Methods and applications of culturing dendritic cells

Abstract

A method for in vitro preparation of cDC1 cells involves co-culturing isolated peripheral blood mononuclear cells with a cytokine composition (composed of FLT-3L, GM-CSF, SCF, and TGF-β) in vitro to obtain cDC1 cells. The method has been verified to be effective in in vitro preparation of cDC1 cells from an initial 6 × 10⁻⁶ cells. 6 PBMCs can obtain cDC1 cells in a quantity of at least 1.41 × 10⁻⁶. 6 23.5% of the cells were XCR1+ / CD11b-, which meets the clinical application requirements for cell quantity.

Description

Technical Field

[0001] This invention relates to a method for preparing biological materials, and more particularly to a method for culturing conventional type I dendritic cells (cDC1) using peripheral blood mononuclear cells (PBMCs). Background Technology

[0002] Dendritic cells (DCs), as the most potent professional antigen-presenting cells (APCs) in the body, play a crucial role in both innate and adaptive immune responses. DCs are central to initiating, regulating, and maintaining immune responses, dominating various functions of the body's immune system. Immature DCs possess strong migratory capabilities, while mature DCs can effectively activate naive T cells. The function of DCs is to efficiently take up, process, and present antigens in the body, providing the recognition basis for the initiation of specific immune responses through helper T cells (Th) and B cells.

[0003] Dendritic cells (DCs) belong to the mononuclear phagocytes (MPs) and can be classified into conventional dendritic cells (cDCs), plasmacytoid DCs (pDCs), and Langerhans cells (LCs) based on their different origins and differentiation pathways. cDCs are further divided into conventional type I dendritic cells (cDC1) and conventional type II dendritic cells (cDC2). The development and differentiation of cDC1 depend on the transcription factors BATF3 and IRF8, and its common surface markers include XCR1, CD141, CLEC9A, and CADM1. cDC1 has been shown to be the only cell capable of transporting intact antigens to lymph nodes and stimulating 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 dendritic cells (DCs) account for only about 0.2% of PBMCs, and cDC1 accounts for only about 2.6% of DCs. Given their extremely low abundance, they cannot be directly obtained through isolation, which greatly hinders their direct application in cell therapy. Therefore, obtaining DCs using in vitro cell differentiation and expansion techniques is a major challenge for their clinical application. Currently, the DCs commonly used in clinical research are monocyte-derived DCs (moDCs, characterized by CD11b) obtained by expanding and differentiating peripheral blood monocytes or hematopoietic stem cells (HSCs). + Clinical studies have found that moDCs have many drawbacks. They cannot effectively migrate to tumor-draining lymph nodes, and they cannot directly present antigens to host T cells. Instead, as a source of antigens, they must be presented by cells responsible for presenting antigens to CD8 cells. + T and CD4 + T cells directly present antigens to the host's cDC1 for transfer and processing, which results in poor clinical treatment efficacy.

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

[0006] One object of the present invention is to provide a method for in vitro preparation of cDC1 cells, thereby increasing the number of obtainable cDC1 cells to meet the needs of clinical applications.

[0007] Another objective of this invention is to provide a method for in vitro preparation of cDC1 cells, thereby improving the efficiency of obtaining cDC1 cells.

[0008] Another object of the present invention is to provide a method for in vitro preparation of cDC1 cells, so as to make the acquisition of cDC1 cells more convenient.

[0009] Another object of the present invention is to provide a method for in vitro preparation of cDC1 cells, and its application in the preparation of dendritic cell vaccines, particularly in the preparation of in vitro specific T cell drugs.

[0010] A method for in vitro preparation of cDC1 cells involves co-culturing isolated peripheral blood mononuclear cells with a cytokine composition in vitro to obtain cDC1 cells.

[0011] The cytokine composition used in the method for in vitro preparation of cDC1 cells according to the present invention consists of FLT-3L, GM-CSF, SCF and TGF-β.

