KR20230080366A - Scaffold assembly for manufacturing cultured meat - Google Patents

Abstract

The present invention includes a biocompatible cross-linked polymer containing a polypeptide and a polysaccharide, and a scaffold for producing cultured meat, a scaffold assembly and a method for manufacturing cultured meat using the same, characterized in that it includes a patterned surface in at least a part of the region As for, it is possible to easily implement cultured meat in which muscle cells and fat cells are mixed like real meat through assembly of a scaffold in which muscle cells are cultured and a scaffold in which fat cells are cultured.

Description

Scaffold assembly for manufacturing cultured meat}

The present invention relates to a scaffold assembly for manufacturing cultured meat and a method for manufacturing cultured meat using the same.

In the production of cultured meat, a scaffold on which cells can attach and grow is an essential element. In addition, in order to produce texture and taste similar to real meat, proper mixing of muscle cells and fat cells is required when manufacturing cultured meat.

However, in the case of cultured meat scaffolds developed so far, most of them contain inedible substances or focus only on muscle cell culture without fat cells, so there is a limitation that the cultured meat produced using the scaffold is different from the actual meat. , In addition to conventional cultured meat manufacturing technology, there are disadvantages in that the manufacturing process is complicated or the price of the material is high.

In addition, materials such as gelatin, cellulose, and agar, which are used as edible scaffolds for conventional cultured meat manufacturing, have low mechanical properties to the extent that they cannot maintain their shape in a cell culture environment during scaffold manufacturing, and it is difficult to fuse fat cells and muscle cells. There is a limit. To solve this problem, there is a way to increase the physical properties of the scaffold by adding a chemical crosslinking agent or to develop and fuse different scaffolds optimized for each cell to mix muscle cells and fat cells. However, this method has limitations in that it causes cytotoxicity or the manufacturing method is complicated and time-consuming.

Therefore, it is necessary to develop a scaffold that can easily and quickly fuse muscle cells and fat cells through a simple manufacturing method while minimizing the burden on cytotoxicity and intake by using only edible materials without chemical crosslinking agents.

The present invention adjusts the physical properties of a hydrogel containing polysaccharides and polypeptides used as edible materials in order to realize cultured meat in which muscle cells and fat cells are mixed in an appropriate ratio like real meat, and prefabricated scan using an edible crosslinking agent. It aims to provide a fold.

The present invention is a scaffold for manufacturing cultured meat that can control the proliferation rate and attachment range of muscle cells and fat cells by controlling the thermal stability and mechanical properties of the hydrogel, as well as the arrangement and differentiation rate of muscle cells through surface patterning. It is an object to provide an assembly.

In addition, an object of the present invention is to provide a method for manufacturing cultured meat in which muscle and fat are easily combined through assembly between a hydrogel in which muscle cells are cultured and a hydrogel in which fat cells are cultured by manufacturing a scaffold in the form of Lego.

In order to solve the above-described technical problem, the present invention provides a scaffold for manufacturing cultured meat, characterized in that it includes a biocompatible crosslinked polymer containing a polypeptide and a polysaccharide, and includes a patterned surface in at least a portion of the region. can

In one embodiment of the present invention, the patterned surface may have a rectilinear embossed pattern in one direction.

In one embodiment of the present invention, the polypeptide may include gelatin. Alternatively, the polypeptide may include an RGD sequence in its main chain or side chain. Alternatively, the polypeptide is cross-linked by an enzyme, and the enzyme may include transglutaminase.

In one embodiment of the present invention, the polysaccharide may include an anionic polysaccharide. Alternatively, the polysaccharide may be crosslinked by divalent metal ions.

In addition, the present invention is a patterned scaffold unit block comprising a biocompatible cross-linked polymer comprising a polypeptide and a polysaccharide; and at least one planarization scaffold unit block comprising a biocompatible crosslinked polymer including a polypeptide and a polysaccharide, wherein the patterned scaffold unit block and the planarization scaffold unit block are assembled to form a patterned first surface. It is possible to provide a scaffold assembly for producing cultured meat, characterized in that it includes a region and a flat second surface region at the same time.

In one embodiment of the present invention, the surface of the patterned scaffold unit block may have a rectilinear embossed pattern in one direction. Alternatively, the patterned scaffold unit block and the planarized scaffold unit block may include the same polypeptide and polysaccharide. Alternatively, the patterned scaffold unit block and the planarized scaffold unit block may have different compressive strengths. Alternatively, the patterned scaffold unit block may have a compressive strength of 10 kPa to 18 kPa, and the flattened scaffold unit block may have a compressive strength of 1 kPa to 6 kPa.

In addition, the present invention provides (S1) preparing a biocompatible polymer solution containing a polypeptide and a polysaccharide; (S2) filling a mold having a pattern on its surface with the biocompatible polymer solution; (S3) in the mold preparing a scaffold precursor by crosslinking the polypeptide in (S4) obtaining a scaffold precursor having a patterned surface by separating the scaffold precursor from within the mold; and (S5) cross-linking the polysaccharide contained in the scaffold precursor having the patterned surface.

In one embodiment of the present invention, prior to the step (S3), gelling the biocompatible polymer solution filled in the mold at a low temperature; may further include. Alternatively, the step (S5) may be performed in an aqueous solution containing divalent metal ions. Alternatively, after the step (S5), (S6) cutting the scaffold to prepare patterned scaffold unit blocks that can be assembled with each other; may further include.

or (S7) preparing a flattened scaffold unit block; and (S8) assembling the patterned scaffold unit block and the flattened scaffold unit block to manufacture an assembly including a patterned first surface region and a flat second surface region at the same time.

