JP7514633B2 - Cell culture additive, medium and cell culture method - Google Patents

Description

The present invention relates to a cell culture additive, a culture medium, and a method for culturing cells.

When stem cells are cultured outside of the body, if they differentiate without maintaining their undifferentiated state, they cannot be differentiated into the desired cells, making it difficult to produce specific tissues or organs. In order to use stem cells in regenerative medicine, it is important to proliferate stem cells while maintaining their undifferentiated state.

One method for culturing stem cells while maintaining their undifferentiated state is to use cytokines. For example, mouse ES cells can be maintained in an undifferentiated state by adding leukemia inhibitory factor (LIF) to the culture medium. Some stem cells, such as primate ES cells and somatic stem cells, cannot maintain their undifferentiated state with LIF alone, and the undifferentiated state can be maintained by further adding fibroblast growth factor (FGF) to the culture medium. However, cytokines are expensive, and there are problems with the raw materials they are harvested from and storage, making them difficult to use continuously. Therefore, there is a demand for technology that can maintain the undifferentiated state of stem cells cheaply and efficiently. Patent documents 1 to 3 disclose technologies for maintaining the undifferentiated state of stem cells.

JP 2018-30787 A International Publication No. 2015/111734 JP 2013-247943 A

Patent Document 1 discloses an agent for maintaining the undifferentiated state of stem cells, which contains elastin peptides derived from the bulbus arteriosus of fish as an active ingredient. However, only one bulbus arteriosus (several grams) can be harvested from one bonito, for example. Patent Document 2 discloses a method for maintaining the undifferentiated state of mesenchymal stem cells using polysaccharides in the form of nanofibers. However, in order to achieve the purpose of maintaining the undifferentiated state in the culture, it is necessary to refine the shape of the nanofibers to have a specific average particle size, average fiber diameter, average fiber length, and average fiber length/average fiber diameter ratio, which is considered to require complicated work. Patent Document 3 discloses a method for maintaining the undifferentiated state of pluripotent stem cells in the culture on a substrate crosslinked with gelatin nanofibers, but the preparation of the substrate requires special operations, such as converting gelatin into nanofibers by electrospinning.

The present invention aims to provide a novel cell culture additive and medium capable of maintaining the undifferentiated state of stem cells, as well as a cell culture method.

The present invention relates to the following [1] to [14].
[1] An additive for cell culture containing crushed fish scales.
[2] The cell culture additive described in [1], wherein the fish scale blasted material is fish scale steam blasted material.
[3] The cell culture additive described in [1] or [2], wherein the fish scales are derived from barramundi.
[4] The cell culture additive according to any one of [1] to [3], which is a liquid or powder.
[5] The cell culture additive according to any one of [1] to [4], which is used for maintaining the undifferentiated state of stem cells.
[6] A culture medium containing crushed fish scales.
[7] The culture medium described in [6], wherein the fish scale crushed material is fish scale steam exploded material.
[8] The medium described in [6] or [7], wherein the fish scales are derived from barramundi.
[9] The medium according to any one of [6] to [8], wherein the fish scale crushed material is contained in an amount of 0.1% by mass or more and 10% by mass or less relative to the mass of the entire medium.
[10] The medium according to any one of [6] to [9], which is used for maintaining the undifferentiated state of stem cells.
[11] The medium described in [10], wherein the stem cells are pluripotent stem cells.
[12] The medium described in [11], wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
[13] A method for culturing cells, which uses the medium according to any one of [6] to [12].
[14] The culture method described in [13], in which the culture is carried out for 4 days or more without subculture.

The present invention provides a novel cell culture additive and medium capable of maintaining the undifferentiated state of stem cells, as well as a cell culture method.

FIG. 1 is a schematic diagram showing an example of a steam explosion apparatus. FIG. 2 is a diagram showing an example of a process for obtaining fish scale exploded material and its refined product from fish scales. FIG. 2 is a diagram showing the process for producing exploded fish scales in the examples. 13 shows optical microscope images of iPS cells obtained in Experiment 1. A: Cells were cultured in undifferentiated maintenance media containing 2.5% by mass, B: 1.25% by mass, C: 0.625% by mass, and D: 0% by mass of crushed fish scales. 13 shows ALP stained images of iPS cell colonies obtained in Experiment 2. A: 2.5% by mass, B: 1.25% by mass, C: 0.625% by mass, D: 0.312% by mass, E: 0% by mass of crushed fish scales. The cells were cultured in undifferentiated maintenance media containing the following: A: 2.5% by mass, B: 1.25% by mass, C: 0.625% by mass, D: 0.312% by mass, E: 0% by mass. This is a graph showing the mRNA expression levels of Oct3/4 (top row), Nanog (middle row), and Sox2 (bottom row) in iPS cells cultured for 7, 14, or 21 days in an undifferentiated maintenance medium containing crushed fish scales, obtained in Experiment 3. These are Oct4 immunostained images (left) and nuclear stained images (right) of iPS cell colonies obtained in Experiment 4. A-1 and A-2 are stained images when cells were cultured in undifferentiated maintenance media containing 2.5% by mass of crushed fish scales, B-1 and B-2 are stained images of 1.25% by mass, C-1 and C-2 are stained images of 0% by mass of crushed fish scales, and D-1 and D-2 are stained images of 0% by mass of crushed fish scales. These are optical microscope images of iPS cells obtained in Experiment 5, subcultured for 14 days after undifferentiation maintenance culture, and then cultured on the first day. A and B were cultured in an undifferentiation maintenance medium containing 1.25% by mass of crushed fish scales, and C and D were cultured in an undifferentiation maintenance medium containing 0% by mass of crushed fish scales. A and B, and C and D are representative colonies of iPS cells cultured under the respective conditions. 13 is a graph showing the mRNA expression levels of Oct3/4 (upper row), Nanog (middle row), and Sox2 (lower row) in iPS cells on the fourth day after passage following 14 days of undifferentiated maintenance culture, obtained in Experiment 6.

[Cell culture additives]
The cell culture additive of the present invention includes crushed fish scales. Stem cells cultured in the presence of the cell culture additive of the present invention tend to maintain an undifferentiated state, so the cell culture additive of the present invention can be used as an additive for maintaining the undifferentiated state of stem cells.

"Stem cells are maintained in an undifferentiated state" means that the stem cells can proliferate while retaining their differentiation ability. Whether or not the stem cells are maintained in an undifferentiated state can be evaluated by detecting the expression level of the mRNA or protein of the stem cell undifferentiation marker, for example, as shown in the Examples described below. Examples of the undifferentiation marker of pluripotent stem cells include Oct3/4, Nanog, Sox2, etc. Examples of methods for measuring the expression level of mRNA include RT-PCR, quantitative PCR, Northern blotting, etc. Examples of methods for measuring the expression level of protein include immunological methods such as ELISA using antibodies, flow cytometry, and Western blotting. When stem cells are cultured in the presence of the cell culture additive according to the present invention, if the expression level of the stem cell undifferentiation marker is maintained at a high level compared to when the cell culture additive is not used, the cell culture additive according to the present invention can be evaluated as having the ability to maintain the undifferentiation of stem cells.

