Stem cell culture method using bilayer composite hydrogel microcarriers

Abstract

To provide a method for culturing stem cells with improved efficiency by using a gel that has improved structural strength and enables long-term and stable release of contents.SOLUTION: A bilayer composite hydrogel microcarrier includes: an inner layer gel structure obtained by ionically crosslinking an inner layer polymer with an inner layer crosslinker; and an outer layer gel structure obtained by covalently crosslinking an outer layer monomer with an outer layer crosslinker, which covers the inner layer gel structure; a first inner layer polymer being sodium alginate, a second inner layer polymer being carboxymethylcellulose, and a nutritional component being located inside the inner layer gel structure. The growth rate of stem cells is improved by controlling the composition of the bilayer composite hydrogel microcarrier, the weight ratio of sodium alginate to carboxymethylcellulose, and the location of the nutritional component. The structural strength can be ensured by controlling the weight percentage of carboxymethylcellulose.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present disclosure relates to bilayer composite hydrogel microcarriers and methods for making and using the same, and in particular to the use of bilayer composite hydrogel microcarriers for stem cell culture. [Background technology]

[0002] Gels have the properties of releasing and delaying the release of loaded contents and are widely used in the field of drug release. However, conventional gels have limitations such as being prone to collapse and lacking structural strength, making it difficult to provide long-term and stable release of contents. Therefore, when using gels for stem cell culture, the stem cell culture efficiency is limited. Summary of the Invention [Problem to be solved by the invention]

[0003] Therefore, in order to improve the efficiency of stem cell culture, it is an issue to be resolved how to provide a gel with improved structural strength and extended release time. [Means for solving the problem]

[0004] In some embodiments of the present disclosure, there is provided a method for culturing stem cells using a bi-layer composite hydrogel microcarrier, the method comprising the steps of: providing stem cells, a bi-layer composite hydrogel microcarrier, and nutritional components; mixing the stem cells, the bi-layer composite hydrogel microcarrier, and nutritional components to obtain a gelling medium; and adding a culture solution to the gelling medium, wherein the bi-layer composite hydrogel microcarrier comprises an inner-layer gel structure obtained by ionically crosslinking an inner-layer polymer with an inner-layer crosslinker; and an outer-layer gel structure obtained by covalently crosslinking an outer-layer monomer with the outer-layer crosslinker, the outer-layer gel structure covering the inner-layer gel structure, the inner-layer polymers comprising a first inner-layer polymer and a second inner-layer polymer, the first inner-layer polymer being sodium alginate and the second inner-layer polymer being carboxymethylcellulose, the weight ratio of sodium alginate to carboxymethylcellulose being 3:2, the weight percentage of the carboxymethylcellulose being greater than 1% when the weight percentage of the inner-layer gel structure is taken as 100%, and the nutritional components being located inside the inner-layer gel structure.

[0005] In some embodiments, the stem cells comprise embryonic stem cells, hematopoietic stem cells, mammary stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory stem cells, adipose stem cells, or a combination thereof.

[0006] In some embodiments, the inner layer gel structure is a plurality of inner layer sheet-like structures connected to each other and separated from each other by a plurality of inner layer pores, and the outer layer gel structure is a plurality of outer layer sheet-like structures connected to each other and separated from each other by a plurality of outer layer pores having pore sizes smaller than the pore size of each of the inner layer pores.

[0007] In some embodiments, the inner layer gel structure is an interpenetrating structure of the first inner layer polymer and the second inner layer polymer.

[0008] In some embodiments, the inner layer polymer and the inner layer crosslinked product have opposite electrical properties.

[0009] In some embodiments, the outer layer monomer comprises N,N-dimethylacrylamide, acrylamide, or a combination thereof.

[0010] In some embodiments, the outer layer crosslinked comprises N,N'-methylenebisacrylamide.

[0011] In some embodiments, the weight percentage of sodium alginate is 0.1% to 5%, where the weight percentage of the inner layer gel structure is 100%.

[0012] In some embodiments, the weight percentage of carboxymethyl cellulose is 1% to 5% when the weight percentage of the inner layer gel structure is 100%.

[0013] In some embodiments, the nutritional ingredients include growth factors, vitamin A acid, penicillin, bovine serum albumin, or combinations thereof.

[0014] In some embodiments of the present disclosure, there is provided a method for culturing stem cells using a bi-layer composite hydrogel microcarrier, the method comprising the steps of: providing stem cells, bi-layer composite hydrogel microcarriers, and nutritional components, the nutritional components comprising a high molecular weight protein having a molecular weight greater than 500 g / mol; mixing the stem cells, bi-layer composite hydrogel microcarriers, and nutritional components to obtain a gelling medium; and adding a culture solution to the gelling medium, wherein the bi-layer composite hydrogel microcarrier comprises an inner-layer gel structure obtained by ionically crosslinking an inner-layer polymer with an inner-layer crosslinker; and an outer-layer gel structure obtained by covalently crosslinking an outer-layer monomer with the outer-layer crosslinker, the outer-layer gel structure covering the inner-layer gel structure, the inner-layer polymer comprising a first inner-layer polymer and a second inner-layer polymer, the first inner-layer polymer being sodium alginate and the second inner-layer polymer being carboxymethylcellulose, the weight ratio of sodium alginate to carboxymethylcellulose being 3:2, and the nutritional components being located inside the inner-layer gel structure.

[0015] In some embodiments, the stem cells comprise embryonic stem cells, hematopoietic stem cells, mammary stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory stem cells, adipose stem cells, or a combination thereof.

[0016] In some embodiments, the inner layer gel structure is a plurality of inner layer sheet-like structures connected to each other and separated from each other by a plurality of inner layer pores, and the outer layer gel structure is a plurality of outer layer sheet-like structures connected to each other and separated from each other by a plurality of outer layer pores having pore sizes smaller than the pore size of each of the inner layer pores.

[0017] In some embodiments, the inner layer gel structure is an interpenetrating structure of the first inner layer polymer and the second inner layer polymer.

[0018] In some embodiments, the inner layer polymer and the inner layer crosslinked product have opposite electrical properties.

[0019] In some embodiments, the outer layer monomer comprises N,N-dimethylacrylamide, acrylamide, or a combination thereof.

[0020] In some embodiments, the outer layer crosslinked comprises N,N'-methylenebisacrylamide.

[0021] In some embodiments, the weight percentage of sodium alginate is 0.1% to 5%, where the weight percentage of the inner layer gel structure is 100%.

[0022] In some embodiments, the weight percent of carboxymethyl cellulose is greater than 1%, where the weight percent of the inner layer gel structure is 100%.

[0023] In some embodiments, the weight percentage of carboxymethyl cellulose is 1% to 5% when the weight percentage of the inner layer gel structure is 100%. [Brief explanation of the drawings]

[0024] The following description of the accompanying drawings will make the above and other objects, features, advantages and embodiments of the present invention more comprehensible. [Figure 1] FIG. 1 shows a gel state diagram corresponding to changes in concentration and temperature when an ABA type triblock copolymer of poly(ε-caprolactone-co-glycolide) and poly(ethylene glycol; Tri-PCG) according to some embodiments of the present disclosure is stored in phosphate buffered saline. [Figure 2] Electron microscope images of two-layer composite hydrogel microcarriers according to some embodiments of the present disclosure are shown, where the top row a-c are the SA-CMC@PDMA-1 group, the second row d-f are the SA-CMC@PDMA-3 group, and the bottom row g-i are the SA-CMC@PAA-1 group. [Figure 3A] The figure shows the water absorption and swelling characteristics of different inner gel structures. [Figure 3B] The water absorption and swelling characteristics of two-layer composite hydrogel microcarriers with different gel structures are shown. [Figure 4A] 1 shows the cumulative release rate of the contents over time at different pH values ​​for two-layer composite hydrogel microcarriers with different gel structures. [Figure 4B] 1 shows the cumulative release rate of the contents over time at different pH values ​​for two-layer composite hydrogel microcarriers with different gel structures. [Figure 5A] 1 shows the cumulative release rate of the contents over time at different pH values ​​for two-layer composite hydrogel microcarriers with different gel structures. [Figure 5B] 1 shows the cumulative release rate of the contents over time at different pH values ​​for two-layer composite hydrogel microcarriers with different gel structures. [Figure 6A] 1 shows the cumulative release rate of the contents over time at different pH values ​​for two-layer composite hydrogel microcarriers with different gel structures. [Figure 6B]1 shows the cumulative release rate of the contents over time at different pH values ​​for two-layer composite hydrogel microcarriers with different gel structures. [Figure 7] FIG. 1 shows viscosity versus temperature plots for gel structures with different weight percentages of sodium alginate and carboxymethylcellulose. [Figure 8A] This shows the mRNA expression ratios corresponding to lipoprotein lipase (LPL) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in adipose stem cells when adipose stem cells are cultured using gel structures with different weight ratios of sodium alginate and carboxymethylcellulose. [Figure 8B] Adipose stem cells were cultured using gel structures with different weight ratios of sodium alginate and carboxymethylcellulose, and the cell growth state diagrams are shown under a microscope after staining the adipose stem cells reddish purple. DETAILED DESCRIPTION OF THE INVENTION

[0025] In order to provide a complete and detailed description of the present invention, the following detailed description of the embodiments and specific examples of the present invention will be provided. However, these are not the only ways to implement or apply the specific examples of the present invention. The disclosed examples can be combined or substituted where beneficial, and one example can be added to another without further description or explanation. In the following description, numerous specific details are provided to enable the reader to fully understand the following examples. However, the embodiments of the present invention may be practiced without these specific details.

