A method for large-scale expansion of human hematopoietic stem cells based on biomimetic microcarriers

Abstract

The present invention provides a method for large-scale expansion of human hematopoietic stem cells based on bionic microcarriers. On the one hand, the large-scale culture can obtain a sufficient number of functional hematopoietic stem cells. On the other hand, the large-scale culture can maintain the stemness of hematopoietic stem cells for a long time and enhance their potential for differentiation into the megakaryocyte system, overcoming the defect that the absolute number of cells obtained by in vitro culture in the prior art is insufficient for treatment. At the same time, the method can directly culture human monocytes, directly collect and culture human monocytes, and avoid the unknown risks brought about by the process of sorting hematopoietic stem and progenitor cells. The method provided by the present invention takes into account the principles of efficiency and economy and has broad experimental and clinical application prospects. The present invention also provides the application of the large-scale culture method in the preparation of blood disease treatment preparations, and the application of the cell products obtained based on the large-scale culture method in the preparation of blood disease treatment agents.

Description

Technical Field

[0001] The present invention relates to the medical field of hematopoietic stem cells, specifically, a method for large-scale expansion of human hematopoietic stem cells based on biomimetic microcarriers, the method including but not limited to being applicable to the expansion of hematopoietic stem cells derived from bone marrow, peripheral blood and umbilical cord blood. Background Art

[0002] Hematopoietic stem cells (HSCs) are stem cells responsible for the production of mature blood cells in the bone marrow. In a normal human body, HSCs must meet the body's production needs for hematopoiesis (e.g., producing more than 500 billion blood cells per day) and precisely regulate the number of each blood cell type in circulation. Most hematopoiesis occurs in the bone marrow and originates from a limited number of HSCs, which are multipotent and capable of self-renewal.

[0003] However, in the current treatment of blood-related diseases, adult hematopoietic stem cell transplantation is subject to limitations such as extremely low matching probability and graft-versus-host disease, making it impossible to cure bone marrow and malignant hematopoietic diseases. Even if umbilical cord blood hematopoietic stem cells are used, they are still limited in number and quality, making them inapplicable to adults and children with larger body weights. Therefore, increasing the number of cells obtained through in vitro culture and increasing the proportion of functional hematopoietic stem cells among them are the primary solutions to improve the bottleneck of clinical application of hematopoietic stem cells.

[0004] Existing methods for expanding human hematopoietic stem cells in vitro rarely involve large-scale culture. The main reason is that the in vitro expansion methods are not ideal and cannot reach the number of cells required for treatment. Among them, the strategy of adding serum and cytokines such as SCF, TPO, Flt3L to the culture medium not only fails to meet the requirements of experiment and treatment in terms of cell number, but also the HSCs obtained through culture quickly differentiate and fail in terms of quality. + cells, umbilical cord blood CD34 + cells and bone marrow CD34 cells from monkeys and dogs + Cells can be expanded, but clinical and phenotypic / transcriptional studies have found that the expanded cells may be multipotent progenitor cells and erythroid / megakaryocytes rather than true long-term stem cells. Therefore, they cannot maintain long-term stemness, resulting in poor subsequent cell therapy effects. Another small molecule compound, pyrimidine indole derivative UM171, requires the help of a batch feed culture system. Under this system, the CD34 + CD45RA -The number of stem cells increased by an average of 35 times. Experiments have shown that it can preferentially support the expansion of long-term stem cells (LT-HSCs). However, transcriptome analysis shows that UM171 inhibits genes related to the differentiation of red blood cells and megakaryocytes, which may lead to difficulties in subsequent cell recovery in cell therapy, especially the reconstruction of the megakaryocyte system. The polymer material polyvinyl alcohol (PVA) has species-specific effects on the culture of hematopoietic stem cells. Researchers have found that the addition of PVA can enable mouse hematopoietic stem cells to continuously self-renew. Under optimal culture conditions, mouse hematopoietic stem cells can expand approximately 200-800 times within 28 days, but the effect on human hematopoietic stem cells is not significant. In terms of 3D culture, the 3D culture of the degradable zwitterionic hydrogel ZTG can effectively expand umbilical cord blood CD34 in vitro + cells, inhibiting stem cell differentiation, but not the reconstitution of the megakaryocyte lineage.