The method of explosion of the fish scales is not particularly limited as long as the fish scales are exploded, but steam explosion is preferable from the viewpoint of being able to explode into low molecular weight materials in a short time. The fish scale steam explosion product can be obtained by subjecting the fish scales to a pressurized steam treatment under saturated steam, and then releasing it to atmospheric pressure and exploding it. When the fish scales are placed in a pressure vessel and steamed with high-pressure, high-temperature steam, the steam penetrates into the tissue of the fish scales. When the pressure is then instantly released, the steam expands rapidly and the fish scales are crushed. The fish scale steam explosion product can be obtained, for example, by the method described in International Publication No. 2017/002767.

Figure 1 shows an outline of a steam explosion device 10 as an example of an explosion device. A method for obtaining exploded fish scales will be specifically illustrated using Figure 1. The steam explosion device 10 may be a known device, and for example, an explosion device manufactured by Nippon Dentsu Co., Ltd. can be used.

The water from which impurities such as sodium, potassium, calcium, and magnesium have been removed by the water purifier 13 is sent to the boiler 12 by the pump 14, where it becomes high-temperature, high-pressure steam.
Fish scales, which are the raw material for the steam explosion product, are fed into the reactor 15 from a hopper 16. At this time, valve 17 is open, and valves 18 and 19 are closed. When a predetermined amount of fish scales is fed into the reactor 15, the valve 17 is closed. When the valve 18 connected to the boiler 12 is opened, high-temperature, high-pressure steam is introduced into the reactor 15, and pressure cooking is performed for a predetermined period of time. After that, the valve 18 is closed.
When the valve 19 is opened, the fish scales are suddenly discharged through the pipe 20 into the receiving tank 21. The fish scales are suddenly exposed to the atmosphere in the receiving tank 21 and are subjected to an explosion treatment. The receiving tank 21 may be equipped with a silencer 22. The steam-exploded fish scales are taken out from the outlet 23.

Fish scales are believed to contain collagen and calcium phosphate (ash) as the main components. Crushed fish scales are believed to contain at least one selected from the group consisting of collagen, gelatin, collagen peptides, and other collagen decomposition products. Collagen is the main protein that constitutes the body of animals, and is composed of three polypeptide chains (alpha chains, beta chains, etc.) with a molecular weight of about 100,000. Collagen is difficult to dissolve in water and is viscous at low temperatures. Gelatin is collagen that has been denatured by heating, changing its three-dimensional structure and molecular weight. Gelatin has a molecular weight of tens of thousands to hundreds of thousands, and dissolves in water when heated and becomes gel-like when cooled. Collagen peptides are gelatin with an even smaller molecular weight. Collagen peptides have a molecular weight of several hundred to several thousand, and have improved water solubility.

During the pressurized steaming process, by changing the conditions such as pressure, time, and temperature, it is possible to obtain exploded products with different collagen states. The processing temperature may be, for example, 150°C to 250°C, or 180°C to 220°C. The processing temperature may be, for example, 30 seconds to 1 hour, or 1 minute to 30 minutes. When the processing conditions are mild, the fish scales are decomposed into relatively high molecular weight substances. At this time, the molecular weight of the collagen contained in the exploded fish scales is large, and for example, a gel-like exploded product containing a lot of gelatin is obtained. When the processing conditions are moderate, the fish scales are decomposed into molecules of medium size. At this time, the collagen contained in the exploded fish scales is of medium size, and a liquid-like exploded product with viscosity is obtained. When the processing conditions are severe, the fish scales are decomposed into low molecular weight substances. At this time, the molecular weight of the collagen contained in the exploded fish scales is small, and for example, a liquid-like exploded product containing a lot of collagen peptides is obtained.

The crushed material may be purified as necessary, for example, by treating it with hydrogen peroxide for decolorization, by using activated carbon for decolorization and deodorization, or by centrifugal separation to remove impurities and undecomposed materials. It may be purified using ion exchange resin, ethanol, etc. The crushed material may be liquid, but may also be solid in consideration of ease of handling during storage and transportation. The solid may be, for example, a powder obtained by drying the liquid. The drying method is not particularly limited, and may be freeze-drying, hot air drying, spray drying, etc.

The raw materials used for industrial production of gelatin and collagen peptides have mainly been animal-derived materials such as cow bones, cowhide, pig skin, and chicken bones. However, zoonotic diseases such as Bovine Spongeform Encephalopathy (BSE) and avian influenza have become a problem. From the viewpoint of infectious diseases, it is preferable to use collagen derived from fish. In addition, although there are restrictions on the use of products containing substances derived from mammals (especially pigs) in some regions, substances derived from fish can be deployed in the halal market.

As for fish scales, the scales of marine or freshwater fish such as barramundi, tilapia, and sea bream can be used as the raw material. In general, the denaturation temperature of collagen in fish is 30°C or less, which is lower than the denaturation temperature of collagen in mammals (37°C to 40°C). As the raw material for the additive, it is preferable to use fish scales containing collagen with a relatively high denaturation temperature. Barramundi, which belongs to the family Acanthidae of the order Perciformes, has a high collagen denaturation temperature of about 37°C and is easy to handle. Barramundi is a large carnivorous fish distributed in the tropical regions of the Indo-Pacific Ocean, and is farmed in Southeast Asia for food. Barramundi scales are generated as waste at processing sites, so they can be obtained inexpensively.

Exploded material made from fish scales produces less odor than exploded material made from fish skin.

Fish scales that have been previously demineralized to chemically remove calcium and other components contained in the scales may be used. There are no particular limitations on the method of demineralization, but an example is a method in which washed fish scales are immersed in dilute hydrochloric acid for several hours to about two days. When demineralized fish scales are used as the raw material, the preferred conditions for the pressurized steaming process may differ from those when non-demineralized fish scales are used as the raw material.

The cells to be cultured are not limited to a particular type, but are preferably eukaryotic and include animal cells. The cells may be stem cells. The stem cells to be maintained in an undifferentiated state are not particularly limited as long as they are undifferentiated cells that have differentiation ability and the ability to self-proliferate while remaining in an undifferentiated state. Stem cells include pluripotent stem cells, multipotent stem cells, unipotent stem cells, etc., depending on their differentiation ability. The stem cells to which the cell culture additive of the present invention is applied are preferably pluripotent stem cells.

