Porous scaffold for cell culture and method for producing same

Abstract

The present invention addresses the problem of providing: a porous scaffold for cell culture, which does not deteriorate even when subjected to a sterilization treatment such as autoclave treatment, and which is capable of culturing various cells; and a method for producing the porous scaffold. Provided are: a porous scaffold for cell culture, which contains a crosslinked aggregate of gelatin and xanthan gum or an analogue thereof and does not change in shape during heat sterilization, and a method for producing the porous scaffold for cell culture, the method comprising: a mixing and foaming step for mixing gelatin with xanthan gum or an analogue thereof to form a mixture; foaming the mixture; and a crosslinking step in which gelatin and xanthan gum or an analog thereof are crosslinked in the obtained foam.

Description

Technical Field

[0001] The present invention relates to a porous scaffold for cell culture and a method for manufacturing the same, and further relates to a food composition in which edible cultured cells are attached to the porous scaffold for cell culture and a method for manufacturing the same. Background Art

[0002] Biological tissues are generally composed of cells and the extracellular matrix produced by the cells themselves. The extracellular matrix exists in a way of surrounding 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, as a structural support, and for the adhesion, proliferation, differentiation, etc. of cells, it is also necessary to replace the scaffold of the extracellular matrix. The scaffold for cell culture needs to activate cell activity, provide enough nutrients to cells, and discharge the effect of excrement, so the form of porous body or high water-containing gel is preferred. Further, in recent years, as the scaffold for regenerative medicine and regenerative tissue being actively studied, the scaffold of biodegradability has also received attention.

[0004] As the biodegradable support mainly used, there are synthetic polymer materials and natural polymer materials.Synthetic polymer materials can, for example, include polyglycolic acid (PGA), polylactic acid (PLA), copolymers (PLGA), polyethylene glycol (PEG), polycaprolactone, etc. of polyglycolic acid and polylactic acid.Natural polymer materials can, for example, include components of extracellular matrix, specifically, collagen, gelatin, alginic acid, hyaluronic acid, agarose, chitosan, fibrin, silk protein, etc.Synthetic polymer materials generally have high mechanical strength, and are excellent in function as structural support, but there is a problem with low affinity of cells.Natural polymer materials generally have high cell affinity, but there is a problem (non-patent literature 1) that mechanical strength is low and easily deformed.So, synthetic polymer materials and natural polymer materials all have problems in either aspect of cell adhesion and mechanical strength.

[0005] Collagen adhesion proteins represented by integrins are expressed on the surface of many cells, and collagen has very strong adhesion to various cells. Therefore, scaffolds using natural polymer materials such as collagen, which mimic the extracellular matrix, have attracted attention. However, as mentioned above, natural polymer materials have low mechanical strength and are easily deformed. In addition, there is a problem of shape change during heat sterilization. Therefore, a scaffold with the characteristics of natural polymer materials, improved mechanical strength, and no shape change during heat sterilization is required.

[0006] Patent Document 1 describes a scaffold material for tissue regeneration consisting of a water-insoluble hydrogel or a sponge obtained by drying collagen, gelatin or other protein molecules cross-linked by (-)-epigallocatechin gallate or other polyphenols. Patent Document 2 describes a scaffold for vascular endothelial cell migration comprising genetically modified gelatin having an amino acid sequence derived from a portion of the amino acid sequence of collagen.

[0007] But, whether the support of patent documents 1 and 2 is not recorded even if sterilization treatments such as autoclave treatment are carried out, it will not deteriorate, and whether various cells can be cultured. In addition, in patent documents 1 and 2, there is no record of the support formed by combining gelatin and xanthan gum etc. at all. As shown in the test of comparative example 2 described later, in the support only comprising gelatin, the adhesion and proliferation of cells are insufficient.

[0008] Non-patent documents 2 and 3 describe the production of porous scaffolds stabilized with a cross-linking agent such as glutaraldehyde from gelatin using a bubble method. However, the present inventors conducted additional experiments but were unable to produce such stable porous scaffolds.

[0009] Furthermore, the denaturation temperature of the scaffolds of non-patent documents 2 and 3 is 73.5 to 87.6°C (Table 1 of non-patent document 2, Table 2 of non-patent document 3), and deteriorates if sterilized by autoclave treatment or the like. There is no record of scaffolds composed of gelatin and xanthan gum in non-patent documents 2 and 3. As shown in the test of Comparative Example 2 described later, in a scaffold containing only gelatin, the adhesion and proliferation of cells are insufficient.

[0010] Furthermore, in recent years, the world's food supply and demand has attracted much discussion as the world's population has increased. For example, the world's population is expected to increase by about 1.3 times in 2050 from 2010 to about 8.64 billion people. It is predicted that due to global warming, the average temperature will rise by about 2°C, and the world's farmland area will increase by about 0.7 billion hectares to about 1.61 billion hectares in 2050. In this way, the expected growth rate of the world's population exceeds the expected growth rate of the world's farmland area.

[0011] Therefore, it is expected to develop a new food, such as a new food obtained by other means such as culture technology. For example, cultured meat is a food produced by cell culture using tissue engineering technology, and in recent years, research and development are being carried out in various research institutes, universities and enterprises. Compared with the large amount of feed consumed by animals and the animal husbandry that requires the use of vast farmland, the manufacturing efficiency of food using the technology of cultured meat is high. The technology of cultured meat can eliminate the influence of diseases such as infectious diseases of animals with problems in animal husbandry, as well as the influence of drugs such as antibiotics given to animals. In addition, by manufacturing under a sterile environment, the influence of bacteria and viruses that may be mixed into animal husbandry foods can be eliminated.

[0012] Patent Document 3 describes an edible composition comprising a three-dimensional porous scaffold, a myotube containing a myotube nucleus, and a plurality of cell types. Examples of the three-dimensional porous scaffold include texturized proteins, non-texturized proteins, polysaccharides, and the like, and in the examples, texturized soybean protein was used. However, when the inventors used soybean protein to culture cells, the proliferation of the cultured cells was not smooth, and a food composition comprising the cultured cells could not be produced.

[0013] 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 obtained by proliferating cultured cells on the surface can be used, and polysaccharides such as pectin and polypeptides such as cathinone are described as edible microcarriers. However, no specific examples are described.

[0014] Prior art literature

[0015] Patent Literature

[0016] Patent Document 1: Japanese Patent Application Publication No. 2008-125916

[0017] Patent Document 2: Japanese Patent Application Publication No. 2013-074936

[0018] Patent Document 3: Japanese Patent Application No. 2020-527054

[0019] Patent Document 4: Japanese Patent Application No. 2017-505138

[0020] Non-patent literature

[0021] Non-patent document 1: Materials, 2014, Vol. 63, No. 9, pp. 684-689

[0022] Non-patent document 2: Materials Science and Engineering C, 48 (2015) 63-70

[0023] Non-patent document 3: Materials Science and Engineering C, 63 (2016) 1-9 Summary of the invention

[0024] The present invention aims to provide a porous scaffold for cell culture that can culture various cells without deterioration even after sterilization treatment such as autoclave treatment, and a method for producing the same. Further, a food composition having edible cultured cells attached to a porous scaffold for cell culture, and a method for producing the same.

[0025] In addition, a scaffold is provided that can appropriately proliferate cell lines (cell strains) that have poor proliferative properties even in known PET resin scaffolds (e.g., Comparative Example 1) and porous scaffolds based on gelatin fibers (e.g., Comparative Example 2).

[0026] The inventors of the present application conducted intensive research to solve the above-mentioned problems of the present invention, and found that a porous scaffold for cell culture containing a cross-linked aggregate prepared by mixing gelatin and xanthan gum and cross-linking unexpectedly preserves the properties of collagen or gelatin such as cell adhesion, proliferation, and differentiation, while greatly improving the mechanical strength that is missing in collagen or gelatin, does not change in shape, and does not deteriorate even after sterilization treatment such as autoclave treatment, so that various cells can be cultured, thereby completing the present invention.

[0027] That is, the present invention is as follows.

[0028] [1] A porous scaffold for cell culture, comprising gelatin and a cross-linked aggregate of xanthan gum or its analogue, and having no change in shape during heat sterilization.