[0005] The mononuclear cells in a single umbilical cord blood bank are stored in 10 8 -10 9 The number range is 10, while the number of cells cultured in the laboratory based on existing technology is 10 4 -10 5 Therefore, the culture method of a small number of cells cannot be applied to a large number of cells (>10 7 At present, most of the schemes for in vitro expansion of hematopoietic stem cells are based on the number range of 1-2×10 5 CD34 positive cells in human umbilical cord blood cells were cultured in vitro. 8 —10 9 There is still no effective expansion protocol for in vitro expansion of the order of magnitude (the number of mononuclear cells in a single umbilical cord blood).

[0006] The defects of existing technologies in the in vitro culture of hematopoietic stem cells are not only insufficient in terms of the number of in vitro expansion, but also reflected in the operational steps. Specifically, at this stage, the schemes for in vitro expansion of hematopoietic stem cells are mostly based on the in vitro culture of CD34-positive cells in human umbilical cord blood cells, which are mostly improvements in the expansion culture medium. Such culture schemes require the use of antibodies and magnetic beads to screen and separate CD34-positive cells in umbilical cord blood, which increases the actual contact during the hematopoietic stem cell culture process. The impact of the use of these reagents on the return of the amplified products to the human body is unknown, which is a certain distance from the actual clinical application needs of reducing in vitro processing, in vitro contact with reagents, and reducing costs.

[0007] In summary, based on the research progress of existing technologies, there is currently no in vitro culture method that can meet the requirements of both the quantity and function of human hematopoietic stem cells. Considering future clinical applications, a simple, efficient and high-quality in vitro large-scale expansion method of human hematopoietic stem cells is needed. Summary of the Invention

[0008] In view of this, the present invention aims to provide a method for large-scale in vitro culture of functional human hematopoietic stem cells and its application in the preparation of medical preparations.

[0009] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0010] A first aspect of the present invention provides a method for large-scale in vitro culturing of functional human hematopoietic stem cells. The method comprises collecting human mononuclear cells, culturing the human mononuclear cells in a bioreactor, and adding three-dimensional microcarriers to the reaction system. Preferably, the concentration of the three-dimensional microcarriers in the bioreactor is 50-200 mg / 60 mL / tank.

[0011] Preferably, the three-dimensional microcarriers are in powder or flake form.

[0012] Preferably, the human mononuclear cells are derived from one of bone marrow, peripheral blood and umbilical cord blood.

[0013] Preferably, the human mononuclear cells are collected from the bone marrow of patients with hematological diseases.

[0014] Preferably, the patient suffers from a bone marrow failure disease, which includes bone marrow failure caused by radiotherapy and chemotherapy, aplastic anemia AA, Fanconi anemia FA, primary immune thrombocytopenia (ITP), etc.

[0015] Preferably, the patient suffers from aplastic anemia.

[0016] Preferably, the mononuclear cells are umbilical cord blood mononuclear cells.

[0017] Preferably, the culturing steps are as follows:

[0018] Freshly isolated human mononuclear cells are resuspended in culture medium and divided into four equal portions, each 60 mL, and added to four pre-cleaned, high-temperature and high-pressure sterilized bioreactors; preferably, the human mononuclear cells are derived from bone marrow, peripheral blood, or umbilical cord blood;

[0019] Add 50-200 mg / 60 mL / can of three-dimensional microcarrier material to each reactor, mix well, and place the reactor in a constant temperature incubator for culture.

[0020] Preferably, the culture medium in step (1) is: StemSpan TM SFEMⅡserum-free medium+10ng / mL hSCF+100ng / mL hTPO+1%PS;

[0021] Preferably, the amount of the three-dimensional microcarrier material added in step (2) is 100 mg / 60 ml.

[0022] Preferably, the culture conditions in step (3) are: 37°C, 5% CO2, 40 rpm dynamic culture for 1 to 7 days

[0023] Optionally, the method further comprises the step of detecting CD34, CD38, CD45RA, CD90, CD49f, CD62 and CD133 on the surface of the cultured cells using immunological detection means;

[0024] Optionally, the method further comprises sorting the immunophenotype as CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 + The steps of the cell;

[0025] Optionally, the three-dimensional microcarrier used in the amplification method is a commercially available three-dimensional microcarrier, preferably a gelatin three-dimensional microcarrier for cells.

[0026] Optionally, the three-dimensional microcarriers used in the amplification method are in the form of particles, sheets and / or aggregates.