Pluripotent stem cells include ES cells, iPS cells, as well as embryonic germ cells (EG cells) derived from primordial germ cells, multipotent germline stem cells (mGS cells) isolated during the establishment and culture process of GS cells produced from testicular tissue, and multipotent adult progenitor cells (MAPCs) detected from bone marrow. The pluripotent stem cells are preferably ES cells or iPS cells.

The pluripotent stem cells may be established from any animal from which pluripotent stem cells can be established. The pluripotent stem cells are, for example, pluripotent stem cells derived from rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; felines such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees, and are preferably pluripotent stem cells derived from primates. When used in regenerative medicine, human iPS cells are preferred.

The cell culture additive of the present invention can be added to a medium, a substrate (base material), or other materials used in cell culture. Stem cells cultured in the presence of the cell culture additive of the present invention tend to maintain an undifferentiated state, so the cell culture additive of the present invention is preferably added to a medium for maintaining the undifferentiated state of stem cells, a substrate used to maintain the undifferentiated state of stem cells, etc.

When the cell culture additive is added to the medium, the concentration of crushed fish scales contained in the medium can be appropriately set within a range that allows cell culture, but from the viewpoint of effectively maintaining the undifferentiated state of stem cells, the final concentration (at the time of use) is, for example, 0.1% by mass or more and 10% by mass or less, preferably 0.5% by mass or more, more preferably 1.0% by mass or more, preferably 8% by mass or less, and more preferably 5% by mass or less, relative to the mass of the entire medium.

The medium can be prepared as a basal medium using a medium commonly used for cell culture, particularly for maintaining the undifferentiated state of stem cells. Examples of the basal medium include media containing components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, and fatty acids), specifically, Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), BGjB medium, CMRL 1066 medium, Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM/F-12), and Glasgow Minimum Essential Medium (MEM). Examples of such medium include Glasgow MEM, Hank's balanced salt solution, and Fischer's medium.

The medium may contain bFGF, LIF, epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, fibronectin, progesterone, selenite, B27 supplements, N2 supplements, ITS supplements, antibiotics, fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, etc., as necessary.

The medium may be a serum-containing medium or a serum-free medium, but from the viewpoint of ensuring the safety of cell transplantation by eliminating xenogeneic components, a serum-free medium is preferable. Here, serum-free medium means a medium that does not contain unconditioned or unpurified serum, and a medium that contains purified blood-derived components or animal tissue-derived components (e.g., growth factors) is considered to be serum-free medium. Examples of such serum-free medium include a medium to which an appropriate amount (e.g., 1-20%) of Knockout Serum Replacement (KSR) (manufactured by Thermo Fisher Scientific) has been added, a medium to which insulin and transferrin have been added, a medium to which cell-derived factors have been added, and the like.

The cell culture additive according to the present invention may be added to a substrate, and the substrate may be used as a coating agent for a culture vessel. The method of coating the culture vessel is not particularly limited, but for example, the part of the culture vessel to which the cells adhere may be immersed in a substrate containing crushed fish scales and incubated. The concentration of crushed fish scales contained in the substrate, the coating time, and the coating temperature may be appropriately set, and for example, a substrate containing 1% by mass of crushed fish scales relative to the total mass of the substrate may be used for incubation at 37°C for 30 minutes. After incubation, the substrate may be removed and a medium may be added, or a medium may be added so as to dilute the substrate. The medium may be a medium containing or not containing crushed fish scales.

[Culture medium]
The medium according to the present invention contains crushed fish scales. The medium containing crushed fish scales can maintain the undifferentiated state of stem cells. The medium containing crushed fish scales may be a medium to which the above-mentioned cell culture additive is added. The crushed fish scales may be the above-mentioned crushed fish scales. The medium preferably contains 0.1% by mass or more and 10% by mass or less of the crushed fish scales relative to the total mass of the medium. The stem cells may be the above-mentioned pluripotent stem cells, and are preferably ES cells or iPS cells.

[Cell culture method]
The cell culture method according to the present invention includes a step of culturing cells using the above-mentioned medium containing crushed fish scales. According to the cell culture method according to the present invention, stem cells can be cultured while maintaining their undifferentiated state.

In the cell culture method according to the present invention, the culture can be continued for 4 days or more without passaging. Proliferating stem cells are likely to lose their undifferentiated state unless they are normally passaged every 2 to 3 days. If stem cells are cultured using a medium containing crushed fish scales, the undifferentiated state is likely to be maintained even if passaging is not performed for a long period of time. The culturing period varies depending on the type of stem cells and the culture conditions, but for example, the cells can be cultured without passaging for 7 days or more, 14 days or more, or 21 days or more while maintaining their undifferentiated state. The period for culturing without passaging is, for example, within 30 days.

According to the cell culture method of the present invention, stem cells can be cultured for a long period of time without subculture. Compared to a method in which subculture is performed every 2 to 3 days, the subculture interval is longer, so a larger number of stem cells can be obtained by culture. Furthermore, even when stem cells are cultured using a medium containing crushed fish scales, they maintain an undifferentiated state and exhibit good differentiation ability even after approximately 80 subcultures, just like conventional stem cell culture methods.

The incubator used for cell culture is not particularly limited as long as it is capable of culturing cells, but examples include dishes, multi-well plates, flasks, petri dishes, chamber slides, tubes, trays, culture bags, roller bottles, etc.

The culture vessel may be either non-adhesive or adhesive, and is selected appropriately depending on the purpose. For the cell-adhesive culture vessel, one treated with a cell support substrate such as an extracellular matrix may be used in order to improve adhesion to cells. Examples of cell support substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin.

Culturing may or may not involve the use of feeder cells. When culturing cells to be used in regenerative medicine, it is preferable not to use feeder cells (feeder-free) from the viewpoint of safety after transplantation.

The cell density when seeding the cells is not particularly limited, but may be any density that allows the cells to be maintained and proliferated. In the case of stem cells, the density may be, for example, 1.0×10 ² to 1.0×10 ⁶ cells/cm ² , preferably 5.0×10 ² to 5.0×10 ⁵ cells/cm ² , and more preferably 1.0×10 ³ to 2.0×10 ⁵ cells/cm ² .

The cell culture conditions may be the same as those normally used for cell culture, and no special control is required. The culture temperature is, for example, about 30 to 40°C, preferably 36 to 37°C. The _CO2 gas concentration is, for example, about 1 to 10%, preferably about 2 to 5%. The medium is preferably replaced once every 2 to 3 days, and may be replaced daily. Medium replacement includes full medium replacement, half medium replacement, and medium addition.

The method for producing the exploded fish scale material shown in Figure 2 will be referred to to illustrate an example of the exploded fish scale material that can be used in the present invention. As the exploded fish scale material, the material immediately after the explosion process may be used, or a purified fraction may be used.

