A method for culturing dendritic cells by proliferating and then differentiating them from peripheral blood, and its use.

Abstract

A method for culturing dendritic cells by first proliferating them from peripheral blood and then differentiating them. Peripheral blood mononuclear cells obtained by isolation are proliferated in vitro with a cytokine composition (consisting of FLT-3L, TPO, SCF, IL3, IL6, and SR1) in the initial stage, and then differentiated (using a cytokine composition consisting of FLT-3L, GM-CSF, IL3, and TGF-β) to obtain cDC1 cells. Verification has shown that the method for preparing cDC1 cells in vitro according to the present invention is effective for the first 2.5 × 10 6 From PBMC to XCR1 + cDC1 cells can be obtained, and their number is at least 0.8 × 10⁶ 6 This represents 32% of PBMCs, which can meet the cell count needs required for clinical application.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing biomaterials, and more particularly to a method for culturing conventional dendritic cell 1 (cDC1) in peripheral blood mononuclear cells (PBMCs). [Background technology]

[0002] Dendritic cells (DCs), as the most functional specialized antigen-presenting cells (APCs) in the body, play a crucial role in inducing both innate and adaptive immune responses. DCs are central to initiating, regulating, and maintaining immune responses, governing various functions of the body's immune system. In vivo, immature DCs possess strong migratory capabilities, while mature DCs can effectively activate early T cells. The function of DCs is to efficiently ingest, process, and present antigens within the body, providing a recognition basis for helper T cells (Th) and B cells to initiate specific immune responses.

[0003] DCs (dendritic cells) are mononuclear phagocytes (MPs) and are classified into conventional DCs (cDCs), plasmacytoid DCs (pDCs), and Langerhans cells (LCs) based on their origin and differentiation pathways. cDCs are further subdivided into type I conventional dendritic cells (cDC1) and type II conventional dendritic cells (cDC2). The development and differentiation of cDC1 are dependent on the transcription factors BATF3 and IRF8, and their surface markers mainly include XCR1, CD141, CLEC9A, and CADM1. cDC1 transports complete antigens to lymph nodes and tumor-specific CD8 +It has been proven that T cells are the only antigen-presenting cells capable of stimulating T cells, and their migration and immune-activating functions play a crucial role in the anti-tumor immune response.

[0004] In peripheral blood, mature DCs account for only about 0.2% of PBMCs, and of those, cDC1 accounts for about 2.6%. Due to its extremely low content, it is difficult to obtain directly through isolation. This significantly hinders the direct application of cDC1 to cell therapy. Therefore, obtaining DCs using in vitro cell differentiation and proliferation techniques is a major challenge in clinical application. In current clinical research, DC cells commonly used are monocyte-derived DCs (moDCs, CD11b) obtained by the proliferation and differentiation of peripheral blood mononuclear cells or hematopoietic stem cells (HSCs). + (expressed as) Clinical studies have revealed that moDCs have many drawbacks. MoDCs cannot effectively migrate to tumor drainage lymph nodes and cannot directly present antigens to T cells in the body. Rather, as a source of antigens, CD8 + T and CD4 + The antigen needs to be transferred and processed by cDC1 cells in the body, which are responsible for directly presenting the antigen to T cells. As a result, there has been a problem in clinical practice where therapeutic effects cannot be obtained.

[0005] Therefore, providing a technology to obtain cDC1 cells in vitro will greatly promote the research, development, and application of DC cell tumor vaccines. [Overview of the Initiative]

[0006] One objective of the present invention is to provide a method for preparing cDC1 cells in vitro to meet the needs of clinical applications by increasing the number of cDC1 cells obtained by culturing dendritic cells by proliferating and then differentiating them from peripheral blood.

[0007] Another object of the present invention is to provide a method for preparing cDC1 cells in vitro by selecting a combination of multiple types of cytokines, culturing dendritic cells by proliferating and then differentiating them from peripheral blood, and improving the efficiency of obtaining cDC1 cells.

[0008] Another object of the present invention is to provide a method for preparing cDC1 cells in vitro in order to obtain cDC1 cells more simply.

