Bionic bone marrow microenvironment assembly for hematopoietic stem cell in-vitro amplification and preparation method

Abstract

The invention discloses a bionic bone marrow microenvironment assembly for hematopoietic stem cell in-vitro amplification and a preparation method. The bone marrow microenvironment assembly comprises a three-dimensional scaffold and osteoblasts (OBs), wherein the three-dimensional scaffold comprises chitosan (CS) and sodium alginate (Alg) which are prepared into mixed gel, and the mixed gel is freeze-dried to prepare the three-dimensional scaffold; wherein the osteoblasts (OBs) are obtained by culturing and induced differentiation of umbilical cord mesenchymal stem cells (MSCs) inoculated on a three-dimensional scaffold, and the osteoblasts (OBs) as stromal cells and the three-dimensional scaffold jointly form the hematopoietic stem cell in-vitro amplification bionic bone marrow microenvironment assembly; in the hematopoietic stem cell in-vitro amplification bionic bone marrow microenvironment assembly, the preparation of the three-dimensional scaffold comprises the processes of freezing, remelting, freezing, crosslinking, freeze-drying and the like, so that a three-dimensional space form with wrinkles on the pore surface and pore size distribution between 70 microns and 400 microns is generated in the three-dimensional scaffold, and the three-dimensional space form is closer to a real stem cell niche; therefore, the practical application effect of the bionic bone marrow microenvironment assembly for in-vitro amplification of the hematopoietic stem cells is further improved.

Description

Technical Field

[0001] This invention relates to the fields of tissue engineering and stem cell culture technology, specifically to a biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells and its preparation method. Background Technology

[0002] Hematopoietic stem cells (HSCs) are adult stem cells with high self-renewal capacity and multi-lineage differentiation potential. HSC transplantation is one of the most effective treatments for malignant tumors such as leukemia and lymphoma, metabolic diseases, autoimmune diseases, and congenital immune diseases that seriously endanger human health. The main sources of HSCs are bone marrow, umbilical cord blood, and peripheral blood. Compared with bone marrow and peripheral blood, umbilical cord blood has advantages such as wide availability, convenient collection, low immunogenicity, and low incidence of graft-versus-host disease. Therefore, umbilical cord blood is indeed an important source of HSCs. However, in practical applications, HSCs derived from umbilical cord blood are limited by the limited absolute number of usable HSCs in a single unit of umbilical cord blood, and the requirement for a certain proportion of the patient's body weight. Therefore, HSCs derived from umbilical cord blood are currently only used in the treatment of pediatric patients and cannot meet the needs of adult patients.

[0003] In vitro culture and expansion of hematopoietic stem cells (HSCs) is the most effective method to solve the above problems and is also one of the hot topics in stem cell clinical application research. The article "Construction and Application of a Biomimetic Microenvironment Supporting In Vitro Expansion of Hematopoietic Stem Cells" proposes a method based on a chitosan-polyphenol derivative three-dimensional scaffold for seeding and culture of umbilical cord mesenchymal stem cells (MSCs), which are then induced to differentiate into osteoblasts (OBs), thus constructing a biomimetic microenvironment component to support the in vitro expansion of hematopoietic stem cells. The article further explores the efficiency, cell viability, and stemness maintenance of this component in the in vitro expansion of hematopoietic stem cells (HSCs). However, the influence of the pore size and pore surface morphology of the three-dimensional scaffold on the in vitro expansion efficiency of hematopoietic stem cells (HSCs) in this biomimetic microenvironment component was not discussed.

[0004] Currently, there is no consensus on the impact of pore size of three-dimensional scaffolds on the in vitro expansion efficiency of hematopoietic stem cells (HSCs). For example, a team from Tsinghua University found in their research on the regulation of hematopoiesis by bone marrow biomimetic scaffolds that scaffolds with larger pore sizes (80 μm) and higher Young's modulus (70 kPa) are more conducive to the expansion of hematopoietic stem cells and progenitor cells (HPCs) and the maintenance of stemness-related phenotypes. Meanwhile, in their research on the fabrication of stem cell culture scaffolds using micro-3D printing technology, the Korean Institute of Technology and Kacheon University developed a three-dimensional bone marrow culture system using a polyurethane scaffold with a pore size of 200 μm. This porous scaffold (porosity 90-95%) can promote high-density cell growth and support umbilical cord blood mononuclear cells. The growth of nucleated cells and primary leukemia cells in the absence of exogenous growth factors; it is generally believed that when the pore size of the three-dimensional scaffold is small, the scaffold has a high specific surface area, increasing the contact points between cells and the scaffold and facilitating cell adhesion, but it may restrict the diffusion of nutrients and oxygen and the removal of metabolic waste, easily leading to local hypoxia or acidosis, and inhibiting cell proliferation during long-term culture; when the pore size of the three-dimensional scaffold is large, it helps to improve the uniform distribution of nutrients inside the scaffold, providing survival support for larger cell aggregates (cell clusters), thereby reducing the formation of cell necrosis cores due to insufficient nutrient supply, but the specific surface area of ​​the three-dimensional scaffold decreases and the number of cell adhesion points decreases, which may affect early colonization. Current understanding suggests that when the pore size of a three-dimensional scaffold is distributed within the range of 150-400 μm, i.e., when the pore size of the scaffold has a wider distribution range, it more closely resembles the structure of an in vivo "stem cell niche," allowing cells to migrate freely and easily form 3D aggregates, promoting cell-cell interactions, enhancing self-renewal, and being more conducive to the expansion of hematopoietic stem cells (HSCs). However, there is currently no data revealing how to prepare three-dimensional scaffolds with a pore size distribution between 150-400 μm. Furthermore, regarding the influence of the pore surface morphology of the three-dimensional scaffold on the in vitro expansion efficiency of hematopoietic stem cells (HSCs), it is generally believed that appropriate surface roughness of the three-dimensional scaffold pores increases the surface area and conformational diversity for protein adsorption (such as fibronectin, hydrin, and collagen). These proteins are ligands for cell adhesion molecules (such as integrins), and are more conducive to cell adhesion than smooth surfaces. Stronger adhesion is crucial for early cell colonization and survival.

[0005] Existing three-dimensional scaffolds prepared by freeze-drying or 3D printing methods generally have the following problems:

[0006] Due to limitations in the fabrication methods, three-dimensional scaffolds often exhibit narrow pore size distribution (e.g., the pore size of the bone marrow biomimetic scaffold researched by the Tsinghua University team is mainly distributed at 80 μm, while the pore size of the miniature 3D-printed stem cell culture scaffold researched by the Korean Institute of Technology and Kacheon University is mainly distributed at 200 μm) and relatively smooth pore surfaces. This negatively impacts the efficiency of in vitro expansion of hematopoietic stem cells. Therefore, further improving existing three-dimensional scaffold fabrication methods to produce three-dimensional scaffolds with a wide pore size distribution and rough pore surfaces is of great significance for the clinical application of in vitro expansion of hematopoietic stem cells. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, this invention provides a biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells and its preparation method. This addresses the problems of narrow pore size distribution and smooth pore surface in existing three-dimensional scaffolds used for in vitro expansion of hematopoietic stem cells. It improves the spatial structure of the three-dimensional scaffold in the biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells, making it closer to the in vivo "stem cell niche". This further improves the practical application effect of the biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells, and provides a new idea and direction for the improvement of the three-dimensional scaffold in the biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells.

