Polymeric composite microcarriers and methods of making the same

Abstract

The present application relates to a kind of polymer composite microcarrier and its preparation method.It includes: microcarrier body, the microcarrier body includes synthetic polymer, the microcarrier body is hollow structure, the axial length of the microcarrier body is greater than the radial length of the microcarrier body;Functional area, the functional area is microporous structure, the functional area is formed on the microcarrier body, and the functional area includes natural macromolecule, or, the functional area includes natural macromolecule and inorganic salt.The polymer composite microcarrier of the present application can make cell adhere to the outer surface of hollow microcarrier, and the internal hollow structure can facilitate the flow of liquid, provide uniform, good microenvironment for large-scale expansion of cell, it is advantageous to improve the number of cell expansion.

Description

[0001] This application claims priority to Chinese Patent Application No. 202211309954.1, entitled "A Polymer Composite Microcarrier and its Preparation Method", filed on October 25, 2022, the entire technical content of which is incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedical engineering technology, and in particular to a polymer composite microcarrier and its preparation method. Background Technology

[0003] Stem cells are cells with the potential for self-renewal, multi-lineage differentiation, or directed differentiation; they are the primitive cells at the source of cell line differentiation. Among them, mesenchymal stem cells, such as bone marrow mesenchymal stem cells and adipose-derived mesenchymal stem cells, have attracted significant attention from researchers due to their advantages, including low immune rejection, availability of autologous tissue, and well-established strategies for inducing differentiation. Therefore, they have wide applications in tissue engineering, developmental biology, tumor research, and even tissue repair in clinical settings. Although stem cells have numerous advantages and have been widely used, a fundamental challenge remains to be addressed in stem cell research:

[0004] In practical applications, stem cells require a certain quantity while ensuring high quality; the cell quantities used in tissue engineering are mostly on the order of 10. 7 Around 10,000 cells are used in clinical application research. 9 Approximately 100 μm. Current stem cell expansion culture typically utilizes microcarriers. Microcarriers are miniature spheres with a diameter of 60 μm to 250 μm suitable for adherent cell growth. Advantages of microcarrier culture include a large surface area / volume (S / V) ratio, resulting in high cell yield per unit volume of culture medium. It combines the advantages of suspension and adherent cultures. During the culture process, cell growth can be observed and adjusted in real-time using an optical microscope, simplifying the detection and control of various environmental factors and ensuring good reproducibility. Culture medium utilization is high, scale-up is easy, cell harvesting is simple, labor intensity is low, and the culture system requires minimal floor space. Compared to traditional amplification methods using culture dishes and flasks, microcarrier culture saves significant space, manpower, reagents, and time costs.

[0005] However, currently used microcarriers are almost all spherical in shape, with cells adhering to the surface for growth. This has revealed some shortcomings, such as: 1. Difficulty in separating cells from the microcarrier. When collecting cells after expansion, substances like trypsin and EDTA are needed, but these reagents significantly affect cell viability; 2. Insufficient nutrient and oxygen supply within cell clusters. After massive cell proliferation, clusters form, creating a barrier between internal and external cells. The microenvironment's nutrient and oxygen levels are lower than in the culture medium, creating a gradient effect from the outside in. Different culture microenvironments not only affect cell proliferation but also the production of cell secretions. In practical applications, not only stem cells require these microenvironments, but cell secretions also have corresponding requirements. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing a novel method for manufacturing and applying microcarriers for cell culture. Cells adhere to the outer surface of the hollow microcarrier, while the internal hollow structure facilitates liquid flow. Furthermore, the hollow microcarrier material exhibits thermosensitive properties, allowing for control of cell adhesion and separation, and facilitating the collection of high-purity cells during the expansion phase. Traditional processes using chemicals such as trypsin can significantly impact cell viability. The microcarrier molding process and cell culture method of this invention are simple and efficient, producing a uniform culture microenvironment that effectively improves the quality and quantity of stem cells during expansion. Moreover, large-scale cell collection can be easily achieved through temperature control in the later stages. This invention is suitable for research and applications in high-throughput drug screening, immunotherapy, stem cell therapy, and tissue engineering.

[0007] A polymer composite microcarrier for cell culture includes: a microcarrier body comprising a synthetic polymer, the microcarrier body having a hollow structure and an axial length greater than its radial length; and functional regions having a microporous structure formed on the microcarrier body, the functional regions comprising natural macromolecules, or comprising natural macromolecules and inorganic salts.

