Cell culture scaffold material, method for producing cell culture scaffold material, cell culture food, cell culture substrate, and cell culture method

Abstract

The present invention provides a cell culture scaffold material that can be suitably used in cell culture foods and can improve the adhesion between cells and the surface of the culture substrate. [Solution] A cell culture scaffold material containing an alkaline extract of coffee seeds. Also, a method for producing a cell culture scaffold material, comprising alkaline extracting coffee seeds, neutralizing the obtained alkaline extract with acid, and drying it. It is preferable to bring the alkaline extract into contact with the surface of the cell culture substrate, neutralize it with acid, and then adsorb or fix the coffee seed-derived protein contained in the alkaline extract to the surface of the cell culture substrate.

Description

Technical Field

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

Background Art

[0002] In recent years, technological developments related to the production of cell-cultured foods such as cultured meat have been progressing. Cell-cultured foods are expected not only to help solve problems such as food crises and food loss, but also to reduce livestock production and contribute to preventing global warming and the like. By the way, as a scaffold material for cell culture for culturing cells, proteins extracted mainly from animals have been conventionally used. Specifically, animal proteins such as collagen, laminin, and gelatin have been widely used to improve the adhesiveness between cells and the surface of the culture substrate.

[0003] In addition, as a scaffold material for cell culture, it has also been proposed to use a scaffold material made of synthetic resin instead of animal protein, and it has been reported that the growth efficiency of cells and the like are improved thereby. However, these scaffold materials have had the problem of not being suitable for the production of cell-cultured foods.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] In other words, while cell-cultured foods are expected to contribute to solving problems such as food crises and food loss, and to reducing livestock production, using animal protein as a scaffold material for cell culture was not appropriate. Furthermore, cell culture scaffolds made of chemically synthesized resins were not suitable for use in the manufacture of cell culture foods containing these scaffolds.

[0006] Therefore, the inventors diligently researched whether there were any plant proteins that could be used in cell culture foods that could be suitably used as a scaffold material for cell culture. They discovered that an extract obtained from the residue after hot water extraction of coffee seeds, much of which are treated as waste, is suitable as a scaffold material, and thus completed the present invention. Specifically, we found that a cell culture scaffold containing an alkaline extract of coffee seeds is suitable for adhering cells during culture.

[0007] Here, an example of an invention using coffee in cell culture is the stem cell culture container described in Patent Document 1. This document states that the stem cell culture container is equipped with a resin film containing synthetic resin as a scaffold material, and that the scaffold material may contain a crosslinking agent, and that caffeic acid can be used as the crosslinking agent. However, this invention uses a resin film containing synthetic resin as a scaffolding material, and does not disclose the use of coffee seeds as a raw material for cell culture scaffolding. Furthermore, there is neither description nor suggestion of cell culture scaffolding containing alkaline extracts of coffee seeds.

[0008] Furthermore, an example of an invention related to scaffolding materials using plant-based proteins is the adhesion enhancer containing edible plant-derived components described in Patent Document 2. However, this invention does not disclose the use of coffee seeds as a raw material for cell culture scaffolds, nor does it describe or suggest cell culture scaffolds containing alkaline extracts of coffee seeds.

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

[0010] To achieve the above objective, the cell culture scaffold material of the present invention is configured to contain an alkaline extract of coffee seeds. Furthermore, it is also preferable that the cell culture scaffold material of the present invention be configured such that the coffee seeds are the residue remaining after hot water extraction treatment of the coffee seeds.

[0011] The present invention provides a method for producing a cell culture scaffold material by alkaline extraction of coffee seeds, neutralizing the resulting alkaline extract with acid, and drying it. Furthermore, it is also preferable that the method for producing the cell culture scaffold material of the present invention involves contacting the alkaline extract with the surface of the cell culture substrate, neutralizing it with acid, and then adsorbing or immobilizing the coffee seed-derived protein contained in the alkaline extract onto the surface of the cell culture substrate. Furthermore, it is also preferable that the method for producing the cell culture scaffold material of the present invention uses coffee seeds as the residue remaining after hot water extraction of coffee seeds.