In one embodiment of the present invention, the flattening scaffold unit block may include a biocompatible cross-linked polymer of a polypeptide and a polysaccharide.

In addition, the present invention includes (S10) filling the above-described scaffold assembly for cultured meat production with a culture solution and inoculating animal cells; (S20) incubating the culture solution inoculated with the animal cells to proliferate the cells; And (S30) organizing the proliferated cells in the culture medium; it is possible to provide a cultured meat manufacturing method comprising a.

In one embodiment of the present invention, the animal cells may include animal myoblasts and animal adipocytes.

In one embodiment of the present invention, animal myoblasts may be inoculated into the patterned scaffold unit block, and animal adipocytes may be inoculated into the flattened scaffold unit block.

In one embodiment of the present invention, the ratio of muscle cells and adipocytes may be adjusted by adjusting the ratio of the area of the patterned scaffold to the area of the flattened scaffold.

In addition, the present invention can provide cultured meat prepared according to the above-described manufacturing method.

The present invention can provide nutrition, texture and flavor similar to real meat by preparing cultured meat in which fat cells and muscle cells are appropriately mixed.

The present invention optimizes the physical properties of the scaffold and assembles a scaffold for culturing fat cells and a scaffold for culturing muscle cells in a desired ratio, thereby realizing various tastes and flavors that vary depending on the meat part, and reducing assembly. Through this, mass production of cultured meat can be easily achieved.

According to the present invention, cultured meat in which muscle and fat are mixed can be prepared through a quick and simple assembly method, and at the same time, the ratio of muscle to fat in the cultured meat can be easily adjusted.

Figure 1 is a schematic diagram showing the assembled cultured meat according to the present invention, showing that different muscle tissue and adipose tissue configurations can be implemented depending on the site.
Figure 2 shows the components of the scaffold assembly including muscle block and fat block configurations having different sensory properties by controlling cell differentiation according to the present invention.
3 is a schematic diagram and a graph showing the relationship between the hydrogel crosslinking degree and mechanical properties by alginate and calcium ion concentrations for controlling the scaffold environment according to the present invention.
Figure 4 is a schematic diagram showing the manufacturing process of the surface-patterned gelatin / alginate scaffold according to an embodiment of the present invention. Step 1 shows the process of fabricating a PDMS stamp to impart a gelatin/alginate hydrogel pattern, and step 2 shows the process of fabricating a gelatin/alginate scaffold with a unidirectional pattern on the surface using a PDMS stamp and a silicone mold.
Figure 5 is a design drawing of a Lego form that can be assembled by setting a base shape, which is a basic form, to produce a prefabricated scaffold, and then transforming the base shape into three forms.
6 shows an example of a scaffold assembly according to the present invention, a patterned gelatin/alginate scaffold in which muscle cells are cultured and fat cells in order to prepare cultured meat in which muscle cells and fat cells are mixed. It shows an example of assembling a cultured flattening scaffold (Smooth gelatin/alginate scaffold).
7 shows confocal microscopy images showing the expression level of MHC analyzed by immunostaining on days 5 and 8 of differentiation in the process of optimizing muscle blocks according to the present invention. Nuclei were stained with DAPI (blue) and MHC were stained with MF20 (red) (Scale bars: 20 μm (first row) and 100 μm (second row)).
Figure 8 is an experiment for optimizing the concentration of alginate and calcium suitable for muscle block production of the present invention, each differentiation in the low alginate concentration and low calcium ion concentration (LALC) and high alginate concentration and low calcium ion concentration (HALC) groups It is a result graph showing the level of MHC expression and the amount of protein in bovine myoblasts.
9 is a graph showing the stiffness of the scaffold according to the differentiation of myoblasts and the change in stiffness according to temperature change.
10 shows the results of analyzing the flavor according to cell proliferation and differentiation by performing gas chromatography after cooking LALC and HALC hydrogels according to the experimental example of the present invention.
11 is a schematic diagram showing the differentiation of adipose-derived mesenchymal stem cells (adMSCs) according to scaffolds (a) and a graph (b) showing the results of quantitative analysis of lipids produced from each scaffold.
Figure 12 shows the results of analyzing fat differentiation according to the scaffold, and the appearance stained in red represents lipids.
13 shows confocal microscopy images of cells stained with neutral lipids.
14 is a graph of the results of examining the effect and flavor on the stiffness of the scaffold according to fat production.
15 shows the results of nutritional analysis of the assembled cultured meat and actual beef according to the present invention.
16 shows the results of flavor analysis of the assembled cultured meat and actual beef according to the present invention.
17 shows the results of texture analysis of the assembled cultured meat and actual beef according to the present invention.
18 is a schematic diagram showing a manufacturing process of a T-bone steak composed of a muscle block and a fat block. It shows the process of assembling muscle and fat blocks to produce sirloin and tenderloin with different ratios of muscle to fat.
19 shows an image of a T-bone steak composed of assembled sirloin and tenderloin.

Hereinafter, with reference to the accompanying drawings and examples, in the technical field to which the present invention pertains to the scaffold for producing cultured meat, the scaffold assembly for producing cultured meat and the method for manufacturing cultured meat using the same, and the cultured meat produced therefrom, according to the present invention, with reference to the accompanying drawings and examples. It is described in detail so that those skilled in the art can easily perform it.

