Cell culture scaffold material, production method for cell culture scaffold material, cell-cultured food product, cell culture substrate, and cell culture method

Abstract

Provided is a cell culture scaffold material that is suitable for use for a cell-cultured food product and improves adhesion between cells and the surface of a culture substrate. A cell culture scaffold material according to the present invention comprises an acid- or alkali-soluble protein derived from tea leaves. The molecular weight of the protein is preferably at least 3,000. The protein preferably also includes glutelin and / or prolamin.

Description

Scaffold for cell culture, method for manufacturing scaffold for cell culture, cell culture food, cell culture substrate, and cell culture method

[0001] The present invention relates to a cell culture technique, and more particularly to a cell culture scaffold for culturing cells for cell-cultured foods.

[0002] In recent years, technological developments related to the production of cell-cultured foods, such as cultured meat, have progressed. Cell-cultured foods are expected to not only help resolve issues such as food shortages and food waste, but also reduce livestock production and contribute to preventing global warming. Conventionally, cell culture scaffolds for culturing cells have primarily been made from proteins extracted from animals. Specifically, animal proteins such as collagen, laminin, and gelatin have been widely used to improve adhesion between cells and the surface of the culture substrate.

[0003] It has also been proposed to use scaffolds made of synthetic resins instead of animal proteins as scaffolds for cell culture, and it has been reported that this improves cell proliferation efficiency, etc. However, these scaffolds have the problem of being unsuitable for the production of cell-cultured foods.

[0004] Japanese Patent Application Laid-Open No. 2022-159216 Special Publication No. 2020-527054

[0005] In other words, while cell-cultured foods are expected to contribute to resolving issues such as food shortages and food waste, as well as reducing livestock production, it was inappropriate to use animal protein in cell culture scaffolds.Furthermore, it was inappropriate to use cell culture scaffolds made from synthetic resins in the production of cell-cultured foods that contain scaffolds.

[0006] The present inventors therefore conducted extensive research into whether there were any plant proteins suitable for use in cell-cultured foods that could be used as scaffolds for cell culture, and discovered that proteins extracted from the residue of hot water extraction of tea leaves, much of which is disposed of as waste, are suitable for use as scaffolds, leading to the completion of the present invention. Specifically, they discovered that a cell culture scaffold made from tea leaf-derived proteins, which are soluble in either acid or alkali, is suitable for cell adhesion culture.

[0007] Here, an example of technology related to scaffolds using vegetable proteins is the adhesion promoter described in Patent Document 1. This invention describes the use of legume seeds, such as soybeans, as vegetable proteins. The examples also describe results using tea extract. However, the specific material used was an extract containing 40% polyphenols obtained by hot water extraction, which did not use proteins soluble in acid or alkali, and thus exhibited poor adhesion between cells and the surface of the culture substrate.

[0008] Furthermore, Patent Document 2 discloses a cultured meat composition using soy protein. However, it does not mention or suggest the use of tea leaf-derived protein as the vegetable protein, nor does it disclose the use of an acid- or alkali-soluble protein as the vegetable protein.

[0009] The present invention has been made in consideration of the above circumstances, and aims to provide a cell culture scaffold that can be suitably used in cell culture foods and that can improve adhesion between cells and the surface of a culture substrate, a method for manufacturing a cell culture scaffold, a cell culture food, a cell culture substrate, and a cell culture method.

[0010] To achieve the above object, the scaffold for cell culture of the present invention is composed of a protein derived from tea leaves, the protein being soluble in acid or alkali. It is also preferable that the molecular weight of the protein in the scaffold for cell culture of the present invention is 3,000 or more.

[0011] The scaffold for cell culture of the present invention is also preferably configured so that the protein contains glutelin and / or prolamin.The scaffold for cell culture of the present invention is also preferably configured so that the tea leaves are residues obtained after hot water extraction of tea leaves.

[0012] It is also preferable that the cell culture scaffold of the present invention is configured such that the residue is one that has been stored at room temperature for one week or more.It is also preferable that the cell culture scaffold of the present invention is configured such that it does not contain catechins.Furthermore, it is also preferable that the cell culture scaffold of the present invention is configured such that the above-mentioned cell culture scaffolds are combined in various ways.

[0013] The method for producing a scaffold for cell culture of the present invention is a method for subjecting tea leaves to an alkaline extraction treatment, neutralizing the resulting tea-leaf-derived protein solution with an acid, and drying the solution. Another preferred method for producing a scaffold for cell culture of the present invention is a method for subjecting tea leaves to an alkaline extraction treatment, and then ultrafiltrating the resulting extract to adjust the tea-leaf-derived protein in the protein solution to have a molecular weight of 3,000 or more.

[0014] In addition, the method for producing a scaffold for cell culture of the present invention is also preferably a method in which the protein solution is brought into contact with the surface of a cell culture substrate, and then the protein solution is neutralized with an acid to adsorb or fix the tea leaf-derived protein in the protein solution to the surface of the cell culture substrate.In addition, the method for producing a scaffold for cell culture of the present invention is also preferably a method in which the tea leaves are residues obtained after hot water extraction of tea leaves.

[0015] The cell culture food of the present invention comprises any one of the scaffolds for cell culture described above and cultured cells. The cell culture substrate of the present invention has any one of the scaffolds for cell culture described above adsorbed or fixed to its surface. The cell culture method of the present invention is a method for culturing cells using any one of the scaffolds for cell culture described above or the cell culture substrate described above.

[0016] According to the present invention, it is possible to provide a cell culture scaffold that can be suitably used in cell culture foods and that can improve adhesion between cells and the surface of a culture substrate, a method for manufacturing a cell culture scaffold, a cell culture food, a cell culture substrate, and a cell culture method.