[0012] The cell culture food of the present invention comprises one of the above-mentioned cell culture scaffold materials and cultured cells. Furthermore, the cell culture substrate of the present invention has a configuration in which any of the above-mentioned cell culture scaffold materials is adsorbed or fixed to its surface. Furthermore, the cell culture method of the present invention is a method of culturing cells using any of the above-mentioned cell culture scaffold materials or cell culture substrates. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide a scaffold material for cell culture, a method for producing the scaffold material for cell culture, a cell culture food, a cell culture substrate, and a cell culture method, which can be suitably used for cell culture foods and can improve the adhesion between cells and the surface of the culture substrate.

Brief Description of the Drawings

[0014] [Figure 1] It is a figure showing the test result for confirming the adhesion between cells and the surface of the culture substrate using a scaffold material for cell culture etc. according to an embodiment of the present invention. [Figure 2] It is a figure showing a graph of the test result for confirming the adhesion between cells and the surface of the culture substrate using a scaffold material for cell culture etc. according to an embodiment of the present invention.

Modes for Carrying Out the Invention

[0015] Hereinafter, embodiments of the scaffold material for cell culture, the method for producing the scaffold material for cell culture, the cell culture food, the cell culture substrate, and the cell culture method of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments and the examples described later.

[0016] The scaffold material for cell culture of this embodiment is characterized by containing an alkaline extract of coffee seeds. In the scaffold material for cell culture of this embodiment, the coffee seeds are preferably the residue after hot water extraction treatment of coffee seeds. Also, as these coffee seeds, those subjected to hot water extraction treatment after roasting can be used. The hot water extraction treatment can be carried out, for example, by a conventional method of dripping coffee or by a method of stirring in hot water such as 90°C and removing the hot water by centrifugation.

[0017] There is a report that the amount of coffee waste in the country exceeds an estimated 800,000 tons / year. The scaffold material for cell culture of this embodiment can effectively utilize food waste and can be suitably used for cell culture foods, and can improve the adhesion between cells and the surface of the culture substrate. However, in the scaffold material for cell culture of the present embodiment, it is also possible to use unroasted coffee seeds, those not subjected to hot water extraction treatment, or those dried without roasting or hot water extraction treatment, etc.

[0018] Moreover, by processing the scaffold material for cell culture of the present embodiment from an alkaline extract of coffee seeds into forms such as films, fibers, non-woven fabrics, sponges, particles, etc., it is possible to obtain scaffold materials suitable for cell culture for various cell culture foods respectively. Furthermore, by coating the culture surface of a culture vessel with an alkaline extract of coffee seeds, it is also possible to obtain a culture vessel equipped with the scaffold material for cell culture of the present embodiment.

[0019] In the scaffold material for cell culture of the present embodiment, examples of alkalis that can be used for alkaline extraction include sodium hydroxide, calcium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, sodium hydrogen carbonate, trisodium phosphate, and alkaline electrolyzed water.

[0020] The manufacturing method of the scaffold material for cell culture of the present embodiment is characterized by subjecting coffee seeds to alkaline extraction treatment and neutralizing the obtained alkaline extract with an acid and drying it. Also, in the manufacturing method of the scaffold material for cell culture of the present embodiment, after bringing the alkaline extract into contact with the surface of the cell culture substrate, it is preferably neutralized with an acid to adsorb or fix the coffee seed-derived protein contained in the alkaline extract on the surface of the cell culture substrate.

[0021] By making the manufacturing method of the scaffold material for cell culture of the present embodiment like this, it is possible to suitably coat the surface of the cell culture substrate with the coffee seed-derived protein contained in the alkaline extract.

[0022] Furthermore, in the manufacturing method of the scaffold material for cell culture of the present embodiment, it is preferable that the coffee seeds are the residue after subjecting the coffee seeds to hot water extraction treatment.

[0023] Alkaline extracts of coffee seeds can be obtained, for example, as follows: The residue remaining after hot water extraction of coffee seeds is mixed with an aqueous sodium hydroxide solution and stirred with a stirrer for a predetermined time. Then, by centrifuging the resulting composition and collecting the supernatant, an alkaline extract of coffee seeds can be obtained.

[0024] Furthermore, in the method for producing cell culture scaffold material according to this embodiment, examples of alkalis that can be used for alkaline extraction include sodium hydroxide, calcium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, trisodium phosphate, and alkaline electrolyzed water. Furthermore, in the method for producing cell culture scaffold material according to this embodiment, examples of acids that can be used to neutralize coffee extract include hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, oxalic acid, tartaric acid, lactic acid, fumaric acid, malic acid, succinic acid, and acidic electrolyzed water.