At this time, unless there is another definition in the technical terms and scientific terms used, they have meanings commonly understood by those of ordinary skill in the art to which this invention belongs, and the gist of the present invention in the following description and accompanying drawings Descriptions of well-known functions and configurations that may be unnecessarily obscure are omitted.

The present invention may be embodied in many different forms and is not limited to the embodiments or figures set forth herein. In order to clearly describe the present invention in the drawings, parts not related to the description are omitted, and similar reference numerals are assigned to similar parts throughout the specification. The drawings are provided as examples so that the spirit of the present invention can be sufficiently conveyed to those skilled in the art to which the present invention belongs, and the present invention is not limited to the drawings presented and may be embodied in other forms. However, the drawings may be exaggerated to clarify the spirit of the present invention.

The singular form used herein may be intended to include the plural form as well, unless the context dictates otherwise.

In addition, the term "includes" in this specification is an open description having the same meaning as the expression "includes", "includes", "has" or "characterized by", and is not further listed. It does not exclude any element, material or process.

As used herein, the term "muscle block" may be used as the same meaning as "patterned scaffold unit block", and "fat block" may be used as the same meaning as "flattened scaffold unit block".

As used herein, the term "scaffold" refers to a support for cell culture, and may be used to refer to "hydrogel".

The present invention is a technology for producing cultured meat that provides nutrition, texture and flavor similar to real meat. Currently, most studies on cultured meat focus on muscle cell content and implementation of an aligned canal structure, so factors that determine food characteristics of meat are being overlooked. Meat has a marbling structure in which muscle and fat tissues are randomly distributed, and the texture, taste, and nutrition of the structure may vary due to the distribution of muscle and fat depending on the tissue. In other words, the coexistence of muscle and fat and the quality of tissues determine the food quality of meat.

Accordingly, the present invention was devised to provide cultured meat that emulates the marbling structure of real meat composed of high-quality muscle and fat tissue and embodies the natural food characteristics of meat.

Hereinafter, the scaffold and scaffold assembly for producing cultured meat according to the present invention will be described in detail.

The present invention may provide a scaffold for preparing cultured meat, characterized in that it includes a biocompatible crosslinked polymer including a polypeptide and a polysaccharide, and includes a patterned surface in at least a portion of the region.

The polypeptide may include an RGD motif having cell adhesion ability. An Arg-Gly-Asp (RGD) sequence may be included in the main chain or side chain, and the polypeptide may include gelatin, fibronectin, or collagen.

In one embodiment of the invention, the polypeptide may be cross-linked by an enzyme. Specifically, the enzyme may include transglutaminase (TG), and more specifically, microbial transglutaminase (mTG). Transglutaminase is an enzyme for cross-linking polymerization of the polypeptide, and cross-linking and polymerization between glutamine residues and lysine residues can be promoted by the action of TG. In addition, TG can contribute to the scalability of assembled cultured meat (ACM) by interconnecting the scaffold unit blocks according to the present invention and acting as an adhesive. Specifically, gelatin can form a hard structure by gelating to create a triple helix structure. At this time, a peptide bond between the triple helix structures is generated by the crosslinking reaction of TG, which contributes to maintaining a stable form in the cell culture environment.

The polysaccharide may include an anionic polysaccharide, and may be an alginate derived from algae that can be ion-crosslinked as an edible biocompatible polymer, but is not limited thereto.

The polypeptide and the polysaccharide can form a network with each other using cell culture medium, culture medium, or water as a solvent, and can be chemically cross-linked or physically bonded to form a three-dimensional hydrogel scaffold. .

In one example, the polysaccharide may be crosslinked by divalent metal ions. Specifically, the divalent metal ion may be a calcium ion. Calcium polysaccharides ion-crosslinked by calcium ions can directly affect the stiffness of a hydrogel scaffold containing the calcium polysaccharide. The mechanical strength of a hydrogel containing alginate as a polysaccharide can be controlled by the concentration of alginate and calcium ions. Referring to FIG. 3, for the same amount of cross-linked gelatin, high concentration alginate (HA, 2 wt%) and low concentration alginate (LA, 0.25 wt%) have a calcium ion concentration (alginate: Ca ²⁺ = 1:1 When the weight ratio of) is high, it can be confirmed that the degree of crosslinking increases due to the interaction with the carboxylate of alginate, which can lead to an increase in the mechanical strength of the hydrogel.

However, when the amount of divalent metal ions is excessive, the ratio of the triple helix structure and the β-sheet structure of the polypeptide may be lowered, and in this case, the mechanical properties of the hydrogel may be lowered. Therefore, in the present invention, the polysaccharide and the divalent metal ion are preferably included in a weight ratio of 1:0.1 to 1:1.3 or 1:0.3 to 1:1.

The present invention aims to control the scaffold environment for producing cultured meat that can exhibit stiffness similar to that of actual adipose tissue and muscle tissue through this fact.

Accordingly, the present invention provides each of the different scaffold environments capable of exhibiting stiffness similar to that of adipose tissue and muscle tissue, which is specifically one or more patterned scaffolds comprising a biocompatible cross-linked polymer comprising a polypeptide and a polysaccharide. unit block; and at least one planarization scaffold unit block comprising a biocompatible crosslinked polymer including a polypeptide and a polysaccharide, wherein the patterned scaffold unit block and the planarization scaffold unit block are assembled to form a patterned first surface. It can be implemented as a scaffold assembly for producing cultured meat, characterized in that it includes a region and a flat second surface region at the same time.