[0029] [2] The porous scaffold for cell culture according to [1], wherein the cross-linked aggregate further contains insoluble cellulose.

[0030] [3] The porous scaffold for cell culture according to [2], wherein the insoluble cellulose is fermented cellulose, citrus fiber or microcrystalline cellulose.

[0031] [4] A method for manufacturing a porous scaffold for cell culture, comprising the following steps: a mixing and foaming step of mixing gelatin and xanthan gum or its analogue to foam; and

[0032] In the cross-linking step, gelatin and xanthan gum or the like are cross-linked in the obtained foam.

[0033] [5] The production method according to [4], further comprising a drying step of drying the product obtained in the cross-linking step.

[0034] [6] The production method according to [4] or [5], wherein in the mixing and foaming step, insoluble cellulose is further added to gelatin and xanthan gum or the like, and the mixture is mixed and foamed.

[0035] [7] The production method according to any one of [4] to [6], wherein in the cross-linking step, cross-linking is performed by enzyme treatment and / or heat treatment.

[0036] [8] A porous scaffold for cell culture, produced by the production method described in any one of [4] to [7].

[0037] [9] A food composition comprising edible cultured cells attached to the porous support for cell culture according to any one of [1] to [3] and [8].

[0038]

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

[0039]

[11] The production method according to

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

[0040] According to the present invention, a porous scaffold for cell culture with greatly improved mechanical strength, no change in shape, no deterioration even after sterilization treatment such as autoclave treatment, and an edible cell culture capable of culturing various cells and a method for producing the same can be provided. Furthermore, a food composition having edible cultured cells attached to a porous scaffold for cell culture and a method for producing the same can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a diagram showing the evaluation of the adhesion and proliferation properties of the porous scaffold for cell culture using HEK293T cells in Test Example 1.

[0042] Figure 2 This is a diagram showing the evaluation of the adhesion and proliferation properties of the porous scaffold for cell culture using primary cells derived from duck liver in Test Example 2.

[0043] Figure 3 This is a diagram showing the evaluation of the adhesion and proliferation properties of the porous scaffold for cell culture using primary cells derived from duck liver in Test Example 3.

[0044] Figure 4 This is a diagram showing the evaluation of the adhesion and proliferation properties of the porous scaffold for cell culture using primary cells derived from duck liver in Test Example 3.

[0045] Figure 5 This is a diagram showing the evaluation of the adhesion and proliferation properties of the porous scaffold for cell culture using primary cells derived from chicken muscle in Test Example 4.

[0046] Figure 6 This is a diagram showing the evaluation of the adhesion and proliferation properties of the porous scaffold for cell culture using primary cells derived from chicken muscle in Test Example 4.

[0047] Figure 7This is a diagram showing the evaluation of the adhesion and proliferation properties of the porous scaffold for cell culture using primary cells derived from duck liver in Test Example 5.

[0048] Figure 8 It is a graph showing the evaluation of the adhesion and proliferation of RL34 cells (rat liver epithelial cell line) using the PET resin scaffold of Comparative Example 1 in Test Example 6, the porous scaffold of gelatin fiber substrate of Comparative Example 2, and the porous scaffold for cell culture of Example 5.

[0049] Fig. 9 The graph shows the evaluation of the adhesion and proliferation properties of the porous scaffold for cell culture of Example 5 using various cell lines and bovine primary cells in Test Example 6.

[0050] Fig.10 The graph shows the evaluation of the adhesion and proliferation properties of the porous scaffold for cell culture of Example 5 using various cell lines and bovine primary cells in Test Example 6.

[0051] Fig.11 This is a diagram showing the shape of the porous stent after the pulverization step in Test Example 7.

[0052] Fig.12 This is a diagram showing the evaluation of the shape comparison of the porous scaffold for cell culture using primary cells derived from duck liver in Test Example 7. DETAILED DESCRIPTION

[0053] 1. Porous Scaffolds for Cell Culture

[0054] The present invention relates to a porous scaffold for cell culture that contains a cross-linked aggregate of gelatin and xanthan gum or the like, and does not change in shape during heat sterilization. The cross-linked aggregate prepared by mixing gelatin and xanthan gum and the like and cross-linking them is formed by further cross-linking an aggregate of a mesh structure of polymer networks entangled with each other while electrostatic interactions (ionic bonds, hydrogen bonds) play a role between gelatin and xanthan gum and the like, respectively, and the gelatin and xanthan gum and the like are associated and generated. Thus, it is believed that the mechanical strength of the porous scaffold for cell culture is greatly improved.

[0055] <Gelatin>

[0056] As gelatin used in the present invention, raw materials containing collagen such as bones and skin parts of mammals such as cattle and pigs, bones, skins, and scale parts of fish such as sharks and tilapia can be used, for example, gelatin obtained by hot water extraction of collagen and then hydrolysis by alkali treatment, acid treatment, enzyme treatment, etc. As the above-mentioned alkali, for example, sodium hydroxide, calcium hydroxide, etc. can be mentioned. As the above-mentioned acid, for example, hydrochloric acid, sulfuric acid, nitric acid, etc. can be mentioned. As the above-mentioned enzyme, as long as it is an enzyme that has the function of cutting the peptide bond of gelatin. It is usually an enzyme called a protease or a protease. Specifically, for example, collagenase, thiol protease, serine protease, acid protease, alkaline protease, metalloprotease, etc. can be mentioned, and they can be used alone or in combination. As the above-mentioned thiol protease, for example, papain, bromelain, ficin from plants, tissue protease from animals, calcium-dependent protease, etc. can be mentioned. As the above-mentioned serine protease, trypsin, cathepsin D, etc. can be mentioned. As the above-mentioned acidic protease, pepsin, chymosin, etc. can be mentioned.

[0057] As gelatin that can be used in the present invention, gelatin obtained by hydrolysis by acid treatment or alkali treatment, gelatin obtained by hydrolysis by enzyme treatment, etc. can be cited. Any gelatin can be used, but in the present invention, since it is desired that gelatin is positively charged, it is necessary to consider the isoelectric points (pI values) of various gelatins. For example, compared with gelatin obtained by hydrolysis by alkali treatment, gelatin obtained by hydrolysis by acid treatment or enzyme treatment undergoes less deamidation during the hydrolysis process, so it has a pI value of 7 to 9 close to collagen. The pI value of alkali-treated gelatin is 5. By adjusting to a pH value lower than these pI values, gelatin is positively charged, so if it is considered that enzyme treatment is sometimes performed in the cross-linking process of gelatin and xanthan gum, it is more preferable to use gelatin obtained by hydrolysis by acid treatment, which is easy to use in the weakly acidic region (pH 5 to 6.5) where the enzyme easily functions. By appropriately adjusting the pH value during the production process, the electrostatic interaction with xanthan gum or its analogue becomes strong, which contributes to the mechanical strength and hardness of the porous scaffold for cell culture of the present invention.

[0058] In addition, the weight average molecular weight of gelatin that can be used in the present invention includes, for example, about 5000 to about 20000, 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 selecting the type of raw material of gelatin, the mechanical strength and hardness of the porous scaffold for cell culture of the present invention can be adjusted to a desired range.

[0059] <Xanthan gum or its analogs>

[0060] The xanthan gum used in the present invention is a polysaccharide produced outside the bacterial cell by Xanthomonas campestris. For example, depending on the type of salts used in the manufacturing process of xanthan gum, xanthan gum in the form of potassium salt, sodium salt or calcium salt can be used. As the preferred weight-average molecular weight of xanthan gum, for example, about 2 million to about 50 million can be cited. Xanthan gum is usually composed of repeating units of 2 molecules of glucose, 2 molecules of mannose and glucuronic acid. Since xanthan gum has an acidic group, it interacts firmly with gelatin, especially gelatin obtained by hydrolysis by acid treatment or enzyme treatment with an isoionic point of pH 7 to 9, which contributes to the mechanical strength and hardness of the porous scaffold for cell culture of the present invention. As an analog of xanthan gum, for example, a derivative of xanthan gum that can form an aggregate with gelatin in the same way as xanthan gum can be cited.

