Method for culturing dendritic cells and use

Abstract

A method for preparing cDC1 cells in vitro, comprising: co-culturing, in vitro, peripheral blood mononuclear cells (PBMCs) obtained by separation and a cytokine composition (composed of FLT-3L, GM-CSF, SCF, and TGF-β) to obtain cDC1 cells. By means of verification, in the method for preparing the cDC1 cells in vitro of the present invention, cDC1 cells can be obtained from starting 6x106 PBMCs, the number of the cDC1 cells is at least: 1.41x106 (23.5%), and the cDC1 cells exhibit XCR1+ / CD11b- features, so that the requirement of clinical application for the number of cells can be met.

Description

Method for culturing dendritic cells and its application Technical Field

[0001] The present invention relates to a method for preparing a biomaterial, and in particular to a method for culturing conventional dendritic cells 1 (cDC1) using peripheral blood mononuclear cells (PBMC). Background Art

[0002] Dendritic cells (DCs), the most powerful professional antigen-presenting cells (APCs) in the body, play a key role in both innate and adaptive immune responses. DCs are central to initiating, regulating, and maintaining immune responses, directing various functions of the immune system. Immature DCs possess strong migratory abilities, while mature DCs can effectively activate naive T cells. DCs function to efficiently absorb, process, and present antigens in the body, providing the recognition basis for the initiation of specific immune responses via helper T cells (Th) and B cells.

[0003] DCs belong to mononuclear phagocytes (MPs). According to their different origins and differentiation pathway characteristics, they can be divided into conventional dendritic cells (cDCs), plasmacytoid DCs (pDCs) and Langerhans cells (LCs). Among them, cDCs are further divided into conventional type I dendritic cells (cDC1) and conventional type II dendritic cells (cDC2). The development and differentiation of cDC1 depends on the transcription factors BATF3 and IRF8, and its common surface markers mainly include XCR1, CD141, CLEC9A and CADM1. cDC1 has been shown to be the only one that can transport intact antigens to lymph nodes and stimulate tumor-specific CD8 + T cells are antigen-presenting cells, and their migration and immune activation functions play a crucial role in anti-tumor immune responses.

[0004] In peripheral blood, mature DCs only account for about 0.2% of PBMCs, and cDC1 accounts for only about 2.6% of DCs. Given that its content is extremely rare, it cannot be directly obtained by separation, which greatly hinders its direct application in cell therapy. Therefore, the use of cell in vitro differentiation and expansion technology to obtain DCs is the main difficulty in its clinical application. In current clinical studies, the DC cells commonly used are monocyte-derived DCs (moDCs, which express CD11b) obtained by expanding and differentiating peripheral blood monocytes or hematopoietic stem cells (HSC). + Clinical studies have found that moDCs have many drawbacks. They cannot effectively migrate to tumor-draining lymph nodes and cannot directly present antigens to host T cells. Instead, they serve as a source of antigens and must be responsible for presenting to CD8 T cells. + T and CD4 + The transfer and processing of cDC1 in the host body by T cells directly presenting antigens results in poor clinical therapeutic effects.

[0005] Therefore, providing a technology that can obtain cDC1 cells through in vitro culture will greatly promote the research, development and application of DC cell tumor vaccines.

[0006] Summary of the Invention

[0007] One object of the present invention is to provide a method for preparing cDC1 cells in vitro, thereby increasing the number of obtainable cDC1 cells and meeting the needs of clinical applications.

[0008] Another object of the present invention is to provide a method for preparing cDC1 cells in vitro to improve the efficiency of obtaining cDC1 cells.

[0009] Another object of the present invention is to provide a method for preparing cDC1 cells in vitro, so as to facilitate the acquisition of cDC1 cells.

[0010] Another object of the present invention is to provide a method for preparing cDC1 cells in vitro, and its application in preparing dendritic cell vaccines, especially in preparing in vitro specific T cell drugs.

[0011] A method for preparing cDC1 cells in vitro comprises co-culturing isolated peripheral blood mononuclear cells with a cytokine composition in vitro to obtain cDC1 cells.

[0012] The cytokine composition used in the method for preparing cDC1 cells in vitro of the present invention consists of FLT-3L, GM-CSF, SCF and TGF-β.

