Cell culture materials

Abstract

The present invention aims to provide a cell culture substrate suitable for cell culture. In particular, it aims to provide a cell culture substrate that can efficiently produce cultured meat. [Solution] The cell culture substrate according to the present invention comprises a three-dimensional structure composed of proteins that facilitate cell adhesion and lipids that provide a hydrophobic environment conducive to cell adhesion. Therefore, animal cells can be cultured using a cell culture carrier equipped with this three-dimensional structure. Furthermore, in the cell culture substrate of the present invention, the sum of the dry mass of proteins and lipids in the dry mass of the cell culture substrate is 80% by mass or more, so animal cells adhere easily to the cell culture substrate, preventing unintended inhibition of culture. Based on the above, the cell culture substrate according to the present invention is a cell culture substrate suitable for cell culture.

Description

Technical Field

[0001] The present invention relates to a cell culture substrate.

Background Art

[0002] In recent years, with the increase in the world's population, an increase in the demand for meat is expected. In order to meet such demand for meat and to respond to the desire to protect livestock animals, research and development of cultured meat produced by culturing animal cells have been promoted.

[0003] For example, it is necessary to culture animal-derived cells (animal cells) such as muscle cells and liver cells on a cell culture substrate so that cultured meat can be produced which is controlled to have a desired shape and texture such as steak meat, sashimi, sliced meat, foie gras, etc.

[0004] As a cell culture substrate that can be used for the production of such cultured meat, preferably a porous material that does not use animal-derived materials (for example, natural polymer polysaccharides such as alginic acid or alginate, glucomannan, cellulose derivatives, amylose, pectin, glucomannan, agarose, carrageenan, locust bean gum, etc., microbial-produced polysaccharides such as bacterial cellulose, xanthan gum, gellan, pullulan, hyaluronic acid, etc., or microbial-produced polyamino acids such as polyglutamic acid, polylysine, etc.) into an edible substrate composed of (a) An adhesion improver containing a non-killing animal-derived component derived from milk or eggs, etc. (Japanese Patent Application Laid-Open No. 2022-159217; Patent Document 1), or (b) An adhesion improver containing a non-killing plant-derived component derived from soybeans, etc. (Japanese Patent Application Laid-Open No. 2022-159216; Patent Document 2), A cell culture substrate kneaded with these, or a cell culture substrate to which these adhesion improvers are applied to the surface of an edible substrate is utilized.

[0005] Furthermore, the inventions described in Patent Documents 1 and 2 are characterized by the addition of the above-mentioned adhesion-enhancing agent to an edible substrate in order to improve adhesion to animal cells. For this reason, Patent Documents 1 and 2 disclose that the mass of the adhesion-enhancing agent relative to the mass of the edible substrate is preferably 0.01% to 50%. In other words, Patent Documents 1 and 2 disclose that the amount of adhesion-enhancing agent contained in 100% by mass of the cell culture substrate is preferably about 33% by mass or less.

[0006] Furthermore, Patent Document 3 discloses a biological material made from defatted soybean tofu as a cell culture substrate. However, Patent Document 3 does not disclose the food components that make up the defatted soybean tofu used. However, according to the food component database published by the Ministry of Education, Culture, Sports, Science and Technology, it is publicly known that the food components that make up tofu are as follows per 100g: protein: 5.3g, lipids: 3.5g, carbohydrates: 2.0g, and other components: approximately 0.35g. In other words, it is publicly known that in 100% of the dry mass of the food components that make up tofu, protein and lipids together account for 78.9% by mass (carbohydrates and other components together account for 21.1% by mass).

[0007] Therefore, since the cell culture substrate (biological material made from defatted soybean tofu) described in Reference 3 consists of tofu from which the lipids have been removed, the combined amount of protein and lipids in 100% of the dry mass of the food components constituting the cell culture substrate is less than 78.9% by mass (however, carbohydrates and other components together are present in amounts greater than 21.1% by mass).

