Use of artemisinin in culturing of resistant DC cells

Abstract

The application relates to an application of artemisinin in tolerance DC cell culture, and provides a tolerance DC cell culture medium with artemisinin, which comprises a basic culture medium, cytokines and artemisinin, wherein the concentration of the artemisinin is 0.5-5 muM. The tolerance DC cell culture medium with artemisinin has reduced expression levels of co-stimulating molecules CD80, CD83, CD86 and HLA-DR, which indicates that the addition of artemisinin is beneficial to the tolerance DC cell culture.

Description

Technical Field

[0001] This invention relates to the field of DC cell culture technology, and in particular to a tolerant DC cell culture medium, culture method and application. Background Technology

[0002] Dendritic cells (DCs) are a type of special immune cell found in the normal human body that has a strong antigen-presenting function.

[0003] Tolerogenic dendritic cells (tolDCs) are a type of dendritic cell capable of inducing immune tolerance. They suppress excessive immune responses by secreting anti-inflammatory cytokines such as IL-10 and TGF-β. Compared to conventional mature DCs, tolDCs typically express lower levels of co-stimulatory molecules (such as CD80 and CD86) and higher levels of repressive molecules, which helps maintain immune tolerance and limits their ability to activate T cells. TolDCs can also induce and expand Treg cells in vivo.

[0004] Due to their strong ability to induce immune tolerance, tolerant dendritic cells (DCs) play an important role in the treatment of autoimmune diseases, inflammatory diseases, and in preventing transplant rejection. Generating tolerant DCs through in vitro modification or differentiation and then reinfusing them into patients holds promise for achieving immunomodulatory therapy for diseases.

[0005] Given the therapeutic effects of tolerant DC cells in some diseases, they need to be induced and cultured in a specific cytokine environment to maintain their tolerant phenotype. How to improve the efficiency of induced and cultured tolerant DC cells to produce clinical-grade products is an urgent problem to be solved. Summary of the Invention

[0006] This invention provides the application of artemisinin in the culture of tolerant DC cells. The main purpose of this invention is to solve the problem of the induction efficiency of tolerant DC cells in existing culture techniques.

[0007] The contents of this invention are as follows:

[0008] A first aspect of the present invention claims protection for the use of artemisinin in the culture of tolerant DC cells.

[0009] Artemisinin, also known as artemisia annua, is a natural sesquiterpene lactone compound extracted from the traditional Chinese medicine Artemisia annua. It has significant antimalarial effects. In addition to its antimalarial properties, artemisinin and its derivatives have shown potential therapeutic effects on other diseases, including lupus erythematosus, pulmonary hypertension, and tumors.

[0010] The applicant of this invention has discovered that artemisinin has an immunomodulatory effect in the culture of DC cells, which can promote the differentiation of regulatory T cells, enhance immune tolerance, and is beneficial to the culture of tolerant DC cells.

[0011] Artemisinin is beneficial for the culture of tolerant dendritic cells (DCs). When applied to the culture medium for tolerant DCs, it can further enhance immune tolerance and plays an important role in the research and potential clinical applications of tolerant DCs.

[0012] As a preferred embodiment of the first aspect of the present invention, the concentration of artemisinin in the culture of tolerant DC cells is 0.5–5 μM.

[0013] As a second aspect of the present invention, the present invention claims protection for a culture medium for tolerant DC cells, comprising: a basal culture medium, cytokines and artemisinin, wherein the concentration of artemisinin is 0.5 to 5 μM.

[0014] Adding artemisinin to the culture medium of tolerant DC cells can further induce immune tolerance, which is beneficial for the culture of tolerant DC cells. Cytokines are used to induce the differentiation of cultured cells into DC cells or tolerant DC cells.

[0015] Preferably, the tolerant DC cell culture medium further includes 5-10% autologous plasma. The addition of autologous plasma can meet the nutrient requirements of cell culture and help cells grow and proliferate in the culture environment.

[0016] Preferably, the cytokines include 100–1000 U / mL GM-CSF, 10–50 ng / mL IL-4, and 100–1000 U / mL TGF-β.

[0017] GM-CSF and IL-4 are used to induce PBMC differentiation into DC cells. TGF-β is a multifunctional cytokine that plays an important role in cell proliferation, differentiation, and immune function. In the culture of tolerant DC cells, it can promote the differentiation of regulatory T cells, thereby enhancing immune tolerance.