[0026] In this context, unless otherwise limited, "a," "an," and "the above" generally refer to one or more. As used herein, the terms "comprise," "include," "have," and similar words should be understood to refer to stated features, regions, integers, steps, operations, elements, and / or components, but not to the exclusion of other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0027] As used herein, the term "about" means that the value of a given quantity varies within 5% of that value (e.g., ±1%, ±2%, ±3%, ±4%, 5% of the value). These values ​​are merely examples and are not intended to be limiting. It should be understood that the term "about" may refer to a percentage of the value of the given quantity as interpreted by one of ordinary skill in the art in light of the teachings herein.

[0028] In this specification, unless otherwise specified, "%" is predefined as weight percent (wt%).

[0029] Although the methods disclosed herein are described using a series of operations or steps, the order in which these operations or steps are presented should not be construed as limiting the present invention. For example, some operations or steps may be performed in a different order and / or simultaneously with other steps. Also, not all operations, steps, and / or features need to be performed to achieve embodiments of the present invention. Furthermore, each operation or step described herein may include multiple sub-steps or actions.

[0030] Some embodiments of the present disclosure provide a two-layer composite hydrogel microcarrier comprising an inner layer gel structure and an outer layer gel structure. The inner layer gel structure is obtained by ionically crosslinking the inner layer polymer with the inner layer crosslinker. The outer layer gel structure is obtained by covalently crosslinking the outer layer monomer with the outer layer crosslinker, and covers the inner layer gel structure.

[0031] The inner layer gel structure of the two-layer composite hydrogel microcarrier is an ionically crosslinked structure, which has limitations such as being susceptible to environmental pH values ​​or collapsing due to weakened ionic forces caused by water absorption and swelling. The covalently bonded outer layer gel structure of the present disclosure provides physical support to the inner layer gel structure, avoiding the problem of the inner layer gel structure becoming unstable and improving the structural strength of the two-layer composite hydrogel microcarrier. Furthermore, the outer layer gel structure may also serve as a barrier layer, further delaying the release time of the loaded contents (e.g., nutrient molecules), reducing the burst release phenomenon, and enabling the loaded contents to be released stably over a long period of time.

[0032] In some embodiments, the outer layer gel structure is in the form of a porous film, covering the inner layer gel structure so as to encase the inner layer gel structure, and has a thickness smaller than that of the inner layer gel structure, and there are mutually attractive van der Waals forces between the outer layer gel structure and the inner layer gel structure, but no covalent bonds exist.

[0033] In some embodiments, the inner gel layer structure is a multi-layer sheet-like structure that is interconnected and separated by a multi-layer pore, and the outer gel layer structure is a multi-layer sheet-like structure that is interconnected and separated by a multi-layer pore having a pore size smaller than that of each of the inner pores. The difference in pore size between the inner and outer gel layers provides the outer gel layer with good structural strength, and the small pore size of the outer gel layer provides the two-layer composite hydrogel microcarrier with a good sustained content release effect.

[0034] In some embodiments, in a cross-sectional view, each inner layer sheet-like structure (or outer layer sheet-like structure) has an arc shape, and the concave surface between them defines an inner layer hole (or outer layer hole). In some embodiments, the diameter of an inner layer hole represents the longest vertical distance between two inner layer sheet-like structures located on opposite sides of the inner layer hole. The diameter of an outer layer hole represents the longest vertical distance between two outer layer sheet-like structures located on opposite sides of the outer layer hole. In some embodiments, the diameter of an inner layer hole is 250 microns to 1000 microns, for example, 250 microns, 500 microns, 750 microns, 1000 microns, or a value within the aforementioned interval. In some embodiments, the diameter of an outer layer hole is 50 microns to 500 microns, for example, 50 microns, 100 microns, 150 microns, 200 microns, 250 microns, 300 microns, 350 microns, 400 microns, 450 microns, 500 microns, or a value within the aforementioned interval. If the pore size is too large, the structure becomes unstable, the release rate of the contents is too fast, and sustained release cannot be achieved, whereas if the pore size is too small, the encapsulated contents are blocked excessively, making it difficult to achieve stable and long-term release of the contents.

[0035] In some embodiments, the thickness of the outer gel structure is between 25 microns and 1000 microns, e.g., 25 microns, 50 microns, 100 microns, 200 microns, 300 microns, 400 microns, 500 microns, 600 microns, 700 microns, 800 microns, 900 microns, 1000 microns, or a value within the aforementioned range. If the thickness is too thick, the release rate of the loaded contents will be too slow, while if the thickness is too thin, the release rate of the loaded contents will be too fast and the structural strength will be insufficient. It can be seen that as the thickness increases, the release of the loaded contents (e.g., lipid-soluble small molecules (molecular weight less than 500 grams / mole and solubility in nonpolar solvents greater than 0.1 micrograms / gram) or high molecular weight proteins (molecular weight greater than at least 500 grams / mole)) can be delayed.

[0036] In some embodiments, the inner layer polymer comprises a first inner layer polymer and a second inner layer polymer, and the inner layer gel structure is an interpenetrating structure between the first inner layer polymer and the second inner layer polymer (i.e., the polymer chains of the first inner layer polymer and the second inner layer polymer cross each other), and the design of the interpenetrating structure can provide good support and stabilize the structural strength of the inner layer gel structure.

[0037] In some embodiments, the inner layer polymer and the inner layer crosslinked product have opposite electrical properties, for example, the inner layer polymer is negatively charged and the inner layer crosslinked product is positively charged, and the inner layer crosslinked product and the inner layer polymer are crosslinked by ionic bonds formed between positive and negative charges to form an inner layer gel structure, or the inner layer polymer is positively charged and the inner layer crosslinked product is negatively charged. If the inner layer polymer has different degrees of dissociation at different pH values, the inner layer gel structure crosslinked by ionic bonds will be pH sensitive, and the strength of the ionic bonds will change with a change in pH value, thereby changing the inner layer gel structure.

[0038] In some embodiments, when the first inner layer polymer is sodium alginate (SA), the second inner layer polymer is carboxymethyl cellulose (CMC). Selecting and using sodium alginate and carboxymethyl cellulose offers advantages such as ease of storage and preparation, safety and non-toxicity, ability to form a gel at room temperature, and ease of absorption by the human body. Furthermore, compared with using sodium alginate alone, the combination of sodium alginate and carboxymethyl cellulose can improve the viscosity and structural strength of the inner layer gel structure. Adjusting the ratio of sodium alginate to carboxymethyl cellulose allows for the adjustment of the pore size of the inner layer pores, providing greater flexibility in selecting the inner layer gel structure. Furthermore, when sodium alginate and carboxymethyl cellulose are selected and used, the inner layer gel structure formed by the selection is relatively dense because sodium alginate and carboxymethyl cellulose contain carboxy groups that are less likely to be liberated in an acidic environment, and relatively loose because they are more likely to be liberated in a basic environment.

[0039] In some embodiments, the weight percent of sodium alginate, relative to the weight percent of the inner layer gel structure, taken as 100%, is between 0.1% and 5%, e.g., 0.1%, 0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, or any interval therebetween. The weight percent of carboxymethylcellulose is 0.1% to 5%, for example, 0.1%, 0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, or any value within the aforementioned intervals. If either of these is too high, the inner gel structure will be too dense, limiting the release efficiency of the inner gel structure. If either of these is too low, the colloidal structure will be too loose and unstable, or no gel will form. Furthermore, as the weight percentage of sodium alginate increases and the weight percentage of carboxymethylcellulose decreases, the pore size of the inner layer pores increases, improving the swelling rate of the inner layer gel structure when absorbing water and the release efficiency of the contents. Conversely, as the weight percentage of sodium alginate decreases and the weight percentage of carboxymethylcellulose increases, the pore size of the inner layer pores decreases, decreasing the swelling rate and release efficiency of the inner layer gel structure (e.g., reducing the release of high molecular weight proteins).

