Method for culturing dendritic cells by first expanding and then differentiating peripheral blood, and use

Abstract

A method for culturing dendritic cells by first expanding and then differentiating peripheral blood. A peripheral blood mononuclear cell (PBMC) which is obtained by separation and a cytokine composition (composed of FLT-3L, TPO, SCF, IL3, IL6 and SR1) are expanded in the early stage in vitro, and then subjected to differentiation (FLT-3L, GM-CSF, IL3 and TGF-β) culture to obtain cDC1s. It is verified that according to the method of the present invention for preparing cDC1s in vitro, XCR1+cDC1s can be obtained from initial 2.5×106 PBMCs, and there are at least 0.8×106 (32％) XCR1+cDC1s, so that the requirements for cell quantity in clinical application can be met.

Description

Method and application of expanding peripheral blood and then differentiating and culturing dendritic cells 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, which first expands peripheral blood and then differentiates and cultures dendritic cells, thereby increasing the number of cDC1 cells that can be obtained 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, selecting a combination of multiple cytokines, first expanding peripheral blood and then differentiating and culturing dendritic cells, thereby improving 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 first cytokine composition in vitro to obtain their precursor cells, and then co-culturing with a second cytokine composition to differentiate and obtain cDC1 cells, namely, the HSC-2D culture method.

[0012] The first cytokine composition used in the method for preparing cDC1 cells in vitro of the present invention is used in the precursor cell expansion stage and consists of FLT-3L, TPO, SCF, IL3, IL6 and SR1.

[0013] The second cytokine composition used in the in vitro method for preparing cDC1 cells of the present invention is used in the differentiation stage of precursor cells into cDC1 cells, and consists of FLT-3L, GM-CSF and TGF-β.

[0014] An embodiment of the first cytokine composition for the method of preparing cDC1 cells in vitro according to the present invention, StemSpan TM -XF medium was supplemented with FLT-3L 50±5 ng / ml, TPO 20±2 ng / ml, SCF 20±2 ng / ml, IL3 10±1 ng / ml, IL6 10±1 ng / ml, and SR1 1.0±0.1 nmol / ml for early cell expansion.

[0015] An embodiment of the second cytokine composition for the method of preparing cDC1 cells in vitro of the present invention, a dendritic cell culture medium (ImunoCult TM Cell differentiation was performed by adding cytokines such as FLT-3L, GM-CSF, and TGF-β to a medium containing 100 μg of FLT-3L, 2.5 μg of GM-CSF, and 10 μg of TGF-β. The amount of TGF-β used was determined experimentally by the present invention; a lower concentration would not be conducive to the differentiation and suspension of cDC1 cells.

[0016] Another embodiment of the second cytokine composition used in the method for preparing cDC1 cells in vitro of the present invention further comprises adding IL-3, and the concentration in the dendritic cell culture medium is 5.0±0.5 ng / ml.

[0017] The in vitro method for preparing cDC1 cells of the present invention also includes the addition of plasma, such as plasma from the same individual from which PBMCs were obtained, in an amount of, for example, 5 v / v%. We tested cell culture without plasma, with platelet lysate replacing plasma, and with 5 v / v% autologous plasma. The results showed that almost no cells survived in the serum-free state, while cells could be expanded and differentiated in the platelet lysate condition, but the number and XCR1 + The proportion was significantly lower than that in the autologous plasma group.

[0018] In the method for preparing cDC1 cells in vitro of the present invention, taking a 6-well plate as an example, the PBMC inoculation amount is based on the cell density, such as: 1-1.5×10 6 cells / ml is appropriate.

[0019] The in vitro method for preparing cDC1 cells of the present invention comprises co-culturing isolated peripheral blood mononuclear cells with an HSC-2D expansion-related cytokine composition in vitro (e.g., culturing in a 37°C ± 0.1°C 5% CO2 cell culture incubator) for 12 days, replacing the culture medium and differentiation-related cytokines, and continuing the culture for another 9 days to obtain cDC1 cells.

[0020] It has been verified that the method of preparing cDC1 cells in vitro of the present invention is to start with 2.5×10 6 The minimum number of cDC1 cells that can be obtained from PBMC is: 0.8×10 6 (32%), and showed XCR1 + / CD11b - Characteristics, can meet the demand for cell quantity in clinical applications.

[0021] 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.

[0022] 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.

[0023] 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).

