Method for preparing dendritic cell vaccine through in-vitro amplification and induction of hematopoietic stem cells

Abstract

The invention discloses a method for preparing a dendritic cell vaccine through in-vitro amplification and induction of hematopoietic stem cells, and belongs to the field of biomedicine.The amplification induction method specifically comprises the following steps that firstly, peripheral blood is collected, the collected peripheral blood is diluted, and then peripheral blood mononuclear cells are separated; according to the invention, the conditions that cells enter an inflammatory emergency state due to a large dose of IL-4 in a traditional scheme, metabolic routes such as cell glycolysis are highly activated, ROS release is caused by oxidative stress, and cell dysfunction is caused by abnormal metabolism are avoided, and a cell combination closest to real DC cell phenotype and function in a human body can be obtained; the antigen presentation of the DC vaccine is improved to the maximum extent, meanwhile, hematopoietic stem cells can be partially differentiated to a myeloid system, DC progenitor cells and DC precursor cells, effective amplification of dozens of times can be achieved in quantity, and the technical bottleneck that the clinical curative effect is limited due to insufficient cell quantity is solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for preparing dendritic cell vaccines by amplifying and inducing hematopoietic stem cells in vitro. Background Art

[0002] Dendritic cell (DC) vaccine is an immune cell therapy that can be used to treat solid tumors. It has the advantages of extensive activation of adaptive immunity, long-term persistence, and high biosafety. As early as 2010, the FDA approved the first DC vaccine product for the treatment of advanced prostate cancer. In recent years, there have been hundreds of DC vaccine-related clinical trials, which shows the medical community's expectations for this therapy. However, despite this, according to existing clinical research results, DC vaccines have not achieved breakthrough therapeutic effects in the treatment of advanced cancer.

[0003] The currently widely used DC vaccine preparation scheme has a history of decades. It starts by collecting monocytes from the patient's peripheral blood, inducing differentiation by adding GM-CSF and IL-4, and finally obtaining DC vaccines for reinfusion through tumor antigen loading and DC cell maturation. This technical route has the following defects: (1) The "dendritic cells" obtained by this method are essentially inflammatory monocyte-derived dendritic cells (moDCs), which are inflammatory cells differentiated from monocytes under external stimulation. These cells are significantly weaker than real DC cells in the human body in terms of antigen presentation ability, lymph homing ability, and T cell activation ability; (2) The cell population prepared by this method is essentially a population of inflammatory monocytes at different differentiation stages, while DC cells in the human body include two major categories: plasmacytoid DC cells and classical DC cells. They have significant functional differences and play different roles in anti-tumor immunity. Therefore, the ideal DC vaccine should contain different DC subset combinations to achieve maximum therapeutic effect; (3) Inducing differentiation of monocytes through the GM-CSF and IL-4 system not only fails to achieve quantitative expansion, but also leads to apoptosis of a considerable number of induced cells. The preparation of a large number of DC vaccine cells is a major bottleneck of this technology. Summary of the Invention

[0004] The purpose of the present invention is to solve the defects in the prior art and to propose a method for in vitro expansion and induction of hematopoietic stem cells to prepare dendritic cell vaccines.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for in vitro expansion and induction of hematopoietic stem cells to prepare a dendritic cell vaccine, wherein the specific steps of the expansion and induction method are as follows:

[0007] Ⅰ: Collect peripheral blood, dilute the collected peripheral blood, and then separate peripheral blood mononuclear cells;

[0008] II: Magnetic bead separation of stem cells and progenitor cells obtained after peripheral blood mononuclear cell separation to remove non-target cells;

[0009] III: Enrich and sort the treated stem cells and precursor cells, and count the cells using a hemocytometer;

[0010] IV: Expanding stem cells and precursor cells, and inducing differentiation of the expanded stem cells and precursor cells.

[0011] As a further embodiment of the present invention, the specific steps of collecting peripheral blood in step I, diluting the collected peripheral blood, and then isolating peripheral blood mononuclear cells are as follows:

[0012] S1.1: Collect peripheral blood and dilute it with PBS buffer at a ratio of 1:1;

[0013] S1.2: Add an equal volume of Ficoll-Paque separation buffer to the centrifuge tube. Slowly add the diluted peripheral blood to the surface of the Ficoll-Paque separation buffer. Place the centrifuge tube in a centrifuge at 500g for 25 minutes.

[0014] S1.3: After centrifugation, remove the centrifuge tube and use a pipette to remove the Ficoll-Paque separation solution and the buffy coat produced by centrifugation of the peripheral blood, and transfer them to a new centrifuge tube.