[0012] An embodiment of a cytokine composition for the method of in vitro preparation of cDC1 cells according to the present invention, using dendritic cell culture medium (ImunoCult). TM FLT-3L, GM-CSF, SCF, and TGF-β were added to the CDC1 Dendritic Cell Medium (ACF Dendritic Cell Medium) to achieve the following concentrations: FLT-3L 100±10 ng / ml, GM-CSF 10±1 ng / ml, SCF 20±2 ng / ml, and TGF-β 10±1 ng / ml. The concentrations of each factor were selected from multiple literature reviews and within the appropriate range. The concentrations of GM-CSF and TGF-β were determined through our experiments. If the concentrations of both are too low, it will not be conducive to the differentiation and suspension of cDC1 cells.

[0013] Another embodiment of the cytokine composition used in the method for in vitro preparation of cDC1 cells according to the present invention further includes the addition of IL-3 at a concentration of 5 ng / ml in the dendritic cell culture medium.

[0014] The method for in vitro preparation of cDC1 cells in this invention also includes the addition of plasma, sourced from the same individual from whom PBMCs were obtained, at a dosage of 5±1 v / v%. We tested cell culture with plasma-free plasma, platelet lysate instead of plasma, and 5% autologous plasma. The results showed that cells using autologous plasma > cells using platelet lysate > serum-free plasma. Cells in the autologous plasma group showed the best growth, faster differentiation, and a higher XCR1 expression ratio.

[0015] The method for in vitro preparation of cDC1 cells according to the present invention, taking a 60mm culture dish as an example, involves adding 1×10⁻⁶ PBMCs. 7 (i.e., 4.35 × 10) 5 cells / cm 2 The seeding amount of PBMCs should be based on cell density, such as 1–1.5 × 10⁻⁶ cells / year. 6 Cells / ml

[0016] The present invention provides a method for in vitro preparation of cDC1 cells, in which peripheral blood mononuclear cells are isolated and co-cultured with a cytokine composition in vitro (e.g., cultured in a 37℃±0.1℃ 5% CO2 cell culture incubator) for 10-14 days, and the upper layer of suspended cells is transferred to a low-adsorption culture dish for further culture for 2-4 days to obtain cDC1 cells. The culture medium and the cytokine composition contained therein remain unchanged.

[0017] The method for in vitro preparation of cDC1 cells of this invention has been verified to be effective, starting with 6 × 10⁻⁶ cells. 6 PBMCs can obtain cDC1 cells in a quantity of at least 1.41 × 10⁻⁶. 623.5% of the cells were XCR1+ / CD11b-, which meets the clinical application requirements for cell quantity.

[0018] The method of this invention produces XCR1. + cDC1 cells account for 70%–85% of the total. The remaining cells do not have the function of antigen presentation and therefore do not require further purification, making the process more convenient and efficient.

[0019] The present invention obtains cDC1 and, after maturation stimulation (GM-CSF, CD40L, R848, Poly I:C, INF-γ and antigenic peptide), prepares it into a cell vaccine drug (formulation) for tumor prevention and treatment.

[0020] The cDC1 prepared in this invention, after being stimulated for maturation (e.g., but not limited to GM-CSF, CD40L, R848, Poly I:C, INF-γ, and antigenic peptides), can be used to activate T-cell specific immunity and for the preparation of in vitro specific T-cell drugs.

[0021] Compared with existing technologies, the advantages of the cDC1 cell preparation method of the present invention are: ① The starting material is PBMC, eliminating the need for magnetic bead separation and simplifying the operation; ② The obtained cDC1 cell (XCR1+ / CD11b-) ratio is higher (greater than 70%); ③ The obtained cDC1 cell quantity is greater (approximately 2 × 10⁻⁶). 6 cDC1 / 6×10 6 PBMC). Attached Figure Description

[0022] Figure 1 Bright-field graphs showing cell morphology at various culture times under different culture conditions;

[0023] Figure 2 Bright-field graphs showing cell morphology at various time points after suspension cells are transferred to low-adsorption dishes for further culture.