[0018] Preferably, the basal culture medium includes one of AIM-V medium, KBM-581 medium, and RPMI-1640.

[0019] Preferably, the artemisinin is 5 μM.

[0020] As a third aspect of the invention, protection is claimed for a culture medium for resistant DC cells, comprising: a basal culture medium, cytokines, artemisinin, and autologous plasma, wherein the concentration of artemisinin is 0.5–5 μM; the cytokines include 100–1000 U / mL GM-CSF, 10–50 ng / mL IL-4, and 100–1000 U / mL TGF-β; and the amount of autologous plasma added is 5–10%.

[0021] As a fourth aspect of the invention, protection is claimed for a method of culturing tolerant DC cells, comprising:

[0022] Obtain the cells to be processed;

[0023] The cells to be treated are seeded in a culture vessel and cultured in basal medium for a first predetermined time. Non-adherent cells are discarded. Then, culture medium as described in either the second or third aspect of the present invention is added and cultured for a second predetermined time. Fresh culture medium is added every 2 to 3 days during the second predetermined culture time.

[0024] After the culture was completed, adherent cells were scraped for testing.

[0025] A preferred method for culturing the tolerant DC cells is as follows: the cells to be treated are PBMCs, the first predetermined culture time is 2-4 hours, and the second predetermined culture time is 7 days.

[0026] Specifically, culture vessels can include 6-well plates, T25 culture flasks, T75 culture flasks, etc., depending on the culture density and culture conditions.

[0027] Beneficial effects:

[0028] This invention provides the application of artemisinin in the culture of tolerant DC cells. The addition of artemisinin enhances immune tolerance and is beneficial for the culture of tolerant DC cells. Cells cultured using artemisinin-containing tolerant DC cell culture medium show reduced expression levels of co-stimulatory molecules CD80, CD83, CD86, and HLA-DR, indicating that the addition of artemisinin is beneficial for the culture of tolerant DC cells. Attached Figure Description

[0029] Figure 1 This is a schematic diagram showing the expression levels of the co-stimulatory molecule CD80 in DC cells in different experimental cases;

[0030] Figure 2 This is a schematic diagram showing the expression level of CD83, a co-stimulatory molecule in DC cells, in different experimental cases.

[0031] Figure 3 This is a schematic diagram showing the expression level of the co-stimulatory molecule CD86 in DC cells in different experimental cases;

[0032] Figure 4 This diagram illustrates the expression levels of the co-stimulatory molecule HLA-DR in DC cells in different experimental cases. Detailed Implementation

[0033] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0034] The inventors of this invention have discovered that applying artemisinin to the culture of tolerant DC cells can increase their immune tolerance, which is beneficial to the culture of tolerant DC cells and provides strong support for subsequent clinical research and use of tolerant DC cells.

[0035] Specifically, artemisinin can be applied to tolerant DC cells, specifically through its application in the corresponding culture medium.

[0036] Specifically, the application of artemisinin in the culture of tolerant DC cells can be achieved through a tolerant DC cell culture medium comprising: basal medium, cytokines, artemisinin, and autologous plasma. The concentration of artemisinin is 0.5–5 μM. The cytokines include 100–1000 U / mL GM-CSF, 10–50 ng / mL IL-4, and 100–1000 U / mL TGF-β. The amount of autologous plasma added is 5–10%.

[0037] More preferably, the culture medium for the tolerant DC cells includes AIM-V medium, 500 U / mL GM-CSF, 50 ng / mL IL-4, 700 U / mL TGF-β, 5 μM artemisinin and 10% autologous plasma.

[0038] This invention also provides a method for culturing the above-mentioned tolerant DC cells, comprising:

[0039] Obtain the cells to be processed;

[0040] The cells to be treated are seeded in a culture vessel and cultured in basal medium for a first predetermined time. Non-adherent cells are discarded. Then, culture medium as described in either the second or third aspect of the present invention is added and cultured for a second predetermined time. Fresh culture medium is added every 2 to 3 days during the second predetermined culture time.

[0041] After the culture was completed, adherent cells were scraped for testing.