[0015] S1.4: Add PBS buffer to the centrifuge tube containing the buffy coat layer and mix thoroughly by pipetting. Place the centrifuge tube containing the buffy coat layer in a centrifuge and centrifuge at 300 g for 5 minutes. After centrifugation, remove the supernatant by aspiration.

[0016] S1.5: Add PBS buffer to the centrifuge tube containing the buffy coat layer again, repeatedly pipette to resuspend the cell pellet, and place the centrifuge tube containing the buffy coat layer back into the centrifuge. Centrifuge at 300 g for 5 minutes. After centrifugation, remove the supernatant.

[0017] S1.6: Add PBS buffer to the centrifuge tube and pipette it evenly to resuspend the cell pellet. Place the cell suspension on a hemocytometer. Count the PBMCs under a microscope and calculate the cell concentration.

[0018] As a further embodiment of the present invention, the stem cells and precursor cells obtained after the separation of peripheral blood mononuclear cells in step II are subjected to magnetic bead sorting to remove non-target cells as follows:

[0019] S2.1: Select a magnetic bead separation kit and transfer the cell suspension to a centrifuge tube. Add 200 μL of magnetic bead separation buffer per 5 × 107 cells. Add the magnetic bead separation buffer provided in the magnetic bead separation kit and resuspend the mixture. Add 50 μL of Biotin-Antibody Cocktail and mix thoroughly. Incubate at 4°C for 10 minutes.

[0020] S2.2: After incubation, add 5 mL of magnetic bead separation buffer to the centrifuge tube and mix thoroughly. Centrifuge the tube at 300 g for 10 minutes. After centrifugation, remove the tube and remove the supernatant.

[0021] S2.3: After the supernatant is removed, add 400 μL of magnetic bead separation buffer per 5 × 107 cells to the centrifuge tube. Gently pipette to resuspend the cells and then add 100 μL of Anti-Biotin MicroBeads. Mix thoroughly and incubate the tube at 4°C for 15 minutes.

[0022] S2.4: After incubation, add 5-10 mL of magnetic bead separation buffer to the centrifuge tube and gently invert to mix. Then place the centrifuge tube in a centrifuge and centrifuge at 300g for 10 minutes. After centrifugation, remove the supernatant.

[0023] S2.5: After removing the supernatant, add 1 mL of magnetic bead sorting buffer to the centrifuge tube and resuspend the cells. Then, place the LS magnetic column in the MACS magnetic separator insert and rinse the LS magnetic column with magnetic bead sorting buffer.

[0024] S2.6: After rinsing, slowly add the resuspended cell suspension to the top of the LS magnetic column, allowing the cell suspension to flow naturally into the LS magnetic column. Then, place a new centrifuge tube at the bottom of the LS magnetic column to collect the filtered cells.

[0025] S2.7: After the cell suspension is completely filtered, rinse the magnetic column twice with 3 mL of magnetic bead separation buffer and continue collecting cells.

[0026] S2.8: After magnetic separation, place the centrifuge tube containing the collected cell suspension in a centrifuge and centrifuge at 300 g for 10 minutes. After centrifugation, remove the supernatant.

[0027] As a further embodiment of the present invention, the magnetic bead separation kit described in S3.1 is specifically a Miltenyi Diamond CD34 Isolation Kit magnetic bead separation kit.

[0028] As a further embodiment of the present invention, the specific steps of enriching and sorting the treated stem cells and precursor cells in step III are as follows:

[0029] S3.1: According to every 5×10 7 Add 200uL of magnetic bead sorting buffer to each cell, add magnetic bead sorting buffer to the cell suspension after aspirating the supernatant, resuspend the cells, add 50uL of CD34 MicroBeads, mix well, and then incubate at 4°C for 30 minutes;

[0030] S3.2: After incubation, add 5 mL of magnetic bead separation buffer to the centrifuge tube and mix thoroughly. Centrifuge the tube at 300 g for 10 minutes. After centrifugation, remove the supernatant.

[0031] S3.3: Add 500 μL of magnetic bead separation buffer to the centrifuge tube and resuspend the cells. Then, place the MS magnetic column in the MACS magnetic separator insert and rinse the MS magnetic column with buffer.

[0032] S3.4: After rinsing, slowly add the resuspended cell suspension to the top of the MS magnetic column, allowing the cell suspension to flow naturally into the MS magnetic column. Then, place a new centrifuge tube at the bottom of the MS magnetic column to collect the filtered cells.