[0061] Examples of the xanthan gum derivatives include deacetylated xanthan gum, low-acetylated xanthan gum, low-pyruvic acid xanthan gum, cationized xanthan gum, cross-linked xanthan gum, and acid-treated xanthan gum.

[0062] The content of xanthan gum or the like in the porous scaffold for cell culture of the present invention is, for example, about 1 to 15% by mass, preferably about 2 to 10% by mass, and more preferably about 3 to 8% by mass relative to gelatin.

[0063] When gellan gum or sodium alginate is used instead of xanthan gum, etc., the mechanical strength and hardness of the obtained porous scaffold for cell culture are not sufficiently improved.

[0064] <Insoluble cellulose>

[0065] The cross-linked aggregates in the porous support for cell culture of the present invention may further contain insoluble cellulose within the range that does not damage the effect of the porous support for cell culture of the present invention. As insoluble cellulose, for example, fermented cellulose, citrus fiber, microcrystalline cellulose or a mixture thereof can be cited. As preferred insoluble cellulose, for example, fermented cellulose, citrus fiber and microcrystalline cellulose can be cited. For example, by including insoluble cellulose such as fermented cellulose, the bubbles prepared in the manufacture of the porous support for cell culture of the present invention can be made more stable, and the mechanical strength of the porous support for cell culture is further improved. It is believed that the improvement in mechanical strength is because the network of polymers generated by the association of gelatin and xanthan gum, etc., is further entangled with the insoluble cellulose.

[0066] The content of insoluble cellulose varies depending on the type of insoluble cellulose, and can 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 can 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.

[0067] <Other ingredients>

[0068] The porous scaffold for cell culture of the present invention can further include other components within the range of not damaging the effect of the porous scaffold for cell culture of the present invention. As other components, for example, thickening polysaccharides, protein degradation products, etc. can be cited. As thickening polysaccharides, for example, deacylated gellan gum (and alkaline earth metal salts), white fungus polysaccharides, agar, mannose, hyaluronic acid, carrageenan, sodium carboxymethylcellulose (CMC-Na), HPMC and other modified cellulose or their mixtures can be cited. As protein degradation products, for example, wheat protein degradation products, soy protein degradation products, collagen peptides or their mixtures can be cited. The content of thickening polysaccharides or protein degradation products, etc., varies according to the type of thickening polysaccharides, and relative to gelatin, for example, about 0.1 to about 30% by mass can be cited, preferably about 0.2 to about 20% by mass can be cited, and more preferably about 0.3 to about 10% by mass can be cited.

[0069] <Coating agent>

[0070] The porous scaffold for cell culture of the present invention can also be further coated with a coating agent. In the case of coating with a coating agent, it is preferred that the cultured cells and the porous scaffold are attached via the coating agent. As the coating agent, for example, a coating agent composed of polypeptides or proteins such as polylysine, a polypeptide containing an RGD peptide, fibronectin, laminin, fibrinogen, hyaluronic acid, chitin, chitosan, chondroitin sulfate or a mixture thereof can be cited. Preferably, polylysine etc. can be cited. By coating with a coating agent, the adhesion of cells to the porous scaffold of edible polysaccharides can be adjusted to promote the proliferation of cells.

[0071] 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, based on the porous scaffold for cell culture.

[0072] In the porous scaffold for cell culture of the present invention, the constituent components are cross-linked with each other, and preferably the constituent components are cross-linked with each other by enzyme treatment and / or heat treatment. The enzyme treatment and heat treatment will be described later.

[0073] The porous scaffold for cell culture of the present invention is characterized by having a porous structure, and the pore size thereof can be, for example, about 10 μm to about 1 mm.

[0074] 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 during heat sterilization by high pressure steam sterilization (autoclave), for example. Specifically, it means that according to the conditions used in the examples, i.e., "sterilization at 120°C for 20 minutes in an autoclave", the scaffold structure does not dissolve and the porous structure does not collapse.

[0075] <Shape of the porous scaffold for cell culture of the present invention>

[0076] The prepared porous scaffold for cell culture of the present invention is preferably molded or cut into a shape suitable for cell culture for use. As the shape, for example, a rectangle, a sphere, a cylinder, a bead, a fine powder, etc. can be cited. As the size, the size suitable for the culture device used can be cited, and the major diameter can be, for example, 0.5 to 20 mm, preferably 1 to 10 mm.

[0077] The porous scaffold for cell culture of the present invention has the characteristic of being able to maintain a certain structure even when crushed. In order to increase the specific surface area of ​​the scaffold, it can also be appropriately crushed and processed into a fine powder. The particle size of the scaffold after crushing can be, for example, 20 to 50 μm, 50 to 100 μm, 100 μm to 2 mm, etc.

[0078] Furthermore, even if the scaffold has the same composition as the porous scaffold for cell culture of the present invention and is processed into a fine powder instead of a porous structure, the effects of the present invention can be exhibited.

[0079] 2. Method for manufacturing porous scaffolds for cell culture

[0080] The porous scaffold for cell culture of the present invention can be manufactured by a method comprising the following steps:

[0081] a mixing and foaming step of mixing gelatin with xanthan gum or the like to make them foam; and

[0082] In the cross-linking step, gelatin and xanthan gum or the like are cross-linked in the obtained foam.

[0083] <Mixing and foaming process>

[0084] In the mixing and foaming process, gelatin and xanthan gum or its analogue are first mixed in water. Specifically, a method of preparing a gelatin aqueous solution and an aqueous solution of xanthan gum, etc. respectively, and then mixing the two aqueous solutions; a method of preparing an aqueous solution of xanthan gum, etc., and then adding gelatin to the aqueous solution and dissolving it; a method of mixing powders of gelatin and xanthan gum, etc., and then dissolving them in water. Xanthan gum is very easy to hydrate, so it is easy to form lumps when it is dissolved in water. In addition, in order to avoid agglomeration before gelatin and xanthan gum, etc. are dissolved in water, it is preferred to prepare a gelatin aqueous solution and an aqueous solution of xanthan gum, etc. respectively, and then mix the two aqueous solutions. In addition, insoluble cellulose, other components, etc. can also be further mixed in the aqueous solution of gelatin and xanthan gum or its analogue. By including insoluble cellulose such as fermented cellulose, bubbles can be made more stable, and the mechanical strength of the porous scaffold for cell culture is further improved.

[0085] As the concentration of gelatin in water, for example, about 1 to about 10% by mass can be cited, preferably about 2 to about 8% by mass can be cited, and more preferably about 3 to about 7% by mass can be cited. The addition amount of xanthan gum or its analog, acid and thickening polysaccharide is as described above. As the temperature for mixing, for example, about 40 to about 95° C. can be cited, preferably about 60 to about 90° C. can be cited, and more preferably about 70 to about 85° C. can be cited.

[0086] It is further preferred to add acid in the mixing and foaming process to adjust the pH to below the isoelectric point of gelatin (in the case of acid-treated gelatin and enzyme-treated gelatin: pI value 7 to 9, in the case of alkali-treated gelatin: pI value 5). Although not limited by the following mechanism, by adding acid when manufacturing the scaffold, the pH is lower than the isoelectric point of gelatin, so that the gelatin can be positively charged and strongly electrostatically interact with the negatively charged xanthan gum. By further performing the following enzyme treatment and / or heat treatment cross-linking process in this state, the cross-linking between gelatin and xanthan gum is promoted, and the mechanical strength of the porous scaffold for cell culture is improved.

[0087] As an acid, for example, citric acid, oxalic acid, maleic acid, malic acid, hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, etc. can be cited. As a preferred acid, citric acid, oxalic acid, maleic acid, malic acid, hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, etc. can be cited, and citric acid, etc. can be more preferably cited. The content of the acid varies according to the type and content of the constituent components of the porous scaffold for cell culture. For example, about 0.1 to about 2 mass % can be cited relative to gelatin, preferably about 0.2 to about 1.5 mass %, and more preferably about 0.4 to about 1 mass %. In addition, the pH of the scaffold material after adding the acid is, for example, pH 3 to 6, preferably pH 3 to 5.

