Cell scaffold for producing cultured meat and production method therefor

Abstract

The present invention relates to a cell scaffold for producing cultured meat, a production method therefor, and a method for producing cultured meat using same. More specifically, the present invention relates to a cell scaffold for producing cultured meat, a production method therefor, and a method for producing cultured meat using same, wherein the cell scaffold is capable of adjusting the contents of muscle tissues and adipose tissues at a desired ratio by reversible physicochemical bonding using a self-healing hydrogel capable of self-healing at a temperature and in a pH range at which cells can be cultured.

Description

Cell support for cultured meat production and method for producing the same

[0001] The present invention relates to a cell support for producing cultured meat, a method for producing the same, and a method for producing cultured meat using the same.

[0002] Meat, regardless of animal species, is a vital source of nutrients such as water, protein, lipids, iron, zinc, and vitamin B12. With limited plant sources, excluding soybeans, our ancestors relied on meat as a crucial food source to supplement essential amino acids. Beyond its nutritional importance, meat has also played a crucial role in the development of diverse cultures and cuisines. Meat is composed of approximately 65-80% water, 16-22% protein, and 1.5-13% fat. The degree to which fat is distributed within the muscles (intramuscular fat, marbling) is a key factor in determining the meat's texture and flavor.

[0003] Despite the nutritional benefits of meat, recent global trends such as carbon neutrality and animal welfare have raised concerns about the high greenhouse gas emissions associated with livestock farming. According to the Food and Agriculture Organization of the United Nations (FAO), the livestock industry consumes a significant portion of the world's agricultural land and accounts for approximately 14.5% of global greenhouse gas emissions (GHG). Furthermore, the wastewater discharged from livestock farming significantly contributes to water and soil pollution.

[0004] To address these issues, interest in artificial meat, created by cultivating stem cells extracted from animals in a laboratory, has recently grown. Cultured meat, also known as "green meat," not only improves animal welfare but also significantly reduces greenhouse gas emissions associated with meat production. Some estimates suggest that this could reduce greenhouse gas emissions by up to 96%, as it reduces large-scale deforestation for livestock production.

[0005] To produce cultured meat that closely resembles real meat, it's crucial to achieve a similar texture. However, the technology to replicate complex marbling structures and produce large-scale meat economically and on a large scale is still lacking. The primary reasons are the high cost of the materials used as cell scaffolds for current cultured meat production, and the lack of technology capable of accurately replicating various meat components, such as muscle, fat, and blood vessels.

[0006] One object of the present invention is to provide a cell scaffold for producing cultured meat. Specifically, the present invention aims to provide a cell scaffold capable of culturing fat cells and muscle cells separately by introducing a polymer capable of forming reversible physicochemical bonds through a self-healing process, and allowing these cells to bond together through a self-healing process to form marbling similar to real meat. Furthermore, since bonding is possible through a self-healing process, the size and content of the fat and muscle portions of cultured meat can be freely controlled, and a cell scaffold for producing cultured meat capable of forming marbling close to real meat, a method for producing the same, and a method for producing cultured meat using the same are also provided.

[0007] Furthermore, the present invention provides a self-healing polymer that incorporates specific chemical or physical properties to enable self-healing while enabling cell culture. For example, a material can self-heal, similar to the healing process in living tissue, by utilizing chemical bonds that can be cut and reformed. This possesses characteristics similar to biological properties and shows significant similarities to the regenerative capabilities of living tissue. Therefore, the present invention aims to use such a self-healing polymer as a cell scaffold for the formation of cultured meat structures, thereby producing cultured meat with a structure similar to that of actual meat.

[0008] Another object of the present invention is to provide a biocompatible self-healing hydrogel. For example, the present invention provides a biocompatible self-healing hydrogel that can be used as a tissue regeneration-inducing scaffold capable of inducing cell attachment or gel degradation.

[0009] One embodiment of the present invention relates to a cell support for cultured meat production comprising a self-healing hydrogel, wherein the self-healing hydrogel comprises at least two or more cross-sections of the self-healing hydrogels, which are identical or different from each other, forming a reversible physicochemical bond through a self-healing process to enable assembly with each other.

[0010] In one embodiment, the cell support for producing cultured meat may include a first self-healing hydrogel in which muscle cells are cultured and a second self-healing hydrogel in which fat cells are cultured.

[0011] In one embodiment, the self-healing hydrogel may form reversible physicochemical bonds through a self-healing process in one or more atmospheres selected from the temperature and pH ranges in which cells can be cultured.

[0012] In one embodiment, the self-healing hydrogel may include a unit (A) derived from chitosan substituted with a boronic acid compound and a unit (B) derived from a polymer containing a hydroxyl group.

[0013] In one embodiment, the unit (A) derived from chitosan substituted with the boronic acid compound may include a compound represented by the following chemical formula 1.

[0014] [Chemical Formula 1]

[0015]

[0016] In the above chemical formula 1, R1 and R2 are each independently hydrogen or Selected from,

[0017] At least one of the above R1 and R2 am.

[0018] In one embodiment, the unit (A) derived from chitosan substituted with the boronic acid compound may have a substitution rate of 10 to 50% of the boronic acid compound according to the following formula 1.

[0019] [Formula 1]

[0020] Substitution rate = (number of moles of amine substituted with boronic acid compound / total number of moles of amine in chitosan) × 100

[0021] In one embodiment, the gel fraction of the self-healing hydrogel may be, but is not limited to, 50 to 90%.

[0022] In one embodiment, the cell support may further include a strength reinforcing material.

[0023] In one embodiment, the cell support may be capable of culturing one or more cells selected from the group consisting of myoblasts, adipocytes, fibroblasts, myocytes, mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), satellite cells, endothelial cells, or embryonic stem cells.

[0024] In one embodiment, the first component of the self-healing hydrogel in which the muscle cells are cultured (proliferated) may have a Young's modulus of 200 to 400 kPa, and the second component of the self-healing hydrogel in which the fat cells are cultured (proliferated) may have a Young's modulus of 50 to 180 kPa, but is not limited thereto.

[0025] Another aspect of the present invention provides cultured meat cultured using a cell support for producing cultured meat according to the above embodiment.

[0026] In one embodiment, the cultured meat may have marbling similar to that of actual meat.

[0027] Another aspect of the present invention provides a method for manufacturing a cell support using a self-healing hydrogel, comprising the steps of mixing a chitosan resin precursor substituted with a boronic acid compound and a polymer containing a hydroxyl group, and then adjusting the pH to induce cross-linking.

[0028] In one embodiment, the step of freezing and then thawing may be further included after the mixing.

[0029] In one embodiment, during the mixing, a strength reinforcing material may be further added.

[0030] Another aspect of the present invention comprises the steps of preparing a first self-healing hydrogel and a second self-healing hydrogel, each having a different mixing ratio of a chitosan resin precursor substituted with a boronic acid compound and a polymer including a hydroxyl group;

[0031] A step of culturing muscle cells in one of the first self-healing hydrogel and the second self-healing hydrogel, and culturing and differentiating fat cells in the other;

[0032] A method for producing cultured meat using a cell support including a self-healing hydrogel is provided.

[0033] In one embodiment, in the step of preparing the first self-healing hydrogel and the second self-healing hydrogel, a strength reinforcing material may be further added.

[0034] In one embodiment, the step of preparing the first self-healing hydrogel and the second self-healing hydrogel may further include a step of freezing and then thawing.

[0035] In one embodiment, the first self-healing hydrogel may have a Young's modulus of 200 to 400 kPa, and the second self-healing hydrogel may have a Young's modulus of 50 to 180 kPa, but is not limited thereto.

[0036] In one embodiment, muscle cells are cultured in one of the first self-healing hydrogel and the second self-healing hydrogel, and fat cells are cultured and differentiated in the other, and then these are stacked or placed in contact with each other, thereby allowing them to assemble together through a self-healing process.

[0037] In one embodiment, the first self-healing hydrogel and the second self-healing hydrogel may be arranged in a stacked or contacting manner so as to be joined to each other (assembly) through a self-healing process, and muscle cells may be cultured in one of the first self-healing hydrogel and the second self-healing hydrogel, and fat cells may be cultured and differentiated in the other.

[0038] Another aspect of the present invention provides a biocompatible self-healing polymer comprising a unit (A) derived from chitosan to which a boronic acid compound comprising a compound represented by the following chemical formula 1 is bound, and a unit (B) derived from a polymer containing a hydroxyl group.

[0039] [Chemical Formula 1]

[0040]

[0041] In the above chemical formula 1, R1 and R2 are each independently hydrogen or Selected from,

[0042] At least one of the above R1 and R2 am.

[0043] In one embodiment, the self-healing polymer may be capable of assembling with each other at a pH of 6.5 to 12.

[0044] In one embodiment, the unit (A) derived from chitosan substituted with the boronic acid compound may have a substitution rate of 10 to 50% of the boronic acid compound according to the following formula 1, but is not limited thereto.