[0008] To achieve the aforementioned objective, the present invention employs the following technical solution: a biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells, comprising a three-dimensional scaffold and umbilical cord mesenchymal stem cells (MSCs); the pore size of the three-dimensional scaffold is distributed between 70-400 μm; the pore surface of the three-dimensional scaffold is wrinkled; umbilical cord mesenchymal stem cells (MSCs) are typically isolated and cultured in vitro from Wharton's jelly tissue of fresh umbilical cord through enzymatic digestion or tissue block adhesion method, and seeded onto the three-dimensional scaffold as stromal cells to constitute the biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells.

[0009] Preferably, the biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells includes a three-dimensional scaffold, umbilical cord mesenchymal stem cells (MSCs), and osteoblasts (OBs); the pore size of the three-dimensional scaffold is distributed between 70-400 μm; the pore surface of the three-dimensional scaffold is wrinkled; the osteoblasts (OBs) are induced to differentiate from umbilical cord mesenchymal stem cells (MSCs) seeded on the three-dimensional scaffold, and the umbilical cord mesenchymal stem cells (MSCs) and osteoblasts (OBs) together serve as stromal cells of the environmental component, constituting the biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells with the three-dimensional scaffold.

[0010] Furthermore, the three-dimensional scaffold materials include chitosan (CS) and sodium alginate (Alg); chitosan (CS) is chemically modified with gallic acid to give the three-dimensional scaffold antioxidant activity during biodegradation.

[0011] A method for preparing a biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells includes the following steps:

[0012] S1. Chemical modification of chitosan (CS): Chitosan (CS) is chemically modified with gallic acid to obtain chitosan polyphenol derivatives;

[0013] S2. Preparation and freezing of three-dimensional scaffold gel: Chitosan polyphenol derivatives and sodium alginate (Alg) were prepared into a gel, injected into a container, and subjected to freezing, thawing, freezing, chemical cross-linking and freeze-drying to obtain a three-dimensional scaffold with pore size distribution between 70-400μm and pore surface wrinkled.

[0014] S3. Umbilical cord mesenchymal stem cell (MSC) seeding or culture: Umbilical cord mesenchymal stem cells (MSCs) are seeded on a three-dimensional scaffold. The MSCs, as stromal cells, together with the three-dimensional scaffold, constitute the biomimetic bone marrow microenvironment component for the in vitro expansion of hematopoietic stem cells.

[0015] Alternatively, umbilical cord mesenchymal stem cells (MSCs) can be seeded on a three-dimensional scaffold for culture and expansion, and then induced to differentiate into osteoblasts (OBs). Umbilical cord mesenchymal stem cells (MSCs) and osteoblasts (OBs) serve as stromal cells, and together with the three-dimensional scaffold, they constitute a biomimetic bone marrow microenvironment component for the in vitro expansion of hematopoietic stem cells.

[0016] Furthermore, the specific methods for chemical modification of chitosan (CS) are as follows:

[0017] S1.1 Dissolve 28 mmol gallic acid and 2.8 mmol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 40 mL of 70% v / v ethanol. Add 2.8 mmol N-hydroxysuccinimide (NHS) to the solution. Stir the solution in an ice bath for 10-20 min, let it stand at room temperature for 1-2 h, and then add 1.5 g chitosan (CS) dispersed in 110 mL of 70% v / v ethanol. Stir the solution in an ice bath for 20-30 min, and then continue stirring at room temperature for 20-24 h.

[0018] S1.2 Filter and collect the precipitate and place it in a container. Wash it three times with 70% v / v ethanol. Add 100 mL of deionized water to the precipitate to make a gel suspension. Put the suspension into a dialysis bag and dialyze with deionized water for 3-4 days. Change the deionized water every 8 hours for the first two days and every 12 hours for the last two days.

[0019] S1.3 The suspension of the dialysis precipitate is dried by vacuum freeze drying to obtain chitosan polyphenol derivative powder.

[0020] Furthermore, the method for preparing the three-dimensional scaffold gel is as follows:

[0021] S2.1 Prepare a 1%-2% w / v sodium alginate (Alg) aqueous solution; determine that the pH value of the sodium alginate (Alg) aqueous solution is maintained between 6.0 and 6.3 at this time;

[0022] S2.2 Add chitosan polyphenol derivative powder at 1%-2% wt to the prepared sodium alginate (Alg) solution and stir at room temperature for 30-40 min.

[0023] Furthermore, the three-dimensional scaffold cryotherapy method is as follows:

[0024] S2.3 First freezing: Place the gel prepared by chitosan polyphenol derivative and sodium alginate (Alg) into a container and freeze at -18 to -20℃ for 20 to 24 hours;

[0025] S2.4, Thawing: Thaw the frozen 3D scaffold at room temperature for 4-6 hours;

[0026] S2.5 Second freezing; the thawed three-dimensional scaffold is frozen at -18 to -20℃ for 120 to 240 hours; then dried at 23 to 27℃ for 20 to 24 hours.

[0027] S2.6 Crosslinking: Remove the three-dimensional scaffold and immerse it in 1.5%-3% w / v calcium chloride aqueous solution for 30-60 min; after immersion, wash the three-dimensional scaffold with phosphate buffered solution (PBS) 3-4 times;

[0028] S2.7, Lyophilization after crosslinking: Pre-freezing: After washing, the three-dimensional scaffold is frozen at -38-42℃ for 4-6 hours; First drying: The temperature is increased from -38-42℃ to -25-30℃ at a rate of 0.3-0.5℃ / min, with a vacuum degree ≤30Pa, and maintained for 20-24 hours; Second drying: The temperature is increased from -25-30℃ to 23-27℃ at a rate of 0.3-0.5℃ / min, with a vacuum degree ≤10Pa, and maintained for 20-24 hours.

[0029] The preferred method for freezing a three-dimensional scaffold is as follows:

[0030] S2.3 First freezing: Place the gel prepared by chitosan polyphenol derivative and sodium alginate (Alg) into a container and freeze at -18 to -20℃ for 20 to 24 hours;

[0031] S2.4 Thawing: Thaw the frozen three-dimensional scaffold in a water bath at 25-35℃ for 2-3 hours;

[0032] S2.5 Second freezing; the thawed three-dimensional scaffold is frozen at -8 to -10℃ for 20 to 24 hours; maintained at -18 to -20℃ for 120 to 240 hours; and then heated to 25℃ and maintained for 24 hours for drying.