[0017]

[0023] Fig. 1 is a table showing the results of Test 1 for confirming the adhesiveness between cells and the surface of a culture substrate using a cell culture scaffold according to an embodiment of the present invention and a cell culture scaffold made from other various protein materials. Fig. 2 is a graph showing the results of Test 1 for confirming the adhesiveness between cells and the surface of a culture substrate using a cell culture scaffold according to an embodiment of the present invention and a cell culture scaffold made from other various protein materials. Fig. 3 is a table showing the results of Test 2 for confirming the effect of the adhesiveness between cells and the surface of a culture substrate based on the molecular weight fraction of a cell culture scaffold according to an embodiment of the present invention. Fig. 4 is a graph showing the results of Test 3 for confirming the effect of three-dimensional culture using a cell culture scaffold according to an embodiment of the present invention.

[0018] Hereinafter, embodiments of the cell culture scaffold, the method for manufacturing the cell culture scaffold, the cell culture food, the cell culture substrate, and the cell culture method of the present invention will be described in detail, but the present invention is not limited to the specific contents of the following embodiments and examples described later.

[0019] The cell culture scaffold of this embodiment is characterized in that it is made of tea leaf-derived proteins, which are acid- or alkali-soluble proteins. Examples of tea leaf-derived proteins include albumin, globulin, glutelin, and prolamin, as shown in Table 1 below.

[0020]

[0021] Furthermore, as described above, examples of proteins derived from tea leaves that are soluble in acid or alkali include albumin, globulin, glutelin, and prolamin, with glutelin and / or prolamin being preferred.

[0022] In the cell culture scaffold of this embodiment, the tea leaves are preferably the residue obtained after hot water extraction of tea leaves. It is also preferable to use the dried residue obtained after hot water extraction of tea leaves. The hot water extraction treatment can be selected as appropriate, but for example, treatment can be performed in heated water (pure water) at 70°C to 90°C for 2 to 180 minutes.

[0023] Generally, the residue left after hot water extraction of tea leaves is used as fertilizer, animal feed, etc., but most of it is disposed of as waste. In this situation, the scaffold for cell culture of the present embodiment uses the residue left after hot water extraction of tea leaves, which can be suitably used for cell culture foods and can improve the adhesion between cells and the surface of the culture substrate.

[0024] When tea leaves are extracted with hot water, albumin is eluted, while other components remain in the residue. By treating this residue with an acid or alkali, a protein solution containing tea leaf-derived proteins such as glutelin components can be obtained as a tea leaf extract. This tea leaf-derived protein solution is sometimes referred to as a tea leaf-derived protein solution before fractionation.

[0025] A culture vessel equipped with the cell culture scaffold of this embodiment can be obtained by coating the culture surface of the culture vessel with the protein solution containing the obtained tea leaf-derived protein. Also, by manufacturing the cell culture scaffold of this embodiment by processing the tea leaf-derived protein into films, particles, fibers, nonwoven fabrics, sponges, etc., it is possible to obtain a cell culture scaffold suitable for cell culture for cell-cultured foods.

[0026] In the scaffold for cell culture of this embodiment, examples of alkalis include sodium hydroxide, calcium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, trisodium phosphate, alkaline electrolyzed water, etc. In addition, in the scaffold for cell culture of this embodiment, examples of acids include hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, oxalic acid, tartaric acid, lactic acid, fumaric acid, malic acid, succinic acid, acidic electrolyzed water, etc.

[0027] In the cell culture scaffold of this embodiment, the tea leaf-derived protein solution obtained by the above-mentioned acid or alkali treatment may be used as is, but the molecular weight of the tea leaf-derived protein is preferably 3,000 or more. The cell culture scaffold of this embodiment, which contains as its main component a tea leaf-derived protein solution after fractionation by ultrafiltration of the tea leaf-derived protein solution to contain only components with a molecular weight of 3,000 or more, can be suitably used in cell culture foods and can provide excellent adhesion between cells and the surface of the culture substrate. In particular, when a high molecular weight protein is contained, it is particularly preferable as a cell culture scaffold because it self-associates between molecules and forms a network through aggregation.

[0028] The cell culture scaffold of this embodiment preferably does not contain catechins. Tea leaves contain catechins such as epigallocatechin, but catechins are known to be cytotoxic. Therefore, a cell culture scaffold obtained by ultrafiltration of a tea leaf-derived protein solution to contain only components with a molecular weight of 3,000 or more has low molecular weight components such as catechins removed, making it suitable for use in cell culture foods.

[0029] The cell culture scaffold of this embodiment is preferably the residue of tea leaves after hot water extraction treatment and storage at room temperature for one week or more, more preferably one month or more, and even more preferably two months or more. Catechins contained in tea leaves are gradually removed from the tea leaves by oxidation. Therefore, if the residue is from a long period of storage at room temperature, the catechins contained in the tea leaves will have decreased.

[0030] The method for producing a scaffold for cell culture of this embodiment is characterized by subjecting tea leaves to an alkaline extraction treatment, neutralizing the resulting tea-leaf-derived protein solution with acid, and drying it. Furthermore, the method for producing a scaffold for cell culture of this embodiment preferably comprises ultrafiltration of the resulting extract after the alkaline extraction treatment of tea leaves, so that the tea-leaf-derived protein in the protein solution has a molecular weight of 3,000 or more.

[0031] Furthermore, in the method for producing a scaffold for cell culture of this embodiment, it is preferable to bring a protein solution into contact with the surface of a cell culture substrate, neutralize the protein solution with an acid, and adsorb or fix the tea leaf-derived protein in the protein solution to the surface of the cell culture substrate. Also, in the method for producing a scaffold for cell culture of this embodiment, it is preferable that the tea leaves are residues obtained after hot water extraction of tea leaves.

[0032] Specifically, the protein solution can be obtained, for example, as follows: The residue after hot water extraction of tea leaves is mixed with an aqueous sodium hydroxide solution and stirred for a predetermined time using a stirrer. The resulting composition is then centrifuged and the supernatant is collected to obtain the protein solution.