[0040] In some embodiments, when the first inner layer polymer is poly(ε-caprolactone-co-glycolic acid)·poly(ethylene glycol) copolymer, the second inner layer polymer is a derivative of poly(ε-caprolactone-co-glycolic acid)·poly(ethylene glycol) copolymer having an acyl group. In one embodiment, the poly(ε-caprolactone-co-glycolic acid)·poly(ethylene glycol) copolymer comprises an ABA triblock copolymer of poly(ε-caprolactone-co-glycolic acid) and poly(ethylene glycol) (Tri-PCG), and the acyl group is an acrylate group, so the derivative of the poly(ε-caprolactone-co-glycolic acid)·poly(ethylene glycol) copolymer having an acyl group is poly(ε-caprolactone-co-glycolic acid)·poly(ethylene glycol) copolymer having an acrylate group (Tri-PCG-acryl). Furthermore, the inner-layer gel structure selectively using Tri-PCG and Tri-PCG-acryl changes its colloidal properties in response to temperature changes within the temperature range applicable to the human body (e.g., converting from a solution to a colloid at 25°C to 37°C), allowing for changes in the viscosity and release capacity of the inner-layer gel structure, while maintaining a relatively stable structure that is resistant to collapse. In some embodiments, the two-layer composite hydrogel microcarrier further includes an inner-layer auxiliary molecule (e.g., dipentaerythritol hexakis(3-mercaptopropionate) (DPMP)) present in the inner-layer gel structure. As the temperature gradually increases, DPMP forms a covalent bond with Tri-PCG-acryl, strengthening the colloidal properties and improving the temperature sensitivity of the inner-layer gel structure.

[0041] In some embodiments, the weight percent of the inner-layer gel structure is 100%, and the sum of the weight percents of Tri-PCG and Tri-PCG-acryl is 15% to 30%, e.g., 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or any value within the aforementioned intervals. If the weight percent is too high, the inner-layer gel structure will be too dense, limiting the release efficiency of the inner-layer gel structure. If the weight percent is too low, the colloidal structure will be too loose and unstable, or will not form a gel.

[0042] In some embodiments, when the inner layer polymer is negatively charged, the inner layer crosslinked product contains calcium ions (Ca 2+ Compared to other cross-linked inner layers, calcium ions have the advantages of easy availability, low cost, and low environmental hazard.

[0043] In some embodiments, the outer layer monomer comprises N,N-dimethylacrylamide (DMAA), acrylamide (AA), or a combination thereof. When DMAA is selected over AA, the outer layer pores of the formed outer layer gel structure have a relatively large pore size, a relatively high release rate of the loaded contents (e.g., an improved release rate of high molecular weight proteins), and the two-layer composite hydrogel microcarrier has a large swelling rate when absorbing water.

[0044] In some embodiments, the outer layer crosslinker comprises N,N'-methylene-bis-acrylamide (BIS), which can form covalent bonds with DMAA or AA to crosslink DMAA to poly(N,N-dimethylacrylamide) (PDMA) or AA to poly(acrylamide) (PAA).

[0045] Some embodiments of the present disclosure provide a method for producing a bilayer composite hydrogel microcarrier, the method comprising the steps of: providing an inner layer polymer and an inner layer crosslinked material; mixing the inner layer polymer and the inner layer crosslinked material to obtain an inner layer gel structure through ionic crosslinking between the inner layer polymer and the inner layer crosslinked material; providing an outer layer monomer and an outer layer crosslinked material; and mixing the inner layer gel structure, the outer layer monomer, and the outer layer crosslinked material to obtain an outer layer gel structure through covalent crosslinking polymerization between the outer layer monomer and the outer layer crosslinked material, with the outer layer gel structure coating the inner layer gel structure, thereby obtaining a bilayer composite hydrogel microcarrier. By mixing the inner layer gel structure, the outer layer monomer, and the outer layer crosslinked material, an outer layer gel structure coating the inner layer gel structure can be formed, and the outer layer gel structure can provide physical support for the inner layer gel structure, improving the structural strength of the bilayer composite hydrogel microcarrier and delaying the release time of the loaded contents, thereby achieving long-term stable release.

[0046] In some embodiments, the step of providing an inner layer polymer and an inner layer crosslinked material comprises providing a first inner layer polymer, a second inner layer polymer, and an inner layer crosslinked material.

[0047] In some embodiments, the first inner layer polymer is sodium alginate (SA) and the second inner layer polymer is carboxymethylcellulose (CMC), which has the advantages of being easy to store and prepare, safe and non-toxic, capable of forming a gel at room temperature, and easily absorbed by the human body.

[0048] In some embodiments, when the first inner layer polymer is poly(ε-caprolactone-co-glycolic acid)-poly(ethylene glycol) copolymer, the second inner layer polymer is a derivative of poly(ε-caprolactone-co-glycolic acid)-poly(ethylene glycol) copolymer having an acyl group. In one embodiment, the poly(ε-caprolactone-co-glycolic acid)-poly(ethylene glycol) copolymer is an ABA triblock copolymer of poly(ε-caprolactone-co-glycolic acid) and poly(ethylene glycol) (Tri-PCG), and the derivative of poly(ε-caprolactone-co-glycolic acid)-poly(ethylene glycol) copolymer having an acyl group is poly(ε-caprolactone-co-glycolic acid)-poly(ethylene glycol) copolymer having an acrylate group (Tri-PCG-acryl). The inner gel structure made of Tri-PCG and Tri-PCG-acryl is relatively dense, and within the temperature range applicable to the human body, its colloidal properties change with changes in temperature (for example, it changes from a solution to a colloid at 25°C to 37°C), which can change the viscosity and release capacity of the inner gel structure, and the structure is relatively stable and does not easily collapse.

[0049] In some embodiments, the inner layer polymer and the inner layer crosslinked material have opposite electrical properties. In some embodiments, when the inner layer polymer is negatively charged, the inner layer crosslinked material contains calcium ions.

[0050] In some embodiments, the step of mixing the inner layer polymer and the inner layer crosslinked product includes mixing a first inner layer polymer, a second inner layer polymer, the inner layer crosslinked product, a radical generator, and water. By adding the radical generator in this step, the radical generator stored in the inner layer gel structure diffuses into the outer layer when the outer layer monomer, outer layer crosslinked product, and catalyst are subsequently added, and interacts with the outer layer monomer, outer layer crosslinked product, and catalyst to initiate a reaction to form the outer layer gel structure in the outer layer. Therefore, by adding the radical generator in this step, it is possible to avoid premature reactions that occur when the radical generator is added simultaneously with the outer layer monomer, outer layer crosslinked product, and catalyst, or a situation in which the outer layer gel structure does not adequately coat the inner layer gel structure.

[0051] In some embodiments, the weight ratio of the first inner layer polymer to the second inner layer polymer is 1:5 to 5:1, e.g., 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, or any value within the aforementioned intervals. When the first inner layer polymer has more releasable negatively charged groups (e.g., carboxyl groups) in a basic environment compared to an acidic environment, the swelling rate of the inner layer gel structure increases as the weight ratio increases.

[0052] In some embodiments, the step of mixing the inner layer polymer and the inner layer crosslinked product includes mixing the first inner layer polymer, the second inner layer polymer, the inner layer crosslinked product, a radical generator, and water.

[0053] In some embodiments, when the total weight percentage of the first inner layer polymer, the second inner layer polymer, the inner layer crosslinked product, the radical generator, and the water is 100%, the weight percentage of sodium alginate is 0.1% to 5%, and the weight percentage of carboxymethylcellulose is 0.1% to 5%. If the weight percentage is too high, the inner gel structure becomes too dense, limiting its release efficiency; if the weight percentage is too low, the colloidal structure becomes too loose and unstable, or no gel can be formed. When the weight percentage of carboxymethylcellulose is greater than 1% (e.g., 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, or a value within the aforementioned range), the bilayer composite hydrogel microcarriers can be provided with sufficient structural strength for continuous stem cell culture for several days without collapse. In some embodiments, when the weight ratio of sodium alginate to carboxymethylcellulose is 3:2, controlling the pore size of the inner gel structure allows for stable release of nutrients at an appropriate rate, thereby ensuring a stable intake of nutrients by stem cells and improving stem cell growth.