[0009] A further object of the present invention is to provide a method for producing cDC1 cells in vitro that can be applied to the preparation of dendritic cell vaccines, particularly the preparation of in vitro specific T cell agents.

[0010] The method for preparing cDC1 cells in vitro is a method for obtaining cDC1 cells, that is, the HSC-2D culture method, in which peripheral blood mononuclear cells obtained by separation are co-cultured with a first cytokine composition in vitro to obtain its progenitor cells, and then co-cultured with a second cytokine composition to differentiate them into cDC1 cells.

[0011] The first cytokine composition used in the method for preparing cDC1 cells in vitro according to the present invention is used in the progenitor cell proliferation stage and is composed of FLT-3L, TPO, SCF, IL3, IL6 and SR1.

[0012] The second cytokine composition used in the method for preparing cDC1 cells in vitro according to the present invention is used in the stage of differentiating progenitor cells into cDC1 cells and is composed of FLT-3L, GM-CSF and TGF-β.

[0013] In an embodiment of the first cytokine composition used in the method for preparing cDC1 cells in vitro according to the present invention, StemSpan TMProgenitor cells are proliferated by adding 50±5 ng / ml of FLT-3L, 20±2 ng / ml of TPO, 20±2 ng / ml of SCF, 10±1 ng / ml of IL3, 10±1 ng / ml of IL6, and 1.0±0.1 nmol / ml of SR1 to XF medium.

[0014] In an embodiment of the second cytokine composition used in the method for preparing cDC1 cells in vitro according to the present invention, dendritic cell medium (ImunoCult TM -ACF Dendritic Cell Medium is used to induce differentiation into cDC1 cells by adding cytokines such as FLT-3L, GM-CSF, and TGF-β so that the concentrations of each cytokine are 50±5 ng / ml for FLT-3L, 2.5±0.2 ng / ml for GM-CSF, and 10±1 ng / ml for TGF-β. The amount of TGF-β used is the result of investigations conducted in this invention, and if the concentration is too low, it is detrimental to the differentiation and suspension of cDC1 cells.

[0015] In another embodiment of the second cytokine composition used in the method for preparing cDC1 cells in vitro according to the present invention, IL-3 is further added, and the concentration in dendritic cell medium is 5.0 ± 0.5 ng / ml.

[0016] The present invention provides a method for preparing cDC1 cells in vitro, further comprising the addition of plasma, for example, from the same individual from which the PBMCs are obtained, with a usage amount of, for example, 5 v / v%. The inventors tested culturing cells without plasma, with platelet lysis instead of plasma, and with 5 v / v% autologous plasma. The results showed that in the serum-free state, most cells did not survive, while under the condition of added platelet lysis, the cells could proliferate and differentiate, but their number and XCR1 + The proportion of cells was significantly lower in the autologous plasma group compared to the autologous plasma group.

[0017] The present invention provides a method for preparing cDC1 cells in vitro, using a 6-well plate as an example, where the PBMC inoculation amount is based on the cell density, for example, 1 to 1.5 × 10⁶. 6 Cells / ml is preferred.

[0018] The method for preparing cDC1 cells of the present invention in vitro is to co-culture the peripheral blood mononuclear cells obtained by separation with a cytokine composition related to HSC-2D proliferation in vitro for 12 days (for example, culture in a cell incubator at 37°C ± 0.1°C and 5% CO2), and then exchange the medium and differentiation-related cytokines, and continue the culture for another 9 days to obtain cDC1 cells in this way.

[0019] As a result of verification, in the method for preparing cDC1 cells of the present invention in vitro, the number of cDC1 cells obtained from the initial 2.5×10 6 PBMC is at least 0.8×10 6 cells (accounting for 32% of PBMC), and shows the characteristics of XCR1 + / CD11b - and it has been verified that it can meet the need for the number of cells in clinical applications.

[0020] In the method of the present invention, among the prepared cell population, XCR1 + cDC1 cells account for 70% - 85%. The remaining cells cannot activate the antigen presentation function, so it is more convenient and efficient without further purification.

