Porous scaffold for cell culture and production method therefor

Abstract

To provide a porous scaffold for cell culture that does not deteriorate even when subjected to sterilization treatment such as autoclaving and enables culture of various cells, and a production method therefor.SOLUTION: The present invention relates to a porous scaffold for cell culture that contains cross-linked aggregates of gelatin and xanthan gum or an analog thereof and does not change in shape during heat sterilization, and a production method for the porous scaffold for cell culture, wherein this production method includes: a mixing and foaming step for mixing gelatin and xanthan gum or an analog thereof, thus foaming the same; and a cross-linking step for cross-linking gelatin and xanthan gum or the analog thereof in the resulting foamed body.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a porous scaffold for cell culture and a method for producing the same, and further to a food composition having edible cultured cells attached thereto on a porous scaffold for cell culture and a method for producing the same. [Background technology]

[0002] Biological tissues are generally composed of cells and the extracellular matrix produced by the cells themselves. The extracellular matrix exists in a manner that surrounds the cells, and its main components are structural proteins (collagen, elastin, keratin, etc.), glycosaminoglycans (hyaluronic acid, chondroitin sulfate, etc.), and cell adhesion molecules (fibronectin, laminin, fibrinogen, etc.). The extracellular matrix plays an important role as a scaffold for cell activities such as cell adhesion, proliferation, and differentiation.

[0003] In cell culture, a scaffold that replaces the extracellular matrix is ​​also required as a structural support and for cell adhesion, proliferation, differentiation, etc. Scaffolds for cell culture are required to activate cell activity, provide sufficient nutrients to cells, and discharge waste products, so they are preferably in the form of a porous body or a highly hydrous gel. Furthermore, biodegradable scaffolds have also been attracting attention as scaffolds for regenerative medicine and regenerated tissues, which have been actively researched in recent years.

[0004] The biodegradable scaffolds that are mainly used include synthetic polymer materials and natural polymer materials. Examples of synthetic polymer materials include polyglycolic acid (PGA), polylactic acid (PLA), copolymers of polyglycolic acid and polylactic acid (PLGA), polyethylene glycol (PEG), polycaprolactone, and the like. Examples of natural polymer materials include components of extracellular matrices, specifically collagen, gelatin, alginic acid, hyaluronic acid, agarose, chitosan, fibrin, fibroin, and the like. Synthetic polymer materials generally have high mechanical strength and excellent function as structural supports, but have a problem of low affinity with cells. Natural polymer materials generally have high cell affinity, but have a problem of low mechanical strength and easy deformation (Non-Patent Document 1). Thus, both synthetic polymer materials and natural polymer materials have problems with either cell adhesiveness or mechanical strength.

[0005] Collagen adhesion proteins such as integrins are expressed on the surface of many cells, and collagen has very strong adhesive properties for various cells. Therefore, scaffolds using natural polymeric materials such as collagen that mimic the extracellular matrix have attracted attention. However, as mentioned above, natural polymeric materials have problems such as low mechanical strength, easy deformation, and shape change during heat sterilization. Therefore, there has been a demand for scaffolds that have the properties of natural polymeric materials, have improved mechanical strength, and do not change shape during heat sterilization.

[0006] Patent Document 1 describes a scaffold material for tissue regeneration consisting of a water-insoluble hydrogel in which collagen, gelatin or other protein molecules are crosslinked with (-)-epigallocatechin gallate or other polyphenols, or a sponge obtained by drying this. Patent Document 2 describes a scaffold for vascular endothelial cell migration that contains genetically modified gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen. However, there is no description as to whether the scaffolds of Patent Documents 1 and 2 can be cultured with various cells without deterioration even when sterilized by autoclaving or the like. Furthermore, Patent Documents 1 and 2 do not describe at all a scaffold combining gelatin and xanthan gum or the like. As shown in the test of Comparative Example 2 described below, the adhesion and proliferation of cells was insufficient in a scaffold containing only gelatin.

[0007] Non-Patent Documents 2 and 3 describe the production of porous scaffolds stabilized with a crosslinking agent such as glutaraldehyde from gelatin using the air bubble method. However, the present inventors conducted follow-up tests but were unable to produce these stable porous scaffolds. Furthermore, the scaffolds of Non-Patent Documents 2 and 3 have a denaturation temperature of 73.5 to 87.6°C (Table 1 of Non-Patent Document 2, Table 2 of Non-Patent Document 3), and deteriorate when subjected to sterilization such as autoclaving. Non-Patent Documents 2 and 3 do not mention at all a scaffold combining gelatin and xanthan gum or the like. As shown in the test of Comparative Example 2 described later, the adhesion and proliferation of cells was insufficient in a scaffold containing only gelatin.

[0008] Furthermore, in recent years, with the increase in the world population, there has been much discussion about the world's food supply and demand. For example, the world population in 2050 is expected to reach approximately 8.64 billion people, an increase of approximately 1.3 times compared to 2010, and the world's agricultural land area in 2050 is predicted to increase by approximately 70 million hectares to approximately 1.61 billion hectares due to an increase in average temperature of approximately 2°C caused by global warming. In this way, the projected rate of increase in the world's population exceeds the projected rate of increase in the world's agricultural land area.

[0009] Therefore, the development of new foods, for example, new foods that utilize other means such as culture technology, is expected. For example, cultured meat is a food produced from cell culture using tissue engineering technology, and research and development has been progressing in various laboratories, universities, and companies in recent years. Cultured meat technology has a high food production efficiency compared to livestock farming, which requires large amounts of feed consumed by animals and the vast farmland required for that. Cultured meat technology can eliminate the effects of diseases such as infectious diseases in animals that can be a problem in livestock farming, and the effects of drugs such as antibiotics administered to animals. In addition, by producing it in a sterile environment, the effects of bacteria and viruses that may be contaminated by livestock foods can be eliminated.

[0010] Patent Document 3 describes an edible composition comprising a three-dimensional porous scaffold, myotubes containing myotube nuclei, and a plurality of cell types. Examples of three-dimensional porous scaffolds include textured proteins, non-textured proteins, polysaccharides, and the like, and textured soybean protein is used in the examples. However, when the present inventors cultured cells using soybean protein, the cultured cells did not grow well, and a food composition containing cultured cells could not be produced.

[0011] Patent Document 4 describes an edible dehydrated food containing cultured animal muscle cells combined with a plant-derived hydrogel. As the plant-derived hydrogel, an edible microcarrier having cultured cells grown on its surface can be used, and as the edible microcarrier, polysaccharides such as pectin and polypeptides such as chalcidone are described. However, no specific examples are given. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] JP 2008-125916 A [Patent Document 2] JP 2013-074936 A [Patent Document 3] Special Publication No. 2020-527054 [Patent Document 4] Special Publication No. 2017-505138 [Non-patent literature]

[0013] [Non-Patent Document 1] Materials Science, 2014, Vol. 63, No. 9, pp. 684-689 [Non-Patent Document 2] Materials Science and Engineering C, 48 (2015) 63-70 [Non-Patent Document 3] Materials Science and Engineering C, 63 (2016) 1-9 Summary of the Invention [Problem to be solved by the invention]

[0014] The present invention aims to provide a porous scaffold for cell culture that is not deteriorated even when subjected to sterilization treatment such as autoclaving and that can culture various cells, and a method for producing the same. Further, the present invention aims to provide a food composition in which edible cultured cells are attached to the porous scaffold for cell culture, and a method for producing the same. Another object of the present invention is to provide a scaffold that allows suitable proliferation of cell lines that show poor proliferation in known scaffolds made of PET resin (e.g., Comparative Example 1) and porous scaffolds based on gelatin fiber (e.g., Comparative Example 2). [Means for solving the problem]

[0015] As a result of intensive research aimed at solving the above-mentioned problems of the present invention, the inventors of the present application have unexpectedly discovered that a porous scaffold for cell culture containing a crosslinked aggregate prepared by mixing and crosslinking gelatin and xanthan gum has significantly improved mechanical strength, which is lacking in collagen or gelatin, while preserving the properties of collagen or gelatin such as cell adhesion, proliferation, and differentiation, does not change shape, does not deteriorate even when subjected to sterilization treatment such as autoclaving, and can culture a variety of cells, thereby completing the present invention.

