Methods and applications of expanding and differentiating peripheral blood into dendritic cells

Abstract

A method for amplifying and differentiating peripheral blood dendritic cells involves first expanding isolated peripheral blood mononuclear cells in vitro with a cytokine composition (composed of FLT-3L, TPO, SCF, IL3, IL6, and SR1), followed by differentiation culture (FLT-3L, GM-CSF, IL3, and TGF-β) to obtain cDC1 cells. The method has been verified to produce cDC1 cells in vitro from an initial 2.5 × 10⁻⁶ cells. 6 PBMC can obtain XCR1 + cDC1 cells, at least 0.8 × 10⁻⁶ 6 32% of the cells were sufficient to meet 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 objective of this invention is to provide a method for in vitro preparation of cDC1 cells, which involves expanding peripheral blood and then differentiating and culturing dendritic cells to increase the number of obtainable cDC1 cells and meet the needs of clinical applications.

[0007] Another objective of this invention is to provide a method for obtaining cDC1 cells in vitro, which involves selecting a combination of multiple cytokines, expanding peripheral blood, and then differentiating and culturing dendritic cells to improve 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 first cytokine composition in vitro to obtain their precursor cells, and then co-culturing them with a second cytokine composition to differentiate them into cDC1 cells, namely the HSC-2D culture method.

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

[0012] The second cytokine composition used in the method for in vitro preparation of cDC1 cells of the present invention is for the differentiation stage of precursor cells into cDC1 cells and consists of FLT-3L, GM-CSF and TGF-β.

[0013] An embodiment of a first cytokine composition for the method of in vitro preparation of cDC1 cells according to the present invention, StemSpan TM 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 were added to XF medium to carry out the initial cell expansion.

[0014] An embodiment of a second 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, and TGF-β were added to CDC1 Dendritic Cell Medium (ACF Dendritic Cell Medium) to achieve the following concentrations: FLT-3L 50±5 ng / ml, GM-CSF 2.5±0.2 ng / ml, and TGF-β 10±1 ng / ml, for cell differentiation. The dosage of TGF-β was determined through trial and error in this invention; a concentration that is too low is not conducive to the differentiation and suspension of cDC1 cells.

[0015] Another embodiment of the second cytokine composition used in the in vitro preparation of cDC1 cells according to the present invention further includes the addition of IL-3 at a concentration of 5.0 ± 0.5 ng / ml in the dendritic cell culture medium.

[0016] This invention provides a method for in vitro preparation of cDC1 cells, which also includes the addition of plasma, such as plasma from the same individual from whom PBMCs were obtained, at a dosage of 5 v / v%. We tested cell culture with no plasma, platelet lysis buffer instead of plasma, and 5 v / v% autologous plasma. The results showed that almost no cells survived in the serum-free state, while cells could expand and differentiate under platelet lysis buffer conditions, but the number and XCR1 levels were significantly reduced. + The proportion was significantly lower than that of the autologous plasma group.

[0017] The method for in vitro preparation of cDC1 cells according to this invention, taking a 6-well plate as an example, uses PBMC seeding amount based on cell density, such as 1-1.5 × 10⁻⁶. 6 The ideal ratio is 1 cell / ml.

[0018] The present invention provides a method for in vitro preparation of cDC1 cells, in which peripheral blood mononuclear cells are isolated and obtained and co-cultured in vitro with a composition of HSC-2D amplification-related cytokines (e.g., cultured in a 37℃±0.1℃ 5% CO2 cell culture incubator) for 12 days, the culture medium and differentiation-related cytokines are replaced, and the cells are cultured for another 9 days to obtain cDC1 cells.

[0019] The method for in vitro preparation of cDC1 cells according to the present invention has been verified to be effective, starting with an initial 2.5 × 10⁻⁶ cells. 6 The minimum number of cDC1 cells that can be obtained from PBMCs is 0.8 × 10⁻⁶. 6 32% (inclusive), and presented XCR1 + / CD11b - Features that can meet the needs of clinical applications for cell quantity.

[0020] 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.

[0021] 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.

[0022] 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.

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

[0024] ① The starting material is PBMC, which does not require magnetic bead separation, making the operation simpler;

[0025] ② The obtained cDC1 cells (XCR1) + / CD11b - The proportion is higher (greater than 70%);

[0026] ③ The obtained XCR1 + The number of cDC1 cells is greater (approximately 0.8 × 10⁻⁶). 6 cDC1 / 2.5×10 6 PBMC). Attached Figure Description

[0027] Figure 1Bright field results of cell morphology at various culture times under multiple cytokine conditions;

[0028] Figure 2 Figure showing the results of flow cytometry detection of the expression of cell surface markers CD34, CD141, and CD14 during the HSC-2D amplification phase under various cytokine conditions;

[0029] Figure 3 A schematic diagram showing the fold expansion of various cell types cultured on Day 12 under multiple cytokine conditions;

[0030] Figure 4 The figure shows the results of flow cytometry detection of the expression of cell surface markers CD34, CD141 and XCR1 during the HSC-2D amplification stage under various factors and cell plating density conditions;

[0031] Figure 5 The figure shows the results of flow cytometry analysis of the expression of CD34 and XCR1, cell surface markers, 9 days after HSC-2D amplification and differentiation under various factors and cell plating density conditions.