[0054] In some embodiments, the total weight percentage of the poly(ε-caprolactone-co-glycolic acid)·poly(ethylene glycol) copolymer and the poly(ε-caprolactone-co-glycolic acid)·poly(ethylene glycol) copolymer derivative having an acyl group is 15% to 30% relative to the total weight percentage of the first inner layer polymer, the second inner layer polymer, the inner layer crosslinker, the radical generator, and water, taken as 100%. If the weight percentage is too high, the inner layer gel structure will be too dense, limiting the release efficiency of the inner layer gel structure. If the weight percentage is too low, the colloidal structure will be too loose and unstable, or no gel will form.

[0055] In some embodiments, the step of mixing the first inner layer polymer, the second inner layer polymer, the inner layer crosslinker, the radical generator, and water further comprises adding dipentaerythritol hexakis(3-mercaptopropionate), bovine serum albumin, or a combination thereof. In some embodiments, the step of mixing the first inner layer polymer, the second inner layer polymer, the inner layer crosslinker, the radical generator, and water comprises mixing a first solution, a second solution, calcium ions, and ammonium sulfate, wherein the first solution comprises poly(ε-caprolactone-co-glycolic acid)·poly(ethylene glycol) copolymer or poly(ε-caprolactone-co-glycolic acid)·poly(ethylene glycol) copolymer and dipentaerythritol hexakis(3-mercaptopropionate) (DPMP), and the second solution comprises a derivative of poly(ε-caprolactone-co-glycolic acid)·poly(ethylene glycol) copolymer having an acyl group and bovine fetal serum albumin.

[0056] As the temperature increases, DPMP tends to form covalent bonds with acyl groups, improving the density of the inner-layer gel structure. Therefore, adding DPMP can improve the sustained-release ability of the inner-layer gel structure as the temperature increases, which is advantageous for human application. In some embodiments, the weight percent of DPMP is 5% to 15%, e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or a value within the aforementioned range, relative to the weight percent of the first solution (100%). Furthermore, adding bovine fetal serum albumin can coat the bilayer composite hydrogel microcarriers. When the bilayer composite hydrogel microcarriers are used for cell culture, the bovine fetal serum albumin can be sustainedly released, providing a long-term source of cell nutrients.

[0057] In some embodiments, the step of mixing the inner layer gel structure, the outer layer monomer, and the outer layer crosslinked product includes mixing the inner layer gel structure, N,N-dimethylacrylamide (DMAA), N,N'-methylenebisacrylamide (BIS), and a catalyst, or mixing the inner layer gel structure, acrylamide (AA), N,N'-methylenebisacrylamide, and a catalyst. The covalent bonding between BIS and DMAA or AA forms an outer layer gel structure, which improves the physical support of the inner layer gel structure and forms denser pores than the inner layer gel structure, thereby extending the release of the loaded contents.

[0058] In some embodiments, the weight percentage of the inner layer gel structure is 50% to 80% (e.g., 50%, 60%, 70%, 80% or a value within the aforementioned interval), the weight percentage of the outer layer monomer is 15% to 40% (e.g., 15%, 20%, 25%, 30%, 35%, 40% or a value within the aforementioned interval), and the weight percentage of the outer layer crosslinked product is 5% to 10% (e.g., 5%, 6%, 7%, 8%, 9%, 10% or a value within the aforementioned interval), where the total weight percentage of the inner layer gel structure, outer layer monomer, and outer layer crosslinked product is 100%. If the weight percentage of the inner layer gel structure or outer layer gel structure is too low, it is difficult to form a bilayer composite hydrogel microcarrier. If the weight percentage of the inner layer gel structure or outer layer gel structure is too high, the release rate of the contents loaded in the bilayer composite hydrogel microcarrier is too low.

[0059] In some embodiments, the weight ratio of the inner layer gel structure to the outer layer monomer is 5:4 to 16:3 (e.g., 5:4, 5:3, 2:1, 8:3, 10:3, 4:1, 16:3, or a value within the aforementioned range). If the weight ratio is too high, the structure of the bilayer composite hydrogel microcarrier becomes unstable, while if the weight ratio is too low, the outer layer of the bilayer composite hydrogel microcarrier becomes too thick, resulting in a too low release rate of the loaded contents.

[0060] In some embodiments, the mixing time for mixing the inner layer gel structure, outer layer monomer, and outer layer crosslinked product is 0.5 to 5 minutes, for example, 0.5, 1, 2, 3, 4, or 5 minutes, or any value within the aforementioned range. By controlling the mixing time, the thickness of the outer layer gel structure can be controlled, thereby controlling the structural strength and release rate of the two-layer composite hydrogel microcarrier.

[0061] Some embodiments of the present disclosure further provide a method for culturing stem cells using the aforementioned bilayer composite hydrogel microcarriers, comprising the steps of providing stem cells, bilayer composite hydrogel microcarriers, and nutrient components; mixing the stem cells, bilayer composite hydrogel microcarriers, and nutrient components to obtain a gelling medium; and adding a culture medium to the gelling medium. The two-layer composite hydrogel microcarrier comprises an inner layer gel structure and an outer layer gel structure. The inner layer gel structure is obtained by ionically crosslinking the inner layer polymer with an inner layer crosslinker. The inner layer polymer comprises a first inner layer polymer and a second inner layer polymer. The first inner layer polymer is sodium alginate, and the second inner layer polymer is carboxymethylcellulose. The weight ratio of sodium alginate to carboxymethylcellulose is 3:2. The weight percentage of carboxymethylcellulose is greater than 1% of the weight percentage of the inner layer gel structure, which is taken as 100%. The outer layer gel structure is obtained by covalently crosslinking the outer layer monomer with the outer layer crosslinker. This inner layer gel structure is coated with nutrients located within the inner layer gel structure. Controlling the weight percentage of carboxymethylcellulose can impart good structural stability to the two-layer composite hydrogel microcarrier. Defining the weight ratio of sodium alginate to carboxymethylcellulose and the location of the nutrients can extend the release time of the loaded nutrients. Therefore, when culturing stem cells using a two-layer composite hydrogel microcarrier, it can be maintained for a long time until collapse, increasing the amount of stem cells cultured per unit area, avoiding burst release of contents, and continuously releasing contents to stem cells, thereby increasing the stem cell culture time and the frequency of adding nutrients, and simplifying the culture process.

[0062] In some embodiments, the stem cells comprise embryonic stem cells, hematopoietic stem cells, mammary stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory stem cells, adipose stem cells, or a combination thereof.

[0063] It is understood that different bilayer composite hydrogel microcarriers with different structural strengths and release rates can be selected and used according to the different characteristics of stem cells. For example, when culturing adipose stem cells, which grow quickly, sodium alginate and carboxymethylcellulose, which have large inner pore sizes, can be selected and used, while when culturing neural stem cells, which grow slowly, Tri-PCG and Tri-PCG-acryl, which have small inner pore sizes, can be selected and used.

[0064] To further illustrate the two-layer composite hydrogel microcarriers, the method for manufacturing the two-layer composite hydrogel microcarriers, and the method for culturing stem cells provided by the embodiments of the present disclosure, the following examples are provided. Please note that the following examples are provided for illustrative purposes only and are not intended to limit the present invention.

[0065] Example 1: Preparation method of double-layer composite hydrogel microcarriers

[0066] 1. Inner layer gel structure

[0067] Below, the processes corresponding to different inner layer polymers are provided.

[0068] 1.1. Sodium alginate and carboxymethylcellulose

[0069] Sodium alginate (SA) and carboxymethylcellulose (CMC) were mixed at different weight ratios (SA:CMC = 4:1, 2:3, 3:2, and 1:4), then added to a 5% calcium chloride (CaCl2) solution containing 1.5% ammonium persulfate (APS). After stirring for 20 minutes, SA and CMC were crosslinked by calcium ions to form inner-layer hydrogels. The SA and CMC concentrations in each group were 2.0% / 0.5%, 1.0% / 1.5%, 1.5% / 1.0%, and 0.5% / 2.0%, respectively. The APS and calcium ion concentrations were 1% and 4%, respectively.

[0070] It is understood that in the process of forming the mixed solution (not limited to the example using sodium alginate and carboxymethylcellulose as the inner layer polymer), depending on the subsequent stem cell culture requirements, culture medium or nutrients (e.g., growth factors) can be added to the mixed solution to make the final concentration of the nutrients 5 milligrams / milliliter to 10 milligrams / milliliter, in preparation for future clinical application.

[0071] 1.2 ABA-type triblock copolymer of poly(ε-caprolactone-co-glycolic acid) and poly(ethylene glycol) (Tri-PCG) and its acrylate-containing derivative (also called Tri-PCG-acryl)

[0072] 1.2.1 Preparation of Tri-PCG

[0073] Poly(ethylene glycol) (PEG) (15.0 g, 10 mmol) was dried under reduced pressure at 120°C for 3 hours. ε-caprolactone (33.3 g, 291 mmol), glycolide (5.87 g, 50.6 mmol), and tin 2-ethylhexanoate (Sn(Oct)2) (149 mg, 367 μmol) as a catalyst were added to the flask containing PEG to obtain a reaction solution.