[0045] [Formula 1]

[0046] Substitution rate = (number of moles of amine substituted with boronic acid compound / total number of moles of amine in chitosan) × 100

[0047] In one embodiment, the self-healing polymer may further comprise a strength reinforcing material.

[0048] In one embodiment, the self-healing polymer may have a Young's Modulus in the range of, but not limited to, 50 to 400 kPa.

[0049] According to one embodiment of the present invention, a biocompatible self-healing hydrogel is biocompatible, capable of cell culture, and capable of self-healing under conditions that allow cell culture. Accordingly, it can provide a preferred support for fat and muscle, respectively. Furthermore, by allowing fat-cultured portions and muscle-cultured portions to self-heal and bond together under conditions that allow cell culture, it is possible to produce cultured meat with marbling similar to actual meat. Furthermore, by allowing fat-cultured portions or muscle-cultured portions to self-heal and bond together, the size of the hydrogel can be increased.

[0050] In addition, the biocompatible self-healing hydrogel and the cell support for producing cultured meat using the same according to one embodiment of the present invention can reattach cut sections without applying a separate chemical method such as an adhesive or additional energy. Therefore, the self-healing hydrogel can grow muscle or fat cells only by diffusion of the material during cell culture, and then reattach them to a desired size or location to produce cultured meat of a large size, or freely control the fat content and marbling within the muscle.

[0051] Figure 1 is an infrared spectrum graph of chitosan ChsB(OH)2, which is a combination of chitosan and 3-carboxybenzeneboronic acid according to Manufacturing Example 1.

[0052] Figure 2 is a graph showing the UV-visible spectrophotometer of chitosan ChsB(OH)2 combined with boronic acid in a standard experiment and a comparative experiment according to Manufacturing Example 1.

[0053] Figure 3 is a graph that verifies the degree of substitution by displaying the intensity of absorbance at 285 nm for different concentrations of 3-carboxybenzeneboronic acid according to Manufacturing Example 1.

[0054] Figure 4 is a photograph showing the self-healing and multimodular responsiveness of N1 hydrogel according to an embodiment. a: Three colored and uncolored pieces fuse to form a large piece that can be lifted without falling apart. The dyes used are Brilliant Blue FCF and Amaranth Red 2. b: A single large hydrogel piece is reformed into three smaller pieces shaped like a heart, a star, and a flower simply by cutting and shaping. c: Liquefaction and gelation of the hydrogel in response to pH changes. d, e: The hydrogel's response to glucose solutions of different concentrations. DMEM stands for Dulbecco's modified Eagle's medium.

[0055] Figures 5 to 7 show cell viability and proliferation in ChsB(OH)2 / PVA hydrogels according to examples. Figure 5 shows cytotoxicity (biocompatibility) tests of hydrogel components in two cell lines using cell counting kit 8 (CCK-8). Low-density polyethylene (0.2 g mL -1 ) and zinc diethyldithiocarbamate were used as negative and positive controls, respectively. Figure 6 shows the results of monitoring the growth of 3T3-L1 cells through light absorption from orange formazan formation of CCK-8 on different hydrogel substrates for one week. Figure 7 shows the results of monitoring the growth of C2C12 cells through light absorption from orange formazan formation of CCK-8 on different hydrogel substrates for one week.

[0056] Figure 8 shows the viability of C2C12 cells cultured on 3T3-L1 and N1 ChsB(OH)2 / PVA hydrogel scaffolds in F0.5 according to the example, compared to the quantitative control, Matrigel, and visualized by LIVE / DEAD staining using an optical microscope. The green (calcein AM) indicated by ① indicates viable cells, and the blue (Hoechst 33342) indicated by ② indicates cell nuclei.

[0057] Figure 9 shows the differentiation of 3T3-L1 into adipose tissue on F0.5 hydrogel and Matrigel according to an example, and C2C12 into muscle tissue on N1 hydrogel and Matrigel. Cells were observed using a fluorescence microscope on day 9 of differentiation. The red (Nile Red) indicated by ① represents lipid droplets, the blue (Hoechst 33342) indicated by ② represents cell nuclei, and the green (Alexa Fluor 488) indicated by ③ represents myosin and F-actin.

[0058] Figure 10 is a photograph of artificial meat manufactured using a self-healing hydrogel according to an embodiment. a: Individual N1 muscle-like cultures and F0.5 fat-like cultures differentiated for 28 days, after frying. b: Artificial meat model formed by alternately layering muscle-like tissues and fat-like tissues to form two bilayers. Size: x 1 cm, y 0.5 cm, z 0.35 cm. Muscle tissues are distinguished from fat tissues by carmine staining.

[0059] The present invention will be described in more detail below. However, the following specific examples or examples are merely references for explaining the present invention in detail, and the present invention is not limited thereto, and may be implemented in various forms.

[0060] Additionally, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0061] Additionally, the singular forms used in the specification and the appended claims are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0062] Additionally, when a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0063] Additionally, unless otherwise specifically defined in this disclosure, when a layer or member is said to be “on” another layer or member, this includes not only cases where the layer or member is in contact with the other layer or member, but also cases where another layer or another member exists between the two layers or two members.

[0064] In addition, the terms “about,” “substantially,” etc. used in this disclosure are used in a meaning close to or at the numerical value when manufacturing and material tolerances inherent to the meanings mentioned are presented, and are used to prevent unscrupulous infringers from unfairly using the disclosure contents in which exact or absolute values ​​are mentioned to aid understanding of the present invention.

[0065] In addition, the term 'self-healing' in the present disclosure means that a polymer bonds by itself by forming a reversible physicochemical bond under specific temperature or pH conditions, rather than by an external factor such as a separate adhesive.

[0066] In addition, the term 'hydrogel' in the present disclosure is also called a hydrogel, and refers to a material having a network structure in which polymers form three-dimensional cross-links through physical bonds such as hydrogen bonds, van der Waals forces, and hydrophobic interactions, or chemical bonds such as covalent bonds, and which does not dissolve in an aqueous environment and can contain a significant amount of water.

[0067] Below, each component of the present invention is described in more detail.

[0068] [Cell support]

[0069] One embodiment of the present invention provides a cell support for cultured meat production comprising a self-healing hydrogel, wherein the self-healing hydrogel comprises at least two or more cross-sections of the self-healing hydrogels, which are identical or different from each other, forming a reversible physicochemical bond through a self-healing process to enable assembly with each other.

[0070] In one embodiment, the self-healing hydrogel may be a self-healing hydrogel capable of self-healing within a temperature and pH range in which cells can be cultured. Furthermore, the self-healing hydrogel may be capable of bonding through reversible physicochemical bonding within the self-healing hydrogel itself, without the need for a separate adhesive. Furthermore, the self-healing hydrogel may be capable of culturing muscle tissue and adipose tissue, and may bond a cell support containing cultured muscle tissue and a cell support containing cultured adipose tissue to each other through a self-healing process without the aid of an adhesive.

[0071] In one embodiment, the cell support for producing cultured meat may include a first self-healing hydrogel in which muscle cells are cultured and a second self-healing hydrogel in which fat cells are cultured.

[0072] In one embodiment, the functional group forming a reversible physicochemical bond through the self-healing process may include one or more selected from the group consisting of a hydrogen bond, an ionic bond, a boronic acid ester bond, a disulfide bond, a host-guest bond, an imine bond, an acylhydrazone bond, and the like, but is not limited thereto.

[0073] In one embodiment, the self-healing hydrogel may be capable of assembling with each other at a temperature of 20°C to 40°C, or 25°C to 28°C, at which cells can be cultured, and may form a reversible physicochemical bond capable of self-healing.

[0074] In one embodiment, the self-healing hydrogel may be capable of assembling with each other at a pH range of 6.5 to 12, or 6.5 to 8, at which cells can be cultured, and may form a reversible physicochemical bond capable of self-healing.

[0075] The above temperature and pH ranges are within the ranges in which cell culture is possible, and may be suitable for use as a cell support within the above ranges, but are not limited thereto.

[0076] According to one embodiment, cell supports can be joined to each other through a self-healing process within a temperature and pH range where cells can be cultured, and thus can be assembled to each other through a self-healing process without the need for a separate adhesive, etc. In addition, among cell supports for culturing muscle tissue and cell supports for culturing adipose tissue, the cross-sections of at least two or more cell supports, which are identical or different from each other, can be laminated or positioned so as to be in contact with each other so that they can be joined to each other through a self-healing process.

[0077] Accordingly, the size and content of the cell supports for culturing muscle tissue and the cell supports for culturing adipose tissue can be freely controlled as desired. Furthermore, it is possible to increase the size of at least two cell supports for culturing muscle tissue by joining them together. It is also possible to increase the size of at least two cell supports for culturing adipose tissue by joining them together.

[0078] In one embodiment, the cell support may be capable of culturing one or more cells selected from the group consisting of myoblasts, adipocytes, fibroblasts, myocytes, mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), satellite cells, endothelial cells, or embryonic stem cells, but is not limited thereto.

[0079] The cell support for producing the above cultured meat may include a first self-healing hydrogel in which muscle cells are cultured and a second self-healing hydrogel in which fat cells are cultured.