[0033] S2.6 Crosslinking: Remove the three-dimensional scaffold and immerse it in 1.5%-3% w / v calcium chloride aqueous solution for 30-60 min; after immersion, wash the three-dimensional scaffold with phosphate buffered solution (PBS) 3-4 times;

[0034] S2.7, Lyophilization after crosslinking: Pre-freezing: After washing, the three-dimensional scaffold is frozen at -38-42℃ for 4-6 hours; First drying: The temperature is increased from -38-42℃ to -25-30℃ at a rate of 0.3-0.5℃ / min, with a vacuum degree ≤30Pa, and maintained for 20-24 hours; Second drying: The temperature is increased from -25-30℃ to 23-27℃ at a rate of 0.3-0.5℃ / min, with a vacuum degree ≤10Pa, and maintained for 20-24 hours.

[0035] Furthermore, umbilical cord mesenchymal stem cells (MSCs) are seeded and cultured on a three-dimensional scaffold for 24-72 hours. The MSCs fully attach to and spread in the pores of the three-dimensional scaffold, forming a biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells. This biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells is then directly seeded into hematopoietic stem cells (HSCs) for in vitro expansion and culture.

[0036] Preferably, umbilical cord mesenchymal stem cells (MSCs) are seeded and cultured on a three-dimensional scaffold for 14-21 days. The MSCs are then expanded in vitro and induced to differentiate into osteoblasts (OBs), forming a biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells. This biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells can be used directly for in vitro expansion of hematopoietic stem cells, or it can be cryopreserved in liquid nitrogen for emergency use.

[0037] Due to the adoption of the above-described technical solution, the present invention has the following beneficial effects: The present invention discloses a method for preparing a biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells. The bone marrow microenvironment component includes a three-dimensional scaffold and osteoblasts (OBs); wherein the three-dimensional scaffold includes chitosan (CS) and sodium alginate (Alg), which are mixed into a gel and then freeze-dried; wherein the osteoblasts (OBs) are cultured and differentiated from umbilical cord mesenchymal stem cells (MSCs) seeded on the three-dimensional scaffold, and together with the three-dimensional scaffold, they constitute the biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells; this biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells... In the bone marrow microenvironment component, the preparation of the three-dimensional scaffold involves freezing, thawing, freezing, cross-linking, and lyophilization. The freezing, thawing, and freezing processes generate a pore size distribution between 70-400 μm in the three-dimensional scaffold, and the three-dimensional scaffold collapses on the pore surface to create wrinkles. The final product is a three-dimensional scaffold with a rough pore surface and a pore size distribution between 70-400 μm, which is closer to the spatial morphology of the "stem cell niche" in vivo. This further improves the practical application effect of the biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells, and provides a new idea and direction for the improvement of the three-dimensional scaffold in the biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells. Attached Figure Description

[0038] Figure 1 A micrograph of three-dimensional scaffold sample A;

[0039] Figure 2 Micrograph of 3D scaffold sample B;

[0040] Figure 3 C-micrograph of a three-dimensional scaffold sample;

[0041] Figure 4 This is a graph showing experimental data on the expansion of mesenchymal stem cells on a three-dimensional scaffold.

[0042] Figure 5 This is a graph showing experimental data on the expansion of hematopoietic stem cells in a biomimetic bone marrow microenvironment component. Detailed Implementation

[0043] The present invention will be explained in detail through the following embodiments. The purpose of disclosing the present invention is to protect all technical improvements within the scope of the present invention.

[0044] The biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells (HSCs) disclosed in this invention simulates the real bone marrow microenvironment and is used for the in vitro expansion of umbilical cord blood hematopoietic stem cells (HSCs). The real bone marrow microenvironment of hematopoietic stem cells (HSCs) mainly includes the three-dimensional physical structure of bone marrow, stromal cells, and their secreted extracellular matrix and cytokines. The specific physical structure, complex biochemical signals, and physical signals of the bone marrow microenvironment jointly regulate the expansion and differentiation of hematopoietic stem cells (HSCs), which is an extremely complex biochemical process. How to achieve the biomimetic construction of the bone marrow microenvironment of hematopoietic stem cells (HSCs) in vitro has always been a key research focus in the field of in vitro expansion technology of hematopoietic stem cells (HSCs). Among them, the physical structure of bone marrow and osteoblasts (OBs) are two crucial factors in constructing the bone marrow microenvironment. The cancellous bone region where bone marrow is located is composed of a three-dimensional network scaffold formed by interconnected trabeculae. Bone marrow tissue fills the gaps between the trabeculae, and they intertwine to form a sponge-like or honeycomb-like microenvironment. This structure has a large number of highly interconnected pores, but the size, shape, and distribution of the pores are not uniform throughout the bone marrow cavity, but rather exhibit a multi-scale spatial distribution. These pores provide a three-dimensional physical space for hematopoietic stem cells (HSCs) and osteoblasts (OBs). Osteoblasts (OBs) can secrete extracellular matrix and cytokines. The extracellular matrix, cytokines, the multi-scale pores of the three-dimensional physical space of bone marrow, and the rough surface structure together play a key regulatory role in the migration, expansion, self-renewal, and maintenance of multi-lineage differentiation potential of hematopoietic stem cells (HSCs) residing within it.

[0045] Existing technologies for constructing biomimetic bone marrow microenvironments for in vitro expansion of hematopoietic stem cells typically use three-dimensional scaffolds to replace the physical bone marrow. Current problems include the narrow pore size distribution range of these scaffolds, which differs significantly from the 10-800 μm scale three-dimensional pore network structure found in real bone marrow. Furthermore, the smooth pore surfaces of the scaffolds hinder cell adhesion and expansion. Additionally, osteoblasts (OBs) are often difficult to obtain when constructing biomimetic bone marrow microenvironments composed of three-dimensional scaffolds and osteoblasts (OBs), making it challenging to directly use OBs to construct such microenvironments.

[0046] Example 1:

[0047] The biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells disclosed in this invention, compared with existing technologies, has a pore-scale distribution and pore surface structure in its three-dimensional scaffold that more closely resembles the real bone marrow microenvironment. The biomimetic bone marrow microenvironment component includes a three-dimensional scaffold with antioxidant activity, mesenchymal stem cells (MSCs), and osteoblasts (OBs). The three-dimensional scaffold material includes chitosan (CS) and sodium alginate (Alg), wherein the chitosan (CS) is chemically modified with gallic acid, exhibiting antioxidant activity during biodegradation. The osteoblasts (OBs) are induced to differentiate into osteoblasts from umbilical cord mesenchymal stem cells (MSCs) seeded on the three-dimensional scaffold through culture and expansion. During the culture and expansion of umbilical cord mesenchymal stem cells (MSCs), the gallic acid-modified chitosan (CS) releases antioxidants during the biodegradation of the three-dimensional scaffold, thereby reducing the level of reactive oxygen species (ROS) within MSCs and promoting their differentiation into osteoblasts (OBs). Furthermore, the gel made from chitosan (CS) and sodium alginate (Alg) undergoes multiple freeze-thaw crosslinking, chemical crosslinking, and freeze-drying processes during the preparation of the three-dimensional scaffold. This process creates highly interconnected pores with a size distribution between 70-400 μm within the scaffold, and the pore surface exhibits a wrinkled appearance (see the instruction manual). Figure 1 This type of three-dimensional scaffold with microstructure more closely resembles the spatial morphology of the "stem cell niche" in vivo, which can further improve the practical application effect of the biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells. At the same time, it provides a new idea and direction for the improvement of the three-dimensional scaffold in the biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells.