The patterned scaffold unit block and the flattened scaffold unit block may include the same polypeptide and polysaccharide, but may exhibit different compressive strengths by controlling the concentration of the polysaccharide and the degree of crosslinking of the polysaccharide.

In one embodiment, the patterned scaffold unit block may have a compressive strength of 9 to 20 kPa, or 10 to 18 kPa. The patterned scaffold unit block can proliferate and differentiate, for example, by culturing muscle cells. The surface of the patterned scaffold unit block may have a rectilinear embossed pattern in one direction, and the embossed pattern may be a micrometer-scale unidirectional pattern, and the pattern is imprinted on the surface so that the cells adhere to the focus ( When forming focal adhesion), they can be aligned on the scaffold surface and proliferate under the influence of the pattern. This can provide a form in which muscle cells are regularly differentiated into aligned myotubes according to a straight embossed pattern different from a general cell culture dish.

Also, in one embodiment of the present invention, the planarized scaffold unit block may have a compressive strength of 1 to 6 kPa. At this time, the flattening scaffold unit block can proliferate and differentiate adipocytes.

The scaffold assembly for producing cultured meat may provide cultured meat in which muscle cells and fat cells are mixed in an appropriate ratio by including a patterned scaffold unit block and a flattened scaffold unit block.

Specifically, the present invention is a fat block based on a low-concentration alginate capable of providing stiffness similar to the Young's modulus of 3 to 4.5 kPa of adipose tissue and a low cross-linking hydrogel (LALC) according to a low calcium ion concentration, Young's modulus of skeletal muscle tissue By designing a muscle block based on a hydrogel (HALC) with a low degree of crosslinking according to a high concentration of alginate and a low calcium ion concentration that can provide stiffness similar to 10 to 12 kPa, It is characterized by implementing cultured meat. In addition, the designed configuration not only provides cultured meat having an appropriate fat texture and muscle tissue ratio, but also has excellent mechanical properties and can stably support cells during cell proliferation and differentiation.

As shown in FIG. 9(a), as myoblasts differentiate, the stiffness of the LALC hydrogel increased, whereas the stiffness of the HALC hydrogel (a hydrogel with a low degree of crosslinking due to high concentration of alginate and low calcium ion concentration) This decrease can be observed. Based on the fact that in hydrogels with high stiffness (>10 kPa), the tendency for cells to degrade the scaffold in order to elongate and differentiate into osteocytes becomes dominant. seems to be

9(c) is a schematic diagram showing how denaturation of collagen and MHC protein occurs when the temperature rises. In particular, the triple helix of collagen is destroyed, resulting in a decrease in the Young's modulus of collagen fibrils, and the aggregation of MHC, resulting in an increase in stiffness.

As shown in FIG. 9 (b), when heat-treated at 60 ° C., it can be observed that the stiffness of the HALC hydrogel increases in a different trend from that of the LALC. As shown in FIG. 9(c), this is expressed during differentiation of muscle proteins such as MHC, which is the main component of muscle tissue, and this MHC protein is denatured by aggregation at a high temperature of 50 ° C. or higher and stiffens muscles to improve meat quality. Considering the fact that HALC has a strong expression of muscle protein, the MHC aggregation effect prevails over the collagen destruction effect, which can be explained by the increase in stiffness upon heating. It can be confirmed that the muscle block according to the present invention successfully mimics real muscle tissue through HALC hydrogel in that it shows a similar aspect to the characteristics of real meat.

Furthermore, the present invention can provide an assembly of muscle and fat blocks that realize nutritional and textural characteristics similar to those of real meat, based on the fact that the sensory characteristics of cultured meat vary according to the degree of cell differentiation.

First, the scaffold for preparing cultured meat includes (S1) preparing a biocompatible polymer solution containing a polypeptide and a polysaccharide; (S2) filling the biocompatible polymer solution into a mold having a pattern on its surface; (S3) preparing a scaffold precursor by crosslinking the polypeptide in the mold; (S4) obtaining a scaffold precursor having a patterned surface by separating the scaffold precursor from within the mold; and (S5) cross-linking the polysaccharide contained in the scaffold precursor having the patterned surface.

In addition, prior to the step (S3), gelation of the biocompatible polymer solution filled in the mold at a low temperature may be further included, and the step (S5) may be performed in an aqueous solution containing divalent metal ions. .

As an example of the method for manufacturing the scaffold for preparing the cultured meat, reference may be made to FIG. 4. Specifically, a PDMS stamp having a linear pattern in a single direction is prepared and a gelatin/alginate polymer solution is poured. After gelation at a certain temperature and for a certain time, incubation at 37 ° C. to generate a triple helix structure in gelatin can form a hard hydrogel structure. A scaffold precursor having a patterned surface may be obtained by removing the scaffold precursor after the completion of the crosslinking reaction from the mold. Thereafter, the precursor may be immersed in a calcium chloride solution to cross-link alginate in the precursor. When the alginate crosslinking is completed (S6), the scaffold can be cut to prepare patterned scaffold unit blocks that can be assembled with each other. The cutting method may be manufactured in the form of three deformed Legos with reference to FIG. 5, but is not limited thereto.

