Culture medium, culture method and application of tolerant DC (Dendritic Cell)

Abstract

The invention provides a culture medium, a culture method and application of tolerant DC cells, and relates to the technical field of cell culture.The culture medium comprises a first basic culture medium, granulocyte-macrophage colony stimulating factors, interleukin 4, transforming growth factors-beta, prostaglandin E2 and plasma; wherein the concentration of the granulocyte-macrophage colony stimulating factor is 100 to 1000 U / mL, the concentration of the interleukin 4 is 10 to 50 ng / mL, the concentration of the transforming growth factor-beta is 100 to 1000 U / mL, the concentration of the prostaglandin E2 is 0.4 to 4 [mu] M, and the concentration of the plasma is 5 to 10%. The method can greatly increase the content of the tolerant DC cells, and is convenient, rapid and stable.

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 tolerant DC cells. Background Art

[0002] Tolerogenic dendritic cells (DCs) are a type of antigen-presenting cells with immunosuppressive properties. These cells induce tolerance in effector cells by presenting antigens, thereby inhibiting the body's excessive immune rejection response. Tolerogenic DCs are generally regarded as semi-mature cells, and their surface markers such as CD80, CD86, and CD40 are expressed at low levels, indicating their special role in immune responses. Currently, researchers mainly explore their application in immune regulation by isolating and inducing tolerant DCs from peripheral blood mononuclear cells (PBMCs). Commonly used induction methods include IL-10 and adenovirus induction. Although these methods have successfully generated tolerant DCs to a certain extent, there are still significant problems in induction efficiency and cell activity.

[0003] First, although the use of cytokines such as IL-10 can effectively promote the generation of tolerant DCs, their induction efficiency is often unsatisfactory and it is difficult to achieve stability in large-scale production. Secondly, although the adenovirus induction method can effectively infect DCs and promote the formation of their tolerance characteristics, this method has problems with safety and controllability. In addition, the production and operation of adenoviruses are complicated, which limits their application potential.

[0004] In summary, although the existing methods for inducing tolerant DCs provide a certain basis for research, they still face problems such as low efficiency and poor stability. Summary of the invention

[0005] The present invention provides a culture medium, a culture method and an application of tolerant DC cells, which are used to solve the defects of the induced culture of tolerant DC cells in the prior art and realize the induced culture of a large number of tolerant DC cells.

[0006] The invention provides a culture medium for tolerant DC cells, comprising a first basic culture medium, granulocyte-macrophage colony stimulating factor, interleukin 4, transforming growth factor-β, prostaglandin E2 and plasma; wherein the concentration of the granulocyte-macrophage colony stimulating factor is 100-1000 U / mL, the concentration of the interleukin 4 is 10-50 ng / mL, the concentration of the transforming growth factor-β is 100-1000 U / mL, the concentration of the prostaglandin E2 is 0.4-4 μM, and the concentration of the plasma is 5-10%.

[0007] According to a culture medium for tolerogenic DC cells provided by the present invention, the first basic culture medium comprises one of AIM-V culture medium, KBM-581 culture medium and RPMI-1640 culture medium.

[0008] According to a culture medium for tolerant DC cells provided by the present invention, the concentration of the granulocyte-macrophage colony stimulating factor is 500 U / mL.

[0009] According to the culture medium for tolerant DC cells provided by the present invention, the concentration of interleukin 4 is 50 ng / mL.

[0010] According to a culture medium for tolerant DC cells provided by the present invention, the concentration of transforming growth factor-β is 700 U / mL.

[0011] The present invention provides a method for culturing tolerant DC cells, the method comprising:

[0012] Obtaining cells to be processed;

[0013] Purifying the cells to be treated to obtain purified cells;

[0014] Based on the above culture medium and the preset first culture time, the purified cells are cultured to obtain target cells.

[0015] According to a method for culturing tolerant DC cells provided by the present invention, the seeding density of the cells to be treated is 3 to 8×10 6 Pieces / mL;

[0016] The cells to be treated are PBMCs.

[0017] According to a method for culturing tolerant DC cells provided by the present invention, purifying the cells to be treated to obtain purified cells comprises:

[0018] The cells to be treated are cultured based on the second basal culture medium, and the suspended cells in the cells to be treated are discarded according to a preset second culture time to obtain purified cells.

[0019] According to a method for culturing tolerant DC cells provided by the present invention, purifying the cells to be treated to obtain purified cells comprises:

[0020] The cells to be processed are sorted to obtain purified cells.

