Functional hematopoietic stem cells and method for their proliferation

Abstract

The present invention provides a method for culturing human hematopoietic stem cells in vitro based on artificial three-dimensional microcarriers. The method can culture bone marrow, umbilical cord blood, and peripheral blood mononuclear cells from different sources. The culturing method of the present invention has a simple culture system, which not only promotes short-term hematopoietic stem cell colony formation and maintains the potential of cells to differentiate into multiple lineages after culture, but also promotes the formation of cobblestone-like hematopoietic zones with long-term hematopoietic capacity of human hematopoietic stem cells, thereby increasing the frequency of long-term culture initiating cells (LTC-ICs). Human hematopoietic stem cells cultured in vitro using the method of the present invention can maintain their stemness for a relatively long period of time, promote the bias towards the megakaryotic lineage, and promote the expansion of the absolute number of megakaryotic stem cells. Based on this, a method for expanding a human megakaryotic-biased hematopoietic stem cell subpopulation is developed, and the culturing method is used to prepare cell therapy preparations. The present invention has broad application prospects and great improvements in both scientific research and clinical practice.

Description

Technical Field

[0001] The present invention relates to the medical field of hematopoietic stem cells, specifically to functional hematopoietic stem cells and methods for their proliferation. Specifically, the present invention relates to a method for culturing functional hematopoietic stem cells in vitro using three-dimensional microcarriers. The method amplifies and obtains megakaryocyte-biased hematopoietic stem cells with therapeutic potential, including but not limited to hematopoietic stem cells suitable for amplifying hematopoietic stem cells derived from bone marrow, peripheral blood, and umbilical cord blood. Background Art

[0002] Hematopoietic stem cells (HSCs) are a type of cell with the potential for self-renewal and multipotential differentiation. They possess complex heterogeneity, primarily manifested in their characteristics of both lineage-biased differentiation and lineage restriction. One hallmark of HSC heterogeneity is the presence of megakaryocyte / platelet-biased HSCs (Mk-biased HSCs). With the recent development and application of single-cell technology, increasing evidence has demonstrated that megakaryocytes can differentiate from HSCs without undergoing a multipotential or bipotential stage, and that this megakaryocyte-biased HSC subpopulation resides at the apical level of the HSC differentiation spectrum.

[0003] Studies of human myeloproliferative neoplasms have revealed that regulatory disturbances at the hematopoietic stem cell level are closely associated with the development and progression of these diseases. Studies have also shown that in adult bone marrow hematopoiesis, human megakaryocyte differentiation can arise directly from multipotent hematopoietic progenitors (MPPs) without passing through the intermediate megakaryocyte-erythroid progenitor (MEP), suggesting that megakaryocyte-biased hematopoietic stem cells exist within the human hematopoietic stem cell pool. However, the phenotypic characteristics of these human megakaryocyte-biased hematopoietic stem cells remain unclear. In bone marrow failure disorders (such as aplastic anemia (AA) and Fanconi anemia (FA)) and immune thrombocytopenia (ITP), megakaryocyte hematopoiesis is most vulnerable to damage and the most difficult to restore during treatment. Furthermore, thrombocytopenia is a common postoperative complication in patients with hematologic malignancies undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Data suggest that secondary thrombocytopenia occurs in over 10% of patients after allo-HSCT, significantly increasing the risk of bleeding and severely impacting long-term survival. These clinical phenomena indicate that megakaryocyte hematopoietic reconstruction is relatively difficult, which may be related to the characteristics of megakaryocytes that are biased towards hematopoietic stem cells.

[0004] Currently, adult hematopoietic stem cell transplantation is limited by extremely low matching probabilities and graft-versus-host disease, making it incurable for bone marrow and hematopoietic malignancies. Even with umbilical cord blood (UCB) HSC transplantation, the insufficient number and poor quality of UCBC HSCs preclude their use in adults and larger children. Therefore, addressing the current bottleneck in HSC availability is the primary solution to improving the clinical application of HSCs. Ex vivo expansion of functional HSCs is crucial for the full realization of HSC-based therapies. Existing methods for in vitro expansion of HSCs primarily utilize the addition of cytokines (such as stem cell growth factor (SCF) and thrombopoietin (TPO), small molecules (SR1, UM171, etc.), macromolecules (polyvinyl alcohol (PVA), and peptides (NOV)) to the culture system. Alternatively, zwitterionic hydrogels (ZTG) have been used to construct three-dimensional cultures for in vitro expansion of HSCs. However, the addition of serum and cytokines such as SCF, TPO, and Flt3L to the culture medium has been shown to be ineffective, resulting in rapid differentiation and exhaustion of HSCs. Purine derivative SR1 can mobilize human peripheral blood CD34 + cells, umbilical cord blood CD34 + cells and bone marrow CD34 cells from monkeys and dogs + The cells play an expansion role, and the stem cell frequency of a single transplant increases 17-fold and can maintain multi-lineage reconstruction capacity. However, 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. The pyrimidine indole derivative UM171 can increase the CD34 + CD45RA - The number of stem cells increased by an average of 35 times, and the expansion of long-term stem cells (LT-HSCs) was preferentially supported. However, transcriptome analysis showed that UM171 inhibited genes related to the differentiation of erythrocytes and megakaryocytes. The polymer material polyvinyl alcohol (PVA) can enable continuous self-renewal of mouse hematopoietic stem cells. Under optimal culture conditions, hematopoietic stem cells can expand by about 200-800 times within 28 days, but the effect on human hematopoietic stem cells is not significant. The regulatory protein NOV can culture frozen umbilical cord blood CD34 for a short time (8 hours) in vitro. + The frequency of functional hematopoietic stem cells (secondary transplantation) increased by 6 times, but there was a lack of attention to the reconstruction of the megakaryocyte lineage. 3D culture of degradable zwitterionic hydrogel ZTG can effectively expand umbilical cord blood CD34 +Cells inhibit stem cell differentiation and increase the frequency of functional hematopoietic stem cells (per transplant) by 73-fold. However, similarly, there has been little attention paid to their reconstitution of the megakaryocyte lineage. Currently, phenotypically identical HSCs cultured in vitro are still a heterogeneous population at the molecular and functional levels. Current methods are insufficient to obtain megakaryocyte-biased HSCs at the top of the differentiation lineage. Regarding three-dimensional culture, prior art reports have reported the use of three-dimensional microcarriers to expand human adherent cells (such as mesenchymal stem cells, lymphoblastoid cells, glioma cells, cervical cancer cells (HeLa), and diploid embryonic fibroblasts (MRC-5)) in vitro. The efficacy and in vivo adaptability of human adherent cells cultured using three-dimensional microcarriers have been improved compared to traditional two-dimensional culture methods. However, three-dimensional culture has not been reported for hematopoietic stem cells, preferably for long-term hematopoietic stem cell culture, and more preferably for human megakaryocyte-biased hematopoietic stem cells, such as suspension cells. Based on current insights, there is no motivation to use three-dimensional microcarriers in suspension cell culture.

[0005] At the same time, the current schemes for in vitro expansion of hematopoietic stem cells are mostly based on the CD34 + Cells are cultured in vitro, which is mostly an improvement in the expansion culture medium. On the one hand, this culture program requires the use of antibodies and magnetic beads to screen and separate CD34 + The impact of these reagents on the reinfusion of amplified cells into the human body is unknown, and falls far short of the actual clinical application requirements (reducing in vitro processing, in vitro exposure to reagents, and reducing costs). Given that current technologies cannot achieve both efficient and effective expansion of hematopoietic stem cells, there remains no effective solution for in vitro expansion and subsequent reinfusion therapy. Summary of the Invention

[0006] In view of this, the present invention aims to propose a method for culturing human hematopoietic stem cells in vitro based on three-dimensional microcarriers. This method can directly culture monocytes, and the culture system is simple. The stem cells cultured by this method can promote the expansion of the absolute number of hematopoietic stem cells, especially megakaryocytes, and directly apply them to the treatment of blood diseases, showing good therapeutic effects.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0008] The first aspect of the present invention provides an in vitro expansion method for functional hematopoietic stem cells based on three-dimensional microcarriers, the method comprising adding three-dimensional microcarriers to an expansion medium, culturing human non-adherent cells, wherein the non-adherent cells are human monocytes, and the functional hematopoietic stem cells are human megakaryocyte-biased hematopoietic stem cells.

[0009] Furthermore, the human monocytes in the expansion method are derived from one of bone marrow, peripheral blood and umbilical cord blood.

[0010] Furthermore, the human monocytes in the expansion method are collected from the bone marrow of patients with blood diseases, preferably patients with bone marrow failure diseases, preferably patients with aplastic anemia.

[0011] Furthermore, in the amplification method, the concentration of the three-dimensional microcarriers in the amplification culture medium is 2.5-5 mg / mL.

[0012] 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.

[0013] Optionally, the three-dimensional microcarriers used in the amplification method are in the form of particles, sheets and / or aggregates.

[0014] 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.

[0015] 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%.

[0016] Optionally, the three-dimensional microcarrier used in the amplification method is a 3D Microslides and / or products having the same or similar structure.

[0017] Furthermore, the amplification method comprises the following steps:

[0018] (1) Resuspend the mononuclear cells from bone marrow, peripheral blood, and umbilical cord blood in expansion medium to a cell concentration of 5×10 4 -1×10 6 Cells / 5-20mg three-dimensional microcarriers;

[0019] (2) Placing the three-dimensional microcarriers into a 24-well plate and irradiating with UV light for 20-30 min;

[0020] (3) The ratio of cells to three-dimensional microcarriers in the culture system is: 5×10 4 -1×10 6For cells / 5-20mg three-dimensional microcarriers, pipette 100μL of cell suspension into the wells to allow the three-dimensional microcarriers to fully absorb the cell suspension. After culturing in an incubator for 2 hours, add 1.9mL of culture medium to a final system of 2mL. Stir the microcarriers to disperse them and evenly distribute them in the culture medium. Collect the cells after culturing.

[0021] Furthermore, in the amplification method, the culture conditions in step (3) are constant temperature culture at 37° C. and 5% CO 2 for 7 days.

[0022] Furthermore, in the amplification method, the amplification medium in step (1) is StemSpan TM SFEM Ⅱ serum-free medium+10ng / mL hSCF+100ng / mL hTPO+1%PS.

[0023] The second aspect of the present invention is a functional hematopoietic stem cell, which is obtained by culturing according to the in vitro expansion method of functional hematopoietic stem cells based on three-dimensional microcarriers of the present invention.

[0024] The third aspect of the present invention is a pharmaceutical preparation comprising functional hematopoietic stem cells cultured according to the in vitro expansion method of functional hematopoietic stem cells based on three-dimensional microcarriers of the present invention and a pharmaceutically acceptable carrier.

[0025] The fourth aspect of the present invention is the use of functional hematopoietic stem cells obtained by culturing the functional hematopoietic stem cells by an in vitro expansion method based on three-dimensional microcarriers, and a pharmaceutical preparation containing the functional hematopoietic stem cells for preparing a preparation for treating blood diseases.

[0026] Furthermore, the blood disease is a bone marrow failure disease.

[0027] Furthermore, the bone marrow failure disease includes bone marrow failure caused by radiotherapy and chemotherapy, aplastic anemia AA, Fanconi anemia FA, and primary immune thrombocytopenia (ITP).

[0028] The fifth aspect of the present invention is the use of three-dimensional microcarriers in the in vitro culture of functional hematopoietic stem cells, wherein the three-dimensional microcarriers are added to the culture medium of human monocytes cultured in vitro, and the ratio of the number of cells in the culture medium to the amount of the three-dimensional microcarriers added is: 5×10 4 -1×10 6 Cells / 5-20 mg three-dimensional microcarrier, the functional hematopoietic stem cells are human megakaryocyte-biased hematopoietic stem cells.

[0029] Furthermore, the mononuclear cells used in the application are derived from peripheral blood, umbilical cord blood or bone marrow.

[0030] Furthermore, the mononuclear cells are collected from the peripheral blood of patients with blood diseases, preferably bone marrow failure diseases, preferably aplastic anemia.