[0074] The reaction mixture was then frozen with liquid nitrogen and dried under reduced pressure to obtain the reaction product. The flask containing the reaction product was then immersed in an oil bath at 160°C for 12 hours to carry out a ring-opening polymerization reaction. The reaction product was then dissolved in 100 mL of chloroform and precipitated with 1,000 mL of diethyl ether. The dissolution and precipitation steps were repeated three times. The supernatant was then removed, and the precipitate was dried under reduced pressure to obtain a white solid, Tri-PCG.

[0075] 1.2.2 Preparation of Tri-PCG-acryl

[0076] Acrylic acid (2.0 mL, 29.2 mmol) was dissolved in 250 mL of dichloromethane (CHCl) to obtain an acrylic acid solution, and N,N'-dicyclohexyl carbodiimide (DCC) (6.24 g, 30.2 mmol) was dissolved in 250 mL of dichloromethane to obtain an N,N'-dicyclohexyl carbodiimide solution. The N,N'-dicyclohexyl carbodiimide solution was then added to the acrylic acid solution in an ice bath and stirred for 1 hour. The volume ratio of the N,N'-dicyclohexyl carbodiimide solution to the acrylic acid solution was 1:1. Next, Tri-PCG (20.3 g, 5.1 mmol) and 4-dimethylaminopyridine (DMAP) (299.5 mg, 2.5 mmol) dissolved in 250 mL of dichloromethane were added to the mixed solution containing acrylic acid and N,N'-dicyclohexylcarbodiimide, and the mixture was stirred at 25°C for 24 hours. The by-product dicyclohexylurea was then removed by filtration. After dichloromethane was evaporated, the polymer chains were extended in chloroform as a good solvent (a solvent with high dissolution capacity for the solute and an interaction parameter with the polymer solute less than 0.5). The polymer chains were then reprecipitated three times in a mixture of n-hexane and ethanol (volume ratio of n-hexane to ethanol: 8 / 2) as a poor solvent (a solvent with low dissolution capacity for the solute and an interaction parameter with the polymer solute close to or greater than 0.5). Subsequently, the resulting solid, yellow-white Tri-PCG-acryl, was obtained by drying under reduced pressure.

[0077] 1.2.3 Preparation of Tri-PCG aqueous solution, Tri-PCG-acryl aqueous solution, and Tri-PCG / DPMP aqueous solution

[0078] To prepare the Tri-PCG aqueous solution, first, Tri-PCG (246.5 mg) was dissolved in 986 microliters of adipose stem cell culture medium (product name: StemPro) containing 10% fetal bovine serum. TM MSC SFM, Product Number: Gibco TM The mixture was dissolved in a 90°C water bath (A1033201). After immersion for 5 seconds in a water bath, it was cooled to room temperature with continuous stirring. The immersion and cooling processes were repeated three times. The mixture was then cooled in a water bath (20-25°C), sonicated for 30 minutes to remove air bubbles, and the pH was adjusted to 7.4 with sodium hydroxide and adipose stem cell culture medium to obtain a 20% Tri-PCG aqueous solution (which can be replaced with the subsequent Tri-PCG / DPMP aqueous solution depending on the colloid requirements).

[0079] To prepare the tri-PCG-acryl aqueous solution, tri-PCG-acryl (165 mg) was dissolved in adipose stem cell culture medium (660 microliters) containing 10% fetal bovine serum and stirred overnight at 4°C. The solution was then sonicated at low temperature (20-25°C) for 30 minutes to remove air bubbles, and the pH was adjusted to 7.4 with sodium hydroxide and adipose stem cell culture medium to obtain a 20% tri-PCG-acryl aqueous solution.

[0080] For the Tri-PCG / DPMP aqueous solution, 90.4 mg of dipentaerythritol hexakis(3-mercaptopropionate; DPMP) was dissolved in 1 mL of acetone to obtain a DPMP solution. The DPMP solution was added to a sample tube containing 750 mg of Tri-PCG and 7 mL of acetone was added to obtain a mixed solution. The mixed solution was added to 63 mL of water, stirred, and sonicated for 30 minutes. The acetone was then removed and the mixture was lyophilized to obtain a Tri-PCG / DPMP mixture (DPMP accounts for approximately 10.8% of the Tri-PCG / DPMP mixture).

[0081] The Tri-PCG / DPMP mixture was then dissolved in 354 microliters of adipose stem cell culture medium containing 10% fetal bovine serum. After immersion in a water bath at approximately 90°C for 5 seconds, the mixture was cooled to room temperature with continuous stirring. This immersion and cooling process was repeated three times. The mixture was then sonicated at low temperature (20-25°C) for 30 minutes to remove air bubbles, and the pH was adjusted to 7.4 with sodium hydroxide and adipose stem cell culture medium to obtain a 20% Tri-PCG / DPMP aqueous solution.

[0082] 1.2.4 Preparation of the inner layer hydrogel

[0083] The Tri-PCG / DPMP aqueous solution and the Tri-PCG-acryl aqueous solution were mixed in the appropriate ratio and then poured into a 5% calcium chloride (CaCl2) solution containing 1.5% ammonium persulfate (APS) to form a mixed solution. The mixture was stirred for 20 minutes, and the Tri-PCG and Tri-PCG-acryl were cross-linked by calcium ions to form an inner-layer gel structure. The reaction concentrations of Tri-PCG / DPMP and Tri-PCG-acryl were 70%-79% and 16%-25%, respectively. The reaction concentrations of APS and calcium ions were 1% and 4%, respectively. The total weight percentage of Tri-PCG and Tri-PCG-acryl in the inner-layer gel structure was 16%-33%, ensuring that the inner-layer hydrogel remained in a colloidal state within the operating temperature range.

[0084] As the temperature increases, the acrylate groups of Tri-PCG and Tri-PCG-acryl, or DPMP and Tri-PCG-acryl, can form covalent bonds and change the gel morphology, so the intimal gel structure prepared using Tri-PCG-acryl and Tri-PCG / DPMP, or Tri-PCG-acryl and Tri-PCG, is temperature-sensitive.

[0085] For example, see Figure 1, which shows the gel state change diagram for different concentrations of Tri-PCG and temperatures (Tri-PCG in this example is stored in phosphate buffered saline (PBS)). It shows that adjusting the Tri-PCG concentration (e.g., corresponding to the sum of the concentrations of Tri-PCG / DPMP and Tri-PCG-acryl in this example) or temperature changes the gel state, and accordingly adjusts the viscosity and release capacity of the gel.

[0086] In another example, a Tri-PCG aqueous solution may be used instead of a Tri-PCG / DPMP aqueous solution and mixed with Tri-PCG-acryl. Because the Tri-PCG / DPMP aqueous solution contains DPMP, which can form a covalent bond with Tri-PCG-acryl upon heating, the intimal gel structure prepared using the Tri-PCG / DPMP aqueous solution is understood to have good temperature sensitivity.

[0087] The inner layer gel structure prepared using "1.1, sodium alginate and carboxymethylcellulose" and "1.2, ABA triblock copolymer of poly(ε-caprolactone-co-glycolic acid) and poly(ethylene glycol) (Tri-PCG) and its acrylate-containing Tri-PCG derivative (also called Tri-PCG-acryl)" are different from those prepared using "1.2, Tri-PCG / DPMP and Tri-PCG-acryl." The inner layer gel structures prepared using Tri-PCG / DPMP and Tri-PCG-acryl are relatively stable and resistant to collapse, but differ in that they require a complex preparation process and the use of organic solvents. The inner layer gel structure prepared using SA and CMC has the advantages of a simple preparation process and environmental friendliness, but has the limitation that its structure is also affected by pH.

[0088] Therefore, the inner layer gel structure can be prepared by selecting and using appropriate components depending on the growth characteristics of the stem cells to be cultured later. For example, SA and CMC, which are easy to prepare, can be selected and used to culture adipose stem cells, which have a relatively fast growth rate, and Tri-PCG / DPMP and Tri-PCG-acryl, which have relatively high structural strength, can be selected and used to culture neural stem cells, which have a relatively slow growth rate.