[0013] An embodiment of a cytokine composition for use in the method for preparing cDC1 cells in vitro of the present invention, a dendritic cell culture medium (ImunoCult TM Cytokines such as FLT-3L, GM-CSF, SCF, and TGF-β were added to the ACF Dendritic Cell Medium (ACF) to achieve a FLT-3L concentration of 100±10 ng / ml, a GM-CSF concentration of 10±1 ng / ml, an SCF concentration of 20±2 ng / ml, and a TGF-β concentration of 10±1 ng / ml. The concentrations of each factor were referenced from multiple literatures, and appropriate concentrations were selected within the usage range. The concentrations of GM-CSF and TGF-β were determined experimentally; too low concentrations of the two were not conducive to the differentiation and suspension of cDC1 cells.

[0014] Another embodiment of the cytokine composition used in the method for preparing cDC1 cells in vitro of the present invention further includes adding IL-3, and the concentration in the dendritic cell culture medium is 5 ng / ml.

[0015] The in vitro cDC1 cell production method of the present invention further includes the addition of plasma, e.g., from the same individual as used to obtain PBMCs, in an amount of 5±1 v / v%. We tested cell culture in the absence of plasma, with platelet lysate replacing plasma, and with 5% autologous plasma. The results showed that cells cultured with autologous plasma exhibited significantly higher growth rates than those cultured with platelet lysate, and those cultured without serum. The cells in the plasma group exhibited the best growth, faster differentiation, and a higher XCR1 expression rate.

[0016] The method for preparing cDC1 cells in vitro of the present invention is as follows: taking a 60 mm culture dish as an example, 1×10 PBMCs are added. 7 (i.e. 4.35×10 5 cells / cm 2 ), the PBMC inoculation volume is based on the cell density, such as 1-1.5×10 6 cells / ml.

[0017] The present invention provides a method for producing cDC1 cells in vitro, wherein isolated peripheral blood mononuclear cells are co-cultured with a cytokine composition in vitro (e.g., cultured in a 37°C ± 0.1°C 5% CO2 cell culture incubator) for 10-14 days, and then the upper layer of suspended cells is transferred to a low-adsorption culture dish and cultured for another 2-4 days to obtain cDC1 cells. The culture medium and the cytokine composition used remain unchanged.

[0018] It has been verified that the method of preparing cDC1 cells in vitro of the present invention is to start with 6×10 6 PBMC can be used to obtain cDC1 cells, the number of which is at least 1.41×10 6The number of cells was 23.5% and showed XCR1+ / CD11b- characteristics, which can meet the cell number requirements of clinical applications.

[0019] The method of the present invention is used to prepare XCR1 + cDC1 cells account for 70% to 85%, and the remaining cells cannot activate the antigen presentation function, so they do not need further purification, which is more convenient and efficient.

[0020] The cDC1 prepared in the present invention is subjected to maturation stimulation (GM-CSF, CD40L, R848, Poly I:C, INF-γ and antigen peptide) to prepare a cell vaccine medicine (preparation) for tumor prevention and treatment.

[0021] The cDC1 prepared in the present invention can be used to activate T cell-specific immunity and be used for the preparation of in vitro specific T cell drugs after maturation stimulation (such as but not limited to GM-CSF, CD40L, R848, Poly I:C, INF-γ and antigen peptides).

[0022] Compared with the prior art, the advantages of the cDC1 cell preparation method of the present invention are: ①. The starting material is PBMC, and magnetic bead separation is not required, which makes the operation simpler; ②. The ratio of cDC1 cells (XCR1+ / CD11b-) obtained is higher (greater than 70%); ③. The number of cDC1 cells obtained is larger (about 2×10 6 cDC1 / 6×10 6 PBMC). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a bright field image of cell morphology at various culture conditions and at various culture times;

[0024] FIG2 is a bright field image of cell morphology of cells transferred from suspension to low-adsorption dish and cultured for various time periods;

[0025] FIG3A is a graph showing the expression of XCR1 marker on the surface of cDC1 cells detected by flow cytometry;

[0026] FIG3B is a graph showing the expression of CD11b marker on the surface of cDC1 cells detected by flow cytometry;

[0027] FIG4A is a graph showing the expression of XCR1 marker on the surface of cDC1 cells in a low-adhesion dish detected by flow cytometry;

[0028] FIG4B is a graph showing the results of flow cytometry detection of CD11b marker expression on the surface of cDC1 cells in a low-adhesion dish;

[0029] FIG5 shows the number of cDC1 cells finally obtained under various culture conditions. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are intended only to illustrate the technical solution of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention may be modified or replaced with equivalents without departing from the spirit and scope of the technical solution of the present invention, and all such modifications or equivalents should be included in the scope of the claims of the present invention.