[0021] The cDC1 prepared in the present invention is manufactured into a cellular vaccine (formulation) for tumor prevention and treatment through maturation stimulation (GM-CSF, CD40L, R848, Poly I:C, INF-γ, and antigenic peptides).

[0022] The cDC1 prepared in the present invention is used to activate the specific immunity of T cells through maturation stimulation (such as, but not limited to, GM-CSF, CD40L, R848, Poly I:C, INF-γ, and antigenic peptides), and is used for the production of in vitro specific T cell drugs.

[0023] Compared with the prior art, the advantages of the cDC1 cell preparation method of the present invention are as follows.

[0024] (1) The initial material is PBMC, which does not require separation by magnetic beads, making the operation easier. (2) Obtained cDC1 cells (XCR1 + / CD11b - The proportion of ) is higher (over 70%), (3) The obtained XCR1 + The number of cDC1 cells is higher (approximately 0.8 × 10⁻⁶). 6 cDC1 / 2.5×10 6 PBMC). [Brief explanation of the drawing]

[0025] [Figure 1] These are bright-field images showing cell morphology at various culture times under multiple cytokine conditions. [Figure 2] This figure shows the expression results of the cell surface markers CD34, CD141, and CD14 at different HSC-2D proliferation stages under multiple cytokine conditions, as detected by flow cytometry. [Figure 3] This is a schematic diagram showing the proliferation ratios of each cell on day 12 of culture under multiple cytokine conditions. [Figure 4] This figure shows the expression results of the cell surface markers CD34, CD141, and XCR1 at the HSC-2D proliferation stage under conditions of multiple factors and cell seeding density, as detected by flow cytometry. [Figure 5] This figure shows the expression results of the cell surface markers CD34 and XCR1 in HSC-2D cells differentiated for 9 days after proliferation under conditions of multiple factors and cell seeding density, as detected by flow cytometry. [Figure 6] This is a schematic diagram illustrating the final percentage and number of XCR1+cDC1 cells obtained under various factors and cell seeding density conditions. [Figure 7A] This figure shows the expression results of the cDC1 cell surface markers CD141 and XCR1 from randomly cultured female subjects, as detected by flow cytometry. [Figure 7B] This figure shows the expression results of the cDC1 cell surface markers CD141 and XCR1 from randomly cultured male subjects, as detected by flow cytometry. [Figure 8] This figure shows the expression results of CD86 and CD83 in cDC1 cells after maturation stimulation, as detected by flow cytometry. [Figure 9] This figure shows the expression results of specific antigen tetramers after co-culture of cDC1-T cells, as detected by flow cytometry. [Modes for carrying out the invention]

[0026] The technical solutions of the present invention will be described in detail below with reference to the figures. The embodiments of the present invention are merely for illustrating the technical solutions of the present invention and are not limiting. The present invention has been described in detail with reference to preferred embodiments, but it should be understood by those skilled in the art that the technical solutions of the present invention can be modified or replaced with equivalent substitutions without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be covered by the claims of the present invention.

[0027] A detailed description of each test method used in the following embodiments of the present invention is as follows.

[0028] 1) Obtaining PBMCs by peripheral blood separation PBMC separation was performed on blood collected immediately after being placed in a heparin sodium anticoagulant tube. The blood collection tube was gently inverted several times to ensure uniform mixing of the whole blood. This was then added in a 1:1 ratio to a centrifuge tube containing Ficoll lymphocyte separatory solution, and centrifuged at 2500 rpm for 15 minutes. After centrifugation, the yellow plasma portion at the top was transferred to a new centrifuge tube and inactivated at 56°C for 30 minutes before use. The white membrane layer between the plasma and Ficoll lymphocyte separatory solution was gently aspirated and placed in a 15 ml centrifuge tube. PBS buffer was added, the mixture was thoroughly mixed, and the tube was centrifuged at 2500 rpm for 10 minutes. After centrifugation, the supernatant was discarded, and the cell pellet at the bottom of the tube was resuspended in erythrocyte lysis buffer and erythrocyte lysis was performed for 10 minutes. Subsequently, the cells were centrifuged at 2500 rpm for 5 minutes. The supernatant was then discarded, the cell pellet was resuspended in PBS buffer, and the cells were counted. Finally, the suspension was centrifuged at 2500 rpm for 5 minutes, the supernatant was discarded, and PBMCs were obtained.