[Fish scale crushed material 1]
As the fish scales, barramundi (Lates calcarifer) scales were used which were washed, demineralized and dried.

The fish scales were placed in a steam explosion device and subjected to pressure steaming treatment at a temperature of 200°C for 15 seconds, 30 seconds or 1 minute at a pressure of 1.45 MPaG. After the fish scales were exploded, fish scale exploded material 1 was obtained. Distilled water was added in an amount 10 times that of each fish scale exploded material 1, and the mixture was heated at 60°C for 10 minutes, then centrifuged and the supernatant was collected. The supernatant became gel-like when cooled to 4°C and was liquid at room temperature. For the purpose of decolorization and deodorization, the supernatant was passed through an activated carbon column and roughly purified. The liquid that passed through the column was placed in a freeze dryer and freeze-dried (fraction 1).

20% hydrochloric acid was added to the sample obtained by freeze-drying the fish scale crushed material 1 obtained by 30 seconds of pressure steaming, and the sample was left in an electric furnace at 110 ° C for 24 hours. This breaks down the protein into amino acids. The acid-decomposed sample was placed in an evaporator and dried. The hydrochloric acid was removed during this process. 0.02N hydrochloric acid was added to make a 50 mL solution, and the solution obtained by removing impurities was subjected to amino acid analysis. The analysis device used was an automatic amino acid analyzer (L-8900BH manufactured by Hitachi High-Tech Science Corporation). When the components were analyzed, it was found to contain hydroxyproline, an amino acid unique to collagen. Furthermore, when the ratio of amino acids per 1000 residues was confirmed, glycine was about 1/3 of the total, and the total of alanine, hydroxyproline, and proline was about 1/3 of the total, showing a ratio unique to collagen. From the above results, it is believed that the fish scale crushed material 1 contains gelatin.

When the pressure steaming treatment time was 15 seconds, the decomposition of the fish scales was insufficient (yield 12.91%). When the pressure steaming treatment time was 30 seconds, the yield was 89.46%, and when the pressure steaming treatment time was 1 minute, the yield was 85.29% (first time) and 65.63% (second time). When the pressure steaming treatment time was 30 seconds and 1 minute, the obtained fish scale exploded material 1 was in a gel state immediately after the explosion treatment. When the pressure steaming treatment time was 1 minute (second time), in addition to the gel-like fish scale exploded material 1, liquid fish scale exploded material 1 was obtained immediately after the explosion treatment.

[Fish scale explosion material 1']
As fish scales, raw scales of barramundi that were only washed and not demineralized were used. The fish scales were placed in a steam explosion device and subjected to pressure steaming treatment at a temperature of 190 ° C for 30 seconds, 1 minute or 2 minutes at a pressure of 1.15 MPaG. After the fish scales were exploded, fish scale explosion product 1' was obtained. Distilled water was added in an amount 10 times that of each fish scale explosion product 1', and the mixture was heated at 60 ° C for 10 minutes, centrifuged, and the supernatant was collected. The supernatant became gel-like when cooled to 4 ° C, and was liquid at room temperature. For the purpose of decolorization and deodorization, the supernatant was passed through an activated carbon column and roughly purified. The column-passed liquid was placed in a freeze-dryer and freeze-dried (fraction 1).

A sample obtained by freeze-drying the exploded fish scale material 1' obtained after one minute of pressurized steaming was subjected to amino acid analysis in the same manner as for the exploded fish scale material 1, and it was found to contain hydroxyproline, an amino acid specific to collagen. Furthermore, when the ratio of amino acids per 1000 residues was confirmed, glycine accounted for approximately 1/3 of the total, and the total of alanine, hydroxyproline, and proline was approximately 1/3 of the total, showing ratios specific to collagen. From these results, it is believed that the exploded fish scale material 1' contains gelatin.

The sample obtained by freeze-drying the fish scale crushed material 1' obtained by pressure steaming for 1 minute was subjected to size exclusion chromatography using a TSKgel G2500PWXL (Tosoh Corporation) column. The results are shown in Table 1.

When the pressure steaming treatment time was 30 seconds, the yield was 32.33%, when the pressure steaming treatment time was 1 minute, the yield was 46.27%, and when the pressure steaming treatment time was 2 minutes, the yield was 54.57%. The fish scale explosion product 1' obtained by pressure steaming treatment for 30 seconds to 2 minutes was liquid immediately after the explosion treatment. Since the moisture content of raw scales is about 49%, the equivalent weight of the raw scales when dried was used as the basis for calculating the yield. When raw scales are used as the raw material, calcium phosphate, the main component of fish scales, is contained in the processed product after pressure steaming treatment, so the yield is lower than when dried fish scales that have been demineralized in advance are used as the raw material.

In addition, when the pressure steaming treatment time was 1 minute and the pressure steaming treatment temperature was 170°C, the yield was 21.29%, when the pressure steaming treatment temperature was 180°C, the yield was 36.64%, when the pressure steaming treatment temperature was 190°C, the yield was 46.27%, and when the pressure steaming treatment temperature was 200°C, the yield was 29.29%. The obtained fish scale exploded material 1' was liquid immediately after the explosion treatment.

[Fish scale explosion 2]
The fish scales were prepared by washing and demineralization in the same manner as for the fish scale explosion product 1. The fish scales were put into a steam explosion device and subjected to a pressurized steaming treatment at a temperature of 200°C for 2, 3, 5, 7.5, 10 or 15 minutes at a pressure of 1.45 MPaG. After the fish scales were exploded, the fish scale explosion product 2 was obtained. After removing the uncrushed fish scales by centrifugation, ethanol was added to the supernatant, and the precipitated components were precipitated by centrifugation. The collected precipitate was dissolved in distilled water, and the resulting aqueous solution was passed through an activated carbon column for the purpose of decolorization and deodorization, and crude purification was performed. The column-passed liquid was placed in a freeze-dryer and freeze-dried (fraction 2A).

A sample obtained by freeze-drying the crushed fish scales 2 obtained by five minutes of pressurized steaming was subjected to amino acid analysis in the same manner as for the exploded fish scales 1, and it was found to contain hydroxyproline, an amino acid specific to collagen. Furthermore, when the ratio of amino acids per 1,000 residues was confirmed, glycine accounted for approximately 1/3 of the total, and the total of alanine, hydroxyproline, and proline was approximately 1/3 of the total, showing ratios specific to collagen. It is believed that the exploded fish scales 2 contains substances derived from collagen (decomposition products). The exploded fish scales 2 also contained substances with molecular weights of 10,000 to 1,000,000.