[0031] In a further embodiment of the present invention, the method for culturing patient monocytes using three-dimensional microcarriers is as follows:

[0032] (1) Resuspend the monocytes in culture medium to a cell concentration of 2×10 6 cells / mL;

[0033] (2) 3D microcarriers at a concentration of 5 mg / ml were plated into a 24-well plate at a concentration of 10 mg / well and irradiated with UV light for 20-30 min;

[0034] (3) 100 μL of cell suspension was added to the wells to allow the three-dimensional microcarriers to fully absorb the cell suspension. After culturing in an incubator for 2 hours, 1.9 mL of culture medium was added to a final system of 2 mL. The microcarriers were stirred to disperse and evenly distribute them in the culture medium. The culture conditions were constant temperature incubation at 37°C and 5% CO2. The cells were collected after 7 days of culture.

[0035] The sixth aspect of the present invention is the use of a method for in vitro expansion of functional hematopoietic stem cells based on three-dimensional microcarriers for preparing human megakaryocyte-biased hematopoietic stem cells, wherein the human megakaryocyte-biased hematopoietic stem cells are enriched in CD34 + CD38 - CD45RA - CD90 + CD49f low in cell subpopulations.

[0036] The seventh aspect of the present invention is the use of three-dimensional microcarriers in the in vitro culture of human hematopoietic stem cells, wherein the human hematopoietic stem cells can maintain their stemness for a relatively long period of time and tend to differentiate into the megakaryocyte lineage.

[0037] The eighth aspect of the present invention is the use of three-dimensional microcarriers in the in vitro culture of human hematopoietic stem cells, wherein the human hematopoietic stem cells have the characteristics of short-term hematopoietic stem cell colony formation and the potential for multi-lineage differentiation is maintained after culture.

[0038] The ninth aspect of the present invention is the use of three-dimensional microcarriers in the in vitro culture of human hematopoietic stem cells, which can induce reprogramming by environmental cytokines and have the function of promoting the activation and differentiation of megakaryocyte lineage.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention overcomes the shortcomings of the existing technology and provides a method for culturing human hematopoietic stem cells in vitro. The culture system is simple, uses a serum-free culture medium, and only requires the addition of two additional cytokines, without the need to add small molecule compounds or large molecule compounds. It meets clinical needs while taking into account the need to reduce costs, and is of great significance for its application in the treatment of a large number of blood disease patients.

[0041] The present invention overcomes the technical bottleneck of long-term hematopoietic stem cell culture that cannot be solved by the existing technology. Surprisingly, it is found that by applying the bionic microcarrier used for adherent cell culture to the field of human hematopoietic stem cell culture, human functional long-term hematopoietic stem cells can be obtained.

[0042] The present invention also unexpectedly discovered that biomimetic microcarriers can maintain the stemness of hematopoietic stem cells in vitro and enhance their potential to differentiate into megakaryocyte lineages. The use of biomimetic microcarriers in vitro to culture hematopoietic stem cells can promote the expansion of the absolute number of megakaryocyte stem cells.

[0043] By analyzing the functional hematopoietic stem cells obtained by in vitro culture based on biomimetic microcarriers, we surprisingly found a specific CD34 + CD38 - CD45RA - CD90 + CD49f low The cell population has been verified to be enriched with megakaryocyte hematopoietic stem cells.

[0044] The present invention uses three-dimensional microcarriers to culture the monocytes of patients with blood diseases and prepare transfusion cell therapy preparations, overcoming the shortcomings of the existing cell therapy field, such as low success rate of allogeneic transplantation and insufficient amount of autologous umbilical cord blood expansion. It is of great significance to the convenience and universality of the treatment of blood diseases and has great value for clinical promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] 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:

[0046] Figure 1 .Umbilical cord blood CD34 in Example 1 and Example 2 + Bubble chart of the changes in the proportion and absolute number of live cells in each subpopulation of cells, normal human bone marrow mononuclear cells, peripheral blood mononuclear cells, and umbilical cord blood mononuclear cells after in vitro culture;

[0047] Figure 2 .Umbilical cord blood CD34 in Example 1 + Typical flow cytometry plots of cell phenotypes after in vitro culture;

[0048] Figure 3 Typical flow cytometry plots of cell phenotypes of normal human bone marrow mononuclear cells, peripheral blood mononuclear cells, and umbilical cord blood mononuclear cells after in vitro culture in Example 2;

[0049] Figure 4 . In Example 3, the blank control group and the three-dimensional microcarrier group were cultured with umbilical cord blood CD34 + Statistical graph of the number of different types of colonies in in vitro cell clone formation experiments;

[0050] Figure 5 . In Example 4, the blank control group and the three-dimensional microcarrier group were cultured with umbilical cord blood CD34 + Statistical graph of the frequency of initial cells in long-term culture of cells forming cobblestone-like hematopoietic regions;

[0051] Figure 6 . Flowchart of the experimental design for limiting dilution transplantation in immunodeficient mice in Example 5, a statistical table of long-term stem cell frequencies for primary and secondary transplants, and a statistical chart of typical flow cytometry plots and engraftment rates for each lineage of cells in mouse bone marrow;

[0052] Figure 7 Typical flow cytometry plots of cell phenotypes before and after long-term in vitro culture (weeks 0, 4, and 6) and statistical plots of the proportion and absolute number of viable cells in each subpopulation over time (weeks 0-7) in Example 6;

[0053] Figure 8 Typical flow cytometry plots of megakaryocytes and megakaryocyte hematopoietic stem cell frequencies in the bone marrow of immunodeficient mice after limiting dilution transplantation in Example 7;

[0054] Figure 9 . RNA-seq sequencing results in Example 8 GO: MF and GSEA enrichment analysis and protein spectrum analysis and real-time fluorescence quantitative PCR analysis and Luminex liquid phase chip multi-factor quantitative analysis diagram;

[0055] Figure 10 .Analysis of 10X Genomics single-cell RNA-seq sequencing results in Example 8;

[0056] Figure 11 .Umbilical cord blood CD34 in Example 9 + CD34 + CD38 - CD45RA - CD90 + Typical flow cytometry plots of CD49f expression distribution in subpopulation cells;

[0057] Figure 12 .Analysis of BD Rhapsody single-cell RNA-seq sequencing results in Example 9;

[0058] Figure 13 .Umbilical cord blood CD34 in Example 9 + Typical flow cytometry plots of specific cell subsets after in vitro culture of cells and bone marrow mononuclear cells;

[0059] Figure 14 . The platelet count in peripheral blood and the typical flow cytometry of megakaryocytes in bone marrow of immunodeficient mice after xenotransplantation in Example 10, as well as the statistical graph of the success rate of megakaryocyte reconstruction;

[0060] Figure 15 . Statistical graph showing the changes in the proportion and absolute number of total cells and each subpopulation of cells after in vitro culture of monocytes from patients with aplastic anemia in Example 12;

[0061] Figure 16 . In Example 12, the CD34 + CD38 - CD49f + Typical flow cytometry plots of cell subpopulations and statistical graphs of absolute number changes;

[0062] Figure 17 Statistical graph of the number of different types of colonies in the in vitro clone formation experiment of monocytes from patients with aplastic anemia after culture in Example 13;

[0063] Figure 18 hCD45 in the bone marrow of immunodeficient mice after transplantation of cultured monocytes from patients with aplastic anemia in Example 14 + Typical flow cytometry graph of proportions and statistical graph of implantation success rate. DETAILED DESCRIPTION

[0064] 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.

[0065] "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.

[0066] 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.

[0067] The present invention will be described in detail below with reference to the embodiments.

[0068] Table 1: Reagents used in the examples of the present invention

[0069]

[0070]

[0071] Example 1: Biomimetic microcarrier-based human umbilical cord blood CD34 + Cell culture and phenotypic analysis

[0072] 1. The culturing of hematopoietic stem cells using three-dimensional microcarriers includes the following steps:

[0073] 1) Fresh human umbilical cord blood was mixed in a high-temperature and high-pressure sterilized glass plasma bottle at a ratio of umbilical cord blood: HESpan = 4:1, and then allowed to settle at room temperature for 45 minutes to fully crosslink and settle the red blood cells in the blood.

[0074] 2) Use a 10ml disposable sterile pipette and a 1ml pipette to carefully aspirate the pale yellow supernatant from the plasma bottle into two 50ml centrifuge tubes. Centrifuge at 1600r / min for 10min and discard the supernatant.

[0075] 3) Add a small amount of ACK red blood cell lysis buffer to the centrifuge tube and mix thoroughly by pipetting. Then add 40 ml of ACK and mix thoroughly by inversion. Incubate in a 37°C water bath for about 25 minutes. Centrifuge at 1500 rpm for 10 minutes and discard the supernatant.

[0076] 4) Wash the cells with an appropriate amount of sterile PBS buffer, centrifuge at 1500 r / min for 10 min, and discard the supernatant. In this step, human umbilical cord blood mononuclear cells are obtained, resuspended in an appropriate amount of PBS buffer, and 10 μl is taken for staining and counting using 0.4% Trypan Blue Solution.

[0077] 5) Resuspend the cells in PBS buffer according to the number of monocytes. Then, add appropriate amounts of FcRBlocking Reagent (to block nonspecific binding sites) and CD34 magnetic beads, mix thoroughly by pipetting, and incubate at 4°C in the dark for 30 minutes. Invert the tube several times every 10 minutes to mix the cells.

[0078] 6) Wash the cells with an appropriate amount of sterile PBS buffer and pass them through LS column to collect CD34 + The cells were centrifuged at 1500 r / min for 10 min and resuspended in an appropriate amount of PBS buffer. 10 μl of the cells were stained and counted using 0.4% Trypan Blue Solution.

[0079] 7) Freshly prepare SFEMⅡ medium containing 1% PS, 100 ng / ml hTPO, and 10 ng / ml hSCF. Set up A. Uncultured group (Fresh) B. Blank control group (Control) C. 3D microcarrier group (Microniche) D. Material control group (PVA, Cytodex3, Cytopore1) E. Small molecule control group (SR1, UM171):

[0080] A. For the uncultured group, umbilical cord blood CD34 + The cells were not cultured and were directly subjected to subsequent flow cytometry analysis;

[0081] B. For the blank control group, freshly isolated CD34 + cells to 2×10 6 / ml, add 1.9ml culture medium / well into a 24-well plate, then add 100μl cell suspension / well to a final system of 2ml / well, gently pipette to mix, and the initial cell concentration is 2×10 5 2 ml of PBS buffer was added to the culture wells at the edge of the plate.

[0082] C. For the three-dimensional microcarrier group, plate the microcarriers into a 24-well plate at 10 mg / well (5 mg / ml) and irradiate with UV light for 20-30 minutes. Pipette 100 μl of cell suspension into the wells to allow the cells and carriers to fully absorb the cell suspension. Add 2 ml of PBS buffer to the culture wells at the edge of the well plate. Place in the incubator for 2 hours, then add 1.9 ml of culture medium to a final volume of 2 ml. Gently stir the microcarriers with a pipette tip to disperse them and evenly distribute them in the culture medium. The initial plated cell concentration is 2×10 5 / hole.

[0083] D. For the material control group, use culture medium to dilute the 10% stock solution of the water-soluble polymer polyvinyl alcohol (PVA) to an appropriate concentration, then add it to the wells that have been pre-laid with culture medium and cell suspension to reach a final concentration of 0.1% as reported in the literature, and repeatedly pipette to mix. According to the instructions, GE materials Cytopore1 and Cytodex3 were hydrated (expanded and dried material powder) and pre-treated with high-pressure sterilization. Then, as much PBS as possible was aspirated from the sterile material, and the material was rinsed with culture medium. The volume of culture medium was adjusted so that the final concentration of the material was 2 mg / ml Cytopore1 and 5 mg / ml Cytodex3 (within the recommended range of use). The material suspension and cell suspension were added to the culture wells and pipette to mix. The initial cell concentration for plating was 2×10 5 / hole.

[0084] E. For the small molecule compound group, dilute the compound stock solution to the appropriate concentration using culture medium. For the positive control, use the final concentration of 1 μM for StemRegenin (SR1) and 40 nM for UM171 as reported in the literature. Keep the mixture away from light. First, add 1.8 ml of culture medium to each well, then pipette 100 μl of cell suspension into the well, then add 100 μl of compound dilution solution, and mix thoroughly by pipetting repeatedly. The initial cell density for plating is 2 × 10 5 / hole.

[0085] 8) For groups B, C, D, and E, 4-6 replicate wells were arranged in a 37°C, 5% CO2 incubator for 7 days. After 7 days, cells were removed and observed under a microscope. Cells were harvested in a clean bench and the cells or cell-material complexes were repeatedly pipetted and mixed. The cells were then filtered through a 30 μm sterile nylon membrane and transferred from the plate to a flow cytometer. The plate and nylon membrane were washed once with an appropriate amount of PBS buffer.