[0033] S3.5: After the cell suspension is filtered, rinse the MS magnetic column three times with buffer and continue to collect cells;

[0034] S3.6: After magnetic separation is complete, remove the MS magnetic column from the MACS magnetic separator and place it in a new centrifuge tube. Add 1 mL of buffer to the MS magnetic column and use the included pump to quickly flush the adsorbed cells and collect them in a centrifuge tube.

[0035] S3.7: Perform magnetic separation on the collected cell suspension again. After the second magnetic separation, add 5-10 mL of magnetic bead separation buffer to the centrifuge tube, mix well, and centrifuge at 300 g for 10 minutes. After centrifugation, remove the supernatant from the centrifuge tube.

[0036] As a further embodiment of the present invention, the specific steps of amplifying the stem cells and precursor cells and inducing differentiation of the amplified stem cells and precursor cells in step IV are as follows:

[0037] S4.1: Add the cell suspension after removing the supernatant to the pre-prepared Advanced DMEM / F-12 medium and resuspend the cells. Then, place the resuspended cell suspension on a hemocytometer for counting.

[0038] S4.2: Add 100 ng / mL FLT3L, 50 ng / mL SCF, 100 ng / mL THPO, 20 ng / mL IL-3, and 20 ng / mL IL-6 to Advanced DMEM / F-12 medium and incubate at 37°C in a 5% CO2 incubator for 5-10 days for expansion.

[0039] S4.3: Remove the Advanced DMEM / F-12 medium from the incubator, aspirate the upper layer of suspended cells in the culture flask, and transfer them to a centrifuge tube. Centrifuge at 300g for 10 minutes. After centrifugation, aspirate the supernatant in the centrifuge tube and then drop the cell suspension in the centrifuge tube onto a hemocytometer for counting before proceeding with downstream differentiation induction.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention uses CD34 magnetic columns to sort and enrich hematopoietic stem cells as induced precursors; uses a configured serum-free Advanced DMEM / F-12 medium, adds 100 ng / mL FLT3L, 50 ng / mL SCF, 100 ng / mL THPO, 20 ng / mL IL-3, and 20 ng / mL IL-6 as a factor combination, and culture and expand in a 37°C 5% carbon dioxide incubator. This avoids the high-dose IL-4 in traditional protocols that causes cells to enter an inflammatory emergency state, as well as the resulting high activation of metabolic pathways such as cell glycolysis, oxidative stress that causes ROS release, and cellular dysfunction due to abnormal metabolism. This can obtain a cell combination that is closest to the phenotype and function of real DC cells in the human body, maximizes the antigen presentation of the DC vaccine, and simultaneously enables hematopoietic stem cells to partially differentiate into myeloid lineage, DC progenitor cells, and DC precursor cells, and can achieve effective expansion of up to dozens of times in number, solving the technical bottleneck of insufficient cell number that limits clinical efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0043] Figure 1 This is a flowchart of the method for in vitro expansion and induction of hematopoietic stem cells to prepare dendritic cell vaccines proposed by the present invention. DETAILED DESCRIPTION

[0044] Reference Figure 1 A method for in vitro expansion and induction of hematopoietic stem cells to prepare a dendritic cell vaccine, wherein the specific steps of the expansion and induction method are as follows:

[0045] Peripheral blood was collected and diluted, and then peripheral blood mononuclear cells were isolated.

[0046] Specifically, peripheral blood was collected and diluted with PBS buffer at a ratio of 1:1. A volume of Ficoll-Paque separation solution equal to the diluted peripheral blood was added to a centrifuge tube, and the diluted peripheral blood was slowly added to the surface of the Ficoll-Paque separation solution. The centrifuge tube was then placed in a centrifuge and centrifuged at a centrifugal force of 500g for 25 minutes. After the centrifugation, the centrifuge tube was removed and a pipette was used to aspirate the buffy coat layer produced by the centrifugation of the Ficoll-Paque separation solution and the peripheral blood. The buffy coat layer was then transferred to a new centrifuge tube and added to the centrifuge tube containing the buffy coat layer. Add PBS buffer and mix it by pipetting. Place the centrifuge tube containing the buffy coat layer in a centrifuge and centrifuge at 300 g for 5 minutes. After centrifugation, remove the supernatant and add PBS buffer to the centrifuge tube containing the buffy coat layer again. Repeat pipetting to resuspend the cell pellet. Place the centrifuge tube containing the buffy coat layer in the centrifuge again and centrifuge at 300 g for 5 minutes. After centrifugation, remove the supernatant and add PBS buffer to the centrifuge tube. Pipet it again to resuspend the cell pellet. Take the cell suspension and drop it on a hemacytometer. Count the number of PBMCs under a microscope and calculate the cell concentration.