[0016] That is, the present invention is as follows. [1] A porous scaffold for cell culture that contains cross-linked aggregates of gelatin and xanthan gum or its analogues and does not change shape when sterilized by heat. [2] The porous scaffold for cell culture described in [1], wherein the crosslinked aggregate further contains insoluble cellulose. [3] The porous scaffold for cell culture described in [2], wherein the insoluble cellulose is fermented cellulose, citrus fiber or microcrystalline cellulose. [4] A method for producing a porous scaffold for cell culture, comprising: A mixing and foaming step of mixing gelatin with xanthan gum or its analogue and foaming the mixture; and The method includes a crosslinking step of crosslinking the gelatin and the xanthan gum or its analogue in the obtained foam.

[0017] [5] The manufacturing method according to [4], further comprising a drying step of drying the product obtained in the crosslinking step. [6] A manufacturing method according to [4] or [5], in which in the mixing and foaming step, insoluble cellulose is further added to the gelatin and xanthan gum or a derivative thereof, and the mixture is foamed. [7] The method according to any one of [4] to [6], wherein in the crosslinking step, crosslinking is carried out by enzyme treatment and / or heat treatment. [8] A porous scaffold for cell culture produced by the manufacturing method described in any one of [4] to [7]. [9] A food composition comprising edible cultured cells attached to the porous scaffold for cell culture described in any one of [1] to [3] and [8].

[10] A method for producing a food composition in which edible cultured cells are attached to a porous scaffold for cell culture described in any one of [1] to [3] and [8], the method comprising a culture step of culturing edible cells together with the porous scaffold for cell culture.

[11] The manufacturing method described in

[10] , further comprising a differentiation step of differentiating edible cultured cells. Effect of the Invention

[0018] The present invention provides an edible porous scaffold for cell culture that has significantly improved mechanical strength, does not change shape, is not deteriorated even when subjected to sterilization treatment such as autoclaving, is capable of culturing various cells, and is capable of producing the same. Furthermore, a food composition in which edible cultured cells are attached to the porous scaffold for cell culture and a method for producing the same are prepared. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the evaluation of adhesiveness and proliferation of a porous scaffold for cell culture using HEK293T cells in Test Example 1. [Diagram 2] FIG. 1 shows the evaluation of adhesiveness and proliferation of a porous scaffold for cell culture using duck liver-derived primary cells in Test Example 2. [Diagram 3] FIG. 13 is a diagram showing the evaluation of adhesiveness and proliferation of a porous scaffold for cell culture using duck liver-derived primary cells in Test Example 3. [Figure 4] FIG. 13 is a diagram showing the evaluation of adhesiveness and proliferation of a porous scaffold for cell culture using duck liver-derived primary cells in Test Example 3. [Diagram 5] FIG. 13 is a diagram showing the evaluation of adhesiveness and proliferation of a porous scaffold for cell culture using chicken muscle-derived primary cells in Test Example 4. [Figure 6] FIG. 13 is a diagram showing the evaluation of adhesiveness and proliferation of a porous scaffold for cell culture using chicken muscle-derived primary cells in Test Example 4. [Figure 7] FIG. 13 is a diagram showing the evaluation of adhesiveness and proliferation of a porous scaffold for cell culture using duck liver-derived primary cells in Test Example 5. [Figure 8] FIG. 13 shows an evaluation of the adhesion and proliferation of RL34 cells (rat-derived liver epithelial cell line) using the PET resin scaffold of Comparative Example 1, the gelatin fiber-based porous scaffold of Comparative Example 2, and the porous scaffold for cell culture of Example 5 in Test Example 6. [Figure 9] FIG. 13 shows the evaluation of adhesiveness and proliferation of the porous scaffold for cell culture of Example 5 using various established cell lines and bovine-derived primary cells in Test Example 6. [Figure 10] FIG. 13 shows the evaluation of adhesiveness and proliferation of the porous scaffold for cell culture of Example 5 using various established cell lines and bovine-derived primary cells in Test Example 6. [Figure 11] FIG. 13 is a diagram showing the shape of a porous scaffold obtained after the grinding process in Test Example 7. [Figure 12] FIG. 13 is a diagram showing an evaluation of the shape comparison of porous scaffolds for cell culture using duck liver-derived primary cells in Test Example 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] 1. Porous scaffolds for cell culture The present invention relates to a porous scaffold for cell culture, which contains a crosslinked aggregate of gelatin and xanthan gum or its analogue and does not change shape when heat sterilized. The crosslinked aggregate prepared by mixing and crosslinking gelatin and xanthan gum, etc., is a crosslinked aggregate in which the gelatin and xanthan gum, etc. associate with each other while electrostatic interactions (ionic bonds, hydrogen bonds) act between the gelatin and xanthan gum, etc., and the mesh-structured polymer networks formed are entangled with each other. It is believed that this greatly improves the mechanical strength of the porous scaffold for cell culture.

[0021] <Gelatin> The gelatin used in the present invention may be gelatin obtained by, for example, extracting collagen from collagen-containing raw materials such as bones and skins of mammals such as cows and pigs, and bones, skins, and scales of fish such as sharks and tilapia with hot water, and then hydrolyzing the collagen by alkali treatment, acid treatment, enzyme treatment, or the like. Examples of the alkali include sodium hydroxide and calcium hydroxide. Examples of the acid include hydrochloric acid, sulfuric acid, and nitric acid. The enzyme may be any enzyme that has the function of cleaving the peptide bonds of gelatin. Generally, it is an enzyme called a proteolytic enzyme or protease. Specific examples of the enzyme include collagenase, thiol protease, serine protease, acid protease, alkaline protease, and metal protease, and these may be used alone or in combination. Examples of the thiol protease include chymopapain, papain, bromelain, and ficin derived from plants, cathepsin derived from animals, and calcium-dependent protease. Examples of the serine protease include trypsin and cathepsin D. Examples of the acidic protease include pepsin and chymosin.

[0022] Gelatin that can be used in the present invention includes gelatin hydrolyzed by acid treatment or alkali treatment, gelatin hydrolyzed by enzyme treatment, etc. Any gelatin may be used, but since it is desirable to positively charge gelatin in the present invention, it is necessary to consider the isoelectric point (pI value) of each type of gelatin. For example, gelatin hydrolyzed by acid treatment or enzyme treatment has a pI value of 7 to 9, which is close to that of collagen, compared with gelatin hydrolyzed by base treatment, because deamidation progresses less during hydrolysis. The pI value of alkali-treated gelatin is 5. Since gelatin becomes positively charged by adjusting the pH to a value lower than these pI values, gelatin hydrolyzed by acid treatment is more preferably used, since it is easy to use in the weak acidic range (pH 5 to 6.5) where enzymes are easily functional, in consideration of the fact that enzyme treatment may be performed in the crosslinking step between gelatin and xanthan gum. By appropriately adjusting the pH during the manufacturing process, the electrostatic interaction with xanthan gum or its analogues becomes stronger, which contributes to the mechanical strength and hardness of the porous scaffold for cell culture of the present invention.

[0023] The weight-average molecular weight of gelatin that can be used in the present invention is, for example, about 5000 to about 20000, and preferably, for example, about 8000 to about 15000, about 10000 to about 17000, about 12000 to about 18000, about 15000 to about 20000, etc. By selecting the weight-average molecular weight of gelatin or the type of raw material for gelatin, the mechanical strength and hardness of the porous scaffold for cell culture of the present invention can be adjusted to a desired range.

[0024] <Xanthan gum or its derivatives> The xanthan gum used in the present invention is a polysaccharide produced extracellularly by Xanthomonas campestris. For example, depending on the type of salt used in the manufacturing process of xanthan gum, xanthan gum in the form of a potassium salt, a sodium salt, a calcium salt, or the like can be used. The preferred weight-average molecular weight of xanthan gum is, for example, about 2 million to about 50 million. Xanthan gum is usually composed of repeating units of two glucose molecules, two mannose molecules, and glucuronic acid. Since xanthan gum has an acidic group, it has a strong electrostatic interaction with gelatin, particularly with gelatin hydrolyzed by acid treatment or enzyme treatment having an isoionic point of pH 7 to 9, and contributes to the mechanical strength and hardness of the porous scaffold for cell culture of the present invention. Examples of analogues of xanthan gum include derivatives of xanthan gum that can form aggregates with gelatin in the same way as xanthan gum. Examples of xanthan gum derivatives include deacetylated xanthan gum, low-acetylated xanthan gum, low-pyruvic acid xanthan gum, cationized xanthan gum, crosslinked xanthan gum, and acid-treated xanthan gum.