[0008] In fact, Patent Document 1 discloses Comparative Examples 2 and 3 which mimic the biological material made from defatted soybean tofu disclosed in Patent Document 3, but in 100% by dry mass of the food components constituting the cell culture substrate disclosed in Comparative Examples 2 and 3, (a) Soy meat used in Comparative Example 2: It contains only 56.5% by mass of protein and lipids combined (however, it contains 43.5% by mass of carbohydrates and other components combined), (i) Soy meat used in Comparative Example 3: Contains only 69.0% by mass of protein and lipids combined (carbohydrates and other components make up 31.0% by mass). [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2022-159217 [Patent Document 2] Japanese Patent Publication No. 2022-159216 [Patent Document 3] Special Publication No. 2004-518771 [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] In the cell culture substrates described in Patent Documents 1 and 2, only about 33 parts by mass or less of adhesion-enhancing agent, which can contribute to the adhesion of animal cells to the cell culture substrate, is present in 100 parts by mass of the cell culture substrate. Therefore, animal cells have difficulty adhering to the cell culture substrates described in Patent Documents 1 and 2, and there is a risk that the culture of animal cells may be unintentionally inhibited.

[0011] Furthermore, the cell culture substrate described in Patent Document 3 has a high carbohydrate content. Much of the carbohydrates contained in defatted soybean tofu are hydrophilic substances such as sugars and dietary fiber found in soybeans. In contrast, animal cells to be cultured are covered with a cell membrane made of phospholipids, and are known to have the characteristic of easily adhering to hydrophobic surfaces and difficult to adhere to hydrophilic surfaces. In other words, the components of the cell culture substrate described in Patent Document 3 have a low percentage of proteins and lipids to which cells easily adhere, but a high percentage of carbohydrates to which cells do not adhere.

[0012] Therefore, animal cells were difficult to adhere to the cell culture substrate described in Patent Document 3, and there was a risk that the culture of animal cells was unintentionally inhibited.

[0013] The present invention aims to provide a cell culture substrate suitable for cell culture. In particular, it aims to provide a cell culture substrate that can efficiently produce cultured meat. [Means for solving the problem]

[0014] The present invention "(Claim 1) A cell culture substrate comprising a three-dimensional structure containing protein and lipid, wherein the sum of the dry mass of protein and lipid in the dry mass of the cell culture substrate is 80% by mass or more." (Claim 2) The cell culture substrate according to claim 1, wherein the mass ratio of the dry mass of lipids in the dry mass of the cell culture substrate to the dry mass of protein in the dry mass of the cell culture substrate is greater than 0 and less than 0.79. (Claim 3) The cell culture substrate according to claim 1 or claim 2, wherein the three-dimensional structure is membrane-like. (Claim 4) The cell culture substrate according to claim 1 or claim 2, wherein the three-dimensional structure has a porous structure. (Claim 5) The cell culture substrate according to claim 3, wherein the three-dimensional structure has a porous structure. That is the case. [Effects of the Invention]

[0015] As a result of further investigation by the applicant, it was found that a large amount of animal cells can be cultured on the cell culture substrate according to the present invention. The reason for this has not been fully elucidated, but the following reasons were considered.

[0016] The cell culture substrate according to the present invention comprises a three-dimensional structure composed of a protein to which cells easily adhere and a lipid that provides a hydrophobic environment to which cells easily adhere. Therefore, animal cells can be cultured by using a cell culture carrier provided with the three-dimensional structure. And since the sum of the dry masses of the protein and lipid in the dry mass of the cell culture substrate according to the present invention is 80% by mass or more, animal cells easily adhere to the cell culture substrate, and culturing is prevented from being inhibited unintentionally. From the above, the cell culture substrate according to the present invention is a cell culture substrate suitable for cell culture.

[0017] Furthermore, the applicant of the present application has found that when the mass ratio of the lipid to the protein in the constituent components of the cell culture substrate is greater than 0 and less than 0.79, a large amount of animal cells can be cultured. From the above, the cell culture substrate according to the present invention is a more suitable cell culture substrate for cell culture.