It is also possible to prepare planarized scaffold unit blocks for fat blocks. Specifically, it can be manufactured in the same way in a silicon mold, except for the patterning process. Thereafter, (S8), an assembly including a patterned first surface region and a flattened second surface region may be manufactured by assembling the patterned scaffold unit block and the flattened scaffold unit block. 1, as an example of cultured meat (ACM), it is shown that it was prepared by assembling 10 flattened scaffold unit blocks and 10 patterned scaffold unit blocks.

Cultured meat that can be assembled according to the present invention can assemble muscle blocks and fat blocks in a desired ratio, thereby implementing various flavors and textures for each meat part, and assembling muscle blocks and fat blocks horizontally or vertically to cultured meat It is easy to mass-produce.

Specifically, the present invention comprises the steps of (S10) filling the scaffold assembly with a culture medium and inoculating animal cells; (S20) incubating the culture solution inoculated with the animal cells to proliferate the cells; And (S30) organizing the proliferated cells in the culture medium; it is possible to provide a cultured meat manufacturing method comprising a.

Alternatively, the present invention comprises the steps of preparing a patterned scaffold block in which muscle cells are cultured by filling a culture solution for culturing myoblasts in a patterned scaffold unit block and incubating to proliferate and differentiate myoblasts; Filling a culture medium for culturing adipocytes in a flattening scaffold unit block, incubating to proliferate and differentiate adipocytes, and then preparing a flattened scaffold block in which adipocytes are cultured; and assembling the patterned scaffold unit block and the flattened scaffold unit block.

In addition, if necessary, a step of applying a coloring agent for muscle fiber coloring may be further included. The colorant is a natural colorant of the carotenoid and anthocyanin series, and is sufficient to impart meat-like color to cultured cells, and is not limited as long as it can be used for food. Specifically, the carotenoid may be lycopene, canthaxanthin, beta carotene, and the like.

Animal cells include animal myoblasts and animal adipocytes, but are not limited as long as they can be used for producing cultured meat. It may include pluripotent stem cells (PSCs) and/or cells differentiated therefrom, non-embryonic stem cells (ESCs), or satellite cells.

As an example, animal myoblasts may be inoculated into the patterned scaffold unit block, and animal adipocytes may be inoculated into the flattened scaffold unit block. At this time, the ratio of muscle cells and fat cells can be adjusted by adjusting the area ratio of the patterned scaffold and the flattened scaffold. Specifically, referring to the nutritional information according to the United States Department of Agriculture (USDA), the protein:fat ratio of sirloin in New York is 1.5 to 3:1, and the protein:fat ratio of tenderloin is 1 to 3.6:1, and one embodiment of the present invention As, the area ratio of the patterned scaffold and the planarized scaffold may be set to 1:1 to 4:1, but this is only an example and is not limited thereto.

In addition, the present invention provides cultured meat prepared according to the above-described manufacturing method. The size of the cultured meat according to the present invention can be freely controlled according to the direction of adhesion and the number of blocks used for assembly, so you can enjoy the same texture as real meat, which has not been reproducible in cultured meat.

The present invention will be described in more detail through the following examples. However, the following examples are only references for explaining the present invention in detail, and the present invention is not limited thereto, and may be implemented in various forms.

(experimental method)

animal cell culture

Bovine primary myoblasts were isolated from harvested gluteal or semitendinous muscle tissue, and bovine adipose tissue-derived mesenchymal stem cells (adMSC) were extracted from intramuscular or subcutaneous adipose tissue. All tissues were collected from 29-31 month old male and female Korean cattle slaughtered at a local slaughterhouse (Kwell LPC, Hongcheon, Korea). All experimental procedures were performed in accordance with the Animal Care and Use Guidelines of Kangwon National University and were approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (IACU approval number KW-220714-1).

Bovine myoblasts and adipose-derived mesenchymal stem cells (adMSC) were subcultured in TPP® tissue culture dishes (Sigma Aldrich). For myoblast growth medium, high-glucose Dulbecco's Modified Eagle's Medium (HG-DMEM, Thermo Fisher Scientific); Peprotech, Rocky Hill, NJ, USA) and 1% (v/v) Penicillin-Streptomycin-Amphotericin (Thermo Fisher Scientific) were used. The adMSC growth medium was low glucose Dulbecco's Modified Eagle's Medium (LG-DMEM, Welgene) supplemented with 10% (v/v) FBS and 1% penicillin-streptomycin-amphotericin (PS). Cell passaging was performed using 1X PBS (Gibco® Life Technologies, USA), 0.025% trypsin ethylene-diamine-tetraacetic acid (EDTA; Gibco® Life Technologies, USA) and growth medium for each cell population.

The seeding densities of cultured bovine myoblasts (passage 3) and adMSC (passage 3) on the hydrogel were 3Х10 ⁴ cells and 2Х10 ⁴ cells per 12 wells of the prepared scaffold, respectively. After proliferating myoblasts for 4 days, the medium was replaced with differentiation medium. In addition, adMSC were proliferated for 3 days, and differentiation was induced by replacing the medium after cell confluency reached about 90%. For myoblast differentiation medium, HG-DMEM supplemented with 2% (v/v) horse serum (Gibco® Horse Serum, Thermo Fischer Scientific) and 1% PS was used. Adipose differentiation medium supplemented with 5% (v/v) FBS, 10 μM insulin (bovine pancreatic insulin, Sigma Aldrich), 1 μM dexamethasone (Sigma Aldrich), 10 μM ciglitizone (Sigma Aldrich) and 100 μM oleic acid (Sigma Aldrich) prepared LG-DMEM was used. Medium was changed every other day.