[0088] Next, the obtained mixture is foamed to prepare a foam. The method for foaming is not particularly limited, and any method may be used as long as it can form foam of about 10 μm to about 1 mm. For example, a mixer or the like may be used to stir vigorously so that air is involved. Alternatively, a foam may be prepared by blowing generated bubbles using a bubble generating device such as a microvalve.

[0089] <Cross-linking process>

[0090] In the obtained foam, gelatin and xanthan gum or the like are cross-linked by a desired method. Examples of the method for cross-linking include enzyme treatment and / or heat treatment.

[0091] As the enzyme that can be used in the enzyme treatment, for example, transglutaminase, laccase, peroxidase, lysine oxidase, protein disulfide isomerase, protein disulfide reductase, sulfhydryl oxidase, lipoxygenase and polyphenol oxidase (tyrosinase) can be mentioned, and preferably transglutaminase and the like can be mentioned. As the method of enzyme treatment, it can be implemented by a conventionally known method for each enzyme used. In the case where the enzyme is transglutaminase, for example, the enzyme reaction can be carried out at 40°C for 1 hour. In addition, the optimal pH of the reaction of the enzyme used in the enzyme treatment is considered, for example, adjusted to pH 4 to 7. In addition, in the case where salts are needed in order to stably act on the enzyme, salts can be added as needed. By enzyme treatment, the insolubilization of gelatin is promoted, and the conformability of the porous scaffold for cell culture is improved.

[0092] When the thickening polysaccharide is a polysaccharide that is gelled by adding an alkaline earth metal salt, the alkaline earth metal salt is added to gelate the polysaccharide in the cross-linking step.

[0093] In addition, when cross-linking is strengthened by heat treatment, there is no particular limitation, and for example, the cross-linking can be strengthened at about 150 to about 210° C., preferably about 160 to about 200° C., for about 1 to 10 minutes. The cross-linking by heat treatment can greatly improve the mechanical strength of the porous scaffold for cell culture, and can also withstand autoclave sterilization.

[0094] <Drying process>

[0095] Even the product obtained in the cross-linking step can be used as a scaffold for cell culture, but in consideration of the time for transportation, it is sometimes better to dry the product. In this case, a drying step can also be performed after the cross-linking step. Examples of the drying step that can be used in the present invention include dry heat drying, reduced pressure drying, and freeze drying.

[0096] In addition, when sterilization (e.g., autoclave) is desired before the scaffold is used for cell culture, it is desirable to further strengthen the crosslinking of gelatin and xanthan gum or its analog. At this time, it is preferred that the product is treated by dry heat drying.

[0097] Dry heat drying is not particularly limited, and can be carried out, for example, at about 40 to about 90° C., preferably about 50 to about 70° C., under normal pressure until dry. By drying, a sponge-like porous scaffold can be obtained.

[0098] In addition, by further performing a heat treatment after the drying step, the crosslinking between gelatin and xanthan gum or its analogues can also be strengthened. The heat treatment is not particularly limited, and for example, it can be performed at about 150 to about 210° C., preferably about 160 to about 200° C. for about 1 to 10 minutes. The crosslinking based on the heat treatment greatly improves the mechanical strength of the porous scaffold for cell culture.

[0099] The obtained porous scaffold for cell culture is molded or cut into a scaffold in a shape for cell culture. The shape is as described above. The porous scaffold for cell culture may also be coated with a coating agent as needed.

[0100] <Crushing process>

[0101] As described above, in order to increase the specific surface area, the porous scaffold for cell culture of the present invention may be subjected to a pulverizing step as appropriate. Specifically, the scaffold may be finely pulverized, chopped, cut using a cutting device, and sieved using a screen, thereby being easily prepared.

[0102] The produced porous scaffold for cell culture may be sterilized before use. The sterilization treatment is not particularly limited, and may be performed by, for example, ethylene oxide gas sterilization, radiation sterilization, high pressure steam sterilization (autoclave), dry sterilization, or the like.

[0103] 3. Food composition

[0104] The food composition of the present invention is a food composition in which edible cultured cells are attached to the above-mentioned porous support for cell culture.

[0105] Since the constituent components of the porous scaffold for cell culture of the present invention are edible, the food composition in which edible cultured cells are attached to the porous scaffold for cell culture can be ingested as a food.

[0106] <Edible Cells>

[0107] Edible cells to be cultured are generally cells of animals other than humans. For example, cells can include mammals such as cows, pigs, sheep, goats, deer, wild boars, horses, rabbits, bears, and whales; birds such as chickens, ducks, turkeys, geese, ostriches, pheasants, and pigeons; reptiles such as crocodiles and alligators; fish such as tuna, salmon, mackerel, sardines, anchovies, perch, catfish, carp, cod, eels, flounder, pufferfish, grouper, narrow-scaled flounder, herring, mahi-mahi, swordfish, Japanese roughy, pike, shark, white-star snapper, flounder, swordfish, tilapia, and trout; crustaceans such as crabs, crayfish, lobsters, shrimps, and prawns; mollusks such as abalone, clams, oysters, scallops, and snails; cephalopods such as cuttlefish, octopus, and squid, and insects such as crickets, locusts, and mealworms (larvae of the family Tenebrio spp.).

[0108] Edible cultured cells can be cells of various organs and tissues of the above-mentioned animals. For example, organs and tissues can include skeletal muscle, smooth muscle, heart, liver, kidney, stomach, intestine, etc. Specifically, myoblasts, satellite cells, adipocytes, endothelial cells, hepatocytes, non-parenchymal liver cells, kidney cells, etc. It should be noted that cells obtained by further differentiation of cultured cells, such as myofibrils, are also included in the cultured cells of the present invention.

[0109] The food composition of the present invention can be produced by the production method described below.

[0110] 4. Method for producing food composition

[0111] The food composition of the present invention can be produced by a production method including a culturing step of culturing edible cells together with the above-mentioned porous scaffold for cell culture.

[0112] <Cultivation process>

[0113] The cells can be cultured using a commonly used culture method suitable for each cell. The cells are cultured together with a porous support for cell culture in an appropriate culture medium. As the appropriate culture medium, it is preferred to select a culture medium suitable for the cells used.

[0114] The cultured cells may be further differentiated to form, for example, myofibrils.

[0115] The mixture generated by culturing cells together with the porous support for cell culture can be used as a food composition by removing the medium and washing as needed. Alternatively, the mixture can be pelleted by centrifugation or the like and washed by rinsing.

[0116] The obtained mixture may be added with a gelling agent, a thickener, etc., and processed and molded into a paste or gel state to prepare a food composition, or may be heated or frozen to prepare a food composition.

[0117] Example

[0118] Hereinafter, the present invention will be described using examples, comparative examples, test examples, etc., but the present invention is not limited to these examples.

[0119] Examples 1 to 19

[0120] Preparation of porous scaffolds for cell culture

[0121] The amount of gelatin described in Tables 1 to 3 is stirred and dissolved in 50 parts by weight of ion exchange water at 80°C. The amount of xanthan gum and thickening polysaccharides (excluding calcium chloride) described in Tables 1 to 3, as well as any insoluble cellulose and other components are stirred and dissolved in 50 parts by weight of ion exchange water at 80°C. Next, the prepared aqueous solution of xanthan gum and thickening polysaccharides is stirred while adding the dissolved gelatin aqueous solution, and the acid is further added. Each solution is adjusted so that the final concentration after mixing is the concentration described in Tables 1 to 3, and stirred. Next, the obtained composition is foamed with a hand mixer. A solution obtained by dissolving the enzyme and the calcium chloride in Example 3 in a small amount of ion exchange water is added to the foaming composition and mixed with a hand mixer until uniform. It is added to a stainless steel pad in a manner that the thickness becomes about 1 cm for molding. The stainless steel pad is placed in a constant temperature layer at 40°C and kept warm for 1 hour. Dry in a constant temperature layer at 60°C for about 15 hours. Furthermore, the scaffold was heated in an oven at 180° C. for 7 minutes and cut into a size of 5 mm×5 mm to prepare a porous scaffold for cell culture.

[0122] In Test Examples 1 to 4, the samples were immersed in a 0.5 mass % calcium chloride aqueous solution and sterilized in an autoclave at 120° C. for 20 minutes before use in cell culture.