An organic solvent (chloroform 4: butanol 1) was added to the aqueous solution of distilled water added to the ethanol precipitate of the exploded fish scales 2 obtained by 5 minutes of pressure steaming, and mixed. This separates the protein. The mixture was centrifuged to separate it into three layers: upper, middle, and lower. It is believed that the upper layer contains polysaccharides, the middle layer contains proteins, and the lower layer contains organic solvents. For the purpose of decolorization and deodorization, the upper layer was removed and passed through an activated carbon column for crude purification. The liquid that passed through the column was placed in a freeze dryer and freeze-dried to a solid form (fraction 2B).
Instead of an organic solvent, calcium chloride may be added in an amount of about 5% by mass of the aqueous solution to separate the protein. In this case, the mixture is centrifuged to separate it into two layers: polysaccharide solution/protein. The polysaccharide solution is then removed and lyophilized.

The upper layer solution was freeze-dried and the resulting sample was subjected to size exclusion chromatography using a TSKgel GMPW _XL column, revealing that the molecular weight (number average molecular weight) was in the range of several hundred to several tens of thousands. The results are shown in Table 2.

When the pressure steaming treatment time was 2 minutes, the yield was 63.66%, when the pressure steaming treatment time was 3 minutes, the yield was 65.14%, when the pressure steaming treatment time was 5 minutes, the yield was 54.53%, when the pressure steaming treatment time was 7.5 minutes, the yield was 53.94%, when the pressure steaming treatment time was 10 minutes, the yield was 44.05%, and when the pressure steaming treatment time was 15 minutes, the yield was 61.31%. The fish scale exploded material 2 obtained by pressure steaming treatment for 5 minutes or more was a viscous liquid immediately after the explosion treatment. This liquid did not gel at room temperature or 4°C. The fish scale exploded material 2 obtained by pressure steaming treatment for 3 minutes or less was liquid, but some of it was gelatinous.

[Fish scale explosion material 2']
As fish scales, raw scales of barramundi that were only washed and not demineralized were used. The fish scales were placed in a steam explosion device and subjected to a pressurized steam treatment at a temperature of 190 ° C. for 5 or 10 minutes at a pressure of 1.15 MPaG. After the fish scales were exploded, fish scale explosion product 2' was obtained. After removing the uncrushed fish scales by centrifugation, ethanol was added to the supernatant, and the precipitated components were precipitated by centrifugation. The collected precipitate was dissolved in distilled water, and the aqueous solution was passed through an activated carbon column for the purpose of decolorization and deodorization, and crude purification was performed. The column filtrate was placed in a freeze dryer, and the precipitate was freeze-dried (fraction 2A).

The precipitate formed by adding ethanol to the exploded fish scale material 2' obtained after five minutes of pressurized steaming was subjected to amino acid analysis in the same manner as for the exploded fish scale material 1, and was found to contain hydroxyproline, an amino acid specific to collagen. Furthermore, when the ratio of amino acids per 1,000 residues was confirmed, glycine was about 1/3 of the total, and the total of alanine, hydroxyproline, and proline was about 1/3 of the total, showing ratios specific to collagen. It is believed that the exploded fish scale material 2' contains substances derived from collagen (decomposition products). The exploded fish scale material 2' also contained substances with molecular weights of 10,000 to 1,000,000.

The ethanol precipitate of the exploded fish scales 2' obtained by pressure steaming for 5 minutes was mixed with distilled water to prepare an aqueous solution, to which calcium chloride (5% by mass of the aqueous solution) was added and heated at 80°C for 1 hour. This separates the protein. The mixture was centrifuged to separate into two layers, an upper layer and a lower layer. The upper layer is believed to contain polysaccharides, and the lower layer to contain protein. The upper layer of the solution was removed and freeze-dried to form a solid (fraction 2B).

When the pressure steaming treatment time was 5 minutes, the yield was 22.28%, and when the pressure steaming treatment time was 10 minutes, the yield was 16.69%. The fish scale exploded material 2' obtained by pressure steaming treatment for 5 or 10 minutes was a viscous liquid. The fish scale exploded material 2' did not gel at room temperature or at 4°C.

When the pressure-steaming temperature was 210°C and the pressure-steaming time was 1 minute, the yield was 17.07%. The resulting exploded fish scale material 2' was a viscous liquid immediately after the explosion process.

[Fish scale explosion 3]
The fish scales were washed and demineralized in the same manner as for the fish scale explosion product 1. The fish scales were placed in a steam explosion device and subjected to a pressurized steam treatment at a temperature of 200°C for 20 or 30 minutes at a pressure of 1.45 MPaG. After the fish scales were exploded, the fish scale explosion product 3 was obtained. Each fish scale explosion product 3 was centrifuged to remove uncrushed fish scale precipitate, and the supernatant was passed through an activated carbon column for crude purification for the purpose of decolorization and deodorization. The column filtrate was freeze-dried (fraction 3).

Fish scale crushed material 3, obtained by 20 minutes of pressurized steaming, was subjected to amino acid analysis in the same manner as fish scale crushed material 1, and was found to contain hydroxyproline, an amino acid specific to collagen. Furthermore, when the ratio of amino acids per 1,000 residues was confirmed, glycine accounted for approximately 1/3 of the total, and the total of alanine, hydroxyproline, and proline was approximately 1/3 of the total, showing ratios specific to collagen.

When liquid explosive fish scale material 3 was subjected to size exclusion chromatography using a TSKgel G2500PWXL (Tosoh Corporation) column, the molecular weight (number average molecular weight) was several hundred to several thousand (low molecular weight). The results are shown in Table 3. From these results, it is believed that explosive fish scale material 3 contains collagen peptides.

When the pressure steaming treatment time was 20 minutes, the yield was 72.75%, and when the pressure steaming treatment time was 30 minutes, the yield was 68.31%. The fish scale exploded material 3 obtained by pressure steaming treatment for 20 or 30 minutes was liquid immediately after the explosion treatment. The fish scale exploded material 3 did not gel at room temperature or at 4°C. Furthermore, no precipitate was obtained when ethanol was added to the liquid fish scale exploded material 3.

[Fish scale crushed material 3']
As fish scales, raw barramundi scales that were washed but not demineralized were used. The fish scales were placed in a steam explosion device and subjected to pressurized steaming treatment at a temperature of 190°C for 20 minutes, 30 minutes or 40 minutes at a pressure of 1.15 MPaG. After the fish scales were exploded, fish scale exploded material 3' was obtained. Each fish scale exploded material 3' was centrifuged to remove uncrushed fish scale precipitate, and the supernatant was passed through an activated carbon column for crude purification for the purpose of decolorization and deodorization. The column filtrate was freeze-dried (fraction 3).

Fish scale crushed material 3' obtained by 20 minutes of pressure steaming was subjected to amino acid analysis in the same manner as fish scale crushed material 1, and it was found to contain hydroxyproline, an amino acid specific to collagen. Furthermore, when the ratio of amino acids per 1000 residues was confirmed, glycine was approximately 1/3 of the total, and the total of alanine, hydroxyproline, and proline was approximately 1/3 of the total, showing ratios specific to collagen.

