play an important role in activating T cells. Low expression of costimulatory molecules makes tDCs unable to effectively activate T cells, thereby inducing immune tolerance. The immune tolerance of tolerogenic DC cells makes them play an important role in clinical application. By modifying or differentiating tolerogenic DC cells in vitro and returning them to patients, it is expected to achieve immune-regulatory treatment of diseases, and has broad application prospects.

[0004] In the existing induction of tolerogenic DC cells, cytokines are used for induction, such as adding IL-10 and transforming growth factor-β (TGF-β), or combining with vitamin DC, so as to make them produce tolerance, but the induction efficiency is low, which cannot meet the large demand of clinical practice. Therefore, how to improve the induction and culture efficiency of tolerogenic DC cells to produce clinical grade products is a problem to be solved. SUMMARY

[0005] The present application provides application of berberine in tolerogenic DC cell culture, and the main purpose of the present application is to solve the problem of induction efficiency of tolerogenic DC cells in the existing culture technology.

[0006] The content of the present application is as follows:

[0007] In the first aspect of the present application, the application of berberine in tolerogenic DC cell culture is protected.

[0008] Berberine, also known as coptis, is a quaternary ammonium alkaloid isolated from traditional Chinese medicine coptis, and is the main effective component of coptis antibacterial. Berberine is now mainly used in the treatment of gastrointestinal inflammation, bacterial dysentery and other intestinal infectious diseases in clinic. In addition, it can also be used for the treatment of conjunctivitis, suppurative otitis media and other diseases.

[0009] And the applicant of the present application found that berberine is beneficial to the culture of tolerogenic DC cells, and its specific application in the culture medium of tolerogenic DC cell culture can further improve immune tolerance and increase the content of tolerogenic DC cells, which plays an important role in the research and potential clinical application of tolerogenic DC cells.

[0010] As a preferred scheme of the first aspect of the present application, the concentration of berberine in the culture of tolerogenic DC cells is 0.25-5 μM.

[0011] As a second aspect of the present application, the present application claims a tolerogenic DC cell culture medium, comprising: including: basal medium, cytokine, berberine and autologous plasma, the concentration of the berberine is 0.25-5 μM, the cytokine includes 100-1000 U / mL GM-CSF, 10-50 ng / mL IL-4, 100-1000 U / mL TGF-β.

[0012] Berberine is added to the culture medium of tolerogenic DC cells, and in the concentration range defined in the scheme of the present application, it can further induce immune tolerance and is beneficial to the culture of tolerogenic DC cells.

[0013] Among them, the tolerogenic DC cell culture medium further includes cytokines, autologous plasma and basal medium, and the remaining components are used to provide necessary nutrients, suitable environment for the culture of tolerogenic DC cells and to induce the differentiation of the cells to be cultured to DC cells or tolerogenic DC cells.

[0014] The further scheme of the tolerogenic DC cell culture medium is that the amount of autologous plasma added is 5-10%.

[0015] The further scheme of the tolerogenic DC cell culture medium is that in the cytokine, 500 U / mL GM-CSF, 50 ng / mL IL-4, 700 U / mL TGF-β are included.

[0016] The further scheme of the tolerogenic DC cell culture medium is that the basal medium includes one of AIM-V medium, KBM-581 medium and RPMI-1640.

[0017] The further scheme of the tolerogenic DC cell culture medium is that the berberine is 5 μM.

[0018] As a third aspect of the present application, the present application also claims a method for culturing tolerogenic DC cells, which is based on the culture of tolerogenic DC cells in the culture medium for tolerogenic DC cells, and is simple, effective and easy to operate, and can realize large-scale induction and culture of tolerogenic DC cells.

[0019] The method for culturing tolerogenic DC cells comprises:

[0020] Obtaining cells to be treated;

[0021] The cells to be treated are inoculated into a culture vessel, adherent culture is carried out for a first predetermined time using a basic culture medium, non-adherent cells are discarded, then the culture medium for tolerogenic DC cells defined in the above scheme is added for continuous culture for a second predetermined time, and the target cells are collected after the culture for detection.

[0022] As the method for culturing tolerogenic DC cells, a preferred scheme is that the cells to be treated are PBMCs, the first predetermined culture time is 1-4 h, and the second predetermined culture time is 7 days, and the culture medium for tolerogenic DC cells is supplemented during the second culture time.