[0080] In one embodiment, the self-healing hydrogel can form marbling like real meat by culturing muscle cells in one of the first self-healing hydrogel and fat cells in the other, and then connecting them together.

[0081] In one embodiment, the self-healing hydrogel may be formed by irregularly connecting a first component of the self-healing hydrogel in which muscle cells are cultured (proliferated) and a second component of the self-healing hydrogel in which fat cells are cultured (proliferated) to form marbling similar to real meat. The self-healing hydrogel may be formed by allowing the first component of the self-healing hydrogel and the second component of the self-healing hydrogel to assemble with each other within a temperature range in which cells are cultured, thereby forming marbling similar to real meat.

[0082] In one embodiment, the self-healing hydrogel may include a unit (A) derived from chitosan substituted with a boronic acid compound and a unit (B) derived from a polymer containing a hydroxyl group.

[0083] In one embodiment, the unit (A) derived from chitosan substituted with the boronic acid compound may include, but is not limited to, a compound represented by the following chemical formula 1.

[0084] [Chemical Formula 1]

[0085]

[0086] In the above chemical formula 1, R1 and R2 are each independently hydrogen or Selected from,

[0087] At least one of the above R1 and R2 am.

[0088] In one embodiment, the unit (A) represented by the above chemical formula 1 may be represented by the following chemical formula 2, but is not limited thereto.

[0089] [Chemical Formula 2]

[0090]

[0091] In one embodiment, the unit (A) derived from the chitosan substituted with the boronic acid compound may have a substitution rate of the boronic acid compound according to the following formula 1 of 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 50% or less, 45% or less, or 40% or less, and may be any value between the above values. For example, it may be 10 to 50%, 15 to 45%, or 20 to 30%, but is not limited thereto. In a range satisfying the above substitution rate range, the amine of the unsubstituted chitosan may be stabilized so as to maintain the boronic acid ester bond of the boronic acid compound. Accordingly, self-healing may be possible.

[0092] [Formula 1]

[0093] Substitution rate = (number of moles of amine substituted with boronic acid compound / total number of moles of amine in chitosan) × 100

[0094] In the above equation 1, the total amine mole number of chitosan refers to the mole number of amines that are not acetylated when chitosan is deacetylated. The total amine mole number of chitosan can be calculated from the content of glucosamine.

[0095] In one embodiment, the self-healing hydrogel may include a unit (A) derived from chitosan substituted with a boronic acid compound and a unit (B) derived from a polymer containing a hydroxyl group in a weight ratio of 0.1 to 0.9:1, 0.15 to 0.9:1, 0.3 to 0.9:1, or 0.6 to 0.9:1, but is not limited thereto.

[0096] In one embodiment, the self-healing hydrogel may have a double network formed through boronic acid ester bonds and hydrogen bonds.

[0097] That is, a double network may be formed through hydrogen bonding between the hydroxyl groups of the polymer containing hydroxyl groups and the boronic acid ester bond formed by the reaction of the above units (A) and (B). When a double network is formed in this way, self-healing may be possible due to the flexible and dynamic covalent bond of the boronic acid-diol ester bond. In addition, mechanical strength may be further enhanced through hydrogen bonding between the hydroxyl groups of the polymer containing hydroxyl groups.

[0098] In the above chemical formula 1, the bond between chitosan and the boronic acid compound can be stabilized through a Lewis acid-base interaction between the amino group of chitosan and the boron of the boronic acid compound, and through this, the boronic acid-diol ester bond, which was previously stabilized only under basic conditions (e.g., pH 8.5 or higher), can form a very stable boronic acid-diol ester bond at neutral pH (even between 6 and 8) by coordinating with the chitosan.

[0099] In the case of the combination of the above chitosan and boronic acid compound, it can be stabilized by combining with the diol group of a polyol polymer compound such as polyvinyl alcohol to form a boronic acid-diol ester bond. This boronic acid-diol ester bond cannot be stabilized and will eventually dissociate if the amine group of the chitosan is absent, but the present invention can form a very stable boronic acid-diol ester bond by chitosan.

[0100] The following is a schematic diagram illustrating stabilization of a boronic acid-diol ester complex by the nitrogen atom of the amine group of chitosan. Here, the diol refers to a substance having a hydroxyl group, and in the present invention, it may refer to a polymer having a hydroxyl group, exemplified by polyvinyl alcohol.

[0101]

[0102] In order to bond the above chitosan and boronic acid compound, a crosslinker can be used, and for example, a zero-length crosslinker can be used. Examples of the crosslinker include, but are not limited to, N,N'-carbonyldiimidazole (CDI), N,N'-Dicyclohexylmethanediimine (DCC), N-Hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (sulfo-NHS), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), etc.

[0103] The above chitosan may have, but is not limited to, a diameter of, for example, 1 to 500 nm, 5 to 300 nm, or 10 to 100 nm, and a length of, but is not limited to, 1 μm to 10 mm, 1 μm to 5 mm, or 5 μm to 5 mm. In addition, the chitosan may have a molecular weight of 100 kDa or more, 150 kDa or more, 160 kDa or more, 170 kDa or more, 180 kDa or more, 190 kDa or more, 200 kDa or more, 250 kDa or more, 300 kDa or more, 310 kDa or more, 500 kDa or less, 450 kDa or less, 400 kDa or less, 390 kDa or less, 380 kDa or less, 370 kDa or less, 360 kDa or less, 350 kDa or less, and may have any value between the above values. For example, those having a molecular weight of 100 to 500 kDa, 150 to 400 kDa, and 190 to 310 kDa may be used, but are not limited thereto.

[0104] Additionally, the chitosan may be deacetylated, and may have a degree of deacetylation of 70% or more, 80% or more, or 90% or less, or any value between the above values. For example, 70 to 90%, 80 to 90%, or the like may be used, but is not limited thereto.

[0105] The above boronic acid compound may preferably have a carboxylic acid group in order to form the boronic acid-diol ester bond, and for example, a boronic acid having a substituent having a carboxylic acid group may be used, and specific compounds may be used, for example, 2-carboxybenzeneboronic acid, 3-carboxybenzeneboronic acid, 4-carboxybenzeneboronic acid, etc., but are not limited thereto.

[0106] The polymer containing the above hydroxyl group is preferably a polymer containing a hydroxyl group and having biocompatibility, and for example, polyvinyl alcohol can be used. The polyvinyl alcohol is not limited, but may have a weight average molecular weight of 10,000 g / mol or more, 100,000 g / mol or less, or any value between the above values. For example, one having a molecular weight of 10,000 to 100,000 g / mol, 30,000 to 100,000 g / mol, 50,000 to 100,000 g / mol, 70,000 to 100,000 g / mol, 80,000 to 100,000 g / mol, or 89,000 to 98,000 g / mol can be used.

[0107] In one embodiment, the cell support may further include a strength reinforcing material as needed. The strength reinforcing material is used to improve the strength of the cell support, and examples thereof include cellulose nanocrystals, cellulose nanofibers, chitosan nanowhiskers, nanochitin, etc. In addition, any other strength reinforcing material that can be commonly used in cell supports may be used without limitation as the strength reinforcing material. The content of the strength guaranteeing material is not limited, but may be used in an amount of, for example, 1 wt% or less, 0.9 wt% or less, 0.5 wt% or less, 0.3 wt% or less, 0.2 wt% or less, or 0.1 wt% or less based on the total weight of the cell support. For example, 0.01 to 1 wt%, 0.05 to 1 wt%, or 0.1 to 0.5 wt% may be used, and any value between the above values ​​may be used. It may not be limited to the above numerical range, as long as it can be used as a content for adjusting the strength so that muscle cells or fat cells have a strength suitable for culturing and differentiating.

[0108] For example, but not limited to, the first component of the self-healing hydrogel in which the muscle cells are cultured (proliferated) may have a Young's Modulus of 200 to 400 kPa, and the second component of the self-healing hydrogel in which the fat cells are cultured (proliferated) may have a Young's Modulus of 50 to 180 kPa. This is preferable because it can provide strength suitable for culturing and differentiating the muscle cells and fat cells, respectively, within the above ranges, but is not limited thereto. The Young's modulus refers to that measured according to the method described below.

[0109] In one embodiment, the gel fraction of the self-healing hydrogel is not limited to, but may be 50% or more, 51% or more, 55% or more, 60% or more, 63% or more, 65% or more, 70% or more, 73% or more, 75% or more, 77% or more, 80% or more, 83% or more, 84% or more, 90% or less, or any value therebetween. For example, it may be 50 to 90%. The gel fraction means that measured by the method described below.

[0110] In one embodiment, the cell support may be in the form of, but is not limited to, a scaffold, microbead, hydrogel, or fiber.

[0111] [Method for manufacturing cell support]

[0112] Another aspect of the present invention provides a method for manufacturing a cell support using a self-healing hydrogel, comprising the steps of mixing a chitosan resin precursor substituted with a boronic acid compound and a polymer including a hydroxyl group, and then adjusting the pH to induce cross-linking.