[0027] Optionally, the three-dimensional microcarriers used in the amplification method are three-dimensional microcarrier particles, the average particle size of the particles is between 50-500 μm, preferably 50-200 μm, 200-400 μm or 400-500 μm; the pore size of the particles is 10-30 μm, preferably 15-25 μm.

[0028] Optionally, the three-dimensional microcarrier used in the amplification method is a three-dimensional microcarrier particle, and the porosity of the particle is greater than 80%, preferably the porosity is 80%, 90% or 95%.

[0029] Optionally, the three-dimensional microcarrier used in the amplification method is a 3D Microslides and / or products having the same or similar structure.

[0030] Optionally, the three-dimensional microcarrier used in the method is in the form of a sheet or powder, and preferably the sheet-shaped three-dimensional biomimetic carrier has a concentration of 10-20 mg / sheet.

[0031] The present invention provides a second aspect of applying the method for large-scale in vitro culture of functional human hematopoietic stem cells provided in the first aspect of the present application to prepare a reagent for improving and / or treating blood diseases;

[0032] Preferably, the blood disease is a bone marrow failure disease;

[0033] Preferably, the bone marrow failure disease is aplastic anemia AA, Fanconi anemia FA, primary immune thrombocytopenia (ITP), etc.

[0034] The third aspect provided by the present invention is the use of the method for large-scale in vitro culture of functional human hematopoietic stem cells provided in the first aspect of the present application in the preparation of a preparation for promoting human megakaryocyte phenotype reconstruction.

[0035] The fourth aspect provided by the present invention is to provide a preparation comprising functional stem cells obtained by the method for large-scale in vitro culture of functional human hematopoietic stem cells provided in the first aspect of the present application and pharmaceutically acceptable excipients.

[0036] Compared to the prior art, the first aspect of the present invention provides a method for large-scale in vitro culturing of functional human hematopoietic stem cells. The method comprises collecting human mononuclear cells, culturing the human mononuclear cells in a bioreactor, and adding three-dimensional microcarriers to the reaction system. Preferably, the concentration of the three-dimensional microcarriers in the bioreactor is 50-200 mg / 60 mL / tank.

[0037] Preferably, the three-dimensional microcarriers are in powder or flake form.

[0038] Preferably, the human mononuclear cells are derived from one of bone marrow, peripheral blood and umbilical cord blood.

[0039] Preferably, the human mononuclear cells are collected from the peripheral blood of patients with hematological diseases.

[0040] Preferably, the patient suffers from a bone marrow failure disease, including bone marrow failure caused by radiotherapy and chemotherapy, aplastic anemia AA, Fanconi anemia FA, primary immune thrombocytopenia (ITP), etc.

[0041] Preferably, the patient suffers from aplastic anemia.

[0042] Preferably, the mononuclear cells are umbilical cord blood mononuclear cells.

[0043] Preferably, the culturing steps are as follows:

[0044] Freshly isolated human mononuclear cells are resuspended in culture medium and divided into four equal portions, each 60 mL, and added to four pre-cleaned, high-temperature and high-pressure sterilized bioreactors; preferably, the human mononuclear cells are derived from bone marrow, peripheral blood, or umbilical cord blood;

[0045] Add 50-200 mg / 60 mL / can of three-dimensional microcarrier material to each reactor, mix well, and place the reactor in a constant temperature incubator for culture.

[0046] Preferably, the culture medium in step (1) is: StemSpan TM SFEMⅡserum-free medium+10ng / mL hSCF+100ng / mL hTPO+1%PS;

[0047] Preferably, the amount of the three-dimensional microcarrier material added in step (2) is 100 mg / 60 ml.

[0048] Preferably, the culture conditions in step (3) are: 37°C, 5% CO2, 40 rpm dynamic culture for 1 to 7 days

[0049] Optionally, the method further comprises the step of detecting CD34, CD38, CD45RA, CD90, CD49f, CD62 and CD133 on the surface of the cultured cells using immunological detection means;

[0050] Optionally, the method further comprises sorting the immunophenotype as CD34+CD38 - CD45RA - CD90+CD49flowCD62L - Steps for CD133+ cells;

[0051] Optionally, the method further comprises the step of pressing the three-dimensional microcarrier into a sheet shape, and the preferred sheet-shaped three-dimensional bionic microcarrier has a weight of 10 mg per sheet.