[0024] Compared with the prior art, the advantages of the cDC1 cell preparation method of the present invention are:

[0025] ① The starting material is PBMC, which does not require magnetic bead separation and is easier to operate;

[0026] ② Obtained cDC1 cells (XCR1 + / CD11b - ) has a higher proportion (greater than 70%);

[0027] ③ Obtained XCR1 + The number of cDC1 cells is larger (about 0.8×10 6 cDC1 / 2.5×10 6 PBMC). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a bright field image of cell morphology at different culture times under various cytokine conditions;

[0029] FIG2 is a graph showing the expression results of cell surface markers CD34, CD141 and CD14 in the HSC-2D expansion stage under the conditions of various cytokines detected by flow cytometry;

[0030] FIG3 is a schematic diagram of the expansion multiples of various cells cultured on Day 12 under various cytokine conditions;

[0031] FIG4 is a graph showing the expression results of cell surface markers CD34, CD141 and XCR1 in the HSC-2D expansion stage under various factors and cell plating density conditions detected by flow cytometry;

[0032] FIG5 is a graph showing the expression of cell surface markers CD34 and XCR1 detected by flow cytometry under conditions of various factors and cell plating densities after HSC-2D expansion and differentiation for 9 days;

[0033] FIG6 is a schematic diagram showing the ratio and number of XCR1+cDC1 cells finally obtained under conditions of various factors and cell plating densities;

[0034] FIG7A is a graph showing the results of flow cytometry detection of the expression of cDC1 cell surface markers CD141 and XCR1 in a randomly cultured female subject;

[0035] FIG7B is a graph showing the results of flow cytometry detection of the expression of cDC1 cell surface markers CD141 and XCR1 in another randomly cultured male subject;

[0036] FIG8 is a graph showing the results of flow cytometry detection of CD86 and CD83 expression in cDC1 cells after maturation stimulation;

[0037] FIG9 is a graph showing the expression results of specific antigen tetramers detected by flow cytometry after cDC1-T cell co-culture. DETAILED DESCRIPTION

[0038] 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.

[0039] The various test methods used in the following examples of the present invention are specifically described as follows:

[0040] 1) Isolation of peripheral blood to obtain PBMC

[0041] 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.

[0042] 2) Flow cytometry detection method

[0043] To identify the cell components during the culture process and the proportion of cDC1 in the ultimately expanded and differentiated suspension cells, this example employed flow cytometry to detect surface markers. The antibodies used were Human XCR1 PE-conjugated Antibody (R&D Systems, FAB8571P-100), APC Mouse Anti-Human CD34 (BD, 555824), PE Mouse Anti-Human CD141 (BD, 559781), APC Mouse Anti-Human CD83 (BD, 551073), PerCP-Cy5.5 Mouse Anti-Human CD86 (BD, 561129), Tritest CD3 / 4 / 8 (BD, 340298), and HLA-A*02:01 GPC3 Tetramer-FVGEFFTDV-APC (MBL, TB-0134-2).

[0044] 3) Method for measuring total cell count

[0045] In this example, the number of cells in each group was calculated by counting all the suspended cells in a 6-well plate after HSC-2D expansion for 12 days, or counting all the suspended cells in a 6-well plate after HSC-2D expansion for 12 days and then differentiation for 9 days.

[0046] Example 1 PBMCs were expanded and then differentiated into cDC1 by HSC-2D and cytokine selection.

[0047] Based on early experiments exploring cDC1 cell culture, several cytokines were initially identified. To further explore cDC1 cell culture conditions, this example first conducted four sets of culture tests with different cytokine concentrations. The isolated PBMCs were evenly plated in the following culture conditions:

[0048] ①.FST36-H group: FLT-3L (100ng / ml), SCF (100ng / ml), TPO (50ng / ml), IL3 (20ng / ml), IL6 (20 ng / ml);

[0049] ②.FST36-L group: FLT-3L (50ng / ml), SCF (20ng / ml), TPO (20ng / ml), IL3 (10ng / ml), IL6 (10ng / ml);

[0050] ③.FST36-H+SR1 group: FLT-3L (100ng / ml), SCF (100ng / ml), TPO (50ng / ml), IL3 (20ng / ml), IL6 (20ng / ml), SR1 (1nmol / ml);

[0051] ④.FST36-H+SR1+VEGF group: FLT-3L (100ng / ml), SCF (100ng / ml), TPO (50ng / ml), IL3 (20ng / ml), IL6 (20ng / ml), SR1 (1nmol / ml), VEGF (2ng / ml).