[0032] Figure 6 A schematic diagram showing the proportion and number of XCR1+cDC1 cells finally obtained under various factors and cell plating density conditions;

[0033] Figure 7A Figure showing the results of flow cytometry analysis of the expression of the surface markers CD141 and XCR1 in cDC1 cells of a randomly cultured female subject;

[0034] Figure 7B Figure showing the results of flow cytometry analysis of the expression of the surface markers CD141 and XCR1 in cDC1 cells from another randomly cultured male subject;

[0035] Figure 8 Figure showing the results of flow cytometry analysis of CD86 and CD83 expression in cDC1 cells after maturation stimulation;

[0036] Figure 9 The image shows the results of flow cytometry analysis of the expression of specific antigen tetramers in co-cultured cDC1-T cells. Detailed Implementation

[0037] 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.

[0038] The specific experimental methods used in the following embodiments of the present invention are described below:

[0039] 1) Obtaining PBMCs from peripheral blood

[0040] 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.

[0041] 2) Flow cytometry detection method

[0042] To identify the cellular components during culture and the proportion of cDC1 in the final expanded and differentiated suspension cells, flow cytometry was used to detect surface markers in this embodiment. 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*02:01GPC3 Tetramer-FVGEFFTDV-APC (MBL, TB-0134-2).

[0043] 3) Methods for measuring total cell count

[0044] In this embodiment, the cell count for each group is calculated as follows: all suspended cells in a 6-well plate amplified by HSC-2D for 12 days are counted, or all suspended cells in a 6-well plate amplified by HSC-2D for 12 days and then redifferentiated for 9 days are counted.

[0045] Example 1: Cytokine selection of cDC1 was obtained by first expanding and then differentiating PBMCs using HSC-2D.

[0046] Based on early experiments exploring cDC1 cell culture, several cytokines were preliminarily identified. To further explore cDC1 cell culture conditions, this example first conducted culture tests with four different concentrations of cytokines. The isolated PBMCs were uniformly seeded into the following culture conditions:

[0047] ①.FST36-H group: FLT-3L (100ng / ml), SCF (100ng / ml), TPO (50ng / ml), IL3 (20ng / ml), IL6 (20ng / ml);

[0048] ②.FST36-L group: FLT-3L (50ng / ml), SCF (20ng / ml), TPO (20ng / ml), IL3 (10ng / ml), IL6 (10ng / ml);

[0049] ③.FST36-H+SR1 group: FLT-3L (100ng / ml), SCF (100ng / ml), TPO (50ng / ml), IL3 (20ng / ml), IL6 (20ng / ml), SR1 (1nmol / ml);

[0050] 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).

[0051] Since dendritic cells (DCs) primarily originate from the differentiation of hepatic chorionic villi (HSCs) and their precursor cells, the initial culture stage is the HSC-2D expansion phase to obtain a sufficient number of DC precursor cells. The culture medium used in this stage is StemSpan. TM -XF (STEMCELL, #100-0073), contains 5 v / v% autologous plasma and 1 w / v% penicillin and streptomycin. Cell plating density is 1×10⁻⁶. 6 / ml, each condition group was plated in two replicate wells of a 6-well plate, with 2ml of culture medium per well, meaning the number of cells plated per well was 2×10. 6 After completion, the cells were cultured in a 37℃±0.1℃ 5% CO2 cell culture incubator for 2 hours, and the medium was changed once. After that, the medium was changed or replenished every 2 to 3 days according to the cell growth status, and the cells were observed and photographed regularly.

[0052] The day of cell plating is recorded as Day 0. As the culture time increases, the cell morphology gradually changes from an adherent state to an increased aggregate size. Cell clusters begin to form around Day 6, at which point the suspended cells are mostly small. As culture continues, the number of small cells gradually decreases. After Day 9, cell clusters become prominent, eventually suspending and dispersing into individual cells, as shown below. Figure 1 As shown. By Figure 1 It can be seen that the cells in the VEGF-added group grew the slowest, while the differences among the other three groups were not significant.