[0052] The present invention provides a second aspect of applying the method for large-scale in vitro culture of functional human hematopoietic stem cells provided in the first aspect of the present application to prepare a reagent for improving and / or treating blood diseases;

[0053] Preferably, the blood disease is a bone marrow failure disease;

[0054] Preferably, the bone marrow failure disease is aplastic anemia AA, Fanconi anemia FA, primary immune thrombocytopenia (ITP), etc.

[0055] The third aspect provided by the present invention is the use of the method for large-scale in vitro culture of functional human hematopoietic stem cells provided in the first aspect of the present application in the preparation of a preparation for promoting human megakaryocyte phenotype reconstruction.

[0056] The fourth aspect provided by the present invention is to provide a preparation comprising functional stem cells obtained by the method for large-scale in vitro culture of functional human hematopoietic stem cells provided in the first aspect of the present application and pharmaceutically acceptable excipients.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The present invention is based on the large-scale culture of the functional hematopoietic stem cells in a bioreactor using three-dimensional microbodies. On the one hand, the large-scale culture method of the present invention has a simple culture system and does not require a magnetic bead sorting step, and can obtain a sufficient number of functional hematopoietic stem cells. On the other hand, the large-scale culture can maintain the stemness of hematopoietic stem cells for a long time and enhance their potential for differentiation into the megakaryocyte lineage. It also takes into account the principles of efficiency and economy and has broad experimental and clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0060] Figure 1 Statistical line graphs showing the proportions and absolute numbers of each subpopulation of cells in the blank control group and microcarrier group after 1, 3, 5, and 7 days of in vitro expansion of umbilical cord blood mononuclear cells in a bioreactor;

[0061] Figure 2 Typical flow cytometry plots of each subpopulation of cells in the blank control group and microcarrier group after 1, 3, 5, and 7 days of in vitro expansion of umbilical cord blood mononuclear cells in a bioreactor in Example 1;

[0062] Figure 3 Typical flow cytometry plots of each subpopulation of umbilical cord blood mononuclear cells in the uncultured group and the microcarrier group after 3 days of bioreactor-scale in vitro expansion in Example 1;

[0063] Figure 4 . Statistical graph showing the absolute fold change in the number of cells in each subpopulation in the microcarrier group compared to the uncultured group after umbilical cord blood mononuclear cells were expanded in vitro on a bioreactor scale for 3 days in Example 1;

[0064] Figure 5 . Schematic diagram of the xenotransplantation process in immunodeficient mice in Example 2, as well as the engraftment level of human CD45+ cells and megakaryocytes in the mouse bone marrow, the reconstruction success rate, representative flow cytometry plots, and long-term stem cell frequency statistics. DETAILED DESCRIPTION

[0065] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0066] "Three-dimensional microcarriers," also known as microcarrier particles or microcarriers, are micron-sized particles that are suitable for the growth of adherent cells. While most adherent cells can only proliferate when attached to a solid substrate, the porous, large surface area, and biocompatibility of microcarriers allow cells to grow within them, creating a three-dimensional culture model. This biomimetic 3D culture model is gaining increasing application.

[0067] The size of the three-dimensional microcarrier particles used in the present invention is preferably between 50-500 μm, and the porosity is generally greater than 80%, for example, 90% or 95%, including but not limited to aggregated, flake, and / or powdered microcarrier particles. The three-dimensional microcarrier used in the examples of the present invention is 3D microcarriers manufactured by Huakan Biotechnology Co., Ltd. F01, the microcarrier is a gelatin microcarrier for cells, and its CDE pharmaceutical excipient registration numbers are F20200000496 and F20210000003. The present invention, by way of full introduction, introduces the method for preparing microcarrier particles and the microcarrier particles prepared therefrom as defined in claims 6-21 of the publication number CN113651989A, published on November 16, 2021, and introduces a cell carrier particle aggregate as defined in claim 1 of the publication number CN109762802B, published on August 3, 2021.

[0068] The present invention will be described in detail below with reference to the embodiments.