[0023] As another aspect of the present application, the present application also claims a kit comprising any one of the culture media for tolerogenic DC cells, which is used for the culture, induction or / and preparation of supernatant exosomes of tolerogenic DC cells.

[0024] The kit comprising the culture medium for tolerogenic DC cells can be used for the culture of tolerogenic DC cells, is convenient and flexible to use, can be produced in large quantities, and is convenient for clinical application. Advantages

[0025] The present application provides the application of berberine in the culture of tolerogenic DC cells, and the addition of berberine improves the immune tolerance, which is beneficial to the culture of tolerogenic DC cells. The expression levels of the costimulatory molecules CD80, CD83, CD86 and HLA-DR of the cells cultured by the culture medium for tolerogenic DC cells containing berberine are reduced, indicating that the addition of berberine is beneficial to the culture of tolerogenic DC cells. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a schematic diagram of the expression levels of the costimulatory molecule HLA-DR in different experimental examples and comparative examples.

[0027] Figure 2 is a schematic diagram of the expression levels of the costimulatory molecule CD80 in different experimental examples and comparative examples.

[0028] Figure 3 is a schematic diagram of the expression levels of the costimulatory molecule CD83 in different experimental examples and comparative examples.

[0029] Figure 4 is a schematic diagram of the expression level of co-stimulatory molecule CD86 in different experimental examples and comparative examples. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and effects of the present application more clear and explicit, the present application is further described in detail below. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0031] The inventors of the present application found that the application of berberine to the culture of tolerogenic DC cells can increase their immune tolerance, which is conducive to the culture of tolerogenic DC cells and provides strong support for the subsequent clinical research and use of tolerogenic DC cells.

[0032] Specifically, the application of berberine to tolerogenic DC cells can be in the corresponding culture medium and the transformed product based on the corresponding culture medium, such as a kit, etc.

[0033] Specifically, the application of berberine to the culture of tolerogenic DC cells can be a tolerogenic DC cell culture medium, which comprises a basic culture medium, cytokines, berberine and autologous plasma.

[0034] In each component, the concentration added is as follows: the concentration of berberine is 0.25-5 μM, which can be specifically 0.25 μM, 1 μM, 2 μM, 4 μM or 5 μM.

[0035] The cytokines include 100-1000 U / mL GM-CSF, 10-50 ng / mL IL-4 and 100-1000 U / mL TGF-β; wherein, GM-CSF (granulocyte-macrophage colony-stimulating factor) promotes the differentiation of monocytes into large macrophage-like cells, thereby enhancing the antigen presentation function of cells and improving the activity and number of DCs. IL-4 (interleukin-4) inhibits the excessive growth of macrophages, thereby guiding the differentiation of monocytes into DCs. GM-CSF and IL-4 synergistically induce PBMCs to differentiate into DCs. TGF-β can promote the transformation of immature DC cells into tolerogenic DCs, rather than immature DC cells.

[0036] Specifically, the added concentration of GM-CSF can be 100 U / mL, 200 U / mL, 400 U / mL, 500 U / mL, 700 U / mL or 1000 U / mL; the added concentration of IL-4 can be 10 ng / mL, 20 ng / mL, 40 ng / mL or 50 ng / mL; and the added concentration of TGF-β can be 100 U / mL, 200 U / mL, 400 U / mL, 500 U / mL, 700 U / mL, 900 U / mL or 1000 U / mL.

[0037] In the tolerance DC cell culture medium, autologous plasma is also included, and the adding amount is 5-10%, preferably 10%. The addition of autologous plasma provides necessary growth factors for the cultured cells, thereby supporting the growth and development of the cells.

[0038] The application also provides a culture method of the tolerance DC cells as described above, comprising:

[0039] Obtaining cells to be treated;

[0040] Seeding the cells to be treated in a culture vessel, carrying out adherent culture for a first predetermined time by using a basic culture medium, discarding non-adherent cells, then adding the tolerance DC cell culture medium to continue culture for a second predetermined time, and collecting the target cells after the culture to carry out detection.

