Medium component composition, method for producing medium component composition, cell proliferation method, and food

Abstract

Provided are a highly functional medium component composition suitable for cell culture, a method for producing the medium component composition, a cell proliferation method, and a food. This medium component composition contains a blood-related component containing a blood-derived component or a processed product thereof, and an immunopotentiator that induces or promotes an immune response. This method for producing a medium component composition includes a step for adding the component that induces or promotes an immune response to the blood-related component and incubating the resulting combination to activate immune cells and platelets contained in the blood-related component and obtain the medium component composition. Also provided are a cell proliferation method in which the medium component composition is used, and a food product obtained using the same.

Description

Medium Component Composition, Method for Producing Medium Component Composition, Cell Proliferation Method, and Food

[0001] The present invention relates to a medium component composition used for cell culture for producing foods and the like, a method for producing the same, a cell proliferation method, and a food. This application claims priority based on U.S. Patent Application No. 63 / 735,313, which was provisionally filed in the United States on December 18, 2024, and the contents thereof are incorporated herein by reference.

[0002] Currently, research is being conducted on foods mainly composed of cultured cells of animals, so-called cellular foods. Cultured meat and the like are typical examples of these foods. Foods produced by cultured cells are expected to be able to obtain animal-derived proteins without taking the lives of animals and to reduce the environmental load during production compared to conventional livestock farming.

[0003] For the current cell culture for food production, the standard culture process is to add serum (especially fetal bovine serum (FBS)) to a basal medium (such as DMEM). However, since fetal bovine serum requires abortion and has a high environmental load itself, it inevitably undermines the advantages of food production using cultured cells. Therefore, the development of alternative materials to FBS and the like is required.

[0004] In addition to serum, components that can be used for cell proliferation, for example, platelet lysate (PL; Platelet lysate) can be considered. PL is a material obtained by crushing platelets contained in plasma and eluting the contents into the plasma. It is known that the PL thus obtained contains abundant growth factors compared to serum and that the material can be obtained using a less invasive blood collection even from adults / adult animals.

[0005] A method for producing PL is disclosed in Patent Document 1. This technology is characterized by comprising the steps of: obtaining a buffy coat and a specific gravity fraction heavier than the buffy coat containing leukocytes and platelets from bovine whole blood treated with an anticoagulant by continuous centrifugation; and incubating the fraction obtained in the previous step at a constant temperature for a certain period of time to promote the interaction between leukocytes and platelets in the fraction and activate them, thereby causing the leukocytes and / or platelets to secrete or release humoral factors, and then recoagulating the blood components containing these humoral factors with a recoagulant, wherein the specific gravity fraction heavier than the buffy coat is a fraction with a higher leukocyte concentration than the buffy coat. This technology provides a novel method for producing a bovine serum composition, and specifically aims to provide an inexpensive method for producing a bovine serum composition containing many factors beneficial to cell proliferation and factors that suppress blood coagulation by cells.

[0006] Patent No. 6212723

[0007] Conventional and most commercially available PLs are derived from humans. Furthermore, their use is primarily for human therapeutic purposes, particularly for cell therapy. Human PL presents challenges in terms of production volume and infectious disease risk, making its application to food production via cell culture difficult. Therefore, conventional PLs have not been used in food production via cell culture, or at least not in practical use. The technology described in Patent Document 1 is commercially available as bovine-derived PL, but it is intended for human culture and, due to processes such as the addition of glass beads, is unsuitable for food production and not for mass propagation. In fact, the PL propagated in Patent Document 1 is not used in the production of cellular foods.

[0008] Given the above background, there are currently no effective alternatives to animal serum as components added to culture media primarily for the purpose of promoting cell proliferation, and no components suitable for food production using cell culture have been found.

[0009] This invention was made in view of the above background, and its purpose is to provide a culture medium component composition with high functionality and suitable for cell culture, a method for producing the culture medium component composition, a method for cell proliferation, and a food product.