[0025] According to the method for manufacturing cell culture scaffolds of this embodiment, it is possible to manufacture cell culture scaffolds that can be suitably used in cell culture foods and that can improve the adhesion between cells and the surface of the culture substrate.

[0026] The cell culture food of this embodiment is characterized by comprising the cell culture scaffold material of this embodiment described above and cultured cells. The cultured cells used in the cell culture food of this embodiment are not particularly limited, but for example, induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells) can be used. According to this embodiment of cell culture food, it is possible to obtain cell culture food obtained by properly adhering and culturing cultured cells on the surface of a cell culture substrate made of plant protein.

[0027] The cell culture substrate of this embodiment is characterized in that the cell culture scaffold material of this embodiment described above is adsorbed or fixed to its surface. The material for this cell culture substrate is not particularly limited, but for example, polyolefin resins such as polyethylene and polypropylene, or polystyrene can be suitably used. Furthermore, surface treatments such as oxidation treatment may be applied to the substrate to further enhance adhesion. According to this embodiment of the cell culture substrate, it is possible to properly adhere cells to the surface of the cell culture substrate.

[0028] The cell culture method of this embodiment is characterized by culturing cells using the cell culture scaffold material or cell culture substrate of this embodiment described above. According to this embodiment of the cell culture method, cells can be properly adhered to the surface of the cell culture substrate, and cells suitable for use in cell culture foods can be obtained. [Examples]

[0029] The following tests were conducted to confirm the effectiveness of the cell culture scaffold material according to the embodiment of the present invention.

[0030] [Example 1] Cell adhesion culture was performed using culture vessels coated with the cell culture scaffold material of this embodiment, and the adhesion of cells to the cell culture scaffold material of this embodiment was evaluated. Specifically, we did the following:

[0031] <Removal of water-soluble components derived from coffee> Coffee seeds were used as a raw material for cell culture scaffolds, and water-soluble components derived from coffee were removed from the coffee seeds by washing them with hot water. Specifically, 1g of coffee seed powder (commercially available drip coffee powder "A Little Luxurious Coffee Shop® Premium Drip Aromatic Deep Roast Kilimanjaro Blend," Ajinomoto AGF Co., Ltd.) and 50mL of ultrapure water were mixed and stirred at 90°C for 3 hours using a hot stirrer (AS ONE Corporation). Next, the obtained solution was centrifuged (4500 × g, 60 minutes) and the supernatant was removed. Furthermore, the same hot water washing procedure was repeated two more times (a total of three times) to obtain coffee seed powder from which water-soluble components had been removed.

[0032] <Alkaline extraction of coffee seeds> The entire amount of coffee seed powder obtained by the above procedure was mixed with 50 mL of a 0.05 M aqueous solution of NaOH (Fujifilm Wako Pure Chemical Industries, Ltd.) and stirred at 60°C for 3 hours using a hot stirrer (AS ONE Corporation). Next, the obtained composition was centrifuged (11000 × g, 60 minutes), and the supernatant was collected to obtain a sample of alkaline extract of coffee seeds containing alkali-soluble components (hereinafter sometimes referred to as coffee extract).

[0033] <Measurement of protein concentration in coffee extract> The protein concentration of coffee extract samples was measured using the BCA method. Specifically, a protein concentration measurement kit (TaKaRa BCA Protein Assay Kit, Takara Bio Inc.) was used. Following the instructions, the prepared BCA reaction solution and the sample to be measured, appropriately diluted with D-PBS (Nacalai Tesque Co., Ltd.), were mixed in equal volumes in a 96-well plate (351172, Corning). After standing at 37°C for 2 hours, the absorbance was measured using a microplate reader (Corona Electric Co., Ltd.) at a primary wavelength of 562 nm and a secondary wavelength of 447 nm to determine the protein concentration. Bovine serum albumin (BSA) included in the kit was used as a calibration curve. As a result, the concentration of coffee seed-derived protein in the coffee extract sample was 1.71 mg / mL.

[0034] <Preparation of coffee seed-derived protein solution> Next, a 0.05 M NaOH aqueous solution was added to the coffee extract sample to adjust the final concentration of coffee seed-derived protein to 1 mg / mL, thereby obtaining a coffee seed-derived protein solution.