[0021] A kit comprising any one of the culture media described above, and used for culturing, inducing and / or preparing supernatant exosomes of tolerogenic DC cells.

[0022] The present invention proposes a culture medium, a culture method and an application thereof for inducing tolerant DC cells. The culture medium comprises a first basal culture medium, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 4 (IL-4), transforming growth factor-β (TGF-β), prostaglandin E2 (PGE2) and plasma, and the content of each component is determined. Through the synergistic effect of these components, the present invention significantly promotes the proliferation, maturation and formation of tolerant DC cells. GM-CSF and IL-4 act together on the differentiation of DC cells, while TGF-β and PGE2 jointly provide an immunosuppressive environment for the generation of tolerant DC cells. In particular, the introduction of PGE2 adds mildness to this immunosuppressive process, ensuring that DC cells significantly reduce the activation of effector cells while maintaining the tolerant characteristics, thereby effectively inducing their differentiation into tolerant DC cells rather than mature DC cells. The present invention greatly improves the induction efficiency of tolerant DC cells by accurately optimizing the concentrations of various factors. Compared with traditional methods, tolerant DC cells with higher numbers and activities can be obtained in a shorter time. In addition, the components of the culture medium are relatively simple and easy to obtain, and are suitable for promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 It is a schematic diagram of the toxicity of different concentrations of prostaglandin E2 to PBMC provided by the present invention;

[0025] Figure 2 Schematic diagram of the expression level of HLA-DR, a co-stimulatory molecule of DC cells in different experimental cases;

[0026] Figure 3 It is a schematic diagram of the expression level of DC cell co-stimulatory molecule CD80 in different experimental examples provided by the present invention;

[0027] Figure 4 It is a schematic diagram of the expression level of DC cell co-stimulatory molecule CD83 in different experimental examples provided by the present invention;

[0028] Figure 5 It is a schematic diagram of the expression level of DC cell co-stimulatory molecule CD86 in different experimental examples provided by the present invention. DETAILED DESCRIPTION

[0029] 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.

[0030] The invention provides a culture medium for tolerant DC cells, which comprises a first basic culture medium, granulocyte-macrophage colony stimulating factor, interleukin 4, transforming growth factor-β, prostaglandin E2 and plasma; wherein the concentration of the granulocyte-macrophage colony stimulating factor is 100-1000 U / mL, the concentration of the interleukin 4 is 10-50 ng / mL, the concentration of the transforming growth factor-β is 100-1000 U / mL, the concentration of the prostaglandin E2 is 0.4-4 μM, and the concentration of the plasma is 5-10%.

[0031] The first basal medium, as the basis of culture, provides the cells with the basic nutrients, pH and osmotic pressure required for growth. In the present embodiment, the first basal medium can be selected from one of AIM-V medium, KBM-581 medium and RPMI-1640. AIM-V medium is a serum-free medium that can be used for the proliferation of dendritic cells, and both KBM-581 and RPMI-1640 can be used for culturing immune cells. In addition, other basal mediums that can be used for culturing immune cells can be used as the first basal medium of the present embodiment, such as AIM-V medium with 10% FBS (Fetal Bovine Serum).

[0032] GM-CSF is an important cytokine that can stimulate the proliferation and maturation of dendritic cells and enhance their antigen presentation ability. In this embodiment, within the concentration range of 100 to 1000U / mL, GM-CSF can effectively increase the number and activity of DC cells, promote their growth and maintain their function. IL-4 is another key cytokine that mainly acts to enhance the maturation and function of dendritic cells. Within the concentration range of 10 to 50ng / mL, IL-4 can effectively promote the antigen presentation ability of DC cells and the generation of tolerant DCs while maintaining their immunoregulatory properties. TGF-β is a potent immunosuppressive factor that can induce DC cells to transition to a tolerant state. It inhibits the production of cytokines and reduces the expression of co-stimulatory molecules. At the same time, within the concentration range of 100 to 1000U / mL, TGF-β can enhance its immunosuppressive properties and effectively promote the formation of tolerant DCs, rather than mature DC cells. Plasma contains a variety of bioactive components, such as growth factors, cytokines, and nutrients, which can provide additional support for cells. The addition of plasma can improve cell survival, enhance cell function, and may promote the tolerance characteristics of DC cells. In the concentration range of 5-10%, plasma can effectively supplement the nutritional needs of cells and provide cells with a culture environment that is closer to the physiological state. Furthermore, the use of autologous plasma can create an environment closer to the body.