[0010] The present invention includes the following embodiments: [1] A culture medium component composition comprising blood-related components, including blood-derived components or processed products thereof, and an immunostimulant that induces or promotes an immune response. [2] The culture medium component composition according to [1] for use in cell culture for food manufacturing, cosmetics, pharmaceuticals, or research and development. [3] The culture medium component composition according to [1] or [2], wherein the immunostimulant is one or more selected from the group consisting of microbial components, nucleic acids, lipids, glycans, peptides, polyphenols, fermentation products, or extracts thereof. [4] The culture medium component composition according to [1] to [3], wherein the immunostimulant is one or more selected from the group consisting of lipopolysaccharides, β-glucans, peptidoglycans, vitamins, minerals, lactic acid bacteria, bifidobacteria, and plant-derived components. [5] The culture medium component composition according to [1] to [4], comprising 1 pg / g or more, 1 pg / mL or more, or 0.01 EU / mL or more of the immunostimulant. [6] The culture medium component composition according to [1] to [5], wherein the blood-related component is derived from an adult animal other than a human. [7] The culture medium component composition according to [1] to [6], wherein the blood-related component is derived from a bird, a fish, or a mammal. [8] The culture medium component composition according to [1] to [7], wherein the blood-related component is from a cattle or a pig. [9] A method for producing the culture medium component composition according to [1] to [8], comprising the step of activating the blood-related component by adding the immunostimulant to the blood-related component and incubating it to obtain the culture medium component composition.

[10] The method for producing the culture medium component composition according to [9], wherein the blood-related component includes platelets, and the activated composition is subjected to freezing and thawing once or more times to dissolve the platelets, thereby obtaining the blood-related component containing the platelet lysate, and thus obtaining the culture medium component composition.

[11] The method for producing the culture medium component composition according to [9] or

[10] , wherein the activation step is performed in an environment of 10°C or lower.

[12] A method for producing the culture medium component composition according to [9] or

[10] , further comprising the step of reducing or removing the immunostimulant after the activation step.

[13] A method for cell proliferation, comprising culturing and growing animal cells using the culture medium component compositions according to [1] to [8].

[14] A cell proliferation method according to

[13] for producing a food product using the animal cells.

[15] A cell proliferation method according to

[13] or

[14] , wherein the blood-related component contained in the culture medium component composition and the animal cells are derived from the same species of animal.

[16] A cell proliferation method according to

[15] , wherein the blood-related component is derived from a bovine and the animal cells are bovine myoblasts, or the blood-related component is derived from a pig and the animal cells are pig muscle tissue-derived cells.

[17] A cell proliferation method according to

[13] to

[16] , wherein the animal cells are cultured and proliferated at a temperature of 10°C or lower.

[18] A food product containing a cellular product obtained by proliferating animal cells using the culture medium component composition according to [1] to [8].

[0011] The present invention provides a culture medium component composition with high functionality suitable for cell culture, a method for producing the culture medium component composition, a method for cell proliferation, and a food product.

[0012] This graph shows the 3-day proliferation evaluation of a culture medium composition for bovine myoblasts. This graph shows the 6-day proliferation evaluation of a culture medium composition for bovine myoblasts. This is a micrograph of the time period shown in Figure 2. This graph shows the proliferation evaluation of Neosera and hPL for bovine myoblasts. This is a photograph showing the effect of a culture medium composition using β-glucan on bovine myoblasts. This graph shows the proliferation evaluation of a culture medium composition derived from porcine blood on porcine cells. This graph shows cell proliferation under temperature conditions for activation of a culture medium composition.

[0013] The culture medium component composition, method for producing the culture medium component composition, cell proliferation method, and food product according to the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments.

[0014] [Culture medium component composition] The culture medium component composition of this embodiment includes blood-related components, including blood-derived components or processed products thereof, and immunostimulants that induce or promote an immune response.

[0015] A culture medium component composition is a composition that can be used as a component of a culture medium for cell culture. For example, it may be the main component of the culture medium, or it may be an addable component. For example, it may be a component that can be added to a liquid culture medium as a component that can promote cell culture. In this embodiment, the culture medium component composition is a composition that can be added to a basal culture medium in addition to, or as a substitute for, serum-derived components that were added to the basal culture medium, such as albumin (serum albumin), particularly FBS components.