Cell differentiation assay

Immunostaining was performed to confirm myotube formation. First, the cells were fixed with 10% neutral buffered formalin and thoroughly washed. Then, 5% (w / v) bovine serum albumin (BSA, Sigma Aldrich), 0.3% (v / v) triton X-100 (Triton® X-100 solution, Sigma Aldrich) and 10% (v / v) Samples were treated overnight with horse serum in 1X PBS to inhibit non-specific antibody binding. MHC antibody (MF 20, DSHB) was diluted 100-fold in a dilution solvent composed of 10% (v/v) horse serum and 2% (w/v) BSA, and samples were treated at room temperature for 2 hours. The sample was washed twice with 1X PBS, then washed once with 0.025% (v/v) triton X-100, and the secondary antibody (Donkey anti-mouse Alexa flour 594, Thermo Fischer) was applied to the samples for 1 hour at room temperature. After washing, the samples were treated for 30 minutes to stain the cell nuclei of the scaffold using DAPI (Sigma Aldrich) diluted 250-fold in 1% (w/v) BSA solution. Then, the stained cells were observed using a confocal laser scanning microscope (LSM 980, Carl Zeiss).

Preparation Example 1. Preparation of gelatin/alginate hydrogel solution

에서 12시간 이상 용해시킨다. 알긴산나트륨(#180947)는 3차 증류수에 각각 4 wt%와 8 wt%가 되도록 정량하여 85

에서 12시간 용해시킨다. 미생물 트랜스글루타미나제(mTG)는 3차 증류수에 각각 2%, 4 wt%가 되도록 정량한 다음 실온에서 볼텍서로 충분히 섞어 mTG 용액을 준비한다. 젤라틴과 알지네이트가 모두 용해되면 두 용액을 1:1 부피 비율로 65 ℃에서 2시간 동안 혼합한다. 이후 mTG 용액을 37 ℃에서 10분 동안 젤라틴/알지네이트 혼합용액에 첨가하여 mTG의 활성이 저해되지 않는 온도 조건에서 두 용액이 충분히 섞이도록 하여, 젤라틴/알지네이트 하이드로겔 용액을 제조한다. Fish gelatin (GELTECH) was quantified to 40 wt% in tertiary distilled water and then 65

dissolved over 12 hours. Sodium alginate (#180947) was weighed to 4 wt% and 8 wt% in tertiary distilled water, respectively, to 85

dissolved for 12 hours. Microbial transglutaminase (mTG) was quantified to 2% and 4 wt% in tertiary distilled water, respectively, and then sufficiently mixed with a vortexer at room temperature to prepare an mTG solution. When both gelatin and alginate are dissolved, the two solutions are mixed in a 1:1 volume ratio at 65 °C for 2 hours. Thereafter, the mTG solution was added to the gelatin/alginate mixed solution at 37° C. for 10 minutes so that the two solutions were sufficiently mixed under a temperature condition in which mTG activity was not inhibited, thereby preparing a gelatin/alginate hydrogel solution.

Example 1. Gelatin/alginate hydrogel preparation and surface patterning

Referring to FIG. 4, the method for preparing the surface-patterned gelatin/alginate hydrogel will be described in detail.

(step 1) A PDMS stamp having a unidirectional pattern with a width of about 300 μm and a height of about 500 μm is fabricated.

에서 2 시간 동안 겔화시킨다. 그 후 겔화된 젤라틴/알지네이트 하이드로겔을 37

에서 12 시간 동안 인큐베이션 하여 젤라틴의 가교가 이루어지도록 한다. 가교 반응이 완료된 하이드로겔을 60

에서 30분간 두어 더 이상 젤라틴의 가교 반응이 진행되지 않도록 mTG를 비활성화 시킨 다음, 몰드에서 하이드로겔을 떼어내어 표면에 너비 약 300 μm, 높이 약 500 μm 의 단일 방향 패턴이 새겨진 젤라틴/알지네이트 하이드로겔을 제작한다. 그 후 염화칼슘 1 wt%, 3 wt% 용액에 각각 1시간 동안 담구어 알지네이트가 가교되도록 한다. (step 2) Place the PDMS stamp on the bottom of the silicon mold with the pattern facing upward, pour the gelatin/alginate hydrogel solution prepared in Preparation Example 1, and

gelled for 2 hours. Then, the gelatin/alginate hydrogel was prepared at 37 °C.

and incubated for 12 hours to cross-link the gelatin. The hydrogel after the cross-linking reaction is 60

for 30 minutes to inactivate mTG so that the crosslinking reaction of gelatin does not proceed any further, and then remove the hydrogel from the mold and form a gelatin/alginate hydrogel with a unidirectional pattern engraved on the surface of about 300 μm in width and about 500 μm in height. produce Then, the alginate is cross-linked by soaking in 1 wt% and 3 wt% calcium chloride solutions for 1 hour, respectively.

Example 2. Preparation of gelatin/alginate scaffold assemblies

Except for the patterning process in the surface-patterned gelatin/alginate scaffold prepared in Example 1, adipocytes were cultured in the flattened gelatin/alginate hydrogel having a smooth surface prepared in the same way in the silicone mold, and the patterned gelatin/alginate hydrogel Myoblasts are cultured and differentiated in alginate hydrogels. Subsequently, a flattened scaffold in which fat cells are proliferated and cultured and a patterned scaffold in which myoblasts are differentiated and cultured are assembled according to the shape to prepare cultured meat in which muscle cells and fat cells are fused.