[0025] The content of xanthan gum or an analogue thereof in the porous scaffold for cell culture of the present invention is, for example, about 1 to about 15% by mass, preferably about 2 to about 10% by mass, and more preferably about 3 to about 8% by mass, relative to the gelatin. When gellan gum or sodium alginate was used instead of xanthan gum etc., the mechanical strength and hardness of the obtained porous scaffold for cell culture were not sufficiently improved.

[0026] <Insoluble cellulose> The crosslinked aggregate in the porous scaffold for cell culture of the present invention may further contain insoluble cellulose to the extent that the effect of the porous scaffold for cell culture of the present invention is not inhibited. Examples of insoluble cellulose include fermented cellulose, citrus fiber, microcrystalline cellulose, and mixtures thereof. Examples of preferred insoluble cellulose include fermented cellulose, citrus fiber, and microcrystalline cellulose. By including insoluble cellulose such as fermented cellulose, the bubbles prepared in the production of the porous scaffold for cell culture of the present invention can be further stabilized, and the mechanical strength of the porous scaffold for cell culture is further improved. The improvement in mechanical strength is believed to be due to the fact that the polymer networks generated by the association of gelatin and xanthan gum, etc., are mutually entangled and further entangled with the insoluble cellulose.

[0027] The content of insoluble cellulose varies depending on the type of insoluble cellulose, but may be, for example, about 0.1 to about 30% by mass, preferably about 0.2 to about 20% by mass, and more preferably about 0.3 to about 10% by mass, relative to gelatin. When the insoluble cellulose is fermented cellulose, the content of insoluble cellulose may be, for example, about 0.1 to about 15% by mass, preferably about 0.2 to about 12% by mass, and more preferably about 0.4 to about 10% by mass, relative to gelatin.

[0028] <Other ingredients> The porous scaffold for cell culture of the present invention may further contain other components within a range that does not inhibit the effect of the porous scaffold for cell culture of the present invention. Examples of other components include thickening polysaccharides, protein hydrolysates, and the like. Examples of thickening polysaccharides include deacylated gellan gum (and alkaline earth metal salts), white wood ear polysaccharides, agar, mannan, hyaluronic acid, carrageenan, sodium carboxymethylcellulose (CMC-Na), modified cellulose such as HPMC, and mixtures thereof. Examples of protein hydrolysates include wheat protein hydrolysates, soy protein hydrolysates, collagen peptides, and mixtures thereof. The content of thickening polysaccharides or protein hydrolysates varies depending on the type of thickening polysaccharide, but may be, for example, about 0.1 to about 30% by mass, preferably about 0.2 to about 20% by mass, and more preferably about 0.3 to about 10% by mass, based on gelatin.

[0029] <Coating agent> The porous scaffold for cell culture of the present invention can also be coated with a coating agent. When coated with a coating agent, it is preferable that the cultured cells and the porous scaffold are attached via the coating agent. Examples of the coating agent include coating agents consisting of polypeptides or proteins such as polylysine, RGD peptide-containing polypeptides, fibronectin, laminin, and fibrinogen, hyaluronic acid, chitin, chitosan, chondroitin sulfate, and mixtures thereof. Preferably, polylysine and the like are used. By coating with a coating agent, it is possible to adjust the adhesion of cells to the porous scaffold of edible polysaccharides and promote cell proliferation. The content of the coating agent is, for example, about 1 to 50% by mass, preferably about 2 to 20% by mass, and more preferably about 3 to 10% by mass, relative to the porous scaffold for cell culture.

[0030] In the porous scaffold for cell culture of the present invention, the components are crosslinked to each other, preferably by enzyme treatment and / or heat treatment. The enzyme treatment and heat treatment will be described later. The porous scaffold for cell culture of the present invention is characterized by having a porous structure, and the pore size is, for example, about 10 μm to about 1 mm.

[0031] In the present invention, "the shape does not change during heat sterilization" means that the shape of the porous scaffold for cell culture does not change when heat sterilized by high pressure steam sterilization (autoclave). Specifically, it means that the scaffold structure does not dissolve and the porous structure does not collapse by "sterilization in an autoclave at 120°C for 20 minutes", which is the condition used in the examples.

[0032] <Shape of the porous scaffold for cell culture of the present invention> The prepared porous scaffold for cell culture of the present invention is preferably molded or cut into a shape suitable for cell culture. Examples of the shape include rectangular, spherical, cylindrical, beads, fine powder, etc. The size includes a size suitable for the culture device to be used, and the major axis is, for example, 0.5 to 20 mm, preferably 1 to 10 mm.

[0033] The porous scaffold for cell culture of the present invention has the characteristic that it can maintain a certain structure even if it is pulverized. In order to increase the specific surface area of ​​the scaffold, it can be appropriately pulverized and processed into fine powder. The particle size of the scaffold after pulverization can be, for example, 20 to 50 μm, 50 to 100 μm, or 100 μm to 2 mm. Furthermore, the effects of the present invention can be achieved even with a scaffold that has the same composition as the porous scaffold for cell culture of the present invention and is processed into a fine powder without being processed into a porous structure.

[0034] 2. Method for producing porous scaffolds for cell culture The porous scaffold for cell culture of the present invention comprises: A mixing and foaming step of mixing gelatin with xanthan gum or its analogue and foaming the mixture; and The foam can be produced by a method including a crosslinking step of crosslinking gelatin with xanthan gum or a derivative thereof in the obtained foam.

[0035] <Mixing foaming process> In the mixing and foaming step, gelatin and xanthan gum or its analogues are mixed in water. Specifically, a method of preparing an aqueous solution of gelatin and an aqueous solution of xanthan gum, etc., and then mixing the two aqueous solutions, a method of preparing an aqueous solution of xanthan gum, etc., and then adding gelatin to this aqueous solution to dissolve it, and a method of mixing gelatin and xanthan gum, etc. in powder form and then dissolving them in water are possible. Xanthan gum is very easy to hydrate, so it is prone to clumping when dissolved in water. In addition, in order to avoid the aggregation of gelatin and xanthan gum, etc. before dissolving in water, a method of preparing an aqueous solution of gelatin and an aqueous solution of xanthan gum, etc., respectively, and then mixing the two aqueous solutions is preferable. In addition, insoluble cellulose, other components, etc. may be further mixed into the aqueous solution of gelatin and xanthan gum or its analogue. For example, by including insoluble cellulose such as fermented cellulose, the bubbles can be more stabilized, and the mechanical strength of the porous scaffold for cell culture is further improved.

[0036] The concentration of gelatin in water is, for example, about 1 to about 10% by mass, preferably about 2 to about 8% by mass, and more preferably about 3 to about 7% by mass. The amounts of xanthan gum or its analog, acid, and thickening polysaccharide to be added are as described above. The mixing temperature is, for example, about 40 to about 95°C, preferably about 60 to about 90°C, and more preferably about 70 to about 85°C.

[0037] More preferably, an acid is added in the mixing and foaming step to adjust the pH to below the isoelectric point of gelatin (pI value of 7 to 9 for acid-treated gelatin and enzyme-treated gelatin, pI value of 5 for alkali-treated gelatin). Although not limited to the following mechanism, by adding an acid during the production of the scaffold and lowering the pH to below the isoelectric point of gelatin, the gelatin can be positively charged and can strongly interact electrostatically with the negatively charged xanthan gum. In this state, the crosslinking between the gelatin and xanthan gum is promoted by further performing the crosslinking step of the following enzyme treatment and / or heat treatment, and the mechanical strength of the porous scaffold for cell culture is improved.