[0048] Example 2:

[0049] Three-dimensional scaffolds were added to 24-well plates and incubated in α-MEM + 10% FBS medium for 12 hours; 2*10 wells were then inoculated. 4 Freshly collected umbilical cord mesenchymal stem cells (MSCs) were cultured on a three-dimensional scaffold for 3 days in α-MEM + 10% FBS medium (with one medium change in between), then cultured in osteogenic induction medium for 14 days, with the medium changed every 3 days. After 14 days of culture, a biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells (referred to as COB-A) was formed, consisting of umbilical cord mesenchymal stem cells (MSCs) + osteoblasts (OBs) + a three-dimensional scaffold. After the preparation of this biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells (HSCs) was completed, freshly collected umbilical cord blood hematopoietic stem cells were directly inoculated for in vitro expansion culture.

[0050] Example 3:

[0051] In practical applications, there are often urgent situations where patients urgently need a biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells (HSCs). However, in Example 2, even with a prepared three-dimensional scaffold, the process of inoculating mesenchymal stem cells (MSCs) onto the scaffold to induce differentiation into osteoblasts (OBs) and constructing the biomimetic bone marrow microenvironment component (COB-A) takes 17-21 days, which cannot meet the urgent needs of patients. To solve this problem, cryopreserving the prepared biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells (referred to as COB-B) is an effective means to achieve a ready-made supply of this component. Therefore, after umbilical cord mesenchymal stem cells (MSCs) are cultured on a three-dimensional scaffold for 14-21 days, they are then treated with a solution containing 10% v / v... The cryopreservation solution of DMSO was frozen in liquid nitrogen for emergency use of the in vitro expansion of hematopoietic stem cell biomimetic bone marrow microenvironment components. The specific cryopreservation procedure was as follows: the prepared in vitro expansion of hematopoietic stem cell biomimetic bone marrow microenvironment components were transferred to sterile 1.5mL cryopreservation tubes, and the cryopreservation tubes were placed at 4℃ for 30 min, -20℃ for 60 min, and -80℃ for 12 h in sequence, and finally frozen in liquid nitrogen.

[0052] Example 4:

[0053] In Example 3, because the cryopreservation of the prepared biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells can affect the activity of mesenchymal stem cells (MSCs) and osteoblasts (OBs), to improve this problem and meet the urgent needs of patients, the following scheme for the in vitro expansion of the biomimetic bone marrow microenvironment component for hematopoietic stem cells was adopted: a three-dimensional scaffold was added to a 24-well plate, and the plate was incubated in α-MEM + 10% FBS culture medium for 12 hours; 2*10 4Freshly collected umbilical cord mesenchymal stem cells (MSCs) are placed on a three-dimensional scaffold and cultured in α-MEM + 10% FBS medium for 1-3 days (if cultured for 3 days, the medium is changed once in between). After 1-3 days, a biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells (referred to as COB-C) is formed, consisting of umbilical cord mesenchymal stem cells (MSCs) and a three-dimensional scaffold. After the preparation of this biomimetic bone marrow microenvironment component for in vitro expansion of hematopoietic stem cells is completed, freshly collected umbilical cord blood hematopoietic stem cells (HSCs) are directly inoculated for in vitro expansion culture. During the in vitro expansion of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) are also expanded simultaneously. Through the antioxidant activity generated by chitosan polyphenol derivatives in the biodegradable three-dimensional scaffold, the reactive oxygen species (ROS) level of MSCs is reduced, thereby inducing and promoting differentiation into osteoblasts (OBs). This biomimetic bone marrow microenvironment component (COB-C) for in vitro expansion of hematopoietic stem cells does not require cryopreservation. It can significantly shorten the preparation time while ensuring the activity of both MSCs and OBs. Therefore, it can meet the urgent needs of patients while ensuring the effectiveness of the biomimetic bone marrow microenvironment component.

[0054] In this invention, a three-dimensional scaffold is an essential physical entity for constructing a biomimetic bone marrow microenvironment component. It should possess a good three-dimensional morphology, appropriate pore size, and mechanical strength to meet the expansion requirements of hematopoietic stem cells (HSCs). Furthermore, the pore surface should be modifiable (non-smooth) and capable of loading cytokines and other compounds that regulate cell fate, enabling it to possess specific functions in regulating cell expansion and differentiation. Therefore, the selection and preparation method of the three-dimensional scaffold material are crucial for the in vitro expansion of the biomimetic bone marrow microenvironment component for hematopoietic stem cells. In this invention, the three-dimensional scaffold material uses chitosan (CS) and sodium alginate (Alg). Chitosan (CS) is a linear natural carbohydrate biopolymer derived from chitin, structurally similar to glycosaminoglycans in the extracellular matrix. Simultaneously, the hydrophilic structure of chitosan (CS) facilitates cell adhesion and expansion. Another important characteristic of chitosan (CS) is its structural multifunctionality. Due to the presence of reactive amino and hydroxyl groups in its molecular chain, it exhibits flexibility in chemical modification, allowing for the preparation of scaffolds with different biological functions through chemical modification. Materials: Sodium alginate (Alg) is a natural anionic polysaccharide composed of varying amounts of α-L-guluronic acid (G) monomers and β-D-mannuronic acid (M) linked by covalent bonds to form linear straight-chain copolymers of various shapes, such as continuous G sequences, continuous M sequences, or alternating MG sequences. The linear length of the copolymer composition and sequences determines the physical properties of sodium alginate. The mechanical characteristics of sodium alginate (Alg) can be controlled by altering the length and molecular weight of the G blocks, as the G block sequences have a rigid structure, while the MG blocks have a more flexible structure. Sodium alginate (Alg) is flexible and can influence the degradation rate. Therefore, by selecting an appropriate sodium alginate (Alg) product, the mechanical properties and degradation rate of the final three-dimensional scaffold can be adjusted. Sodium alginate (Alg) has the ability to crosslink with divalent cations (such as divalent calcium ions) to form ion hydrogel networks. By controlling the crosslinking time, the mechanical properties and biodegradation rate of the three-dimensional scaffold can also be adjusted. Sodium alginate (Alg) also has advantages such as good biocompatibility, easy gelation, low biotoxicity, and low price, and has been widely used in the biomedical field.