The powder sample obtained by freeze-drying the crushed fish scales 3' obtained by 20 minutes of pressurized steaming was subjected to size exclusion chromatography using a TSKgel G2500PWXL (Tosoh Corporation) column. The molecular weight (number average molecular weight) was several hundred to several thousand (low molecular weight). The results are shown in Table 4. From these results, it is believed that the crushed fish scales 3' contain collagen peptides.

When the pressure steaming treatment time was 20 minutes, the yield was 12.30%, when the pressure steaming treatment time was 30 minutes, the yield was 17.40%, and when the pressure steaming treatment time was 40 minutes, the yield was 13.47%. The fish scale exploded material 3' obtained by pressure steaming treatment for 20 to 40 minutes was liquid immediately after the explosion treatment. The fish scale exploded material 3' did not gel at room temperature or at 4°C. Furthermore, no precipitate was obtained when ethanol was added to the liquid fish scale exploded material 3'.

In addition, when the pressure steaming treatment time was 1 minute and the pressure steaming treatment temperature was 220°C, the yield was 5.18%, when the pressure steaming treatment temperature was 230°C, the yield was 17.35%, and when the pressure steaming treatment temperature was 240°C, the yield was 4.42%. The obtained fish scale exploded material 3' was a viscous liquid immediately after the explosion treatment.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.

[Fish scale explosion material]
The manufacturing method of the exploded fish scales used in the examples is shown in FIG. 3. Barramundi fish scales were used as the fish scales. The fish scales were stored frozen, so they were thawed under running water and then washed. In this process, all the fish skin and meat pieces were removed, and the viscous substances attached to the scales were also removed. The washing was considered to be complete when the attached substances were visually removed.

250 grams of fish scales were placed in a steam explosion device and subjected to pressure steaming treatment at a temperature of 190°C for 5 minutes at a pressure of 1.15 MPaG. After the fish scales were exploded, 690 to 1000 mL of exploded fish scales were obtained. The resulting exploded fish scales did not solidify at room temperature or 4°C. The exploded fish scales were centrifuged at 3000 rpm for 10 minutes and separated into uncrushed fish scale precipitate (solid) and supernatant (liquid). The supernatant was collected in a separate container to prevent the precipitate from being mixed in. For the purpose of decolorization and deodorization, the supernatant was passed through an activated carbon column and roughly purified. Ethanol was added to the column effluent to a final concentration of 85% by volume and stirred with a glass rod. The container was covered with parafilm and left to stand overnight at 4°C or room temperature. The solution was centrifuged at 3000 rpm for 10 minutes and the precipitate was collected. The precipitate was pre-frozen at -20°C for 2 hours. Once the precipitate was sufficiently frozen, it was placed in a freeze-dryer and freeze-dried. It was visually confirmed that freeze-drying was complete within about 72 hours. In the experiments described below, freeze-dried powder samples were used as the crushed fish scales. In the following examples, the concentration of the crushed fish scales added to the medium is the concentration relative to the mass of the entire medium.

[Stem cell pre-culture]
The culture vessel was coated with mouse embryonic fibroblasts (MEF, Sigma-Aldrich, PMEF-CFL-P1) in advance. The culture medium used was DMEM (GIBCO) supplemented with 10% (v/v) fetal bovine serum (FBS, Equitech-Bio Inc.) and 0.5% (v/v) penicillin-streptomycin (GIBCO). MEFs were treated with 10 μg/ml mitomycin C (Sigma-Aldrich) at 37° C. for 3 hours and then frozen and stored. After thawing, the cells were seeded at 3×10 ⁵ cells/well on a gelatin (Sigma-Aldrich)-coated 6-well dish and left for 6 hours to overnight before use.

Human iPS cells (253G0, Kyoto University) were prepared as stem cells. The culture medium used was DMEM/F12 supplemented with 20% (v/v) knockout serum replacement (KSR, GIBCO), 100 μM Non Essential Amino Acid (NEAA), 20 mM L-glutamine (GIBCO), 100 μM 2-mercaptethanol (Sigma-Aldrich), and 0.5% (v/v) penicillin-streptomycin (Wako, 168-23191). The cells were co-cultured with MEFs in an incubator (Sanyo) at 37° C. under a 5% CO ₂ atmosphere, and passaged every 4-5 days.

[Observation under phase contrast microscope]
Colonies were screened using a phase-contrast microscope (OLYMPUS IX71). Colonies in which small cells with a large nucleus/cytoplasm ratio occupied the majority of the colonies, the colony margins were clear, and the colonies contained almost no abnormally large cells or spindle-shaped cells were determined to be undifferentiated colonies.

[Analysis of undifferentiated state by alkaline phosphatase (ALP) staining]
For ALP staining, an AP color development kit (Bio-Rad, 1706432) was used. The specific procedure is as follows.
1) The dish to which the human iPS cells had adhered was washed once with 0.02 M PBS (pH 7.4) at 37° C.
2) 4% PFA (Paraformaldehyde, Wako) was added and left at room temperature for 20 minutes to fix the cells.
3) 0.02 M PBS (pH 7.4) was added at room temperature, and the mixture was allowed to stand for 5 minutes. This procedure was repeated three times.
4) 10 μL of Reagent A and 10 μL of Reagent B in an AP color development kit (Bio-Rad) were mixed with 1 mL of pre-diluted color development buffer, and the mixture was poured into a dish and allowed to stand at room temperature for 15 minutes.
5) Distilled water was added, and the plate was left to stand at 37° C. for 5 minutes. This operation was repeated three times to stop the color reaction and wash the plate.
6) Observation was performed at 400x magnification using a microscope (OLYMPUS IX71).

[Analysis of undifferentiated state by real-time PCR method]
[1] First, RNA was extracted according to the following procedure.
1) 1.0 mL of Trizol (TRI Reagent, Molecular Research Center, Inc.) was added to the dish to which the iPS cells were attached, and the cells were detached by pipetting.
2) The cell suspension was transferred to a 1.5 mL microtube and allowed to stand at room temperature for 5 minutes.
3) 0.2 mL of chloroform (Wako) was added, mixed for 30 seconds, and then allowed to stand at room temperature for 10 minutes.
4) The mixture was centrifuged at 13,500 rpm for 15 minutes at 4° C. using a small refrigerated tabletop centrifuge (Centrifuge 5415R, Eppendorf) to separate the mixture into three layers: a colorless and transparent upper layer (aqueous phase), a white middle layer, and a red lower layer (organic phase). The upper layer (aqueous phase) was transferred to a new tube.
5) 0.5 mL of isopropyl alcohol (Wako) was added and mixed for 5 minutes, and then the mixture was allowed to stand at room temperature for 10 minutes.
6) The mixture was centrifuged at 13,500 rpm for 10 minutes at 4° C., and the supernatant was removed.
7) 70% ethanol was added, mixed for 5 minutes, centrifuged at 8000 rpm for 10 minutes and 4° C. for 5 minutes, and the supernatant was removed.
8) The sample tube was inverted and dried in an incubator.
9) 30 μL of RNase-free water was added and pipetted.
10) The amount of RNA was measured using an absorbance meter (NanoDrop Lite UV-Vis Spectrophotometer, Thermo Fisher Scientific).