[0086] 9) After centrifugation at 1500 r / min for 10 min, discard the supernatant and resuspend the cells in an appropriate amount of PBS buffer. Take 10 μl of the harvested cell suspension and stain with 0.4% Trypan Blue Solution to count the living cells.

[0087] 10) Centrifuge at 1500r for 10 min and discard the supernatant. Adjust the cell concentration per flow cytometry tube to 2×10 5 -1×10 6 Between 1 and 2 days, add 100 μl of the prepared antibody mixture (APC-conjugated anti-Human CD34, PE-Cy7-conjugated anti-Human CD38, APC-H7-conjugated anti-Human CD45RA, Percp-Cy5.5-conjugated anti-Human CD90, and BV510-conjugated anti-Human CD49f flow cytometry antibody mixture prepared in PBS buffer containing 2% FBS), mix thoroughly by pipetting, and incubate at 4°C in the dark for 30-60 minutes. On day 0, perform the same antibody treatment on the uncultured group (A).

[0088] 11) After incubation, add 1 ml of PBS buffer to each tube to wash the cells. Centrifuge at 1500 r / min for 10 min, discard the supernatant, resuspend the cells in an appropriate amount of PBS buffer, and stain with DAPI before loading.

[0089] 12) Cells were detected using a BD FACS CANTO II flow cytometer and analyzed by gating using FlowJo 10 software.

[0090] 2. Experimental conclusions:

[0091]

[0092] Table 2:

[0093] Table 3:

[0094]

[0095] Depend on Figure 1 、 Figure 2 The data shown in Table 2-3 show that the original HSC subsets: CD34 + CD38 - CD45RA - CD90 + Subpopulations and CD34 + CD38 - CD45RA - CD90 + CD49f +The average proportion of subpopulations in living cells was 0.82% and 0.01% in A. uncultured group; 4.70% and 4.13% in C. three-dimensional microcarrier group (the absolute numbers of the two stem cell subpopulations in C. three-dimensional microcarrier group were 5.8 times and 843 times that of A. uncultured group, respectively); 1.58% and 0.11% in B. blank control group; 1.85% and 0.16% in PVA of material control group, 0.41% and too little in Cytopore1, and 0.09% and too little in Cytodex3 of material control group; 2.33% and 0.14% in SR1 of small molecule compound group, and 7.92% and 0.13% in UM171 of small molecule compound group E.

[0096] Based on the above data, it can be seen that umbilical cord blood CD34 + The cells were cultured in vitro under three-dimensional microcarrier conditions, which significantly increased the number of primitive HSC subsets: CD34 + CD38 - CD45RA - CD90 + Subpopulations and CD34 + CD38 - CD45RA - CD90 + CD49f + The proportion and absolute number of subpopulations were statistically significant (p<0.01); the three-dimensional microcarriers could significantly increase the CD34 + CD38 - CD45RA - CD90 + CD49f + The proportion and absolute number of subpopulations (the absolute numbers of each group were 843, 228, 380, 296, and 139 times that of the uncultured group, respectively).

[0097] Example 2 Cultivation and phenotypic analysis of human monocytes from bone marrow, peripheral blood, and umbilical cord blood based on three-dimensional biomimetic microcarriers

[0098] 1. Collection of mononuclear cells from umbilical cord, bone marrow, and peripheral blood

[0099] 1.1 Isolation of umbilical cord blood mononuclear cells:

[0100] 1) Fresh human umbilical cord blood was mixed in a high-temperature and high-pressure sterilized glass plasma bottle at a ratio of umbilical cord blood: HESpan = 4:1, and then allowed to settle at room temperature for 45 minutes to fully crosslink and settle the red blood cells in the blood.

[0101] 2) Use a 10ml disposable sterile pipette and a 1ml pipette to carefully aspirate the pale yellow supernatant from the plasma bottle into two 50ml centrifuge tubes. Centrifuge at 1600r / min for 10min and discard the supernatant.

[0102] 3) Add a small amount of ACK red blood cell lysis buffer to the centrifuge tube and mix thoroughly by pipetting. Then add 40 ml of ACK and mix thoroughly by inversion. Incubate in a 37°C water bath for about 25 minutes. Centrifuge at 1500 rpm for 10 minutes and discard the supernatant.

[0103] 4) Wash the cells with an appropriate amount of sterile PBS buffer, centrifuge at 1500 r / min for 10 min, and discard the supernatant. In this step, human umbilical cord blood mononuclear cells are obtained, resuspended in an appropriate amount of PBS buffer, and 10 μl is taken for staining and counting using 0.4% Trypan Blue Solution.

[0104] 1.2 Isolation of mononuclear cells from bone marrow and peripheral blood:

[0105] 5) Collect human bone marrow samples or peripheral blood samples in anticoagulant tubes, centrifuge at 1200 rpm for 5 minutes, and discard the upper serum.

[0106] 6) Add the same amount of sterile PBS buffer as the centrifuged sample to the remaining sample to dilute the sample and mix thoroughly by pipetting.

[0107] 7) Take another sterile 15ml centrifuge tube, mark it, and add 2 / 3 of the volume of the lymphocyte separation medium after diluting the sample with PBS. Use a disposable pipette to carefully transfer the diluted sample onto the surface of the lymphocyte separation medium, taking care not to break the interface and keep it clear. Centrifuge at 1800r / min for 20min (speed increase 3, speed decrease 3).

[0108] 8) After centrifugation, the liquid surface is separated into three layers. Carefully aspirate the middle buffy coat layer with a 1 ml pipette tip into a 15 ml sterile centrifuge tube. Add 6-8 times the volume of PBS buffer, pipette to mix thoroughly, and centrifuge at 1500 rpm for 10 min.

[0109] 9) After centrifugation, the supernatant was discarded and human bone marrow or peripheral blood-derived mononuclear cells were obtained. The cells were resuspended in an appropriate amount of PBS and mixed thoroughly by pipetting. 10 μl of the cells were taken and stained with 0.4% Trypan Blue Solution for counting.

[0110] 2. The culture method comprises the following steps:

[0111] Freshly prepared SFEMⅡ medium containing 1% PS, 100 ng / ml hTPO, and 10 ng / ml hSCF. Set up A. Uncultured group (Fresh) B. Blank control group (Control) C. 3D microcarrier group (Microniche) D. Small molecule compound group (SR1 and UM171):

[0112] A. For the uncultured group, human umbilical cord blood, bone marrow, or peripheral blood mononuclear cells isolated through steps 1) to 9) above were not cultured and were directly subjected to subsequent flow cytometry analysis;

[0113] B. For the blank control group, resuspend freshly isolated human umbilical cord blood, bone marrow or peripheral blood mononuclear cells in culture medium to 2×10 6 / ml, add 1.9ml culture medium / well into a 24-well plate, then add 100μl cell suspension / well to a final system of 2ml / well, gently pipette to mix, and the initial cell concentration is 2×10 5 2 ml of PBS buffer was added to the culture wells at the edge of the plate.

[0114] C. For the three-dimensional microcarrier group, plate the microcarriers into a 24-well plate at 10 mg / well (5 mg / ml) and irradiate with UV light for 20-30 minutes. Pipette 100 μl of cell suspension into the wells to allow the cells and carriers to fully absorb the cell suspension. Add 2 ml of PBS buffer to the culture wells at the edge of the well plate. Place in the incubator for 2 hours, then add 1.9 ml of culture medium to a final volume of 2 ml. Gently stir the microcarriers with a pipette tip to disperse them and evenly distribute them in the culture medium. The initial plated cell concentration is 2×10 5 / hole.

[0115] D. For the small molecule compound group, dilute the compound stock solution to the appropriate concentration using culture medium. For the positive control, use the final concentration of 1 μM for StemRegenin (SR1) and 40 nM for UM171 as reported in the literature. Keep the mixture away from light during the preparation process. First, add 1.8 ml of culture medium to each well, then pipette 100 μl of the mononuclear cell suspension into the well, then add 100 μl of the compound diluent and mix thoroughly by pipetting repeatedly. The initial cell density for plating is 2 × 10 5 / hole.

[0116] 11) The subsequent steps are the same as steps 8) to 12) in Example 1.

[0117] 3. Experimental conclusions:

[0118] Table 4:

[0119]

[0120] Depend on Figure 1 、 Figure 3 As shown in Table 4, for normal human bone marrow-derived mononuclear cells (BM MNCs), peripheral blood-derived mononuclear cells (PB MNCs) and umbilical cord blood-derived mononuclear cells (UCB MNCs), compared with the blank control group and the small molecule compound group, the three-dimensional microcarrier can stably maintain and expand the original HSC subsets: CD34 +CD38 - CD45RA - CD90 + Subpopulations and CD34 + CD38 - CD45RA - CD90 + CD49f + The absolute numbers of the two primitive HSC subsets in BM MNCs increased by 6-15 times and 8-16 times compared with the uncultured group, respectively; the absolute numbers of the two primitive HSC subsets in PB MNCs increased by 90.6 times and 8.6 times compared with the uncultured group, respectively; the original HSC subset CD34 in UCB MNCs increased by 1.3 times and 2.8 times compared with the uncultured group, respectively. + CD38 - CD45RA - CD90 + The absolute number of the cultured group increased by 37.5 times compared with the uncultured group.

[0121] The above results indicate that, compared with currently known peripheral blood expansion methods, three-dimensional microcarriers have significant advantages and are capable of expanding human hematopoietic stem cells from multiple sources (bone marrow, peripheral blood, and umbilical cord blood).

[0122] Example 3 Analysis of the Effect of Three-Dimensional Microcarriers on Promoting Human Hematopoietic Stem Cell Clone Formation

[0123] 1. Experimental methods:

[0124] 1) Umbilical cord blood hCD34 + The cell culture and cell harvesting methods of the cell microcarrier plate were the same as steps 1) to 9) of Example 1, and the grouping was the same as that of B. blank control group and C. three-dimensional microcarrier group in Example 1.

[0125] 2) Add appropriate amount of IMDM medium to each tube of cells and resuspend them to adjust the cell concentration to 4×10 4 cells / ml is 4×10 3 hCD34 + After culture, cells / 100 μl system.

[0126] 3) Add the adjusted cell suspension to H4434 semi-solid medium thawed overnight at 4°C at a ratio of 1:10 (100 μl of cell suspension per ml of H4434 semi-solid medium). Vortex thoroughly to mix and allow bubbles to float to the surface and disappear. Set up two groups, solvent control and microcarrier experimental groups, respectively. Plate the cells in 6-well plates, with 4-5 replicate wells per group.

[0127] 4) Use a screw-cap syringe with a Syringes needle to draw up the semisolid cell suspension and add 1 ml to each of the two center wells of a 6-well plate. Gently shake to evenly distribute the semisolid suspension across the entire bottom surface of the plate. Seal the plate with PBS buffer and carefully place in a 37°C, 5% CO2 incubator.

[0128] 5) After 12-16 days of culture, observe the culture plates daily under a microscope. Remove the plates when the colony density is appropriate and count and photograph the number of burst-forming units of erythrocytes (BFU-E), colony-forming units of erythrocytes (CFU-E), colony-forming units of granulocytes and macrophages (CFU-GM), and colony-forming units of granulocytes, erythrocytes, macrophages, and megakaryocytes (CFU-GEMM).

[0129] 2. Experimental conclusions:

[0130] Depend on Figure 4 It can be seen that the umbilical cord blood CD34 + The number of BFU-E, CFU-E, CFU-GM and CFU-GEMM colonies and the total number of colonies all increased significantly to varying degrees, with no obvious tendency towards lineage differentiation. These results indicate that three-dimensional microcarriers can promote the short-term formation of hematopoietic stem cell colonies when culturing human hematopoietic stem cells in vitro, and that the potential for multi-lineage differentiation of cells is maintained after culture.

[0131] Example 4 Analysis of the Effect of Three-Dimensional Microcarriers on Promoting Long-Term Hematopoietic Cloning of Human Hematopoietic Stem Cells

[0132] 1. Experimental methods:

[0133] (1) Preparation of stromal cells

[0134] 1) Resuscitate frozen mouse bone marrow stromal M2-10B4 cells and resuspend the cells in fresh culture medium (high glucose 1640 + 10% FBS + 1% PS). Remove a small amount of cells and stain with 0.4% Trypan Blue Solution before counting viable cells.