[0038] Examples of the acid include citric acid, oxalic acid, maleic acid, malic acid, hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, etc. Preferred examples of the acid include citric acid, oxalic acid, maleic acid, malic acid, hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, etc., and more preferably citric acid, etc. The content of the acid varies depending on the type and content of the constituent components of the porous scaffold for cell culture, and is, for example, about 0.1 to about 2% by mass, preferably about 0.2 to about 1.5% by mass, and more preferably about 0.4 to about 1% by mass, based on gelatin. The pH of the scaffold material after the addition of the acid is, for example, pH 3 to 6, and preferably pH 3 to 5.

[0039] The mixture is then foamed to prepare a foam. The foaming means is not particularly limited, and any means may be used as long as it can form bubbles of, for example, about 10 μm to about 1 mm. For example, a mixer or the like may be used to vigorously stir the mixture so as to incorporate air. A foam can also be prepared by blowing bubbles generated using a bubble generator such as a microvalve. A foam can also be prepared by blowing bubbles generated using a bubble generator such as a microvalve.

[0040] <Crosslinking process> The gelatin and xanthan gum or its analogue are crosslinked in the resulting foam by a desired method, such as an enzyme treatment and / or a heat treatment. Examples of the enzyme that can be used in the enzyme treatment include transglutaminase, laccase, peroxidase, lysyl oxidase, protein disulfide isomerase, protein disulfide reductase, sulfhydryl oxidase, lipoxygenase, and polyphenol oxidase (tyrosinase), and preferably transglutaminase. The enzyme treatment can be carried out by a conventional method for each enzyme used. When the enzyme is transglutaminase, the enzyme reaction can be carried out at 40°C for 1 hour. In addition, the pH is adjusted to, for example, pH 4 to 7, taking into consideration the optimal reaction pH of the enzyme used in the enzyme treatment. In addition, when salts are required to stably act on the enzyme, salts can be added as necessary. The enzyme treatment promotes the insolubilization of gelatin, and improves the shape retention of the porous scaffold for cell culture.

[0041] When the thickening polysaccharide is one that is gelled by the addition of an alkaline earth metal salt, the alkaline earth metal salt is added in the crosslinking step to cause gelation. Furthermore, when crosslinking is strengthened by heat treatment, the temperature is not particularly limited, but may be, for example, about 150 to about 210° C., preferably about 160 to about 200° C., for about 1 to 10 minutes. Crosslinking by heat treatment greatly improves the mechanical strength of the porous scaffold for cell culture, and enables it to withstand autoclave sterilization.

[0042] <Drying process> Although the product obtained in the crosslinking step can be used as a scaffold for cell culture, it may be better to dry the product in consideration of the labor of transportation, etc. In this case, a drying step may be performed following the crosslinking step. Examples of drying steps that can be used in the present invention include dry heat drying, vacuum drying, and freeze drying.

[0043] Furthermore, if it is desired to sterilize the scaffold (e.g., by autoclaving) before use in cell culture, it is desirable to further strengthen the crosslinking between the gelatin and the xanthan gum or its analogues, in which case the product is preferably treated by dry heat drying. The dry heat drying is not particularly limited, but can be carried out, for example, under normal pressure at about 40 to about 90° C., preferably about 50 to about 70° C. until dry. By carrying out the drying, a sponge-like porous product can be obtained.

[0044] Furthermore, by further carrying out a heat treatment following the drying step, crosslinking between gelatin and xanthan gum or its analogues can be strengthened. The heat treatment is not particularly limited, but can be carried out, for example, at about 150 to about 210°C, preferably about 160 to about 200°C, for about 1 to 10 minutes. Crosslinking by the heat treatment greatly improves the mechanical strength of the porous scaffold for cell culture.

[0045] The obtained porous scaffold for cell culture is molded or cut into a scaffold having a shape to be used for cell culture. The shape is as described above. If necessary, the porous scaffold for cell culture can also be coated with a coating agent.

[0046] <Crushing process> As described above, the porous scaffold for cell culture of the present invention may be subjected to a pulverization process as appropriate in order to improve the specific surface area. Specifically, the scaffold can be easily prepared by pulverizing, cutting, and sieving the scaffold with a cutting device and sieving the scaffold with a mesh.

[0047] The produced porous scaffold for cell culture can be sterilized before use. The sterilization method is not particularly limited, but can be, for example, ethylene oxide gas sterilization, radiation sterilization, high pressure steam sterilization (autoclave), dry sterilization, etc.

[0048] 3. Food Composition The food composition of the present invention is a food composition in which edible cultured cells are attached onto the porous scaffold for cell culture described above. Since the components of the porous scaffold for cell culture of the present invention are edible, a food composition having edible cultured cells attached to the porous scaffold for cell culture can be ingested as a food.

[0049] <Edible cells> The edible cells to be cultured are generally cells of animals other than humans. For example, the cells may be cells of mammals such as cows, pigs, sheep, goats, deer, wild boars, horses, rabbits, bears, whales, etc.; birds such as chickens, ducks, turkeys, geese, ostriches, pheasants, pigeons, etc.; reptiles such as crocodiles and alligators; tuna, salmon, mackerel, sardines, anchovies, sea bass, catfish, carp, cod, eels, flounders, pufferfish, groupers, halibut, herring, These include fish such as dolphin fish, swordfish, bream, pike, shark, snapper, flounder, swordfish, tilapia, and trout; crustaceans such as crabs, crayfish, lobsters, shrimp, and prawns; mollusks such as abalone, clams, oysters, scallops, and snails; cephalopods such as cuttlefish, octopus, and squid, and insects such as crickets, grasshoppers, and mealworms (mealworm larvae).

[0050] The edible cultured cells may be cells of various organs and tissues of the above animals. For example, the organs and tissues include skeletal muscle, smooth muscle, heart, liver, kidney, stomach, intestine, etc. Specific examples include myoblasts, satellite cells, adipocytes, endothelial cells, hepatic parenchymal cells, non-parenchymal hepatic cells, kidney cells, etc. Note that the cultured cells further differentiated, such as myofibril, are also included in the cultured cells of the present invention. The food composition of the present invention can be produced by the production method described below.

[0051] 4. Method for producing food composition The food composition of the present invention can be produced by a production method including a culture step of culturing edible cells together with the above-mentioned porous scaffold for cell culture.

[0052] <Culture process> The cells are cultured by a commonly used culture method suitable for each cell. The cells are cultured in a suitable medium together with a porous scaffold for cell culture. As the suitable medium, it is preferable to select a medium suitable for the cells to be used. The cultured cells can also be further differentiated, for example into myofibrils.

[0053] The mixture produced by culturing the cells together with the porous scaffold for cell culture can be used as a food composition, optionally after removing the medium and washing, or it can be pelleted by centrifugation or the like and washed by rinsing. A gelling agent, thickening agent, etc. can be added to the resulting mixture, and the mixture can be processed and shaped into a paste or gel to form a food composition, or the mixture can be heated or frozen to form a food composition. EXAMPLES

[0054] The present invention will be described below with reference to examples, comparative examples, test examples, etc., but the present invention is not limited to these examples.

[0055] Examples 1 to 19 Preparation of porous scaffolds for cell culture While stirring at 80°C, gelatin in the amount shown in Tables 1 to 3 was stirred and dissolved in 50 parts by weight of ion-exchanged water. While stirring at 80°C, xanthan gum and thickening polysaccharides (except calcium chloride) in the amounts shown in Tables 1 to 3, and optionally insoluble cellulose and other components, were stirred and dissolved in 50 parts by weight of ion-exchanged water. Next, while stirring the prepared aqueous solution of xanthan gum and thickening polysaccharides, etc., the dissolved gelatin aqueous solution was added thereto, and further, acid was added to adjust the final concentration after mixing of each solution to the concentration shown in Tables 1 to 3, and stirred. Next, the obtained composition was foamed with a hand mixer. A solution in which the enzyme and, in Example 3, calcium chloride were dissolved in a small amount of ion-exchanged water was added to the foamed composition and mixed with a hand mixer until it was uniform. The mixture was added to a stainless steel pad to a thickness of about 1 cm and molded. The stainless steel pad was placed in a constant temperature bath at 40°C and kept warm for 1 hour. The mixture was dried in a constant temperature bath at 60°C for about 15 hours. The scaffold was then heated in an oven at 180° C. for 7 minutes, and cut into pieces measuring 5 mm×5 mm to prepare porous scaffolds for cell culture.