[0008] This invention discloses a polymer composite microcarrier for cell culture. Compared to traditional spherical polymer composite microcarriers, the polymer composite microcarrier of this application has a hollow, elongated cylindrical structure, preventing cells from clustering on the surface of the polymer composite microcarrier and thus avoiding insufficient internal nutrient and oxygen supply. The polymer composite microcarrier of this invention allows cells to adhere to the outer surface of the hollow microcarrier, while the internal hollow structure facilitates liquid flow, providing a uniform and favorable microenvironment for large-scale cell expansion, which is beneficial for increasing the number of cells expanded.

[0009] In one embodiment, the microcarrier body further includes a temperature-sensitive material.

[0010] The polymer composite microcarriers of this invention further contain thermosensitive substances, giving the formed microcarriers a thermosensitive effect. This allows for temperature-dependent control of cell adhesion and separation, facilitating the collection of high-purity cells during the later stages of expansion. Compared to traditional cell collection methods, this invention eliminates the need to collect the polymer composite microcarriers along with the cells after cell culture. Therefore, it avoids the need to reassess the impact of the microcarrier material on subsequent experiments and clinical applications, saving time, effort, and costs. Furthermore, because the polymer composite microcarriers of this invention have a long cylindrical structure, cells do not adhere tightly to the surface of traditional spherical microcarriers, preventing difficulties in cell separation. Therefore, when collecting cells after cell expansion, it is not necessary to use reagents such as trypsin or EDTA to digest the cells from the microcarrier surface. Consequently, the viability of the cells cultured and collected by this invention is largely unaffected by reagents, resulting in high cell quality.

[0011] Cell culture using the polymer composite microcarrier of the present invention is a simple and efficient method with a uniform culture microenvironment, which can effectively improve the quality and quantity of stem cells during the expansion phase, and allows for convenient large-scale cell collection by adjusting the temperature later.

[0012] In one embodiment, the temperature-sensitive material is iron oxide microparticles and poly(N-isoacrylamide).

[0013] In one embodiment, the axial length of the polymer composite microcarrier is 500 μm to 1000 μm.

[0014] In one embodiment, the diameter of the polymer composite microcarrier is 60 μm to 250 μm.

[0015] In one embodiment, the synthetic polymer comprises one of polylactic acid, polylactic acid-glycolic acid copolymer, polyglycolic acid, and polycaprolactone, or a combination of two or more of the above.

[0016] In one embodiment, the functional region includes natural macromolecules, which include one or a combination of two or more of gelatin, collagen, fibrinogen, and chitosan.

[0017] In one embodiment, the synthetic polymer is methacrylated gelatin.

[0018] In one embodiment, the natural macromolecule is chitosan.

[0019] In one embodiment, the functional region includes natural macromolecules and inorganic salts, wherein the inorganic salts are sodium chloride or potassium chloride.

[0020] A method for preparing a polymer composite microcarrier includes the following steps:

[0021] S11. Dissolve the polymer, natural macromolecules and temperature-sensitive materials in a solvent to form solution A;

[0022] S12. Inject the A solution into the middle needle of the coaxial needle syringe, and simultaneously inject distilled water into the inner needle and the outer needle of the coaxial needle syringe.

[0023] S13. Connect the inner needle of the coaxial needle to the inner solution channel of the microfluidic chip, connect the middle needle of the coaxial needle to the middle solution channel of the microfluidic chip, and connect the outer needle of the coaxial needle to the outer solution channel of the microfluidic chip.

[0024] S14. Solution A is dispensed from the middle solution channel of the microfluidic chip, and distilled water is dispensed from the inner solution channel and the outer solution channel of the microfluidic chip.

[0025] S15. The microfluidic chip is provided with multiple auxiliary channels, which intersect and communicate with the outer solution channel respectively. The multiple auxiliary channels are spaced apart on the microfluidic chip on the side away from the coaxial needle. Distilled water flows into or out of the outer solution channel through the auxiliary channels.

[0026] S16. The synthetic polymer in the middle solution channel of the microfluidic chip is precipitated and formed into a polymer composite microcarrier with a hollow and porous structure, including the functional area and the microcarrier body.

[0027] In one embodiment, the concentration of the synthetic polymer in the solvent ranges from 20 g / L to 60 g / L.

[0028] In one embodiment, the concentration of the natural macromolecule in the solvent ranges from 0.05% to 1.50%.

[0029] In one embodiment, the concentration of the temperature-sensitive material in the solvent ranges from 0.05% to 3.00%.

[0030] In one embodiment, the solvent is hexafluoroisopropanol.

[0031] In one embodiment, the flow rate of solution A in the intermediate solution channel is 10 mL / h to 30 mL / h.

[0032] In one embodiment, the flow rate of distilled water in the inner solution channel is 3 mL / h to 10 mL / h.