[0042] Specifically, using PBMCs as the culture medium, after initial culturing in basal medium for 2-4 hours, monocytes—one of the precursor cells of dendritic cells—adhere to the bottom of the culture dish, while other cell types (such as lymphocytes) remain in suspension. By washing away the non-adherent cells, monocytes can be initially enriched.

[0043] The basal culture medium referred to can be any one of AIM-V medium, KBM-581 medium, or RPMI-1640.

[0044] Subsequently, the preliminarily enriched monocytes were added to the culture medium of this invention. Cytokines and artemisinin were used to induce the monocytes to differentiate into dendritic cells and promote the formation of immune tolerance. The addition of autologous plasma further ensured cell growth. Cells cultured using this culture medium and method exhibited improved immune tolerance.

[0045] To better illustrate the technical effects of the present invention, the present invention constructed the following experimental examples and comparative examples for testing.

[0046] Experimental Example 1

[0047] The culture medium for tolerant DC cells is AIM-V medium;

[0048] The complete culture medium contains cytokines: 500 U / mL GM-CSF, 50 ng / mL IL-4, and 700 U / mL TGF-β.

[0049] Complete culture media do not contain artemisinin;

[0050] The complete culture medium includes 10% autologous plasma.

[0051] Experiment Example 2

[0052] The culture medium for tolerant DC cells is AIM-V medium;

[0053] The complete culture medium contains cytokines: 500 U / mL GM-CSF, 50 ng / mL IL-4, and 700 U / mL TGF-β.

[0054] The complete culture medium includes 0.5 μM artemisinin;

[0055] The complete culture medium includes 10% autologous plasma.

[0056] Experimental Example 3

[0057] The culture medium for tolerant DC cells is AIM-V medium;

[0058] The complete culture medium contains cytokines: 500 U / mL GM-CSF, 50 ng / mL IL-4, and 700 U / mL TGF-β.

[0059] The complete culture medium includes 5 μM artemisinin;

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

[0061] Comparative Example 1

[0062] The standard culture medium for DC cells is AIM-V medium;

[0063] The complete culture medium contains cytokines: 500 U / mL GM-CSF, 50 ng / mL IL-4, and 700 U / mL TNF-α.

[0064] The complete culture medium includes 10% autologous plasma.

[0065] The culture methods for Experimental Examples 1-3 and the comparative examples are as follows:

[0066] S1: Human peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation.

[0067] S2: Inoculate PBMCs into culture dishes at a density of 3–8 × 10⁶. 6 Cells were cultured at a density of 1 cell / mL, and AIM-V basal medium was added for adherent culture for 2 hours. Then, the non-adherent cells were discarded, and the cells were cultured in the above complete medium for another 7 days. Fresh complete medium was added every 2-3 days.

[0068] S3: After culture, adherent cells were scraped using a cell scraper, and flow cytometry was used for immunophenotypic analysis of tolerant DCs. Specifically, the expression of co-stimulatory molecules CD80, CD83, CD86, and HLA-DR was detected. The test results are as follows: Figure 1-4 As shown in Table 1.

[0069] Table 1. Percentage of positive co-stimulatory molecules in DC cells of each group (%)

[0070] Group HLA-DR CD80 CD83 CD86 Comparative Example 1 96.10±0.46 98.20±0.46 91.33±0.06 72.03±0.15 Experimental Example 1 <![CDATA[56.80±3.05 a ]]> <![CDATA[67.70±1.61 a ]]> <![CDATA[92.63±0.12 a ]]> <![CDATA[38.97±0.15 a ]]> Experiment Example 2 <![CDATA[51.90±2.09 a ]]> <![CDATA[61.60±2.18 a,b ]]> <![CDATA[86.50±0.30 a,b ]]> <![CDATA[37.33±0.50 a,b ]]> Experimental Example 3 <![CDATA[49.33±3.11 a,b ]]> <![CDATA[61.07±2.95 a,b ]]> <![CDATA[87.30±0.26 a,b ]]> <![CDATA[37.57±0.49 a,b ]]>

[0071] Note: a indicates a significant difference compared to Comparative Example 1, P < 0.05; b indicates a significant difference compared to Experimental Example 1, P < 0.05.