[0123] In Test Examples 5 and 6, the samples were immersed in ion exchange water and sterilized by autoclave at 121° C. for 20 minutes before use in cell culture.

[0124] The mechanical strength and porous structure (pore size: about 100 to about 1000 μm) were maintained even after autoclaving. In addition, even when ion exchange water of 40 times the dry mass of the porous scaffold for cell culture was added, the structure of the porous scaffold for cell culture was maintained.

[0125] [Table 1]

[0126]

[0127] 1) Total amount of fermented cellulose preparations, including xanthan gum

[0128] 2) The content of cellulose in fermented cellulose preparations

[0129] 3) Content of CMC-Na in fermented cellulose preparation [Table 2]

[0130]

[0131] 1) Total amount of fermented cellulose including xanthan gum in the fermented cellulose preparation 2) Content of fermented cellulose in the fermented cellulose preparation 3) Content of CMC-Na in the fermented cellulose preparation

[0132] [Table 3]

[0133]

[0134] 1) Total amount of added xanthan gum in the fermented cellulose preparation

[0135] 2) The content of fermented cellulose in the fermented cellulose preparation

[0136] 3) CMC-Na content in fermented cellulose preparations

[0137] The following components were used for the components described in Tables 1 to 3.

[0138] Gelatin: Sansupport (registered trademark) P-203 (derived from pigs, acid-treated) (manufactured by Sanrongyuan FFI Co., Ltd.), Sansupport (registered trademark) P-204 (derived from pigs, acid-treated) (manufactured by Sanrongyuan FFI Co., Ltd.), Sansupport (registered trademark) P-205 (derived from fish, acid-treated) (manufactured by Sanrongyuan FFI Co., Ltd.), and Sansupport (registered trademark) P-206 (derived from cattle, acid-treated) (manufactured by Sanrongyuan FFI Co., Ltd.)

[0139] Xanthan gum: Sansupport (registered trademark) P-207 (manufactured by Sanrongyuan FFI Co., Ltd.)

[0140] Fermented cellulose preparation: Sansupport (registered trademark) P-212 (manufactured by Sanyogen FFI Co., Ltd.) (manufactured by Sanyogen FFI Co., Ltd.: containing 50.0% fermented cellulose, 33.0% xanthan gum, and 17.0% CMC-Na)

[0141] Sodium alginate: Sansupport (registered trademark) P-208 (manufactured by Sanrongyuan FFI Co., Ltd.)

[0142] Soybean polysaccharides: Sansupport (registered trademark) P-214 (manufactured by Sanrongyuan FFI Co., Ltd.)

[0143] Mannan: Sansupport (registered trademark) P-209 (manufactured by Sanrongyuan FFI Co., Ltd.)

[0144] Agar: Sansupport (registered trademark) P-210 (manufactured by Sanrongyuan FFI Co., Ltd.)

[0145] Wheat protein decomposition product: Sansupport (registered trademark) P-211 (manufactured by Sanrongyuan FFI Co., Ltd.)

[0146] Citrus fiber: Sansupport (registered trademark) P-213 (manufactured by Sanrongyuan FFI Co., Ltd.)

[0147] Microcrystalline cellulose: Sansupport (registered trademark) P-215 (manufactured by Sanrongyuan FFI Co., Ltd.)

[0148] Transglutaminase: Activa TG-K and Activa TG-K Shinayaka (manufactured by Ajinomoto Co., Ltd.)

[0149] Laccase: Laccase (Amano Enzyme Co., Ltd.)

[0150] Comparative Example 1

[0151] PET resin bracket

[0152] BelloCell Chip (CESCO BIOENGINEERING Co. Ltd) was cut into 5 mm×5 mm and used. Hereinafter, the stent made of the present PET resin is also referred to as a PET stent.

[0153] Comparative Example 2

[0154] Porous scaffolds based on gelatin fibers

[0155] Genocel (registered trademark) (Nikke Medical Co., Ltd.), a gelatin fiber substrate having a special nonwoven fabric structure that can maintain its shape even during cell culture, was used. Hereinafter, the porous scaffold of the gelatin fiber substrate is also referred to as a gelatin fiber scaffold.

[0156] Test Example 1

[0157] Evaluation of the adhesion and proliferation abilities of porous scaffolds for cell culture using HEK293T cells

[0158] <Materials>

[0159] Cells: HEK293T (human embryonic kidney cells RCB2202; RIKEN)

[0160] 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.))

[0161] ATP measurement kit CellTiter-Glo@Luminescent Cell Viability Assay (Promega Corporation)

[0162] PET resin stent of Comparative Example 1

[0163] The porous scaffold for cell culture of Example 1

[0164] <Cell culture>

[0165] The PET resin scaffold of Comparative Example 1 and the porous scaffold for cell culture of Example 1 were cut into 5 mm × 5 mm pieces and heated in 0.5% CaCl 2 The autoclaved scaffold was washed with 10% FBS 1% PSAI-MEM and CaCl 2 The solution was fully replaced with culture medium, and 4 PET resin scaffolds of Comparative Example 1 and 1 porous scaffold for cell culture of Example 1 were placed in each well of a 96-well plate (n=6). Figure 1 HEK293T cells were prepared in 10% FBS 1% PSA I-MEM (number of cells / well as indicated in the table), and 100 μL of the cells were seeded in each well. The seeded plate was incubated at 37°C and 5% CO 2 Static culture was carried out under the environment of.

[0166] <Measurement of viable cell count after culture by ATP measurement>

[0167] The number of viable cells was measured after 1 day and 4 days of culture, and the adhesion and proliferation abilities of the cells were evaluated.

[0168] The number of viable cells after culture is measured using CellTiter-Glo@Luminescent Cell ViabilityAssay according to the protocol included with the kit. After cell inoculation, transfer the scaffolds after 1 day or 4 days to a new 96-well plate, add 50μL PBS and 50μL CellTiter-Glo@Regent to the scaffolds, stir thoroughly with a vortex mixer to dissolve the cells, and extract ATP. Transfer 100μL of the resulting lysate to a new 96-well plate, and measure the luminescence using a multiplate reader (Glomax (Promega)) to measure the luminescence based on the amount of ATP. Use a standard curve made from a sample with a known number of cells to calculate the number of viable cells from the luminescence obtained. The measurement results are shown in Figure 1 (n=6, error bars: standard deviation).

[0169] <Results and Investigation>

[0170] The number of viable cells of HEK293T cells on the first and fourth days of culture was measured. The results confirmed that in the case of HEK293T cells, in either the PET resin scaffold of Comparative Example 1 or the porous scaffold for cell culture of Example 1 (hereinafter also referred to as the scaffold of Example 1) used in the BelloCell culture device (CESCO), the number of viable cells after culture increased with the increase in the number of inoculated cells on the first day of culture, indicating that a certain number of cells adhered to the scaffold at any inoculation amount. In addition, it was confirmed that the number of viable cells increased from the first to the fourth day of culture when any scaffold was used ( Figure 1 ). Thus, it was confirmed that the porous scaffold for cell culture of the present invention has adhesion ability and cell proliferation ability relative to HEK293T cells. In addition, it can be confirmed that for HEK293T cells, the scaffold developed in this study has the same cell adhesion ability and cell proliferation ability as the commercially available PET scaffold that has been used as a scaffold for culture.

[0171] Test Example 2

[0172] Evaluation of the adhesion and proliferation abilities of porous scaffolds for cell culture using primary cells from duck liver price

[0173] <Materials>

[0174] Cells: Primary cells from duck liver (self-made: cells isolated from liver tissue obtained by dissecting ducks, placed in 10% serum 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.), and cultured at 37°C)

[0175] Culture medium: 10% serum 1% PSAI-MEM (10% serum, Integriculture Co., Ltd.), 1% penicillin-streptomycin-amphotericin B (PSA) (Fujifilm Wako Pure Chemical Industries, Ltd.), I-MEM (food composition culture medium, Integriculture Co., Ltd.)