[0033] In one embodiment, the flow rate of distilled water in the outer solution channel is 10 mL / h to 50 mL / h.

[0034] In one embodiment, the flow rate of distilled water in the auxiliary flow channel is 30 mL / h to 100 mL / h.

[0035] In one embodiment, after step S16, the following step is further included:

[0036] S17. After the polymer composite microcarrier is formed, the polymer composite microcarrier is frozen in the freezing liquid of a cryostat, with a layer thickness of 500μm to 1000μm. The uniformly cut microparticles are collected, washed with distilled water, and then put into use.

[0037] A method for preparing a polymer composite microcarrier includes the following steps:

[0038] S21. Dissolve the polymer, natural macromolecules, inorganic salts and temperature-sensitive materials in a solvent to form solution B;

[0039] S22. Inject the B solution into the middle needle of the coaxial needle syringe, and simultaneously inject distilled water into the inner needle and the outer needle of the coaxial needle syringe.

[0040] S23. Connect the inner needle of the coaxial needle to the inner solution channel of the microfluidic chip, connect the middle needle of the coaxial needle to the middle solution channel of the microfluidic chip, and connect the outer needle of the coaxial needle to the outer solution channel of the microfluidic chip.

[0041] S24. Solution B is exited from the middle solution channel of the microfluidic chip, and distilled water is exited from the inner solution channel and the outer solution channel of the microfluidic chip.

[0042] S25. The microfluidic chip is placed within the blue light irradiation range, and the synthetic polymer of solution B in the middle layer solution channel of the microfluidic chip is formed into a hollow microcarrier body by light irradiation.

[0043] S26. The microfluidic chip is provided with multiple auxiliary channels, which intersect and communicate with the outer solution channel respectively. The multiple auxiliary channels are spaced apart on the microfluidic chip on the side away from the coaxial needle. Distilled water flows into or out of the outer solution channel through the auxiliary channels. The inorganic salt in the hollow microcarrier body is continuously dissolved in the distilled water. The hollow microcarrier body is formed into the polymer composite microcarrier with functional areas and a microporous structure that connects the inside and outside.

[0044] In one embodiment, the concentration of the synthetic polymer in the solvent ranges from 3% to 15%.

[0045] In one embodiment, the concentration of the natural macromolecule in the solvent ranges from 0.05% to 1.50%.

[0046] In one embodiment, the concentration of the inorganic salt in the solvent ranges from 0.5% to 3.0%.

[0047] In one embodiment, the concentration of the temperature-sensitive material in the solvent ranges from 0.05% to 3.00%.

[0048] In one embodiment, the solvent is distilled water.

[0049] In one embodiment, the flow rate of solution B in the intermediate solution channel is 10 mL / h to 30 mL / h.

[0050] In one embodiment, the flow rate of distilled water in the inner solution channel is 3 mL / h to 10 mL / h.

[0051] In one embodiment, the flow rate of distilled water in the outer solution channel is 10 mL / h to 50 mL / h.

[0052] In one embodiment, the flow rate of distilled water in the auxiliary flow channel is 30 mL / h to 100 mL / h.

[0053] In one embodiment, a blue light lamp is also included, the distance between the blue light lamp and the microfluidic chip ranging from 5cm to 25cm.

[0054] In one embodiment, after step S26, the following step is further included:

[0055] S27. After the polymer composite microcarrier is formed, it is frozen in the freezing liquid of a cryostat, with a layer thickness of 500μm to 1000μm. The uniformly cut microparticles are collected, washed with distilled water, and then put into use. Attached Figure Description

[0056] Figure 1 This is a three-dimensional schematic diagram of a microfluidic chip for manufacturing a polymer composite microcarrier according to the present invention;

[0057] Figure 2 This is a three-dimensional schematic diagram of a polymer composite microcarrier according to the present invention;

[0058] Figure 3 This is a schematic diagram illustrating the large-scale application of a polymer composite microcarrier according to the present invention.

[0059] Figure 4 Schematic diagram of the application scenario of traditional microcarriers;

[0060] Figure 5 This is a schematic diagram illustrating the application scenario 2 of traditional microcarriers;

[0061] Figure 6 This is a process flow diagram for preparing one type of polymer composite microcarrier according to the present invention;

[0062] Figure 7 This is a process flow diagram for preparing another polymer composite microcarrier according to the present invention;

[0063] Figure 8 A radial cross-sectional view of the polymer composite microcarrier as observed by scanning electron microscopy;

[0064] Figure 9 This is a scanning electron microscope view of the outer surface structure of the polymer composite microcarrier after cells have been grown and cultured on the polymer composite microcarrier.