[0072] Figure 1This diagram illustrates the expression levels of the co-stimulatory molecule CD80 in dendritic cells (DCs) across different experimental cases. CD80 is a crucial co-stimulatory molecule in T cell activation, and its positivity rate reflects whether tolerant cells receive sufficient co-stimulatory signals upon antigen stimulation. Low CD80 expression may lead to insufficient T cell activation, thereby promoting the formation of tumor immune tolerance. Figure 1 The expression levels of the co-stimulatory molecule CD80 in Experimental Example 2 (containing 0.5 μM artemisinin) and Experimental Example 3 (containing 5 μM artemisinin) were significantly lower than those in Comparative Example 1 (conventional DC) and Experimental Example 1 (without artemisinin).

[0073] Figure 2 This diagram illustrates the expression levels of CD83, a co-stimulatory molecule in dendritic cells (DCs), in different experimental cases. CD83 is an important marker of DC maturation, and its positivity rate reflects the maturation status and antigen-presenting capacity of DCs. A low CD83 positivity rate may indicate insufficient DC maturation, limited antigen-presenting capacity, resulting in insufficient T cell activation and promoting immune tolerance. Figure 2 The results showed that the expression level of the co-stimulatory molecule CD83 in Experiment 2 (containing 0.5 μM artemisinin) and Experiment 3 (containing 5 μM artemisinin) was significantly lower than that in Comparative Example 1 (conventional DC) and Experiment 1 (without artemisinin).

[0074] Figure 3 This diagram illustrates the expression levels of the co-stimulatory molecule CD86 in dendritic cells (DCs) across different experimental cases. The CD86 positivity rate reflects whether tolerant cells receive sufficient co-stimulatory signals upon antigen stimulation. A low CD86 positivity rate may indicate weaker T cell activation capacity, contributing to immune tolerance. Figure 3 The results showed that the expression level of the co-stimulatory molecule CD86 in Experiment 2 (containing 0.5 μM artemisinin) and Experiment 3 (containing 5 μM artemisinin) was significantly lower than that in Comparative Example 1 (conventional DC) and Experiment 1 (without artemisinin).

[0075] Figure 4 This diagram illustrates the expression levels of the co-stimulatory molecule HLA-DR in dendritic cells (DCs) from different experimental cases. HLA-DR is a molecule in the human leukocyte antigen (HLA) system, and the HLA-DR positivity rate reflects the ability of these cells to present antigens to T cells. Low HLA-DR expression may lead to T cells' inability to effectively recognize tumor cells, thereby promoting the formation of tumor immune tolerance. Figure 4 The results showed that the expression level of the co-stimulatory molecule HLA-DR in Experiment 2 (containing 0.5 μM artemisinin) and Experiment 3 (containing 5 μM artemisinin) was significantly lower than that in Control Example 1 (conventional DC); the expression level of the co-stimulatory molecule HLA-DR in Experiment 3 (containing 5 μM artemisinin) was significantly lower than that in Experiment 1 (without artemisinin).

[0076] In summary, the above test results indicate that artemisinin is beneficial for the culture of tolerant DC cells.

[0077] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions 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 invention.

Claims

1. A culture medium for tolerant DC cells, characterized in that, include: The basal culture medium, cytokines, and artemisinin, wherein the concentration of artemisinin is 0.5-5 μM, and the cytokines include 100-1000 U / mL GM-CSF, 10-50 ng / mL IL-4, and 100-1000 U / mL TGF-β.

2. The DC cell culture medium according to claim 1, characterized in that, The culture medium for the tolerant DC cells also includes 5-10% autologous plasma.

3. The tolerant DC cell culture medium according to claim 1, characterized in that, The basal culture medium includes one of AIM-V medium, KBM-581 medium, and RPMI-1640.

4. The tolerant DC cell culture medium according to claim 1, characterized in that, The artemisinin was 5 μM.

5. A method for culturing tolerant DC cells, characterized in that, include: Obtain cells to be processed, wherein the cells to be processed are PBMCs; The cells to be treated are seeded into a culture vessel and cultured in basal medium for a first predetermined time. Non-adherent cells are discarded. Then, the culture medium as described in any one of claims 1-4 is added and cultured for a second predetermined time. Fresh culture medium is added every 2-3 days during the second predetermined culture time. After the culture was completed, adherent cells were scraped for testing.

6. The method for culturing tolerant DC cells according to claim 5, characterized in that, The first predetermined culture time is 2-4 hours, and the second predetermined culture time is 7 days.

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