[0016] The culture medium component composition may be used for cell culture in food manufacturing, cosmetics, pharmaceuticals, or research and development. Because the culture medium component composition of this embodiment has high functionality in promoting cell culture, it can be used for any type of cell culture. However, due to its high productivity and versatility, it is particularly preferred for use in food manufacturing, cosmetics, pharmaceuticals (medical or other medical purposes), or research and development. Furthermore, as will be described later, its origin eliminates ethical issues in its use, poses a low risk of infection, and offers high productivity, making it particularly suitable for use in food manufacturing.

[0017] (Blood-related components) Blood-related components are components that include components derived from blood or processed products thereof. Blood-derived components refer to blood or components obtained from blood, and broadly include, for example, blood that has been fractionated, purified, processed, or obtained therefrom.

[0018] Blood-related components are preferably those contained in whole blood (components derived from blood), mainly serum (liquid, low-density, or minute components separated into the supernatant in the crude fraction). Serum is preferred because it contains components that promote cell proliferation in cell culture, as well as other components that are advantageous for cell culture, such as immune components. Components contained in plasma include serum, platelets, and their derivatives.

[0019] Blood-related components preferably include platelets or platelet-derived components, and more preferably include platelet lysate (PL). The PL may be obtained by chemical or physical dissolution or pulverization, regardless of its form or manufacturing method, as long as it contains components obtained by dissolving or pulverizing platelets. Details will be described later in the manufacturing method section, but for example, platelets contained in plasma can be crushed to elute their contents into the plasma, and this can be used. As for the crushing, mainly physical crushing methods can be used. Alternatively, the action of crushing platelets by freezing and thawing may also be used.

[0020] The blood-related components can be derived from any organism, but it is preferable that they be from an organism other than a human. Using blood from a non-human organism makes it suitable for various applications, such as food production. It is also preferable that the blood-related components are derived from birds, fish, or mammals. The blood of these animals is readily available as a material.

[0021] The growth stage (age, months, etc.) of the organism from which the blood-related components are derived is irrelevant; fetuses, juveniles, and adults can all be used. In this embodiment, it is preferable that the components be derived from adult animals, and it is particularly preferable that they be derived from adult animals other than humans. This embodiment can be carried out without any problems even with blood-related components derived from adult animals. By using adult animals, for example, abortion is not required when producing fetal serum of mammals, and the process can be carried out without the environmental burden associated with animal abortion. Furthermore, blood may be collected from living organisms, or blood may be collected from slaughtered organisms and used.

[0022] The blood-related components are preferably derived from cattle or pigs, and are particularly preferably derived from adult cattle or pigs. As shown in the examples described below, the inventors have obtained a highly functional culture medium component composition by using blood-related components derived from adult cattle or pigs.

[0023] (Immunostimulants) Immunostimulants are substances that induce or promote an immune response. Any substance conventionally known to induce or promote an immune response can be used as an immunostimulant. The immunostimulant may be a single type of substance or a combination of multiple types of substances.

[0024] The immunostimulant is preferably one or more selected from the group consisting of, for example, microbial components, nucleic acids, lipids, glycans, peptides, polyphenols, fermentation products, or extracts thereof.

[0025] The immunostimulant is more preferably one or more selected from the group consisting of lipopolysaccharides, β-glucans, peptidoglycans, vitamins, minerals, lactic acid bacteria, bifidobacteria, and plant-derived components. Many of these components are known to induce or promote immune responses and have been conventionally used in foods. For example, among vitamins and minerals, vitamin C, vitamin D, and zinc are widely used as nutrient supplements and are positioned under Japan's "Nutrient Functional Foods" system, with standard amounts and labeling methods being published. Lactic acid bacteria or bifidobacteria (probiotics) or sterilized lactic acid bacteria (postbiotics) are widely distributed as general foods (e.g., yogurt, beverages, supplements, etc.), and in the field of immunology, functional foods using plasma lactic acid bacteria (Lactococcus lacticis JCM5805) are known as an example (e.g., notification numbers F184, G777 in the Consumer Affairs Agency database). As for plant-derived ingredients, for example, common herbs and plant-based materials are used as general food products, while ginseng, echinacea, garlic, and elderberry (after heating) are sold as food products (e.g., tea, extract, supplements).