[0029] 2) Flow cytometry detection method To identify cellular components during the culture process and the proportion of cDC1 in the ultimately proliferated and differentiated suspension cells, this example used flow cytometry to detect cell 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).

[0030] 3) Method for measuring the total number of cells In this embodiment, the method for calculating the number of cells in each group involves counting all suspension cells in a 6-well plate grown with HSC-2D for 12 days, or counting all suspension cells in a 6-well plate grown with HSC-2D for 12 days followed by differentiation for 9 days.

[0031] Example 1: Selection of cytokines for differentiation of PBMCs into cDC1 after proliferation with HSC-2D. Based on previous experiments in the culture and investigation of cDC1 cells, several cytokines were preliminaryly identified. To further investigate the culture conditions for cDC1 cells, this example first performed culture tests with four groups of cytokines at different concentrations. PBMCs obtained by isolation were evenly seeded under the following culture conditions.

[0032] (1) FST36-H group: FLT-3L (100ng / ml), SCF (100ng / ml), TPO (50ng / ml), IL3 (20ng / ml), IL6 (20ng / ml), (2) FST36-L group: FLT-3L (50ng / ml), SCF (20ng / ml), TPO (20ng / ml), IL3 (10ng / ml), IL6 (10ng / ml), (3) FST36-H+SR1 group: FLT-3L (100ng / ml), SCF (100ng / ml), TPO (50ng / ml), IL3 (20ng / ml), IL6 (20ng / ml), SR1 (1nmol / ml), (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).

[0033] Since DC cells are mainly derived from the differentiation of HSCs and their progenitor cells, the initial culture was in the HSC-2D proliferation phase in order to obtain a sufficient number of DC progenitor cells. The culture medium used at that stage was StemSpan. TM-XF (STEMCELL, #100-0073) was used, supplemented with 5 v / v% autologous plasma and 1 w / v% penicillin-streptomycin. The cell seeding density was 1 × 10⁻⁶. 6 The ratio was / ml, and in each condition group, 2 ml of medium was used per well to seed two overlapping wells of a 6-well plate, meaning the number of cells seeded in each well was 2 × 10⁶. 6 After completion, the plates were placed in a cell incubator at 37°C ± 0.1°C and 5% CO2, cultured for 2 hours, then the culture medium was changed once. Subsequently, the culture medium was changed or replenished every 2-3 days according to the cell growth status, and observations and photographs were taken regularly.

[0034] The day the cells were seeded was designated as day 0. As the culture time progressed, the cell morphology gradually increased and clustered from an initial adherent state. From day 6 of culture, cell clusters gradually formed, and at this time, the suspension cells were mostly small, and their number gradually decreased as the culture progressed. As shown in Figure 1, after day 9, the cell clusters became clear, and thereafter, the cell clusters changed from an adherent state to a suspension state and dispersed into individual cells. As can be seen from Figure 1, the cell growth in the group to which VEGF was added was the slowest, and there was no significant difference among the other three groups.

[0035] After 12 days of growth culture, flow cytometry analysis was performed on suspension cells in the culture wells for CD34 (HSC surface marker), CD141 (DC surface marker), and CD14 (monocyte surface marker). Comparing the FTS36-H group with the FTS36-L group, CD141 expression in the FTS36-L group (43.08%) was significantly higher than in the FTS36-H group (25.21%). Comparing the FTS36-H group with the FTS36-H+SR1 group, CD141 expression in the FTS36-H+SR1 group (43.68%) was higher than in the FTS36-H group (25.21%) in all cases (see Figure 2). CD141 expression was also high in the FTS36-H+SR1+VEGF group, but CD34 expression was slightly lower than in the other groups, and cell growth was slower, so there was no significant advantage. CD34 expression in each group was significantly increased compared to PBMCs, and proliferation rates reached over 40-fold (see Figure 3). This indicates that this combination of factors is effective in promoting HSC proliferation, and the proliferation rates in the FTS36-L and FTS36-H+SR1 groups were higher than the other two groups. Since little amplification of CD14 was observed, it was suggested that the culture conditions were unfavorable for monocyte proliferation.