[0018] Also, when the cell culture substrate is in the form of a membrane, cell culture can be efficiently performed on the cell culture substrate. And by culturing muscle cells or liver cells on the cell culture substrate, it is easy to produce cultured meat having a flat plate shape imitating steak meat or the like with animal cells cultured along the main surface thereof. From the above, the cell culture substrate according to the present invention is an even more suitable cell culture substrate for cell culture. In particular, by using the cell culture substrate according to the present invention, cultured meat can be efficiently produced.

[0019] And since the cell culture substrate according to the present invention has a porous structure, the surface area of the cell culture substrate is increased, and cell culture can be performed. From the above, the cell culture substrate according to the present invention is an even more suitable cell culture substrate for cell culture.

Brief Description of the Drawings

[0020] [Figure 1]This is an electron microscope image of the cell culture substrate prepared in Comparative Example 1, in which animal cells were cultured, after a total culture period of 12 days. [Figure 2] This is an electron microscope image of the cell culture substrate prepared in Example 1, with animal cells being cultured, after a total culture period of 12 days. Note that the main surface of the cell culture substrate shown in this electron microscope image is obscured by the presence of animal cells being cultured on that surface. [Modes for carrying out the invention]

[0021] In this invention, various configurations can be appropriately selected, such as the following configuration. Unless otherwise specified, the various measurements described in this invention are performed under normal pressure and a temperature of 25°C. Unless otherwise specified, the various measurement results described in this invention are obtained by measurement to a value one decimal place smaller than the desired value, and the desired value is calculated by rounding this value. For example, if the desired value is to be expressed to the first decimal place, the value is obtained to the second decimal place by measurement, and the obtained second decimal place value is rounded to the first decimal place, and this value is used as the desired value. In addition, the upper and lower limits exemplified in this invention can be combined arbitrarily.

[0022] The cell culture substrate according to the present invention comprises a three-dimensional structure containing proteins and lipids (hereinafter sometimes abbreviated as "three-dimensional structure").

[0023] The type of three-dimensional structure can be selected as appropriate, but one example is a three-dimensional structure formed by the Ramsden phenomenon, where proteins and lipids undergo intermolecular bonding, concentration, and coagulation. Familiar examples of this include the film that forms on the surface of hot milk and yuba (tofu skin). Therefore, such three-dimensional structures produced by the Ramsden phenomenon can be used as cell culture substrates.

[0024] Furthermore, while it can be clearly seen that the three-dimensional structure produced by the Ramsden phenomenon is formed by intermolecular bonding between proteins and lipids, it is impossible or impractical to directly identify the detailed distribution of proteins and lipids constituting the structure or the specific nature of the intermolecular entanglement.

[0025] The type of protein used in this invention can be appropriately selected depending on the type of cells to be cultured and the culture conditions. For example, proteins found in animal bodily fluids such as milk, proteins found in the meat, hides, tendons, bones, or organs of livestock such as cows, pigs, or chickens, proteins found in fish and shellfish, proteins found in plant seeds and fruits such as soybeans, and proteins produced by insects and microorganisms can be used.

[0026] Furthermore, the type of lipid used in this invention is not limited to those that can be used to prepare a cell culture substrate. For example, lipids contained in animal bodily fluids such as milk, lipids contained in the meat, fat, or organs such as the liver of livestock such as cows, pigs, or chickens, lipids contained in fish and shellfish, lipids contained in the seeds and fruits of plants such as soybeans, and lipids produced by insects and microorganisms can be used.

[0027] In this invention, a three-dimensional structure is a structure having a three-dimensional shape with length in the vertical direction, the horizontal direction perpendicular to the vertical direction, and the height direction perpendicular to both the vertical and horizontal directions. The shape of the three-dimensional structure according to this invention is appropriately selected depending on the type of cells to be cultured and the culture conditions. For example, it can be membrane-like (porous film-like or non-porous film-like), granular (spherical or rod-like, etc.), fibrous, fabric-like (nonwoven, woven, knitted) composed of intertwined fibers, foam sheet-like, block-like, or irregularly shaped.