[0033] In the method for producing a scaffold for cell culture of this embodiment, alkalis that can be used include sodium hydroxide, calcium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, trisodium phosphate, alkaline electrolyzed water, etc. In the method for producing a scaffold for cell culture of this embodiment, acids that can be used include hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, oxalic acid, tartaric acid, lactic acid, fumaric acid, malic acid, succinic acid, acidic electrolyzed water, etc.

[0034] Furthermore, in the method for manufacturing a scaffold for cell culture of this embodiment, it is preferable to store the residue after hot water extraction of tea leaves at room temperature for one week or more before subjecting the residue to alkaline extraction treatment, more preferably to store the residue at room temperature for one month or more before subjecting the residue to alkaline extraction treatment, and even more preferably to store the residue at room temperature for two months or more before subjecting the residue to alkaline extraction treatment.

[0035] According to the method for manufacturing a scaffold for cell culture of this embodiment, it is possible to manufacture a scaffold for cell culture that can be suitably used in cell culture foods and that can improve the adhesion between cells and the surface of the culture substrate.

[0036] The cell-cultured food product of this embodiment is characterized by including the above-described cell culture scaffold of this embodiment and cultured cells. The cultured cells in the cell-cultured food product of this embodiment are not particularly limited, but may include, for example, induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). According to this cell-cultured food product of this embodiment, it is possible to obtain a cell-cultured food product in which cultured cells are appropriately adhered and cultured on the surface of a cell culture substrate made of plant protein.

[0037] The cell culture substrate of this embodiment is characterized in that the cell culture scaffold of this embodiment described above is adsorbed or immobilized on the surface. The material of this cell culture substrate is not particularly limited, but suitable materials include polyolefin resins such as polyethylene and polypropylene, and polystyrene. Furthermore, the substrate may be subjected to a surface treatment such as oxidation treatment to further enhance adhesiveness. The cell culture substrate of this embodiment allows cells to be properly adhered to the surface of the cell culture substrate.

[0038] The cell culture method of this embodiment is characterized by culturing cells using the above-described cell culture scaffold of this embodiment or the above-described cell culture substrate of this embodiment. According to such a cell culture method of this embodiment, cells can be appropriately adhered to the surface of the cell culture substrate, and cells suitable for use in cell-cultured foods can be obtained.

[0039] Tests to confirm the effects of the cell culture scaffolds according to the embodiments of the present invention were carried out as follows: [Test 1] Test 1 was carried out to confirm the adhesiveness between cells and the surface of the culture substrate when using the cell culture scaffolds according to the embodiments of the present invention and cell culture scaffolds made of various other protein materials.

[0040] [Example 1] Cell adhesion culture was carried out using a culture vessel coated with the scaffold for cell culture of the present embodiment, and the adhesiveness of the cells to the scaffold for cell culture of the present embodiment was evaluated. Specifically, the procedure was as follows.

[0041] <Extraction of tea leaf-derived proteins> Tea leaves were extracted with hot water, and the residue was then subjected to alkaline extraction to obtain a tea leaf extract sample containing tea leaf-derived proteins. Sencha tea was used as the tea leaves, and the residue after hot water extraction was dried to obtain dried tea leaves.

[0042] Next, 5 g of the dried tea leaves were mixed with 70 mL of a 0.05 M aqueous solution of NaOH (Fujifilm Wako Pure Chemical Industries, Ltd.), and the mixture was stirred for 3 hours at 60° C. using a hot stirrer (AS ONE Corporation). The resulting composition was then centrifuged (6,000×g, 60 minutes), and the supernatant was collected to obtain Sample 1, a tea leaf extract containing tea leaf-derived proteins (tea leaf-derived protein solution before fractionation).

[0043] <Measurement of Protein Concentration in Tea Leaf Extract> The protein concentration of tea leaf extract Sample 1 was measured by the BCA method. Specifically, a protein concentration measurement kit (TaKaRa BCA Protein Assay Kit, Takara Bio Inc.) was used. According to the manufacturer's instructions, the prepared BCA reaction solution and the target substance, appropriately diluted with phosphate buffer (Nacalai Tesque, Inc.), were mixed in equal amounts in a 96-well plate (351172, Corning Incorporated). After incubation at 37°C for 2 hours, the absorbance was measured using a microplate reader (Corona Electric Co., Ltd.) at a dominant wavelength of 562 nm and a sub-wavelength of 447 nm to determine the protein concentration. The bovine serum albumin (BSA) included in the kit was used for the calibration curve. The tea leaf-derived protein concentration in tea leaf extract Sample 1 was found to be 20.4 mg / mL.

[0044] <Preparation of Components with a Molecular Weight Cutoff of 10,000 or More by Ultrafiltration> Next, tea leaf extract Sample 1 was ultrafiltered to prepare tea leaf-derived proteins with a molecular weight of 10,000 or more. Specifically, tea leaf extract Sample 1 was placed in an ultrafiltration filter (Nanosep®, molecular weight cutoff 10,000, Cytiva) at 500 μL / container, and centrifuged (10,000×g, 40 minutes) to remove low molecular weight components.

[0045] At this time, an equal volume of 0.05 M NaOH aqueous solution to the volume of liquid transferred to the low molecular weight fraction was added to the high molecular weight component side, and centrifugation was repeated six times. Then, 150 μL of the resulting high molecular weight component liquid was mixed with 350 μL of D-PBS (14249-95, Nacalai Tesque, Inc.) to obtain Sample 2, a tea leaf extract (tea leaf-derived protein solution after fractionation) from which low molecular weight components had been removed. The calculated residual rate of low molecular weight components was 0.033% before ultrafiltration. Furthermore, the tea leaf-derived protein concentration of the high molecular weight component was measured by the BCA method and found to be 2.22 mg / mL.