[0089] 2. Outer-layer Gel Structure and Two-layer Composite Hydrogel Microcarriers

[0090] Next, N,N-dimethylacrylamide (DMAA) or acrylamide (AA) was mixed as the outer layer monomer, N,N'-methylenebisacrylamide (BIS) was mixed as the outer layer crosslinker, and tetramethylethylenediamine (TEMED) was mixed as the catalyst. The inner layer hydrogel (prepared in Section 1.1, sodium alginate and carboxymethylcellulose) was then added and mixed for different times to crosslink the DMAA or AA with the BIS to form poly(N,N-dimethylacrylamide) (PDMA) or poly(acrylamide) (PAA). The resulting bilayer composite hydrogel microcarriers had different outer layer gel structure thicknesses or different outer layer components. The reaction concentrations of DMAA or AA were 1 mol / L, BIS was 2.2 mol / L, and TEMED was 15 mg / mL.

[0091] Specifically, during the mixing process, APS in the inner hydrogel layer diffused outward and provided radicals to initiate the reaction that formed the outer gel layer. The outer layer monomers covalently bonded to BIS and PDMA or PAA with the APS radicals and TEMED as a catalyst to form the outer gel layer.

[0092] Finally, the unreacted outer layer monomer was removed by stirring and washing with water.

[0093] Example 2: Stem cell culture method using two-layer composite hydrogel microcarriers

[0094] First, 1x10 7 An adipose stem cell suspension containing adipose stem cells (i.e., adipose stem cells suspended in adipose stem cell culture medium) containing 10 ...

[0095] Next, 400 microliters of adipose stem cell culture medium was dropped onto the surface of the stem cell-mixed gel and cultured in an environment containing 5% carbon dioxide at 37°C. Every two days, 200 microliters of the adipose stem cell culture medium (supernatant) was removed and replaced with fresh adipose stem cell culture medium. After culturing until the bilayer composite hydrogel microcarriers ruptured, the adipose stem cells were collected with PBS and other culture components were removed.

[0096] It is understood that providing an outer layer gel structure provides physical stability to the inner layer gel structure, preventing the inner layer gel structure from swelling and bursting due to water absorption or structural collapse in the presence of alkali, while also preventing excessive release and leakage of nutritional components and maintaining stable release of nutritional components.

[0097] Example 3, Efficacy Test

[0098] First, two-layer composite hydrogel microcarriers were prepared according to the method described in Example 1 above, and the specific components, weight percentages and formation time of the outer layer gel structure (i.e., the mixing time in "2. Outer layer gel structure and two-layer composite hydrogel microcarriers") were adjusted to compare the differences in structural properties and content release efficiency between different two-layer composite hydrogel microcarriers. The conditions for each group are exemplified as follows:

[0099] [Table 1]

[0100] 1. Gel morphology under electron microscope

[0101] First, the bilayer composite hydrogel microcarriers of each group were observed under different magnifications using a scanning electron microscope. The results are shown in Figure 2. The top row a-c are the SA-CMC@PDMA-1 group, the second row d-f are the SA-CMC@PDMA-3 group, and the bottom row g-i are the SA-CMC@PAA-1 group.

[0102] As can be seen from Figure 2, the inner and outer layers of the two-layer composite hydrogel microcarrier are interconnected sheet-like structures, the sheet structures are spaced apart by three-dimensional pores, and the boundaries between the inner and outer layers are clearly visible. Furthermore, the pore sizes of the inner and outer gel structures are different, with the pore size of the outer gel structure being smaller than that of the inner gel structure.

[0103] Furthermore, by comparing Figure c (SA-CMC@PDMA-1 group) and Figure f (SA-CMC@PDMA-3 group), it was found that when the crosslinking time of the outer layer increased from 1 minute to 3 minutes, the thickness of the outer layer gel structure increased from approximately 100 microns to approximately 450 microns.

[0104] Furthermore, when comparing Figure b (SA-CMC@PDMA-1 group) and Figure e (SA-CMC@PDMA-3 group), it was found that as the concentration of SA decreased, a significant change was observed in the pore size of the inner and outer layers, and the higher the proportion of SA, the larger the pore size of the inner layer pores.

[0105] As can be seen from Figure 2, the double-layered composite hydrogel microcarrier has a double-layered framework structure, with the pores of the inner gel layer being relatively large, which is beneficial for nutrient adsorption, and the outer gel layer being relatively dense, which provides physical support for the inner gel layer, preventing the inner gel layer from collapsing and excessive release of nutrients. Therefore, the double-layered composite hydrogel microcarrier can provide a scaffold for the three-dimensional growth of stem cells, retain nutrients for a long time, increase the number of stem cells cultured, and extend the time that stem cells can be cultured sustainably. The relevant effectiveness will be further verified below.

[0106] 2.Water absorption and expansion

[0107] To test the water absorption and swelling of the single-layer gel structure (single-layer gel) and the double-layer composite hydrogel microcarriers, the inner-layer gel structures (four groups, with SA and CMC concentrations of 0.5% SA / 2.0% CMC, 1.0% SA / 1.5% CMC, 1.5% SA / 1.0% CMC, and 2.0% SA / 0.5% CMC, respectively) and the double-layer composite hydrogel microcarriers (SA-CMC@PDMA-1 group, SA-CMC@PDMA-2 group, SA-CMC@PDMA-3 group, and SA-CMC@PAA-1 group) were air-dried, and the air-dried inner-layer gel structures and air-dried double-layer composite hydrogel microcarriers (hereinafter referred to as samples) were weighed and subjected to a swelling test. The procedure was as follows:

[0108] First, the sample was placed in an acidic solution (0.5 M hydrochloric acid) with a pH of 1.2 for 2 hours, and then transferred to a basic solution (1 M trihydroxymethylaminomethane) with a pH of 7.4 until the weight reached equilibrium or decreased (indicating the start of dissolution). During this process, the sample was taken out of the solution every 30 minutes, and weighed after removing the remaining solution. The swelling ratio (SR) at each time point was calculated using the following formula:

[0109] SR=[(m0-mt) / m0]

[0110] SR is the swelling ratio of the bilayer composite hydrogel microcarrier, mt is the mass of the bilayer composite hydrogel microcarrier at time t (swollen state), and m0 is the initial mass of the hydrogel.

[0111] See Figure 3A for the swelling rate trends of the inner-layer gel structures obtained from the above tests, and see Figure 3B for the swelling rate trends of the double-layer composite hydrogel microcarriers.

[0112] Figure 3A shows the water absorption and swelling characteristics of different inner-layer gel structures. The reason why the swelling rates of the four inner-layer gel structures are low in an acidic environment is that SA and CMC contain abundant carboxyl groups (-COOH), but in an acidic solution, the carboxyl groups are not liberated, the electrostatic repulsion between polymers is low, the gel network structure is dense, and the hydrophilicity of SA and CMC is reduced. Therefore, the swelling rate of the inner-layer gel structure (single-layer gel) is lower in an acidic solution than in a basic solution. We also observed that the swelling rate decreases as the concentration of CMC decreases in an acidic solution.

[0113] In contrast, in a basic environment, the swelling rate increased over time, then gradually increased and then decreased. Specifically, the carboxyl groups were in the free state (COO - ), forming electrostatic repulsion between the carboxyl groups, which increases with time. As the repulsion gradually increases, the monolayer gel expands, widening the network gap, changing its structure, increasing its hydrophilicity, absorbing water molecules, and improving the expansion rate. In the later stage of the reaction, with the change in structure, the ionic bridges formed by the carboxyl groups and calcium ions gradually become unstable, the concentration of the carboxyl groups decreases, further weakening the crosslinking reaction between the carboxyl groups and calcium ions, and gradually causing the monolayer gel to collapse.

[0114] Figure 3B shows the water absorption and swelling characteristics of two-layer composite hydrogel microcarriers with different gel structures. Compared to the single-layer gel (inner layer gel structure only) in Figure 3A, which has a water absorption and swelling time (time to absorb water before collapse) of 4.5 hours, Figure 3B shows that the water absorption and swelling time of the two-layer composite hydrogel microcarriers is significantly longer (12 hours), and the overall swelling rate is higher than that of the single-layer gel. In other words, the inner layer gel structure of the two-layer composite hydrogel microcarriers is sensitive to pH, while the outer layer gel structure is not. Therefore, even in a basic environment, the outer layer gel structure maintains its integrity and is less likely to collapse.

[0115] In addition, when comparing the SA-CMC@PDMA-2 group (outer layer crosslinking time was 2 h) and the SA-CMC@PDMA-3 group (outer layer crosslinking time was 3 h), it was found that the collapse of the gel could be delayed by extending the outer layer crosslinking time to increase the thickness of the outer layer gel structure.

[0116] Comparing the SA-CMC@PDMA-1 group (1.5% SA / 1.0% CMC) and the SA-CMC@PDMA-2 group (0.5% SA / 2.0% CMC), the group with a lower SA concentration showed a relatively lower swelling rate after 4 hours. Therefore, reducing the SA concentration in a basic environment can be used as a means to extend the release time of nutrients.