[0041] Specifically, in the application, PBMC is used as the cells to be treated, which has the advantages of convenient sampling, sufficient quantity, and easy culture and operation in vitro. In other schemes, bone marrow-derived hematopoietic stem cells can also be used, but their collection is limited.

[0042] After obtaining PBMC, the PBMC can be washed with PBS buffer first, and then cultured for a first predetermined culture time for separation and purification.

[0043] Specifically, the separation and purification scheme of the application is as follows: seeding PBMC in a culture vessel, using the adherent ability of mononuclear cells to separate, removing non-adherent cells by washing to preliminarily enrich mononuclear cells, and realizing separation and purification. In this stage, the culture time can be 1-4h, preferably 2h.

[0044] In the adherent stage, the basic culture medium can be selected from the basic culture medium defined in the application, such as AIM-V culture medium, KBM-581 culture medium, and RPMI-1640 culture dish.

[0045] Specifically, the culture vessel can be a 6-well plate, a T25 culture bottle, a T75 culture bottle, etc., which is selected according to the culture density and culture condition.

[0046] The separated and purified mononuclear cells are added to the culture medium of the application, and cultured for a second predetermined time, specifically 7 days. During the culture, fresh culture solution is supplemented according to the culture condition to ensure the growth requirement of the cells. Specifically, the supplementing time is once every 2-3 days.

[0047] After the culture is completed, the adherent cells are scraped by using a cell scraper to carry out detection.

[0048] The cytokines and berberine are used to induce monocytes to differentiate into dendritic cells and promote the formation of immune tolerance, and the addition of autologous plasma can better ensure the growth of cells. The cells cultured by the culture medium and the culture method have improved immune tolerance performance.

[0049] In order to better illustrate the technical effects of the technical solutions of the present application, the present application constructs the following experimental examples and comparative examples for testing.

[0050] Experimental Example 1

[0051] The tolerance DC cell culture medium of Experimental Example 1 comprises:

[0052] AIM-V culture medium;

[0053] The cytokines in the complete culture medium include 500U / mL GM-CSF, 50 ng / mL IL-4, and 700U / mL TGF-β;

[0054] The complete culture medium includes 10% autologous plasma

[0055] Experimental Example 2

[0056] The tolerance DC cell culture medium of Experimental Example 2 comprises:

[0057] AIM-V culture medium;

[0058] The cytokines in the complete culture medium include 500U / mL GM-CSF, 50 ng / mL IL-4, and 700U / mL TGF-β;

[0059] The complete culture medium includes 0.25μM berberine;

[0060] The complete culture medium includes 10% autologous plasma.

[0061] Experimental Example 3

[0062] The tolerance DC cell culture medium of Experimental Example 3 comprises:

[0063] AIM-V culture medium;

[0064] The cytokines in the complete culture medium include 500U / mL GM-CSF, 50 ng / mL IL-4, and 700U / mL TGF-β;

[0065] The complete culture medium includes 5μM berberine;

[0066] The complete culture medium includes 10% autologous plasma.

[0067] Comparative Example 1

[0068] The conventional DC cell culture medium of Comparative Example 1 is AIM-V medium;

[0069] The complete culture medium includes cytokines, 500 U / mL GM-CSF, 50 ng / mL IL-4, 700 U / mL TNF-α.

[0070] The complete culture medium includes 10% autologous plasma.

[0071] The culture method of Experimental Examples 1-3 and Comparative Example is as follows:

[0072] S1: Human peripheral blood mononuclear cells (PBMCs) are separated by Ficoll density gradient centrifugation;

[0073] S2: The PBMCs are inoculated into culture vessels at a density of 3-8 x 10 6 / mL, and AIM-V basic culture medium is added for adherent culture for 2 h, and then the non-adherent cells are discarded, and the above complete culture medium is added to continue to culture the adherent cells for 7 days, and fresh complete culture medium is supplemented every 2-3 days.

[0074] S3: After the culture is completed, the adherent cells are scraped off using a cell scraper, and flow cytometry is used for tolerance DC immunophenotype analysis, specifically for detecting the expression of co-stimulatory molecules HLA-DR, CD80, CD83, and CD86, and the test results are shown in Figures 1-4.