[0065] Figure 10 This is a comparison of cell proliferation when the polymer composite microcarrier prepared in one embodiment of the present invention is used as a cell culture carrier and when a conventional culture dish is used as a cell culture carrier.

[0066] The correspondence between the reference numerals and the component names is as follows:

[0067] 101-Inner flow channel inlet; 102-Intermediate flow channel inlet; 103-Outer flow channel inlet; 104-Auxiliary flow channel inlet; 105-Auxiliary flow channel outlet; 301-Microcarrier body; 302-Cell; 401-Traditional spherical microcarrier. Detailed Implementation

[0068] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0069] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0070] The preparation methods of polymer composite microcarriers according to some embodiments of the present invention are described below with reference to the accompanying drawings.

[0071] Example 1

[0072] This embodiment provides a method for preparing polymer composite microcarriers using extraction precipitation molding technology. Figure 1 This is a three-dimensional schematic diagram of the microfluidic chip for manufacturing polymer composite microcarriers according to the present invention. The microfluidic chip includes a three-layer coaxial needle in the front half and a multi-stage auxiliary flow channel in the rear half for extraction and precipitation. The coaxial needle includes an inner solution channel 101, a middle solution channel 102, and an outer solution channel 103. In this embodiment, the pore size of the inner solution channel is 30 μm, the pore size of the middle solution channel is 150 μm, and the pore size of the outer solution channel is 350 μm. A syringe pump sprays distilled water from the inner and outer solution channels of the coaxial needle in a stable flow manner. The flow rate of distilled water in the inner solution channel 101 is 3 mL / h, and the flow rate of distilled water in the outer solution channel 103 is 10 mL / h. Polylactic acid (PLA), gelatin, chitosan, iron oxide microparticles, and poly(N-isopropylacrylamide) are dissolved in hexafluoroisopropanol to form solution A. Solution A is then pushed out through the intermediate solution channel 102 at a flow rate of 10 mL / h. The mass concentrations of PLA in hexafluoroisopropanol are: PLA 50 g / L, gelatin 20 g / L, chitosan 0.05%, iron oxide microparticles 1%, and poly(N-isopropylacrylamide) 0.05%. Because hexafluoroisopropanol has good solubility in water, while PLA is almost insoluble in water at room temperature, the PLA, gelatin, and chitosan solutes gradually precipitate and form porous hollow microtubes as they pass through the auxiliary flow channel in the latter half of the channel. Distilled water flows in from the inlet 104 of the auxiliary flow channel and out from the outlet 105. To ensure a certain concentration difference in the latter half and improve extraction efficiency, a multi-stage auxiliary flow channel was designed to achieve efficient flow of distilled water within it. The flow rate of distilled water in the auxiliary flow channel is 30 mL / h. After continuous hollow tube forming, it is frozen in the freezing solution of a cryostat, with a layer thickness set at 500 μm, thus ensuring that the axial length of the cut polymer composite microcarriers is 500 μm. The uniformly cut microparticles are then collected, washed with distilled water, and ready for cell culture. The three-dimensional structure of the polymer composite microcarrier is shown below. Figure 2 As shown.

[0073] It should be noted that hexafluoroisopropanol is used as the corresponding solvent in this embodiment. Hexafluoroisopropanol has good solubility and volatility, and can be well miscible with water. It is easy to remove, has low toxicity, and is suitable for cell culture.

[0074] It should be noted that distilled water is used as the extractant in this embodiment, mainly because it has no toxic side effects. Using any other solvent as the extractant will leave residues and cause significant damage to cells.

[0075] It should be noted that in this embodiment, the liquid in each solution channel of the microfluidic chip can flow continuously, which enables continuous production of hollow microcarriers with more stable size and quality.

[0076] It should be noted that in this embodiment, after the continuous hollow tube is formed, it is frozen in the freezing liquid of the cryostat. The freezing liquid that comes with the cryostat can be used, or distilled water can be used as the freezing liquid. Water can also freeze the sample on the sample stage at low temperatures.

[0077] like Figure 3 As shown, during large-scale cell culture, the polymer composite microcarriers of this invention exhibit clustering due to the adhesion of cells 302. In this case, due to the presence of the hollow internal structure, the culture medium can penetrate well into the cluster through capillary action, providing sufficient nutrients and oxygen to the cells inside, overcoming the difficulty of cells inside traditional spherical microcarriers obtaining nutrients and oxygen.