[0135] 2) Plate the cell suspension into a T75 cell culture flask or a 10 cm cell culture dish. After overnight culture, observe the attachment of the stromal cells.

[0136] 3) When the stromal cells have proliferated to the desired number, discard the culture supernatant in a clean bench. Add an appropriate amount of PBS buffer and gently shake to wash twice. Then, add 3 mL of 0.25% Trypsin-EDTA solution and shake thoroughly to mix. When most of the fibrous adherent cells are suspended and detached and their morphology becomes rounded, add an appropriate amount of FBS to terminate the digestion.

[0137] 4) The cells were repeatedly pipetted to collect the cell suspension, and a small amount of cells were taken out and stained with 0.4% Trypan Blue Solution before counting the viable cells.

[0138] 5) Centrifuge at 1000r for 10 min, discard the supernatant, wash twice with appropriate amount of PBS buffer, resuspend the cells and transfer them to flow cytometry tube, adjust the concentration of M2-10B4 stromal cells to 10 7 / mL, irradiated with an X-ray source at a dose of 8000 cGy. After irradiation, the solution was centrifuged at 1000 r for 10 min and the supernatant was discarded.

[0139] 6) Freshly prepare human long term culture media (HLTCM) with a final concentration of 10 -6 mol / L Hydrocortisone, the stromal cells were resuspended in the above medium and the viable cells were counted again. The final stromal cell concentration was adjusted to 1.2×10 5 / mL for use. Now the M2-10B4 stromal cells are ready.

[0140] (2) Matrix cell covering

[0141] 1) In a clean bench, use a spray gun to add 50 μL of collagen solution to each of the central 60 wells of a flat-bottomed 96-well plate. Gently shake the plate to spread it evenly, then place it flat at room temperature to dry, so that the collagen is evenly spread on the bottom of the plate. After about 1 hour, seal it with sealing film and store it at 4°C for one week before use. The collagen plate coating is now complete.

[0142] 2) Before plating the stromal cells, use a dispenser to gently add 100 μL of PBS buffer to each well of the collagen plate, let it stand for a while, and then aspirate it. Wash twice in total to fully wash away the floating acid generated on the collagen surface and avoid the effect of the acidic environment on the stromal cells.

[0143] 3) The X-ray irradiated stromal cell suspension was thoroughly pipetted and mixed, and 100 μL of M2-10B4 cell suspension was added to each well on the collagen layer of the well plate. The 96-well plate was gently shaken to evenly spread on the collagen surface, that is, each well contained 1.2×10 4 After irradiation, the irradiated M2-10B4 stromal cells were added to the edge of the well plate with PBS buffer, and cultured in a 37°C, 5% CO2 constant temperature cell culture incubator for 24 hours. The adhesion status of the stromal cells was observed, and the upper layer of cells was added if the state was good.

[0144] (3) Place the upper layer of cells

[0145] 1) Umbilical cord blood hCD34 +The method for harvesting cells cultured on microcarrier well plates was the same as steps 1 to 9 in Example 1, and the grouping was the same as in Example 1. B. Blank control group (Control) C. Three-dimensional microcarrier group (Microniche)

[0146] 2) Before laying the upper layer of cells, use a pipette to gently aspirate 50 μL of supernatant from each well of the matrix cell plate.

[0147] Resuspend the cells in HLTCM medium to the appropriate cell density. Then, dilute the cells serially according to a pre-determined top layer cell concentration gradient (typically 1000, 500, 250, 125, and 63 starting cells / well, with 12 replicate wells per concentration gradient). The seeding volume is 150 μL, and the final volume per well of a 96-well plate is 250 μL. Incubate the cells in a 37°C, 5% CO2 incubator for 5-6 weeks.

[0148] (4) Long-term cultivation stage

[0149] 1) During long-term culture, half-volume medium changes should be performed weekly, with particular attention paid to the cell density and growth status of the underlying M2-10B4 stromal cells. Considering evaporation during long-term culture, half-volume medium changes should be performed by aspirating approximately 40 μL per well and adding approximately 60 μL of freshly prepared HLTCM human long-term medium. Be extremely gentle when aspirating the medium, taking care not to disturb the stromal cell layer by touching the bottom of the plate.

[0150] 2) After 5-6 weeks of culture, the 96-well plates were observed under a microscope for cobblestone formation. A well was considered positive if cobblestone-like primitive hematopoietic areas with >10 cell clones were present. Microscopic photographs were taken and the probability of cobblestone-like primitive hematopoietic areas at different cell concentrations was calculated. The statistical data were used to calculate the frequency of long-term culture-initiating cells (LTC-ICs) with long-term hematopoietic function in different groups using ELDA software.

[0151] 2. Experimental conclusions:

[0152] Depend on Figure 5 It can be seen that the frequency of LTC-IC in the blank control group is 1 / 1218, and in the three-dimensional microcarrier group is 1 / 662. Based on this data, it can be seen that three-dimensional microcarrier in vitro culture can promote the formation of cobblestone-like hematopoietic areas with long-term hematopoietic ability of human hematopoietic stem cells, increase the frequency of LTC-IC, and functionally maintain the stemness characteristics of primitive HSCs.

[0153] Example 5 Verification of the Effect of Three-Dimensional Microcarriers on Amplifying Human HSC in Vitro

[0154] 1. Immunodeficient Mouse Transplantation Experiment

[0155] 1) On the day of transplantation, NOG immunodeficient mice were irradiated with X-rays at a dose of 120 cGy, and transplantation was performed 4 hours after the end of irradiation.

[0156] 2) Umbilical cord blood hCD34 + The method for harvesting cells cultured on microcarrier well plates was the same as steps 1 to 9 in Example 1, and the grouping was the same as in Example 1: A. Fresh group B. Control group C. Microniche group.

[0157] 3) Wash twice with sterile PBS buffer in a clean bench, centrifuge at 1500 r / min for 10 min at 4°C, and discard the supernatant.

[0158] 4) Dilute the cells to the appropriate concentration using sterile PBS buffer according to the cell number gradient as follows:

[0159] A. Uncultured group: 100,000, 50,000, 10,000, 1,000, 100, 10 CD34 + cell;

[0160] B. Blank control group 50000, 10000, 2000, 400, 80, 16 corresponding to the initial CD34 + cell;

[0161] C. The three-dimensional microcarrier groups of 100,000, 20,000, 4,000, 800, 160, and 32 correspond to the initial CD34 + cell;

[0162] NOG immunodeficient mice were injected into the tail vein at 200 μL per mouse.

[0163] 5) Take 10 μL tail blood at the 4th, 8th, 12th and 16th week after transplantation to detect human CD45 + Implantation rate.

[0164] 6) 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.

[0165] 7) 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 50% of the bone marrow cells from each mouse for secondary transplantation. 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.

[0166] 8) Prepare an antibody mixture using PBS buffer containing 2% FBS. Add an appropriate amount of the antibody mixture to the flow cytometry tube to label the cells. Mix thoroughly by pipetting and incubate at 4°C in the dark for 30-60 minutes.

[0167] 9) After incubation, add 1 mL of PBS buffer to each tube to wash the cells. Centrifuge at 1500 rpm for 10 min at 4°C, discard the supernatant, and resuspend in 100-200 μL of PBS buffer. Analyze on a BD LSRII / LSRFortessa / CantoII flow cytometer and analyze the expression of human CD45 in mouse bone marrow using FlowJo 10 software. + The cell ratio was determined to be a successful transplantation if there was an obvious positive group. The transplantation success rate of each cell dose transplantation group was calculated to calculate the long-term stem cell frequency. The mouse CD45 - human CD45 + Human CD33 in cells + 、CD19 + 、CD34 + CD38 - 、CD34 + CD38 + 、CD3 + 、CD56 + CD45 - human CD45 - CD235a + 、CD71 + The cell ratio is used to determine the differentiation potential of each lineage of implanted HSCs. 2. Experimental conclusion:

[0168] Depend on Figure 6 The displayed data showed that the long-term stem cell frequency in the three-dimensional microcarrier group (1 / 2394) was significantly higher than that in the blank control group (1 / 16624) (with significant difference, p<0.001) and the uncultured group (1 / 11994) (with significant difference, p<0.01); the long-term stem cell frequency in the three-dimensional microcarrier group was 6.94 times that of the control group and 5.01 times that of the uncultured group, respectively.

[0169] The average implantation levels of the three-dimensional microcarrier group in the 20,000 and 100,000 cell dose groups were 24.37% and 29.45%, respectively, while the average implantation level of the uncultured group in the highest cell dose group of 100,000 cells was 24.45%, and the average implantation level of the control group in the highest cell dose group of 50,000 cells was 8.11%. It can be seen that the cell transplantation implantation level after three-dimensional microcarrier culture is higher, and fewer cells are required, that is, the transplantation success rate is higher.

[0170] Depend on Figure 6Flow cytometry analysis of myeloid, B lymphocyte, T lymphocyte, natural killer cell, and red blood cell surface molecule markers in mouse bone marrow (mouse CD45 - human CD45 + Human CD33 in cells + 、CD19 + 、CD34 + CD38 - 、CD34 + CD38 + 、CD3 + 、CD56 + CD45 - human CD45 - CD235a + 、CD71 + ) detection, in the three-dimensional microcarrier group of mice with successful xenotransplantation, normal differentiation and reconstruction of various lineages (myeloid, B lymphoid, T lymphoid, natural killer cells, and red blood cells) were observed, without obvious partial differentiation. Compared with the uncultured group and the control group, the number of human CD45 + There was no significant difference in the proportion of each lineage within the cells.

[0171] Figure 6 The experimental steps of secondary transplantation and the results of the success rate of secondary transplantation were further given: the secondary transplantation success rate of the three-dimensional microcarrier group (22.2%) was higher than that of the uncultured group (16.3%) and the control group (3.3%).

[0172] The above results indicate that the three-dimensional microcarrier group has the function of expanding human functional long-term hematopoietic stem cells in vitro. The three-dimensional microcarrier in vitro culture system can improve the transplantation success rate and has broad prospects.

[0173] Example 6 Analysis of the Effect of Three-Dimensional Microcarriers on Promoting Human Megakaryotic Stem and Progenitor Cells

[0174] 1. Experimental methods:

[0175] 1) Freshly prepare SFEMⅡ culture medium containing 1% PS, 100ng / ml hTPO, 100ng / ml hSCF, and umbilical cord blood hCD34 + The cell culture method on the microcarrier plate was the same as steps 1 to 7 of Example 1. The grouping was the same as in Example 1: A. Fresh group B. Control group C. 3D microcarrier group D. Small molecule compound group (UM171). Multiple replicate wells were arranged for each group and cultured in a constant temperature incubator at 37°C and 5% CO2.

[0176] 3) Every two weeks, observe the cell status under a microscope. Repeatedly pipette and mix the cells or the cell-material complex in a clean bench. Filter through a 30 μm sterile nylon membrane and transfer from the well plate to a flow tube. Wash the well plate and nylon membrane once with an appropriate amount of PBS buffer.

[0177] 4) After centrifugation at 1500 r / min for 10 min, discard the supernatant and resuspend the cells in an appropriate amount of PBS buffer. Take 10 μl of the harvested cell suspension and stain with 0.4% Trypan Blue Solution to count the living cells.

[0178] 5) Based on the count results, remove an appropriate amount of cells for flow cytometry analysis, CFC assay, CFU-Mk assay, RNA cryopreservation, and subculture until the end of the 8th week of culture.

[0179] 6) Cells used for flow cytometry analysis:

[0180] ① Centrifuge at 1500r for 10min and discard the supernatant. Adjust the cell concentration per flow cytometry tube to 2×10 5 -1×10 6 Between the two groups, add 100 μl of the prepared antibody mixture (APC-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, FITC-conjugated anti-Human CD71, BV421-conjugated anti-Human CD110, BV510-conjugated anti-Human CD41a flow cytometry antibody mixture prepared in PBS buffer containing 2% FBS), pipette thoroughly to mix, and incubate at 4°C in the dark for 30-60 minutes. + The cells were treated with the same antibody.

[0181] ②After incubation, add 1 ml of PBS buffer to each tube to wash the cells, centrifuge at 1500r for 10 min, discard the supernatant, resuspend the cells in an appropriate amount of PBS buffer, and stain with DAPI before loading.

[0182] ③ BD FACS CANTOⅡ / LSRⅡ / AriaIII flow cytometer was used for detection and FlowJo 10 software was used to gate and analyze the cell ratio and absolute number.