[0033] Prostaglandin E2 (PGE2) is a lipid mediator with immunomodulatory effects. It can promote the maturation of dendritic cells, and its introduction helps maintain the tolerance state and prevent excessive immune response. It effectively regulates the function of DC cells, giving them better tolerance characteristics.

[0034] To verify the toxic effect of PGE2 on cells, this example also uses PBMC (peripheral blood mononuclear cell) which is often used as a source of induced differentiation tolerance DC cells for testing. AIM-V medium containing 10% FBS (Fetal Bovine Serum) is used as the first basal medium. Figure 1 As shown, PBMCs were cultured for 3 days using a first basal medium containing different concentrations of PGE2, and then the cell survival rate of each group was calculated based on the absorbance value of CCK-8 cell viability detection. PGE2 was non-toxic to PBMCs within a certain concentration range (0-4 μM) (P>0.05); but when the concentration was 8 μM, PGE2 had a significant toxic effect on PBMCs (P<0.05), so the concentration range of PGE2 in this embodiment was 0.4-4 μM.

[0035] Based on the culture medium of the above-mentioned tolerant DC cells, immature DC cells can be induced to culture, thereby obtaining tolerant DC cells. In one embodiment, the cells to be treated, such as PBMC, containing immature DC cells are directly cultured and induced based on the culture medium of the tolerant DC cells to obtain tolerant DC cells. In order to improve the induction efficiency of the above-mentioned culture medium and maximize the induction and proliferation effect of the culture medium on tolerant DC cells, the present invention also provides a culture method for tolerant DC cells. The method comprises:

[0036] S10, obtaining cells to be processed.

[0037] First, cells to be treated are obtained, and cells to be treated refer to cells or cell groups that have the ability to differentiate into tolerant DC cells, such as PBMC. Taking PBMC as an example, Ficoll method (density gradient centrifugation method) can be used to extract PBMC from peripheral blood.

[0038] S20, purifying the cells to be processed to obtain purified cells.

[0039] Specifically, since the cells to be treated contain many different types of cells, the cells to be treated need to be purified to improve the induction efficiency. The cell density affects the efficiency of the culture medium in supporting cell growth and subsequent differentiation and proliferation. The cell density selected in this embodiment is 3 to 8×10 6 Pieces / mL.

[0040] The first purification method provided in this embodiment is to culture the cells to be treated based on the second basal culture medium, and discard the suspended cells in the cells to be treated according to a preset second culture time to obtain purified cells.

[0041] Specifically, the second culture time refers to the time for culturing the treated cells, and the second culture time is 1 to 4 hours, preferably 2 hours as the second culture time. The second basal culture medium is a culture medium independently selected relative to the first basal culture medium, and one of the above-mentioned AIM-V culture medium, KBM-581 culture medium, etc. can be selected. Early DC cells are in an easy-to-adhere state, and PBMC also contains some suspended growth cells. By determining whether they are attached to the wall for a short time, PBMC can be coarsely screened to obtain purified cells including early DC cells, thereby increasing the content of tolerant DC cells after induced culture and improving the induction efficiency.

[0042] Taking PBMC as the cells to be treated as an example, the PBMC cells are resuspended in the above-mentioned second culture medium preheated at 37°C and inoculated in a culture vessel, such as a 6-well plate, a T25 culture flask, or a T75 culture flask. Then, the cells are cultured in a carbon dioxide incubator for 2 hours. After 2 hours, the suspended cells are discarded, and the retained adherent cells are purified cells. For ease of use, the subsequent experimental groups in this embodiment adopt the first purification method.

[0043] The second purification method provided in this embodiment is to sort the cells to be treated to obtain purified cells. The cells to be treated are sorted by marker proteins on the surface of monocytes, such as Ly6C on the surface of mouse monocytes and CD14 on the surface of human monocytes, so as to obtain monocytes with a higher concentration as purified cells. The sorting methods include magnetic bead sorting, flow sorting, etc. The sorted cells are used as purified cells for subsequent induction culture.

[0044] S30, culturing the purified cells based on the culture medium of the tolerogenic DC cells and a preset first culture time to obtain target cells.

[0045] Specifically, the first culture time is the culture time of the purified cells using the culture medium of the tolerogenic DC cells, preferably 5 to 10 days, and further, the second culture time is 7 days. After culturing the purified cells for 7 days based on the culture medium of the tolerogenic DC cells, the cells in the culture vessel are collected to obtain target cells containing tolerogenic DC cells.