[0026] Food ingredients containing β-glucan include paramylon (β-1,3-glucan) derived from Euglena, which is a functional food product that will be approved in 2025 to display the claim of "maintaining immune function." β-glucan derived from yeast and microalgae is also increasingly used in foods and supplements in Japan. Other ingredients containing β-glucan include Aureobazidium culture solution and curdlan, which are listed as components in the food additive list. β-glucan and peptidoglycan have been reported to improve macrophage activity and have a synergistic effect with LPS (glycolipid). In this embodiment, a food additive containing β-glucan may be added as an immunostimulant. For example, purified Aureobazidium culture solution can be used as a component containing β-glucan.

[0027] In this embodiment, it is also preferable to use lipopolysaccharide (LPS) as an immunostimulant. Lipopolysaccharide is a type of substance (glycolipid) composed of lipids and polysaccharides, and in particular refers to the component of the peptidoglycan outer membrane of the cell wall outer membrane of Gram-negative bacteria, including Escherichia coli, Salmonella, and Bordetella pertussis. Lipopolysaccharide has a structure in which a sugar chain is attached to a lipid (lipid A), and the sugar chain portion is known to be composed of core polysaccharide (oligosaccharide also called R core) and O side chain polysaccharide (O specific polysaccharide). Lipid A is known as a component of endotoxin and is composed of two glucosamine (carbohydrate / sugar) units to which acyl chains (fatty acids) are attached, and usually each sugar has one phosphate group. The core polysaccharide consists of 5 to 8 carbon sugars and is constant depending on the bacterial species. The O side chain polysaccharide has a basic structure consisting of about 3 to 5 types of 5 to 6 carbon sugars, and is known to differ from strain to strain even in the same bacterial species. Lipopolysaccharide (LPS) is known as a receptor for TLR4, which is involved in immunity, and is known to promote the secretion of inflammatory cytokines through intracellular signaling. While LPS has a high immune-enhancing effect, it is also cytotoxic when secreted during cell death and is sometimes called an endotoxin. On the other hand, LPS has low toxicity when ingested orally, so it is used in immune-boosting foods and supplements. When antigens and LPS are administered simultaneously, the immune response is enhanced, so it is also used as a type of adjuvant to enhance vaccine efficacy. The preparation and properties of LPS are disclosed, for example, in Japanese Patent No. 4043533. In this embodiment, LPS prepared from various microorganisms can be used as appropriate. It is particularly preferable to use LPS prepared from the cell wall of Gram-negative bacteria. In this embodiment, LPS can be easily manufactured by preparing it from the cell wall of Gram-negative bacteria, making it suitable for mass production for food manufacturing.

[0028] The culture medium component composition of this embodiment preferably contains an immunostimulant of 1 pg / g or more, 1 pg / mL or more, or 0.01 EU / mL or more. By including the immunostimulant in the above amount, blood-related components, such as cell proliferation components contained in PL, can be activated, thereby enhancing functionality such as cell proliferation. The culture medium component composition may also contain other components used in cell culture, such as components added to the culture medium described later.

[0029] Furthermore, the components contained in the culture medium component composition of the embodiment of this application, or in a culture medium containing it, can be detected by various component analyses. For example, detection can be performed by spectroscopic analysis, mass spectrometry, chromatography, or elemental analysis. For example, to detect LPS components, Limulus Color KY Test Wako Reagent (Fujifilm Wako Pure Chemical Industries, Ltd.) can be used, and an endotoxin test using a toxinometer or plate reader can be used. To detect β-glucan, an SLP reagent such as SLP-HS Single Reagent Set II (Fujifilm Wako Pure Chemical Industries, Ltd.) and a test using a toxinometer can be used.

[0030] [Method for producing the culture medium component composition] The method for producing the culture medium component composition according to this embodiment is the method for producing the culture medium component composition described above, and includes the step of activating the blood-related components by adding the immunostimulator to the blood-related components and incubating them to obtain the culture medium component composition.

[0031] As for blood-related components, the aforementioned components can be used as appropriate, but as for preparation methods, for example, blood to which citric acid has been added and used as an anticoagulant can be used. This blood can be fractionated by centrifugation. Centrifugation can be performed by conventional centrifugation or density gradient centrifugation. The main liquid fraction (containing plasma as the main liquid component) obtained by density gradient centrifugation with sucrose or dextran can be used. Since sucrose and dextran are edible, they can be suitably used to prepare ingredients for food production.