[0055] In the embodiments of this invention, during the construction of a biomimetic bone marrow microenvironment component, the expansion culture of mesenchymal stem cells (MSCs) and the induction of differentiation into osteoblasts (OBs), intracellular reactive oxygen species (ROS) have a significant impact on the differentiation of MSCs into osteoblasts (OBs). Current research shows that only unregulated ROS levels are harmful, while regulating basal levels of ROS plays an important role in maintaining stem cell function (such as expansion, differentiation, or survival). As a second messenger regulating function, ROS is beneficial for maintaining cell function. For mesenchymal stem cells... Low levels of reactive oxygen species (ROS) can promote differentiation into osteoblasts (OBs). Therefore, in the preparation process of the three-dimensional scaffold for the in vitro expansion of hematopoietic stem cells into a biomimetic bone marrow microenvironment component disclosed in this invention, the flexibility of chitosan (CS) in chemical modification is utilized. After chemical modification with gallic acid, chitosan polyphenol derivatives are obtained. The antioxidant activity generated by the biodegradation of chitosan polyphenol derivatives is used to reduce the level of reactive oxygen species (ROS) in the three-dimensional scaffold mesenchymal stem cells (MSCs), thereby promoting the differentiation of mesenchymal stem cells (MSCs) into osteoblasts (OBs).

[0056] In this embodiment of the invention, the three-dimensional scaffold gel preparation process is as follows: First, a 2% w / v sodium alginate (Alg) aqueous solution is prepared, and the pH value of the sodium alginate (Alg) solution is maintained between 6.0 and 6.3. Then, chitosan polyphenol derivative powder of equal weight to sodium alginate in the 2% w / v sodium alginate (Alg) solution is added to the prepared sodium alginate (Alg) aqueous solution and stirred at room temperature for 30-40 min. During the above three-dimensional scaffold gel preparation process, since the pH value of the sodium alginate (Alg) solution is maintained at 6.0-6.3 (weakly acidic), the carboxyl groups (-COO) of sodium alginate (Alg) are... - The amino group (-NH2) of chitosan polyphenol derivatives is in an ionized state (negatively charged), while the amino group (-NH2) of chitosan polyphenol derivatives is partially ionized. Because the chitosan polyphenol derivative powder is positively charged, when it is added to a negatively charged sodium alginate (Alg) solution, a strong electrostatic interaction occurs between the positive and negative charges. This leads to the rapid formation of a three-dimensional ionic cross-linked network between the chitosan molecular chains and the sodium alginate molecular chains. This cross-linking effect is dominant and far exceeds the solubility tendency of the chitosan polyphenol derivative in weakly acidic aqueous solutions. Therefore, the chitosan polyphenol derivative powder will not be completely molecularly dispersed in the sodium alginate (Alg) solution. Instead, it forms microscopic chitosan polyphenol derivative gel agglomerates in the sodium alginate (Alg) solution through electrostatic bonding (electrostatic attraction to form ionic bonds). Prolonged stirring promotes the wetting and initial dispersion of the chitosan polyphenol derivative powder. The stirring shear force breaks up the gel agglomerates and distributes them evenly throughout the solution, ultimately obtaining a two-component gel mixture with uniform component distribution and high viscosity. This two-component gel mixture forms a three-dimensional scaffold with specific multi-scale pores and surface wrinkles during the subsequent freeze-drying process.

[0057] In this embodiment of the invention, the entire freeze-drying process of the three-dimensional scaffold employs a two-freeze-one-thaw method. The aim is to control the size of ice crystal growth during the two freezing processes, thereby generating a pore size distribution between 70-400 μm within the three-dimensional scaffold. The specific process is explained as follows: During the first freezing process, several crystal nuclei are uniformly generated in the bicomponent gel mixture in the initial freezing stage. As these nuclei grow into ice crystals, water is released from the bicomponent gel mixture, compressing and reducing the distribution space between sodium alginate (Alg) and chitosan (CS) molecular chains, thus further promoting freeze-crosslinking between chitosan and sodium alginate. After the first freezing and subsequent thaw, the freeze-crosslinking between sodium alginate and chitosan does not completely disappear. Therefore, the thawed bicomponent gel mixture actually forms a one-time molded three-dimensional scaffold with a porous structure, although the pores contain pure water. During the second freezing process, the pure water in the pores is unlikely to generate crystal nuclei. In the initial stage of the second freezing, the number of crystal nuclei is small and they are randomly generated. Therefore, before the second freezing is completely finished, the time it takes for each crystal nucleus to grow into an ice crystal is not the same, with earlier crystal nuclei forming... Ice crystals have ample time to grow larger, while those formed later in the crystallization process grow smaller in a shorter time. This allows the pore structure of the three-dimensional scaffold in the two-component gel mixture to be modified by the ice crystal size during secondary freezing, resulting in a secondary structure with a wider pore size distribution. This secondary structure is further fixed during subsequent freeze-drying and chemical cross-linking, ultimately yielding a three-dimensional scaffold with a wide pore size distribution. The secondary freezing process of the three-dimensional scaffold involves high freezing temperatures and long durations, effectively constituting a freeze-drying process. Since the freeze-drying temperature is higher than the eutectic point temperature, the three-dimensional scaffold undergoes drying shrinkage during the secondary freezing. The drying shrinkage rates of sodium alginate and chitosan in the gel are inconsistent, ultimately forming wrinkles on the pore surface of the three-dimensional scaffold. The secondary freeze-dried three-dimensional scaffold is then chemically cross-linked using CaCl2 solution, followed by freeze-drying to fix the pore size and surface wrinkles. The number of freeze-drying cycles and the chemical cross-linking time using CaCl2 solution can be adjusted and controlled to regulate the biodegradation rate of the three-dimensional scaffold.

[0058] Example 5:

[0059] The three-dimensional scaffold sample A is prepared by the following method, specifically including the following steps:

[0060] S1. Chitosan (CS) Chemical Modification: Chitosan (CS) is chemically modified with gallic acid to obtain chitosan polyphenol derivatives; the specific process is as follows:

[0061] S1.1 Dissolve 28 mmol gallic acid and 2.8 mmol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 40 mL of 70% v / v ethanol, and add 2.8 mmol N-hydroxysuccinimide (NHS) to the solution; stir the solution in an ice bath for 20 min, let it stand at room temperature for 2 h, and then add 1.5 g chitosan (CS) dispersed in 110 mL of 70% v / v ethanol; stir the solution in an ice bath for 30 min, and then continue stirring at room temperature for 24 h;

[0062] S1.2 Filter and collect the precipitate and place it in a container. Wash it three times with 70% v / v ethanol. Add 100 mL of deionized water to the precipitate to make a gel suspension. Put the suspension into a dialysis bag and dialyze with deionized water for 4 days. Change the deionized water every 8 hours for the first two days and every 12 hours for the last two days.

[0063] S1.3 The dialysis suspension was dried by vacuum freeze-drying to obtain chitosan polyphenol derivative powder.