[0036] Based on the above, we determined that the appropriate cytokine condition is FTS36-L + SR1.

[0037] Example 2: Selection of cell seeding density for obtaining cDC1 by growing PBMCs with HSC-2D and then differentiating them. The PBMCs obtained by isolation were seeded at different densities under the following culture conditions.

[0038] (1) FST36S-H group: FLT-3L (100ng / ml), SCF (100ng / ml), TPO (50ng / ml), IL3 (20ng / ml), IL6 (20ng / ml), SR1 (1nmol / ml), (2) FST36S-L group: FLT-3L (50ng / ml), SCF (20ng / ml), TPO (20ng / ml), IL3 (10ng / ml), IL6 (10ng / ml), SR1 (1nmol / ml).

[0039] The seeding densities were 0.5 × 10⁻⁶ each. 6 / ml, 1.25 × 10 6 / ml, 2.5 × 10 6 The culture medium was / ml, and in each condition group, seeds were seeded in two replication wells of a 6-well plate, with 2 ml of medium per well. The initial culture was the HSC-2D growth stage. The medium used at this stage was StemSpan. TM -XF(STEMCELL,#100-0073), after 12 days of growth, the culture medium is changed to ImunoCult TM -The medium was changed to ACF Dendritic Cell Medium (STEMCELL, #10986), and differentiation culture was continued for 9 days. The cytokines used for differentiation were FLT-3L (50 ng / ml), GM-CSF (2.5 ng / ml), IL3 (5 ng / ml), and TGF-β (10 ng / ml). Both media were supplemented with 5% autologous plasma and 1 w / v% penicillin-streptomycin. After seeding was complete, the plates were placed in a cell incubator at 37°C ± 0.1°C and 5% CO2 for 2 hours, followed by one medium change. Thereafter, the medium was changed or replenished every 2-3 days according to the cell growth status, and observations and photographs were taken regularly.

[0040] After 12 days of growth culture, flow cytometry analysis was performed on suspension cells in the culture wells for CD34, CD141, and XCR1. CD141 is expressed in several types of DC cells, but XCR1 is expressed only in cDC1 cells; therefore, XCR1 was selected for characterization of cDC1 cells. At this time, 0.5 × 10⁶ cells were obtained. 6 Cell growth was slow in the seeding density group of / ml, and cells did not suspend, making flow cytometry measurements impossible. 1.25 × 10 6 Comparing the two groups with densities of 2.5 × 10⁻¹⁰ / ml, the FST36S-L group had a higher CD34 expression rate (19.75%), while the FST36S-H group had 10.16%, but there was no difference in the XCR1 proportion between the two groups. 6Comparing the two groups by density ( / ml), the FST36S-L group had a higher CD141 expression rate (32.98%), while the FST36S-H group had 11.57%, but there was almost no difference in the XCR1 proportions between the two groups (see Figure 4).

[0041] After 12 days of growth culture, the differentiation medium was changed, and differentiation culture was continued for another 9 days. Cells were then harvested, and flow cytometry analysis of CD34 and XCR1 in suspension cells in the culture wells was performed. There were no significant differences in CD34 expression among the groups, and XCR1 expression was 0.5 × 10⁶. 6 Aside from the FST36S-H group (71.82%) with a concentration of / ml, all other groups were able to reach approximately 80% (see Figure 5). The final XCR1 + When the number of cDC1 cells was analyzed and compared (see Figure 6), it was 2.5 × 10⁶. 6 At a seeding density of / ml, 1.25 × 10 6 Compared to / ml, the number of cells obtained did not double, but only increased slightly, at 0.5 × 10⁻⁶. 6 At a seeding density of / ml, 1.25 × 10 6 Compared to / ml, the cell count in the FST36S-H group was excessively low at lower densities, while the FST36S-L group had 0.5 × 10⁶ cells. 6 At a seeding density of / ml, 1.25 × 10 6 Compared to / ml, the proportion of cells ultimately recovered was nearly twice as high, therefore, 1.25 × 10 6 The seeding density of / ml was presumed to be the most appropriate density. Furthermore, although the cell count in the FST36S-H group was slightly higher than that of the FST36S-L group, the difference was small, and considering cost and other factors, FST36S-L was deemed the most appropriate for XCR1 + These were the most favorable conditions for the proliferation culture of cDC1 cells.