[0032] The step of adding the immunostimulant to the blood-related components and incubating them is performed to activate the blood-related components, particularly the components contained in the blood-related components that are effective for cell culture. For example, this includes the activation of immune cells and platelets. Activation broadly refers to the promotion of functions effective for cell culture, and in particular, the promotion of immune function and cell proliferation function.

[0033] The incubation conditions can be selected as appropriate. For example, the temperature may be 10°C or lower, or it may be above 10°C. That is, if the incubation temperature is low, such as 10°C or lower, it is possible to prevent deterioration of quality when handling food. Furthermore, processing at 10°C or lower is preferable because it is below the temperature permitted as a processing temperature under the Food Sanitation Law. When processing at 10°C or lower, processing may be done at 4°C or lower, preferably around 4°C. Also, if the incubation temperature is 10°C or higher, which is a high culture condition, it can be set to the temperature at which the reaction of enzymes, etc., is most activated. When processing at 10°C or higher, it is more preferable to be 18°C ​​or higher, even more preferable to be between 18 and 40°C, and particularly preferable to be around 37°C. In the present invention, as shown in the examples, it has been confirmed that the culture medium component composition has the same effect regardless of whether incubation is performed at low temperature or high culture condition, that is, when cell culture is performed using the culture medium component composition activated at 4°C and 37°C, respectively, cell proliferation can be performed equally well. The time is preferably 5 minutes or more, more preferably 1 hour or more, and even more preferably 8 hours (overnight) or more.

[0034] In the incubation described above, it is preferable to incubate in a system containing 1 pg / g or more, 1 pg / mL or more, or 0.01 EU / mL or more of the immunostimulant, more preferably 0.1 μg / mL or more, and even more preferably 1 μg / mL or more. In this embodiment, the blood-derived component is a component containing plasma and platelets, and LPS is added to a system in which plasma is the main liquid component so that the concentration after addition is 1 μg / mL, and incubated overnight at 37°C.

[0035] Furthermore, it is preferable that the blood-related components include platelets or components derived from platelets. It is more preferable that the platelet-derived components be platelet lysates. To include platelet lysates, it is preferable to dissolve platelets in the activated composition. The process of dissolving platelets is not limited to any particular physical or chemical method. Physical methods include physical crushing or pulverization, and platelet pulverization by repeated freeze-thaw cycles. Chemical methods include the use of compounds, enzymes, etc., for thawing.

[0036] In this embodiment, platelets are crushed by repeatedly freezing and thawing the activated composition. Preferably, the process further includes a step of dissolving the platelets by freezing and thawing once or more times to obtain the blood-related components containing the platelet lysate, which are then used to make a culture medium component composition. It is more preferable to perform the freezing and then thawing process to dissolve the platelets two or more times, and particularly preferable to perform it four or more times. The freezing and thawing steps can be performed at temperatures at which the liquid components of the culture medium component composition freeze and thaw, respectively. For example, they may be performed at the set temperature of a freezer (-80°C) or an incubator (37°C). Thawing may also be performed at 4°C. Quarantine standards and other regulations sometimes stipulate that processing should be performed at temperatures not exceeding 10°C, and that blood that is not frozen should be stored at 4°C or below. Therefore, thawing may be performed at 4 to 10°C or below, particularly at 4°C, according to these standards. In this embodiment, freezing is performed at -80°C and thawing at 37°C, and the freezing and thawing steps are repeated four times.

[0037] Furthermore, a product that has undergone sterilization or disinfection in any of the processes can be used. Various sterilization methods can be selected, but for example, pasteurization (heat sterilization), filter sterilization, or sterilization or disinfection by gamma rays or electron beams can be used. In this embodiment, when obtaining blood-derived components, the collected blood, or the fraction obtained by crude fractionation from the blood, is sterilized by gamma rays or electron beams. In particular, a product in which a calcium salt has been added in any of the above processes to remove components coagulated by calcium can be used. As a calcium salt, CaCl ２ These can be suitably used. These can be performed, for example, in the process of obtaining blood-related components or on the obtained culture medium component composition. In addition to the above steps, there may be further steps of fractionation or purification. In particular, when used for a specific purpose of this embodiment, such as cell culture for pharmaceuticals, these steps may be included to reduce or remove components other than those necessary from the final product to be manufactured from the cell culture. For example, there may be a step after the activation step to reduce or remove the immunostimulant that induces or promotes the immune response. As such a removal step, conventional methods known as methods for removing endotoxins using a spin column can be used.