[0176] ATP measurement kit CellTiter-Glo@Luminescent Cell Viability Assay (Promega Corporation)

[0177] PET resin stent of Comparative Example 1

[0178] Porous scaffolds for cell culture of Examples 1 to 6

[0179] <Cell culture>

[0180] The PET resin scaffold of Comparative Example 1 and the porous scaffolds for cell culture of Examples 1 to 6 were cut into 5 mm × 5 mm pieces and autoclaved in a 0.5% CaCl2 solution at 120°C for 20 minutes. The autoclaved scaffolds were washed with 10% serum and 1% PSAI-MEM, and the CaCl2 solution was added. 2 The solution was fully replaced with culture medium, and 4 PET resin scaffolds of Comparative Example 1 and 1 porous scaffold for cell culture of Examples 1 to 6 were placed in each well of a 96-well plate (n=6). 4 Primary cells derived from duck liver were prepared in 10% serum and 1% PSAI-MEM (cell / cell suspension adjusted to 100 μL), and 100 μL was inoculated in each well. The inoculated plate was statically cultured at 37°C and 5% CO2.

[0181] <Measurement of viable cell count after culture by ATP measurement>

[0182] The number of viable cells was measured after 1 day and 4 days of culture, and the adhesion and proliferation abilities of the cells were evaluated.

[0183] The number of viable cells after culture is measured using CellTiter-Glo@Luminescent Cell ViabilityAssay according to the protocol included with the kit. After cell inoculation, transfer the scaffolds after 1 day or 4 days to a new 96-well plate, add 50μL PBS and 50μL CellTiter-Glo@Regent to the scaffolds, stir thoroughly with a vortex mixer to dissolve the cells, and extract ATP. Transfer 100μL of the resulting lysate to a new 96-well plate, and measure the luminescence using a multiplate reader (Glomax (Promega)). Measure the luminescence based on the amount of ATP. Use a standard curve made from a sample with a known number of cells to calculate the number of viable cells from the luminescence obtained. The measurement results are shown in Figure 2 (n=6, error bars: standard deviation).

[0184] <Results and Investigation>

[0185] The number of live cells on the first and fourth days of culture of primary cells from duck liver was measured. As a result, live cells were confirmed on the first and fourth days of culture in the porous scaffolds for cell culture of Examples 1 to 6 (hereinafter, also referred to as scaffolds of Examples 1 to 6, respectively). In addition, an increase in the number of cells was found on the first to fourth days of culture ( Figure 2 ). Therefore, it is shown that the porous scaffold for cell culture of the present invention has cell adhesion ability and cell proliferation ability in primary cells from duck liver.

[0186] On the other hand, for the PET resin scaffold of Comparative Example 1, although living cells were confirmed, no increase in the number of cells was confirmed from culture day 1 to day 4. Therefore, it is speculated that the combination of cell species, culture scaffold, and culture method affects the culture results, suggesting that the PET scaffold may not be suitable for the culture of primary cells from duck liver in the 96-well plate performed in this experiment.

[0187] On the other hand, for the porous scaffold for cell culture of the present invention, cell proliferation was observed, suggesting that the versatility of the scaffold may be higher than that of the PET scaffold. The porous scaffold for cell culture of the present invention is based on gelatin and is compounded with xanthan gum, etc., thereby improving the thermal and mechanical stability of the scaffold. Since gelatin contains an amino acid sequence (RGD sequence) bound by proteins such as integrins related to cell adhesion, it is believed that the scaffold composed of gelatin with high cell adhesion has improved versatility than the PET scaffold.

[0188] Test Example 3

[0189] Evaluation of the adhesion and proliferation abilities of porous scaffolds for cell culture using primary cells from duck liver price Test Example 3 is a test of stirring culture using a stirring container.

[0190] <Materials>

[0191] Cells: Primary cells from duck liver (self-made: cells isolated from liver tissue obtained by dissecting ducks, placed in 10% serum 1% PSAI-MEM (10% serum, Integriculture Co., Ltd.), and cultured at 37°C)

[0192] Culture medium: 10% serum 1% PSAI-MEM (10% serum, Integriculture Co., Ltd.), 1% penicillin-streptomycin-amphotericin B (PSA) (Fujifilm Wako Pure Chemical Industries, Ltd.), I-MEM (food composition culture medium, Integriculture Co., Ltd.)

[0193] Cedex Bio (Kit reagents: Glucose Bio, Glutame V2 Bio, Lactate Bio, NH3 Bio; Roche Diagnostics Co., Ltd.)

[0194] Crystal Violet Dye Nucleus Count Kit(CESCO BIOENGINEERING Co.Ltd)

[0195] Corning Microcarriers (Corning International Inc.)

[0196] PET resin stent of Comparative Example 1

[0197] Porous scaffolds for cell culture of Examples 1 to 6

[0198] <Cell culture>

[0199] The PET resin scaffold of Comparative Example 1 and the porous scaffolds for cell culture of Examples 1 to 6 were cut into 5 mm × 5 mm pieces and heated in 0.5% CaCl 2 The autoclaved scaffold was washed with 10% serum 1% PSAI-MEM and CaCl 2 The solution was fully replaced with the culture medium, and in the stirring culture system, 9 PET resin scaffolds of Comparative Example 1 were placed in each culture container, 9 porous scaffolds for cell culture of Examples 1 to 6 were placed in each culture container, and 3 mg of Corning microcarriers were placed in each culture container. Primary cell suspensions (5×10 6 Cells) were inoculated with 10% serum and 1% PSA I-MEM, and the cells (5×10 6 After inoculation, cells (liquid volume 5 mL) / culture container were placed in the container. 2The solution was stirred at 5 to 120 rpm and cultured for 9 days.

[0200] <Evaluation of cell adhesion and proliferation ability based on measurement of total cell number after culture>

[0201] The total number of cells was measured after 1 day and 9 days of culture, and the adhesion and proliferation abilities of the cells were evaluated.

[0202] The total number of cells after culture was measured using the Crystal Violet Dye Nucleus Count Kit, and the measurement operation 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 to calculate the number of cells per scaffold. The measurement results are shown in Figure 3 (n=3, error bars: standard deviation).

[0203] <Evaluation of metabolic activity by measuring metabolites in culture medium>

[0204] The measurement of metabolites in the culture medium was performed using Cedex Bio (n=1). For glucose and glutamate, the evaluation based on the increase or decrease of metabolites was performed by calculating the consumption of the metabolites obtained by the measurement, and for lactic acid and ammonia, the evaluation based on the increase or decrease of metabolites was performed by calculating the cumulative value of the generated amount. The measurement results are shown in Figure 4 .

[0205] <Results and Investigation>

[0206] The cell adhesion ability and cell proliferation ability of the porous scaffold for cell culture of the present invention were evaluated by a stirring culture system. The total cell counts of the primary cells from duck liver on the first and ninth days of culture were measured. As a result, for the porous scaffold for cell culture of the present invention, an increase in the number of cells was confirmed from the first to the ninth day of culture ( Figure 3 ). In addition, since only the total cell number can be evaluated in the Crystal Violet Dye Nucleus Count Kit, the consumption and accumulation of metabolites were investigated. As a result, it was found that the increase in the total cell number showed appropriate consumption of glucose and glutamine, and no abnormalities were found in the accumulation of lactic acid and ammonia. Therefore, it is suggested that the increase in the total cell number depends on the increase in the number of living cells ( Figure 4). In the experiment without scaffold (no scaffold), no increase in total cell number and glucose consumption were found, confirming that primary cells from duck liver cannot survive without an appropriate scaffold. This shows that the culture scaffold developed in this study has cell adhesion and cell proliferation capabilities in primary cells from duck liver.

[0207] Corning microcarriers have been reported on the manufacturer's homepage to have actual results in the culture of human mesenchymal stem cells. It is believed that Corning microcarriers may function as a positive control in this experiment and are added to the experiment. Although Corning microcarriers were found to proliferate from the 1st to the 9th day of culture, the number of cells after 9 days was significantly less 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 is superior to Corning microcarriers in primary cells from duck liver. In addition, when compared with the PET scaffold, it is also confirmed that the porous scaffold for cell culture of the present invention has the same or higher cell proliferation ability, indicating that it is also superior to the commercially available non-edible culture scaffolds of Corning microcarriers and PET.