[0028] In particular, when the three-dimensional structure is membrane-like, cell culture can be efficiently performed on a cell culture substrate equipped with the three-dimensional structure. Furthermore, by culturing muscle cells or liver cells on the cell culture substrate, the cells are cultured along the main surface, making it easier to produce cultured meat with a flat plate shape that mimics steak, which is preferable.

[0029] Furthermore, the three-dimensional structure according to the present invention preferably has a porous structure having multiple openings. Having a porous structure increases the surface area, allowing for cell culture. Therefore, a cell culture substrate equipped with a three-dimensional structure having a porous structure is an even more suitable cell culture substrate for cell culture.

[0030] The size of the pores in the three-dimensional structure can be adjusted as appropriate, but in order to provide a cell culture substrate that can culture a larger quantity of animal cells, the average pore diameter in its dry state is preferably 300 μm or less, more preferably 200 μm or less, more preferably 100 μm or less, more preferably 50 μm or less, and most preferably 30 μm or less. The lower limit can be adjusted as appropriate, but it is practical and preferable for the average pore diameter to be 1 μm or more. The average pore diameter of the three-dimensional structure can be measured using an electron microscope image of the structure. The average value of the pore diameters (in μm) of 15 pores visible in the electron microscope image is taken as the average pore diameter (in μm) of the three-dimensional structure. If the shape of the pore is not circular, the length of the longest line segment obtained by overlapping both ends on the outer circumference of the pore is taken as the pore diameter of the pore.

[0031] When the three-dimensional structure is in the form of a membrane, fabric, or foam sheet, its composition, such as basis weight and thickness, can be adjusted as appropriate. For example, the basis weight of the three-dimensional structure can range from 1 to 5000 g / m². 2 It can be 5-2500g / m² 2 It can be 10-1000g / m² 2 It can be such that the thickness can be 0.01 to 5 mm, 0.05 to 2.5 mm, or 0.1 to 1 mm. The thickness and basis weight of the three-dimensional structure can be measured as follows. (1) Immerse the three-dimensional structure to be measured in pure water at 25°C for 3 hours. (2) After removing the three-dimensional structure from the pure water, a paper towel is placed on one main surface for 3 seconds, and then on the other main surface for 3 seconds to remove any excess water droplets from the surface. (3) For the three-dimensional structure after removing unwanted water droplets, the 1m of its main surface (the widest surface of the three-dimensional structure) 2 Calculate the mass per unit area, and use this calculated value to determine the basis weight (unit: g / m²) of the three-dimensional structure. 2 Similarly, for the three-dimensional structure after removing unwanted water droplets, measure the distance in the direction perpendicular to its main surface using a micrometer (Mitutoyo Corporation, VL-50S-B, measuring force: 6.5 cm). 2 The length measured using a load of 0.01 N per unit area is defined as the thickness of the three-dimensional structure (in mm).

[0032] The three-dimensional structure according to the present invention can be used as a cell culture substrate on its own, but it may also be a cell culture substrate formed by laminating or compounding a substrate onto the three-dimensional structure. The type of substrate can be selected as appropriate, but it is preferable to use an edible substrate such as an edible sheet mainly composed of starch, gels or fibers such as gelatin, or these three-dimensional structures, so that the cultured meat can be ingested and digested together with the cell culture substrate. When cells cultured on the three-dimensional structure are used for cell assays of pharmaceuticals or for regenerative medicine applications such as prosthetic materials or cell fragments for transplantation, the substrate does not have to be made of edible components, and substrates made of polylactic acid resins or chitin / chitosan-derived plastics that have biocompatibility and biodegradability can also be selected.

[0033] The shape of the substrate used can be selected as appropriate, and can be a film (porous or non-porous film), a fabric (nonwoven, woven, or knitted), a foamed sheet, particulate, short fiber, or an amorphous gel. The method of laminating the three-dimensional structure and the substrate can be adjusted as appropriate, but methods such as simply stacking them, welding and integrating them by placing them in an ultrasonic welding device, heat sealing device, or solvent, or bonding and integrating them using a binder can be employed.

[0034] Alternatively, a cell culture substrate consisting of a substrate and a three-dimensional structure may be prepared by coating the three-dimensional structure with a substrate such as a gel, or by embedding the three-dimensional structure in a substrate such as a gel.