[0047] The stem cells and precursor cells obtained after separation of peripheral blood mononuclear cells are magnetically sorted and non-target cells are removed.

[0048] Specifically, a magnetic bead sorting kit was selected, and the cell suspension was dropped into a centrifuge tube. According to the requirement of adding 200uL magnetic bead sorting buffer per 5×107 cells, the magnetic bead sorting buffer in the magnetic bead sorting kit was added, and the mixture was resuspended. Then 50uL Biotin-Antibody Cocktail was added and mixed evenly. Then, it was placed in an environment of 4°C for incubation for 10 minutes. After the incubation, 5mL magnetic bead sorting buffer was added to the centrifuge tube and mixed evenly. The centrifuge tube was placed in a centrifuge and centrifuged at 300g for 10 minutes. After the centrifugation was completed, the centrifuge tube was taken out and the supernatant was removed. After the supernatant was removed, 400uL magnetic bead sorting buffer was added per 5×107 cells. The magnetic bead sorting buffer was added to the centrifuge tube, and the resuspended cells were precipitated by gently blowing, and then 100uL was added. Anti-BiotinMicroBeads, mix well, and then incubate the centrifuge tube at 4°C for 15 minutes. After the incubation is complete, 5-10 mL of magnetic bead separation buffer is added to the centrifuge tube and gently inverted to mix. The centrifuge tube is then placed in a centrifuge and centrifuged at 300 g for 10 minutes. After centrifugation, the supernatant is aspirated. After aspirating the supernatant, 1 mL of magnetic bead separation buffer is added to the centrifuge tube and the cells are resuspended. The LS magnetic column is then placed in the MACS magnetic separator insertion position and the LS magnetic column is rinsed with magnetic bead separation buffer. After rinsing, the resuspended cell suspension is slowly added to the top of the LS magnetic column to allow the cell suspension to flow naturally into the LS magnetic column. A new centrifuge tube is then placed at the bottom of the LS magnetic column to collect the filtered cells. After all the cell suspension is filtered, the magnetic column is rinsed twice with 3 mL of magnetic bead separation buffer and cells are collected. After magnetic separation is complete, the centrifuge tube containing the collected cell suspension is placed in a centrifuge and centrifuged at 300 g for 10 minutes. After centrifugation, the supernatant is aspirated.

[0049] It should be further explained that the model of the magnetic bead separation kit is the Miltenyi Diamond CD34 Isolation Kit magnetic bead separation kit.

[0050] The treated stem cells and precursor cells were enriched and sorted, and the cells were counted using a hemocytometer.

[0051] Specifically, according to every 5×10 7Add 200uL magnetic bead sorting buffer to each cell, add magnetic bead sorting buffer to the cell suspension from which the supernatant has been removed, resuspend the cells, add 50uL CD34 MicroBeads, mix well, and then incubate at 4°C for 30 minutes. After incubation, add 5mL magnetic bead sorting buffer to the centrifuge tube, mix well, and then place the centrifuge tube in a centrifuge and centrifuge at 300g for 10 minutes. After centrifugation, remove the supernatant, add 500uL magnetic bead sorting buffer to the centrifuge tube, resuspend the cells, and then place the MS magnetic column in the MACS magnetic separator insertion position. Rinse the MS magnetic column with buffer. After rinsing, slowly add the resuspended cell suspension to the top of the MS magnetic column to allow the cell suspension to flow naturally into the MS magnetic column, and then place the MS magnetic column under the MS magnetic column. Place a new centrifuge tube in the mouth to collect the filtered cells. After the cell suspension is filtered, use buffer to repeatedly rinse the MS magnetic column 3 times and continue to collect cells. After the magnetic separation is completed, remove the MS magnetic column from the MACS magnetic separator and place it on a new centrifuge tube. Add 1 mL of buffer to the MS magnetic column, and then use the matching push pump to quickly flush the adsorbed cells and collect them in the centrifuge tube. Magnetic separation is performed again on the collected cell suspension. After the second magnetic separation is completed, 5-10 mL of magnetic bead sorting buffer is added to the centrifuge tube, mixed, and centrifuged at 300 g centrifugal force for 10 minutes. After centrifugation, the supernatant in the centrifuge tube is aspirated.