Experimental Example 1. Flavor evaluation for patterned scaffold (muscle block) optimization

All samples (① Bare LALC, ② LALC+myoblast, ③ Bare HALC, ④ LALC+myoblast) were baked at 180 °C for 5 minutes, and then GC-MS analysis (Agilent 8890 GC system-Agilent 5677B MSD, Agilent Technologies) was performed. , confirmed the effect of cell myogenesis on the Maillard response. Cell growth affects the flavor of cultured meat by increasing the content of proteins that participate in the Maillard or Mailard reaction.

The results are shown in FIG. 10 . Benzaldehyde and butylated hydroxytoluene, which are Maillard reaction products found in cooked beef, were detected. Benzaldehyde showed a 1.8-fold higher peak area with cells compared to scaffolds without cells in the case of LALC hydrogels, but 5-fold higher peak areas with cells compared to scaffolds without cells in the case of HALC hydrogels. It can be seen that muscle generation was remarkably well achieved in the HALC hydrogel. In addition, dibutylhydroxytoluene was detected only on the scaffold with cells, and the peak area was 2.6 times higher in the HALC hydrogel than in the LALC hydrogel. These results also suggest that the degree of muscle formation can affect the content of Maillard reaction products and consequently have a significant impact on the flavor of cultured meat.

Based on the flavor information obtained from the Flavor Extract Manufacturers Association of the United States (FEMA) list and the Good Scents Company (TGSC) information system, specific flavors corresponding to each compound were identified. Benzaldehyde can counteract bitter almond, burnt sugar, and malt flavors, and dibutylhydroxytoluene has been found to have a toasted cereal-like flavor.

In addition, among muscle protein-related genes, the expression of MYH7 gene, known to enrich sensory characteristics such as tenderness, juiciness, and flavor of beef, was expressed more than twice as high in HALC hydrogel, maximizing fundamental differentiation according to the present invention to improve meat quality of cultured meat. It was confirmed that can be greatly improved (not shown).

Through the results of the above experimental examples, it was confirmed that the HALC hydrogel of the present invention can be optimized to a muscle block rich in muscle protein and appetizing flavor by promoting myogenic differentiation.

Experimental Example 2. Fat differentiation analysis for flattening scaffold (fat block) optimization

After 3 days of growth, the medium was replaced to initiate adipose differentiation. Upon initiation of adipogenesis, fibroblast-like adMSCs begin to change to a round shape and form abundant lipid droplets inside the cytoplasm. Lipid droplet formation was investigated on each scaffold by performing fluorescence staining of lipid droplets using LipidTOX® (HCS LipidTOX® Red Neutral Lipid Stain, Invitrogen) diluted 200-fold with Oil Red O staining kit (abcam). The results are shown in Figures 11 to 14. As shown in FIG. 14 (a), it can be confirmed that the stiffness increased as more adipocytes differentiated in the LALC hydrogel compared to the HALC hydrogel.

In addition, as shown in FIG. 14 (b), through GC analysis performed to confirm the fatty flavor in each sample, the LALC flavor peak area increased by 150% compared to the cell-free hydrogel, and in HALC, significant change was not observed. Through this, it was confirmed that LALC hydrogel can be optimized for fat block with rich lipid content and fat flavor.

Experimental Example 3. Manufacturing and characterization of cultured meat (ACM) assembled with muscle blocks and fat blocks

에서 1시간 동안 배양하여, 조립된 배양육(ACM)을 준비하였다. 이후 조립된 배양육과 실제 소고기의 영양, 식감 및 풍미를 비교하였다. 근육 조직과 지방 조직의 비율은 쇠고기의 절단 부위에 따라 다르므로 근육:지방 비율이 3:1인 쇠고기 부위를 비교군으로 사용하였다.In order to implement the structure of each beef part, assembled cultured meat was prepared by first attaching muscle blocks and fat blocks vertically at a ratio of 3:1. Assembled cultured meat (ACM) is prepared by spreading a solution of mTG (30% (w/v) mTG in distilled water) on each surface of the block, stacking the blocks vertically, and then wrapping the block tightly with PVC wrap.

Incubated for 1 hour to prepare assembled cultured meat (ACM). Then, the nutrition, texture, and flavor of the assembled cultured meat and the actual beef were compared. Since the ratio of muscle tissue to fat tissue differs depending on the cut part of beef, beef parts with a muscle:fat ratio of 3:1 were used as a comparison group.

Nutritional analysis was conducted by OATC INC, a Korea Laboratory Accreditation Scheme (KOLAS) certification institution. Samples were prepared by digesting ACM and beef with sulfuric acid to measure the protein content. Digested samples were distilled with 40% NaOH solution and titrated with 0.1N HCl solution. Protein content was calculated by the following equation.

[Equation 1]

Carbohydrate content was calculated by subtracting the contents of protein, ash, fat and moisture measured in the sample from the total mass of the sample. Fat content was measured with a flame ionization detector. The nutritional analysis results are shown in FIG. 15 .

Texture profile analysis (TPA) is TXA? It was performed on grilled ACM and grilled beef using a Multi-axis Micro-Texture analyzer (YEONJIN S-Tech Corporation). The results are shown in FIG. 17 . Compared with real beef, ACM showed a more elastic texture with higher cohesiveness, elasticity and elasticity. Also, the hardness, stickiness and chewiness of ACM were slightly lower than that of beef, indicating the softness of ACM.