[0053] After 12 days of expansion culture, suspension cells from the culture wells were 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 observed 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 significantly 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 in other groups, and its cell growth was slower, therefore it did not have a significant advantage. CD34 expression was significantly higher in all groups compared to PBMCs, with a fold increase of over 40-fold (see...). Figure 3 The results indicate that the combination of factors is effective in amplifying HSCs, and the amplification fold of the FTS36-L and FTS36-H+SR1 groups is higher than that of the other two groups. CD14 showed almost no amplification, indicating that the culture conditions were unfavorable for monocyte proliferation.

[0054] In summary, the appropriate cytokine condition is FTS36-L+SR1.

[0055] Example 2: Selection of cell plating density for cDC1 obtained by first expanding PBMCs with HSC-2D and then differentiating them.

[0056] The isolated PBMCs were plated at different densities under the following culture conditions:

[0057] ①.FST36S-H group: FLT-3L (100ng / ml), SCF (100ng / ml), TPO (50ng / ml), IL3 (20ng / ml), IL6 (20ng / ml), SR1 (1nmol / ml);

[0058] ②.FST36S-L group: FLT-3L (50ng / ml), SCF (20ng / ml), TPO (20ng / ml), IL3 (10ng / ml), IL6 (10ng / ml), SR1 (1nmol / ml).

[0059] The board density is 0.5×10 6 / ml, 1.25×10 6 / ml, 2.5×10 6 / ml, each condition group was plated in two replicate wells of a 6-well plate, with 2ml of culture medium per well. The initial culture was the HSC-2D amplification phase. The culture medium used in this phase was StemSpan. TM -XF (STEMCELL, #100-0073), after 12 days of amplification, the culture medium was changed to ImmunoCult. TM -ACFDendritic Cell Medium (STEMCELL, #10986) was cultured for differentiation for 9 days. The following cytokines were used for differentiation: FLT-3L (50 ng / ml), GM-CSF (2.5 ng / ml), IL3 (5 ng / ml), and TGF-β (10 ng / ml). Both culture media contained 5% autologous plasma and 1 w / v of penicillin and streptomycin. After plating, the cells were incubated at 37℃±0.1℃ in a 5% CO2 cell culture incubator for 2 hours, with the medium changed once. Thereafter, the medium was changed or replenished every 2-3 days according to the cell growth, and the cells were observed and photographed regularly.

[0060] After 12 days of expansion culture, suspension cells from the culture wells were analyzed by flow cytometry for CD34, CD141, and XCR1. Since CD141 is expressed in various types of dendritic cells (DCs), while XCR1 is expressed only in cDC1 cells, XCR1 was subsequently selected to represent the characteristics of cDC1 cells. At this point, 0.5 × 10⁻⁶ cells were cultured. 6 The / ml plating density group could not be analyzed by flow cytometry because cell growth was slow and there was no cell suspension. Comparison with 1.25×10⁶ 6 Between the two groups with a density of 2.5 × 10⁶ / ml, the FST36S-L group had a higher CD34 expression rate (19.75%), while the FST36S-H group had 10.16%. There was no difference in the XCR1 ratio between the two groups. 6 Between the two groups with a CD141 expression density of 1 / ml, the FST36S-L group had a higher CD141 expression rate (32.98%), while the FST36S-H group had 11.57%. The XCR1 expression rate was almost identical between the two groups (see [link to relevant documentation]). Figure 4 ).

[0061] After 12 days of amplification culture, the differentiation medium was replaced, and differentiation culture was continued for another 9 days. Cells were then harvested, and the suspension cells in the culture wells were analyzed by flow cytometry for CD34 and XCR1 expression. There was little difference in CD34 expression among the groups, but XCR1 expression was significantly different (except for 0.5 × 10⁻⁶). 6 Except for the FST36S-H group (71.82%), all other groups achieved around 80% (see [link to data]). Figure 5 The number of XCR1+cDC1 cells obtained was analyzed and compared (see...). Figure 6 From this, we can see 2.5 × 10 6 / ml and 1.25×10 6 Compared to a plate density of / ml, the number of cells obtained was only slightly higher, not doubled; while 0.5×10 6 / ml and 1.25×10 6 Compared to the plate-laying density of 0.5 × 10⁶ cells / ml, the FST36S-H group had too few cells at low densities, while the FST36S-L group had 0.5 × 10⁶ cells / ml. 6 / ml and 1.25×10 6 Compared to the plate density of / ml, the final harvested cell count was nearly twice as high, therefore, it is estimated to be 1.25×10⁻⁶. 6 The optimal plating density is 1 / ml. Furthermore, although the cell count in the FST36S-H group was slightly higher than that in the FST36S-L group, the difference was small, and considering cost, FST36S-L was the optimal condition for the expansion and culture of XCR1+cDC1 cells.