[0052] Since DC cells are mainly derived from the differentiation of HSC and its precursor cells, in order to obtain a sufficient number of DC precursor cells, the initial culture is the HSC-2D expansion stage. The culture medium used in this stage is StemSpan TM -XF (STEMCELL, #100-0073), containing 5v / v% autologous plasma and 1w / v% penicillin and streptomycin. The cell plating density was 1×10 6 Each condition group was plated in duplicate wells of a 6-well plate with 2 ml of culture medium per well, i.e., the number of cells plated per well was 2 × 10 6 After completion, the cells were placed in a 37°C ± 0.1°C 5% CO2 cell culture incubator and cultured for 2 hours, followed by a change of medium. Thereafter, the medium was changed or replenished every 2 to 3 days according to the cell growth status, and observation and photography were performed regularly.

[0053] The day cells were plated was designated Day 0. As culture time increased, the cells gradually increased in size and aggregated, shifting from an initially adherent state. Cell clusters began to form on Day 6, with the suspended cells primarily presenting small cells. These small cells gradually decreased in number as culture progressed. After Day 9, cell clusters became prominent, which then became suspended and dispersed into individual cells, as shown in Figure 1. As shown in Figure 1, the group supplemented with VEGF showed the slowest cell growth, with no significant differences observed in the other three groups.

[0054] After 12 days of expansion culture, suspended cells in the culture wells were collected and analyzed by flow cytometry for CD34 (HSC surface marker), CD141 (DC surface marker), and CD14 (monocyte surface marker). Comparing the FTS36-H and FTS36-L groups, we can see that the expression of CD141 in the FTS36-L group (43.08%) was significantly higher than that in the FTS36-H group (25.21%). Comparing the FTS36-H and FTS36-H+SR1 groups, the expression of CD141 in the FTS36-H+SR1 group (43.68%) was higher than that in the FTS36-H group (25.21%) (see Figure 2). Although CD141 expression was also high in the FTS36-H+SR1+VEGF group, its CD34 expression was slightly lower than that in the other groups, and its cell growth was slower, thus not having a significant advantage. CD34 expression in each group was significantly elevated compared to PBMC, with expansion folds exceeding 40-fold (see Figure 3), demonstrating that this factor combination is effective in expanding HSCs. Furthermore, the expansion folds in the FTS36-L and FTS36-H+SR1 groups were higher than in the other two groups. CD14 expression was almost absent, indicating that these culture conditions were not conducive to the proliferation of monocytes.

[0055] In summary, the appropriate cytokine condition should be FTS36-L+SR1.

[0056] Example 2 PBMCs were expanded and then differentiated via HSC-2D to obtain cDC1 and the cell plating density was selected.

[0057] The isolated PBMCs were plated at different densities in the following culture conditions:

[0058] ①.FST36S-H group: FLT-3L (100ng / ml), SCF (100ng / ml), TPO (50ng / ml), IL3 (20ng / ml), IL6 (20ng / ml), SR1 (1nmol / ml);

[0059] ②.FST36S-L group: FLT-3L (50ng / ml), SCF (20ng / ml), TPO (20ng / ml), IL3 (10ng / ml), IL6 (10ng / ml), SR1 (1nmol / ml).

[0060] The plating density was 0.5×10 6 / ml, 1.25×10 6 / ml, 2.5×10 6 / ml, each condition group was plated in duplicate wells of a 6-well plate, with 2ml of culture medium per well. The initial culture was the HSC-2D expansion stage. The culture medium used in this stage was StemSpan TM -XF (STEMCELL, #100-0073), after 12 days of expansion, the culture medium was changed to ImunoCult TM Differentiation culture was continued for 9 days using ACF Dendritic Cell Medium (STEMCELL, #10986). The following cytokines were used for differentiation: FLT-3L (50 ng / ml), GM-CSF (2.5 ng / ml), IL-3 (5 ng / ml), and TGF-β (10 ng / ml). Both media contained 5% autologous plasma and 1% penicillin / streptomycin. After plating, cells were incubated in a 37°C ± 0.1°C, 5% CO2 incubator for 2 hours, followed by medium exchange. Thereafter, the medium was exchanged or supplemented every 2-3 days depending on cell growth. Cells were observed and photographed regularly.