[0113] In one embodiment, when mixing the chitosan resin precursor substituted with the boronic acid compound and the polymer including a hydroxyl group, the Young's modulus of the cell support can be controlled within a desired range by controlling the mixing ratio. In one embodiment, the content of the chitosan resin precursor substituted with the boronic acid compound: the polymer including a hydroxyl group may be a weight ratio of 0.1 to 0.9:1, but is not limited thereto. For example, the weight ratio may be 0.15 to 0.9:1, 0.3 to 0.9:1, or 0.6 to 0.9:1.

[0114] Additionally, the Young's modulus may be adjusted to a desired range by adding additional strength reinforcing materials as needed. The strength reinforcing materials are not limited as long as they are generally applicable to cell supports, and the content thereof may be used in a range of, for example, 1 wt% or less or 0.5 wt% or less of the total weight of the cell support, but may be used in a content for adjusting the desired strength, and is not limited thereto.

[0115] Additionally, by further including a freezing and thawing step of the self-healing hydrogel manufactured as needed, the mechanical strength, for example, Young's modulus, can be adjusted to a desired range. Including this freezing and thawing process can induce hydrogen bonding between polyvinyl alcohol chains, thereby increasing the mechanical properties of the hydrogel.

[0116] In one example, the first component of the self-healing hydrogel in which muscle cells are cultured (proliferated) may be adjusted to have a Young's modulus of 200 to 400 kPa, and the second component of the self-healing hydrogel in which fat cells are cultured (proliferated) may be adjusted to have a Young's modulus of 50 to 180 kPa. This range is preferable because it facilitates cell culture and provides a texture similar to actual meat, but is not limited thereto.

[0117] [Cultured meat and its production method]

[0118] Another aspect of the present invention provides cultured meat cultured using a cell support according to the above embodiment.

[0119] As described above, the cell support according to one embodiment of the present invention is capable of self-healing in a temperature and pH range where cell culture is possible, and thus, each cell support in which muscle cells and fat cells are cultured can assemble with each other through a self-healing process, and can form marbling like real meat.

[0120] In addition, mechanical strength, such as Young's modulus, can be easily controlled by adjusting the mixing ratio of the chitosan resin precursor substituted with a boronic acid compound and the polymer containing a hydroxyl group, adding a strength reinforcing agent, or repeating freeze-thawing. Accordingly, cell supports having strengths favorable for culturing muscle cells and strengths favorable for culturing fat cells can be manufactured, respectively, and cultured meat formed with marbling through integration through self-healing before or after cell culture can be provided.

[0121] Another aspect of the present invention comprises the steps of preparing a first self-healing hydrogel and a second self-healing hydrogel, each having a different mixing ratio of a chitosan resin precursor substituted with a boronic acid compound and a polymer including a hydroxyl group;

[0122] A step of culturing muscle cells in one of the first self-healing hydrogel and the second self-healing hydrogel, and culturing and differentiating fat cells in the other;

[0123] A method for producing cultured meat using a cell support including a self-healing hydrogel is provided.

[0124] In one embodiment, the first self-healing hydrogel and the second self-healing hydrogel may have different mixing ratios of the chitosan resin precursor substituted with the boronic acid compound and the polymer including a hydroxyl group. For example, the first self-healing hydrogel may be mixed so that the Young's modulus is 200 to 400 kPa, and the second self-healing hydrogel may be mixed so that the Young's modulus is 50 to 180 kPa.

[0125] In one embodiment, the method may further include a step of freezing and then thawing the first self-healing hydrogel and the second self-healing hydrogel to control mechanical strength, and may further include a strength reinforcing material as needed.

[0126] In one embodiment, by culturing muscle cells in one of the first self-healing hydrogel and the second self-healing hydrogel, and culturing and differentiating fat cells in the other, and then stacking or arranging them in contact with each other, the self-healing process allows for assembly, thereby producing cultured meat in which the content of muscle tissue and fat tissue can be adjusted to a desired ratio.

[0127] Alternatively, the first self-healing hydrogel and the second self-healing hydrogel may be arranged in a stacked or contacting manner so that they are joined (assembled) to each other through a self-healing process, and muscle cells may be cultured in one of the first self-healing hydrogel and the second self-healing hydrogel, and fat cells may be cultured and differentiated in the other.

[0128] The cultured meat manufactured by the manufacturing method according to the above embodiment can have the content of muscle tissue and fat tissue adjusted to a desired ratio, which can be adjusted by a person skilled in the art.

[0129] [Biocompatible self-healing polymer]

[0130] Another aspect of the present invention provides a biocompatible self-healing polymer comprising a unit (A) derived from chitosan substituted with a boronic acid compound and a unit (B) derived from a polymer containing a hydroxyl group.

[0131] In one embodiment, the unit (A) derived from chitosan substituted with the boronic acid compound may include, but is not limited to, a compound represented by the following chemical formula 1.

[0132] [Chemical Formula 1]

[0133]

[0134] In the above chemical formula 1, R1 and R2 are each independently hydrogen or Selected from,

[0135] At least one of the above R1 and R2 am.

[0136] In one embodiment, the unit (A) derived from chitosan substituted with a boronic acid compound represented by the above chemical formula 1 may be represented by the following chemical formula 2, but is not limited thereto.

[0137] [Chemical Formula 2]

[0138]

[0139] In one embodiment, the unit (A) derived from the chitosan substituted with the boronic acid compound may have a substitution rate of the boronic acid compound according to the following formula 1 of 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 50% or less, 45% or less, or 40% or less, and may be any value between the above values. For example, it may be 10 to 50%, 15 to 45%, or 20 to 30%, but is not limited thereto. In a range satisfying the above substitution rate range, the amine of the unsubstituted chitosan may be stabilized so as to maintain the boronic acid ester bond of the boronic acid compound. Accordingly, self-healing may be possible.

[0140] [Formula 1]

[0141] Substitution rate = (number of moles of amine substituted with boronic acid compound / total number of moles of amine in chitosan) × 100

[0142] In the above equation 1, the total amine mole number of chitosan refers to the mole number of amines that are not acetylated when chitosan is deacetylated. The total amine mole number of chitosan can be calculated from the content of glucosamine.

[0143] In one embodiment, the self-healing hydrogel may include a unit (A) derived from chitosan substituted with a boronic acid compound and a unit (B) derived from a polymer containing a hydroxyl group in a weight ratio of 0.1 to 0.9:1, 0.15 to 0.9:1, 0.3 to 0.9:1, or 0.6 to 0.9:1, but is not limited thereto.

[0144] The self-healing polymer may be capable of assembling with each other at a pH of 6.5 to 12, but is not limited thereto. In addition, the self-healing may be exhibited at various temperature ranges, and from the perspective of biocompatibility, the self-healing process may be capable of assembling with each other even at a biocompatible temperature. For example, the self-healing process may be capable of assembling with each other at a temperature of 20°C or higher, 25°C or higher, 26°C or higher, 27°C or higher, 50°C or lower, 45°C or lower, 40°C or lower, 37°C or lower, or 25 to 37°C.

[0145] In one embodiment, the self-healing polymer may be formed by a double network through boronic acid ester and hydrogen bonding.

[0146] In one embodiment, the self-healing polymer may be, but is not limited to, a hydrogel.

[0147] In one embodiment, the self-healing polymer may be one in which the Young's modulus can be adjusted to a range of 50 to 400 kPa by controlling the ratio of the unit (A) and the unit (B). As long as the Young's modulus range can be satisfied, there is no limitation, but the unit (A) and the unit (B) derived from a polymer including a hydroxyl group may be included in a weight ratio of 0.1 to 0.9:1, 0.15 to 0.9:1, 0.3 to 0.9:1, or 0.6 to 0.9:1.

[0148] In one embodiment, the self-healing polymer may further include a reinforcing material to control its mechanical strength. The type and content of the reinforcing material are the same as described above, and thus further description is omitted.

[0149] In one embodiment, the self-healing polymer may also be used as a tissue regeneration-inducing scaffold capable of inducing cell attachment or gel degradation.

[0150] In one embodiment, the self-healing polymer may be used as a carrier for a bioactive substance, and the bioactive substance may be, for example, one or a mixture of two or more selected from the group consisting of antibiotics, anticancer agents, analgesics, anti-inflammatory agents, antiviral agents, antibacterial agents, proteins, peptides, nucleic acids, polysaccharides, lipids, carbohydrates, steroids, extracellular matrix materials, and cells, but is not limited thereto.

[0151] In one embodiment, the delivery speed of the bioactive substance can be controlled depending on the type and concentration of the bioactive substance carrier chemically or physically bonded to the self-healing polymer, the physical strength of the self-healing polymer, the chemical properties, the decomposition rate, etc. The carrier refers to a bioactive substance that can be delivered into a living body by carrying or chemically bonding the bioactive substance, and depending on the site of use or injection method, it can be injected into a living body or released at a predetermined site at a predetermined time consistently.

[0152] For example, the self-healing polymer may be applied to, but is not limited to, biocompatible dressings, adhesives for surgical site closure, films, sutures, supports for artificial skin, etc.