[0046] <Preparation of Tea Leaf-Derived Protein Solution> Next, 0.05 M NaOH aqueous solution was added to tea leaf extract sample 2 so that the final concentration of the high molecular weight component tea leaf-derived protein was adjusted to 1 mg / mL.

[0047] <Coating with tea leaf-derived protein solution> Next, 50 μL of the prepared tea leaf-derived protein solution was placed in the wells of a 96-well plate (351172, Corning) and allowed to stand for 1 hour at 37° C. Furthermore, 50 μL of 0.05 M HCl aqueous solution was added to the wells and allowed to stand for 1 hour at 37° C. After that, the tea leaf-derived protein solution and HCl aqueous solution were removed from the wells.

[0048] The wells were then washed with 100 μL of D-PBS (14249-95, Nacalai Tesque, Inc.) The above process was repeated twice to coat the wells with the tea leaf-derived protein solution.

[0049] Cell adhesion culture and evaluation of adhesiveness Next, cell culture was performed using well plates coated with the tea leaf protein solution as described above, and the adhesiveness of the tea leaf protein solution between the cells and the surface of the culture substrate was evaluated. Specifically, mouse myoblast cells (C2C12, KAC Co., Ltd.) were used as the cells.

[0050] The cell culture medium used was DMEM high-glucose (043-30085, Fujifilm Wako Pure Chemical Industries, Ltd.). Cells cultured in a cell culture medium containing 10% bovine serum (S-FBS-NL-025, Cosmo Bio Co., Ltd.) were centrifuged, and the supernatant was removed.

[0051] After removing the supernatant, a cell culture medium containing no bovine serum was added to the cells, and the cell density was adjusted to 2 × 10 ４ Next, 100 μL of the cell suspension was placed in a well of a well plate coated with the tea leaf protein solution, and incubated at 37°C in 5% CO ２ The cells were cultured in an incubator for 24 hours as adherent cells.

[0052] The cultured cells were then stained and photographed, and the cell adhesion area ratio was calculated. Crystal violet (031-04852, Fujifilm Wako Pure Chemical Industries, Ltd.) was used as the cell staining solution, and the concentration of crystal violet was adjusted to 0.2% using a 20% aqueous ethanol solution. After 24 hours of culture, the cell culture medium was removed from the wells, and 100 μL of the cell staining solution was added to the wells and allowed to stand at room temperature for 10 minutes.

[0053] Next, the cell staining solution was removed from the wells, and the wells were washed with 100 μL of deionized water. This cell staining with the cell staining solution and washing of the wells were repeated twice. The wells were then air-dried at room temperature to remove moisture.

[0054] Next, the cells adhered to the well bottom were photographed using an optical microscope (CKX-41, Olympus Corporation) to obtain images. The images were then binarized using image analysis software (cellSens Dimension, Olympus Corporation) to extract stained cells and calculate the cell adhesion area ratio (extracted area / (image width × image height), unit: pixel).

[0055] Each treatment in Example 1 was performed three times to calculate the average and standard deviation of the cell adhesion area ratio. Similarly, for Comparative Examples 1 to 9 described below, each treatment in each comparative example was performed three times to calculate the average and standard deviation of the cell adhesion area ratio.

[0056] Comparative Example 1 Cell adhesion culture was performed using a culture vessel coated with a gelatin solution, and the adhesiveness of cells to a scaffold for cell culture made of gelatin was evaluated. Specifically, the procedure was as follows.

[0057] <Preparation of Gelatin Solution> Gelatin (892301, Nippi Corporation) was used as the gelatin solution, and prepared using deionized water so that the final gelatin concentration was 2 mg / mL.

[0058] <Coating with gelatin solution> Next, 100 μL of the prepared gelatin solution was placed in the wells of a 96-well plate (351172, Corning) and allowed to stand at 37° C. for 1 hour, after which the gelatin solution was removed from the wells.

[0059] The wells were then washed with 100 μL of D-PBS (14249-95, Nacalai Tesque, Inc.). The above process was repeated twice to coat the wells with the gelatin solution. Then, cell adhesion culture was performed in the same manner as in Example 1, and the cell adhesiveness was evaluated.

[0060] Comparative Example 2 Cell adhesion culture was performed using a culture vessel coated with a soybean-derived protein solution, and the adhesiveness of cells to the cell culture scaffold made of soybean-derived protein was evaluated. Specifically, the procedure was as follows.

[0061] <Preparation of soybean-derived protein solution> Soybean A (14619-37, Sunbright Co., Ltd.) was dissolved in 1 M NaCl aqueous solution. The resulting soybean-derived protein solution was sterilized by filtration using a membrane filter with a pore size of 0.2 μm (S6534, Sartorius Stedim Biotech GmbH), and then adjusted with 1 M NaCl aqueous solution so that the final concentration of the soybean-derived protein was 1 mg / mL.

[0062] <Coating with soybean-derived protein solution> Next, 50 μL of the soybean-derived protein solution prepared above was placed in a well of a 96-well plate (351172, Corning) and allowed to stand for 1 hour at 37° C. Furthermore, 50 μL of deionized water was added to the well, and the plate was allowed to stand for 1 hour at 37° C. After that, the soybean-derived protein solution and deionized water were removed from the well.

[0063] The wells were then washed with 100 μL of D-PBS (14249-95, Nacalai Tesque, Inc.). The above process was repeated twice to coat the wells with the soybean-derived protein solution. Cell adhesion culture was then performed in the same manner as in Example 1, and the cell adhesiveness was evaluated.

[0064] Comparative Example 3 Cell adhesion culture was performed using a culture vessel coated with a soybean-derived protein solution, and the adhesiveness of cells to the cell culture scaffold made of soybean-derived protein was evaluated. Specifically, the procedure was as follows.