[0031] The various test methods used in the following examples of the present invention are specifically described as follows:

[0032] 1) Isolation of peripheral blood to obtain PBMC

[0033] Freshly collected blood in a sodium heparin tube is used for PBMC isolation. After gently inverting the tube several times, the mixed whole blood is added to a centrifuge tube containing Ficoll lymphocyte separation buffer in a 1:1 ratio and centrifuged at 2,500 rpm for 15 minutes. After centrifugation, the upper yellow plasma layer is transferred to a fresh centrifuge tube and inactivated at 56°C for 30 minutes before use. Gently aspirate the buffy coat layer into a 15ml centrifuge tube, add PBS buffer, mix well, and centrifuge at 2,500 rpm for 10 minutes. After centrifugation, discard the liquid and use red blood cell lysis buffer to dislodge the cell pellet at the bottom of the tube. Lyse the red blood cells for 10 minutes. After lysis, centrifuge the lysed cells at 2,500 rpm for 5 minutes. Resuspend the cell pellet in PBS buffer and count the cells. Finally, centrifuge the suspension at 2,500 rpm for 5 minutes, discard the supernatant, and obtain the PBMCs.

[0034] 2) Flow cytometry detection method

[0035] To identify the proportion of cDC1 in the differentiated suspension cells, this example used flow cytometry to detect surface markers. The antibody used was Human XCR1 PE-conjugated Antibody (R&D Systems, FAB8571P-100), BD Pharmingen TM PE Mouse Anti-Human CD11b (BD, 555388).

[0036] 3) Method for measuring total cell count

[0037] In this example, the number of cells in each group was calculated by taking the number of cells cultured in the low-adsorption culture dish for 2 days and adding the number of suspended cells taken from the original 60 mm dish for each flow cytometry test, and the two were combined to record the total number of cells harvested.

[0038] Example 1 cDC1 cells were obtained by direct differentiation of PBMC.

[0039] Methods: First, the isolated PBMCs were evenly plated into the following culture conditions: ①. FGS group: FLT-3L (100 ng / ml), GM-CSF (10 ng / ml), SCF (20 ng / ml); ②. FGST group: FLT-3L (100 ng / ml), GM-CSF (10 ng / ml), SCF (20 ng / ml), TGF-β (10 ng / ml); ③. FGS3T group: FLT-3L (100 ng / ml), GM-CSF (10 ng / ml), SCF (20 ng / ml), TGF-β (10 ng / ml), IL-3 (5 ng / ml); the culture medium was ImunoCult TM -ACF Dendritic Cell Medium (STEMCELL, #10986), containing 5% autologous plasma and 1 w / v% penicillin and streptomycin. The number of cells plated was 6 × 10 6 After completion, place the cells in a 37°C ± 0.1°C 5% CO2 incubator and culture for 2 hours, then change the medium once. Observe and photograph regularly.

[0040] The day cells were plated was designated Day 0. As differentiation culture progressed, the cells gradually increased in size and aggregated, then began to float. On Day 6, suspended cells began to appear. These cells varied in size and morphology, with small cells being the majority. On Day 10, the number of suspended cells gradually increased, becoming more uniform and generally assuming a large oval shape. On Day 13, the cell morphology was nearly identical to that on Day 10, as shown in Figure 1.

[0041] On Day 6 of differentiation culture, the upper suspension cells were transferred to a low-adhesion 60mm dish for continued culture. The second day of culture was designated as suspension culture Day 1. As shown in Figure 2, after culture in the low-adhesion dish, the number of small cells gradually decreased. On Day 2, the number of cells was higher. Afterward, a small number of cell aggregates began to form, and the number gradually decreased. Because the FGS group suspended cells more slowly, low-adhesion dishes were not used for this group.

[0042] As shown, there were no significant differences in cell morphology between the FGST and FGS3T groups. DC differentiation was observed in both groups after one week of culture, but the FGS3T group appeared to be the most numerous on Day 10. The FGS group grew the slowest, with cells only beginning to appear in suspension on Day 10. Suspension cells maintained their optimal state after 2-3 days of culture in low-adhesion dishes.

[0043] Example 2 Detection of cDC1 cell surface markers by flow cytometry

[0044] Figure 3A shows the results of flow cytometry detection of XCR1 marker expression on the surface of cDC1 cells. As shown in Figure 3A, the XCR1+ ratio was highest on Day 10, and the ratios of the three groups were almost the same, all reaching above 70%. On Day 17, the FGST group decreased from 74.42% to 42.09%, and the FGS3T group decreased from 73.86% to 38.96%, a decrease of nearly 50%. Due to the slower differentiation, the FGS group was still in the stage of high XCR1 expression (66.29%).