[0069] Example 1 A method for large-scale expansion of human functional hematopoietic stem cells

[0070] 1. Experimental process:

[0071] 1) Collect a bag of 60-200mL of neonatal umbilical cord blood (containing mononuclear cells ranging from 10 8 -10 9 ) Place the plasma in a clean and sterile 200 ml bottle, add hydroxyethyl starch (HES) to the cord blood at a volume ratio of 1:4, shake well, and let it stand at room temperature for 45 minutes to 1 hour to fully settle the red blood cells in the blood, thereby achieving the purpose of initially removing most of the red blood cells;

[0072] 2) Use a 10ml disposable sterile pipette to gently aspirate the supernatant of the settled cord blood, place it in a 50ml centrifuge tube, and centrifuge at 1600 rpm for 10 minutes;

[0073] 3) Discard the supernatant and resuspend the cells in culture medium (SFEM + 10 ng / mL SCF + 100 ng / mL TPO + 1% PS). Count the cells and add four equal portions of the cell suspension to four pre-cleaned, autoclaved, 125 mL to 1 L bioreactors of equal volume. Each culture volume is 60 mL. Fill the volume up to 60 mL with the above culture medium.

[0074] 4) Prepare 3D F01, product model is F01-100;

[0075] 5) Set up A. microcarrier group, B. blank control group, C. uncultured group

[0076] A. Add 10-200 mg microcarriers / 5×10 7 -2×10 8 Mix the mononuclear cells / 60 mL culture medium three-dimensional microcarrier material;

[0077] B. Blank control group without three-dimensional microcarrier material;

[0078] C. Uncultured cells do not need to be cultured in a bioreactor. After separation and resuspension in step 2, they can be directly labeled with antibodies for detection (same as step 6A)

[0079] The bioreactor culture bottles of the microcarrier group and the blank control group were placed in a 5% CO2, 37°C constant temperature incubator and cultured dynamically at 40 rpm;

[0080] 6) On the 1st, 3rd, and 5th day, after properly mixing the liquid in the tank, take 10 ml from each tank;

[0081] A. Microcarrier group was mixed by pipetting, and the material was filtered through a 500-mesh yellow filter. The filtrate was centrifuged (1500 rpm, 10 min, 4°C) to lyse the red cells. After washing the red cells, the cells were resuspended and counted. The supernatant was discarded after centrifugation (1500 rpm, 10 min, 4°C). The labeled antibodies were resuspended in an appropriate amount of PBS: FITC-anti-CD34 (Biolegend, 343604), PECy7-anti-CD38 (Biolegend, 356608), APCH7-anti-CD45RA (BD, 560674), PercpCy5.5-anti-CD90 (BD, 561557), BV510-anti-CD49f (BD, 563271), PE-anti-CD62L (BD, 555544), APC-anti-CD133 (BD, 566596). The volume of the added antibodies was calculated according to the antibody instructions. After 20-30 minutes, the antibody was washed away with an appropriate amount of PBS and the solution was resuspended in 2% FBS / PBS for flow cytometry detection.

[0082] B. The blank control group does not require membrane filtration, and other procedures are consistent with the microcarrier group;

[0083] Each time 10 ml was taken, 10 ml of fresh culture medium was added to continue dynamic culture.

[0084] 7) On the 7th day, collect all the culture fluid from each tank, label it with antibodies and perform flow cytometry detection, following the same procedure as step 6.

[0085] 2. Experimental conclusions:

[0086] When mononuclear cells were cultured in a bioreactor, both the absolute number of cells and the proportion of stem and progenitor cell groups in the three-dimensional microcarrier group were significantly higher than those in the blank control group. Specifically:

[0087] from Figure 1 It can be seen that with the extension of culture time, the absolute number and proportion of cells in the three-dimensional microcarrier group and the blank control group changed to varying degrees:

[0088] A. The absolute number of each cell subset, CD34 + 、CD34 + CD38 - 、CD34 + CD38 - CD45RA - CD90 + The cell proliferation peaked on day 5, with a peak of 3.97×10 7 , 2.86×10 7 and 1.55×10 7 , CD34 + CD38 - CD45RA - CD90 + CD49f low The absolute number of cells in the population increased over time and reached a maximum of 1.18×10 on the 7th day. 6 , CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 + The absolute number of cells in the population reached a peak of 2.53×10 on the 5th day. 5 , CD34 + CD38 - CD45RA - CD90 + CD62L - CD133 +The absolute number of cells in the population showed an increasing trend, which was equivalent on the 3rd and 5th days and reached a peak of 5.85×10 on the 7th day. 5 .