[0064] S2. Preparation and Freezing of Three-Dimensional Scaffold Gel: Chitosan polyphenol derivatives were prepared into a gel with sodium alginate (Alg), injected into a container, and subjected to freezing, thawing, freezing, chemical cross-linking, and lyophilization to obtain a three-dimensional scaffold with a pore size distribution between 70-400 μm and a wrinkled pore surface; the specific process is as follows:

[0065] S2.1 Prepare a 2% w / v sodium alginate (Alg) aqueous solution; determine the pH value of the sodium alginate (Alg) solution at this time, and adjust it to maintain it between 6.0 and 6.3 with citrate buffer.

[0066] S2.2. Chitosan polyphenol derivative powder was added to the prepared sodium alginate (Alg) solution at 2% wt and stirred at room temperature for 40 min.

[0067] S2.3 First freezing: Chitosan polyphenol derivative and sodium alginate (Alg) aqueous solution were placed in a 24-well plate and frozen at -18℃ for 24 hours;

[0068] S2.4, Thawing: Thaw the frozen three-dimensional scaffold at 22°C for 5 hours;

[0069] S2.5, Second freezing; The thawed three-dimensional scaffold was frozen at -18℃ for 120 hours; and then dried at 22℃ for 24 hours.

[0070] S2.6 Chemical crosslinking: The three-dimensional scaffold was removed and immersed in 2.0% w / v calcium chloride aqueous solution for 40 min; the immersed three-dimensional scaffold was washed 3 times with phosphate buffered solution (PBS);

[0071] S2.7, Lyophilization after crosslinking: Pre-freezing: After washing, the three-dimensional scaffold is frozen at -40℃ for 5 hours; First drying: The temperature is increased from -40℃ to -25℃ at a rate of 0.4℃ / min, with a vacuum degree ≤30Pa, and maintained for 24 hours; Second drying: The temperature is increased from -25℃ to 25℃ at a rate of 0.4℃ / min, with a vacuum degree ≤30Pa, and maintained for 24 hours.

[0072] See the attached instruction manual for a micrograph of sample A of the three-dimensional scaffold. Figure 1 .

[0073] Example 6:

[0074] The three-dimensional scaffold sample B was prepared using the following method, specifically including the following steps:

[0075] S1. Chitosan (CS) Chemical Modification: Chitosan (CS) is chemically modified with gallic acid to obtain chitosan polyphenol derivatives; the specific process is as follows:

[0076] S1.1 Dissolve 28 mmol gallic acid and 2.8 mmol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 40 mL of 70% v / v ethanol, and add 2.8 mmol N-hydroxysuccinimide (NHS) to the solution; stir the solution in an ice bath for 20 min, let it stand at room temperature for 2 h, and then add 1.5 g chitosan (CS) dispersed in 110 mL of 70% v / v ethanol; stir the solution in an ice bath for 30 min, and then continue stirring at room temperature for 24 h;

[0077] S1.2 Filter and collect the precipitate and place it in a container. Wash it three times with 70% v / v ethanol. Add 100 mL of deionized water to the precipitate to make a gel suspension. Put the suspension into a dialysis bag and dialyze with deionized water for 4 days. Change the deionized water every 8 hours for the first two days and every 12 hours for the last two days.

[0078] S1.3 The suspension of the dialysis precipitate is dried by vacuum freeze drying to obtain chitosan polyphenol derivative powder.

[0079] S2. Preparation and Freezing of Three-Dimensional Scaffold Gel: Chitosan polyphenol derivatives were prepared into a gel with sodium alginate (Alg), injected into a container, and subjected to freezing, thawing, freezing, chemical cross-linking, and lyophilization to obtain a three-dimensional scaffold with a pore size distribution between 70-400 μm and a wrinkled pore surface; the specific process is as follows:

[0080] S2.1 Prepare a 2% w / v sodium alginate (Alg) aqueous solution; determine that the pH value of the sodium alginate (Alg) solution is maintained between 6.0 and 6.3 at this time;

[0081] S2.2 Add 2% wt of chitosan polyphenol derivative powder to the prepared sodium alginate (Alg) solution and stir at room temperature for 40 min;

[0082] S2.3 First freezing: Place the chitosan polyphenol derivative and sodium alginate (Alg) aqueous solution into a container and freeze at -18℃ for 24 hours;

[0083] S2.4 Thawing: Thaw the frozen three-dimensional scaffold in a 35°C water bath for 2 hours;

[0084] S2.5 Second freezing; the thawed three-dimensional scaffold was frozen at -10℃ for 24 hours; then maintained at -18℃ for 120 hours; and finally dried at 25℃ for 24 hours.

[0085] S2.6 Chemical crosslinking: The three-dimensional scaffold was removed and immersed in 2% w / v calcium chloride aqueous solution for 50 min; the immersed three-dimensional scaffold was washed 4 times with phosphate buffered solution (PBS);

[0086] S2.7, Lyophilization after chemical cross-linking: Pre-freezing: After washing, the three-dimensional scaffold is frozen at -40℃ for 6 hours; First drying: The temperature is increased from -40℃ to -25℃ at a rate of 0.4℃ / min, with a vacuum degree ≤30Pa, and maintained for 24 hours; Second drying: The temperature is increased from -25℃ to 23℃ at a rate of 0.4℃ / min, with a vacuum degree ≤30Pa, and maintained for 24 hours.

[0087] See the attached instruction manual for a micrograph of sample B of the three-dimensional scaffold. Figure 2 .

[0088] Example 7:

[0089] The three-dimensional scaffold sample C is prepared by the following method, specifically including the following steps:

[0090] S1. Chitosan (CS) Chemical Modification: Chitosan (CS) is chemically modified with gallic acid to obtain chitosan polyphenol derivatives; the specific process is as follows:

[0091] S1.1 Dissolve 28 mmol gallic acid and 2.8 mmol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 40 mL of 70% v / v ethanol, and add 2.8 mmol N-hydroxysuccinimide (NHS) to the solution; stir the solution in an ice bath for 20 min, let it stand at room temperature for 2 h, and then add 1.5 g chitosan (CS) dispersed in 100 mL of 70% v / v ethanol; stir the solution in an ice bath for 30 min, and then continue stirring at room temperature for 24 h;

[0092] S1.2 Filter and collect the precipitate and place it in a container. Wash it three times with 70% v / v ethanol. Add 100 mL of deionized water to the precipitate to make a gel suspension. Put the suspension into a dialysis bag and dialyze with deionized water for 4 days. Change the deionized water every 8 hours for the first two days and every 12 hours for the last two days.

[0093] S1.3 The suspension of the dialysis precipitate is dried by vacuum freeze drying to obtain chitosan polyphenol derivative powder.

[0094] S2. Three-dimensional scaffold preparation: Utilizing the physical property that chitosan derivatives are soluble in acidic solutions and insoluble in alkaline solutions, porous chitosan polyphenol derivative scaffolds are prepared.

[0095] S2.1 Preparation of chitosan derivative solution: Dissolve 2.0 g of chitosan derivative in 100 mL of 0.1% v / v glacial acetic acid;

[0096] S2.2, One-time freeze drying: Inject into a container and freeze at -20℃ for 24h; keep the temperature constant and maintain a vacuum of ≤30Pa for 24h; heat from -18℃ to 25℃ at a heating rate of 0.4℃ / min, maintain a vacuum of ≤30Pa for 24h.