Flavor analysis was prepared by baking at 180 ° C. for 10 minutes (minutes), and it was observed that the color of the two samples changed from red to tan due to the Maillard reaction. The flavor analysis results shown in FIG. 16 indicate that burnt flavor and roasted flavor derived from the Maillard reaction were detected in both groups. ACM detected high levels of volatile compounds with roast beef-like flavors such as butterscotch and caramel flavors. Fat flavor was also detected in all samples due to the rich lipid content of ACM and beef. These results indicate that ACM can provide a similar flavor to slaughtered beef with increased protein and lipid content.

What has been described above is only an example for carrying out the scaffold for producing cultured meat and the method for manufacturing cultured meat using the scaffold according to the present invention, but is not limited thereto, and does not deviate from the gist of the present invention as claimed in the following claims. Anyone with ordinary knowledge in the technical field to which the present invention belongs will say that the technical spirit of the present invention exists to the extent that various changes can be made.

Claims (25)

A scaffold for producing cultured meat, characterized in that it includes a biocompatible crosslinked polymer including a polypeptide and a polysaccharide, and includes a patterned surface in at least a part of the region. According to claim 1,
The patterned surface has a linear embossed pattern in one direction, a scaffold for producing cultured meat. According to claim 1,
The polypeptide is a scaffold for producing cultured meat comprising gelatin. According to claim 1,
The polypeptide comprises a RGD sequence in the main chain or side chain, scaffold for producing cultured meat. According to claim 1,
The polysaccharide comprises an anionic polysaccharide, a scaffold for producing cultured meat. According to claim 1,
The polypeptide is cross-linked by an enzyme, a scaffold for producing cultured meat. According to claim 6,
The enzyme comprises a transglutaminase, a scaffold for producing cultured meat. According to claim 1,
The polysaccharide is a scaffold for producing cultured meat, which is crosslinked by divalent metal ions. at least one patterned scaffold unit block comprising a biocompatible cross-linked polymer comprising a polypeptide and a polysaccharide; and
at least one planarized scaffold unit block comprising a biocompatible cross-linked polymer comprising a polypeptide and a polysaccharide;
The scaffold assembly for producing cultured meat, wherein the patterned scaffold unit block and the flattened scaffold unit block are assembled together to include a patterned first surface region and a flat second surface region at the same time. According to claim 9,
The scaffold assembly for producing cultured meat, wherein the surface of the patterned scaffold unit block has a linear embossed pattern in one direction. According to claim 9,
The scaffold assembly for producing cultured meat, wherein the patterned scaffold unit block and the flattened scaffold unit block contain the same polypeptide and polysaccharide. According to claim 9,
The scaffold assembly for producing cultured meat, wherein the patterned scaffold unit block and the flattened scaffold unit block have different compressive strengths. According to claim 9,
The patterned scaffold unit block has a compressive strength of 10 kPa to 18 kPa, scaffold assembly for producing cultured meat. According to claim 13,
The flattening scaffold unit block has a compressive strength of 1 kPa to 6 kPa, scaffold assembly for producing cultured meat. (S1) preparing a biocompatible polymer solution containing a polypeptide and a polysaccharide;
(S2) filling the biocompatible polymer solution into a mold having a pattern on its surface;
(S3) preparing a scaffold precursor by crosslinking the polypeptide in the mold;
(S4) obtaining a scaffold precursor having a patterned surface by separating the scaffold precursor from within the mold; and
(S5) cross-linking the polysaccharide included in the scaffold precursor including the patterned surface;
Scaffold manufacturing method for producing cultured meat comprising a. According to claim 15,
Prior to the step (S3), gelling the biocompatible polymer solution filled in the mold at a low temperature; further comprising a scaffold manufacturing method for preparing cultured meat. According to claim 15,
The step (S5) is performed in an aqueous solution containing divalent metal ions, a scaffold manufacturing method for producing cultured meat. According to claim 15,
After the step (S5), (S6) cutting the scaffold to prepare patterned scaffold unit blocks that can be assembled with each other; further comprising a scaffold manufacturing method for producing cultured meat. According to claim 15,
(S7) preparing a flattened scaffold unit block; and
(S8) assembling the patterned scaffold unit block and the flattened scaffold unit block to manufacture an assembly including a patterned first surface region and a flat second surface region at the same time;
A scaffold manufacturing method for producing cultured meat further comprising a. According to claim 19,
The flattening scaffold unit block comprises a biocompatible cross-linked polymer of a polypeptide and a polysaccharide, scaffold manufacturing method for producing cultured meat. (S10) filling the culture medium into the scaffold assembly for preparing cultured meat according to any one of claims 9 to 14 and inoculating animal cells;
(S20) incubating the culture solution inoculated with the animal cells to proliferate the cells; and
(S30) organizing the proliferated cells in the culture medium;
Cultured meat manufacturing method comprising a. According to claim 21,
Wherein the animal cells include animal myoblasts and animal adipocytes. According to claim 21,
A method for producing cultured meat, wherein animal myoblasts are inoculated into the patterned scaffold unit block, and animal adipocytes are inoculated into the flattened scaffold unit block. According to claim 21,
The cultured meat manufacturing method of controlling the ratio of muscle cells and fat cells by adjusting the ratio of the area of the patterned scaffold and the area of the flattened scaffold. Cultured meat prepared by the manufacturing method according to any one of claims 21 to 24.

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