[2] Subsequently, DNA removal and reverse transcription of RNA were performed using PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio).
1) DNA Removal Reaction The following genomic DNA removal reaction solution (per reaction) was prepared on ice.
5x gDNA Eraser Buffer 2.0μL
gDNA Eraser 1.0 μL
Total RNA 1.0 μg
RNase Free dH2O _remaining amount
Total 10.0 μL
After reaction at 42°C for 2 minutes (DNA removal), the mixture was stored at 4°C.

2) Reverse Transcription Reaction The following reverse transcription reaction solution was prepared on ice.
Reaction solution of 1) above (genomic DNA removal reaction solution) 10.0 μL
5x PrimeScript Buffer 4.0μL
PrimeScript RT Enzyme Mix I 1.0 μL
RT Primer Mix 1.0 μL
RNase Free _dH2O 4.0μL
Total 20.0 μL
After reacting at 37°C for 15 minutes and at 85°C for 5 seconds, the mixture was stored at 4°C.
Thermal Cycler Dice Touch (Takara Bio, Model: TP350) was used for the above 1) DNA removal reaction and 2) reverse transcription reaction.

[3] Real-time PCR
After reverse transcription, real-time PCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio).
1) The following PCR reaction solution (per reaction) was prepared.
TB Green Premix Ex Taq II (2x) 12.5 μL
PCR Forward Primer (10 μM) 1 μL
PCR Reverse Primer (10 μM) 1 μL
The above reverse transcription reaction solution (cDNA solution) 2 μL
Sterile purified water 8.5 μL
Total 25μL

2) The PCR reaction was carried out according to the standard shuttle PCR protocol described below.
Hold (1 Cycle) 95°C 30 seconds
2STEP PCR (40 cycles) 95℃ 5 seconds
60℃ 30-60 seconds Dissociation

After the PCR reaction was completed, the amplification curve and melting curve were confirmed, and a calibration curve was created. The expression levels were compared using the comparative Ct method. A Thermal Cycler Dice Real Time System (Takara Bio, Model: TP850) was used for real-time PCR. The sequences of the primers used in real-time PCR are shown in Table 5.

[Analysis of undifferentiated cells using immunostaining method]
1) The dish to which the human iPS cells had adhered was washed once with 0.02 M PBS (pH 7.4) at 37° C.
2) 4% PFA (Wako, 162-16065) was added and left at room temperature for 20 minutes to fix the cells.
3) The PFA was removed by adding 0.02 M PBS (pH 7.4) and leaving the plate at room temperature for 5 minutes to wash the plate, which was repeated three times.
4) 0.02 M PBS containing 0.25% Triton X-100 (Wako, A16046) was added, and the mixture was allowed to stand at room temperature for 15 minutes to perform antigen activation treatment.
5) 0.02 M PBS (pH 7.4) was added and the mixture was allowed to stand for 4 minutes. This operation was repeated three times.
6) Blocking solution I (1.5% goat normal serum (Vector Laboratories, S-1000)/0.02 M PBS, pH 7.4) was added, and the mixture was allowed to stand at room temperature for 1 hour or more.
7) A primary antibody solution (a solution obtained by diluting Oct4 antibody (Santa Cruz Biotechnology, product number SC-5279) 300-fold with blocking solution I) was added, and the mixture was allowed to stand overnight at 4°C.
8) The membrane was washed with 0.02 M PBS (pH 7.4) by leaving the membrane at room temperature for 5 minutes. This washing procedure was repeated three times.
The following operations were carried out in a darkroom.
9) A secondary antibody solution prepared in advance (a solution obtained by diluting goat anti-mouse IgG 488 (Invitrogen, A-21121) 1000-fold with blocking solution I, containing 10000-fold diluted DAPI) was added, and the mixture was allowed to stand at room temperature for 1 hour to carry out a secondary antibody reaction.
10) The membrane was washed three times with 0.02 M PBS (pH 7.4) by leaving the membrane at room temperature for 5 minutes.
11) A drop of mounting medium was placed on the sample and the sample was sealed with a cover glass.
12) The sample was observed under a fluorescence microscope (KEYENCE BZ-X700) at 400x magnification.

(Experiment 1)
iPS cells were cultured in a medium containing 2.5%, 1.25%, 0.625% or 0% by mass of crushed fish scales. In the presence of feeder cells, the cells were seeded at 3×10 ⁵ cells/well on a gelatin-coated 6-well dish and cultured at 37° C. and 5% CO _2. The same medium as that used in the preculture was used as the basal medium. The medium was replaced every day and cultured for 14 days without subculture.

Phase contrast optical microscope images of iPS cells on the 14th day of culture are shown in Figure 4. The cells in the colonies of iPS cells cultured in media containing 2.5% and 1.25% by mass of crushed fish scales were small and round in shape, and are considered to have maintained an undifferentiated state (Figures 4A and B). The cells in the colonies of iPS cells cultured in media containing 0.625% by mass of crushed fish scales were large and polygonal and flat, but most of the cells were considered to have maintained an undifferentiated state (Figure 4C). Most of the iPS cells cultured in media containing no crushed fish scales were differentiated (Figure 4D).

(Experiment 2)
Using the same method as in Experiment 1, iPS cells cultured for 14 days were stained with ALP using a medium containing 2.5 mass%, 1.25 mass%, 0.625 mass%, 0.312 mass%, or 0 mass% of exploded fish scales. The ALP stained images are shown in FIG. 5. Alkaline phosphatase is highly expressed in all pluripotent stem cells, including ES cells, EG cells, and iPS cells, and is used as an undifferentiation marker that shows high activity in an undifferentiated state. Histological staining of ALP makes it easy to determine whether pluripotent stem cells are differentiated or undifferentiated. Lightly stained areas indicate differentiation, while darkly stained areas indicate undifferentiation.