[0065] <Preparation of soybean-derived protein solution> Soybean B (920, Sunbright Co., Ltd.) was dissolved in 1 M NaCl aqueous solution. The resulting soybean-derived protein solution was sterilized by filtration using a membrane filter with a pore size of 0.2 μm (S6534, Sartorius Stedim Biotech GmbH), and then adjusted with 1 M NaCl aqueous solution so that the final concentration of the soybean-derived protein was 1 mg / mL.

[0066] <Coating with soybean-derived protein solution> Next, 50 μL of the soybean-derived protein solution prepared above was placed in a well of a 96-well plate (351172, Corning) and allowed to stand for 1 hour at 37° C. Furthermore, 50 μL of deionized water was added to the well, and the plate was allowed to stand for 1 hour at 37° C. After that, the soybean-derived protein solution and deionized water were removed from the well.

[0067] The wells were then washed with 100 μL of D-PBS (14249-95, Nacalai Tesque, Inc.). The above process was repeated twice to coat the wells with the soybean-derived protein solution. Cell adhesion culture was then performed in the same manner as in Example 1, and the cell adhesiveness was evaluated.

[0068] Comparative Example 4 Cell adhesion culture was performed using a culture vessel coated with a wheat-derived protein solution, and the adhesiveness of cells to the cell culture scaffold made of wheat-derived protein was evaluated. Specifically, the procedure was as follows.

[0069] <Preparation of wheat-derived protein solution> Wheat A (V-75, Glico Nutrition Foods Co., Ltd.) was dissolved in 0.05 M NaOH aqueous solution. The resulting wheat-derived protein solution was sterilized by filtration using a membrane filter with a pore size of 0.2 μm (S6534, Sartorius Stedim Biotech GmbH), and then adjusted to a final wheat-derived protein concentration of 1 mg / mL using 0.05 M NaOH aqueous solution.

[0070] <Coating with wheat-derived protein solution> Next, 50 μL of the wheat-derived protein solution prepared above was placed in a well of a 96-well plate (351172, Corning) and allowed to stand for 1 hour at 37° C. Furthermore, 50 μL of 0.05 M HCl aqueous solution was added to the well and allowed to stand for 1 hour at 37° C. After that, the wheat-derived protein solution and HCl aqueous solution were removed from the well.

[0071] The wells were then washed with 100 μL of D-PBS (14249-95, Nacalai Tesque, Inc.). The above process was repeated twice to coat the wells with the wheat-derived protein solution. Cell adhesion culture was then performed in the same manner as in Example 1, and the cell adhesiveness was evaluated.

[0072] Comparative Example 5 Cell adhesion culture was performed using a culture vessel coated with a wheat-derived protein solution, and the adhesiveness of cells to the cell culture scaffold made of wheat-derived protein was evaluated.

[0073] <Preparation of wheat-derived protein solution> Wheat B (GMG K-75, Glico Nutrition Foods Co., Ltd.) was dissolved in 0.05 M NaOH aqueous solution. The resulting wheat-derived protein solution was sterilized by filtration using a membrane filter with a pore size of 0.2 μm (S6534, Sartorius Stedim Biotech GmbH), and then adjusted to a final wheat-derived protein concentration of 1 mg / mL using 0.05 M NaOH aqueous solution.

[0074] <Coating with wheat-derived protein solution> Next, 50 μL of the wheat-derived protein solution prepared above was placed in a well of a 96-well plate (351172, Corning) and allowed to stand for 1 hour at 37° C. Furthermore, 50 μL of 0.05 M HCl aqueous solution was added to the well and allowed to stand for 1 hour at 37° C. After that, the wheat-derived protein solution and HCl aqueous solution were removed from the well.

[0075] The wells were then washed with 100 μL of D-PBS (14249-95, Nacalai Tesque, Inc.). The above process was repeated twice to coat the wells with the wheat-derived protein solution. Cell adhesion culture was then performed in the same manner as in Example 1, and the cell adhesiveness was evaluated.

[0076] Comparative Example 6 Cell adhesion culture was performed using a culture vessel coated with a rice-derived protein solution, and the adhesiveness of cells to the cell culture scaffold made of rice-derived protein was evaluated. Specifically, the procedure was as follows.

[0077] <Preparation of rice-derived protein solution> Rice (P70, Oryza Oil & Fat Chemical Co., Ltd.) was dissolved in 0.05 M NaOH aqueous solution. The resulting rice-derived protein solution was sterilized by filtration using a 0.2 μm pore membrane filter (S6534, Sartorius Stedim Biotech GmbH), and then adjusted to a final rice-derived protein concentration of 1 mg / mL using 0.05 M NaOH aqueous solution.

[0078] <Coating with rice-derived protein solution> Next, 50 μL of the prepared rice-derived protein solution was placed in the wells of a 96-well plate (351172, Corning) and allowed to stand for 1 hour at 37° C. Furthermore, 50 μL of 0.05 M HCl aqueous solution was added to the wells and allowed to stand for 1 hour at 37° C. After that, the rice-derived protein solution and HCl aqueous solution were removed from the wells.

[0079] The wells were then washed with 100 μL of D-PBS (14249-95, Nacalai Tesque, Inc.). The above process was repeated twice to coat the wells with the rice-derived protein solution. Cell adhesion culture was then performed in the same manner as in Example 1, and the cell adhesiveness was evaluated.

[0080] Comparative Example 7 Cell adhesion culture was performed using a culture vessel coated with a pea-derived protein solution, and the adhesiveness of cells to the cell culture scaffold made of pea-derived protein was evaluated. Specifically, the procedure was as follows.