[0183] 2. Experimental conclusions:

[0184] By analyzing the surface markers of stem cell subsets and megakaryocyte subsets, we further verified the unexpected effect of 3D microcarriers in culturing functional hematopoietic stem cells. The results showed that:

[0185] Compared with B. blank control group (control) and D. small molecule compound group (UM171), C. three-dimensional microcarrier group CD34 + CD38 - CD45RA - CD90 + The proportion of stem cell population has remained high since week 4, with CD34 + CD38 - CD45RA - CD90 + CD49f + The proportion of stem cell population reached its peak at week 4, and the corresponding cumulative absolute number was also significantly higher (p<0.0001); while the megakaryocyte progenitor CD34 + CD38 + CD45RA - CD71 + CD110 + and CD34 + CD38 + CD41 + The proportion of subpopulations reached its peak at the 6th week, indicating that 3D microcarriers can maintain the stemness of human hematopoietic stem cells in vitro for a long time and promote the bias of megakaryocyte lineage at the hematopoietic stem cell level (see Figure 7 ).

[0186] Example 7 Verification of the Effect of Three-Dimensional Microcarriers on the Expansion of Human Megakaryotic Hematopoietic Stem Cells in Vitro

[0187] 1. Immunodeficient mouse transplantation experimental method:

[0188] The specific steps are consistent with steps 1) to 6) of Example 5, and the same three-dimensional microcarrier group (microniche), uncultured group (fresh) and blank control group (control) are set.

[0189] 1) After transplantation, the mouse bone marrow cells were added to an appropriate amount of PBS buffer and centrifuged at 1500r for 10 minutes at 4°C. The supernatant was discarded and the cells were resuspended in an appropriate amount of PBS buffer. 100μl of the prepared antibody mixture was added to the flow cytometry tube to label the cells. After pipetting and mixing, the cells were incubated at 4°C in the dark for 30-60 minutes. The antibody mixture was prepared in PBS buffer containing 2% FBS: APC-Cy7-conjugated anti-Mouse CD45, APC-conjugated anti-Mouse CD41, Percp-Cy5.5-conjugated anti-Mouse CD42d, 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-conjugated anti-HumanCD61, Alexa Fluor700-conjugated anti-Human CD235a, BV711-conjugated anti-HumanCD71 flow cytometry antibody mixture;

[0190] 2) After incubation, 1 mL of PBS buffer was added to each tube to wash the cells. The cells were centrifuged at 1500 r / min for 10 min at 4°C, and the supernatant was discarded. The cells were resuspended in 100-200 μL of PBS buffer and stained with DAPI before loading. The cells were loaded onto a BD Aria III flow cytometer and gated using FlowJo10 software to detect mouse CD45 in mouse bone marrow. - CD41 - CD42d - CD61 - human CD45 - Human CD41a + 、CD41a + CD61 + CD42b - 、CD41a + CD61 + CD42b + Cell ratio, mouse CD45 - human CD45 + CD34 + CD38 + Human CD41a +、CD71 + CD110 + 、CD71 - CD110 + The cell ratio was used to determine the reconstruction of the transplanted megakaryocyte lineage, and the reconstruction success rate of each megakaryocyte phenotype in each cell dose transplantation group was calculated to calculate the frequency of long-term megakaryocyte-biased stem cells.

[0191] 2. Experimental conclusions:

[0192] Depend on Figure 8 It can be seen that the umbilical cord blood CD34 + Compared with the fresh group and the blank control group, the expression of mouse CD45 - CD41 - CD42d - CD61 - human CD45 - Megakaryocyte CD41a represents three phenotypes of cells with different maturation levels + 、CD41a + CD61 + CD42b - 、CD41a + CD61 + CD42b + The flow cytometry positive groups were clearly divided, and the reconstruction success rate was improved. According to the ELDA calculation of the three phenotypic megakaryocyte groups, it was found that the frequency of functional megakaryocytes tending to be long-term hematopoietic stem cells in the three-dimensional microcarrier culture group was higher than that in the uncultured group. + 、CD41a + CD61 + CD42b - 、CD41a + CD61 + CD42b + The calculated frequencies of the three-dimensional microcarrier group were 3.36 (p<0.05), 2.85 and 2.72 times that of the uncultured group, respectively. This result indicates that the biomimetic microcarrier has the ability to expand functional human megakaryocyte-biased hematopoietic stem cells.

[0193] Example 8: In vitro culture based on three-dimensional microcarriers regulates cytokine reprogramming and immune cell subsets

[0194] 1. Analysis object:

[0195] Umbilical cord blood hCD34 +The methods of culturing cells on the three-dimensional microcarrier plate and harvesting cells were the same as steps 1 to 9 of Example 1, and the grouping was the same as that of B. blank control group and C. three-dimensional microcarrier group in Example 1.

[0196] Among them, the objects of PCR, transcriptome sequencing and 10x Genomics single-cell sequencing were CD34 cells obtained by magnetic bead sorting after two groups of culture. + The Luminex liquid suspension array was used to detect the supernatant excluding cells after the two groups of cultures, and the protein spectrum quantitative analysis was performed on the total cells after the two groups of cultures.

[0197] 2. The specific analysis method is:

[0198] (1) Real-time fluorescence quantitative PCR:

[0199] 1) Cellular RNA was extracted using the RNeasy Micro Kit (QIAGEN), and the quantity and quality of RNA were detected using a NanoDrop 2000 fluorescence spectrometer (ThermoFisher Scientific).

[0200] 2) Refer to the Invitrogen M-MLV Reverse Transcriptase instructions. The mRNA reverse transcription reaction system is as follows:

[0201]

[0202] 3) In a Nuclease-free EP tube, mix Oligo(dT)18, Total RNA, dNTPs, and Nuclease-free water according to the table above. Heat the mixture in a thermostatic metal bath at 65°C for 5 minutes and place on ice.

[0203] 4) After a brief centrifugation, add 5× First-Strand Buffer and 0.1 M DTT (RNase inhibitor can be added optionally) to the EP tube, mix gently, and heat in a thermostatic metal bath at 37°C for 2 min.

[0204] 5) Add 1 μl of M-MLV RT to a Nuclease-free EP tube and mix thoroughly by pipetting gently. Incubate in a thermostatic metal bath at 37°C for 50 min. Terminate the reaction by heating in a thermostatic metal bath at 70°C for 15 min.

[0205] 6) Add 180 μl of nuclease-free water to the reverse transcribed cDNA, mix thoroughly, and store at -20°C.

[0206] 7) Using the SYBR Green dye method (refer to the Takara SYBR@Premix Ex Taq™ (Tli RNaseHPlus) instructions, 10 μl for a 384-well plate system), the real-time quantitative PCR reaction system is as follows:

[0207]

[0208] 8) Vortex the reaction mixture and carefully add each well to the corresponding 384-well PCR plate. After brief centrifugation, place in the Q6 Real-Time PCR instrument and set the reaction conditions as follows:

[0209]

[0210] 9) The melting curve program was the instrument default, and the experimental data were analyzed according to the ΔΔCt method.

[0211] (2) Transcriptome sequencing analysis

[0212] Prepare 1×10 6 CD34 + Cells were used for sequencing. RNA extraction, library preparation, sequencing, and data analysis were performed by Novogene (Beijing, China). 3 μg of RNA from each sample of three replicates in each group was used as the starting material. UltraTM RNA Library Prep Kit for (NEB, USA) to generate sequencing libraries. Sequencing was performed on the cBotCluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After mapping to the reference genome and quantification, differentially expressed genes were analyzed using DESeq2 (R package, 1.10.1). The criteria for establishing |Log2(FoldChange)|>1 and q value<0.05 were considered significant. Significantly changed genes were used for subsequent GO analysis and KEGG enrichment analysis. The GO term results of molecular function (MF) were summarized and clustered based on semantic similarity metrics using the online tool REVIGO.

[0213] (3) Gene set enrichment analysis (GSEA)

[0214] The total gene list obtained by transcriptome sequencing was used for GSEA analysis. The reference gene set was searched from the GSEA gene set database (http: / / www.gsea-494msigdb.org / gsea / msigdb / index.jsp), specifically:

[0215] KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION,

[0216] GRAHAM_NORMAL_QUIESCENT_VS_NORMAL_DIVIDING_UP, GOBP_CELL_CYCLE_ARREST,

[0217] REACTOME_FACTORS_INVOLVED_IN_MEGAKARYOCYTE_DEVELOPMENT_AND_PLATELET_PRODUCTION, GO_MEGAKARYOCYTE_DEVELOPMENT

[0218] (4) Quantitative analysis of protein spectrum

[0219] Prepare 1×10 6 Total cells. Protein extraction, trypsinization, mass spectrometry (MS), and data analysis were performed by Novogene (Beijing, China). Cells were lysed after trypsin digestion to extract proteins. Peptides were separated using a homemade analytical column using a linear gradient elution. The separated peptides were then analyzed using a Q Exactive HF mass spectrometer (Thermo Fisher). The raw data were individually searched in the homo_sapiens_uniprot_2019.01.18 database using the search engine Proteome Discoverer 2.2 (PD 2.2, Thermo). Protein families and pathways were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG).

[0220] (5) Detection using Luminex liquid suspension chip

[0221] Luminex liquid suspension array assays were performed by Huaying Biopharmaceutical Technology Co., Ltd. (Shanghai, China). The Bio-Plex Pro Human Chemokine Panel 40-plex kit was used according to the manufacturer's instructions. Conditioned medium from three replicates of each group was incubated in a 96-well plate embedded with magnetic beads for 1 hour, followed by the addition of detection antibodies and a 30-minute incubation. Streptavidin-PE was then added to each well for a 10-minute reaction, and data were read using the Bio-Plex MAGPIX system (Bio-Rad).

[0222] (6) 10x Genomics single-cell sequencing (scRNA-seq)

[0223] Prepare about 1×10 4 CD34 + Cells were used for sequencing. Library construction, sequencing, and matrix data were completed by Novogene (Beijing, China). For experiments using the 10X Genomics platform, Chromium Single Cell 3'Library & Gel Bead Kit v2 (PN-120237), Chromium Single Cell 3'Chip kit v2 (PN-120236), and Chromium i7 Multiplex Kit (PN-120262) were used according to the manufacturer's instructions in the Chromium Single Cell 3'Reagents Kitsv2 User Guide. The library was run on an Illumina Hiseq PE150 sequencing run. Reads were calibrated according to the GRCh38 reference assembly (v2.2.0, 10X Genomics). Novogene used the 10X Cell Ranger software package (v2.1.0, 10X Genomics) for post-processing and quality control. Subsequent analyses of Cell Ranger and Seura were performed based on this output gene expression matrix. Seurat R-package (4.0.1) was used for data normalization, dimensionality reduction, clustering, and differential expression.

[0224] 3. Experimental conclusions:

[0225] The differences in mRNA profiles between the three-dimensional microcarrier group and the blank control group were analyzed based on transcriptome sequencing, and the following conclusions were drawn: 273 genes were upregulated in microcarrier cultured cells, Figure 9 As shown in a, GO functional annotation and KEGG pathway enrichment analysis revealed that several significantly differentially expressed gene sets and pathways were regulated by microcarrier culture, including chemokine- and cytokine-related pathways.

[0226] Based on gene set enrichment analysis (GSEA), we evaluated the gene sets related to cytokine-cytokine receptor interactions, stemness-related gene sets (quiescence, cell arrest), and megakaryocyte lineage development reported in the literature and databases, and obtained the following conclusions: Figure 9 The five gene sets shown in b were all enriched in the three-dimensional microcarrier group.

[0227] KEGG enrichment analysis based on protein spectrum data was performed by Figure 9 c The following conclusions were obtained: focal adhesion and platelet-activating protein were activated in microcarrier-cultured cells, which was consistent with the RNA-seq results, indicating that three-dimensional microcarrier culture in vitro promoted megakaryocyte lineage activation. Figure 9d Real-time quantitative PCR again confirmed that most genes in the cytokine-cytokine receptor pathway were upregulated after microcarrier culture.

[0228] Based on Luminex liquid suspension chip detection, the differences of 40 chemokines in blank control and three-dimensional microcarrier culture cells were quantitatively compared. Figure 9 The following conclusions were drawn: the levels of most of the tested factors were increased in the 3D microcarrier group. These results suggest that the cytokine profile is reprogrammed in the 3D microcarrier culture environment.