[0052] The stem cells and precursor cells are expanded, and the expanded stem cells and precursor cells are induced to differentiate.

[0053] Specifically, the cell suspension from which the supernatant was removed was added to a pre-prepared Advanced DMEM / F-12 medium, and the cells were resuspended. The resuspended cell suspension was then dropped onto a hemocytometer for counting. 100 ng / mL FLT3L, 50 ng / mL SCF, 100 ng / mL THPO, 20 ng / mL IL-3, and 20 ng / mL IL-6 were added to the Advanced DMEM / F-12 medium, respectively, and the cells were cultured in a 37°C 5% carbon dioxide incubator for 5-10 days for expansion. The Advanced DMEM / F-12 medium was removed from the incubator, the upper suspended cells in the culture flask were aspirated, and transferred to a centrifuge tube, which was then centrifuged at 300 g for 10 minutes. After centrifugation, the supernatant in the centrifuge tube was aspirated, and the cell suspension in the centrifuge tube was then dropped onto a hemocytometer for counting, and then downstream differentiation induction was performed.

Claims

1. A method for in vitro expansion and induction of hematopoietic stem cells to prepare a dendritic cell vaccine, characterized in that: The specific steps of the amplification induction method are as follows: Ⅰ: Collect peripheral blood, dilute the collected peripheral blood, and then separate peripheral blood mononuclear cells; II: Magnetic bead separation of stem cells and progenitor cells obtained after peripheral blood mononuclear cell separation to remove non-target cells; III: Enrich and sort the treated stem cells and precursor cells, and count the cells using a hemocytometer; IV: Expanding stem cells and precursor cells, and inducing differentiation of the expanded stem cells and precursor cells.

2. The method for preparing a dendritic cell vaccine by in vitro expansion and induction of hematopoietic stem cells according to claim 1, characterized in that: The specific steps of collecting peripheral blood as described in step I, diluting the collected peripheral blood, and then isolating peripheral blood mononuclear cells are as follows: S1.1: Collect peripheral blood and dilute it with PBS buffer at a ratio of 1:1; S1.2: Add an equal volume of Ficoll-Paque separation buffer to the centrifuge tube. Slowly add the diluted peripheral blood to the surface of the Ficoll-Paque separation buffer. Place the centrifuge tube in a centrifuge at 500g for 25 minutes. S1.3: After centrifugation, remove the centrifuge tube and use a pipette to remove the Ficoll-Paque separation solution and the buffy coat produced by centrifugation of the peripheral blood, and transfer them to a new centrifuge tube. S1.4: Add PBS buffer to the centrifuge tube containing the buffy coat layer and mix thoroughly by pipetting. Place the centrifuge tube containing the buffy coat layer in a centrifuge and centrifuge at 300 g for 5 minutes. After centrifugation, remove the supernatant by aspiration. S1.5: Add PBS buffer to the centrifuge tube containing the buffy coat layer again, repeatedly pipette to resuspend the cell pellet, and place the centrifuge tube containing the buffy coat layer back into the centrifuge. Centrifuge at 300 g for 5 minutes. After centrifugation, remove the supernatant. S1.6: Add PBS buffer to the centrifuge tube and pipette it evenly to resuspend the cell pellet. Place the cell suspension on a hemocytometer. Count the PBMCs under a microscope and calculate the cell concentration.