[0081] <Preparation of pea-derived protein solution> Peas (E 86 HV, Sunbright Co., Ltd.) were dissolved in 1 M NaCl aqueous solution. The resulting pea-derived protein solution was sterilized by filtration using a membrane filter with a pore size of 0.2 μm (S6534, Sartorius Stedim Biotech GmbH), and then adjusted with 1 M NaCl aqueous solution so that the final concentration of the pea-derived protein was 1 mg / mL.

[0082] <Coating with pea-derived protein solution> Next, 50 μL of the pea-derived protein solution prepared above was placed in a well of a 96-well plate (351172, Corning) and allowed to stand for 1 hour at 37° C. Furthermore, 50 μL of deionized water was added to the well, and the plate was allowed to stand for 1 hour at 37° C. After that, the pea-derived protein solution and deionized water were removed from the well.

[0083] The wells were then washed with 100 μL of D-PBS (14249-95, Nacalai Tesque, Inc.). The above process was repeated twice to coat the wells with the pea-derived protein solution. Cell adhesion culture was then performed in the same manner as in Example 1, and the cell adhesiveness was evaluated.

[0084] Comparative Example 8: Cell adhesion culture was performed using an untreated culture vessel that was not coated with a protein solution, and the cell adhesion was evaluated. A 96-well plate (351172, Corning) was used as the untreated culture vessel. Cell adhesion culture was then performed in the same manner as in Example 1, and the cell adhesion was evaluated.

[0085] Comparative Example 9 Cell adhesion culture was performed using a culture vessel coated with an egg white peptide solution, and the adhesiveness of cells to a cell culture scaffold made of egg white peptide was evaluated. Specifically, the procedure was as follows.

[0086] <Preparation of egg white peptide solution> Egg white peptide (EP-1, Kewpie Corporation) was dissolved in deionized water. The egg white peptide solution was sterilized by filtration using a membrane filter with a pore size of 0.2 μm (S6534, Sartorius Stedim Biotech GmbH), and then adjusted with deionized water to a final egg white peptide concentration of 1 mg / mL.

[0087] <Coating with egg white peptide solution> Next, 50 μL of the prepared egg white peptide solution was placed in a well of a 96-well plate (351172, Corning) and allowed to stand for 1 hour at 37° C. Furthermore, 50 μL of deionized water was added to the well, and the plate was allowed to stand for 1 hour at 37° C. After that, the egg white peptide solution and deionized water were removed from the well.

[0088] The wells were then washed with 100 μL of D-PBS (14249-95, Nacalai Tesque, Inc.). The above process was repeated twice to coat the wells with the egg white peptide solution. Cell adhesion culture was then performed in the same manner as in Example 1, and the cell adhesiveness was evaluated.

[0089] The results are shown in Figures 1 and 2. The average cell adhesion area ratio of the well plates coated with the cell culture scaffold made of tea leaf-derived protein in Example 1 was 45.7%, with a standard deviation of 4.8. The average cell adhesion area ratio of the well plates coated with the cell culture scaffold made of gelatin in Comparative Example 1 was 39.3%, with a standard deviation of 4.9.

[0090] The average cell adhesion area ratio of well plates coated with the scaffold for cell culture made of soybean-derived protein in Comparative Example 2 was 16.2%, with a standard deviation of 0.6. The average cell adhesion area ratio of well plates coated with the scaffold for cell culture made of soybean-derived protein in Comparative Example 3 was 10.5%, with a standard deviation of 0.8.

[0091] The average cell adhesion area ratio of well plates coated with the scaffold for cell culture made of wheat-derived protein in Comparative Example 4 was 7.6%, with a standard deviation of 1.5. The average cell adhesion area ratio of well plates coated with the scaffold for cell culture made of wheat-derived protein in Comparative Example 5 was 7.7%, with a standard deviation of 0.2.

[0092] The average cell adhesion area ratio of well plates coated with the cell culture scaffold made of rice-derived protein in Comparative Example 6 was 15.7%, with a standard deviation of 2.9. The average cell adhesion area ratio of well plates coated with the cell culture scaffold made of pea-derived protein in Comparative Example 7 was 24.5%, with a standard deviation of 1.8.

[0093] The average cell adhesion area ratio of well plates not coated with the scaffold for cell culture of Comparative Example 8 was 28.6%, with a standard deviation of 3.1. The average cell adhesion area ratio of well plates coated with the scaffold for cell culture made of egg white peptide of Comparative Example 9 was 43.9%, with a standard deviation of 0.6.

[0094] These results show that the well plate coated with the cell culture scaffold material made of tea leaf-derived protein in Example 1 was able to improve the cell adhesion area rate by approximately 60% compared to the well plate not coated with the cell culture scaffold material in Comparative Example 8.

[0095] Furthermore, it was found that a well plate coated with the cell culture scaffold made of tea leaf-derived protein in Example 1 achieved a higher cell adhesion area ratio than a well plate coated with a cell culture scaffold made of gelatin using animal protein in Comparative Example 1, or a well plate coated with a cell culture scaffold made of egg white peptide in Comparative Example 9. Thus, it was revealed that the cell culture scaffold of this embodiment can further improve the adhesion between cells and the surface of the culture substrate, even though it is made of plant protein, without being inferior to scaffolds made of animal protein.

[0096] [Test 2] Test 2 was carried out as follows to confirm the effect of the adhesiveness between cells and the surface of the culture substrate based on the molecular weight fraction of the cell culture scaffold according to an embodiment of the present invention.

[0097] [Example 2] A tea leaf extract containing tea leaf-derived proteins and not subjected to ultrafiltration was prepared. Specifically, tea leaf-derived proteins were extracted in the same manner as in Example 1, and Sample 3, a tea leaf extract in which the tea leaf-derived protein components had not been subjected to ultrafiltration (tea leaf-derived protein solution before fractionation), was obtained.