[0229] Based on the dimensionality reduction and UMAP visualization analysis of 10x Genomics single-cell sequencing (scRNA-seq) results, the following conclusions were obtained: Figure 10 Nine cell clusters (C0-8) were annotated based on their corresponding canonical marker genes. Among them, C1, C6, and C8 showed a higher proportion of cells originating from the microcarrier group than the blank control group. Based on the enrichment of megakaryocyte (Mk) and erythroid (Er) lineage marker genes, such as IGTA2B (CD41), VWF, ITGB3 (CD61), NFE2, GATA1, and TFRC (CD71), as well as stem cell marker genes TAL1, IGTA6 (CD49f), and HES1, C6 was annotated as a Mk and Er stem cell progenitor. These results are consistent with the promotion of megakaryocyte lineage activation by three-dimensional microcarrier culture.

[0230] The typical marker genes of each cluster were searched in the CellMarker database, and the other clusters were defined as C0: granulocyte-monocyte progenitor 1 (GMP1); C1: CD4 + Plasmacytoid dendritic cells (pDC); C2: multipotent lymphocyte progenitor 1 (MLP1); C3: multipotent lymphocyte progenitor 2 (MLP2); C4: Pre-NK / T, C5: unknown; C7: unknown; C8: granulocyte-monocyte progenitor 2 (GMP2). In addition, Figure 9 The cytokine pathway genes shown in a (RNA-seq) and d (qRT-PCR) were mainly expressed in C1, with the main contribution from cells obtained from three-dimensional microcarrier group culture.

[0231] In addition, based on CellPhoneDB cell communication ( Figure 10 k) Analysis of cytokine-receptor communication within each cell cluster revealed that C1 communicated extensively with other cell clusters. These results suggest that a specific cell population (C1) in the 3D microcarrier cell culture system triggers environmental cytokine reprogramming, potentially contributing to megakaryocyte lineage activation and differentiation.

[0232] The above results indicate that three-dimensional bionic microcarriers cultured in vitro have the function of regulating cytokine reprogramming and immune cell subsets.

[0233] Example 9: Bionic microcarriers promote the effect of specific cell subpopulations, namely megakaryocyte hematopoietic stem cells, in vitro culture

[0234] 1. Analysis object:

[0235] Umbilical cord blood hCD34 + The cell culture and cell harvesting methods of the cell microcarrier plate were the same as steps 1 to 9 of Example 1, and the grouping was the same as that of Example 1, B. blank control group and C. microcarrier group.

[0236] The BD Rhapsody single-cell sequencing targets were CD34 cells sorted by FACS ArialIII flow cytometer after two groups of cultured cells were sorted. + CD38 - CD45RA - CD90 + cell.

[0237] 2. Specific analysis methods:

[0238] 2.1 Flow cytometry analysis method:

[0239] 1) Umbilical cord blood hCD34 + After the cells were cultured on the microcarrier plate and harvested, the cell concentration per flow cytometry tube was adjusted to 2×10 5 -1×10 6 Between the two groups, 100 μL of the prepared 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, BV510-conjugated anti-human CD49f, PE-conjugated anti-human CD62L, APC-conjugated anti-human CD133 flow cytometry antibody mixture prepared in PBS buffer containing 2% FBS) was added, pipetted thoroughly to mix, and then incubated at 4°C in the dark for 30-60 minutes. + The cells were treated with the same antibody.

[0240] 2) After incubation, add 1 ml of PBS buffer to each tube to wash the cells. Centrifuge at 1500 r / min for 10 min, discard the supernatant, resuspend the cells in an appropriate amount of PBS buffer, and stain with DAPI before loading.

[0241] 3) BD FACS CANTOⅡ / AriaIII flow cytometer was used for detection and FlowJo 10 software was used to gate and analyze the cell ratio and absolute number.

[0242] 2.2BD Rhapsody single-cell sequencing analysis:

[0243] 1) BD FACS AriaIII flow cytometry sorter was used to sort 2×10 4 CD34 + CD38-CD45RA-CD90 + cell.

[0244] 2) Add appropriate amount of AbSeq antibody against CD34, CD38, CD45RA, CD90, and CD49f, incubate on ice for 30 minutes, and add appropriate amount of BD Pharmingen TM Wash the cells 2-3 times with Stain Buffer (FBS) by centrifugation.

[0245] 3) Use a low-adsorption pipette tip to draw up 620 μL of pre-chilled Sample Buffer (Cat. No. 650000062) and gently pipette the cells to mix. Add 3.1 μL of 2 mM Calcein AM (BD Biosciences, Cat. No. 564061) and 3.1 μL of 0.3 mM Draq7 (BD Biosciences, Cat. No. 564904) and gently pipette to mix approximately 10 times. Incubate at 37°C in the dark for 5 minutes, then draw up 10 μL of the sample for viable cell counting. Place the remaining cells on ice until ready for use.

[0246] 4) Set the P1200M pipette to Prime / Treat mode and process the cartridge as follows:

[0247] ①100% ethanol, 700 μL

[0248] ②Air

[0249] ③Cartridge Wash Buffer 1, 700μL, room temperature 1min

[0250] ④Air

[0251] ⑤Cartridge Wash Buffer 1, 700μL, room temperature 10min

[0252] ⑥Air

[0253] ⑦Cartridge Wash Buffer 2, 700 μL, room temperature ≤ 4 hours

[0254] 5) Loading cells and magnetic beads: Set the P1200M pipette to Prime / Treat mode, aspirate 700 μL of air into the cartridge, then switch the P1200M pipette to Cell Load mode to load the cells. Carefully remove the loaded cartridge and immediately place it in the BD Scanner for counting.

[0255] 6) Loading Magnetic Beads: Set the P1200M pipette to Prime / Treat mode, aspirate 700 μL of air into the cartridge, and immediately set the P1200M pipette to Bead Load mode to load the magnetic beads. Let stand for 3 minutes before scanning.

[0256] 7) Washing magnetic beads: Set the P1200M pipette to the Wash mode and treat the cartridge twice with 720 μL of pre-cooled sample buffer before scanning.

[0257] 8) Lyse cells, recover captured beads, and wash magnetic beads: Set the P1200M pipette to Lysis mode and aspirate 550 μL of Lysis Buffer containing DTT. After 2 minutes, slide the front slider to "BEADS" and the left slider to "RETRIEVAL." Set the P5000M pipette to Retrieval mode. After 30 seconds, slide the front slider to "0" and add 4950 μL of Lysis Buffer containing DTT. Slide the front slider to "OPEN," remove the 5 mL low-binding tube, and immediately place it on a 15 mL magnetic rack for 1 minute. Wash the beads immediately. Scan the cartridge again.

[0258] 9) Reverse Transcription: Prepare 200 μL (1 library) of cDNA Mix (containing 40 μL RT Buffer, 20 μL dNTPs, 10 μL RT 0.1M DTT, 12 μL Bead RT / PCR Enhancer, 10 μL RNase Inhibitor, 10 μL Reverse Transcriptase, and 98 μL nuclease-free water) and incubate on a metal shaker at 1200 rpm, 37°C, for 45 min.

[0259] 10) Exonuclease I Treatment: Prepare 200 μL (1 library) of Exonuclease I Mix (containing 20 μL Exonuclease I Buffer, 10 μL Exonuclease I, and 170 μL nuclease-free water). Incubate on a metal shaker at 1200 rpm at 37°C for 30 min. Incubate on a metal block at 80°C for 20 min without shaking. Incubate on ice for 1 min, remove from a magnetic rack, and discard the supernatant. Resuspend in 200 μL of pre-chilled Bead Resuspension Buffer (Cat. No. 650000066) and store at 4°C.

[0260] 11) Construction of RPE for WTA library:

[0261] Prepare Random Primer Mix: For 1 library: 20 μL WTA Extension Buffer, 20 μL WTA Extension Primers, 134 μL nuclease-free water.

[0262] Place the beads on a magnetic rack and discard the supernatant. Resuspend in 75 μL Elution Buffer, incubate at 95°C for 5 minutes (without shaking), and remove the supernatant from the magnetic rack. Wash the beads with 200 μL Elution Buffer and discard the supernatant from the magnetic rack. Resuspend in Random Primer Mix and follow the steps below:

[0263] Incubate at 95°C for 5 minutes (without shaking);

[0264] Incubate at 37°C for 5 minutes (shaking at 1200 rpm);

[0265] Incubate at 25°C for 15 minutes (shaking at 1200 rpm).

[0266] Prepare Primer Extension Enzyme Mix. For 1 library: 8 μL 10 mM dNTPs, 12 μL Bead RT / PCR Enhancer, and 6 μL WTA Extension Enzyme. Follow the steps below:

[0267] Incubate at 25°C for 10 minutes (shaking at 1200 rpm);

[0268] Incubate at 37°C for 15 minutes (shaking at 1200 rpm);

[0269] Incubate at 45°C for 10 minutes (shaking at 1200 rpm);

[0270] Incubate at 55°C for 10 minutes (shaking at 1200 rpm);

[0271] Discard the supernatant from the magnetic rack. Resuspend in Elution Buffer and incubate at 95°C for 5 minutes (without shaking). Shake at 1200 rpm for 10 seconds. Remove the supernatant from the magnetic rack and resuspend the beads in 200 μL of pre-chilled Bead Resuspension Buffer. Store at 4°C.

[0272] 12) PCR1 for WTA+AbSeq+SMK library construction,

[0273] Step 12) uses the following reagents:

[0274] Component For 1library(μL) PCR Master Mix(Cat.No.91-1118) 100 Universal Oligo(Cat.No.650000074) 20 Bead RT / PCR Enhancer(Cat.No.91-1082) 12 Sample Tag PCR1 Primer(Cat.No.91-1088) 1.2 AbSeq PCR1 Primer(Cat.No.91-1086) 12 Total 145.2

[0275] The reaction procedure is as follows:

[0276]

[0277] 13) RPE product purification: Prepare 50 mL of 80% ethanol and AMPure XP magnetic beads at room temperature and vortex thoroughly for 1 minute. Pipette 360 μL of AMPure XP magnetic beads into 200 μL of RPE product, mix thoroughly by pipetting, and incubate at room temperature for 10 minutes. Remove the supernatant from the magnetic rack. While maintaining the magnetic rack, carefully add 1 mL of 80% anhydrous ethanol and, after 30 seconds, remove the supernatant. Repeat this step once, dry at room temperature for 5 minutes, resuspend in 40 μL of Elution Buffer, incubate at room temperature for 2 minutes, and remove the supernatant from the magnetic rack.

[0278] 14) Prepare RPE PCR Mix: For 1 library: 60μL PCR MasterMix, 10μL UniversalOligo, 10μL WTA Amplification Primer;

[0279] The reaction procedure is as follows:

[0280]

[0281] 15) Purify RPE PCR products and AbSeq+SMK PCR1 products, quantify using Qubit, and measure library fragment size using 2100;

[0282] 16) Perform WTA Index PCR: For 1 library: 60 μL PCR MasterMix, 5 μL LibraryForward Primer, 5 μL nuclease-free water;

[0283] The reaction procedure is as follows:

[0284]

[0285] 17) Purify the WTA Index PCR product, quantify it using Qubit, and measure the library fragment size using 2100;

[0286] 18) Perform AbSeq Index PCR: For 1 library: 25 μL PCR MasterMix, 2 μL LibraryForward Primer, 18 μL nuclease-free water;

[0287] The reaction procedure is as follows:

[0288]

[0289] 19) Purify AbSeq Index PCR products, quantify using Qubit, and measure library fragment size using 2100;

[0290] 20) Perform Sample Tag PCR2: For 1 library: 25 μL PCR Master Mix, 2 μL Universal Oligo, 3 μL Sample Tag PCR2 Primer, 15 μL nuclease-free water;

[0291] The reaction procedure is as follows:

[0292]

[0293] 21) Purify the Sample Tag PCR2 product and quantify it using Qubit;

[0294] 22) Perform Sample Tag Index PCR: For 1 library: 25 μL PCR Master Mix, 2 μL Library Forward Primer, 18 μL nuclease-free water;

[0295] The reaction procedure is as follows:

[0296]

[0297] 23) Purify the Sample Tag Index PCR product, quantify it using Qubit, and measure the library fragment size using 2100;

[0298] 24) BD SeqGeq software was used to analyze the single-cell sequencing results.