[0062] In summary, the optimal conditions for differentiation of PBMCs after HSC-2D amplification are determined as follows:

[0063] ①. The HSC-2D amplification stage uses FST36S-L, namely: 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), StemSpan. TM -XF medium;

[0064] ② During the differentiation phase, FG3T was used: 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. 6 The ideal volume is around / ml.

[0065] Example 3: Stability of cDC1 method obtained by differentiating PBMCs after HSC-2D amplification.

[0066] To verify the reproducibility of the above culture method, two volunteers (one male and one female) were randomly selected for XCR1 testing. + cDC1 cell culture assay. Cells were differentiated using FST36S-L for 12 days, followed by FG3T for 9 days, for a total culture period of 21 days. The cell density was approximately 1.25 × 10⁻⁶ cells / year. 6 / ml. The final flow cytometry analysis results of the harvested cells are as follows: Figure 7A and Figure 7B As shown. Figure 7A For female subjects, their XCR1 + The proportion was 76.16%; Figure 7B For male subjects, their XCR1 + The proportion was 71.95%. This indicates that the method is stable, reproducible, and has universal significance.

[0067] Example 4: PBMCs were amplified by HSC-2D and differentiated to obtain the antigen presentation function of cDC1.

[0068] Methods: To verify whether XCR1+cDC1 cells obtained from in vitro culture possess antigen-presenting function, cDC1 cells underwent maturation stimulation and antigen loading tests. Resuscitated cDC1 cells were plated 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, TNF-α 5 ng / ml (experimental tests showed that IL-1β and TNF-α could be removed, as they had almost no effect on DC cell maturation stimulation). The culture medium used was ImmunoCult. TM -ACF Dendritic Cell Medium was incubated overnight (16 to 24 hours, not exceeding 48 hours) in a 37℃±0.1℃ 5% CO2 cell culture incubator. The next day, 20ug / ml of GPC3 antigen peptide (FVGEFFTDV) was added and cultured for another 2 hours. Since cDC1 cells will become adherent if plated in ordinary culture dishes after cryopreservation and thawing, this step involves plated cells in low-adsorption culture dishes or, after HSC-2D amplification and differentiation culture, directly performing maturation stimulation in the original culture dish without cryopreservation.

[0069] After maturation stimulation and antigen loading, cDC1 cells should express surface markers such as CD83 and CD86. CD83 is a marker of DC cell maturation, and CD86, as a co-stimulatory factor, demonstrates that DC cells have the function of activating T cells. Flow cytometry analysis of the expression of CD83 and CD86 on the surface of cDC1 cells revealed that after maturation stimulation, the expression of CD83 in our cultured cDC1 cells increased from 9.73% in immature dendritic cells (iDCs) to 50.48% in mature dendritic cells (mDCs), while the expression of CD83 in PBMCs was only 0.31%, indicating that cDC1 cells had reached a mature state. The expression of the co-stimulatory factor CD86 reached over 40% in both iDC and mDC states, a significant increase compared to 7.95% in PBMCs, demonstrating its function of activating T cells (see [link to relevant documentation]). Figure 8 ).

[0070] T cell culture was performed as follows: One day before co-culture, PBMCs were plated in culture flasks coated with OKM25 (FUKUKO, Japan), and an appropriate amount of OKM100 medium (FUKUKO, Japan), 5% autologous plasma, and 1 w / v penicillin-streptomycin were added. DC-T co-culture was divided into two groups: ①. T cell control group: the same number of T cells without DC cells as controls; 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 and then plated. This ratio can range from 1:5 to 1:20, with 1:10 being a commonly recommended ratio. After plating, the cells were incubated for three days in a 37℃±0.1℃ 5% CO2 cell culture incubator before flow cytometry analysis.

[0071] If antigen-specific T cells are produced, they will express the corresponding antigen tetramer (Tetramer). Therefore, 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 group T cells, a small population of GPC3 Tetramer-positive cells was generated in both CD4+ T cells and CD8+ T cells, with the CD4+ / GPC3Tetramer+ ratio being 3.76% and the CD8+ / GPC3Tetramer+ ratio being 4.06%. This indicates that the cDC1 cells cultured by this method can perform antigen presentation and be activated to generate antigen-specific T cells.

Claims

1. A method of producing cDC1 cells in vitro, characterized in that, Peripheral blood mononuclear cells obtained from isolation were expanded and differentiated in vitro. They were expanded and cultured in FST36S-L medium for 12 days and then differentiated and cultured in FG3T medium for 9 days to obtain cDC1 cells. The FST36S-L medium consisted of: 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, StemSpan™-XF medium containing 5 v / v% plasma and 1 w / v% penicillin and streptomycin. The FG3T medium consisted of: 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, ImunoCult™-ACF Dendritic Cell Medium containing 5 v / v% plasma and 1 w / v penicillin-streptomycin.

Images