[0098] A 0.05 M NaOH solution was then added to the solution of Sample 3 to adjust the final concentration of tea leaf-derived protein to 1 mg / mL. Cell adhesion culture was then performed in the same manner as in Example 1, and the adhesion between the cells and the surface of the culture substrate using the tea leaf-derived protein solution was evaluated. The concentration of tea leaf-derived protein in Sample 3 was measured by the BCA method and found to be 8.63 mg / mL.

[0099] Each treatment in Example 2 was performed three times to calculate the average and standard deviation of the cell adhesion area ratio. Similarly, each treatment in Examples 3 and 4 and Comparative Examples 10 and 11 described below was performed three times to calculate the average and standard deviation of the cell adhesion area ratio.

[0100] Example 3 A tea leaf extract containing tea leaf-derived proteins with a molecular weight of 3,000 or greater was prepared. Specifically, using an ultrafiltration filter with a molecular weight cutoff of 3,000 (NanoSep®, Cytiva), tea leaf-derived proteins with a molecular weight of 3,000 or greater were prepared from Sample 3 of the tea leaf extract of Example 2 in the same manner as the ultrafiltration in Example 1, and Sample 4 was obtained as a tea leaf extract (tea leaf-derived protein solution after fractionation) from which low molecular weight components had been removed.

[0101] Next, 0.05 M NaOH aqueous solution was added to the solution of Sample 4 to adjust the final concentration of tea leaf-derived protein to 1 mg / mL. Then, similar to Example 1, cell adhesion culture was performed to evaluate the adhesion between the cells and the surface of the culture substrate using the tea leaf-derived protein solution. The concentration of the tea leaf-derived protein in the high molecular weight component contained in Sample 4 was measured by the BCA method and found to be 2.13 mg / mL.

[0102] Example 4 A tea leaf extract containing tea leaf-derived proteins with a molecular weight of 10,000 or greater was prepared. Specifically, using an ultrafiltration filter (NanoSep®, Cytiva) with a molecular weight cutoff of 10,000, tea leaf-derived proteins with a molecular weight of 10,000 or greater were prepared from Sample 3 of the tea leaf extract of Example 2 in the same manner as the ultrafiltration in Example 1, and Sample 5, a tea leaf extract from which low molecular weight components had been removed (tea leaf-derived protein solution after fractionation), was obtained.

[0103] Next, 0.05 M NaOH aqueous solution was added to the solution of Sample 5 to adjust the final concentration of tea leaf-derived protein to 1 mg / mL. Then, similar to Example 1, cell adhesion culture was performed to evaluate the adhesion between the cells and the surface of the culture substrate using the tea leaf-derived protein solution. The concentration of the tea leaf-derived protein in the high molecular weight component contained in Sample 5 was measured by the BCA method and found to be 1.37 mg / mL.

[0104] [Comparative Example 10] As in Comparative Example 1, a gelatin solution was prepared and coated, and cell adhesion culture was performed using a well plate coated with the gelatin solution, and the adhesiveness of cells to a cell culture scaffold made of gelatin was evaluated.

[0105] Comparative Example 11 As in Comparative Example 8, cell adhesion culture was carried out using an untreated well plate that was not coated with a protein solution, and the cell adhesiveness was evaluated.

[0106] The results are shown in Figures 3 and 4. The average cell adhesion area ratio of the well plates coated with the cell adhesion scaffold made of tea leaf-derived protein before fractionation in Example 2 was 50.9%, with a standard deviation of 3.4. The average cell adhesion area ratio of the well plates coated with the cell adhesion scaffold made of tea leaf-derived protein with a molecular weight cutoff of 3,000 in Example 3 was 63.4%, with a standard deviation of 2.2.

[0107] The average cell adhesion area ratio of well plates coated with the cell adhesion scaffold made of tea leaf-derived protein with a molecular weight cutoff of 10,000 in Example 4 was 58.7%, with a standard deviation of 5.4. The average cell adhesion area ratio of well plates coated with the cell culture scaffold made of gelatin in Comparative Example 10 was 39.2%, with a standard deviation of 4.4. The average cell adhesion area ratio of well plates not coated with the cell culture scaffold in Comparative Example 11 was 20.2%, with a standard deviation of 2.5.

[0108] These results demonstrate that a well plate coated with a cell culture scaffold made of tea leaf-derived protein before fractionation in Example 2 can improve the cell adhesion area rate compared to a well plate coated with a cell culture scaffold made of gelatin in Comparative Example 10 or a well plate not coated with a cell culture scaffold in Comparative Example 11.

[0109] Furthermore, it was found that well plates coated with a scaffold for cell culture made from tea leaf-derived proteins after fractionation in Examples 3 and 4 had a molecular weight cutoff of 3,000 or more, which removed catechins that have a negative effect on cells and water-soluble components that reduce cell adhesion, resulting in a higher cell adhesion area ratio than well plates coated with a scaffold for cell culture made from tea leaf-derived proteins before fractionation in Example 2.

[0110] [Test 3] Test 3 for confirming the effect of three-dimensional culture using the scaffold for cell culture according to an embodiment of the present invention was carried out as follows.

[0111] [Example 5] Cells were cultured using microcarriers coated with the scaffold for cell culture of the present embodiment, and the adhesiveness of the cells to the scaffold for cell culture of the present embodiment was evaluated. Specifically, the procedure was as follows.

[0112] <Preparation of tea leaf-derived protein solution> A 0.05 M aqueous NaOH solution was added to Sample 5 of the tea leaf-derived protein solution after fractionation obtained in Example 4 (containing tea leaf-derived proteins with a molecular weight of 10,000 or more) so that the final concentration of the high molecular weight components of the tea leaf-derived proteins was 1 mg / mL.