[0078] During the routine culture phase, the culture medium temperature is maintained at a normal 37°C. At this temperature, the surface of the polymer composite microcarrier body 301 is weakly hydrophobic, allowing cells 302 to adhere and grow well. In the later collection phase, the culture medium temperature can be lowered to 4°C. Under these low-temperature conditions, the surface of the polymer composite microcarrier becomes weakly hydrophilic, causing cells to detach from the microcarrier surface, facilitating the collection of high-purity cells. If traditional spherical microcarriers also incorporate temperature-sensitive materials, even with reduced cell adhesion to the microcarrier surface during collection, cells cannot detach from the microcarrier in large quantities and rapidly. This is because, after extensive expansion, cells 302 grow in clusters, encapsulating the spherical microcarrier 401. Figure 4 , Figure 5 As shown, separation of microcarriers is quite difficult under these conditions. However, the polymer composite microcarriers of this invention are much larger in the axial dimension than in the radial dimension, thus preventing cells from effectively encapsulating them and achieving a good separation effect.

[0079] Example 2

[0080] This embodiment provides a second method for preparing polymer composite microcarriers using extraction precipitation molding technology. In this embodiment, the pore size of the inner solution channel is 50 μm, the pore size of the middle solution channel is 250 μm, and the pore size of the outer solution channel is 1000 μm. A syringe pump sprays distilled water from the inner and outer solution channels of the coaxial needle in a stable flow manner. The flow rate of distilled water in the inner solution channel 101 is 10 mL / h, and the flow rate of distilled water in the outer solution channel 103 is 50 mL / h. Polylactic acid, gelatin, chitosan, iron oxide microparticles, and poly(N-isopropylacrylamide) are dissolved in hexafluoroisopropanol to form solution A. Solution A is then pushed out from the middle solution channel 102, where the flow rate of solution A is 30 mL / h. The concentrations of polylactic acid (PLA) in hexafluoroisopropanol (H2SO4) were 60 g / L, gelatin 30 g / L, chitosan 1.5%, iron oxide microparticles 3%, and poly(N-isopropylacrylamide) 1.5%. Distilled water flowed at a rate of 100 mL / h in the auxiliary flow channel. After forming continuous hollow tubes, they were frozen in the freezing solution of a cryostat, with a layer thickness of 1000 μm, resulting in polymer composite microcarriers with an axial length of 1000 μm. The uniformly cut microparticles were then collected, washed with distilled water, and ready for use in cell culture.

[0081] Example 3

[0082] This embodiment provides a third method for preparing polymer composite microcarriers using extraction precipitation molding technology. In this embodiment, the pore size of the inner solution channel is 50 μm, the pore size of the middle solution channel is 250 μm, and the pore size of the outer solution channel is 1000 μm. A syringe pump sprays distilled water from the inner and outer solution channels of the coaxial needle in a stable flow manner. The flow rate of distilled water in the inner solution channel 101 is 6 mL / h, and the flow rate of distilled water in the outer solution channel 103 is 25 mL / h. Polylactic acid, gelatin, chitosan, iron oxide microparticles, and poly(N-isopropylacrylamide) are dissolved in hexafluoroisopropanol to form solution A. Solution A is then pushed out from the middle solution channel 102, where the flow rate of solution A is 15 mL / h. The concentrations of polylactic acid (PLA) in hexafluoroisopropanol (H2SO4) were 60 g / L, collagen 25 g / L, chitosan 0.75%, iron oxide microparticles 1.5%, and poly(N-isopropylacrylamide) 0.75%. Distilled water flowed at a rate of 50 mL / h in the auxiliary flow channel. After forming continuous hollow tubes, they were frozen in the freezing solution of a cryostat, with a layer thickness set at 850 μm, resulting in polymer composite microcarriers with an axial length of 850 μm. The uniformly cut microparticles were then collected, washed with distilled water, and ready for use in cell culture.

[0083] Example 4

[0084] This embodiment provides a method for preparing polymer composite microcarriers using photopolymerization molding technology. Figure 1 This is a three-dimensional schematic diagram of a microfluidic chip used to manufacture the polymer composite microcarrier of the present invention. The microfluidic chip includes a three-layer coaxial needle in the front half and a multi-stage flow channel in the rear half for extraction and precipitation. The coaxial needle includes inner, middle, and outer solution channels. In this embodiment, the pore size of the inner solution channel is 30 μm, the pore size of the middle solution channel is 150 μm, and the pore size of the outer solution channel is 350 μm. A syringe pump sprays distilled water from the inner and outer solution channels of the coaxial needle in a stable flow manner. The flow rate of distilled water in the inner solution channel 101 is 3 mL / h, and the flow rate of distilled water in the outer solution channel 103 is 10 mL / h. Methacrylated gelatin, chitosan, sodium chloride ferric oxide microparticles, and poly(N-isopropylacrylamide) are dissolved in distilled water to form solution B. Solution B is pushed out from the middle solution channel 102, and the flow rate of solution B in the middle solution channel 102 is 10 mL / h. The mass concentration range of the methacrylated gelatin is 3%, the mass concentration of chitosan is 0.05%, the mass concentration of sodium chloride is 0.5%, the mass concentration of iron oxide microparticles is 1%, and the concentration of poly(N-isopropylacrylamide) is 0.05%.