[0117] Finally, when comparing the SACMC@PDMA-1 group (PDMA) and the SA-CMC@PAA-1 group (PAA), it was found that, compared with PAA, PDMA, with its outer gel structure, exhibited a higher swelling rate and a higher water absorption and swelling capacity.

[0118] 3. Release behavior of contents

[0119] 3.1 Test Method

[0120] To test the content release behavior of each group of double-layer composite hydrogel microcarriers, a content release test was carried out according to the following procedure.

[0121] Three different ingredients with different molecular weights and solubility were mixed with different groups of bilayer composite hydrogel microcarriers. Each ingredient was loaded into the bilayer composite hydrogel microcarriers. The ingredients were selected from the lipid-soluble ingredient, tretinoin (TR), the water-soluble ingredient, penicillin (AM), and bovine serum albumin (BS), respectively. The vitamin A acid concentration in the bilayer composite hydrogel microcarriers was 0.5 mg / L, the penicillin concentration was 100 mg / mL, and the weight percentage of bovine serum albumin in the bilayer composite hydrogel microcarriers was 5%. The loaded bilayer composite hydrogel microcarriers were immersed in an acidic solution with a pH of 1.2 for 2 hours and then transferred to a weakly basic solution with a pH of 7.4 at 36.5°C ± 0.5°C. The bilayer composite hydrogel microcarriers were then weighed and immersed in 100 mL of PBS. They were then subjected to rotary shaking at an appropriate speed at a temperature of 36.5°C ± 0.5°C. A fixed amount of PBS was extracted at regular intervals, and the content concentration was measured using a UV spectrophotometer (TR measurement wavelength: 350 nm, AM measurement wavelength: 463 nm, BS measurement wavelength: 595 nm). An equal amount of PBS was then replenished. The cumulative release rate Qn of the contents of the bilayer composite hydrogel microcarriers was then calculated using the following equation. The results are summarized in Figures 4A-6B.

[0122] Qn(%)=(V0Cn+VC(n-1)) / (W)×100

[0123] Cn and C(n-1) are the concentrations of the contents sampled n times and n-1 times, V0 is the initial volume of PBS, V is the sampling volume, and W is the weight of the contents loaded on the bilayer composite hydrogel microcarriers.

[0124] 3.2, Results analysis

[0125] To better understand the effects of the components, weight percentage, and outer layer crosslinking time on the release behavior of the contents in the double-layer composite hydrogel microcarriers, we will later compare Figure 4A (SA-CMC@PDMA-2) with Figure 4B (SA-CMC@PDMA-3), Figure 5A (SA-CMC@PDMA-1) with Figure 5B (SA-CMC@PDMA-2), and Figure 6A (SA-CMC@PDMA-1) with Figure 6B (SA-CMC@PAA-1).

[0126] 3.2.1 Comparison of content release behavior depending on outer layer thickness

[0127] First, referring to Figures 4A and 4B, the two-layer composite hydrogel microcarriers differ between the two groups in that the outer layer crosslinking time is extended from 2 minutes (SA-CMC@PDMA-2 group) to 3 minutes (SA-CMC@PDMA-3 group), increasing the thickness of the outer layer gel structure.

[0128] As can be seen from Figures 4A and 4B, in the initial acidic environment, the two types of bilayer composite hydrogel microcarriers exhibited good sustained-release effects for both lipid-soluble vitamin A acid (TR) and bovine serum albumin (BS), but not for water-soluble penicillin (AM). Due to AM's small molecular weight and easy diffusion in solution, a severe release effect occurred for AM. The SA-CMC@PDMA-2 group achieved a high 2-hour release rate of approximately 90%. The SA-CMC@PDMA-3 group, with its relatively thick outer layer, achieved a high 2-hour release rate of approximately 65% ​​due to increased diffusion resistance. This indicates that the thickness of the outer layer still has a release-delaying effect on the water-soluble component AM in the acidic environment.

[0129] In a weakly alkaline environment, the inner gel layer absorbed water and began to swell, causing the network structure to collapse, gradually releasing vitamin A acid (TR) and bovine serum albumin (BS). In the SA-CMC@PDMA-2 group, the release rate of TR reached over 90% after 36 hours, while the release rate of BS, which has a relatively large molecular structure, reached only approximately 64%, indicating that the bilayer structure design is more effective in sustaining the release of stem cell culture medium components (BS) than fat-soluble components (TR). In contrast, in the SA-CMC@PDMA-3 group, which has a relatively thick outer layer, the release rate of TM decreased to approximately 80% after 36 hours, while the release rate of BS reached only 35%, demonstrating its ability to delay and control the release of fat-soluble components (TR) and stem cell culture medium components (BS) in a weakly alkaline environment.

[0130] Therefore, as can be seen from Figures 4A and 4B, increasing the thickness of the outer layer can improve the diffusion resistance and further significantly delay the release of the contents.

[0131] 3.2.2 Comparison of content release behavior depending on the concentration of the inner layer composition

[0132] See Figures 5A and 5B, the two-layer composite hydrogel microcarriers between the two groups differ in the weight percentage of the inner layer polymer (1.5% SA / 1.0% CMC for the SA-CMC@PDMA-1 group and 0.5% SA / 2.0% CMC for the SA-CMC@PDMA-2 group).

[0133] The results in Figures 5A and 5B show that in the initial acidic environment, the sustained-release effect of the bilayer hydrogels did not obviously change depending on the composition of the inner layer gel structure, and it had a good sustained-release effect for both fat-soluble vitamin A acid (TR) and bovine serum albumin (BS). However, a significant release effect still occurred for water-soluble penicillin (AM), with the release rate reaching 70% to 90% after 2 hours.

[0134] In a weakly basic environment, there was no significant difference in the release rates of TR and AM between the two groups, but SA-CMC@PDMA-2 with a relatively low concentration of SA (as exemplified by the fact that the pore size of SA-CMC@PDMA-2 is obviously smaller than that of SA-CMC@PDMA-1 in "1. Gel morphology under electron microscope") had a better control effect on the release of BS.

[0135] Compared to the polymeric BS, the lipid-soluble TR and water-soluble penicillin AM are both small molecules. As can be seen from Figures 5A and 5B, changing the composition of the inner gel layer does not significantly affect the small molecule content. However, decreasing the concentration of SA can slow the diffusion rate of the polymer content and reduce the release rate.

[0136] 3.2.3 Comparison of content release behavior depending on the outer layer composition

[0137] See Figures 6A and 6B. The two groups of double-layered composite hydrogel microcarriers differ in the composition of the outer layer monomer (PDMA for the SA-CMC@PDMA-1 group and PAA for the SA-CMC@PAA-1 group).

[0138] The results in Figures 6A and 6B show that there is no significant difference in the release rates of lipid-soluble vitamin A acid (TR) and water-soluble penicillin (AM) from the two groups of bilayer composite hydrogel microcarriers, but the release rate of bovine serum albumin (BS) significantly decreases when the outer layer polymer component is changed from PDMA to PAA, which has a lower swelling ratio and smaller pore size.

[0139] In other words, when PAA is selected and used as a component of the outer gel structure, compared to the selection and use of PDMA, it does not have a significant effect on the small molecule content, but it can slow the diffusion rate of the polymer content and reduce the release rate.

[0140] 4.Structural strength

[0141] To test the relationship between the content of specific components in the gel structure and the structural strength, a single-layer gel structure was prepared based on the formulation in Table 2 below, substantially similar to step 1.1 of Example 1, and the relationship between the viscosity of the single-layer gel structure and temperature was measured. The results are shown in Figure 7.

[0142] [Table 2]

[0143] Figure 7 shows that the viscosity of the gel structure increases with increasing concentration of carboxymethyl cellulose (CMC) at 37.5°C. It can be seen that as the viscosity of the gel structure increases, the structural strength also increases accordingly. Therefore, as the concentration of CMC increases, the structural strength of the gel structure also improves accordingly.

[0144] When the viscosity is close to or higher than 1 Pascal·second (Pa·s) (e.g., SA is 5 wt% and CMC is 2 wt% in Group 1, and SA is 5 wt% and CMC is 1 wt% in Group 2), the structural strength of the gel structure can be suitable for culturing stem cells without easily rupturing.

[0145] In contrast, if the viscosity of the gel structure is too low (for example, SA is 5 wt% and CMC is 0.1 wt% in Group 3), there is a relatively high risk of the gel structure bursting when the gel structure is used for culturing stem cells.