[0035] <Coating with coffee seed-derived protein> Next, 50 μL of the prepared coffee seed-derived protein solution was placed into the wells of a 96-well plate (351172, Corning) and left to stand at 37°C for 1 hour. Furthermore, 50 μL of a 0.05 M aqueous solution of HCl (Nacalai Tesque Co., Ltd.) was added to the wells, and after standing at 37°C for 1 hour, the coffee seed-derived protein solution and the HCl aqueous solution were removed from the wells.

[0036] Then, the wells were washed with 100 μL of D-PBS (14249-95, Nacalai Tesque Co., Ltd.). By repeating the above process twice, coffee seed-derived protein was coated onto the wells.

[0037] <Cell adhesion culture> Next, cell culture was performed using well plates coated with coffee seed-derived protein as described above. Specifically, mouse-derived myoblasts (C2C12, K.A.C. Co., Ltd.) were used as the cells.

[0038] DMEM high-glucose (043-30085, Fujifilm Wako Pure Chemical Corporation) was used as the cell culture medium. 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.

[0039] After removing the supernatant from the cell suspension, a cell culture medium without bovine serum was added, resulting in a cell density of 2 × 10⁶. 4 The cells were diluted to a concentration of cells / mL to prepare a cell suspension. Next, 100 μL of cell suspension was placed in the wells of a well plate coated with coffee seed-derived protein, and the cells were cultured for adhesion in a 5% CO2 incubator at 37°C for 24 hours.

[0040] <Evaluation of cell adhesion> Next, we evaluated the adhesion of cells cultured using well plates coated with coffee seed-derived protein to the culture substrate surface. Then, the cultured cells were 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 a 20% ethanol aqueous solution was prepared to achieve a concentration of 0.2% of Crystal Violet. Then, after 24 hours of incubation, the cell culture medium was removed from the wells, 100 μL of cell staining solution was added to the wells, and the mixture was left to stand at room temperature for 10 minutes.

[0041] Next, the cell staining solution was removed from the wells, and the wells were washed with 100 μL of deionized water. This process of staining cells with the cell staining solution and washing the wells was repeated twice. Then, the wells were air-dried at room temperature to remove moisture.

[0042] Next, images of cells adhering to the bottom surface of the wells were obtained by photographing them using an optical microscope (CKX-41, Olympus Corporation). Then, by binarizing the images acquired using image analysis software (cellSens Dimension, Olympus Corporation), stained cells were extracted, and the cell adhesion area ratio (extracted area / (image width × image height), in pixels) was calculated.

[0043] The treatment described in Example 1 was repeated three times, and the average and standard deviation of the adhesion area ratio of the cells was calculated. Similarly, for Reference Example 1 and Comparative Examples 1-4, which will be described later, the treatments for Reference Example 1 and each Comparative Example were performed three times, and the average and standard deviation of the adhesion area ratio of the cells was calculated.

[0044] [Reference example 1] Cell adhesion culture was performed using culture vessels coated with gelatin, an animal protein, and the adhesion of cells to a cell culture scaffold containing gelatin was evaluated. Specifically, we did the following:

[0045] <Preparation of gelatin solution> As the gelatin solution, gelatin (892301, Nippi Corporation) was used, and deionized water was used to prepare the solution so that the final gelatin concentration was 2 mg / mL.

[0046] <Gelatin coating> Next, 100 μL of the prepared gelatin solution was placed into the wells of a 96-well plate (351172, Corning), and after standing at 37°C for 1 hour, the gelatin solution was removed from the wells.

[0047] The wells were also washed with 100 μL of D-PBS (14249-95, Nacalai Tesque Co., Ltd.). The gelatin was coated into the wells by repeating the above process twice. Then, similar to Example 1, cell adhesion culture was performed and the cell adhesion was evaluated.

[0048] [Comparative Example 1] Cell adhesion was evaluated by performing adherent culture of cells using untreated culture vessels that were not coated with cell culture scaffold materials such as proteins. A 96-well plate (351172, Corning) was used as the untreated culture vessel. Then, similar to Example 1, cell adhesion culture was performed and the cell adhesion was evaluated.