[0042] Based on the above, the appropriate conditions for propagating PBMCs with HSC-2D and then differentiating them are as follows.

[0043] (1) During the HSC-2D proliferation stage, FST36S-L, i.e., FLT-3L (50 ng / ml), SCF (20 ng / ml), TPO (20 ng / ml), IL3 (10 ng / ml), IL6 (10 ng / ml), SR1 (1 nmol / ml), and StemSpan TM - Use XF medium. (2) During the differentiation stage, FG3T, namely FLT-3L (50 ng / ml), GM-CSF (2.5 ng / ml), IL3 (5 ng / ml), TGF-β (10 ng / ml), and ImunoCult TM - Use ACF Dendritic Cell Medium. Cell seeding density is 1.25 × 10⁶ 6 Approximately / ml is preferable.

[0044] Example 3: Stability of a method for obtaining cDC1 by propagating PBMCs with HSC-2D and then differentiating them. To verify the reproducibility of the above culture method, two volunteers (one male and one female) were randomly selected and subjected to XCR1 + A cDC1 cell culture test was performed. The cells were differentiated in FST36S-L for 12 days, then in FG3T for 9 days, for a total of 21 days of culture. The cell seeding density was approximately 1.25 × 10⁶. 6 The result was / ml. The flow cytometry analysis results of the finally recovered cells are shown in Figures 7A and 7B. Figure 7A shows a female subject and her XCR1 + The percentage was 76.16%, and Figure 7B shows the male subjects, and their XCR1 + The success rate was 71.95%. This demonstrated that the method is consistently reproducible and has universal significance.

[0045] Example 4: Antigen presentation function of cDC1 obtained by growing PBMCs with HSC-2D and then differentiating them. Method: XCR1 obtained by in vitro culture +To verify whether cDC1 cells possess antigen-presenting function, maturation stimulation and antigen loading tests were performed on cDC1 cells. Thawed cDC1 cells were seeded under the following conditions: GM-CSF 20 ng / ml, CD40L 1 μg / ml, R848 10 μg / ml, Poly I:C 20 μg / ml, INF-γ 10 ng / ml, IL-1β 10 ng / ml, and TNF-α 5 ng / ml (of the above factors, experiments showed that IL-1β and TNF-α could be excluded, and both had little effect on the maturation stimulation of DC cells). The culture medium was still ImunoCult. TM - Using ACF Dendritic Cell Medium, cells were cultured overnight (16-24 hours, not exceeding 48 hours) in a cell incubator at 37°C ± 0.1°C and 5% CO2. The following day, 20 ug / ml GPC3 antigenic peptide (FVGEFFTDV) was added, and the cells were cultured for another 2 hours. Since cDC1 cells are thawed after cryopreservation, seeding them in a normal petri dish would result in wall adhesion. Therefore, in this step, cells were either seeded in a low-adhesion petri dish, or, after the completion of HSC-2D post-proliferation differentiation culture, maturation stimulation was performed directly in the original petri dish without cryopreservation.