[0035] However, to make the cell culture substrate more suitable for cell culture, it is preferable to use the three-dimensional structure as a cell culture substrate on its own.

[0036] The applicant has found that a large quantity of animal cells can be cultured on the cell culture substrate according to the present invention. Although the reason for this has not been fully elucidated, the following reasons are considered.

[0037] The sum of the dry masses of proteins and lipids contained in the cell culture substrates described in Patent Documents 1 and 2 is low (approximately 33% by mass or less). Furthermore, the sum of the dry masses of proteins and lipids contained in the cell culture substrate described in Patent Document 3 is low, and it also contains a high percentage of carbohydrates, which make it difficult for cells to adhere. As a result, animal cells do not adhere well to the cell culture substrates described in the conventional technologies such as Patent Documents 1 to 3, and there is a risk that the culture of animal cells may be unintentionally inhibited.

[0038] In contrast, in the cell culture substrate according to the present invention, the sum of the dry masses of proteins and lipids in the dry mass of the cell culture substrate is 80% by mass or more, so that animal cells adhere easily to the cell culture substrate and the culture is not unintentionally inhibited. For this reason, the cell culture substrate according to the present invention is a cell culture substrate suitable for cell culture. To make the cell culture substrate more suitable for culturing animal cells, the sum of the dry masses of proteins and lipids in the dry mass of the cell culture substrate is preferably 85% by mass or more, preferably 90% by mass or more, and most preferably 95% by mass or more.

[0039] Furthermore, in order to enable the cultivation of a large amount of animal cells, the cell culture substrate according to the present invention preferably contains less than 20% by mass of carbohydrates in its dry mass. To make the cell culture substrate more easily capable of culturing animal cells, it is preferable that the carbohydrate content be 15% by mass or less, preferably 10% by mass or less, more preferably 5% by mass or less, and most preferably the cell culture substrate contains no carbohydrates at all.

[0040] Furthermore, in order to enable the cultivation of a large amount of animal cells, the cell culture substrate according to the present invention preferably has a mass ratio of the dry mass of lipids in the dry mass of the cell culture substrate to the dry mass of proteins in the dry mass of the cell culture substrate that is greater than 0 and less than 0.79. This mass ratio can be appropriately adjusted to ensure that the cell culture substrate can cultivate a large amount of animal cells, but it is preferably 0.70 or less, more preferably 0.50 or less, and most preferably 0.30 or less. Since the cell culture substrate according to the present invention has a three-dimensional structure containing proteins and lipids, the lower limit of this mass ratio will be greater than 0. This lower limit of the mass ratio can be appropriately adjusted, but it is practical and preferable to have it be 0.05 or more.

[0041] These dry masses are determined by subjecting the cell culture substrate to a near-infrared analyzer (near-infrared spectroscopy) used to determine the nutritional content of food products.

[0042] The types of cells that can be cultured using the cell culture substrate of the present invention can be appropriately selected depending on the type of cells to be cultured and the culture conditions. For example, muscle cells and organ cells such as liver cells from animals such as cattle, pigs, or birds, as well as muscle cells and organ cells from fish and shellfish, can be cultured. Known methods and conditions can be used for the culture method and culture conditions.

[0043] The method for producing the cell culture substrate of the present invention will be described with an example. The method for producing the cell culture substrate of the present invention can be appropriately selected, for example, (Step 1) A step to prepare a dispersion in which proteins and lipids are dispersed in a dispersion medium. (Step 2) A step of forming a three-dimensional structure of the protein and the lipid by removing the dispersion medium contained in the dispersion. (Step 3) Step of recovering the three-dimensional structure, This method provides a way to produce a cell culture substrate comprising a three-dimensional structure containing proteins and lipids produced by the Ramsden phenomenon.