[0024] Figure 3A Figure showing the results of flow cytometry detection of XCR1 marker expression on the surface of cDC1 cells;

[0025] Figure 3B Figure showing the results of flow cytometry detection of CD11b marker expression on the surface of cDC1 cells;

[0026] Figure 4A Figure showing the results of flow cytometry detection of XCR1 marker expression on the surface of cDC1 cells in a low-adsorption dish;

[0027] Figure 4B Figure showing the results of flow cytometry detection of CD11b marker expression on the surface of cDC1 cells in a low-adsorption dish;

[0028] Figure 5 This is a graph showing the final number of cDC1 cells obtained under various culture conditions. Detailed Implementation

[0029] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0030] The specific experimental methods used in the following embodiments of the present invention are described below:

[0031] 1) Obtaining PBMCs from peripheral blood

[0032] Fresh blood collected from heparin sodium anticoagulant tubes was used for PBMC separation. After gently inverting the blood collection tube several times, the mixed whole blood was added 1:1 to a centrifuge tube containing Ficoll lymphocyte separation medium and centrifuged at 2,500 rpm for 15 minutes. After centrifugation, the supernatant yellow plasma was transferred to a new centrifuge tube and inactivated at 56°C for 30 minutes. The white membrane layer was gently aspirated into a 15 ml centrifuge tube, PBS buffer was added, and the mixture was centrifuged at 2,500 rpm for 10 minutes. After centrifugation and discarding the liquid, the cell pellet at the bottom of the tube was aspirated using erythrocyte lysis buffer and lysed for 10 minutes. After lysis, the lysed cells were centrifuged at 2,500 rpm for 5 minutes. The cell pellet was then resuspended in PBS buffer and counted. Finally, the suspension was centrifuged at 2,500 rpm for 5 minutes, the supernatant was discarded, and the PBMCs were obtained.

[0033] 2) Flow cytometry detection method

[0034] To identify the proportion of cDC1 in differentiated suspension cells, flow cytometry was used to detect surface markers in this embodiment. The antibody used was Human XCR1 PE-conjugated Antibody (R&D Systems, FAB8571P-100), manufactured by BD Pharmaceuticals. TM PE Mouse Anti-Human CD11b (BD, 555388).

[0035] 3) Methods for measuring total cell count

[0036] In this embodiment, the cell count of each group is calculated as follows: the number of cells cultured in the low-adsorption culture dish for 2 days is added to the number of suspended cells taken from the original 60mm dish for each flow cytometry detection, and the two are combined to record the total number of cells harvested at the end.

[0037] Example 1: cDC1 cells were obtained by direct differentiation of PBMCs.

[0038] Methods: First, the isolated PBMCs were uniformly 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 -ACFDendritic Cell Medium (STEMCELL, #10986), all containing 5% autologous plasma, penicillin-streptomycin 1 w / v, cell plate count 6 × 10⁻⁶. 6 / 60mm culture dish. After completion, incubate in a 37℃±0.1℃ 5% CO2 cell culture incubator for 2 hours, then change the medium once, and observe and photograph regularly.

[0039] The day of cell plating is recorded as Day 0. As the differentiation culture time increases, the cell morphology changes from an adherent state in the early stages to an increased aggregation, and then gradually floats. Suspended cells begin to appear on Day 6; at this time, the suspended cells vary in size and shape, with small cells being the most numerous. On Day 10, the number of suspended cells gradually increases, and their morphology becomes more uniform, with most being large oval. On Day 13, the cell morphology is almost identical to that of Day 10. Figure 1 As shown.

[0040] On Day 6 of differentiation culture, the upper layer of suspension cells was transferred to a 60mm low-adsorption culture dish for continued culture. The second day of culture was designated as Day 1 of suspension culture. Figure 2 As shown, after culturing in the low-adsorption dish, the number of small cells gradually decreased, with more cells observed on Day #2. Subsequently, a small number of cell clusters appeared and gradually decreased in number. The FGS group did not exhibit this characteristic in the low-adsorption dish due to its slower cell resuscitation.

[0041] Therefore, from a morphological perspective, there was no significant difference between the FGST and FGS3T groups, and both groups showed DC cell differentiation after one week of culture. However, the FGS3T group appeared to have the highest number of cells at Day 10. The FGS group grew the slowest, with cells only beginning to suspend at Day 10. Suspension cells maintained their optimal state for 2-3 days in low-absorption dishes.