[0085] Methacrylated gelatin was cross-linked and fixed into hollow microtubes after irradiation with blue light. The wavelength of the blue light was 405 nm, and the irradiated area was... Figure 1 As shown at point A, the distance between the lamp and point A is 15 cm. During the passage through the multi-stage auxiliary flow channel in the latter half, sodium chloride in the colloid continuously dissolves in the water, forming micropores connecting the inside and outside of the hollow microtube. The flow rate of distilled water in the auxiliary flow channel is 30 mL / h. After the continuous hollow tube is formed, it is frozen in the freezing solution of a cryostat, with a layer thickness set at 500 μm, thus ensuring that the axial length of the cut microcarrier is 500 μm. The uniformly cut microparticles are then collected, washed with distilled water, and ready for cell culture. The three-dimensional structure of the polymer composite microcarrier is shown below. Figure 2 As shown.

[0086] During routine culture, the culture medium temperature is maintained at the normal level of 37°C. At this temperature, the surface of the microcarrier is weakly hydrophobic, allowing cells to adhere and grow well. Figure 8 and Figure 9 As shown; in the later stage of culture and collection, the temperature of the culture medium can be lowered to 4℃. Under low temperature conditions, the surface of the microcarrier becomes weakly hydrophilic, and the cells will detach from the surface of the microcarrier, making it convenient to collect high-purity cells.

[0087] Example 5

[0088] This embodiment provides a second method for preparing polymer composite microcarriers using photopolymerization molding technology. The microfluidic chip includes a three-layer coaxial needle in the front half and a multi-stage flow channel in the rear half for extraction and precipitation. The coaxial needle includes inner, middle, and outer solution channels. In this embodiment, the pore size of the inner solution channel is 50 μm, the pore size of the middle solution channel is 250 μm, and the pore size of the outer solution channel is 1000 μm. A syringe pump sprays distilled water from the inner and outer solution channels of the coaxial needle in a stable flow manner. The flow rate of distilled water in the inner solution channel 101 is 10 mL / h, and the flow rate of distilled water in the outer solution channel 103 is 50 mL / h. Methacrylated gelatin, chitosan, sodium chloride ferric oxide microparticles, and poly(N-isopropylacrylamide) are dissolved in distilled water to form solution B. Solution B is then pushed out from the middle solution channel 102, where the flow rate of solution B is 30 mL / h. The mass concentration range of the methacrylated gelatin is 15%, the mass concentration of chitosan is 1.5%, the mass concentration of sodium chloride is 3%, the mass concentration of iron oxide microparticles is 3%, and the concentration of poly(N-isopropylacrylamide) is 1.5%.

[0089] Methacrylated gelatin was cross-linked and fixed into hollow microtubes after irradiation with blue light. The wavelength of the blue light was 405 nm, and the irradiated area was as follows: Figure 1As shown at point A, the distance between the lamp and point A is 5 cm. During the passage through the multi-stage auxiliary flow channel in the latter half, sodium chloride in the colloid continuously dissolves in the water, forming micropores that connect the inside and outside of the hollow microtube. The flow rate of distilled water in the auxiliary flow channel is 100 mL / h. After the continuous hollow tube is formed, it is frozen in the freezing solution of a cryostat, with a layer thickness set at 1000 μm, resulting in a 1000 μm axial length for the cut microcarriers. The uniformly cut microparticles are then collected, washed with distilled water, and ready for use in cell culture.

[0090] Example 6

[0091] This embodiment provides a third method for preparing polymer composite microcarriers using photopolymerization molding technology. The microfluidic chip includes a three-layer coaxial needle in the front half and a multi-stage flow channel in the rear half for extraction and precipitation. The coaxial needle includes inner, middle, and outer solution channels. In this embodiment, the pore size of the inner solution channel is 50 μm, the pore size of the middle solution channel is 250 μm, and the pore size of the outer solution channel is 1000 μm. A syringe pump sprays distilled water from the inner and outer solution channels of the coaxial needle in a stable flow manner. The flow rate of distilled water in the inner solution channel 101 is 7 mL / h, and the flow rate of distilled water in the outer solution channel 103 is 25 mL / h. Methacrylated gelatin, chitosan, sodium chloride ferric oxide microparticles, and poly(N-isopropylacrylamide) are dissolved in distilled water to form solution B. Solution B is then pushed out from the middle solution channel 102, where the flow rate of solution B is 20 mL / h. The mass concentration range of the methacrylated gelatin is 7.5%, the mass concentration of chitosan is 0.75%, the mass concentration of sodium chloride is 0.75%, the mass concentration of iron oxide microparticles is 1.5%, and the concentration of poly(N-isopropylacrylamide) is 0.75%.