[0044] or, (Step 1) A step to prepare a dispersion in which proteins and lipids are dispersed in a dispersion medium. (Step 2) A step of removing the dispersion medium contained in the dispersion to form a film of the three-dimensional structure of the protein and the lipid on the surface of the dispersion. (Step 3) A step of recovering the film of the three-dimensional structure from the liquid surface. This method provides a way to produce a cell culture substrate consisting of a membrane with a three-dimensional structure containing proteins and lipids produced by the Ramsden phenomenon.

[0045] Let's explain step 1.

[0046] The method for preparing the dispersion can be selected as appropriate. Commercially available dispersions, such as those containing pre-dispersed proteins and lipids in water, such as milk or soy milk, can be used, or a dispersion prepared by dispersing proteins and lipids in a dispersion medium such as water can be used.

[0047] The concentrations of proteins and lipids in the dispersion are adjusted as appropriate to prepare the three-dimensional structure. Additives such as salts, acids, or emulsifiers may be added to the dispersion to facilitate the preparation of the three-dimensional structure, as well as fillers to adjust the mechanical properties of the structure, and physiologically active substances such as growth factors to promote cell culture. Furthermore, seasonings such as monosodium glutamate may be added to the dispersion to prepare a cell culture substrate with a good taste. Additionally, foaming agents such as sodium bicarbonate may be added to the dispersion, or foaming treatments such as injecting bubbles may be performed. By using a dispersion containing such foaming agents or a dispersion that has undergone foaming treatment, a three-dimensional structure with a porous structure can be prepared.

[0048] While thermal denaturation of proteins is not a necessary requirement for the Ramsden phenomenon described above, it is possible to induce thermal denaturation in the proteins present in the dispersion by preheating the dispersion. By using a dispersion in which protein insolubilization has been promoted in advance by thermal denaturation, it is possible to create three-dimensional structures with superior water resistance and strength.

[0049] Steps 2 and 3 will now be explained.

[0050] The method for removing the dispersion medium from the dispersion can be selected as appropriate, but examples include leaving it in the atmosphere to allow the dispersion medium to volatilize, exposing it to reduced pressure to allow the dispersion medium to volatilize, and heating to evaporate the dispersion medium. The processing conditions in each method, such as heating temperature and processing time, should be adjusted as appropriate to prepare the three-dimensional structure.

[0051] This process forms a three-dimensional structure in the dispersion (for example, on the surface of the liquid), and the three-dimensional structure according to the present invention can be obtained by recovering it from the dispersion. In particular, by recovering the film formed on the surface of the dispersion, a film of the three-dimensional structure can be obtained.

[0052] Furthermore, the recovered three-dimensional structure may then be subjected to a step in which protein components are crosslinked. Methods for this crosslinking include thermal crosslinking by heating, and crosslinking reactions using crosslinking agents such as formalin or peptide synthesis reagents. By applying this crosslinking treatment, a cell culture substrate with high rigidity and excellent long-term shape retention in the culture medium can be realized. As a result, it is preferable to produce a cell culture substrate that allows for more efficient cell culture due to its superior long-term shape retention in the culture medium.

[0053] Furthermore, the recovered three-dimensional structure, or the three-dimensional structure after being subjected to the crosslinking process, may then be subjected to a process to remove a portion of the lipids from the three-dimensional structure. As a method for removing the portion of the lipids, one example is to immerse the three-dimensional structure in an organic solvent that dissolves lipids, such as ethanol or an aqueous ethanol solution. By removing the portion of the lipids, a cell culture substrate can be realized that suppresses the elution of lipids into the culture medium during culture. As a result, it is preferable to produce a cell culture substrate that is more suitable for use in cell culture, in which the effect of lipids on cells during culture is suppressed.