[0146] Therefore, the gel structures of groups 1 and 2, when used as inner layer gel structures (weight percentage of CMC greater than 1 wt%), can provide good structural strength to the bilayer composite hydrogel microcarriers and serve as a growth scaffold for stem cells.

[0147] 5. Stem Cell Culturing Efficiency

[0148] First, two groups of bilayer composite hydrogel microcarriers with different weight ratios of SA and CMC, SA-CMC@PDMA-1 (SA is 1.5% and CMC is 1%) and SA-CMC@PDMA-2 (SA is 0.5% and CMC is 2%) in Table 1, were selected and used to culture adipose stem cells in a manner similar to that in Example 2. At the same time, the culture conditions were divided into groups with and without the addition of bovine serum albumin (BSA) as a nutrient component, and synchronous observations were performed.

[0149] Later, the following two parts were analyzed to observe whether the growth efficiency of adipose stem cells was affected when two-layer composite hydrogel microcarriers with different component ratios were used for culturing adipose stem cells.

[0150] The first part was mRNA analysis. After culturing the adipose stem cells for up to 7 days, the mRNA expression ratio of a protein specific to adipose stem cells (lipoprotein lipase; LPL) and a general cellular protein (glyceraldehyde-3-phosphate dehydrogenase; GAPDH) was analyzed to determine the growth status of the adipose stem cells. A higher ratio indicates a higher adipose stem cell content. The results are shown in Figure 8A.

[0151] Figure 8A shows that SA-CMC@PDMA-1 (SA 1.5% + CMC 1%) has better adipose stem cell growth efficiency compared with SA-CMC@PDMA-2 (SA 0.5% and CMC 2 wt%), regardless of whether BSA is added or not.

[0152] The second part is a microscopic observation. The number of cells grown under a microscope after culturing the adipose stem cells from each group for 7 days was observed. Adipose stem cells were recognized by CD44 monoclonal antibody and stained with Oil Red O. A higher percentage of the cells showed a higher adipose stem cell content. The results are shown in Figure 8B.

[0153] Figure 8B shows that when macromolecular nutrients (BSA) were added, the percentage of adipose stem cells was higher in SA-CMC@PDMA-1 (SA 1.5% + CMC 1%) than in SA-CMC@PDMA-2 (SA 0.5% and CMC 2 wt%). In other words, when nutrients were added, high stem cell culture efficiency could be achieved by adopting the weight ratio conditions of SA-CMC@PDMA-1.

[0154] In some embodiments of the present disclosure, the method for culturing stem cells using a two-layer composite hydrogel microcarrier provides physical support for the inner layer gel structure by designing the inner and outer layer gel structures, preventing the structure from being affected by acid-base balance, improving diffusion resistance, and delaying the release of nutrients. By defining the weight ratio of sodium alginate and carboxymethylcellulose and the location of nutrients, the pore size of the inner layer gel structure can be controlled, adjusting and controlling the rate at which nutrients are released from the two-layer composite hydrogel microcarrier, ensuring stable uptake of nutrients by stem cells and improving stem cell growth. Controlling the weight percentage of carboxymethylcellulose ensures the structural strength of the two-layer composite hydrogel microcarrier, allowing it to serve as a scaffold for stem cell growth.

[0155] Although the present disclosure has been disclosed as above by way of embodiments, the embodiments are not intended to limit the present disclosure, and those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure is determined by the content specified in the following claims.

Claims

1. providing stem cells, bilayer composite hydrogel microcarriers, and nutritional components; mixing the stem cells, the bilayer composite hydrogel microcarriers, and the nutritional components to obtain a gelling medium; adding a culture medium to the gelling medium; Including, The two-layer composite hydrogel microcarrier comprises: an inner layer gel structure obtained by ionically crosslinking the inner layer polymer with an inner layer crosslinked material; an outer layer gel structure obtained by covalently crosslinking an outer layer monomer with an outer layer crosslinked product, the outer layer gel structure covering the inner layer gel structure; the inner layer polymer comprises a first inner layer polymer and a second inner layer polymer, the first inner layer polymer is sodium alginate and the second inner layer polymer is carboxymethyl cellulose, the weight ratio of the sodium alginate to the carboxymethyl cellulose is 3:2, the weight percentage of the carboxymethyl cellulose is greater than 1% when the weight percentage of the inner layer gel structure is 100%, and the pore size of the inner layer pores is 250 microns to 1000 microns; the outer layer monomer comprises N,N-dimethylacrylamide, acrylamide, or a combination thereof; the thickness of the outer layer gel structure is 25 microns to 1000 microns, and is smaller than the thickness of the inner layer gel structure; the pore size of the outer layer pores is 50 microns to 500 microns, and is smaller than the pore size of the inner layer pores; The nutritional components are located within the inner layer gel structure and are released from the two-layer composite hydrogel microcarriers. A method for culturing stem cells using bilayer composite hydrogel microcarriers.

2. 2. The method of claim 1, wherein the stem cells comprise embryonic stem cells, hematopoietic stem cells, mammary stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory stem cells, adipose stem cells, or a combination thereof.

3. the inner layer gel structure is a plurality of inner layer sheet-like structures connected to each other and separated from each other by a plurality of inner layer pores, The method of claim 1 , wherein the outer layer gel structure is a plurality of outer layer sheet-like structures connected to each other and separated from each other by a plurality of outer layer pores.

4. The method of claim 1 , wherein the inner layer gel structure is an interpenetrating structure of the first inner layer polymer and the second inner layer polymer.

5. The method of claim 1 , wherein the inner layer polymer and the inner layer crosslinked product have opposite electrical properties.

6. The method of claim 1 , wherein the outer layer crosslinked product comprises N,N′-methylenebisacrylamide.

7. 10. The method of claim 1, wherein the weight percent of the carboxymethyl cellulose is between 1% and 3.25%, where the weight percent of the inner layer gel structure is 100%.

8. 10. The method of claim 1, wherein the nutritional components include growth factors, vitamin A acid, bovine serum albumin, or combinations thereof.

9. providing stem cells, bilayer composite hydrogel microcarriers, and a nutritional component comprising a macromolecular protein having a molecular weight greater than at least 500 grams / mole; mixing the stem cells, the bilayer composite hydrogel microcarriers, and the nutritional components to obtain a gelling medium; adding a culture medium to the gelling medium; Including, The two-layer composite hydrogel microcarrier comprises: an inner layer gel structure obtained by ionically crosslinking the inner layer polymer with an inner layer crosslinked material; an outer layer gel structure obtained by covalently crosslinking an outer layer monomer with an outer layer crosslinked product, the outer layer gel structure covering the inner layer gel structure; the inner layer polymer comprises a first inner layer polymer and a second inner layer polymer, the first inner layer polymer is sodium alginate, the second inner layer polymer is carboxymethyl cellulose, the weight ratio of the sodium alginate to the carboxymethyl cellulose is 3:2, the weight percentage of the sodium alginate is 1.5% to 5% when the weight percentage of the inner layer gel structure is 100%, and the pore size of the inner layer pores is 250 microns to 1000 microns; the outer layer monomer comprises N,N-dimethylacrylamide, acrylamide, or a combination thereof; the thickness of the outer layer gel structure is 25 microns to 1000 microns, and is smaller than the thickness of the inner layer gel structure; the pore size of the outer layer pores is 50 microns to 500 microns, and is smaller than the pore size of the inner layer pores; The nutritional components are located within the inner layer gel structure and are released from the two-layer composite hydrogel microcarriers. A method for culturing stem cells using bilayer composite hydrogel microcarriers.

10. 10. The method of claim 9, wherein the stem cells comprise embryonic stem cells, hematopoietic stem cells, mammary stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory stem cells, adipose stem cells, or a combination thereof.

11. the inner layer gel structure is a plurality of inner layer sheet-like structures connected to each other and separated from each other by a plurality of inner layer pores, 10. The method of claim 9, wherein the outer layer gel structure is a plurality of outer layer sheet-like structures connected to each other and separated from each other by a plurality of outer layer pores.

12. The method of claim 9 , wherein the inner layer gel structure is an interpenetrating structure of the first inner layer polymer and the second inner layer polymer.

13. The method of claim 9 , wherein the inner layer polymer and the inner layer crosslinked product have opposite electrical properties.

14. The method of claim 9, wherein the outer layer crosslinked product comprises N,N'-methylenebisacrylamide.

15. 10. The method of claim 9, wherein the weight percent of the carboxymethyl cellulose is greater than 1%, based on 100% weight percent of the inner layer gel structure.

16. 16. The method of claim 15, wherein the weight percent of the carboxymethyl cellulose is 1% to 5%, based on 100% weight percent of the inner layer gel structure.

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