[0092] Methacrylated gelatin was cross-linked and fixed into hollow microtubes after irradiation with blue light. The wavelength of the blue light was 405 nm, and the irradiated area was as follows: Figure 1 As shown at point A, the distance between the lamp and point A is 10 cm. During the passage through the multi-stage auxiliary flow channel in the latter half, sodium chloride in the colloid continuously dissolves in the water, forming micropores that connect the inside and outside of the hollow microtube. The flow rate of distilled water in the auxiliary flow channel is 60 mL / h. After the continuous hollow tube is formed, it is frozen in the freezing solution of a cryostat, with a layer thickness set at 750 μm, thus ensuring that the axial length of the cut microcarriers is 750 μm. The uniformly cut microparticles are then collected, washed with distilled water, and ready for use in cell culture.

[0093] Example 7

[0094] This embodiment is a comparative experiment on the effect of the polymer composite microcarrier of the present invention obtained by any of the preparation methods in Examples 1 to 6 on cell proliferation culture.

[0095] Take bone marrow mesenchymal stem cells as an example.

[0096] Experimental group: The polymer composite microcarrier culture of the present invention (hereinafter referred to as "3D culture"), with an initial cell number of 500,000;

[0097] Control group: Cultured in petri dishes (hereinafter referred to as "2D culture"), with an initial cell count of 500,000.

[0098] Three parallel experimental samples were set up for both the experimental group and the control group. The same stem cell culture medium was used during the culture process. The pH value of the stem cell culture medium was 7.2±0.3. The medium was changed every 48 hours and placed in the same incubator (37℃, 5% carbon dioxide concentration).

[0099] The cell culture curves of both are as follows: Figure 10 As shown, the control group in 2D culture initially grew rapidly, but when the space in the culture dish was filled and the cells were in complete contact with each other, contact inhibition occurred. Not only did the number of cells stop increasing, but apoptosis even occurred. The experimental group provided the cells with sufficient growth space and ensured good nutrition and oxygen supply, so the cells continued to grow steadily.

[0100] The average number of cells in the experimental group was 1.25 million after 48 hours, 3.12 million after 96 hours, 4.78 million after 144 hours, and 5.92 million after 192 hours.

[0101] The average number of cells in the control group was 780,000 after 48 hours, 1.68 million after 96 hours, 2.32 million after 144 hours, and 1.98 million after 192 hours.

[0102] Therefore, it can be seen that the polymer composite microcarrier of the present invention has significant advantages over culture dish culture in the culture of bone marrow mesenchymal stem cells.

[0103] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0104] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A polymeric composite microcarrier for use in cell culture, characterized in that, include: The microcarrier body comprises a synthetic polymer selected from one or more of polylactic acid, polylactic acid-glycolic acid copolymer, polyglycolic acid, and polycaprolactone. The microcarrier body has a hollow structure, and the axial length of the microcarrier body is greater than the radial length of the microcarrier body. The functional region has a microporous structure and is formed on the microcarrier body. The functional region includes natural macromolecules, which are selected from one or more of gelatin, collagen, fibrinogen, and chitosan. Alternatively, the synthetic polymer is methacrylated gelatin, and the functional region includes natural macromolecules and inorganic salts, wherein the natural macromolecules are chitosan and the inorganic salts are sodium chloride or potassium chloride; The microcarrier body also includes a temperature-sensitive material; The preparation method of the polymer composite microcarrier includes the following steps: S11. Dissolve the polymer, natural macromolecules and temperature-sensitive materials in a solvent to form solution A; S12. Inject the A solution into the middle needle of the coaxial needle syringe, and simultaneously inject distilled water into the inner needle and the outer needle of the coaxial needle syringe. S13. Connect the inner needle of the coaxial needle to the inner solution channel of the microfluidic chip, connect the middle needle of the coaxial needle to the middle solution channel of the microfluidic chip, and connect the outer needle of the coaxial needle to the outer solution channel of the microfluidic chip. S14. Solution A is dispensed from the middle solution channel of the microfluidic chip, and distilled water is dispensed from the inner solution channel and the outer solution channel of the microfluidic chip. S15. The microfluidic chip is provided with multiple auxiliary channels, which intersect and communicate with the outer solution channel respectively. The multiple auxiliary channels are spaced apart on the microfluidic chip on the side away from the coaxial needle. Distilled water flows into or out of the outer solution channel through the auxiliary channels. S16. The synthetic polymer in the middle solution channel of the microfluidic chip is precipitated and formed into a polymer composite microcarrier with a hollow and porous structure, including the functional area and the microcarrier body.