[0299] 3. Experimental conclusions:

[0300] (1) Generally speaking, CD49f expression is divided into CD49f negative (CD49f - ), CD49f weak positive (CD49f low) and CD49f strong positive (CD49f high) three subgroups, while CD49f positive (CD49f + ) includes CD49f low and CD49f high, which are Figure 11 and Figure 13 It is known that in human umbilical cord blood CD34 + cells or bone marrow mononuclear cells, CD34 + CD38 - CD45RA - CD90 + The distribution of CD49f expression in the phenotype was different in the uncultured group, blank control group and three-dimensional microcarrier group: In the uncultured group and three-dimensional microcarrier group, CD49f + CD49f low In the blank control group, CD49f high was the main subpopulation, and this phenomenon was more obvious in cultured bone marrow mononuclear cells. + cells or bone marrow mononuclear cells, which

[0301] CD34 + CD38 - CD45RA - CD90 + CD49f low The proportion and absolute number of subpopulation cells were increased compared with the uncultured group and the blank control group.

[0302] (2) Based on previous literature on stem cell and megakaryocyte marker genes, primitive stem cell marker genes tend to be enriched in the C2 subpopulation; multipotent progenitor cell marker genes that are biased towards the megakaryocyte lineage are enriched in the C3 subpopulation; and megakaryocyte progenitor cell marker genes tend to be enriched in the C3 and C4 subpopulations. Visualization of pseudo-time analysis suggests that the C2 subpopulation is at the top of the hematopoietic hierarchy.

[0303] Depend on Figure 12 The results of single-cell sequencing showed that k-means clustering analysis showed that CD34 + CD38 - CD45RA - CD90 + The cells were divided into 4 subpopulations, which clearly matched the distribution of CD49f: CD49f - (C1), CD49 flow(C2) and CD49f high The 3D microcarrier group contained nearly all C2 cells and a subset of C3 cells, while the blank control group contained mostly C1 and C4 cell subsets. Therefore, C2 is considered an important subpopulation for maintaining HSC function mediated by 3D microcarriers. Among the marker genes of the C2 subpopulation, the inventors' team discovered two surface markers: SELL (CD62L) and PROM1 (CD133).

[0304] (3) By Figure 13 By using umbilical cord blood CD34 + Flow cytometry analysis of cells and bone marrow mononuclear cells revealed a specific CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 + The cell population was only contained in the uncultured group and the three-dimensional microcarrier group, but was almost lost in the blank control group cells. This phenomenon was more obvious in cultured bone marrow mononuclear cells.

[0305] The above results indicate that the three-dimensional microcarrier culture in vitro maintains the natural subpopulation of human megakaryocyte-biased hematopoietic stem cells that is easily lost in blank control culture (also present in uncultured cells), and this subpopulation is enriched in CD34 + CD38 - CD45RA - CD90 + CD49f low CD62L - CD133 + In the cell population, the combination of surface molecular markers of the cell population can be used as surface markers of human megakaryocyte-biased hematopoietic stem cells.

[0306] Example 10: Verification of the Effect of Three-Dimensional Microcarriers on In Vitro Expansion of Specific Cell Subpopulations Based on Immunodeficient Mouse Medullary Cavity Transplantation Experiment

[0307] 1. Experimental Methods

[0308] Bone marrow transplantation experiment in immunodeficient mice

[0309] 1) On the day of transplantation, NOG immunodeficient mice were irradiated with X-rays at a dose of 120 cGy to establish an immunodeficient mouse model. Transplantation was performed 4 hours after the end of irradiation.

[0310] 2) Umbilical cord blood hCD34 +The cell harvesting method of the three-dimensional microcarrier plate culture cells was the same as steps 1) to 7) of Example 1. The grouping was the same as in Example 1. A. Fresh group C. Three-dimensional microcarrier group (Microniche). The same batch of untreated umbilical cord blood hCD34 + Wash cells twice with sterile PBS buffer in a clean bench, centrifuge at 1500 rpm for 10 min at 4°C, and discard the supernatant.

[0311] 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 thoroughly by pipetting and incubate at 4°C in the dark for 30-60 minutes. (For flow cytometry, prepare the FITC-conjugated anti-HumanCD34, PE-Cy7-conjugated anti-Human CD38, APC-H7-conjugated anti-Human CD45RA, Percp-Cy5.5-conjugated anti-Human CD90, and PE-conjugated anti-Human CD49f antibody mixture in PBS buffer containing 2% FBS.)

[0312] 4) After incubation, add 1 mL of PBS buffer 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. Load the BD AriaIII flow cytometer with an appropriate amount of sterile PBS buffer as the receiving fluid. Perform two-way DAPI sorting on the two groups of cells. - CD34 + CD38 - CD45RA - CD90 + CD49flow (CD49flow) or DAPI - CD34 + CD38 - CD45RA - CD90 + CD49fneg&high (nonCD49flow) cells.

[0313] 5) Adjust the concentration to an appropriate level according to the number of cells obtained using sterile PBS buffer (200 cells / 25 μL / mouse), and inject the solution into the medullary cavity of anesthetized NOG immunodeficient mice.

[0314] 6) Take 10 μL tail blood at the 4th, 8th, 12th and 16th week after transplantation and detect human CD45 by flow cytometry + Implantation rate, take 15 μL tail blood and add 10 5The samples were diluted with CBC diluent (1:7) and tested by automatic hematology analyzer.

[0315] 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.

[0316] 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.

[0317] 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 CD71 Add appropriate amount of antibody mixture to each flow cytometry tube to label cells, mix thoroughly by pipetting, and incubate at 4°C in the dark for 30-60 min.

[0318] 10) After incubation, add 1 mL of PBS buffer to each tube to wash the cells. Centrifuge at 1500 r / min for 10 min at 4°C, discard the supernatant, resuspend in 100-200 μL of PBS buffer, and stain with DAPI before loading. Detect mouse bone marrow CD45 in mouse bone marrow using BD AriaIII flow cytometer and FlowJo 10 software. - CD41 - CD42d -CD61 - human CD45 - Human CD41a + The proportion of cells was used to determine the reconstruction of the transplanted megakaryocyte lineage, and the success rate of megakaryocyte phenotype reconstruction in each group was calculated.

[0319] Experimental conclusion:

[0320] Depend on Figure 14 The blood routine results (right) show that at 16 weeks after transplantation, CD49f low The number of platelets in the peripheral blood of recipient mice was significantly higher than that of nonCD49f low There was no significant difference in platelet level between the uncultured group and the 3D microcarrier group.

[0321] Figure 14 Flow cytometry showed that compared with nonCD49f low Recipient mice, uncultured group and 3D microcarrier group had more CD49f low Reconstitution of mCD45 in recipient mice - mCD41 - mCD42d - mCD61 - hCD45 - hCD41a + Megakaryocytes (with obvious megakaryocyte-positive population), and three-dimensional microcarriers CD49f low Group (7 / 9) and uncultured CD49f low The success rate of megakaryocyte reconstruction was improved compared with the group (4 / 7), and the three-dimensional microcarrier nonCD49f low Group (4 / 7) and uncultured CD49f low The success rate of megakaryocyte reconstruction was similar in both groups (4 / 7).

[0322] Based on the above experimental data, it was concluded that megakaryocyte hematopoietic stem cells were enriched in CD34 in both physiological state and three-dimensional microcarrier culture system. + CD38 - CD45RA - CD90 + CD49f low cell population, and three-dimensional microcarrier culture expanded this functional human megakaryocyte-biased hematopoietic stem cell subset in vitro.

[0323] Example 11 Collection of bone marrow mononuclear cells from patients with aplastic anemia

[0324] 1. Experimental subjects: AA aplastic anemia, NSAA non-severe aplastic anemia

[0325] The age, gender, disease, and number of cells collected of the experimental subjects are as follows:

[0326] Sample No. age gender Disease diagnosis Collect monocyte count AA1 35 F AA <![CDATA[3.31×10 6 ]]> AA2 40 F AA <![CDATA[5.15×10 6 ]]> AA3 32 F AA <![CDATA[5.75×10 6 ]]> AA4 13 F NSAA <![CDATA[1.63×10 6 ]]> AA5 9 M NSAA <![CDATA[2.76×10 6 ]]> AA6 38 F NSAA <![CDATA[5.9×10 6 ]]> AA-7 44 F AA <![CDATA[2.36×10 6 ]]> AA-8 31 F AA <![CDATA[5.8×10 6 ]]> AA-9 25 M AA <![CDATA[4×10 6 ]]>

[0327] 2. The culture method comprises the following steps:

[0328] 1) Collect the patient's bone marrow sample in an anticoagulant tube, centrifuge at 1200r / min for 5 minutes, and discard the upper serum.

[0329] 2) Add the same amount of sterile PBS buffer as the centrifuged sample to the remaining sample to dilute the sample and mix thoroughly by pipetting.

[0330] 3) Take another sterile 15ml centrifuge tube, mark it, and add 2 / 3 of the volume of the lymphocyte separation medium after diluting the sample with PBS. Use a disposable pipette to carefully transfer the diluted sample onto the surface of the lymphocyte separation medium, taking care not to break the interface and keep it clear. Centrifuge at 1800r / min for 20min (speed increase 3, speed decrease 3).

[0331] 4) After centrifugation, the liquid surface is separated into three layers. Carefully aspirate the middle buffy coat layer with a 1 ml pipette tip into a 15 ml sterile centrifuge tube. Add 6-8 times the volume of PBS buffer, pipette to mix thoroughly, and centrifuge at 1500 rpm for 10 min.

[0332] 5) After centrifugation, the supernatant was discarded to obtain mononuclear cells, which were resuspended in an appropriate amount of PBS and mixed thoroughly by pipetting. 10 μl of the cells were taken and stained with 0.4% Trypan Blue Solution for counting.

[0333] 6) Freshly prepared IMDM medium containing 10% fetal bovine serum (FBS), 1% PS, 100 ng / ml hTPO, 100 ng / ml hSCF, 100 ng / ml hFlt3L, 100 ng / ml hIL-3, 100 ng / ml hIL-6, and 100 ng / ml hEPO was used to resuspend the freshly isolated monocytes and culture the cells in a 37°C, 5% CO2 incubator.

[0334] 3. Experimental conclusions:

[0335] After culture, the living cells were counted, washed thoroughly with PBS buffer, and prepared into a single-cell suspension for later use; or resuspended with cell freezing solution and stored in liquid nitrogen using the gradient freezing method.

[0336] Example 12 Analysis of the Effect of Three-Dimensional Microcarriers on the Expansion of Phenotypic Hematopoietic Stem Cells from Patients with Aplastic Anemia

[0337] 1. Culture method and flow cytometry method:

[0338] 1) Resuspend the patient bone marrow mononuclear cells isolated in Example 11 to 2×10 6 / ml for future use.

[0339] 2) Set up A. 3D microcarrier group (microniche) and B. blank control group (control)

[0340] A. For the three-dimensional microcarrier group, the carriers were plated into a 24-well plate at a concentration of 5 mg / well (2.5 mg / ml) and irradiated with UV light for 20-30 min. 100 μl of cell suspension (i.e., 2×10 5 Add monocytes) into the wells to allow the cells and carriers to fully absorb the cell suspension. Add 2 ml of PBS buffer to the culture wells at the edge of the well plate. After placing it in the incubator for 2 hours, add 1.9 ml of culture medium to a final system of 2 ml. Gently stir the microcarriers with a pipette tip to disperse them and evenly distribute them in the culture medium. The initial cell concentration for plating is 2 × 10 5 / hole.

[0341] B. For the blank control group, add 1.9 ml of culture medium to each well, then pipette 100 μl of cell suspension into the well and mix thoroughly by pipetting repeatedly.

[0342] 2) The cells were placed in a 37°C, 5% CO2 constant temperature incubator and cultured for 12 days.

[0343] 3) After the culture is completed, the cells or the cell-material complex are repeatedly pipetted and mixed, and filtered through a 30 μm sterile nylon membrane while being transferred from the well plate to a flow tube. The well plate and nylon membrane are washed once with an appropriate amount of PBS buffer.

[0344] 4) After centrifugation at 1500 r / min for 10 min, discard the supernatant and resuspend the cells in an appropriate amount of PBS buffer. Take 10 μl of the harvested cell suspension and stain with 0.4% Trypan Blue Solution to count the living cells.