[0075] Figure 1 is a schematic diagram of the expression level of DC cell co-stimulatory molecule HLA-DR in different experimental examples and comparative examples. HLA-DR is a molecule in the human leukocyte antigen (HLA) system, and the HLA-DR positive rate reflects the ability of these cells to present antigens to T cells. Low HLA-DR expression can lead to T cells being unable to effectively recognize tumor cells, thereby promoting the formation of tumor immune tolerance. As shown in Figure 1, the expression level of co-stimulatory molecule HLA-DR in Experimental Example 2 and Experimental Example 3 is significantly lower than that in Comparative Example 1 and Experimental Example 1.

[0076] Figure 2 is a schematic diagram of the expression level of DC cell co-stimulatory molecule CD80 in different experimental examples. As shown in Figure 2, the expression level of co-stimulatory molecule CD80 in Experimental Example 2 and Experimental Example 3 is significantly lower than that in Comparative Example 1 and Experimental Example 1.

[0077] Figure 3 is a schematic diagram of the expression level of the co-stimulatory molecule CD83 of DC cells in different experimental examples. CD83 is an important marker of the maturation of dendritic cells (DCs), and the positive rate thereof reflects the maturation state and antigen presentation ability of DCs. A low CD83 positive rate can mean that the maturation of DCs is insufficient, the antigen presentation ability is limited, and the activation of T cells is insufficient, thereby promoting immune tolerance. As shown in Figure 3, the expression level of the co-stimulatory molecule CD83 of the experimental example 2 and the experimental example 3 is significantly lower than that of the comparative example 1 and the experimental example 1.

[0078] Figure 4 is a schematic diagram of the expression level of the co-stimulatory molecule CD86 of DC cells in different experimental examples. As shown in Figure 4, the expression level of the co-stimulatory molecule CD86 of the experimental example 2 and the experimental example 3 is significantly lower than that of the comparative example 1 and the experimental example 1.

[0079] In summary, the above test results show that berberine is conducive to the culture of tolerogenic DC cells, and the above culture medium can increase the content of tolerogenic DC cells, which is conducive to the mass production of tolerogenic DC cells and provides support for subsequent clinical applications.

[0080] The above-described embodiments are merely used to illustrate the technical solutions of the present application, but not to limit the present application; even though the present application has been described in detail with reference to the foregoing embodiments, those ordinarily skilled in the art should understand: the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced by equivalents; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Application of berberine in tolerogenic DC cell culture.

2. Application of berberine in tolerogenic DC cell culture according to claim 1, wherein the concentration of berberine in the tolerogenic DC cell culture is 0.25-5 μM.

3. A tolerance DC cell culture medium, characterized in that, Comprising: a basic medium, cytokines, berberine and autologous plasma, wherein the concentration of berberine is 0.25-5 μM, and the cytokines comprise 100-1000 U / mL GM-CSF, 10-50 ng / mL IL-4, 100-1000 U / mL TGF-β.

4. The DC cell culture medium according to claim 3, characterized in that, The amount of autologous plasma added is 5-10%.

5. The tolerance DC cell culture medium according to claim 3, characterized in that, The cytokines comprise 500 U / mL GM-CSF, 50 ng / mL IL-4, 700 U / mL TGF-β.

6. The tolerance DC cell culture medium of claim 3, wherein, The basic medium comprises one of AIM-V medium, KBM-581 medium, RPMI-1640.

7. The tolerance DC cell culture medium of claim 3, wherein, The berberine is 5 μM.

8. A method for culturing tolerance DC cells, characterized by, Comprising: obtaining cells to be treated; seeding the cells to be treated in a culture vessel, adherent culture for a first predetermined time using a basic medium, discarding non-adherent cells, then adding the tolerogenic DC cell culture medium according to any one of claims 3-7 for a second predetermined time, and collecting the target cells for detection after the culture is completed.

9. The method of claim 8, wherein the tolerogenic DC cells are generated by the method of any one of claims 1-7. The cells to be treated are PBMC, the first predetermined culture time is 2-4 h, and the second predetermined culture time is 7 days, and during the second culture time, the tolerogenic DC cell culture medium is also supplemented.

10. A kit characterized in that, The kit comprises the tolerogenic DC cell culture medium according to any one of claims 3-7, and the kit is used for the culture, induction or / and supernatant exosome preparation of tolerogenic DC cells.

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