[0054] The three-dimensional structures prepared as described above can be used as cell culture substrates as is. However, depending on the intended use of the cell culture substrate and the cell culture conditions, secondary processing such as adjusting the thickness to obtain the desired shape, punching or cutting, or compounding with a separately prepared substrate may be performed to manufacture the cell culture substrate. [Examples]

[0055] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0056] (Comparative Example 1) Unsweetened soy milk manufactured by Kikkoman Soy Foods Co., Ltd. was used. The food components of the unsweetened soy milk were as follows per 100 mL: protein 4.2 g, fat 3.3 g, carbohydrates 1.7 g, and other components 0.5 g. 80 mL of unsweetened soy milk and 0.2 g of sodium bicarbonate, a foaming agent, were added to a 10 cm diameter Teflon® petri dish. Next, the unsweetened soy milk was physically foamed by applying the transducer of an ultrasonic device. Then, the Teflon® petri dish containing the foamed unsweetened soy milk was placed in a dryer (heating temperature: 90°C) and heated to completely decompose the sodium bicarbonate, causing further foaming. The mixture was then left to stand for 10 minutes in this state. Subsequently, a porous membrane, a three-dimensional structure formed by the Ramsden phenomenon, where proteins and lipids form intermolecular bonds, was scooped up from the surface of the unsweetened soy milk. Furthermore, a beaker was prepared with a platform inside, and filled with pure water so that the water level was lower than the platform. The three-dimensional structure was then placed on the platform inside the beaker, and the beaker was covered with a lid. After that, it was placed in a microwave oven (300W output, 1 minute) to subject the three-dimensional structure to thermal crosslinking treatment by moist heat heating. Furthermore, the three-dimensional structure was cut out into a circular shape with a diameter of 1.3 cm. The cut-out three-dimensional structure was then impregnated in an ethanol solution containing peptide synthesis reagents (1.52 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.92 g of N-hydroxysuccinimide, and ethanol diluted to 40 mL) at room temperature for 4 hours to perform peptide crosslinking treatment on the three-dimensional structure. Then, to clean the three-dimensional structure, it was immersed in 99.5% ethanol at room temperature for 10 minutes. This cleaning process was repeated three times. The washed three-dimensional structure was dried to obtain a porous membrane (average pore diameter: 116 μm) in which the three-dimensional structure was formed by intermolecular bonding between proteins and lipids, and this was used as a cell culture substrate. Furthermore, the sum of the dry masses of protein and lipids in the dry mass of the cell culture substrate was 77.3% by mass. In addition, the mass ratio of the dry mass of lipids in the dry mass of the cell culture substrate to the dry mass of protein in the dry mass of the cell culture substrate was 0.79.

[0057] (Example 1) into pure water, • Additive-free soy protein (manufactured by Kosei Co., Ltd.) 20.0 parts by mass, Canola oil (manufactured by Riken Agricultural Chemicals Co., Ltd.) 4.0 parts by mass, • Food-grade emulsifier (polysorbate 80) 0.5 parts by mass, A dispersion was prepared by adding [the specified ingredient]. The food components of the dispersion were as follows per 100g of the prepared dispersion: 17.5g of protein, 4.7g of lipids, 0.58g of carbohydrates, and 0.7g of other components. Except for using the prepared dispersion instead of unsweetened soy milk, the procedure was the same as in Comparative Example 1 to obtain a porous membrane (average pore size: 29 μm) with a three-dimensional structure formed by the Ramsden phenomenon, where proteins and lipids form a three-dimensional structure through intermolecular bonding. This was then used as a cell culture substrate. Furthermore, the sum of the dry masses of protein and lipids in the dry mass of the cell culture substrate was 94.5% by mass. In addition, the mass ratio of the dry mass of lipids in the dry mass of the cell culture substrate to the dry mass of protein in the dry mass of the cell culture substrate was 0.27.