[0097] S2.3 Deacidification treatment: The three-dimensional scaffold was removed and immersed in 2% w / v NaOH solution for deacidification for 4 hours; the deacidified three-dimensional scaffold was washed 4 times with phosphate buffered solution (PBS);

[0098] S2.4 Secondary freeze-drying: The deacidified three-dimensional scaffold is frozen at -20℃ for 24h; the temperature remains constant and the vacuum degree is ≤30Pa, and this is maintained for 24h; the temperature is increased from -18℃ to 25℃ at a rate of 0.4℃ / min, and the vacuum degree is ≤30Pa, and this is maintained for 24h.

[0099] See the attached instruction manual for C-micrographs of the three-dimensional scaffold sample. Figure 3 .

[0100] Expansion experiments of mesenchymal stem cells (MCSs) in three-dimensional scaffolds:

[0101] To evaluate the expansion effect of mesenchymal stem cells (MSCs) on three-dimensional scaffold samples A, B, and C, a control group (sample C) was established, and the experimental groups were the groups cultured using samples A and B. The expansion of MSCs on the three-dimensional scaffolds in each group was measured using the CCK-8 assay. The specific method was as follows: first, MSCs were isolated, and then... 4Mesenchymal stem cells (MSCs) were seeded at a density of cells / well on a 24-well 3D scaffold and divided into three groups: sample C control group, sample A culture group, and sample B culture group. MSCs were cultured in 1 mL of serum-free SFEM medium containing 50 ng / mL SCF, 50 ng / mL LTPO, and 20 ng / mL Flt-3 in a 37°C, 5% CO2 incubator. After culturing in the 24-well plates for 1, 3, 5, and 7 days, a CCK-8 working solution was prepared by mixing α-MEM medium and CCK-8 solution at a ratio of 9:1. 200 μL of this CCK-8 working solution was added to the MSCs and incubated for 2 hours. Then, 100 μL of the sample solution was taken and the absorbance was measured at 450 nm to detect cell expansion. Results are shown in the appendix to the instruction manual. Figure 4 The differences in mesenchymal stem cells (MSCs) on three-dimensional scaffold samples A, B, and C were not significant at 1 and 3 days of culture. With the extension of culture time, the proliferation rate of mesenchymal stem cells (MSCs) on samples A and B was significantly higher than that of the control group of sample C.

[0102] Experiments on the expansion of hematopoietic stem cells (HSCs) in a biomimetic bone marrow microenvironment component:

[0103] To evaluate the expansion effect of hematopoietic stem cells (HSCs) in a biomimetic bone marrow microenvironment component, a three-dimensional scaffold sample control group (C) was established, while the experimental groups were the biomimetic bone marrow microenvironment component COB-A culture group, COB-B (cryopreserved) culture group, and COB-C culture group. The overall expansion fold of the hematopoietic stem cell population was determined by directly counting the change in the number of nucleated cells before and after expansion. The specific method was as follows: firstly, hematopoietic stem cells (HSCs) were isolated, and the isolated CD34... + Cells at 1*10 4 Cells were seeded at a density of 1 cell / well on 24-well plates using a 3D scaffold or a biomimetic bone marrow microenvironment component, and divided into four groups: 3D scaffold sample C control group, COB-A culture group, COB-B (cryopreserved) culture group, and COB-C culture group. Hematopoietic stem cells (HSCs) were cultured in 1 mL of serum-free SFEM medium containing 50 ng / mL SCF, 50 ng / mL TPO, and 20 ng / mL Flt-3 in a 37°C, 5% CO2 incubator. On days 4, 7, 10, and 14, half the medium was replaced, and the number of nucleated cells (NCs) before and after expansion was directly counted. The expansion fold of total cells in each group during culture is detailed in the instruction manual. Figure 5On day 4 of culture, the total cell expansion fold of each group was similar, with no significant difference. After day 7 of culture, the difference between the total cell expansion fold of each culture group and the control group began to increase. On day 14 of culture, the COB-A culture group had the highest total cell expansion fold, which was 208.6+ / -21.6 times (*p<0.05). The COB-B (cryopreserved) culture group and the COB-C culture group were similar, which were 188.3+ / -18.3 times (*p<0.05) and 185.7+ / -15.6 times (*p<0.05), respectively, both of which were significantly higher than the 56.3+ / -7.8 times (*p<0.05) of the three-dimensional scaffold sample C control group.

[0104] Based on the above experimental results, a three-dimensional scaffold with a pore size distribution between 70-400 μm and a wrinkled pore surface is more conducive to the expansion of mesenchymal stem cells (MSCs). A biomimetic bone marrow microenvironment component composed of a three-dimensional scaffold with a pore size distribution between 70-400 μm and a wrinkled pore surface, and mesenchymal stem cells (MSCs) or mesenchymal stem cells (MSCs) + osteoblasts (OBS) can significantly improve the in vitro expansion effect of hematopoietic stem cells (HSCs).

[0105] Additional notes: All experiments were repeated three times. Data are expressed as mean plus or minus standard deviation. Statistical analysis between two groups was performed using t-tests, and statistical analysis between three or more groups was performed using ANOVA.

[0106] The parts of this invention not described in detail are prior art.

Claims

1. A hematopoietic stem cell in vitro expansion biomimetic bone marrow microenvironment assembly, characterized in that, The three-dimensional scaffold, umbilical cord mesenchymal stem cells (MSCs); the three-dimensional scaffold has a pore size distribution of 70-400 μm; the pore surface of the three-dimensional scaffold is corrugated; the umbilical cord mesenchymal stem cells (MSCs) are obtained by separation and culture of Wharton's jelly tissue blocks of umbilical cord, are inoculated on the three-dimensional scaffold, and are used as stromal cells to form the in-vitro expansion of hematopoietic stem cell biomimetic bone marrow microenvironment components together with the three-dimensional scaffold.

2. The in vitro expanded hematopoietic stem cell biomimetic bone marrow microenvironment assembly of claim 1, wherein, The three-dimensional scaffold, umbilical cord mesenchymal stem cells (MSCs), and osteoblasts (OBs); the osteoblasts (OBs) are obtained by induction and differentiation of umbilical cord mesenchymal stem cells (MSCs) inoculated on the three-dimensional scaffold; the umbilical cord mesenchymal stem cells (MSCs) and the osteoblasts (OBs) are used as stromal cells to form the in-vitro expansion of hematopoietic stem cell biomimetic bone marrow microenvironment components together with the three-dimensional scaffold.

3. The in vitro expanded hematopoietic stem cell-bionic bone marrow microenvironment assembly of claim 1, wherein, The three-dimensional scaffold material comprises chitosan (CS) and sodium alginate (Alg); the chitosan (CS) is chemically modified by gallic acid, so that the three-dimensional scaffold has antioxidant activity during biodegradation.