[0113] Next, 0.1 g of microcarriers (3779, Corning) and 0.5 mL of the prepared tea leaf protein solution were placed in a 1.5 mL sample tube, and 0.5 mL of 0.05 M HCl aqueous solution was added to the sample tube. The tube was stirred overnight at room temperature, then centrifuged (300 × g, 3 minutes) to remove the supernatant. 1 mL of D-PBS (14249-95, Nacalai Tesque) was then added, and the mixture was centrifuged (300 × g, 3 minutes) to remove the supernatant. This process was repeated three times to coat the microcarrier surface with the tea leaf protein solution.

[0114] Three-dimensional cell culture and evaluation of adhesiveness Next, cells were cultured using microcarriers coated with the tea leaf protein solution, and the adhesiveness between the cells and the microcarrier surface was evaluated. Specifically, mouse myoblast cells (C2C12, KAC Co., Ltd.) were used.

[0115] The cell culture medium used was DMEM high-glucose (043-30085, Fujifilm Wako Pure Chemical Industries, Ltd.). Cells were cultured in a cell culture medium containing 10% bovine serum (S-FBS-NL-025, Cosmo Bio Co., Ltd.) and centrifuged to remove the supernatant. Cell culture medium (DMEM high-glucose) without bovine serum was added to the cells from which the supernatant had been removed, and the cells were cultured at a cell density of 0.3 × 10. ４ The solution was diluted to 1000 cells / mL to prepare a cell suspension.

[0116] Next, 30 mL of the cell suspension was placed in a bioreactor (BWV-S03A, Able Co., Ltd.), and 300 microcarriers coated with the tea leaf-derived protein solution were added at 300 cells / mL. The cells were incubated at 37°C in 5% CO ２ After leaving the mixture in the incubator for 1 hour, the rotation speed of the bioreactor was set to 55 rpm, and stirring culture was carried out for 4 days.

[0117] Next, the cell suspension containing the microcarriers was centrifuged (300 × g, 3 minutes), the supernatant was removed, and the cells were washed with 2 mL of D-PBS (14249-95, Nacalai Tesque, Inc.), after which 1 mL of Accutase (12679-54, Nacalai Tesque, Inc.) was added and the cells were incubated at 37°C in 5% CO ２ The cells were detached from the microcarriers by leaving the plate in an incubator for 4 minutes.

[0118] Then, 1 mL of cell culture medium (DMEM high-glucose) was added and stirred, and the mixture was left to stand to allow the microcarriers to settle, after which the supernatant was collected. The number of viable cells in the supernatant was measured using a cell counter (NC-200, Chemometec).

[0119] Comparative Example 12: Three-dimensional cell culture was performed using microcarriers that were not coated with tea leaf-derived protein solution, and cell adhesion was evaluated. Microcarriers (3779, Corning) were used as the three-dimensional culture carrier. Then, three-dimensional cell culture was performed in the same manner as in Example 5, and cell adhesion was evaluated.

[0120] The results are shown in Figure 5. The number of cells on the microcarrier coated with the scaffold for cell culture made of tea leaf-derived protein in Example 5 was 183.2 x 10 ４ The cell count of the uncoated microcarriers of Comparative Example 12 was 47.8 x 10 ４ cells / reactor.

[0121] These results demonstrate that the cell count of microcarriers coated on the surface with the cell culture scaffold made of tea leaf-derived protein in Example 5 was approximately 3.8 times higher than that of the uncoated microcarrier in Comparative Example 12. Thus, it has become clear that the cell culture scaffold of this embodiment can further improve the adhesion between cells and the surface of the microcarrier in three-dimensional culture, even when the scaffold is made of plant protein.

[0122] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the present invention. For example, it is possible to use tea leaves that have not been subjected to hot water extraction treatment.

[0123] The present invention effectively utilizes the residue after hot water extraction of tea leaves, much of which is considered waste, and can be suitably used in cell culture foods.It can also be suitably used when producing a cell culture scaffold material that can improve adhesion between cells and the surface of the culture substrate.

[0124] The contents of the documents cited in this specification and the Japanese application which is the basis for the Paris priority of this application are hereby incorporated by reference in their entirety.

Claims

1. A scaffold for cell culture, comprising a protein derived from tea leaves, the protein being soluble in acid or alkali.

2. A scaffold for cell culture according to claim 1, characterized in that the molecular weight of said protein is 3,000 or more.

3. A scaffold for cell culture according to claim 1 or 2, characterized in that the protein contains glutelin and / or prolamin.

4. A scaffold for cell culture according to claim 1 or 2, characterized in that the tea leaves are the residue after tea leaves have been extracted with hot water.

5. A scaffold for cell culture according to claim 1 or 2, characterized in that the residue has been stored at room temperature for one week or more.

6. A scaffold for cell culture according to claim 1 or 2, characterized in that it does not contain catechins.

7. A method for producing a scaffold for cell culture, comprising subjecting tea leaves to an alkaline extraction treatment, neutralizing the resulting tea leaf-derived protein solution with acid, and drying the resulting solution.

8. A method for producing a scaffold for cell culture according to claim 7, characterized in that after the alkaline extraction treatment of the tea leaves, the resulting extract is ultrafiltered to make the tea leaf-derived protein in the protein solution have a molecular weight of 3,000 or more.

9. A method for producing a scaffold for cell culture according to claim 7 or 8, characterized in that after the protein solution is brought into contact with the surface of the cell culture substrate, the protein solution is neutralized with an acid, and the tea leaf-derived protein in the protein solution is adsorbed or fixed to the surface of the cell culture substrate.

10. A method for producing a scaffold for cell culture according to claim 7 or 8, characterized in that the tea leaves are the residue after tea leaves have been subjected to a hot water extraction treatment.

11. A cell culture food comprising the scaffold for cell culture according to claim 1 or 2 and cultured cells.

12. A cell culture substrate having the scaffold material for cell culture according to claim 1 or 2 adsorbed or fixed to its surface.

13. A cell culture method comprising culturing cells using the scaffold for cell culture according to claim 1 or 2, or the cell culture substrate according to claim 12.

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