[0046] cDC1 cells that have undergone maturation stimulation and antigen loading can express surface markers such as CD83 and CD86. CD83 is a maturation marker for DC cells, and CD86 has been shown to have the function of activating T cells as a co-stimulator. Flow cytometry detection of CD83 and CD86 expression on the surface of cDC1 cells revealed that after maturation stimulation, CD83 expression in cultured cDC1 cells increased from 9.73% in immature iDCs (immature dendritic cells) to 50.48% in mature mDCs (mature dendritic cells). On the other hand, CD83 expression in PBMCs was only 0.31%, indicating that cDC1 cells had already reached a mature state at this point. The expression of the co-stimulator CD86 reached over 40% in both iDC and mDC states, a significant improvement compared to 7.95% in PBMCs, and it possessed the function of activating T cells (see Figure 8).

[0047] For T cell culture, PBMCs were seeded in culture flasks coated with OKM25 (FUKUKO, Japan) the day before co-culture, and an appropriate amount of OKM100 medium (FUKUKO, Japan), 5% autologous plasma, and 1 w / v% penicillin-streptomycin were added. The DC-T co-culture was divided into two groups: (1) T cell control group: the same number of T cells, excluding DC cells; (2) DC-T co-culture group: a group of cells seeded by mixing mature and antigen-loaded cDC1 cells with T cells from the same subject in a DC:T ratio of 1:5. The ratio range may be 1:5 to 1:20, but a ratio of 1:10 is generally recommended. After seeding was complete, the cells were placed in a cell incubator at 37°C ± 0.1°C and 5% CO2 and cultured for 3 days before flow cytometry analysis was performed.

[0048] When antigen-specific T cells produce the corresponding antigen tetramer, they can express the CD4 tetramer, and thus flow cytometry can determine CD4 + T and CD8 + The expression status of GPC3 Tetramer in T cells was analyzed (see Figure 9). Compared to T cells in the control group, CD4 + T cells and CD8 + In all T cells, a small group of GPC3 Tetramer-positive cells is generated, and CD4 + / GPC3 Tetramer + The proportion is 3.76%, and CD8 + / GPC3 Tetramer + The percentage was 4.06%. This indicated that cDC1 cells cultured using this method could perform antigen presentation, become activated, and produce T cells with antigen specificity.

Claims

1. This method involves obtaining cDC1 cells by co-culturing peripheral blood mononuclear cells obtained by isolation with a cytokine composition in vitro. The aforementioned co-culture is Peripheral blood mononuclear cells obtained by isolation are co-cultured in vitro with a first cytokine composition to first obtain their progenitor cells, and The process includes co-culturing the aforementioned progenitor cells with a second cytokine composition and differentiating them to obtain 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 first cytokine composition is prepared by StemSpan TM - Add to XF medium, A method for preparing cDC1 cells in vitro according to claim 1.

3. The second cytokine composition is used in ImmunoCult TM - Add to ACF Dendritic Cell Medium. A method for preparing cDC1 cells in vitro according to claim 1.

4. In the first cytokine composition, the concentrations of each cytokine are as follows: FLT-3L is 50±5 ng / ml, TPO is 20±2 ng / ml, SCF is 20±2 ng / ml, IL3 is 10±1 ng / ml, IL6 is 10±1 ng / ml, and SR1 is 1.0±0.1 nmol / ml. A method for preparing cDC1 cells in vitro according to claim 1.

5. In the second cytokine composition, the concentrations of each cytokine are as follows: FLT-3L is 50 ± 5 ng / ml, GM-CSF is 2.5 ± 0.2 ng / ml, IL-3 is 5.0 ± 0.5 ng / ml, and TGF-β is 10 ± 1 ng / ml. A method for preparing cDC1 cells in vitro according to claim 1.

6. Add more plasma. A method for preparing cDC1 cells in vitro according to claim 1.

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

8. The inoculation density of peripheral blood mononuclear cells is 1–1.5 × 10⁻⁶. ６ The number of cells per ml is A method for preparing cDC1 cells in vitro according to claim 1.

9. Peripheral blood mononuclear cells obtained by isolation are co-cultured in vitro with a cytokine composition, followed by 12 days of cell proliferation culture and 9 days of differentiation culture to obtain cDC1 cells. A method for preparing cDC1 cells in vitro according to claim 1.

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

11. Use of the method for preparing cDC1 cells in vitro according to claim 1 in the production of an in vitro specific T cell drug.

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