[0056] In Test Examples 1 to 4, the plates were immersed in a 0.5% by mass aqueous calcium chloride solution and sterilized in an autoclave at 120° C. for 20 minutes before use in cell culture. In Test Examples 5 and 6, the plates were immersed in ion-exchanged water and sterilized in an autoclave at 121° C. for 20 minutes before use in cell culture. Even after autoclaving, the mechanical strength and porous structure (pore size: about 100 to about 1000 μm) were maintained. Furthermore, even when ion-exchanged water was added in an amount 40 times the dry mass of the porous scaffold for cell culture, the structure of the porous scaffold for cell culture was maintained.

[0057] [Table 1]

[0058] [Table 2]

[0059] [Table 3]

[0060] The ingredients listed in Tables 1 to 3 are the following types of gelatin used. Gelatin: SunSupport (registered trademark) P-203 (pork-derived, acid-treated) (manufactured by San-Ei Gen F.F.I. Co., Ltd.), SunSupport (registered trademark) P-204 (pork-derived, acid-treated) (manufactured by San-Ei Gen F.F.I. Co., Ltd.), SunSupport (registered trademark) P-205 (fish-derived, acid-treated) (manufactured by San-Ei Gen F.F.I. Co., Ltd.) and SunSupport (registered trademark) P-206 (bovine-derived, acid-treated) (manufactured by San-Ei Gen F.F.I. Co., Ltd.) Xanthan gum: Sun Support (registered trademark) P-207 (manufactured by San-Ei Gen F.F.I. Co., Ltd.) Fermented cellulose preparation: Sansupport (registered trademark) P-212 (manufactured by San-Ei Gen F.F.I. Co., Ltd.) (manufactured by San-Ei Gen F.F.I. Co., Ltd.: contains 50.0% fermented cellulose, 33.0% xanthan gum, and 17.0% CMC-Na) Sodium alginate: Sun Support (registered trademark) P-208 (manufactured by San-Ei Gen F.F.I. Co., Ltd.) Soybean polysaccharide: Sun Support (registered trademark) P-214 (manufactured by San-Ei Gen F.F.I. Co., Ltd.) Mannan: Sun Support (registered trademark) P-209 (manufactured by San-Ei Gen F.F.I. Co., Ltd.) Agar: Sansupport (registered trademark) P-210 (manufactured by San-Ei Gen F.F.I. Co., Ltd.) Wheat protein hydrolysate: Sun Support (registered trademark) P-211 (manufactured by San-Ei Gen F.F.I. Co., Ltd.) Citrus fiber: Sun Support (registered trademark) P-213 (manufactured by San-Ei Gen F.F.I. Co., Ltd.) Microcrystalline cellulose: Sansupport (registered trademark) P-215 (manufactured by San-Ei Gen F.F.I. Co., Ltd.) Transglutaminase: Activa TG-K and Activa TG-K Shinayaka (manufactured by Ajinomoto Co., Inc.) Laccase: Laccase (Amano Enzyme Co., Ltd.)

[0061] Comparative Example 1 PET resin scaffolding BelloCell Chip (CESCO BIOENGINEERING Co. Ltd.) was cut to 5 mm × 5 mm and used. This PET resin scaffold is hereinafter also referred to as PET scaffold.

[0062] Comparative Example 2 Gelatin fiber-based porous scaffolds We used Genocel (registered trademark) (manufactured by Nikke Medical Co., Ltd.), a gelatin fiber substrate with a special nonwoven structure that can maintain its shape even during cell culture. This gelatin fiber substrate porous scaffold is hereinafter also referred to as gelatin fiber scaffold.

[0063] Test Example 1 Evaluation of adhesion and proliferation ability of porous scaffolds for cell culture using HEK293T cells <Material> Cells: HEK293T (human embryonic kidney cells RCB2202; RIKEN) Culture medium: 10% FBS 1% PSA I-MEM (10% fetal bovine serum (FBS) (Fujifilm Wako Pure Chemical Industries, Ltd.), 1% penicillin-streptomycin-amphotericin B (PSA) (Fujifilm Wako Pure Chemical Industries, Ltd.), I-MEM (food composition medium, IntegriCulture Co., Ltd.)) ATP measurement kit CellTiter-Glo@Luminescent Cell Viability Assay (Promega Corporation) Comparative Example 1: PET resin scaffolding Porous scaffold for cell culture according to Example 1

[0064] <Cell culture> The PET resin scaffold of Comparative Example 1 and the porous scaffold for cell culture of Example 1 were cut to 5 mm x 5 mm and were then filled with 0.5% CaCl 2 The scaffolds were autoclaved in the solution at 120°C for 20 minutes. The autoclaved scaffolds were washed with 10% FBS, 1% PSA, I-MEM, and then washed with CaCl 2The solution was thoroughly replaced with the medium, and 4 scaffolds made of the PET resin of Comparative Example 1 per well and 1 porous scaffold for cell culture of Example 1 per well were placed in each well of a 96-well plate (n = 6). For HEK293T cells, they were prepared in a state of being suspended in 10% FBS 1% PSA I-MEM (the number of cells per well described in FIG. 1), and 100 μL was seeded into each well. The plate after seeding was statically cultured under the environment of 37 °C and 5% CO 2 2.

[0065] <Measurement of viable cell count after culture by ATP measurement> The viable cell counts after 1 day and 4 days of culture were measured to evaluate the cell adhesion ability and proliferation ability. The measurement of the viable cell count after culture was performed using the CellTiter-Glo@Luminescent Cell Viability Assay according to the protocol attached to the kit. After cell seeding, the scaffolds after 1 day or 4 days were transferred to a new 96-well plate, 50 μL of PBS and 50 μL of CellTiter-Glo@Regent were added to the scaffolds, and they were thoroughly stirred with a vortex mixer to lyse the cells and extract ATP. 100 μL of the obtained lysate was transferred to a new 96-well plate, and the luminescence amount based on the ATP amount was measured by measuring the luminescence amount with a multiplate reader (Glomax (Promega)). The viable cell count was calculated from the obtained luminescence amount using a calibration curve prepared from samples with known cell counts. The measurement results are shown in FIG. 1 (n = 6, error bar: standard deviation).

[0066] <Results and discussion> As a result of measuring the number of live cells on the first and fourth days of culture of HEK293T cells, in the case of HEK293T cells, in both the PET resin scaffold of Comparative Example 1 used in the BelloCell culture vessel (CESCO) and the porous scaffold for cell culture of Example 1 (hereinafter also referred to as the scaffold of Example 1), an increase in the number of live cells after culture was confirmed according to an increase in the number of seeded cells on the first day of culture, and it was shown that a certain number of cells adhered to the scaffold in both cases of seeding amount. In addition, it was confirmed that the number of live cells increased from the first to fourth days of culture in both cases of the scaffolds used (Figure 1). From this, it was confirmed that the porous scaffold for cell culture of the present invention has adhesion ability and cell proliferation ability for HEK293T cells. In addition, it was confirmed that the scaffold developed in this study has cell adhesion ability and cell proliferation ability equivalent to those of the PET scaffold already commercially available as a scaffold for culture for HEK293T cells.

[0067] Test Example 2 Evaluation of the adhesion and proliferation capacity of porous scaffolds for cell culture using primary cells derived from duck liver <Material> Cells: Primary cells derived from duck liver (self-produced: cells were isolated from liver tissue obtained by dissection of ducks, placed in 10% serum, 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.) and cultured at 37°C) Culture medium: 10% serum, 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.), 1% penicillin-streptomycin-amphotericin B (PSA) (Fujifilm Wako Pure Chemical Industries, Ltd.), I-MEM (food composition medium, Integriculture Co., Ltd.) ATP measurement kit CellTiter-Glo@Luminescent Cell Viability Assay (Promega Corporation) Comparative Example 1: PET resin scaffolding Porous scaffolds for cell culture according to Examples 1 to 6

[0068] <Cell culture> The PET resin scaffold of Comparative Example 1 and the porous scaffolds for cell culture of Examples 1 to 6 were cut to 5 mm x 5 mm and were then washed with 0.5% CaCl 2In solution, autoclaving was performed at 120 °C for 20 minutes. The autoclaved scaffolds were washed with 10% serum, 1% PSA I-MEM, and CaCl 2 solution was thoroughly replaced with the medium, and 4 scaffolds made of PET resin of Comparative Example 1 per well and 1 porous scaffold for cell culture of Examples 1 to 6 per well were placed in each well of a 96-well plate (n = 6). For primary cells derived from duck liver, they were suspended in 10% serum, 1% PSA I-MEM (adjusted to 8×10 4 cells / cell suspension 100 μL), and 100 μL was seeded into each well. The seeded plate was statically cultured under the environment of 37 °C and 5% CO 2 .