[0153] The present invention will be described in more detail based on the following examples and comparative examples. However, the following examples and comparative examples are merely illustrative examples for further explaining the present invention, and the present invention is not limited by the following examples and comparative examples.

[0154] The following physical properties were measured as follows.

[0155] [Spectroscopy]

[0156] Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra of the freeze-dried ChsB(OH)2 resin precursor and resin were measured using a Nicolet iS50 FTIR spectrometer (Thermo Scientific, USA) with a smart iTR diamond / ZnSe ATR accessory (face angle 45°) in the range of 4000–650 cm -1 2 cm in range -1 It was recorded by scanning 128 times at a scan interval of .

[0157] The UV-Vis spectra of ChsB(OH)2 samples were measured using a UV-visible-near-infrared (NIR) spectrophotometer (Jasco V-770, USA). The UV data were used to confirm the grafting of 3-carboxybenzeneboronic acid onto chitosan and to calculate the degree of boronic acid moiety substitution (DS) according to the following equation.

[0158] DS = [BA] / [GlnN] × 100

[0159] Here, [BA] is the boronic acid moiety content (mmol g -1 ) represents the glucosamine (chitosan repeating unit) content in the sample. At this time, the average degree of deacetylation is 80% (4.718 mmol g -1 ) was assumed.

[0160] [Gel Fraction (%)]

[0161] The gel fraction of the manufactured self-healing hydrogel (polymer) was measured from the freeze-dried weight before and after immersing the manufactured self-healing hydrogel in ion-exchanged water.

[0162] That is, the manufactured self-healing hydrogel (polymer) was freeze-dried until the weight became constant, and the weight was measured, which was designated as Wi. Then, it was immersed in ion-exchanged water until the weight became constant, and then taken out, freeze-dried until the weight became constant, and the weight was measured, which was designated as We.

[0163] The gel fraction was calculated using the following formula.

[0164] Gel fraction = (We / Wi) × 100

[0165] [Swelling]

[0166] To determine the degree of swelling, the self-healing hydrogel (polymer) was weighed and its weight (Wd) was measured, and the resin was immersed in excess ion-exchanged water at room temperature to swell until equilibrium was reached. Thereafter, the swollen resin was weighed and its weight (Ws) in the swollen state was measured to determine the equilibrium swelling degree (ESD) using the following formula.

[0167] ESD (%)=(Ws-Wd) / Wd ×100%

[0168] [Mechanical properties]

[0169] The compressive mechanical properties of the fabricated self-healing hydrogel (resin) samples were evaluated at room temperature using a universal testing machine (UTM, Instron 5943, UK) equipped with a 1 kN load cell. The resin samples were cut into 0.5 cm × 0.5 cm × 0.5 cm cubes and placed on a support plate. The compression test was performed at a feed rate of 5 mm / min. Data were collected every 0.1 s, and Young's modulus, which is the compressive elastic modulus (E), was measured as the slope of the linear region of the stress-strain curve. In addition, the compressive modulus was measured when the strain was 30% and 50%, respectively.

[0170] [Response to changes in glucose concentration and pH of resin]

[0171] The stabilized resin was exposed to an acidic pH by adding HCl solution (0.1 M), which dissociated the boronic acid-diol ester bonds. The pH was then raised to 7–8 using NaOH solution (0.1 M), which reformed the boronic acid-diol ester bond network. Furthermore, the response of the resin to aqueous glucose solutions (0.45 wt% and 5 wt%) was also investigated. Glucose, a saccharide with a polyol group, competes with the boronic acid-diol ester bonds and is an essential component of mammalian cell culture media (used at 0.45 wt%). It was expected that low glucose concentrations would not cause significant destruction of the resin.

[0172] [Meat cultivation on resin-based scaffolds]

[0173] 1. Cell lines and cell treatment

[0174] 3T3-L1 is a mouse fibroblast cell line that can differentiate into adipocytes. C2C12 is a mouse myoblast cell line that can rapidly differentiate, form contractile sarcomeres, and produce muscle proteins. Both cell lines were obtained from ATCC (https: / / www.atcc.org / ) and were handled according to their guidelines.

[0175] 2. Cell viability and proliferation (biocompatibility / cytotoxicity of resin scaffolds)

[0176] The cytotoxicity of ChsB(OH)2 and PVA, which are resin components, was evaluated using Cell Counting Kit-8 (CCK-8) based on the ISO-10993-5 and ISO-10993-5-12 standards against C2C12 myoblasts and 3T3-L1 fibroblasts. 0.2 g mL of lyophilized test material was used. -1 The extract was added to the cell culture media and cultured for 24 h at 37°C with shaking at 100 rpm, and then tested on the target cell lines. Growth media and low-density polyethylene (LDPE, 0.2 g mL -1 ) and zinc diethyldithiocarbamate (100 μM) extracts were used as blank, negative, and positive controls, respectively. Cells were cultured at 10 4 cells mL -1The cells were cultured on polystyrene 96-well cell plates, proliferated in growth media for 24 h, and then incubated in extracts for 24 h. The cells were then washed three times with phosphate-buffered saline (PBS) and incubated in DMEM (Dulbecco's Modified Eagle Medium) containing 10 v% of CCK-8 (Cell Counting Kit-8) at 37 °C for 1 h to allow in-vitro transfer of mitochondrial dehydrogenase to convert the triazine salt of CCK-8 to orange formazan. The amount of formazan was measured using a microplate reader (Hidex Sense) using absorbance at 450 nm (optical density OD 450). Cell viability was compared against the blank using the following formula:

[0177] Viability (%) = (100% × OD) 450e ) / OD 450b

[0178] OD here 450e is the measured OD 450 value of the extract of the test sample, and OD 450 b is the measured OD 450 value of the bin.

[0179] Additionally, cell proliferation on the resin scaffolds was monitored for 1 week using CCK-8. Cells were seeded 10 on 0.5 cm high resin scaffolds in 24-well cell plates pre-equilibrated with growth media (cell culture medium for cell proliferation). 6 cells mL -1was grafted. 500 μL of growth media was added to the top of the resin scaffold and refreshed daily to maintain healthy cell growth and proliferation. OD 450 values ​​were measured as described above. However, before measurement, the reacted CCK-8 solution was transferred from the culture plate to a new 96-well cell plate to prevent interference from the resin scaffold. Fresh growth media was then added to the cell culture to continue the experiment. An increasing OD 450 value over time indicates cell proliferation.

[0180] Cell viability was visually confirmed using the LIVE / DEAD Viability / Cytotoxicity Kit for mammalian cells (Thermo Fisher Scientific L-3224). Cells cultured on the resin scaffold (10 6 cells mL -1 ) were washed three times with PBS, and then stained with calcein acetoxymethyl ester (calcein AM, which stains live cells; 2 μM in PBS) and ethidium homodimer-1 (EthD-1, which stains dead cells; 4 μM in PBS) for 30 min in the dark at 37°C. Then, cell nuclei were stained with Hoechst 33342 (5 μg mL in PBS -1 ) were stained for 5 min in the dark at 37 °C and washed once with PBS. Stained cells were observed using a laser fluorescence microscope (LSM 780, Carl Zeiss AG, Germany) with the following parameters: calcein AM, absorption 495 nm, emission 515 nm (green); EthD-1, absorption 528 nm, emission 617 nm (red); Hoechst 33342, absorption 350 nm, emission 461 nm (blue).

[0181] Cell viability and proliferation were measured using Corning Matrigel Matrix (Product No. 354230, 5 mg mL) which mimics the extracellular matrix and serves as a positive control. -1 ) was compared with.

[0182] [Manufacturing Example 1] Synthesis of boronic acid-substituted chitosan (resin precursor)

[0183]

[0184] Chitosan (5.0 g; Sigma-Aldrich, medium molecular weight Chitosan 448877 product, viscosity molecular weight M V 190-310 kDa, 0.0236 mol as glucosamine monomer, 80% deacetylation) was dissolved in ion-exchanged water (500 mL). The pH of the solution was adjusted to 4-4.5 using HCl (0.1 M). 3-Carboxybenzeneboronic acid (1.958 g, 0.0118 mol, 50 mol% ratio with respect to glucosamine) was added to the chitosan solution. Cross-linking agents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride (EDC HCl) (2.262 g, 0.0118 mol) and N-hydroxysuccinimide (NHS) (1.358 g, 0.0118 mol), were added to the reaction mixture, and the reaction was carried out at room temperature for 48 h. Boric acid-conjugated chitosan [ChsB(OH)2] is a regenerated cellulose dialysate membrane with a molecular weight of 10 KDa (Spectra / Por from Spectrum Labs). TM The product was purified using a lyophilized extract and preserved through lyophilization.

[0185] As a result, the successful grafting of 3-carboxybenzeneboronic acid onto chitosan was confirmed through FTIR spectroscopy, as shown in Fig. 1. The spectrum of ChsB(OH)2 shows characteristic phenyl group signals, which are due to aromatic ring stretching (1580 cm -1) and three adjacent CH non-planar strains of the m-substituted aromatic ring (748 and 705 cm -1 ) is included. In addition, C=O stretching vibration (1604 cm -1 ) confirmed the formation of amide bonds in ChsB(OH)2.