[0345] 5) Centrifuge at 1500 rpm for 10 min, discard the supernatant, adjust the cells to an appropriate concentration in each flow cytometry tube, add 100 μl of the prepared antibody mixture (APC-conjugated anti-Human CD34, PE-Cy7-conjugated anti-Human CD38, PE-conjugated anti-Human CD49f flow cytometry antibody mixture prepared in PBS buffer containing 2% FBS), mix thoroughly by pipetting, and incubate at 4°C in the dark for 30-60 min.

[0346] 6) After incubation, add 1 ml of PBS buffer to each tube to wash the cells. Centrifuge at 1500 r / min for 10 min, discard the supernatant, and resuspend the cells in an appropriate amount of PBS buffer. Stain with DAPI before loading.

[0347] 7) BD FACS CANTOⅡ flow cytometer was used for detection and FlowJo 10 software was used to gate and analyze the cell ratio and absolute number.

[0348] 3. Experimental conclusions:

[0349] Depend on Figure 15 and Figure 16 It is known that after the bone marrow mononuclear cells of patients with aplastic anemia were cultured in vitro on three-dimensional microcarriers, the stem cell progenitor cells CD34 + CD38 - Subpopulations and stem cells CD34 + CD38 - CD49f + The proportion and absolute number of subpopulations were higher than those in the blank control group and were statistically significant. The results showed that the addition of three-dimensional microcarriers to in vitro culture has the ability to expand the phenotypic hematopoietic stem cells of patients with aplastic anemia.

[0350] Example 13 Analysis of the Effect of Three-Dimensional Microcarriers on Promoting Hematopoietic Stem Cell Cloning in Patients with Aplastic Anemia

[0351] 1. The CFC method is as follows:

[0352] Methods for culturing and harvesting bone marrow mononuclear cells from patients with aplastic anemia on three-dimensional microcarrier plates were used in conjunction with Examples 11-12. The grouping was the same as in Example 11: A. three-dimensional microcarrier group (microniche), B. blank control group (control).

[0353] 1) Add appropriate amount of IMDM medium to resuspend the cultured cells and adjust the cell concentration to 5×10 4 After mononuclear culture, cells / 100μl system.

[0354] 2) Add the adjusted cell suspension to H4434 semi-solid medium thawed overnight at 4°C at a ratio of 1:10 (100 μl of cell suspension per ml of H4434 semi-solid medium). Vortex thoroughly to mix and allow air bubbles to float to the surface and dissipate. Plate the cells in a 6-well plate, with 4-5 replicate wells per group.

[0355] 3) Use a screw-cap syringe with a Syringes needle to draw up the semisolid cell suspension and add 1 ml to each of the two center wells of a 6-well plate. Gently shake to evenly distribute the semisolid suspension across the entire bottom surface of the plate. Seal the plate with PBS buffer and carefully place in a 37°C, 5% CO2 incubator for incubation.

[0356] 4) After 14-16 days of culture, observe the culture plates daily under a microscope. Remove the plates when the colony density is appropriate and count and photograph the number of burst-forming units of erythrocytes (BFU-E), colony-forming units of erythrocytes (CFU-E), colony-forming units of granulocytes and macrophages (CFU-GM), and colony-forming units of granulocytes, erythrocytes, macrophages, and megakaryocytes (CFU-GEMM).

[0357] 2. Experimental conclusions:

[0358] The experimental results are shown in Figure 17 Compared with the blank control group, the number of CFU-E colonies and the total number of BFU-E colonies in the bone marrow mononuclear cells of aplastic anemia patients after three-dimensional microcarrier culture were significantly increased (p<0.001), and the total number of BFU-E colonies were significantly increased (p<0.01). There was no significant difference in the number of BFU-E and CFU-GM colonies compared with the blank control group, and there was no obvious tendency of biased differentiation.

[0359] The above results indicate that three-dimensional microcarriers can promote the formation of erythroid colony units when culturing hematopoietic stem cells from patients with aplastic anemia in vitro, and the potential of cells to differentiate into multiple lineages is maintained after culture.

[0360] Example 14 Verification of the Effect of Bionic Microcarriers on In Vitro Expansion of HSCs in Patients with Aplastic Anemia

[0361] 1. Experimental Methods

[0362] Immunodeficient mouse transplantation experiment

[0363] 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.

[0364] 2) The cell harvesting method of bone marrow mononuclear cells cultured on three-dimensional microcarrier well plates from patients with aplastic anemia was the same as in Examples 11-12, and the grouping was the same as in Example 11: A. three-dimensional microcarrier group (microniche), B. blank control group (control).

[0365] 3) Wash twice with sterile PBS buffer in a clean bench, centrifuge at 1500 rpm for 10 min at 4°C, and discard the supernatant. Resuspend in an appropriate amount of PBS buffer. Take 10 μl of the harvested cell suspension and stain with 0.4% Trypan Blue Solution to count viable cells.

[0366] 4) Choose the appropriate transplantation method based on the number of cells: If the number of cells is large, dilute the cells to 1×10 6 / ml concentration, and NOG immunodeficient mice were injected into the tail vein, 200 μL (2×10 5If the number of cells is small, intramedullary injection is used. The cells are diluted to 5×10 4 The mice were anesthetized by intraperitoneal injection of 150 μL 1.25% 2,2,2-tribromoethanol. The hair at the joint between the tibia and femur of the mice was shaved with a razor. The cells were aspirated with a 29G insulin needle and injected into the tibia along the patella. 25 μL (5×10 4 Add appropriate amount of Baynoxin to the drinking water of mice to prevent Gram-positive, Gram-negative bacteria and mycoplasma infection.

[0367] 5) Take 10 μL tail blood at the 4th, 8th, 12th and 16th week after transplantation to detect human CD45 + Implantation rate.

[0368] 6) 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.

[0369] 7) 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.

[0370] 8) Prepare an antibody mixture using PBS buffer containing 2% FBS. Add an appropriate amount of the antibody mixture to the flow cytometry tube to label the cells. Mix thoroughly by pipetting and incubate at 4°C in the dark for 30-60 minutes.

[0371] 9) After incubation, add 1 mL of PBS buffer to each tube to wash the cells. Centrifuge at 1500 rpm for 10 min at 4°C, discard the supernatant, and resuspend in 100-200 μL of PBS buffer. Analyze on a BD LSRII / LSRFortessa / CantoII flow cytometer and analyze the expression of human CD45 in mouse bone marrow using FlowJo 10 software. + The cell ratio was determined to be a successful transplantation if there was an obvious positive group. The transplantation success rate of each cell dose transplantation group was calculated to calculate the long-term stem cell frequency. The mouse CD45 - human CD45 + Human CD33 in cells + 、CD19 + 、CD3 + 、CD56 + CD45 - human CD45- CD235a + The cell ratio was used to determine the differentiation potential of each lineage of transplanted HSCs.

[0372] Experimental conclusion:

[0373] Depend on Figure 18 It can be seen that positive engraftment of hematopoietic cells from patients with aplastic anemia cultured on three-dimensional microcarriers can be observed in xenotransplantation of immunodeficient mice, while no human CD45 + Cell implantation (implantation success rate was 3 / 10 in the three-dimensional microcarrier group and 0 in the blank control group). The above results indicate that microcarriers can amplify long-term hematopoietic stem cells with in vivo hematopoietic function in patients with aplastic anemia in vitro.

[0374] 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 in vitro expansion of functional hematopoietic stem cells based on three-dimensional microcarriers, characterized in that: The method comprises adding a three-dimensional microcarrier to an expansion medium to culture human non-adherent cells, wherein the non-adherent cells are human mononuclear cells, the functional hematopoietic stem cells are human megakaryocyte-biased hematopoietic stem cells, and the surface marker of the human megakaryocyte-biased hematopoietic stem cells is CD34. + CD38 - CD45RA - CD90 + CD49f low ; The three-dimensional microcarrier is 3DTableTrix® Microslide® F01 of Huakan Biotechnology Co., Ltd.

2. The amplification method according to claim 1, characterized in that The human mononuclear cells are derived from one of bone marrow, peripheral blood and umbilical cord blood.

3. The amplification method according to claim 1, characterized in that The human mononuclear cells are collected from the blood of patients with hematological diseases.

4. The amplification method according to claim 3, characterized in that The blood disease patient is a bone marrow failure disease patient.

5. The amplification method according to claim 3 or 4, characterized in that The blood disease patient is a patient with aplastic anemia.

6. The amplification method according to any one of claims 1 to 4, characterized in that In the method, the concentration of the three-dimensional microcarriers in the expansion medium is 2.5-5 mg / mL.

7. The amplification method according to claim 5, characterized in that In the method, the concentration of the three-dimensional microcarriers in the expansion medium is 2.5-5 mg / mL.

8. The amplification method according to any one of claims 1 to 4, characterized in that The steps include: (1) Resuspend mononuclear cells from bone marrow, peripheral blood, and umbilical cord blood in expansion medium to a cell concentration of 5×10 4 -1×10 6 cell; (2) Place the three-dimensional microcarriers into a 24-well plate and irradiate with UV light for 20-30 minutes; (3) The ratio of cells to three-dimensional microcarriers in the culture system is: (5×10 4 -1×10 6 ) cells / (5-20 mg) three-dimensional microcarriers, aspirate 100 μL of cell suspension and add it into the wells to allow the three-dimensional microcarriers to fully absorb the cell suspension. After culturing in an incubator for 2 hours, add 1.9 mL of culture medium to a final system of 2 mL. Stir the microcarriers to disperse them and evenly distribute them in the culture medium. Collect the cells after culturing.

9. The amplification method according to claim 8, characterized in that The culture conditions in step (3) are constant temperature culture at 37°C and 5% CO2 for 7 days.

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

11. A functional hematopoietic stem cell, characterized in that: Obtained by culturing according to any one of claims 1 to 10.

12. A pharmaceutical preparation, characterized in that The preparation comprises the functional hematopoietic stem cells according to claim 11 and a pharmaceutically acceptable carrier.

13. Use of the functional hematopoietic stem cells according to claim 11 or the pharmaceutical preparation according to claim 12 in preparing a preparation for treating blood diseases, characterized in that: The blood disease is a bone marrow failure disease.

14. Use of the functional hematopoietic stem cells according to claim 11 or the pharmaceutical preparation according to claim 12 in preparing a preparation for treating blood diseases, characterized in that: The blood disease is primary immune thrombocytopenia (ITP).

15. The use according to claim 13, characterized in that The bone marrow failure disease is selected from bone marrow failure caused by radiotherapy and chemotherapy, aplastic anemia AA, and Fanconi anemia FA.

16. Use of three-dimensional microcarriers in the in vitro culture of functional hematopoietic stem cells, characterized in that: The application is to add the three-dimensional microcarriers to the culture medium for culturing human mononuclear cells in vitro, and the ratio of the number of cells in the culture medium to the amount of the three-dimensional microcarriers added is: 5×10 4 -1×10 6 Cells / 5-20mg three-dimensional microcarrier, the functional hematopoietic stem cells are human megakaryocyte-biased hematopoietic stem cells, and the surface marker of the human megakaryocyte-biased hematopoietic stem cells is CD34 + CD38 - CD45RA - CD90 + CD49f low ; The three-dimensional microcarrier is 3D TableTrix® Microslide® F01 produced by Huakan Biotechnology Co., Ltd.

17. Use according to claim 16, characterized in that The human mononuclear cells are derived from peripheral blood, umbilical cord blood or bone marrow.

18. The use according to claim 16, characterized in that The human mononuclear cells are collected from the bone marrow of patients with hematological diseases.

19. The use according to claim 18, characterized in that The human mononuclear cells are collected from the bone marrow of patients with bone marrow failure diseases.

20. The use according to claim 18, characterized in that The mononuclear cells are collected from the bone marrow of patients with aplastic anemia.

21. The use according to claim 16, characterized in that The method of culturing patient mononuclear cells using three-dimensional microcarriers is as follows: (1) Resuspend the mononuclear cells in culture medium to a cell concentration of 2×10 6 cells / mL; (2) 3D microcarriers with a concentration of 5 mg / ml were plated into a 24-well plate at a rate of 10 mg / well and irradiated with UV light for 20-30 min; (3) Pipette 100 μL of cell suspension into the wells to allow the three-dimensional microcarriers to fully absorb the cell suspension. After culturing in the incubator for 2 hours, add 1.9 mL of culture medium to a final system of 2 mL. Stir the microcarriers to disperse them and evenly distribute them in the culture medium. The culture conditions are constant temperature incubation at 37°C and 5% CO2. After culturing for 7 days, collect the cells.

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