4. A method for preparing an in vitro expanded biomimetic bone marrow microenvironment assembly of hematopoietic stem cells according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: S1, chitosan (CS) chemical modification: chitosan (CS) is chemically modified by gallic acid to obtain a chitosan polyphenol derivative; S2, three-dimensional scaffold gel preparation and freeze-drying: the chitosan polyphenol derivative and sodium alginate (Alg) are prepared into a hydrogel, which is injected into a container, and then subjected to freezing, thawing, freezing, chemical cross-linking, and freeze-drying to obtain a three-dimensional scaffold with a pore size distribution of 70-400 μm and a corrugated pore surface; S3, umbilical cord mesenchymal stem cell (MSCs) inoculation and culture: umbilical cord mesenchymal stem cells (MSCs) are inoculated on the three-dimensional scaffold; the umbilical cord mesenchymal stem cells (MSCs) are used as stromal cells to form the in-vitro expansion of hematopoietic stem cell biomimetic bone marrow microenvironment components together with the three-dimensional scaffold; or umbilical cord mesenchymal stem cells (MSCs) are inoculated on the three-dimensional scaffold for culture and expansion, and are induced and differentiated into osteoblasts (OBs); the umbilical cord mesenchymal stem cells (MSCs) and the osteoblasts (OBs) are used as stromal cells to form the in-vitro expansion of hematopoietic stem cell biomimetic bone marrow microenvironment components together with the three-dimensional scaffold.

5. The method of claim 4, wherein the preparation of the in vitro expanded hematopoietic stem cell biomimetic bone marrow microenvironment assembly is characterized by, The specific method for chitosan (CS) chemical modification is as follows: S1.1, 28 mmol of gallic acid and 2.8 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) are dissolved in 40 mL of 70% v / v ethanol, 2.8 mmol of N-hydroxysuccinimide (NHS) is added to the solution, the solution is stirred in an ice bath for 10-20 min, is left to stand at room temperature for 1-2 h, and then 1.5 g of chitosan (CS) dispersed in 110 mL of 70% v / v ethanol is added; the solution is stirred in an ice bath for 20-30 min, and then is continuously stirred at room temperature for 20-24 h; S1.2, the precipitate is collected by filtration, is placed in a container, is washed with 70% v / v ethanol for three times, 100 mL of deionized water is added to the precipitate to prepare a gel-like suspension, the suspension is loaded into a dialysis bag, and is dialyzed with deionized water for 3-4 days; the deionized water is replaced every 8 h for the first two days, and is replaced every 12 h for the last two days. S1.3, the suspension after dialysis is dried by vacuum freeze-drying method to obtain chitosan polyphenol derivative powder.

6. The method of claim 4, wherein the preparation of the in vitro expanded hematopoietic stem cell biomimetic bone marrow microenvironment assembly is characterized by, The three-dimensional scaffold gel preparation method is: S2.1, preparing 1%-2% w / v sodium alginate (Alg) aqueous solution; the PH value of the sodium alginate (Alg) solution at this time is maintained at 6.0-6.3; S2.2, chitosan polyphenol derivative powder is added to the prepared sodium alginate (Alg) solution at 1%-2% wt, and stirred at room temperature for 30-40 min.

7. The method of claim 4, wherein the preparation of the in vitro expanded hematopoietic stem cell biomimetic bone marrow microenvironment assembly is characterized by, The three-dimensional scaffold freezing method is: S2.3, first freezing: the gel prepared from chitosan polyphenol derivative and sodium alginate (Alg) is placed in a container and frozen at-18--20℃ for 20-24h; S2.4, thawing: the frozen three-dimensional scaffold is thawed at room temperature for 4-6h; S2.5, second freezing: the thawed three-dimensional scaffold is frozen at-18--20℃ for 120-240h; then dried at 23-27℃ for 20-24h; S2.6, crosslinking: the three-dimensional scaffold is taken out and soaked in 1.5%-3% w / v calcium chloride aqueous solution for 30-60min; the soaked three-dimensional scaffold is washed with phosphate buffer solution (PBS) for 3-4 times; S2.7, crosslinking and freeze-drying: pre-freezing: the washed three-dimensional scaffold is frozen at-38--42℃ for 4-6h; first drying: the temperature is raised from-38--42℃ to-25--30℃ at a temperature raising speed of 0.3-0.5℃ / min, the vacuum degree is ≤30Pa, and the temperature is maintained for 20-24h; second drying: the temperature is raised from-25-30℃ to 23-27℃ at a temperature raising speed of 0.3-0.5℃ / min, the vacuum degree is ≤10Pa, and the temperature is maintained for 20-24h.

8. The method of claim 4, wherein the preparation of the in vitro expanded hematopoietic stem cell biomimetic bone marrow microenvironment assembly is characterized by, The three-dimensional scaffold freezing method is: S2.3, first freezing: the gel prepared from chitosan polyphenol derivative and sodium alginate (Alg) is placed in a container and frozen at-18--20℃ for 20-24h; S2.4, thawing: the frozen three-dimensional scaffold is thawed at room temperature for 4-6h; S2.5, second freezing: the thawed three-dimensional scaffold is frozen at-18--20℃ for 20-24h; then dried at 23-27℃ for 20-24h; S2.6, crosslinking: the three-dimensional scaffold is taken out and soaked in 1.5%-3% w / v calcium chloride aqueous solution for 30-60min; the soaked three-dimensional scaffold is washed with phosphate buffer solution (PBS) for 3-4 times; S2.7, crosslinking and freeze-drying: pre-freezing: the washed three-dimensional scaffold is frozen at-38--42℃ for 4-6h; first drying: the temperature is raised from-38--42℃ to-25--30℃ at a temperature raising speed of 0.3-0.5℃ / min, the vacuum degree is ≤30Pa, and the temperature is maintained for 20-24h; second drying: the temperature is raised from-25-30℃ to 23-27℃ at a temperature raising speed of 0.3-0.5℃ / min, the vacuum degree is ≤10Pa, and the temperature is maintained for 20-24h.

9. The method of claim 4, wherein the preparation of the in vitro expanded hematopoietic stem cell biomimetic bone marrow microenvironment assembly is characterized by, The umbilical cord mesenchymal stem cells (MSCs) are inoculated and cultured on the three-dimensional support for 24-72 hours, and the umbilical cord mesenchymal stem cells (MSCs) are fully attached and spread in the pores of the three-dimensional support, thereby forming the in-vitro expansion of the hematopoietic stem cell biomimetic bone marrow microenvironment assembly.

10. The method of claim 4, wherein the preparation of the in vitro expanded hematopoietic stem cell biomimetic bone marrow microenvironment assembly is characterized by, The umbilical cord mesenchymal stem cells (MSCs) are inoculated and cultured on the three-dimensional support for 14-21 days, and are differentiated into osteoblasts (OBs) through induction, thereby forming the in-vitro expansion of the hematopoietic stem cell biomimetic bone marrow microenvironment assembly.

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