[0069] <Measurement of viable cell count after culture by ATP measurement> The viable cell counts after 1 day and 4 days of culture were measured to evaluate the cell adhesion ability and proliferation ability. The measurement of the viable cell count after culture was performed using the CellTiter-Glo@Luminescent Cell Viability Assay according to the protocol attached to the kit. After cell seeding, the scaffolds after 1 day or 4 days were transferred to a new 96-well plate, 50 μL of PBS and 50 μL of CellTiter-Glo@Regent were added to the scaffolds, and they were thoroughly stirred with a vortex mixer to lyse the cells and extract ATP. 100 μL of the obtained lysate was transferred to a new 96-well plate, and the luminescence amount based on the ATP amount was measured by measuring the luminescence amount with a multiplate reader (Glomax (Promega)). The viable cell count was calculated from the obtained luminescence amount using a calibration curve prepared from samples with known cell counts. The measurement results are shown in Figure 2 (n = 6, error bar: standard deviation).

[0070] <Results and discussion> As a result of measuring the number of live cells on the first and fourth days of culture of duck liver-derived primary cells, live cells could be confirmed on the first and fourth days of culture for the porous scaffolds for cell culture of Examples 1 to 6 (hereinafter also referred to as the scaffolds of Examples 1 to 6, respectively). In addition, an increase in the number of cells was observed from the first to fourth days of culture (FIG. 2). Therefore, it was demonstrated that the porous scaffold for cell culture of the present invention has cell adhesion ability and cell proliferation ability in duck liver-derived primary cells.

[0071] On the other hand, although live cells were confirmed in the PET resin scaffold of Comparative Example 1, no increase in cell number was confirmed from the first to fourth days of culture. Therefore, it was presumed that the combination of cell type, culture scaffold, and culture method affects the culture results, suggesting that the PET scaffold may not be suitable for the culture of duck liver-derived primary cells in the 96-well plate used in this test.

[0072] On the other hand, cell proliferation was observed in the porous scaffold for cell culture of the present invention, suggesting the possibility of greater versatility than PET scaffolds. The porous scaffold for cell culture of the present invention is based on gelatin, and is composited with xanthan gum, etc., to improve the thermal and mechanical stability of the scaffold. Since the amino acid sequence (RGD sequence) to which proteins such as integrins involved in cell adhesion bind is contained in gelatin, it is believed that the fact that it is based on gelatin, which has high cell adhesiveness, makes it more versatile than PET scaffolds.

[0073] Test Example 3 Evaluation of the adhesion and proliferation capacity of porous scaffolds for cell culture using primary cells derived from duck liver Test Example 3 is a test using agitation culture in an agitation vessel. <Material> Cells: Primary cells derived from duck liver (self-produced: cells were isolated from liver tissue obtained by dissection of ducks, placed in 10% serum, 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.) and cultured at 37°C) Culture medium: 10% serum, 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.), 1% penicillin-streptomycin-amphotericin B (PSA) (Fujifilm Wako Pure Chemical Industries, Ltd.), I-MEM (food composition medium, Integriculture Co., Ltd.) Cedex Bio (Kit Reagents: Glucose Bio, Glutame V2 Bio, Lactate Bio, NH3 Bio; Roche Diagnostics) Crystal Violet Dye Nucleus Count Kit(CESCO BIOENGINEERING Co. Ltd) Corning Microcarriers (Corning International Inc.) Comparative Example 1: PET resin scaffolding Porous scaffolds for cell culture according to Examples 1 to 6

[0074] <Cell culture> The PET resin scaffold of Comparative Example 1 and the porous scaffolds for cell culture of Examples 1 to 6 were cut to 5 mm x 5 mm and were then washed with 0.5% CaCl 2 The scaffolds were autoclaved at 120°C for 20 minutes in a 10% serum, 1% PSA I-MEM solution and washed with CaCl 2 The solution was thoroughly replaced with the medium, and the PET resin scaffolds of Comparative Example 1 were placed in a stirring culture system at 9 pieces per culture vessel, the porous scaffolds for cell culture of Examples 1 to 6 were placed in 9 pieces per culture vessel, and the Corning microcarriers were placed in 3 mg per culture vessel. The duck liver-derived primary cells were suspended in 10% serum, 1% PSA I-MEM (5 × 10 6 cells) and the vessels were seeded with cells (5 × 10 6 Cells (volume 5 mL) / culture vessel). After seeding, culture at 37°C and 5% CO 2 The culture was carried out for 9 days with the solution agitated at 5 to 120 rpm.

[0075] <Evaluation of cell adhesion and proliferation ability by measuring total cell count after culture> The total cell count was measured after 1 and 9 days of culture to evaluate the adhesion and proliferation ability of the cells. The total cell number after culturing was measured using the Crystal Violet Dye Nucleus Count Kit, and the measurement procedure was performed according to the protocol attached to the kit. The cells were lysed using Crystal Violet Dye, and the nuclei were extracted. The number of nuclei contained in the obtained extract was counted, and the number of cells per scaffold was calculated. The measurement results are shown in Figure 3 (n = 3, error bars: standard deviation).

[0076] <Evaluation of metabolic activity by measuring metabolic products in the culture medium> Measurement of metabolic products in the culture medium was performed using Cedex Bio (n=1). Evaluation of increases and decreases in metabolic products was performed by calculating the integrated values ​​of the consumption amount of the metabolic products obtained by measurement for glucose and glutamic acid, and the production amount of lactate and ammonia. The measurement results are shown in Figure 4.

[0077] <Results and Discussion> The cell adhesion ability and cell proliferation ability of the porous scaffold for cell culture of the present invention were evaluated using a stirred culture system. As a result of measuring the total cell number on the first and ninth days of culture of duck liver-derived primary cells, an increase in the cell number was confirmed from the first to ninth days of culture for the porous scaffold for cell culture of the present invention (Figure 3). In addition, since the Crystal Violet Dye Nucleus Count Kit can only evaluate the total cell number, the consumption and accumulation of metabolic products were examined. In those in which an increase in the total cell number was observed, appropriate consumption of glucose and glutamine was observed, and no abnormalities were observed in the accumulation of lactic acid and ammonia. Therefore, it was suggested that the increase in the total cell number was due to an increase in live cells (Figure 4). In the test in which a scaffold was not inserted (without a scaffold), no increase in the total cell number or consumption of glucose was observed, and it was confirmed that duck liver-derived primary cells cannot survive without an appropriate scaffold. From these findings, it was shown that the culture scaffold developed in this study has cell adhesion ability and cell proliferation ability in duck liver-derived primary cells.

[0078] The manufacturer's website reports that the Corning microcarrier has a proven track record in culturing human mesenchymal stem cells. The Corning microcarrier was added to the test in this experiment, considering the possibility that it could function as a positive control. Although proliferation was observed on the Corning microcarrier from the first to ninth days of culture, the number of cells after nine days was clearly lower than that of the PET scaffold and the porous scaffold for cell culture of the present invention. This shows that the porous scaffold for cell culture of the present invention has an advantage over the Corning microcarrier in primary cells derived from duck liver. In addition, when compared with the PET scaffold, the porous scaffold for cell culture of the present invention was confirmed to have the same or higher cell proliferation ability, and was shown to have an advantage over the Corning microcarrier and the commercially available non-edible culture scaffold of PET.