2. The polymer composite microcarrier according to claim 1, characterized in that, The temperature-sensitive material is iron oxide microparticles and poly(N-isoacrylamide).

3. The polymer composite microcarrier according to claim 1, characterized in that, The axial length of the polymer composite microcarrier is 500 μm to 1000 μm; and / or The diameter of the polymer composite microcarrier is 60 μm to 250 μm.

4. The polymer composite microcarrier according to claim 1, characterized in that, In the preparation process of the polymer composite microcarrier, the concentration of the synthesized polymer in the solvent ranges from 20 g / L to 60 g / L; and / or The concentration of the natural macromolecule in the solvent ranges from 0.05% to 1.50%; and / or The concentration range of the temperature-sensitive material in the solvent is 0.05% to 3.00%; and / or The solvent is hexafluoroisopropanol; and / or The flow rate of solution A in the intermediate layer solution channel is 10 mL / h to 30 mL / h; and / or The flow rate of distilled water in the inner solution channel is 3 mL / h to 10 mL / h; and / or The flow rate of distilled water in the outer solution channel is 10 mL / h to 50 mL / h; and / or The flow rate of distilled water in the auxiliary flow channel is 30 mL / h to 100 mL / h; and / or Following step S16, the following steps are also included: S17. After the polymer composite microcarrier is formed, the polymer composite microcarrier is frozen in the freezing liquid of a cryostat, with a layer thickness of 500μm to 1000μm. The uniformly cut microparticles are collected, washed with distilled water, and then put into use.

5. The polymer composite microcarrier according to claim 1, characterized in that, The preparation method of the polymer composite microcarrier includes the following steps: S21. Dissolve the polymer, natural macromolecules, inorganic salts and temperature-sensitive materials in a solvent to form solution B; S22. Inject the B solution into the middle needle of the coaxial needle syringe, and simultaneously inject distilled water into the inner needle and the outer needle of the coaxial needle syringe. S23. Connect the inner needle of the coaxial needle to the inner solution channel of the microfluidic chip, connect the middle needle of the coaxial needle to the middle solution channel of the microfluidic chip, and connect the outer needle of the coaxial needle to the outer solution channel of the microfluidic chip. S24. Solution B is exited from the middle solution channel of the microfluidic chip, and distilled water is exited from the inner solution channel and the outer solution channel of the microfluidic chip. S25. The microfluidic chip is placed within the blue light irradiation range, and the synthetic polymer of solution B in the middle layer solution channel of the microfluidic chip is formed into a hollow microcarrier body by light irradiation. S26. The microfluidic chip is provided with multiple auxiliary channels, which intersect and communicate with the outer solution channel respectively. The multiple auxiliary channels are spaced apart on the microfluidic chip on the side away from the coaxial needle. Distilled water flows into or out of the outer solution channel through the auxiliary channels. The inorganic salt in the hollow microcarrier body is continuously dissolved in the distilled water. The hollow microcarrier body is formed into the polymer composite microcarrier with functional areas and a microporous structure that connects the inside and outside.

6. The polymer composite microcarrier according to claim 5, characterized in that, During the preparation of the polymer composite microcarrier, The concentration range of the synthesized polymer in the solvent is 3% to 15%; and / or The concentration of the natural macromolecule in the solvent ranges from 0.05% to 1.50%; and / or The concentration of the inorganic salt in the solvent ranges from 0.5% to 3.0%; and / or The concentration range of the temperature-sensitive material in the solvent is 0.05% to 3.00%; and / or The solvent is distilled water; and / or The flow rate of solution B in the intermediate layer solution channel is 10 mL / h to 30 mL / h; and / or The flow rate of distilled water in the inner solution channel is 3 mL / h to 10 mL / h; and / or The flow rate of distilled water in the outer solution channel is 10 mL / h to 50 mL / h; and / or The flow rate of distilled water in the auxiliary flow channel is 30 mL / h to 100 mL / h; and / or It also includes a blue light, wherein the distance between the blue light and the microfluidic chip ranges from 5cm to 25cm; and / or Following step S26, the following steps are also included: S27. After the polymer composite microcarrier is formed, it is frozen in the freezing liquid of a cryostat, with a layer thickness of 500μm to 1000μm. The uniformly cut microparticles are collected, washed with distilled water, and then put into use.

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