[0061] After 12 days of expansion culture, the suspended cells in the culture wells were collected for flow cytometry analysis of CD34, CD141, and XCR1. Since CD141 is expressed on various types of DC cells, while XCR1 is only expressed on cDC1 cells, XCR1 was subsequently selected to reflect the characteristics of cDC1 cells. At this time, 0.5×10 6 / ml plating density group had slower cell growth and no cell suspension, so flow cytometry could not be performed. 6 In the two groups with different cell density, the FST36S-L group had a higher CD34 expression ratio (19.75%), while the FST36S-H group had a 10.16% expression ratio. There was no difference in the XCR1 ratio between the two groups. 6 In the two groups with different cell density, the FST36S-L group had a higher CD141 expression ratio (32.98%), while the FST36S-H group had a CD141 expression ratio of 11.57%. There was almost no difference in the XCR1 ratio between the two groups (see Figure 4).

[0062] After 12 days of expansion culture, the differentiation medium was replaced and the cells were harvested after 9 days of differentiation culture. The suspended cells in the culture wells were taken for flow cytometry analysis of CD34 and XCR1. There was no significant difference in the expression of CD34 among the groups. The expression of XCR1 was not different except for 0.5×10 6 / ml FST36S-H group (71.82%), the other groups can reach around 80% (see Figure 5). Analyzing and comparing the number of XCR1+cDC1 cells finally obtained (see Figure 6), we can see that 2.5×10 6 / ml and 1.25×10 6 / ml plating density, the number of cells obtained was only slightly higher and did not double; while 0.5×10 6 / ml and 1.25×10 6 / ml plating density, the number of cells in the FST36S-H group was too small at low density, and the number of cells in the FST36S-L group was 0.5×10 6 / ml and 1.25×10 6 / ml plating density, the final harvested cell number ratio is nearly 2 times, so it is estimated that 1.25×10 6 The optimal plating density was 100 μg / ml. Furthermore, although the number of cells in the FST36S-H group was slightly higher than that in the FST36S-L group, the difference was small and, considering the overall cost, FST36S-L was the optimal condition for the expansion and culture of XCR1+cDC1 cells.

[0063] In summary, the optimal conditions for differentiation of PBMC after HSC-2D expansion are as follows:

[0064] ①. FST36S-L was used in the HSC-2D expansion stage, namely: FLT-3L (50ng / ml), SCF (20ng / ml), TPO (20ng / ml), IL3 (10ng / ml), IL6 (10ng / ml), SR1 (1nmol / ml), StemSpan TM -XF medium;

[0065] ②FG3T in the differentiation stage: FLT-3L (50 ng / ml), GM-CSF (2.5 ng / ml), IL3 (5 ng / ml), TGF-β (10 ng / ml), ImunoCult TM -ACF Dendritic Cell Medium. Cell plating density: 1.25×10 6 / ml is appropriate.

[0066] Example 3 Stability of the method for obtaining cDC1 by differentiating PBMC after expansion via HSC-2D.

[0067] To verify the repeatability of the above culture method, two volunteers (one male and one female) were randomly selected and XCR1 + cDC1 cell culture test: FST36S-L was used for differentiation for 12 days and then FG3T for 9 days, for a total of 21 days, and the cell plating density was approximately 1.25×10 6 / ml. The flow cytometry results of the final harvested cells are shown in Figures 7A and 7B. Figure 7A shows a female subject whose XCR1 + The proportion was 76.16%; Figure 7B shows a male subject whose XCR1 + The ratio is 71.95%, which shows that this method is stable and repeatable and has universal significance.

[0068] Example 4 PBMCs were expanded by HSC-2D and then differentiated into cDC1 to obtain antigen presenting function.

[0069] Methods: To verify whether the in vitro cultured XCR1+ cDC1 cells have antigen presentation function, cDC1 cells were subjected to maturation stimulation and antigen loading test. Resuscitated cDC1 cells were plated in the following conditions: GM-CSF 20ng / ml, CD40L 1μg / ml, R848 10μg / ml, Poly I:C 20ug / ml, INF-γ 10ng / ml, IL-1β 10ng / ml, TNF-α 5ng / ml (the above factors have been experimentally tested and can be removed IL-1β and TNF-α, which have little effect on DC cell maturation stimulation). The culture medium was still ImunoCult®. TM -ACF Dendritic Cell Medium, culture overnight (16 to 24 hours, no more than 48 hours) in a 37°C ± 0.1°C 5% CO2 cell culture incubator. The next day, add 20ug / ml GPC3 antigen peptide (FVGEFFTDV) and continue culturing for 2 hours. Since cDC1 cells will become adherent if plated on a regular culture dish after cryopreservation and resuscitation, this step is to plate the cells on a low-attachment culture dish or, after HSC-2D expansion and differentiation culture, perform maturation stimulation directly in the original culture dish without freezing.