[0186] The above bond was also confirmed by UV-Vis spectroscopy in Fig. 2, which showed the appearance of a phenyl ring peak at 285 nm, as shown in Fig. 2. In addition, the degree of boronic acid substitution was determined using ultraviolet-visible spectroscopy (UV-Vis spectroscopy) based on a calibration curve for 3-carboxybenzeneboronic acid, as shown in Fig. 3. As a result, the substitution degree was determined to be 21.2 mol%.

[0187] [Comparative Example 1] Synthesis of an Alginate-Based Self-Healing Resin Receptor for Cell Culture

[0188] AlgB(OH)2, which is a boronic acid-bonded alginate, was prepared by grafting 3-aminophenylboronic acid onto alginate in the presence of EDC·HCl and NHS as follows. Sodium alginate (5 g, 0.025 mol based on monomer) was dissolved in ion-exchanged water (500 mL), and then EDC·HCl (4.8 g, 0.025 mol), NHS (2.88 g, 0.025 mol), and 3-aminophenylboronic acid (1.95 g, 0.0125 mmol) were added. The mixture was stirred at room temperature for 48 h, then dialyzed with distilled water for 1 week, and lyophilized after replacing the water at least 7 times.

[0189] AlgB(OH)2 could form a stable resin with PVA only at an alkaline pH of 10, regardless of the ratio between the two components. At physiological pH 7 to 7.5, the resin began to flow and quickly turned into a fluid liquid.

[0190] [Examples 1 to 10]

[0191] Synthesis of a Boronic Acid Ester-Based Self-Healing Hydrogel Resin

[0192] The freeze-dried ChsB(OH)2 prepared in the above Preparation Example 1 was dissolved in an acetic acid aqueous solution (0.2 M, pH 4.76) at 3 wt% (Solution A). Polyvinyl alcohol (PVA) powder (MW 89 × 10 3 98 × 10 3 g / mol, >99 mol% hydrolyzed, atactic hydroxy side chains) was dissolved in deionized water at 90°C at 10 wt% (Solution B). In Examples 3 and 8, 0.1 wt% of spray-dried cellulose nanocrystals was further added to Examples 2 and 7, respectively.

[0193] To form a hydrogel, solution A was added to solution B at different mass ratios as described in Table 1, and the pH was adjusted to 7.5 using 1.0 M NaOH solution to induce cross-linking.

[0194] To remove air bubbles trapped in the resin and obtain a uniform texture, the resin was mixed in a mixer, then stabilized at 4°C for 24 hours to induce complete cross-linking of the polymer network. Examples 6 to 10 were placed in a freezer at -20°C for 6 hours and then thawed at 20°C for 12 hours to improve the mechanical properties. This process induces additional cross-linking through hydrogen bonding between the PVA chains within the resin, forming a double network through boronic acid esters and hydrogen bonds within the resin. The resin was purified by dialyzing with ion-exchanged water and Dulbecco's phosphate buffered saline (PBS 1X, pH 7.4, for cell culture) using a regenerated cellulose membrane with a molecular weight of 10 KDa until completely free of byproducts.

[0195] <Evaluation of gel fraction and swelling degree>

[0196] In Table 1 below, the sample names N indicate that no freeze / thaw treatment was performed, F indicates that a freeze / thaw process was added, and C indicates that 0.1 wt% of cellulose nanocrystals were added as a strength reinforcing agent to the entire hydrogel resin. The numbers indicate the weight ratio of ChsB(OH)2 to PVA.

[0197] The physical properties of the hydrogel resins manufactured in Examples 1 to 10 were evaluated.

[0198] SampleChsB(OH)2 / PVA mass ratioGel fraction (%)Equilibrium degree of swelling (%)Example 1F0.50.1575.6 ± 2.59145 ± 2.52Example 2F10.383.1 ± 3.30153 ± 7.57Example 3F1C0.384.4 ± 1.20152 ± 1.73Example 4F20.664.6 ± 4.65188 ± 6.66Example 5F30.957.7 ± 3.18196 ± 3.61Example 6N0.50.1566.9 ± 2.23201 ± 5.13Example 7N10.373.9 ± 2.73182 ± 4.16Example 8N1C0.377.1 ± 1.81185 ± 6.03Example 9N20.663.8 ± 3.94231 ± 6.03Example 10N30.951.8 ± 2.65237 ± 5.86

[0199] As shown in Table 1, ChsB(OH)2 solutions and PVA solutions were mixed at various volume ratios over a physiological pH range of 7.0–7.5, forming self-healing hydrogels via boronic acid-diol bonds. These bonds were stabilized by chitosan nitrogen atoms (N series). Freeze-thaw treatment induces hydrogen bonds between PVA chains, mechanically strengthening the hydrogels (F series). <Mechanical Property Evaluation>

[0200] Additionally, as can be seen in Table 2, the formation of a hydrogen bonding network between PVA chains resulted in a high gel fraction and low swelling ratio.

[0201] SampleCompressive modulus (MPa)at 30% strain Stress (MPa)at 50% strain Stress (MPa)Example 1F0.50.160.100.39Example 2F10.420.220.92Example 3F1C0.610.341.60Example 4F20.100.070.23Example 5F3---Example 6N0.50.080.050.26Example 7N10.210.130.47Example 8N1C0.250.150.17Example 9N20.070.040.37Example 10N3---

[0202] In Table 2 above, F3 of Example 5 and N3 of Example 10 were not strong enough to measure mechanical properties, so they could not be measured. Also, as can be seen from the table above, the compressive strength properties of the hydrogels could be changed by adjusting the mass ratio of ChsB(OH)2 and PVA. This ability is advantageous in promoting tissue growth and differentiation by creating a scaffold with an elasticity system that matches a specific type of tissue. The compressive elasticity showed the highest value when ChsB(OH)2 and PVA were mixed at an appropriate mass ratio of 0.3.

[0203] Self-healing and multi-module responsiveness assessment

[0204] Next, self-healing and multi-module responsiveness were evaluated.

[0205] The dynamic nature of the boronic acid-diol bond allows for efficient self-healing of the ChsB(OH)2 / PVA hydrogel, leading to efficient fusion of incompatible interfaces. As shown in Fig. 4, when the N1 hydrogel of Example 7 was prepared from three separately prepared specimens (Brilliant Blue FCF dyed / unstained / Amaranth Red 2 dyed) with independently prepared dyes, and contacted with each other, after 5 min, the three separate pieces were smoothly connected, resulting in a hydrogel that could be lifted without fragmentation (see Fig. 4a).

[0206] This sealing-healing capability enabled easy shape modification of the hydrogel. For example, a piece of N1 hydrogel shaped like a teddy bear's head was cut, completely destroyed, and placed in a heart, star, or flower-shaped mold. After a certain period of time, it reshaped into a heart, star, or flower shape that fit the mold. No additional adhesive was required, confirming the reusability of the self-healing polymer (Fig. 4b).

[0207] The N1 hydrogel also responded to changes in pH and glucose. Lowering the pH from alkaline to acidic resulted in hydrogel collapse due to pH-dependent boronic acid-diol bond dissociation between ChsB(OH)2 and PVA chains. Raising the pH back to 7.5 resulted in gel reformation and restoration of stability (Fig. 4c).

[0208] Furthermore, when the N1 hydrogel came into contact with a 5 wt% glucose solution, the gel began to flow due to the strong competitive bonding between the monosaccharides and the boronic acid, and soon became freely fluid. However, when placed in DMEM medium with a much lower glucose concentration (0.45 wt%), the hydrogel remained intact, which may impart essential properties to its function as a tissue-loading scaffold (Fig. 4d, e).

[0209] Evaluation as a scaffold for tissue culture

[0210] Next, the feasibility of using self-healing hydrogels as scaffolds for tissue culture was evaluated. Cell lines were cultured using the hydrogels prepared in Examples 1 to 10.

[0211] Cell line culture evaluation was performed using the Cell Counting Kit-8 (CCK-8) to investigate the growth and proliferation of a cell line in hydrogels with other cell lines. For this experiment, hydrogels showing similar proliferation rates to Matrigel (positive control) were selected for each individual cell line. The following hydrogels and their corresponding compressive strengths were used: N1 (207 kPa) and N1C (250 kPa) for the C2C12 cell line, and N0.5 (80.2 kPa), F0.5 (161 kPa), and F2 (104 kPa) for the 3T3-L1 cell line.