3. The method for preparing a dendritic cell vaccine by in vitro expansion and induction of hematopoietic stem cells according to claim 2, characterized in that: The specific steps for magnetic bead separation of stem cells and precursor cells obtained after separation of peripheral blood mononuclear cells in step II to remove non-target cells are as follows: S2.1: Select the magnetic bead sorting kit, take the cell suspension and drop it into the centrifuge tube, and then add 5×10 7 Add 200uL of magnetic bead sorting buffer to the cells, add the magnetic bead sorting buffer in the magnetic bead sorting kit, resuspend the mixture, add 50uL of Biotin-Antibody Cocktail, mix well, and then incubate at 4°C for 10 minutes; S2.2: After incubation, add 5 mL of magnetic bead separation buffer to the centrifuge tube and mix thoroughly. Centrifuge the tube at 300 g for 10 minutes. After centrifugation, remove the tube and remove the supernatant. S2.3: After the supernatant is removed, 7 Add 400uL of magnetic bead sorting buffer to the cells, add magnetic bead sorting buffer to the centrifuge tube, and gently pipette to resuspend the cell pellet, then add 100uL of Anti-Biotin MicroBeads, mix well, and then incubate the centrifuge tube at 4°C for 15 minutes; S2.4: After incubation, add 5-10 mL of magnetic bead separation buffer to the centrifuge tube and gently invert to mix. Place the tube in a centrifuge and centrifuge at 300 g for 10 minutes. After centrifugation, aspirate the supernatant. S2.5: After removing the supernatant, add 1 mL of magnetic bead sorting buffer to the centrifuge tube and resuspend the cells. Then, place the LS magnetic column in the MACS magnetic separator insert and rinse the LS magnetic column with magnetic bead sorting buffer. S2.6: After rinsing, slowly add the resuspended cell suspension to the top of the LS magnetic column, allowing the cell suspension to flow naturally into the LS magnetic column. Then, place a new centrifuge tube at the bottom of the LS magnetic column to collect the filtered cells. S2.7: After the cell suspension is completely filtered, rinse the magnetic column twice with 3 mL of magnetic bead separation buffer and continue collecting cells. S2.8: After magnetic separation, place the centrifuge tube containing the collected cell suspension in a centrifuge and centrifuge at 300 g for 10 minutes. After centrifugation, remove the supernatant.

4. The method for preparing a dendritic cell vaccine by in vitro expansion and induction of hematopoietic stem cells according to claim 3, characterized in that: The magnetic bead separation kit model described in S3.1 is specifically the Miltenyi Diamond CD34 Isolation Kit magnetic bead separation kit.

5. The method for preparing dendritic cell vaccine by in vitro expansion and induction of hematopoietic stem cells according to claim 1, characterized in that: The specific steps for enriching and sorting the treated stem cells and precursor cells in step III are as follows: S3.1: According to every 5×10 7 Add 200uL of magnetic bead sorting buffer to each cell, add magnetic bead sorting buffer to the cell suspension after aspirating the supernatant, resuspend the cells, add 50uL of CD34 MicroBeads, mix well, and then incubate at 4°C for 30 minutes; S3.2: After incubation, add 5 mL of magnetic bead separation buffer to the centrifuge tube and mix thoroughly. Centrifuge the tube at 300 g for 10 minutes. After centrifugation, remove the supernatant. S3.3: Add 500 μL of magnetic bead separation buffer to the centrifuge tube and resuspend the cells. Then, place the MS magnetic column in the MACS magnetic separator insert and rinse the MS magnetic column with buffer. S3.4: After rinsing, slowly add the resuspended cell suspension to the top of the MS magnetic column, allowing the cell suspension to flow naturally into the MS magnetic column. Then, place a new centrifuge tube at the bottom of the MS magnetic column to collect the filtered cells. S3.5: After the cell suspension is filtered, rinse the MS magnetic column three times with buffer and continue to collect cells; S3.6: After magnetic separation is complete, remove the MS magnetic column from the MACS magnetic separator and place it in a new centrifuge tube. Add 1 mL of buffer to the MS magnetic column and use the included pump to quickly flush the adsorbed cells and collect them in a centrifuge tube. S3.7: Perform magnetic separation on the collected cell suspension again. After the second magnetic separation, add 5-10 mL of magnetic bead separation buffer to the centrifuge tube, mix well, and centrifuge at 300 g for 10 minutes. After centrifugation, remove the supernatant from the centrifuge tube.

6. The method for preparing dendritic cell vaccines by in vitro expansion and induction of hematopoietic stem cells according to claim 1, characterized in that: The specific steps of amplifying the stem cells and precursor cells and inducing differentiation of the amplified stem cells and precursor cells in step IV are as follows: S4.1: Add the cell suspension after removing the supernatant to the pre-prepared Advanced DMEM / F-12 medium and resuspend the cells. Then, place the resuspended cell suspension on a hemocytometer for counting. S4.2: Add 100 ng / mL FLT3L, 50 ng / mL SCF, 100 ng / mL THPO, 20 ng / mL IL-3, and 20 ng / mL IL-6 to Advanced DMEM / F-12 medium and incubate at 37°C in a 5% CO2 incubator for 5-10 days for expansion. S4.3: Remove the Advanced DMEM / F-12 medium from the incubator, aspirate the upper layer of suspended cells in the culture flask, and transfer them to a centrifuge tube. Centrifuge at 300g for 10 minutes. After centrifugation, aspirate the supernatant in the centrifuge tube and then drop the cell suspension in the centrifuge tube onto a hemocytometer for counting before proceeding with downstream differentiation induction.

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