[0070] After maturation stimulation and antigen loading, cDC1 cells should express surface markers such as CD83 and CD86. CD83 is a marker of DC maturation, while CD86, as a co-stimulatory factor, demonstrates that DCs can activate T cells. Flow cytometry analysis of CD83 and CD86 expression on cDC1 cell surfaces revealed that after maturation stimulation, CD83 expression in our cultured cDC1 cells increased from 9.73% in immature dendritic cells (iDCs) to 50.48% in mature dendritic cells (mDCs). CD83 expression in PBMCs was only 0.31%, indicating that cDC1 cells had reached maturity. Expression of the co-stimulatory factor CD86 reached over 40% in both iDC and mDC, a significant increase from 7.95% in PBMCs, indicating that cDC1 cells possess T cell activation function (see Figure 8).

[0071] T cells were cultured as follows: one day prior to co-culture, PBMCs were plated onto OKM25 (FUKUKO, Japan)-coated culture flasks and supplemented with OKM100 medium (FUKUKO, Japan), 5% autologous plasma, and 1% penicillin / streptomycin. DC-T co-cultures were divided into two groups: 1. T cell control group, consisting of the same number of T cells without DCs; and 2. DC-T co-culture group: mature and antigen-loaded cDC1 cells were mixed with T cells from the same subject at a DC:T ratio of 1:5, with a range of 1:5 to 1:20, and a typical ratio of 1:10 was recommended. After plating, cells were cultured in a 37°C ± 0.1°C, 5% CO2 incubator for three days before flow cytometry analysis.

[0072] If antigen-specific T cells are produced, they will express the corresponding antigen tetramer (Tetramer), so flow cytometry was used to analyze the expression of GPC3 Tetramer in CD4+T and CD8+T cells (see Figure 9). Compared with the control T cells, a small group of GPC3 Tetramer-positive cells were produced in both CD4+T and CD8+T cells, with a CD4+ / GPC3 Tetramer+ ratio of 3.76% and a CD8+ / GPC3 Tetramer+ ratio of 4.06%. This indicates that the cDC1 cells cultured by this method can present antigens and activate the generation of antigen-specific T cells.

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 co-cultivation comprises: The separated peripheral blood mononuclear cells are co-cultured with the first cytokine composition in vitro to obtain the precursor cells thereof, and The precursor cells are then co-cultured with a second cytokine composition to differentiate into cDC1 cells; The first cytokine composition consists of FLT-3L, TPO, SCF, IL3, IL6 and SR1; The second cytokine composition consists of FLT-3L, GM-CSF, IL3 and TGF-β.

2. The method for preparing cDC1 cells in vitro according to claim 1, characterized in that The first cytokine composition is added to StemSpan TM -XF medium.

3. The method for preparing cDC1 cells in vitro according to claim 1, characterized in that The second cytokine composition is added to ImmunoCult TM -ACF Dendritic Cell Medium.

4. The method for preparing cDC1 cells in vitro according to claim 1, characterized in that The first cytokine composition has a cytokine concentration of FLT-3L 50±5ng / ml, TPO 20±2ng / ml, SCF 20±2ng / ml, IL3 10±1ng / ml, IL6 10±1ng / ml and SR1 1.0±0.1nmol / ml.

5. The method for preparing cDC1 cells in vitro according to claim 1, characterized in that The second cytokine composition has the following cytokine concentrations: FLT-3L 50±5 ng / ml, GM-CSF 2.5±0.2 ng / ml, IL-3 5.0±0.5 ng / ml, and TGF-β 10±1 ng / ml.

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

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

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

9. The method for preparing cDC1 cells in vitro according to claim 1, characterized in that The isolated peripheral blood mononuclear cells were co-cultured with a cytokine composition in vitro, firstly subjected to 12 days of cell expansion culture, and then subjected to 9 days of differentiation culture to obtain cDC1 cells.

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

11. 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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