[0208] Test Example 4

[0209] Adhesion and proliferation capacity of porous scaffolds for cell culture using primary cells from chicken muscle evaluate

[0210] Test Example 4 is a test of stirring culture using a stirring container.

[0211] <Materials>

[0212] Cells: Primary cells from chicken muscle (self-made: cells isolated from liver tissue obtained by dissecting chickens, placed in 10% serum 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.), and cultured at 37°C)

[0213] Culture medium: 10% serum 1% PSAI-MEM (10% serum, Integriculture Co., Ltd.), 1% penicillin-streptomycin-amphotericin B (PSA) (Fujifilm Wako Pure Chemical Industries, Ltd.), I-MEM (food composition culture medium, Integriculture Co., Ltd.)

[0214] Cedex Bio (Kit reagents: Glucose Bio, Glutame V2 Bio, Lactate Bio, NH3 Bio; Roche Diagnostics Co., Ltd.)

[0215] Crystal Violet Dye Nucleus Count Kit(CESCO BIOENGINEERING Co.Ltd)

[0216] PET resin stent of Comparative Example 1

[0217] Porous scaffold for cell culture of Example 5

[0218] <Cell culture>

[0219] The PET resin scaffold of Comparative Example 1 and the porous scaffold for cell culture of Example 5 were cut into 5 mm × 5 mm pieces and heated in 0.5% CaCl 2 The autoclaved scaffold was washed with 10% serum 1% PSAI-MEM and CaCl 2 The solution was fully replaced with the culture medium, and in the stirring culture system, 9 PET resin scaffolds of Comparative Example 1 and 9 porous scaffolds for cell culture of Example 5 were placed in each culture container. Primary cells from chicken muscle were suspended in (5×10 6 Cells) 10% serum 1% PSAI-MEM, cells were seeded in containers (5×10 6 After inoculation, the cells were cultured at 37°C and 5% CO 2 The solution was stirred at 5 to 120 rpm and cultured for 9 days.

[0220] <Evaluation of cell adhesion and proliferation ability by measuring the total number of cells after culture>

[0221] The total number of cells was measured after 1 day and 9 days of culture, and the adhesion and proliferation abilities of the cells were evaluated.

[0222] The total number of cells after culture was measured using the Crystal Violet Dye Nucleus Count Kit, and the measurement operation was performed according to the protocol attached to the kit. Crystal Violet Dye was used to fully stir with a vortex mixer to dissolve the cells and extract the nuclei. The number of nuclei contained in the obtained extract was counted to calculate the number of cells per scaffold. The measurement results are shown in Figure 5 (n=3, error bars: standard deviation).

[0223] <Evaluation of metabolic activity by measuring metabolites in culture medium>

[0224] The measurement of metabolites in the culture medium was performed using Cedex Bio (n=1). For glucose and glutamate, the evaluation based on the increase or decrease of metabolites was performed by calculating the consumption of the metabolites obtained by the measurement, and for lactic acid and ammonia, the evaluation based on the increase or decrease of metabolites was performed by calculating the cumulative value of the generated amount. The measurement results are shown in Figure 6 .

[0225] <Results and Investigation>

[0226] The cell adhesion and cell proliferation abilities of the porous scaffold for cell culture of the present invention were evaluated by the stirring culture system. The total cell counts of primary cells from chicken muscle on the first and ninth days of culture were measured, and the results showed that a significant increase in the number of cells was observed in the porous scaffold for cell culture of the present invention from the first to ninth days of culture ( Figure 5 ). In addition, the consumption and accumulation of metabolites were investigated, and the results showed that the scaffolds with increased total cell numbers showed appropriate consumption of glucose and glutamine, and no abnormalities were found in the accumulation of lactic acid and ammonia ( Figure 6 ). Therefore, it is suggested that the increase in the total cell number depends on the increase in the viable cells. Thus, it is shown that the culture scaffold developed in this study also has cell adhesion and cell proliferation capabilities in primary cells from chicken muscle.

[0227] Test Example 5

[0228] Evaluation of the adhesion and proliferation abilities of porous scaffolds for cell culture using primary cells from duck liver price

[0229] <Materials>

[0230] Cells: Primary cells from duck liver (homemade: cell sheets were cut out from liver tissue obtained by dissecting ducks, placed in 10% serum 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.), and cultured at 37°C)

[0231] 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 culture medium, Integriculture Co., Ltd.)

[0232] Cell Culture Plate Multiwell Plate,with Cover 24Wells(Corning)

[0233] Crystal Violet Dye Nucleus Count Kit(CESCO BIOENGINEERING Co.Ltd)

[0234] PET resin stent of Comparative Example 1

[0235] Porous scaffolds for cell culture of Examples 5 and 13 to 19

[0236] <Cell culture>

[0237] 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 × 5 mm pieces and autoclaved at 121°C × 20 minutes in a state of being immersed in ion exchange water. The autoclaved scaffolds were washed with 10% serum 1% PSA I-MEM and fully replaced with culture medium. The PET resin scaffolds of Comparative Example 1 and the porous scaffolds for cell culture of Examples 5 and 13 to 19 were placed in each well of the Cell Culture Plate Multiwell Plate, with Cover 24 Wells at a rate of 3 per well. For primary cells from duck liver, 2.4 × 10 5 Cells were seeded in 10% serum 1% PSA I-MEM (1 mL / well) at 37°C and 5% CO2 for 1 or 7 days. The culture medium was replaced by aspirating all the culture medium after 1 day and 4 days and replacing it with fresh culture medium.

[0238] <Evaluation of cell adhesion and proliferation based on measurement of total cell number after culture>

[0239] The total number of cells was measured after 1 day and 7 days of culture to evaluate the adhesion and proliferation of the cells.

[0240] The total number of cells after culture was measured using the Crystal Violet Dye Nucleus Count Kit, and the measurement operation was performed according to the protocol attached to the kit. The cell solution was dissolved using Crystal Violet Dye, and the 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 was displayed on Figure 7 (n=6, error bars: standard deviation).

[0241] <Results and Investigation>

[0242] Depend on Figure 7It can be seen that compared with the cell adhesion and increase in the total cell number of the PET resin scaffold of Comparative Example 1 from the 1st day to the 7th day of culture, the cell adhesion and increase in the total cell number of the porous scaffolds for cell culture of Examples 5 and 13 to 19 (hereinafter also recorded as the scaffolds of Examples 5 and 13 to 19, respectively) from the 1st day to the 7th day of culture were significant.

[0243] Specifically, even when citrus fiber and microcrystalline cellulose are used as insoluble cellulose, it is shown that the adhesion and proliferation of the same cells are promoted (Examples 13 and 14). In addition, even when gelatin from fish and gelatin from cattle are used as gelatin, it is shown that the adhesion and proliferation of the same cells are promoted (Examples 15 and 16). Further, even when laccase is used as an enzyme, it is shown that the adhesion and proliferation of the same cells are promoted (Example 17). Even when gum ghatti is used as a thickening polysaccharide, even when other ingredients (protein decomposition products) are used, it is shown that the adhesion and proliferation of the same cells are promoted (Examples 18 and 19).