[0038] [Method for cell proliferation] The cell proliferation method of this embodiment involves culturing and proliferating animal cells using the culture medium component composition described above.

[0039] The cell proliferation method of this embodiment is preferably a method for producing food using the cells. The culture medium component composition of this embodiment is highly functional and suitable for the production of the composition and for large-scale cell proliferation, and is therefore suitable for use in food production.

[0040] Specifically, in the case of a method for manufacturing food, it is preferable to produce cultured meat cells, so the animal cells to be cultured are preferably muscle-related cells. For example, myoblasts may be used.

[0041] The blood-related component contained in the culture medium component composition and the animal cells may be derived from the same species of animal or from different species of animals. It is more preferable that the blood-related component and the animal cells are derived from the same species of animal. By using those derived from the same species of animal, cell growth can be performed more efficiently.

[0042] It is also preferable that the blood-related component is derived from cattle and the animal cells are bovine myoblasts. It is also preferable that the blood-related component is derived from pigs and the animal cells are cells derived from pig muscle tissue. The cells derived from pig muscle tissue may be, for example, fibroblasts derived from pig muscle tissue. The inventors have carried out cell growth using the above components in cattle and pigs and obtained good results.

[0043] The step of culturing and growing animal cells may use those conventionally known as appropriate. For example, a medium obtained by adding a culture medium component composition to a conventional cell culture medium may be used as the medium. Also, a medium obtained by adding a culture medium component composition to a so-called basal medium may be used as the medium. In this case, a serum-free medium using a culture medium component composition instead of albumin (such as FBS) can be obtained. The serum-free medium can be a chemically defined culture medium (or a completely synthetic medium). In this embodiment, a method including the step of culturing and growing animal cells is also referred to as a cell culture method.

[0044] The addition amount of the culture medium component composition may be developed so as to contain the same amount as albumin, for example, FBS or the like. For example, the culture medium component composition may be contained in the medium at a final concentration of 5 to 25% by mass, for example, approximately 20% by mass.

[0045] In this embodiment, culturing means incubating cells under conditions suitable for their growth or maintenance. Incubating, in the case of human cells, is preferably at 37 °C and 5% CO ２Although it can be carried out under certain atmospheres, in this embodiment, it may be carried out at 10°C or lower, or may be carried out at 4°C. In this embodiment, since cell growth is carried out efficiently, it has a sufficient growth rate even at 10°C or lower which is recognized as a processing temperature by the Food Hygiene Law. When "cultivation" is accompanied by growth, "cultivation" is understood to be the production of the grown cells. In this specification, "cultivation" can be carried out in a serum-free medium.

[0046] In this embodiment, the medium actually used for cultivation may be referred to as a culture medium. The culture medium can be prepared by adding necessary components to a basal medium. The necessary components can be a pH adjuster, a sugar source such as glucose, antibiotics (for example, penicillin and streptomycin, etc.), essential amino acids such as glutamine, and culture additives such as insulin, transferrin, selenium (for example, sodium selenite) and ethanolamine. The culture medium can be a liquid medium.