[0058] (Methods for culturing animal cells) The procedures in this section were performed using aseptic techniques in a clean bench, and all instruments and reagents other than the prepared cell culture substrates were sterile. Cell culture substrates, cut to match the shape of the well bottoms, were laid on the well bottoms of 24-well plates that had been treated with a low-adhesion cell surface treatment. Next, 1 mL of phosphate-buffered saline (PBS) was added to each well. The mixture was then allowed to stand for 30 minutes to allow the cell culture substrate to absorb the PBS. After removing PBS from the wells, 1 mL of 70 v / v% ethanol aqueous solution was injected into each well. The cell culture substrate was then sterilized by allowing it to stand for 30 minutes. Finally, after removing the ethanol aqueous solution from the well, the well and the cell culture substrate were washed with PBS, and then any excess PBS was removed from the well. In the wells of the 24-well plate prepared as described above, a cell culture substrate was laid on the bottom surface of each well. In these wells, 1 mL of a suspension of adherent animal cells (mouse fibroblast cell line NIH3T3) suspended in DMEM medium (containing 10 v / v% fetal bovine serum and 1 v / v% penicillin-streptomycin) was seeded (cell count: 1.5 × 10⁵ cells / mL). The 24-well plates were then placed in an incubator at 37°C under a 5% CO2 atmosphere, and adherent animal cells were cultured overnight on a cell culture substrate. Subsequently, the cell culture substrate, along with the cultured adherent animal cells, was transferred to the bottom of the wells of a 6-well plate treated with a low-adhesion surface, and 3 mL of DMEM medium was added to each well. Culture was continued for another 11 days. During this period, the DMEM medium was completely replaced every 3 days.

[0059] (Method for identifying animal cells cultured on a cell culture substrate) The cell culture carriers, after being subjected to the (animal cell culture method) described above, were immersed in a 10 v / v% neutral buffered formalin aqueous solution and then incubated at room temperature for 30 minutes. After that, the cell culture carriers were removed from the neutral buffered formalin aqueous solution and washed with PBS. Next, the animal cells adhering to the cell culture substrate were subjected to dehydration and freeze-drying, electron microscope images were taken, and the form of the animal cells cultured on the main surface of the cell culture substrate was visually confirmed.

[0060] (Evaluation results) Comparative Example 1: As shown in Figure 1, after a total culture period of 12 days, the animal cells cultured on the cell culture substrate had rounded edges and were clustered together locally without extending pseudopods. Furthermore, in electron microscope images, more than half of the main surface of the cell culture substrate was exposed. This indicates that the cultured animal cells did not exhibit good adhesion to the cell culture substrate prepared in Comparative Example 1. Therefore, it was found that efficient culture of animal cells is not possible with the cell culture substrate prepared in Comparative Example 1.

[0061] Example 1: As shown in Figure 2, after a total culture period of 12 days, the cells cultured on the cell culture substrate were in a state with their pseudopods extended (spindle-shaped). Furthermore, in the electron microscope image, the main surface of the cell culture substrate was obscured by the presence of animal cells cultured on that surface. This indicates that the cultured animal cells showed good adhesion to the cell culture substrate prepared in Example 1. Therefore, it was found that the cell culture substrate prepared in Example 1 allows for efficient culture of animal cells.

[0062] From the above, it has been found that the cell culture substrate according to the present invention is a suitable cell culture substrate for cell culture. Furthermore, by using the cell culture substrate according to the present invention, it is possible to efficiently produce cultured meat. [Industrial applicability]

[0063] Animal cells such as muscle cells and liver cells can be cultured on the cell culture substrate of the present invention. Therefore, cultured meat can be provided by using the cell culture substrate according to the present invention. Furthermore, cells cultured on the cell culture substrate of the present invention can be used for cell assays of pharmaceuticals and for regenerative medicine applications such as prosthetic materials and cell fragments for transplantation. [Explanation of Symbols]

[0064] 1...Cell culture substrate 2...Open hole 3. Animal cells cultured on the main surface of a cell culture substrate.

Claims

1. A cell culture substrate comprising a three-dimensional structure containing proteins and lipids, A cell culture substrate in which the sum of the dry masses of proteins and lipids in the dry mass of the cell culture substrate is 80% by mass or more.

2. The cell culture substrate according to claim 1, wherein the mass ratio of the dry mass of the lipid in the dry mass of the cell culture substrate to the dry mass of the protein in the dry mass of the cell culture substrate is greater than 0 and less than 0.

79.

3. The cell culture substrate according to claim 1 or claim 2, wherein the three-dimensional structure is in the form of a membrane.

4. The cell culture substrate according to claim 1 or claim 2, wherein the three-dimensional structure has a porous structure.

5. The cell culture substrate according to claim 3, wherein the three-dimensional structure has a porous structure.

Images