Stem cells cultured using a medium containing 2.5% by mass of crushed fish scales had slightly smaller colonies, but the entire colony was stained and was considered to maintain an undifferentiated state (Figure 5A). Stem cells cultured using a medium containing 1.25% by mass of crushed fish scales had the entire colony stained and was considered to maintain an undifferentiated state (Figure 5B). Stem cells cultured using a medium containing 0.625% by mass of crushed fish scales had differentiated cells at the periphery of the colony, but most cells were considered to maintain an undifferentiated state (Figure 5C). Stem cells cultured using a medium containing 0.312% by mass of crushed fish scales had differentiated cells at the center of the colony (Figure 5D). Stem cells cultured using a medium not containing crushed fish scales had enlarged colonies and differentiated cells were found at the periphery of the colony (Figure 5E).

(Experiment 3)
In the same manner as in Experiment 1, iPS cells were cultured without subculture for 7, 14 or 21 days using a medium containing 2.5 mass%, 1.25 mass%, 0.625 mass% or 0 mass% of crushed fish scales. After the culture, the mRNA expression levels of Oct3/4, Nanog and Sox2 were detected by real-time PCR. The results are shown in FIG. 6. Oct3/4, Nanog and Sox2 are representative undifferentiated markers of pluripotent stem cells.

The mRNA expression level of iPS cells on the 4th day cultured in a medium containing no crushed fish scales was set to 1. The iPS cells cultured in a medium containing crushed fish scales maintained high mRNA expression levels of Oct3/4, Nanog, and Sox2 even after long periods of culture, especially for 14 and 21 days (Figure 6). The expression of these undifferentiated markers was lower in iPS cells cultured in a medium containing no crushed fish scales than in iPS cells cultured in a medium containing crushed fish scales.

(Experiment 4)
Using the same method as in Experiment 1, iPS cells were cultured for 14 days using a medium containing 2.5 mass%, 1.25 mass%, 0.625 mass%, or 0 mass% of crushed fish scales, and immunostained with an anti-Oct4 antibody to confirm the protein expression level of Oct4. The staining results are shown in Figure 7. Oct4 is a transcription factor with a POU domain, and is known to play an important role in early embryo development and maintaining the pluripotency of ES cells.

iPS cells cultured in medium containing crushed fish scales had a higher proportion of Oct4-positive cells than iPS cells cultured in medium not containing crushed fish scales (Figure 7).

The results of Experiments 3 and 4 showed that iPS cells cultured in medium containing crushed fish scales, especially in medium containing 1.25% and 2.5% crushed fish scales by mass, had higher mRNA transcription and protein expression levels of undifferentiated markers than iPS cells cultured in medium not containing crushed fish scales, and that the undifferentiated state of stem cells was well maintained.

(Experiment 5)
In the same manner as in Experiment 1, iPS cells were cultured for 14 days using a medium containing 1.25% or 0% by mass of crushed fish scales, and then passaged. In the presence of feeder cells, cells were seeded at 3×10 ⁵ cells/well on a gelatin-coated 6-well dish and cultured at 37° C. and 5% CO _2. The medium used was the same as that used in the preculture, and did not contain crushed fish scales. Phase contrast optical microscope images of colonies on the first day after passage are shown in FIG. 8.

iPS cells cultured in a medium containing 1.25% by mass of crushed fish scales formed colonies after passage, and morphological characteristics suggest that the undifferentiated state was maintained (Figures 8A and B). iPS cells cultured in a medium not containing crushed fish scales did not form colonies after passage, and many of the cells had a flattened shape, and morphological characteristics suggest that the undifferentiated state was not maintained (Figures 8C and D).

(Experiment 6)
The cells subcultured in Experiment 5 were cultured continuously until the fourth day, and the mRNA expression level of the undifferentiated marker was analyzed. The results are shown in FIG. 9. The mRNA expression level of the iPS cells on the fourth day cultured in a medium containing no crushed fish scales was set to 1. The iPS cells cultured in a medium containing crushed fish scales maintained high expression levels of undifferentiated markers after subculture, especially Oct3/4 and Sox2, compared to the iPS cells cultured in a medium containing no crushed fish scales. The high expression level of undifferentiated markers was particularly notable in the iPS cells cultured in a medium containing 1.25% by mass of crushed fish scales.

The embodiments and examples disclosed herein should be considered to be illustrative and not restrictive in all respects. The scope of the present invention is indicated by the claims rather than the above description, and is intended to include all modifications within the meaning and scope of the claims.

10 Steam explosion device, 12 Boiler, 13 Water purifier, 14 Pump, 15 Reactor, 16 Hopper, 17, 18, 19 Valve, 20 Pipe, 21 Receiving tank, 22 Silencer, 23 Outlet.

Claims (12)

An additive for cell culture containing a component of crushed fish scales ,
The components are
A process for subjecting the fish scales to pressure steaming treatment at a temperature of 150°C to 250°C for 3 minutes to 15 minutes to obtain the fish scale exploded product;
A precipitation step in which ethanol is added to the fish scale crushed material to precipitate the components,
The fish scales are from barramundi,
The cell culture additive is used to maintain the undifferentiated state of stem cells,
The cell culture additive, wherein the stem cells are induced pluripotent stem cells . The cell culture additive according to claim 1, wherein the fish scales are steam-exploded fish scales. The cell culture additive according to claim 1 or 2, wherein the pressure steaming treatment is carried out under conditions of 150°C to 250°C and 5 minutes to 15 minutes. The cell culture additive according to any one of claims 1 to 3, which is in the form of a liquid or powder. The cell culture additive according to any one of claims 1 to 4, wherein the pressure steaming treatment is carried out under conditions of 190°C to 200°C and 5 minutes to 15 minutes. A medium containing ingredients of crushed fish scales .
The components are
A process for subjecting the fish scales to pressure steaming treatment at a temperature of 150°C to 250°C for 3 minutes to 15 minutes to obtain the fish scale exploded product;
A precipitation step in which ethanol is added to the fish scale crushed material to precipitate the components,
The fish scales are from barramundi,
The fish scale crushed material contains 0.625% by mass or more and 2.5% by mass or less based on the mass of the entire medium,
The medium is used for maintaining the undifferentiated state of stem cells,
The medium, wherein the stem cells are induced pluripotent stem cells . The medium according to claim 6, wherein the exploded fish scales are steam-exploded fish scales. The medium according to claim 6 or 7, wherein the pressure steaming treatment is carried out under conditions of 150°C to 250°C and 5 minutes to 15 minutes. The medium according to any one of claims 6 to 8 , wherein the pressure steaming treatment is carried out under conditions of 190 ° C. to 200 ° C. and 5 minutes to 15 minutes . The medium according to any one of claims 6 to 9 , wherein the fish scale crushed material is contained in an amount of 1.25% by mass or more and 2.5% by mass or less relative to the mass of the entire medium . A method for culturing cells, comprising using the medium according to any one of claims 6 to 10 . The culture method according to claim 11 , wherein the culture is carried out for 4 days or more without subculture.

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