[0047] The culture medium can be appropriately selected depending on the type of cells to be grown. Examples of basal media include S-clone SF-3 medium, F12 medium, StemSpan (Stem Cell Technologies), STEMα (STEM ALPHA), StemPro-34 serum-free medium (Gibco Invitrogen), StemPro MSC serum-free medium (Invitrogen), HSC-CFU medium (Miltenyl Biotech), S-Clone serum-free medium (SF-02, SF-03, CM-B, SF-B) (Sanko Junyaku), HPGM medium (Sanko Junyaku), AIM V medium (Invitrogen), Marrow MAX bone marrow medium (Invitrogen), and KnockOut. DMEM / F-12 medium (Invtrogen), Stemline hematopoietic stem cell proliferation medium (Sigma), SYN serum-free medium (SYN H, SYN B) (AbCys SA), SPE IV medium (AbCys SA), MyeloCult medium (StemCell Technologies), HPG serum-free medium (Lonza), UltraCULTURE medium (Lonza), Opti-MEM medium (Gibco Invitrogen, etc.), MEM medium (Gibco Invitrogen, etc.), MEMα (Gibco Invitrogen, etc.), DMEM medium (Gibco Invitrogen, etc.), IMDM medium (Gibco Mediums such as Invitrogen (Gibco Invitrogen, etc.), PRMI1640 medium (Gibco Invitrogen, etc.), Ham F-12 medium (Gibco, etc.), RD medium, etc., can be used, but are not limited to these. Various synthetic media for cultured meat production may also be used.

[0048] The culture medium includes a basal medium. The culture medium may contain, for example, one or more, or all of, selected from insulin, tranfuferin (apo), sodium selenite, and ethanolamine. The culture medium may contain HEPES, sodium pyruvate, vitamins, amino acids, heparin, heparan sulfate, chondroitin sulfate, etc. The culture medium may contain antibiotics (e.g., penicillin and streptomycin). The culture medium may contain glutamine. The culture medium may contain, for example, insulin, tranfuferin (apo), sodium selenite, ethanolamine, and antibiotics, and may also broadly contain buffer components such as HEPES.

[0049] The albumin-free culture medium used in the cell proliferation method of this embodiment contains less than 0.1 (w / v)% or less than 0.001 (w / v)% of albumin, or is completely albumin-free as far as detectable.

[0050] The cell proliferation method of this embodiment may further include growing animal cells under conditions sufficient for maintenance and / or proliferation. In this embodiment, for example, it may include growing animal cells to a 2x, 10x, 50x, 100x, or 500x increase from the start of culture.

[0051] [Food] The food containing the cellular product of this embodiment includes the cellular product obtained by growing animal cells using the culture medium component composition.

[0052] Cellular products, for example, when muscle cells are used as animal cells, are tissues containing proliferated muscle cells. In this case, the food is preferably cultured meat (artificial meat). Foods include not only ordinary foods but also functional foods. In terms of form, they can be various edible substances such as food and beverages. When manufacturing food, it is preferable that the materials used in the manufacturing process are food or food additives. In this embodiment, since the blood-related components, immunostimulants, or other components used in the manufacturing of the food can all be composed of food or food additives, the food can be suitably manufactured.

[0053] The food contains the immunostimulant at a concentration of 1 pg / g or more, 1 pg / mL or more, or 0.01 EU / mL or more. Specifically, it is the immunostimulant contained in the culture medium component composition and present in the culture medium during cell proliferation. By detecting the immunostimulant in the food, it can be confirmed that the food was manufactured using the cell proliferation method and the culture medium component composition.

[0054] [Effects of this embodiment] This embodiment provides a culture medium component composition with high functionality and suitability for cell culture, a method for producing the culture medium component composition, a cell proliferation method, and a food product.

[0055] Platelet lysates (PL) are being investigated as a potential substitute for serum and albumin, as they are expected to be effective in promoting cell proliferation. Compared to serum, they are known to be rich in growth factors, and even when derived from adult animals, they can be obtained using minimally invasive blood collection, which aligns with the advantages of cell-based foods. In addition, human PL promotes the proliferation of human muscle cells and mesenchymal stem cells, making it highly applicable to cell-based food manufacturing processes that often utilize muscle cells and mesenchymal stem cells. However, there are problems with using human PL in food, even indirectly, and it is difficult to procure large quantities through blood collection from humans. As a result, human PL has conventionally been used only for therapeutic purposes in humans, such as cell therapy, and its use in large-scale industrial applications and mass production, such as food manufacturing, has not been considered.

[0056] Regarding platelet-derived (PL) products from non-human sources, Japan Biomedical's Neosera is commercially available as an adult-derived PL. However, although Neosera is an adult-derived PL, it is intended for use in culturing human MSCs, and therefore has no track record of use in the production of cell-based foods. In addition, Neosera's manufacturing process includes stirring with glass material to activate platelets, but this is a physical activation, and its effect is limited, making it unsuitable for continuous, large-scale activation in conjunction with cell proliferation.