[0089] B. The proportion of each cell subset in living cells, CD34 + 、CD34 + CD38 - 、CD34 + CD38 - CD45RA - CD90 + The change trends of the two groups were similar, and both reached their peaks on the 5th day; CD34 + CD38 - CD45RA - CD90 + CD49f low The proportion of population cells showed an increasing trend, which was equivalent on the 3rd and 5th days and reached a peak on the 7th day; CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 + The proportion of population cells showed no significant difference on the 3rd, 5th and 7th days, reaching the peak; CD34 + CD38 - CD45RA - CD90 + CD62L - CD133 + The number of cells reached its peak on the 7th day.

[0090] from Figure 2 and Figure 3 It can be seen that in CD34 + 、CD34 + CD38 - 、CD34 + CD38 - CD45RA - CD90 + 、CD34 + CD38 - CD45RA - CD90 + CD49f + and CD34 + CD38 - CD45RA - CD90 + CD49f lowThere were significant differences in the cell density centers of each subpopulation in the microcarrier group compared with the blank control group or the uncultured group. The cells in the microcarrier group were clearly clustered, and the density centers migrated toward the stemness subpopulation. + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 + There were also significant differences in the subpopulations of the microcarrier group compared with the blank control.

[0091] From Table 1 and Figure 4 It can be seen that after 3 days of bioreactor-scale expansion, the absolute number of several cell subpopulations with different phenotypes in the microcarrier group increased significantly compared with before culture. + 、CD34 + CD38 - 、CD34 + CD38 - CD45RA - CD90 + 、CD34 + CD38 - CD45RA - CD90 + CD49f + 、CD34 + CD38 - CD45RA - CD90 + CD49f low and CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 + The cell numbers were 4.50 times, 44.45 times, 75.28 times, 1083 times, 1705 times, 1765 times and 1950 times that of the uncultured group, respectively.

[0092] Table 1 Absolute number of each cell subset in different culture groups

[0093]

[0094] The above results show that the in vitro large-scale expansion of hematopoietic stem cells in a bioreactor based on three-dimensional microcarriers is feasible in terms of quantity, efficiency and types of large-scale expansion of cells.

[0095] Example 2: Functional experiment to verify large-scale expansion of cells in vitro based on three-dimensional microcarriers - megakaryocyte reconstruction experiment in immunodeficient mice

[0096] Immunodeficient mouse transplantation test for megakaryocyte reconstruction:

[0097] 1) On the day of transplantation, NOG immunodeficient mice were irradiated with 120 cGy of X-rays. Transplantation was performed 4 hours after the end of irradiation.

[0098] 2) Umbilical cord blood mononuclear cells were isolated and cultured in a bioreactor according to the same method as in Example 1, steps 1-5 (microcarrier assembly). After 3 days of culture, cells were harvested: the cells were mixed by pipetting in a cleanroom, filtered through a 500-mesh yellow filter to remove material, and the filtrate was centrifuged (1500 rpm, 10 min, 4°C) to lyse the red blood cells, then resuspended and counted. The cells were washed twice with sterile PBS buffer in a cleanroom, centrifuged at 1500 rpm for 10 min at 4°C, and the supernatant discarded.

[0099] 3) Resuspend the cells in an appropriate amount of PBS buffer and add 100 μL of the prepared antibody mixture to the flow cytometry tube to label the cells. Mix by pipetting and incubate at 4°C in the dark for 30-60 minutes. (For flow cytometry, prepare the following antibody mixture: FITC-conjugated anti-Human CD34, PE-Cy7-conjugated anti-Human CD38, APC-H7-conjugated anti-Human CD45RA, Percp-Cy5.5-conjugated anti-Human CD90, PE-conjugated anti-Human CD49f, AlexaFluor 700-conjugated anti-Human CD62L, and APC-conjugated anti-Human CD133 in PBS buffer containing 2% FBS.)

[0100] 4) After incubation, 1 mL of PBS buffer was added to each tube to wash the cells. Centrifuge at 1500 r / min for 10 min at 4°C, discard the supernatant, resuspend in an appropriate amount of PBS buffer, and stain with DAPI before loading. The cells were loaded onto a BD Aria III flow cytometer, using an appropriate amount of sterile PBS buffer as the receiving fluid. Two-way sorting of DAPI-CD34 was performed on the two groups of cells. + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 +

[0101] (CD62L -CD133 + )or

[0102] DAPI - CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 - &CD62L + CD133 - & + (nonCD62L - CD133 + )cell.