[0079] Test Example 4 Evaluation of the adhesion and proliferation capabilities of porous scaffolds for cell culture using primary cells derived from chicken muscle Test Example 4 is a test using agitation culture in an agitation vessel. <Material> Cells: Primary cells derived from chicken muscle (own product: cells were isolated from liver tissue obtained by dissection of a chicken, placed in 10% serum, 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.) and cultured at 37°C) Culture medium: 10% serum, 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.), 1% penicillin-streptomycin-amphotericin B (PSA) (Fujifilm Wako Pure Chemical Industries, Ltd.), I-MEM (food composition medium, Integriculture Co., Ltd.) Cedex Bio (Kit Reagents: Glucose Bio, Glutame V2 Bio, Lactate Bio, NH3 Bio; Roche Diagnostics) Crystal Violet Dye Nucleus Count Kit(CESCO BIOENGINEERING Co. Ltd) Comparative Example 1: PET resin scaffolding Porous scaffold for cell culture according to Example 5

[0080] <Cell culture> The PET resin scaffold of Comparative Example 1 and the porous scaffold for cell culture of Example 5 were cut to 5 mm x 5 mm and were then filled with 0.5% CaCl 2 The scaffolds were autoclaved at 120°C for 20 minutes in a 10% serum, 1% PSA I-MEM solution and washed with CaCl 2 The solution was thoroughly replaced with the medium, and 9 PET resin scaffolds of Comparative Example 1 and 9 porous scaffolds for cell culture of Example 5 were placed in a stirred culture system per culture vessel. Chicken muscle-derived primary cells were suspended in 10% serum, 1% PSA I-MEM (5 × 10 6 cells) and the vessels were seeded with cells (5 × 10 6 cells / culture vessels). After seeding, 37℃, 5%CO 2 The culture was carried out for 9 days with the solution agitated at 5 to 120 rpm.

[0081] <Evaluation of cell adhesion and proliferation ability by measuring total cell count after culture> The total cell count was measured after 1 and 9 days of culture to evaluate the adhesion and proliferation ability of the cells. The total cell count after culturing was measured using the Crystal Violet Dye Nucleus Count Kit, and the measurement procedure was performed according to the protocol attached to the kit. Using Crystal Violet Dye, the cells were thoroughly stirred in a vortex mixer to dissolve the cells and extract the nuclei. The number of nuclei contained in the obtained extract was counted, and the number of cells per scaffold was calculated. The measurement results are shown in Figure 5 (n = 3, error bars: standard deviation).

[0082] <Evaluation of metabolic activity by measuring metabolic products in the culture medium> Measurement of metabolic products in the culture medium was performed using Cedex Bio (n=1). Evaluation of increases and decreases in metabolic products was performed by calculating the integrated values ​​of the consumption amount of the metabolic products obtained by measurement for glucose and glutamic acid, and the production amount of lactate and ammonia. The measurement results are shown in Figure 6.

[0083] <Results and Discussion> The cell adhesion and cell proliferation capabilities of the porous scaffold for cell culture of the present invention were evaluated using a stirred culture system. As a result of measuring the total cell count on the first and ninth days of culture of primary cells derived from chicken muscle, a significant increase in the cell count was confirmed from the first to ninth days of culture for the porous scaffold for cell culture of the present invention (Figure 5). In addition, when the consumption and accumulation of metabolic products were examined, the cells that showed an increase in the total cell count showed appropriate consumption of glucose and glutamine, and no abnormalities were observed in the accumulation of lactic acid and ammonia (Figure 6). Therefore, it was suggested that the increase in the total cell count was due to an increase in live cells. From these findings, it was shown that the culture scaffold developed in this study has cell adhesion and cell proliferation capabilities even for primary cells derived from chicken muscle.

[0084] Test Example 5 Evaluation of the adhesion and proliferation capacity of porous scaffolds for cell culture using primary cells derived from duck liver <Material> Cells: Primary cells derived from duck liver (self-produced: Cell fragments were excised from liver tissue obtained by dissection of ducks, placed in 10% serum, 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.) and cultured at 37°C) Culture medium: 10% serum, 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.), 1% penicillin-streptomycin-amphotericin B (PSA) (Fujifilm Wako Pure Chemical Industries, Ltd.), IMEM (food composition medium, Integriculture Co., Ltd.) Cell Culture Plate Multiwell Plate, with Cover 24 Wells(Corning) Crystal Violet Dye Nucleus Count Kit(CESCO BIOENGINEERING Co. Ltd) Comparative Example 1: Porous scaffold made of PET resin Porous scaffolds for cell culture according to Examples 5 and 13 to 19

[0085] <Cell culture> The PET resin scaffold of Comparative Example 1 and the porous scaffolds for cell culture of Examples 5 and 13 to 19 were cut into 5 mm x 5 mm pieces, and autoclaved at 121°C for 20 minutes while immersed in ion-exchanged water. The autoclave-sterilized scaffolds were washed with 10% serum, 1% PSA I-MEM, and the medium was thoroughly replaced. The PET resin scaffold of Comparative Example 1 and the porous scaffolds for cell culture of Examples 5 and 13 to 19 were placed in each well of a Cell Culture Plate Multiwell Plate, with Cover 24 Wells, with 3 pieces per well. For duck liver-derived primary cells, 2.4 x 10 5 Cells were seeded at 1 mL per well. Incubation was continued at 37°C, 5% CO 2 The culture was carried out for 1 or 7 days under the above conditions. The medium was replaced by removing the entire medium after 1 and 4 days and replacing it with fresh medium.

[0086] <Evaluation of cell adhesion and proliferation by measuring total cell count after culture> The total cell count was measured after 1 and 7 days of culture to evaluate the adhesion and proliferation of the cells. The total cell number after culturing was measured using the Crystal Violet Dye Nucleus Count Kit, and the measurement procedure was performed according to the protocol attached to the kit. The cell solution was dissolved using Crystal Violet Dye, and nuclei were extracted. The number of nuclei contained in the obtained extract was counted, and the total number of cells per scaffold was calculated. The calculated total number of cells is shown in Figure 7 (n = 6, error bar: standard deviation).

[0087] <Results and Discussion> As can be seen from Figure 7, compared with the cell adhesion and increase in total cell number from the first to seventh days of culture in the PET resin scaffold of Comparative Example 1, the cell adhesion and increase in total cell number from the first to seventh days of culture in the porous scaffolds for cell culture of Examples 5 and 13 to 19 (hereinafter also referred to as the scaffolds of Examples 5 and 13 to 19, respectively) were both significant. Specifically, it was shown that the use of citrus fiber and microcrystalline cellulose as insoluble cellulose promoted the same adhesion and proliferation of cells (Examples 13 and 14). It was also shown that the use of fish-derived gelatin and bovine-derived gelatin as gelatin promoted the same adhesion and proliferation of cells (Examples 15 and 16). It was also shown that the use of laccase as an enzyme promoted the same adhesion and proliferation of cells (Example 17). It was also shown that the use of gaddy gum and other components (protein hydrolysates) as thickening polysaccharides promoted the same adhesion and proliferation of cells (Examples 18 and 19).

[0088] Test Example 6 Evaluation of the adhesion and proliferation capabilities of porous scaffolds for cell culture using various cell lines and primary bovine cells <Material> Cells: RL34 cells (rat-derived hepatic epithelial cell line), HepG2 cells (human hepatoma-derived cell line), C2C12 cells (mouse skeletal muscle-derived myoblast cell line), porcine skeletal muscle satellite cell-derived cell line, primary cells derived from bovine longissimus muscle (own product: cell pieces were cut from the longissimus muscle obtained by dissection of a cow, placed in I-MEM medium (Integriculture Co., Ltd.) supplemented with 10% serum and 1% PSA, and cultured at 37°C), primary cells derived from bovine intermuscular fat (own product: isolated from intermuscular fat tissue obtained by dissection of a cow, placed in I-MEM medium (Integriculture Co., Ltd.) supplemented with 10% serum and 1% PSA, and cultured at 37°C), primary cells derived from bovine biceps muscle (own product: isolated from biceps muscle tissue obtained by dissection of a cow, placed in I-MEM medium (Integriculture Co., Ltd.) supplemented with 10% serum and 1% PSA, and cultured at 37°C) Culture medium: 10% FBS (SERENA Europe GmbH), 1% penicillin-streptomycin-amphotericin B (PSA) (FUJIFILM Wako Pure Chemical Corporation), D-MEM medium (D-MEM (High Glucose) with L-Glutamine, Phenol Red and Sodium Pyruvate) (FUJIFILM Wako Pure Chemical Corporation) Cell Culture Plate Multiwell Plate, with Cover 24 Wells(Corning) Crystal Violet Dye Nucleus Count Kit(CESCO BIOENGINEERING Co. Ltd) Comparative Example 1: PET resin scaffolding Gelatin fiber-based porous scaffold of Comparative Example 2 Porous scaffold for cell culture according to Example 5