[0049] [Comparative Example 2] Cell adhesion culture was performed using culture vessels coated with cabbage-derived proteins contained in cabbage extract, and the adhesion of cells to a cell culture scaffold material containing alkaline cabbage extract (hereinafter sometimes referred to as cabbage extract) was evaluated. Specifically, we did the following:

[0050] <Alkaline extraction of cabbage> Cabbage (core) was mixed with a 0.05 M NaOH aqueous solution and stirred at 60°C for 3 hours using a hot stirrer (AS ONE Corporation). Next, the obtained composition was centrifuged (11000 × g, 60 minutes), and the supernatant was collected to obtain a sample of cabbage extract containing alkali-soluble components.

[0051] <Preparation of cabbage-derived protein solution> A 0.05 M NaOH aqueous solution was added to the obtained cabbage extract sample to adjust the final concentration of cabbage-derived protein to 1 mg / mL, thereby obtaining a cabbage-derived protein solution.

[0052] <Cabbage-derived protein coating> Next, 50 μL of the prepared cabbage-derived protein solution was placed into the wells of a 96-well plate (351172, Corning) and left to stand at 37°C for 1 hour. Furthermore, 50 μL of 0.05 M HCl aqueous solution was added to the wells and allowed to stand at 37°C for 1 hour, after which the cabbage-derived protein solution and the HCl aqueous solution were removed from the wells.

[0053] Then, the wells were washed with 100 μL of D-PBS (14249-95, Nacalai Tesque Co., Ltd.). By repeating the above process twice, the wells were coated with cabbage-derived protein. Then, similar to Example 1, cell adhesion culture was performed and the cell adhesion was evaluated.

[0054] [Comparative Example 3] Cell adhesion culture was performed using culture vessels coated with carrot-derived proteins contained in carrot extract, and the adhesion of cells to a cell culture scaffold material containing alkaline carrot extract (hereinafter sometimes referred to as carrot extract) was evaluated. Specifically, we did the following:

[0055] <Alkaline extraction of carrots> Carrots (core portion) were mixed with a 0.05 M NaOH aqueous solution and stirred at 60°C for 3 hours using a hot stirrer (AS ONE Corporation). Next, the obtained composition was centrifuged (11000 × g, 60 minutes), and the supernatant was collected to obtain a sample of carrot extract containing alkali-soluble components.

[0056] <Preparation of carrot-derived protein solution> A 0.05 M NaOH aqueous solution was added to the obtained carrot extract sample to prepare a carrot-derived protein solution with a final concentration of 1 mg / mL.

[0057] <Carrot-derived protein coating> Next, 50 μL of the prepared carrot-derived protein solution was placed into the wells of a 96-well plate (351172, Corning) and left to stand at 37°C for 1 hour. Furthermore, 50 μL of 0.05 M HCl aqueous solution was added to the wells and allowed to stand at 37°C for 1 hour, after which the carrot-derived protein solution and the HCl aqueous solution were removed from the wells.

[0058] Then, the wells were washed with 100 μL of D-PBS (14249-95, Nacalai Tesque Co., Ltd.). By repeating the above process twice, carrot-derived protein was coated onto the wells. Then, similar to Example 1, cell adhesion culture was performed and the cell adhesion was evaluated.

[0059] [Comparative Example 4] Cell adhesion culture was performed using culture vessels coated with citrus-derived proteins contained in citrus extracts, and the adhesion of cells to a cell culture scaffold material containing alkaline citrus extracts (hereinafter sometimes referred to as citrus extracts) was evaluated. Specifically, we did the following:

[0060] <Alkaline extraction of citrus fruits> Citrus fruits (exocarp of Iyokan orange) were mixed with a 0.05 M NaOH aqueous solution and stirred at 60°C for 3 hours using a hot stirrer (AS ONE Corporation). Next, the obtained composition was centrifuged (11000 × g, 60 minutes), and the supernatant was collected to obtain a sample of citrus extract containing alkali-soluble components.

[0061] <Preparation of citrus-derived protein solution> A 0.05 M NaOH aqueous solution was added to the obtained citrus extract sample to prepare a solution with a final concentration of citrus-derived protein of 1 mg / mL, thereby obtaining a citrus-derived protein solution.

[0062] <Coating with citrus-derived protein> Next, 50 μL of the prepared citrus-derived protein solution was placed into the wells of a 96-well plate (351172, Corning) and left to stand at 37°C for 1 hour. Furthermore, 50 μL of 0.05 M HCl aqueous solution was added to the wells and allowed to stand at 37°C for 1 hour, after which the citrus-derived protein solution and the HCl aqueous solution were removed from the wells.