[0103] 5) Adjust the concentration of the suspension to the following concentration gradient using sterile PBS buffer according to the number of cells obtained: 1000, 500, 100, and 50 cells / 25 μL cell suspension / mouse, and inject the suspension into the medullary cavity of anesthetized NOG immunodeficient mice.

[0104] 6) Take 10 μL tail blood at the 4th, 8th, 12th and 16th week after transplantation and detect human CD45 by flow cytometry + The implantation rate was determined by taking 15 μL of tail blood and adding it into 105 μL of CBC diluent (1:7) for detection by automatic blood cell analyzer.

[0105] 7) At week 16, mice were sacrificed by CO2 inhalation and soaked in 75% alcohol for 10 minutes. The hind limbs, including the bilateral femurs, tibias, and iliac bones, were removed in a clean bench. The muscle tissue was stripped clean with sterile gauze. PBS buffer was drawn up with a 1 ml syringe to flush out the bone marrow cells into a flow cytometer, which was then temporarily stored on ice.

[0106] 8) Filter the collected mouse bone marrow cells through a 30 μm sterile nylon membrane to prepare a single-cell suspension. Centrifuge at 1500 × 10 min at 4°C. Discard the supernatant and resuspend in an appropriate amount of PBS buffer. Remove an appropriate amount of cells and transfer them to a flow cytometer for flow cytometry analysis. Centrifuge the remaining cells and freeze them in freezing medium at -80°C.

[0107] 9) Use PBS buffer containing 2% FBS to prepare an antibody mixture (APC-Cy7-conjugated anti-Mouse CD45, APC-conjugated anti-Mouse CD41, Percp-Cy5.5-conjugated anti-MouseCD42d, BV786-conjugated anti-Mouse CD61, PE-Cy7-conjugated anti-Mouse Ter119, BV605-conjugated anti-Mouse CD71, FITC-conjugated anti-Human CD45, BV510-conjugated anti-Human CD41a, PE-conjugated anti-Human CD42b, BV650-conjugatedanti-Human CD61, Alexa Fluor700-conjugated anti-Human CD235a, BV711-conjugatedanti-Human Add appropriate amount of antibody mixture to each flow cytometry tube to label the cells, mix thoroughly by pipetting, and incubate at 4°C in the dark for 30-60 min.

[0108] 10) After incubation, add 1 mL of PBS buffer to each tube to wash the cells, centrifuge at 1500 r for 10 min at 4°C, discard the supernatant, resuspend in 100-200 μL of PBS buffer, and stain with DAPI before loading. BD Aria III flow cytometer was used to detect mouse CD45 in mouse bone marrow using FlowJo10 software. - CD41 - CD42d - CD61 - human CD45 - Human CD41a + The cell ratio was used to determine the reconstruction of the transplanted megakaryocyte lineage and the success rate of megakaryocyte phenotype reconstruction in each group was calculated. - human CD45 + Cell ratio to determine the level of engraftment.

[0109] Experimental conclusion:

[0110] mCD45 was detected in the bone marrow cells of the recipient mice 16 weeks after transplantation. - hCD45 - mCD41 - mCD42d - mCD62 - hCD41a+ The proportion of subpopulation cells is not less than 0.01% as the standard for successful reconstruction of megakaryocytes, mCD45 - hCD45 + The proportion of subpopulation cells should be no less than 0.01% as the standard for successful engraftment of human leukocytes.

[0111] Depend on Figure 5 It can be seen that at different cell transplantation doses (1000, 500, 100, 50 cells / 25 μL cell suspension / mouse) obtained by the large-scale culture method of this application, the mouse bone marrow was successfully implanted with human CD45 + cells and can reconstitute megakaryocyte CD41a + Among them, transplantation of 1000, 500, and 100 cell doses of CD62L - CD133 + The success rate of mouse megakaryocyte reconstruction by subpopulation cells was higher than that by transplanting nonCD62L - CD133 + Subpopulations of cells in mice; based on megakaryocytes

[0112] mCD45 - hCD45 - mCD41 - mCD42d - mCD62 - hCD41a + Phenotype ELDA calculation, we know CD62L - CD133 + The frequency of functional megakaryocytes in the group is biased towards long-term hematopoietic stem cells compared with nonCD62L - CD133 + The group with nonCD62L transplantation increased by 3.19 times (p<0.05). - CD133 + Reconstitution of mouse myeloid and lymphoid lineages by subpopulation cells (CD45 + The success rate of transplantation is higher than that of CD62L - CD133 + Subpopulations of cells in mice, based on hCD45 + Phenotyping was performed by ELDA to calculate the frequency of long-term hematopoietic stem cells, nonCD62L - CD133 + Compared with CD62L - CD133 + The serum creatinine level in the control group increased by 11.85 times (p<0.0001).