[0046] To verify the culture medium and culture method of the tolerance DC cells, the culture medium of the comparative example and each group of experimental examples in the present embodiment comprises the first basal culture medium AIM-V culture medium, 500U / mL GM-CSF, 50ng / mL IL-4 and 10% autologous plasma. The comparative example adopts conventional DC cell culture medium, and the content of the prostaglandin E2 is 0, and comprises 700U / mL TNF-α. In experimental example 1, 700U / mL TGF-β is included. In experimental example 2, 700U / mL TGF-β and 0.4μM prostaglandin E2 are included. In experimental example 3, 700U / mL TGF-β and 4μM prostaglandin E2 are included.

[0047] To further increase the number of cells in this embodiment, in order to reduce the damage to the proteins on the surface of the tolerant DC cells, a cell scraper was used to scrape the cells. The obtained target cells were analyzed and identified.

[0048] This example uses multicolor flow cytometry to analyze the expression levels of costimulatory molecules on the surface of target cells, including HLA-DR, CD80, CD83 and CD86. In tolerant DC cells, their expressions are maintained at medium to low levels. After analysis and identification, the results are shown in the following table.

[0049] Table 1. Proportion of costimulatory molecules positive in target cells (%)

[0050]

[0051]

[0052] The superscript a in the table indicates that there is a significant difference compared with Comparative Example 1, P < 0.05; the superscript b indicates that there is a significant difference compared with Experimental Example 1, P < 0.05.

[0053] like Figures 2 to 5 As shown, compared with the comparative example and experimental example 1, the expression level of co-stimulatory molecules in the target cells of experimental examples 2 to 3 was significantly reduced, and the content of tolerant DC cells was significantly increased. Figures 2 to 5 As shown in Table 1, the expression level of co-stimulatory molecules was significantly reduced under the induction culture of the medium containing prostaglandin E2. Therefore, the culture medium provided in this embodiment can successfully induce the increase of the content of tolerant DC cells without damaging the cells. This method is convenient, fast and stable, and can meet the needs of large-scale tolerant DC cells.

[0054] The present invention also provides a kit, which includes the culture medium as described above, and the kit can be used in inducing tolerant DC cells, preparing supernatant exosomes, etc. Through the culture medium, the content of tolerant DC cells can be efficiently increased, based on the fact that this exosome is rich in a variety of tolerant DC cell secretion factors.

[0055] 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 tolerant DC cells, characterized in that: include: A first basal culture medium, granulocyte-macrophage colony stimulating factor, interleukin 4, transforming growth factor-β, prostaglandin E2 and plasma; wherein the concentration of the granulocyte-macrophage colony stimulating factor is 100-1000 U / mL, the concentration of the interleukin 4 is 10-50 ng / mL, the concentration of the transforming growth factor-β is 100-1000 U / mL, the concentration of the prostaglandin E2 is 0.4-4 μM, and the concentration of the plasma is 5-10%.

2. The culture medium for tolerant DC cells according to claim 1, characterized in that The first basic culture medium includes one of AIM-V culture medium, KBM-581 culture medium and RPMI-1640 culture medium.

3. The culture medium for tolerant DC cells according to claim 1, characterized in that: The concentration of the granulocyte-macrophage colony stimulating factor is 500 U / mL.

4. The culture medium for tolerant DC cells according to claim 1, characterized in that: The concentration of interleukin-4 is 50 ng / mL.

5. The culture medium for tolerant DC cells according to claim 1, characterized in that: The concentration of transforming growth factor-β is 700 U / mL.

6. A method for culturing tolerant DC cells, characterized in that: The method comprises: Obtaining cells to be processed; Purifying the cells to be treated to obtain purified cells; Based on the culture medium described in any one of claims 1 to 5 and the preset first culture time, the purified cells are cultured to obtain target cells.

7. The culture method according to claim 6, characterized in that: The seeding density of the cells to be treated is 3 to 8×10 6 Pieces / mL; The cells to be treated are PBMCs.

8. The culture method according to claim 6, characterized in that: Purifying the cells to be treated to obtain purified cells comprises: The cells to be treated are cultured based on the second basal culture medium, and the suspended cells in the cells to be treated are discarded according to a preset second culture time to obtain purified cells.

9. The culture method according to claim 6, characterized in that: Purifying the cells to be treated to obtain purified cells comprises: The cells to be processed are sorted to obtain purified cells.

10. A kit, characterized in that: The kit comprises the culture medium according to any one of claims 1 to 5, and is used for the culture, induction and / or supernatant exosome preparation of tolerogenic DC cells.

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