[0212] First, 10 in a 24-well plate 6 Cells were seeded on hydrogels (~0.3 cm high) pre-equilibrated with growth medium at 1 mL-1 of cells. Growth medium (500 μL) was then added to the top of the seeded hydrogels to facilitate cell attachment, and the cells were then incubated at 37 °C with 5% CO2 for 3 h. After aspirating the medium, the C2C12-seeded hydrogels were carefully separated from the wells with a spatula, inverted, and attached to 3T3-L1-seeded hydrogels in another well plate using the hydrogels' self-healing ability. Six combinations of self-healing hydrogels were evaluated. Cultures were incubated at 37 °C with 5% CO2 on top of growth medium (500 μL), and the medium was changed daily to maintain optimal cell growth and proliferation. Considering the slower growth rate of the 3T3-L1 cell line compared to the C2C12 cell line, the growth medium was supplemented with bovine calf serum (BCS 10 v%), which is optimized for the growth of 3T3-L1 fibroblasts. The growth and proliferation of both cell lines in co-culture were assessed using CCK-8 as described for individual cell cultures. Matrigel (5 mg mL-1), which has no self-healing ability, was used as a negative control.

[0213] 1. Cell viability and proliferation

[0214] All hydrogel components, consisting of ChsB(OH)2 and PVA, showed no observable toxicity in 3T3-L1 and C2C12 cell lines. Cell viability under all treatment conditions complied with the ISO 10993-5 standard and was comparable to the negative control (Fig. 5).

[0215] We also investigated the effect of hydrogel stiffness on cell proliferation. Soft hydrogels (F0.5, N0.5, and F2) promoted proliferation of the 3T3-L1 fibroblast cell line before differentiation (Fig. 6), whereas the C2C12 myoblast cell line grew better on stiffer hydrogels (N1 and N1C) (Fig. 7). Growth of both cell lines on these hydrogels was similar to that on Matrigel (positive control).

[0216] According to the LIVE / DEAD staining results, the viability of 3T3-L1 cultured on F0.5 hydrogel and C2C12 cultured on N1 hydrogel were almost the same as those of the positive control, Matrigel (Fig. 8). In other words, it can be seen that almost no cells died. In Table 7 above, the green (calcein AM) indicated by ① represents viable cells, and the blue (Hoechst 33342) indicated by ② represents cell nuclei.

[0217] 2. Preparation of differentiation medium and differentiation of C2C12 and 3T3-L1 cell lines

[0218] Next, we looked at cell differentiation.

[0219] To differentiate 3T3-L1 cells into adipocytes, differentiation medium was prepared with the following composition: 90% DMEM, 10% FBS, 1 μM Dexamethasone, 0.5 mM methyl isobutyl xanthine (IBMX), 1 μg / ml Insulin. After removing the growth medium from 3T3-L1 cells that had sufficiently grown, differentiation medium was applied. Depending on the day of observation, differentiation medium was applied for at least 7 days, 14 days, or 21 days, and the differentiation medium was replaced once every 2-3 days.

[0220] Similarly, to differentiate C2C12 cells into myoblasts, a differentiation medium was prepared with the following composition: 98% DMEM, 2% Donor equine serum (DES), 1 uM Insulin. After removing the growth medium from C2C12 cells that had grown sufficiently, differentiation medium was applied. Depending on the day of observation, differentiation medium was applied for at least 7 to 14 days, or 21 days, and the differentiation medium was replaced once every 2-3 days.

[0221] 3T3-L1 cells differentiated into adipocyte-like tissues with accumulation of Nile Red-stained lipid droplets on F0.5 hydrogels and Matrigel. Similarly, C2C12 cells changed their morphology from a fibroblast-like stem-like shape to a muscle-like shape on N1 hydrogels and Matrigel (Fig. 9). That is, 3T3-L1 differentiated into adipocyte-like tissues on F0.5 hydrogels and Matrigel, and C2C12 differentiated into muscle-like tissues on N1 hydrogels and Matrigel. Cells were observed using a fluorescence microscope on day 9 of differentiation. Red (Nile Red), indicated by ①, represents lipid droplets, blue (Hoechst 33342), indicated by ②, represents cell nuclei, and green (Alexa Fluor 488), indicated by ③, represents myosin and F-actin.

[0222] 3. Production of cultured meat

[0223] Cultures of adipose (3T3-L1) and muscle (C2C12) tissues were cultured in 6-well culture dishes to produce artificial meat prototypes. Hydrogel cell scaffolds, no more than 0.3 cm thick and sized to fit the well, were prepared by mixing ChsB(OH)2 (3 wt%, pH 4–5) and PVA (10 wt%) solutions in appropriate ratios and adjusting the pH to 7.4–7.5. Freeze / thaw treatment can be applied to the hydrogels. 24 h prior to cell seeding, scaffolds were equilibrated by impregnating them with growth media appropriate for each cell type.

[0224] Cells (10) were seeded on the hydrogel scaffolds in each well (N1 for C2C12, F0.5 for 3T3-L1). 6 (1 mL of cell / mL suspension) were seeded. Cells were cultured until 100% confluent to ensure sufficient intercellular signaling for differentiation.

[0225] The fused 3T3-L1 cells were induced to differentiate into adipocytes for 3 days using adipocyte differentiation medium and then maintained in adipocyte maintenance medium, which was replaced daily. The fused C2C12 cells were induced to differentiate into myocytes and maintained with daily medium replacement. The composition of the growth, differentiation, and maintenance medium is described in "2. Preparation of Differentiation Medium for C2C12 and 3T3-L1 Cell Lines and Differentiation" above.

[0226] On day 28 after differentiation, mature adipose and muscle tissue cultures were used to assemble meat prototypes via scaffold self-healing. After removing the culture medium, fat and muscle layers were assembled side by side into a sandwich structure (Figure 10), edge-to-edge bacon / pork belly structures, or customized structures mimicking complexly marbled beef. Assembly was achieved within 2 to 5 minutes at room temperature without any external adhesives or stimuli. The assembled meats were confirmed to be refrigerated or cooked as desired.

[0227] Differentiated N1 muscle morphotype cultures and F0.5 fat morphotype cultures exhibited the appearance of muscle and fat of real raw meat after 28 days (Fig. 10 a). Muscle and fat tissues were distinguished using carmine, a common food dye that imparts a red color to processed meat. Both cultures were fried in sunflower oil at 150°C, and the resulting cooked artificial meat had a texture similar to beef jerky or bacon. Finally, cell-laden hydrogel sheets were alternately layered up to 0.75 μm thick, resulting in thick meat slices (0.35 cm thick) capable of creating customized patterns within the fat and muscle structures (Fig. 10 b). This demonstrates the potential for creating large-area artificial meat without the aid of advanced technologies such as 3D printing.

[0228] <Results of Edibility and Stability Test>

[0229] 1. Hydrogel weight evaluation

[0230] To analyze the composition of the tissue culture, the weights of N1 and F0.5 hydrogels that had reached a completely equilibrium state were measured before cell seeding. On day 28 after differentiation, the culture medium of the corresponding cultures (N1 containing C2C12 cells and F0.5 containing 3T3-L1 cells) was removed, washed with distilled water, and the surface moisture was removed, and the weight (Ws) was measured. The cultures were then freeze-dried to obtain the dry contents, and the weight of the dried hydrogel (Wd) was measured. The dried tissue contents were obtained by subtracting the weight of the dried hydrogel and water from the total weight of the culture, assuming that there was no hydrogel degradation during the culture period. The equilibrium swelling degree (ESD) was calculated by the following equation.

[0231] ESD (%)=(Ws-Wd) / Wd ×100%

[0232] 2. Analysis of hydrogel composition

[0233] Elemental analyses of the dried contents of the cultures were performed for hydrogen (H), carbon (C), nitrogen (N), and sulfur (S) using a Unicube elemental analyzer (Elementar, Germany), and for oxygen (O) using a Flash 2000 elemental analyzer (Thermo Fisher Scientific, USA). For boron content analysis, lyophilized samples (~0.1 g) were ground in liquid nitrogen and then dispersed in distilled water (100 mL) by initial sonication (40% amplitude, 5 s on / 2 s off cycle, room temperature, 3 min). The dispersions were then analyzed using an iCAP 6500 Duo inductively coupled plasma-atomic emission spectrometer (IOP-AES) spectrometer.

[0234] Below are the experimental results.

[0235] After differentiation of 3T3-L1 / F0.5 cultures as adipocytes / cell support and C2C12 / N1 cultures as muscle cells / cell support, their compositions were measured.

[0236] The weight composition of the differentiated 3T3-L1 / F0.5 culture was confirmed to be composed of 19.5% moisture, 9.68% dried hydrogel, and 70.84% ​​dried tissue. In addition, the weight composition of the differentiated C2C12 / N1 culture was confirmed to be composed of 73.29% moisture, 4.82% dried hydrogel, and 21.89% dried tissue.

[0237] Adipose tissue, which stores neutral fat, contains less water than muscle, whereas muscle tissue retains water, which is important for electrolyte transport and contraction. After 28 days of differentiation, the dry hydrogel contents of C2C12 / N1 and 3T3-L1 / F0.5 cultures were calculated to be 4.82 wt% and 9.65 wt%, respectively. This indicates that as the cell and tissue content increases during cell proliferation and differentiation, the volume occupied by the hydrogel gradually decreases.

[0238] Additionally, the elemental composition of the final culture, including the scaffold and tissue, was measured, revealing significant changes after cell growth and differentiation compared to the initial hydrogel. The results are presented in Table 3.