[0113] The results showed that megakaryocyte hematopoietic stem cells were enriched in CD34 in the microcarrier three-dimensional culture system. + CD38- CD45RA - CD90 + CD49f low CD62L - CD133 + cell population, and the three-dimensional microcarrier reactor cultured mononuclear cells on a large scale in vitro to expand this functional human megakaryocyte-biased hematopoietic stem cell subset.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for large-scale in vitro culture of functional human hematopoietic stem cells, characterized in that: The method comprises the steps of collecting human mononuclear cells, culturing the human mononuclear cells using a bioreactor, and adding a three-dimensional microcarrier to the reaction system; the method further comprises the steps of sorting cells with an immunophenotype of CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 + The three-dimensional microcarrier is 3D TableTrix® Microslide® F01 of Huakan Biotechnology Co., Ltd.

2. The method according to claim 1, characterized in that The concentration of the three-dimensional microcarriers in the bioreactor is 50-200 mg / 60 mL / tank.

3. The method according to claim 2, characterized in that The human mononuclear cells are derived from bone marrow, peripheral blood or umbilical cord blood.

4. The method according to claim 3, characterized in that The human mononuclear cells are collected from the bone marrow of patients with bone marrow failure diseases.

5. The method according to claim 3, wherein The human mononuclear cells were collected from the bone marrow of patients with primary immune thrombocytopenia (ITP).

6. The method according to claim 4, characterized in that The bone marrow failure disease is selected from the group consisting of: bone marrow failure caused by radiotherapy and chemotherapy, aplastic anemia AA, and Fanconi anemia FA.

7. The method according to claim 3, characterized in that The mononuclear cells are single umbilical cord blood mononuclear cells, and the number of cells is 10 8 -10 9 Quantity range.

8. The method according to any one of claims 1 to 7, characterized in that: The culturing comprises the following steps: (1) Freshly isolated human mononuclear cells were resuspended in culture medium and divided into four equal portions, each 60 mL, and added to four bioreactors that had been cleaned and sterilized at high temperature and high pressure; (2) Add 50-200 mg / 60 mL / can of three-dimensional microcarrier material to each reactor and mix thoroughly; (3) Place the mixed reactor in a constant temperature incubator for cultivation.

9. The method according to claim 8, characterized in that Step (1) culture medium: StemSpan TM SFEM Ⅱserum-free medium+10ng / mL hSCF+100ng / mL hTPO+1%PS.

10. The method according to claim 8, characterized in that In step (2), 100 mg / 60 mL / can of three-dimensional microcarrier material was added to each reactor.

11. The method according to claim 8, characterized in that The culture conditions of step (3) are: 37° C., 5% CO 2 , and dynamic culture at 40 rpm for 1 to 7 days.

12. The method according to any one of claims 1-7, 9-11, characterized in that: The three-dimensional microcarriers used in the method are in powder or sheet form.

13. The method according to claim 12, characterized in that The sheet-shaped three-dimensional biomimetic carrier has a content of 10 or 20 mg per sheet.

14. Use of the method for large-scale in vitro culture of functional human hematopoietic stem cells according to any one of claims 1 to 13 in the preparation of a reagent for treating blood diseases, characterized in that: The blood disease is a bone marrow failure disease.

15. Use of the method for large-scale in vitro culture of functional human hematopoietic stem cells according to any one of claims 1 to 13 in preparing a reagent for treating blood diseases, characterized in that: The blood disease is immune thrombocytopenia (ITP).

16. The use according to claim 14, characterized in that The bone marrow failure disease is selected from the group consisting of: bone marrow failure caused by radiotherapy and chemotherapy, aplastic anemia AA, and Fanconi anemia FA.

17. Use of the method for large-scale in vitro culture of functional human hematopoietic stem cells according to any one of claims 1 to 13 in the preparation of a preparation for promoting reconstitution of human megakaryocyte phenotype.

18. A preparation comprising functional stem cells obtained by the method for large-scale in vitro culture of functional human hematopoietic stem cells according to any one of claims 1 to 13 and pharmaceutically acceptable excipients.

Images