[0045] Figure 3B shows flow cytometry analysis of CD11b expression on the surface of cDC1 cells. As shown in Figure 3B, CD11b expression remained consistently high in the FGS group, exceeding 40%, while expression was very low in the other two groups, particularly in the FGS3T group, which reached 1.65% on Day 6 and 1.21% on Day 10, significantly lower than the FGS group. Since CD11b is the primary surface marker of moDCs, the FGST and FGS3T groups significantly outperformed the FGS group in terms of surface marker expression.

[0046] After 4 days of culture, the cells transferred to the low-adhesion dish on Day 6 of differentiation showed that their XCR1 + The proportion increased compared to Day 6, with the FGST group increasing from 37.62% to 64.02%, and the FGS3T group increasing from 39.52% to 53.99%. At the same time, CD11b expression remained at a low level (see Figures 4A and 4B), indicating that low-adsorption dishes are conducive to the differentiation of cells into cDC1. However, the culture time of cells in low-adsorption dishes should not be too long, otherwise not only will the cell number be greatly reduced, but the expression of XCR1 will also be significantly reduced. As shown in Figure 4A, Day 11 flow cytometry results, the FGST group decreased from 64.02% to 25.67%, and the FGS3T group decreased from 53.99% to 37.82%, a decrease of nearly 50%. In other words, the number of days for suspension cells to be cultured in low-adsorption dishes should not exceed 4 days.

[0047] This shows that on Day 10 of differentiation culture, XCR1 + The ratio was the highest and dropped significantly on Day 17, which means that the culture period is preferably 2 weeks. There was no significant difference between the FGST and FGS3T groups. + The ratio is also high, but its CD11b + It is also higher, which means that most of the cells in the FGS group are moDCs, so the cytokines in the FGS group cannot be used for the culture of cDC1 cells.

[0048] Example 3 Number of cDC1 cells

[0049] The final number of cells harvested in the FGS group was 1.03×10 6 The FGST group was 1.41×10 6 and 1.81×10 in the FGS3T group. 6 The number of PBMCs in the original plate was 6×10 6 Therefore, the final cell yields were 17.12% in the FGS group, 23.5% in the FGST group, and 30.17% in the FGS3T group.

[0050] It can be seen that the number of cDC1s finally obtained by using the cytokine combination of the FGS3T group of this example is significantly higher than that of the other two groups, and the cytokine combination of the FGST group is better than that of the FGS group.

Claims

1. A method for preparing cDC1 cells in vitro, characterized in that The isolated peripheral blood mononuclear cells are co-cultured with a cytokine composition in vitro to obtain cDC1 cells; The cytokine composition consists of FLT-3L, GM-CSF, SCF and TGF-β.

2. The method for preparing cDC1 cells in vitro according to claim 1, characterized in that IL-3 was also added for co-culture.

3. The method for preparing cDC1 cells in vitro according to claim 2, characterized in that The IL-3 dosage is 5 ng / ml.

4. The method for preparing cDC1 cells in vitro according to claim 1, characterized in that The culture medium used was dendritic cell culture medium containing 100±10 ng / ml FLT-3L, 10±1 ng / ml GM-CSF, 20±2 ng / ml SCF and 10±1 ng / ml TGF-β.

5. The method for preparing cDC1 cells in vitro according to claim 1, characterized in that Plasma was also added.

6. The method for preparing cDC1 cells in vitro according to claim 5, characterized in that The amount of plasma used is 5 v / v%.

7. The method for preparing cDC1 cells in vitro according to claim 1, characterized in that The seeding concentration of peripheral blood mononuclear cells was 1-1.5×10 6 cells / ml.

8. The method for preparing cDC1 cells in vitro according to claim 1, characterized in that The isolated peripheral blood mononuclear cells are co-cultured with the cytokine composition in vitro for a total of 12-14 days to obtain cDC1 cells. During this period, the upper layer of suspended cells are transferred to a low-adsorption culture dish to continue culturing for 2-3 days to obtain cDC1 cells.

9. Use of the method for preparing cDC1 cells in vitro according to claim 1 in preparing dendritic cell vaccines.

10. Use of the method for preparing cDC1 cells in vitro according to claim 1 in preparing specific T cell drugs for in vitro use.

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