[0239] Table 3 below shows the elemental composition of dried samples of hydrogel precursors, hydrogels, and tissue cultures (cell-containing hydrogels). Units are weight percent.

[0240] Dried sampleFormula / CompositionHydrogen (H)Carbon (C)Nitrogen (N)Oxygen (O)Sulfur (S)Theoretical valueExperimental valueTheoretical valueExperimental valueTheoretical valueExperimental valueChitosan(C6H 11 O4N) 0.8 (C8H 13 O5N) 0.2b) 6.786.5245.3345.278.268.4439.6339.530NDPVAC2H4O9.158.5954.5354.270ND36.3236.310NDChsB(OH)2(C 13 H 16 O7NB) 0.1696 (C6H 11 O4N) 0.6304 (C8H 13 O5N) 0.2 c) 6.346.4846.8246.957.26.8538.737.850NDN1 hydrogelChsB(OH)2 / PVA weight ratio 0.3 / 0.77.317.1349.4848.54.715.0237.8837.670NDF0.5 hydrogelChsB(OH)2 / PVA weight ratio 0.15 / 0.857.927.8551.1551.823.153.1237.3638.030NDC2C12 / N1 culture d) -10.00-19.60-3.44-66.00-0.253T3-L1 / F0.5 culture d) -7.16-61.74-0.87-29.69-ND

[0241] In Table 3 above b) Based on a deacetylation degree of 80 mol%. c) Based on an average degree of boric acid-N substitution of 21.2 mol% by UV-visible photometry. d)Samples after 28 days of cell differentiation. 3T3-L1 / F0.5 cultures showed increased carbon content due to the accumulation of long-chain hydrocarbon triglycerides by adipocytes, while C2C12 / N1 cultures had high nitrogen and oxygen contents derived from muscle proteins. Calculation results showed that the elemental compositions of differentiated C2C12 and 3T3-L1 closely matched those of muscle and adipocytes in the database (see Table 4). This suggested that self-healing hydrogels capable of dynamic bonding could promote normal muscle and fat development. Safety assessment of meat prototypes through boron content showed that 28-day wet cultures contained <0.07 wt% boron (equivalent to 0.4 wt% boronic acid). This meets the maximum usage requirement for boronic acid as a food additive, which suggests the potential safe consumption of meat products.

[0242] Table 4 below shows the calculated elemental composition of dried muscle and fat tissues 28 days after differentiation. Compositions are in wt%.

[0243] TissueHydrogen (H)Carbon (C)Nitrogen (N)Oxygen (O)Phosphorus (P)Sulfur (S)Db a) Calc b) DbCalcDbCalcDbCalcDbCalcDbCalcMuscle (C2C12)10.1510.6313.1313.243.253.172.472.240.200.330.3Adipose (3T3-L1)7.147.0662.3863.090.60.5629.3428.550.0600.060

[0244] In Table 4 above, a)Db, element% data were obtained from the IT'IS Foundation database (https: / itis.swiss / virtual-population / tissue-properties / database / elements / ) for element composition of various tissue types, and weight% was calculated and reported therefrom. b) Calc, a value calculated using the weight ratio of the tissue in the dry content. As described above, the present invention has been described through specific matters and limited examples, but these are provided only to help a more general understanding of the present invention, and the present invention is not limited to the above examples, and those skilled in the art to which the present invention pertains can make various modifications and variations from this description.

[0245] Therefore, the idea of ​​the present invention should not be limited to the described embodiments, and all things that are equivalent or equivalent to the following claims as well as the claims are considered to fall within the scope of the idea of ​​the present invention.

Claims

1. A cell support for cultured meat production comprising a self-healing hydrogel, The self-healing hydrogel is a cell support for producing cultured meat, wherein the cross-sections of at least two or more self-healing hydrogels, which are identical or different from each other, form reversible physicochemical bonds through a self-healing process, enabling assembly.

2. In paragraph 1, A cell support for cultured meat production, wherein the cell support for cultured meat production comprises a first self-healing hydrogel in which muscle cells are cultured and a second self-healing hydrogel in which fat cells are cultured.

3. In paragraph 1, The above self-healing hydrogel is a cell support for producing cultured meat, which forms reversible physicochemical bonds through a self-healing process in one or more atmospheres selected from the temperature and pH ranges in which cells can be cultured.

4. In paragraph 1, The above self-healing hydrogel is a cell support for producing cultured meat, comprising a unit (A) derived from chitosan substituted with a boronic acid compound and a unit (B) derived from a polymer containing a hydroxyl group.

5. In paragraph 4, A cell support for producing cultured meat, wherein the unit (A) derived from chitosan substituted with the above boronic acid compound comprises a compound represented by the following chemical formula 1. [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are each independently hydrogen or Selected from, At least one of the above R1 and R2 am.

6. In paragraph 5, A cell support for producing cultured meat, wherein the unit (A) derived from chitosan substituted with the above boronic acid compound has a substitution rate of 10 to 50% of the boronic acid compound according to the following formula 1. [Formula 1] Substitution rate = (number of moles of amine substituted with boronic acid compound / total number of moles of amine in chitosan) × 100 7. In paragraph 1, A cell support for cultured meat production, wherein the gel fraction of the self-healing hydrogel is 50 to 90%.

8. In paragraph 1, A cell support for cultured meat production, wherein the cell support further includes a strength reinforcing material.

9. In paragraph 1, The above cell support is a cell support for producing cultured meat capable of culturing one or more cells selected from the group consisting of myoblasts, adipocytes, fibroblasts, myocytes, mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), satellite cells, endothelial cells, or embryonic stem cells.

10. In paragraph 2, A cell support for producing cultured meat, wherein the first component of the self-healing hydrogel in which the muscle cells are cultured (proliferated) has a Young's modulus of 200 to 400 kPa, and the second component of the self-healing hydrogel in which the fat cells are cultured (proliferated) has a Young's modulus of 50 to 180 kPa.

11. Cultured meat cultured using any one of the cell supports selected from clauses 1 to 10.

12. In paragraph 11, The above cultured meat is cultured meat that has formed marbling similar to actual meat.

13. A method for manufacturing a cell support using a self-healing hydrogel, comprising the steps of mixing a chitosan resin precursor substituted with a boronic acid compound and a polymer containing a hydroxyl group, and then adjusting the pH to induce cross-linking.

14. In paragraph 13, A method for producing a cell support, further comprising a step of freezing and then thawing after the above mixing.

15. In paragraph 13, A method for manufacturing a cell support, wherein a strength reinforcing material is further added during the above mixing.

16. A step of preparing a first self-healing hydrogel and a second self-healing hydrogel, each having a different mixing ratio of a chitosan resin precursor substituted with a boronic acid compound and a polymer containing a hydroxyl group; A step of culturing muscle cells in one of the first self-healing hydrogel and the second self-healing hydrogel, and culturing and differentiating fat cells in the other; A method for producing cultured meat using a cell support comprising a self-healing hydrogel.

17. In paragraph 16, A method for producing cultured meat, wherein a strength reinforcing material is further added in the step of preparing the first self-healing hydrogel and the second self-healing hydrogel.

18. In paragraph 16, A method for producing cultured meat, wherein the step of preparing the first self-healing hydrogel and the second self-healing hydrogel further includes a step of freezing and then thawing.

19. In paragraph 16, A method for producing cultured meat, wherein the first self-healing hydrogel has a Young's modulus of 200 to 400 kPa, and the second self-healing hydrogel has a Young's modulus of 50 to 180 kPa.

20. In paragraph 16, A method for producing cultured meat, wherein muscle cells are cultured in one of the first self-healing hydrogel and the second self-healing hydrogel, and fat cells are cultured and differentiated in the other, and then these are stacked or placed in contact with each other, thereby causing assembly through a self-healing process.

21. In paragraph 16, A method for producing cultured meat, wherein the first self-healing hydrogel and the second self-healing hydrogel are arranged in a stacked or contacting manner so as to be joined to each other (assembly) through a self-healing process, muscle cells are cultured in one of the first self-healing hydrogel and the second self-healing hydrogel, and fat cells are cultured and differentiated in the other.

22. A biocompatible self-healing polymer comprising a unit (A) derived from chitosan to which a boronic acid compound comprising a compound represented by Chemical Formula 1 is bonded, and a unit (B) derived from a polymer containing a hydroxyl group. [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are each independently hydrogen or Selected from, At least one of the above R1 and R2 am.

23. In paragraph 22, The above self-healing polymer is a self-healing polymer capable of assembling with each other at a pH of 6.5 to 12.

24. In paragraph 22, A self-healing polymer in which the unit (A) derived from chitosan substituted with the above boronic acid compound has a substitution rate of 10 to 50% of the boronic acid compound according to the following formula 1. [Formula 1] Substitution rate = (number of moles of amine substituted with boronic acid compound / total number of moles of amine in chitosan) × 100 25. In paragraph 22, A polymer wherein the self-healing polymer further comprises a strength reinforcing material.

26. In paragraph 22, The above self-healing polymer is a self-healing polymer having a Young's Modulus in the range of 50 to 400 kPa.

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