[0089] <Cell culture> The PET resin scaffold of Comparative Example 1 and the porous scaffold for cell culture of Example 5 were cut into 5 mm x 5 mm pieces, and autoclaved at 121 ° C for 20 minutes while immersed in ion-exchanged water. The scaffolds sterilized by autoclaving were washed with 10% FBS 1% PSA D-MEM, and the medium was thoroughly replaced. The PET resin scaffold of Comparative Example 1, the gelatin fiber-based porous scaffold of Comparative Example 2, and the porous scaffold for cell culture of Example 5 were placed in each well of the Cell Culture Plate Multiwell Plate, with Cover 24 Wells, 3 pieces per well. The various cells used in the test were prepared in a state of being suspended in 10% FBS 1% PSA D-MEM, and 2.4 × 10 5 Cells were seeded at 1 mL per well. Incubation was continued at 37°C, 5% CO 2 The culture was carried out for 1 or 7 days under the above conditions. The medium was replaced by removing the entire medium after 1 and 4 days and replacing it with fresh medium.

[0090] <Evaluation of cell adhesion and proliferation ability by measuring total cell count after culture> The total cell count was measured after 1 and 7 days of culture to evaluate the adhesion and proliferation ability of the cells. The total cell number after culturing was measured using the Crystal Violet Dye Nucleus Count Kit, and the measurement procedure was performed according to the protocol attached to the kit. The cells were dissolved using Crystal Violet Dye, and nuclei were extracted. The number of nuclei contained in the obtained extract was counted, and the total number of cells per scaffold was calculated. The calculated total cell numbers are shown in Figures 8 to 10 (n = 6, error bars: standard deviation).

[0091] <Results and Discussion> 8 shows the test results of adhesion and proliferation of RL34 cells (rat-derived liver epithelial cell line) using the PET resin scaffold of Comparative Example 1, the gelatin fiber-based porous scaffold of Comparative Example 2, and the porous scaffold for cell culture of Example 5. As can be seen from FIG. 8, the PET resin scaffold of Comparative Example 1 and the gelatin fiber-based porous scaffold of Comparative Example 2 had similar cell adhesion and proliferation, but the porous scaffold for cell culture of Example 5 had significantly higher cell adhesion and proliferation. 9 and 10 show the results of comparative experiments using various cell lines and primary bovine cells, between the PET resin scaffold of Comparative Example 1 and the porous scaffold for cell culture of Example 5. In the experiments using all of the cell lines and primary bovine cells, the porous scaffold for cell culture of Example 5 showed an increase in cell adhesion and proliferation that was equivalent to or superior to that of the PET resin scaffold of Comparative Example 1.

[0092] Test Example 7 Evaluation of comparative shapes of porous scaffolds for cell culture using primary cells derived from duck liver <Material> Cells: Primary cells derived from duck liver (self-produced: Cell fragments were excised from liver tissue obtained by dissection of ducks, placed in 10% serum, 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.) and cultured at 37°C) Culture medium: 10% serum, 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.), 1% penicillin-streptomycin-amphotericin B (PSA) (Fujifilm Wako Pure Chemical Industries, Ltd.), I-MEM (food composition medium, Integriculture Co., Ltd.) Crystal Violet Dye Nucleus Count Kit(CESCO BIOENGINEERING Co. Ltd) LUNA-II™ Automated Cell Counter (Aligned Genetics Inc.) Porous scaffold for cell culture according to Example 5

[0093] <Cell culture> The porous scaffold for cell culture of Example 5 was cut into 5 mm x 5 mm pieces and sterilized with ethylene oxide gas (EOG). The sterilized scaffold was washed with 10% serum, 1% PSA, I-MEM, and the medium was thoroughly replaced. 2.5 g of dry weight each of the scaffold cut into 5 mm x 5 mm pieces of Example 5 or the scaffold subjected to the crushing process of Example 5 (crushed into 1 to 2 mm pieces) was placed in a 500 mL container. The various cells used in the test were prepared in a state of being suspended in 10% serum, 1% PSA, I-MEM, and 4.2 x 10 7 The cells were seeded per vessel (volume: 200 mL per vessel). Incubated at 37°C, 5% CO 2 The cells were cultured for 1 or 15 days under the above conditions. The medium was replaced by removing the entire medium every day and replacing it with fresh medium.

[0094] <Evaluation of cell adhesion and proliferation ability by measuring total cell count after culture> Total cell number after 15 days in culture was assessed using a LUNA-II™ automated cell counter. After 15 days of culture, the cells were detached from the scaffolds and a cell suspension was extracted. The number of cells in the extract was counted, and the total number of cells per scaffold was calculated.

[0095] <Results and Discussion> FIG. 11 shows an image of the porous scaffold for cell culture of Example 5 after the pulverization process. This image shows that the 1-2 mm scaffold maintains its porous structure even after the pulverization process. Therefore, the pulverization process is a process that simply reduces the size of the porous scaffold while maintaining the perforated structure and surface structure. Since the surface area of ​​a porous scaffold increases as it becomes smaller, the pulverization process is also a process that increases the surface area of ​​a porous scaffold of the same mass. This can also be expected to increase the area available for cell culture.

[0096] FIG. 12 shows the shape comparison results of the porous scaffolds for cell culture in Example 5 using duck liver-derived cells. As can be seen from FIG. 12, it was shown that more cells can be cultured when using a scaffold that has been subjected to a crushing process compared to when a scaffold cut to 5 mm x 5 mm is used. From FIG. 11, it is assumed that the surface area of ​​a porous scaffold that has been subjected to a crushing process is increased compared to a porous scaffold of the same mass that has not been subjected to a crushing process, and the surface area available for cell culture is increased. It is considered that the results of FIG. 12 are the results shown in FIG. 11.

[0097] The embodiments and examples disclosed herein are illustrative in all respects and are not restrictive. The scope of the present invention is defined by the claims, not the above description, and includes all modifications within the meaning and scope of the claims. [Industrial Applicability]

[0098] The present invention provides a porous scaffold for cell culture that is not deteriorated even when subjected to sterilization treatment such as autoclaving and that can culture various cells, and a method for producing the same. Furthermore, a food composition in which edible cultured cells are attached to the porous scaffold for cell culture and a method for producing the same are prepared.

Claims

1. A porous scaffold for cell culture, which contains a cross-linked aggregate of gelatin and xanthan gum or its analogue, and which does not change shape when heat sterilized.

2. 10. The porous scaffold for cell culture of claim 1, wherein the crosslinked aggregate further comprises insoluble cellulose.

3. 3. The porous scaffold for cell culture according to claim 2, wherein the insoluble cellulose is fermented cellulose, citrus fiber or microcrystalline cellulose.

4. A method for producing a porous scaffold for cell culture, comprising the steps of: A mixing and foaming step of mixing gelatin and xanthan gum or an analog thereof and foaming the mixture; and The method includes a crosslinking step of crosslinking the gelatin and the xanthan gum or its analogue in the obtained foam.

5. The method according to claim 4, further comprising a drying step of drying the product obtained in the crosslinking step.

6. 6. The method according to claim 4 or 5, wherein in the mixing and foaming step, insoluble cellulose is further added to the gelatin and xanthan gum or its analogue, and the mixture is foamed.

7. The method according to any one of claims 4 to 6, wherein in the crosslinking step, crosslinking is carried out by enzyme treatment and / or heat treatment.

8. A porous scaffold for cell culture, produced by the method according to any one of claims 4 to 7.

9. A food composition comprising the porous scaffold for cell culture according to any one of claims 1 to 3 and 8, on which edible cultured cells are attached.

10. A method for producing a food composition having edible cultured cells attached thereto, the method comprising a culture step of culturing edible cells together with the porous scaffold for cell culture according to any one of claims 1 to 3 and 8.

11. The method according to claim 10, further comprising a differentiation step of differentiating the edible cultured cells.

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