[0063] Then, the wells were washed with 100 μL of D-PBS (14249-95, Nacalai Tesque Co., Ltd.). By repeating the above process twice, citrus-derived proteins were coated onto the wells. Then, similar to Example 1, cell adhesion culture was performed and the cell adhesion was evaluated.

[0064] 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 material containing coffee extract from Example 1 was 34.2%, with a standard deviation of 0.2. Furthermore, the average cell adhesion area ratio of the well plates coated with the gelatin-containing cell culture scaffold material of Reference Example 1 was 39.2%, with a standard deviation of 4.4.

[0065] Furthermore, the average cell adhesion area ratio of the well plates in Comparative Example 1 that were not coated with the cell culture scaffold material was 20.2%, with a standard deviation of 2.5. Furthermore, the average cell adhesion area ratio of the well plates coated with the cell culture scaffold material containing cabbage extract in Comparative Example 2 was 9.7%, with a standard deviation of 2.3. Furthermore, the average cell adhesion area ratio of the well plates coated with the cell culture scaffold material containing carrot extract in Comparative Example 3 was 8.5%, with a standard deviation of 1.7. Furthermore, the average cell adhesion area ratio of the well plates coated with the cell culture scaffold material containing citrus extract in Comparative Example 4 was 8.0%, with a standard deviation of 0.3.

[0066] These results indicate that the well plate coated with the cell culture scaffold material containing coffee extract in Example 1 showed no significant difference in adhesion area ratio compared to the well plate coated with the cell culture scaffold material containing gelatin in Reference Example 1, which used animal protein, and that it could achieve roughly the same effect.

[0067] Furthermore, the well plate coated with the cell culture scaffold material containing coffee extract in Example 1 showed an improvement of approximately 70% in the cell adhesion area ratio compared to the well plate without the cell culture scaffold material coating in Comparative Example 1. Furthermore, the well plates coated with the cell culture scaffold material containing coffee extract from Example 1 showed a 3.5 to 4.3 times improvement in cell adhesion area compared to the well plates of Comparative Examples 2 to 4.

[0068] Thus, it was found that the cell culture scaffold material containing coffee extract according to this embodiment, while containing plant-based proteins, is comparable to scaffold materials containing animal-based proteins in its ability to improve adhesion between cells and the surface of the culture substrate. Furthermore, it was revealed that the cell culture scaffold material containing coffee extract according to this embodiment can obtain a superior cell adhesion effect compared to the other plant protein-containing scaffold materials described above.

[0069] The present invention is not limited to the embodiments and examples described above, and it goes without saying that various modifications can be made within the scope of the present invention. For example, it is possible to make appropriate modifications, such as using coffee seeds that have not undergone hot water extraction treatment. [Industrial applicability]

[0070] This invention can be effectively utilized to produce cell culture scaffolds that can be suitably used in cell culture foods and improve the adhesion between cells and the surface of the culture substrate, by making effective use of the residue left after hot water extraction of coffee seeds, which is often considered waste.

Claims

1. A cell culture scaffold material characterized by containing an alkaline extract of coffee seeds.

2. The cell culture scaffold material according to claim 1, characterized in that the coffee seeds are the residue after hot water extraction treatment of coffee seeds.

3. A method for producing a cell culture scaffold, characterized by subjecting coffee seeds to an alkaline extraction treatment, neutralizing the resulting alkaline extract with acid, and drying it.

4. A method for producing a cell culture scaffold according to claim 3, characterized in that the alkaline extract is brought into contact with the surface of the cell culture substrate, and then neutralized with an acid to adsorb or fix the coffee seed-derived protein contained in the alkaline extract to the surface of the cell culture substrate.

5. The method for producing a cell culture scaffold according to claim 3 or 4, characterized in that the coffee seeds are the residue after hot water extraction of the coffee seeds.

6. A cell culture food characterized by comprising a cell culture scaffold material according to claim 1 or 2 and cultured cells.

7. A cell culture substrate characterized in that the cell culture scaffold material described in claim 1 or 2 is adsorbed or fixed to the surface.

8. A cell culture method characterized by culturing cells using the cell culture scaffold material described in claim 1 or 2, or the cell culture substrate described in claim 7.

Images