[0042] Example 2: Flow cytometry detection of cDC1 cell surface markers

[0043] Figure 3A The image shows the results of flow cytometry detection of XCR1 marker expression on the surface of cDC1 cells, as shown below. Figure 3A As shown in the figure, the proportion of XCR1+ was highest on Day 10, and the proportions in the three groups were almost the same, all reaching over 70%. On Day 17, the proportion in the FGST group decreased from 74.42% to 42.09%, and the proportion in 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 a stage of high XCR1 expression (66.29%).

[0044] Figure 3B The image shows the results of flow cytometry analysis of CD11b marker expression on the surface of cDC1 cells. Figure 3B As shown, CD11b expression remained consistently high in the FGS group, exceeding 40%, while the other two groups exhibited very low expression, particularly the FGS3T group, which showed 1.65% on Day 6 and 1.21% on Day 10, significantly lower than the FGS group. Since CD11b is a major surface marker of moDC, from the perspective of surface marker expression, the FGST and FGS3T groups are significantly superior to the FGS group.

[0045] Cells differentiated on Day 6 and transferred to low-adsorption dishes showed increased XCR1 levels after 4 days of culture. + The proportions increased compared to Day 6, with the FGST group increasing from 37.62% to 64.02% and the FGS3T group from 39.52% to 53.99%, while CD11b expression remained at a low level throughout (see [link to study]). Figure 4A and Figure 4B This indicates that low-adsorption dishes are beneficial for cell differentiation towards cDC1. However, the culture time of cells in low-adsorption dishes should not be too long, otherwise not only will the cell number decrease significantly, but the expression of XCR1 will also be significantly reduced. Figure 4A The flow cytometry results on Day 11 showed that 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%. This means that the suspension cells should not be cultured in low-adsorption dishes for more than 4 days.

[0046] This shows that during Day 10 of differentiation culture, XCR1 + The proportion was highest at Day 17, then dropped significantly, indicating that a two-week culture period is recommended. There was no significant difference between the FGST and FGS3T groups. Although the FGS group had XCR1... + The proportion is also relatively high, but its CD11b + The FGS group is also relatively high, which means that most of the FGS group are moDCs. Therefore, FGS group cytokines cannot be used for the culture of cDC1 cells.

[0047] Example 3: Number of cDC1 cells

[0048] The final cell count harvested from the FGS group was 1.03 × 10⁻⁶. 6 The number of FGST groups was 1.41 × 10⁻⁶. 6 The number of FGS3T groups was 1.81 × 10⁻⁶. 6 Each original PBMC has 6 × 10⁶ units. 6 Therefore, the final cell yields were 17.12% for the FGS group, 23.5% for the FGST group, and 30.17% for the FGS3T group.

[0049] It can be seen that the number of cDC1 obtained by the cytokine combination of the FGS3T group in this embodiment is significantly higher than that of the other two groups, and the cytokine combination of the FGST group is superior to that of the FGS group.

Claims

1. A method for in vitro preparation of cDC1 cells, characterized in that, The isolated peripheral blood mononuclear cells were evenly plated into any of the following culture conditions: 1) FGST group: 100±10 ng / ml FLT-3L, 10±1 ng / ml GM-CSF, 20±2 ng / ml SCF and 10±1 ng / ml TGF-β, culture medium was ImunoCult™-ACF Dendritic Cell Medium containing 5 v / v% plasma and 1 w / v% penicillin and streptomycin. 2) FGS3T group: 100±10 ng / ml FLT-3L, 10±1 ng / ml GM-CSF, 20±2 ng / ml SCF, 10±1 ng / ml TGF-β and 5 ng / ml IL-3, culture medium was ImunoCult™-ACF Dendritic Cell Medium containing 5 v / v% plasma and 1 w / v% penicillin and streptomycin. After 6 days of differentiation culture in a culture dish, the upper layer of suspended cells was transferred to a low-adsorption culture dish for further culture. After 4 days of culture in the low-adsorption culture dish, cDC1 cells were obtained.

2. The method for in vitro preparation of cDC1 cells according to claim 1, characterized in that... The concentration of peripheral blood mononuclear cells inoculated was 1~1.5×10⁻⁶. 6 Cells / ml

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