[0244] Test Example 6

[0245] Adhesion and adhesion of porous scaffolds for cell culture using various cell lines and primary cells from bovine Evaluation of proliferation capacity

[0246] <Materials>

[0247] Cells: RL34 cells (rat liver epithelial cell line), HepG2 cells (human liver cancer cell line), C2C12 cells (mouse skeletal muscle myoblast cell line), porcine skeletal muscle satellite cell line, primary cells from bovine longissimus muscle (homemade: cell pieces were cut out from the longissimus muscle obtained by dissecting the cow, placed in I-MEM medium (manufactured by Integriculture Co., Ltd.) supplemented with 10% serum and 1% PSA, and cultured at 37°C), bovine intermuscular fat Primary cells from bovine biceps (self-made: isolated from intermuscular adipose tissue obtained by dissecting a bovine, placed in I-MEM medium (manufactured by Integriculture Co., Ltd.) supplemented with 10% serum and 1% PSA, and cultured at 37°C), primary cells from bovine biceps (self-made: isolated from biceps tissue obtained by dissecting a bovine, placed in I-MEM medium (manufactured by Integriculture Co., Ltd.) supplemented with 10% serum and 1% PSA, and cultured at 37°C)

[0248] Culture medium: 10% FBS (SERENA Europe GmbH), 1% penicillin-streptomycin-amphotericin B (PSA) (Fujifilm Wako Pure Chemical Industries, Ltd.), D-MEM (D-MEM (High Glucose) with L-Glutamine, Phenol Red and Sodium Pyruvate) (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0249] Cell Culture Plate Multiwell Plate,with Cover 24Wells(Corning)

[0250] Crystal Violet Dye Nucleus Count Kit(CESCO BIOENGINEERING Co.Ltd)

[0251] PET resin stent of Comparative Example 1

[0252] Porous scaffold of gelatin fiber substrate of Comparative Example 2

[0253] Porous scaffold for cell culture of Example 5

[0254] <Cell culture>

[0255] The PET resin scaffold of Comparative Example 1 and the porous scaffold for cell culture of Example 5 were cut into 5 mm × 5 mm pieces and treated with an autoclave at 121°C × 20 minutes in a state of being immersed in ion exchange water. The autoclaved scaffold was washed with 10% FBS 1% PSA D-MEM and fully replaced with the culture medium. The PET resin scaffold of Comparative Example 1, the porous scaffold of the gelatin fiber substrate 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 at a rate of 3 per well. Various cells used in the experiment were prepared in a state of being suspended in 10% FBS 1% PSA D-MEM to form 2.4×10 5 The cells were inoculated at 1 mL / well. 2 The culture was carried out for 1 day or 7 days under the environment of . The culture medium was replaced by withdrawing all the culture medium after 1 day and 4 days and replacing it with fresh culture medium.

[0256] <Evaluation of cell adhesion and proliferation ability based on measurement of total cell number after culture>

[0257] The total number of cells was measured after 1 day and 7 days of culture, and the adhesion and proliferation abilities of the cells were evaluated.

[0258] The total number of cells after culture was measured using the Crystal Violet Dye Nucleus Count Kit, and the measurement operation 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 total number of cells per scaffold was calculated. The calculated total number of cells was displayed on Figure 8 to Figure 10 (n=6, error bars: standard deviation).

[0259] <Results and Investigation>

[0260] Figure 8 The results of the test on the adhesion and proliferation of RL34 cells (rat liver epithelial cell line) using the PET resin scaffold of Comparative Example 1, the porous scaffold of gelatin fiber substrate of Comparative Example 2, and the porous scaffold for cell culture of Example 5 are shown in FIG. Figure 8 It can be seen that the cell adhesion and proliferation properties are equivalent in the PET resin scaffold of Comparative Example 1 and the porous scaffold of gelatin fiber substrate of Comparative Example 2, but the cell adhesion and proliferation properties are significantly different in the porous scaffold for cell culture of Example 5.

[0261] Fig. 9 and Fig.10 The results of comparative experiments using various cell lines and primary cells from bovine using the PET resin scaffold of Comparative Example 1 and the porous scaffold for cell culture of Example 5 are shown in FIG. In any experiment with the cell lines and primary cells from bovine, it was found that the porous scaffold for cell culture of Example 5 was equivalent to or superior to the PET resin scaffold of Comparative Example 1 in terms of increased cell adhesion and proliferation.

[0262] Test Example 7

[0263] Comparative evaluation of the shape of porous scaffolds for cell culture using primary cells from duck liver

[0264] <Materials>

[0265] Cells: Primary cells from duck liver (homemade: cell sheets were cut out from liver tissue obtained by dissecting ducks, placed in 10% serum 1% PSA I-MEM (10% serum, Integriculture Co., Ltd.), and cultured at 37°C)

[0266] 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 culture medium, Integriculture Co., Ltd.)

[0267] Crystal Violet Dye Nucleus Count Kit(CESCO BIOENGINEERING Co.Ltd)

[0268] LUNA-II (trademark) automatic cell counter (Aligned Genetics Inc)

[0269] Porous scaffold for cell culture of Example 5

[0270] <Cell culture>

[0271] The porous scaffold for cell culture of Example 5 was cut into 5 mm×5 mm. Sterilize with ethylene oxide gas (EOG). Wash the sterilized scaffold with 10% serum 1% PSAI-MEM, fully replace with culture medium, and put 2.5 g of dry weight of the scaffold of Example 5 cut into 5 mm×5 mm or the scaffold of Example 5 that has undergone the crushing process (crushed into 1-2 mm) in a 500 mL container. Prepare various cells used in the experiment in a state suspended in 10% serum 1% PSAI-MEM to form 4.2×10 7 The cells were inoculated in a 200 mL / container format (liquid volume: 200 mL / container). 2 The culture was carried out for 1 day or 15 days under the environment of . The culture medium was replaced by withdrawing all the culture medium every day and replacing it with fresh culture medium.

[0272] <Evaluation of cell adhesion and proliferation ability based on measurement of total cell number after culture>

[0273] The total cell number after 15 days of culture was evaluated using a LUNA-II (trademark) automated cell counter.

[0274] After 15 days of culture, cells were detached from the scaffolds, and a cell suspension was extracted. The number of cells contained in the obtained extract was counted to calculate the total number of cells per scaffold.

[0275] <Results and Investigation>

[0276] Fig.11An image of the porous scaffold for cell culture of Example 5 after the crushing process is shown in FIG. According to the image, even after the crushing process, the 1-2 mm scaffold maintains a porous structure. Therefore, the crushing process is a process of simply reducing the size of the porous scaffold while maintaining the open-pore structure or surface structure. Since the surface area increases as the porous scaffold becomes smaller, the crushing process is also a process that results in increasing the surface area of ​​the porous scaffold of the same mass. This can also be assumed to increase the area that can be cultured with cells.

[0277] Fig.12 The shape comparison results of the porous scaffold for cell culture in Example 5 using cells derived from duck liver are shown in FIG. Fig.12 It can be seen that more cells can be cultured when using a scaffold that has been subjected to a pulverization step than when using a scaffold cut into 5 mm×5 mm. Fig.11 The porous scaffolds that have been pulverized have a larger surface area than the porous scaffolds of the same mass that have not been pulverized, and it is assumed that the surface area that can be used for cell culture has increased. Fig.12 The result is shown Fig.11 the envisioned result.

[0278] The embodiments and examples disclosed this time are all illustrative and non-limiting. The scope of the present invention is indicated by the scope of the claims rather than the above description, and includes all changes within the meaning and scope equivalent to the scope of the claims.

[0279] Industrial Applicability

[0280] According to the present invention, a porous scaffold for cell culture that does not deteriorate even after sterilization treatment such as autoclave treatment and can culture various cells, and a method for producing the same can be provided. Further, a food composition having edible cultured cells attached to a porous scaffold for cell culture and a method for producing the same can be provided.

Claims

1. A porous scaffold for cell culture, comprising gelatin and a cross-linked aggregate of xanthan gum or its analogues, and having no change in shape during heat sterilization.

2. The porous scaffold for cell culture according to claim 1, wherein: The cross-linked coacervate also contains insoluble cellulose.

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 manufacturing method is a method for manufacturing a porous scaffold for cell culture, comprising the following steps: a mixing and foaming step of mixing gelatin with xanthan gum or the like to make them foam; and In the cross-linking step, gelatin and xanthan gum or the like are cross-linked in the obtained foam.

5. The manufacturing method according to claim 4, wherein: The method further includes a drying step of drying the product obtained in the cross-linking step.

6. The manufacturing method according to claim 4 or 5, wherein: In the mixing and foaming step, insoluble cellulose is further added to gelatin and xanthan gum or the like, and the mixture is mixed and foamed.

7. The production method according to any one of claims 4 to 6, wherein In the cross-linking step, cross-linking is performed by enzyme treatment and / or heat treatment.

8. A porous scaffold for cell culture, produced by the production method according to any one of claims 4 to 7. 9 . A food composition comprising edible cultured cells attached to the porous support for cell culture according to claim 1 .

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

11. The manufacturing method according to claim 10, wherein: The method further includes a differentiation step of differentiating the edible cultured cells.

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