[0057] Conventional PL-containing preparations have been used in human cell therapy and other applications, and it was recognized that the co-inclusion of components such as LPS with PL was undesirable. Specifically, when administered into the bloodstream, LPS induces an immune response and becomes a pyrogen (having pyrogenic properties), so its inclusion in applications aiming for transplantation, such as regenerative medicine, or in pharmaceuticals was not envisioned. Therefore, it was difficult to develop PL preparations containing both PL and LPS. On the other hand, LPS poses no problem when consumed as food and is found in seaweed and tap water. Here, the inventors succeeded in simplifying the activation process to enhance the function of PL by deliberately using LPS to activate immune cells and platelets. Furthermore, the inventors considered that activating PL by adding an edible substance that induces activation would be simpler for continuous large-scale production, and developed a method that allows activation simply by adding an immunostimulant.

[0058] Furthermore, since raw material costs are the largest source of costs in the production of cellular foods, an appropriate combination of PL (particulate matter) and cells is necessary to obtain high yields with fewer raw materials. The inventors have also found that combining PL and cells derived from the same animal is even more efficient. Due to the effects of these materials and combinations, it is expected that they can be used as excellent additives in the production of various cellular foods.

[0059] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0060] [Test Example 1: Preparation of Culture Medium Component Compositions from Bovine or Pig Blood] Bovine PL (bPL) and Pig PL (pPL) were prepared using whole blood containing an anticoagulant, purchased from Tokyo Shibaura Organ Co., Ltd. Each whole blood sample was layered in a sucrose aqueous solution of appropriate concentration, and the supernatant fraction containing platelets was recovered by density gradient centrifugation. This supernatant fraction contained plasma and platelets and was used as the blood-related component in this embodiment. As the activating agent, LPS (lipopolysaccharide) from E. coli, Serotype EH100 (Ra) (TLRGRADE(R)) (Ready-to-Use) ALX-581-010 (Enzo Biochem Inc.) was used. When adding LPS to blood-related components, the concentration after addition to the plasma was set to 1 μg / mL, and the plasma was left overnight at 37°C to attempt to activate immune cells and platelets. Since these plasma samples were likely to contain bacteria, they were subsequently sterilized by gamma irradiation.

[0061] To convert platelets, which are blood-related components, into platelets (PL), the platelets were frozen at -80°C and thawed at 37°C four times. Since PL contains coagulation components such as fibron, after freezing and thawing, CaCl was added. ２ By adding [a certain substance], these components were coagulated, and the coagulated components were removed. The resulting PL-containing component was used as the culture medium component composition of this embodiment.

[0062] [Test Example 2: Culture of Bovine Cells] The culture medium composition obtained in Test Example 1 was used to culture bovine myoblasts, and its effect on cell proliferation was evaluated. In this case, the additives for each condition (FBS, Neosera, bPL, pPL) were all used as culture medium by adding 20% ​​to DMEM containing antibiotics.

[0063] Figure 1 is a graph showing the 3-day proliferation evaluation of culture medium component compositions for bovine myoblasts. In the figure, bPL represents bovine PL, pPL represents porcine PL, and bPL+LPS and pPL+LPS represent culture medium component compositions incubated with an immunostimulant (LPS). The vertical axis shows the number of cells counted at the start of culture and at the time of subculturing on day 3, indicating how many times the number increased after 3 days compared to the start. The proliferation rate was measured when bovine myoblasts were cultured for 3 days with each condition of culture medium added without changing the medium. Neosera (Japan Biomedical Co., Ltd.) is bPL activated with a glass material.

[0064] The culture medium composition containing bPL in this embodiment showed growth comparable to that of Neosera, and it was found that the growth efficiency was further improved by adding LPS. In addition, although the growth rate of pPL derived from pigs was lower than that of other bovine-derived conditions such as bPL, a certain level of growth was observed.

[0065] Figure 2 is a graph showing the evaluation of the proliferation of bovine myoblasts over 6 days using different culture medium compositions. The cells were subcultured on Day 3, and the cumulative proliferation rate was calculated after an additional 3 days of culture. A decrease in proliferation was observed in bPL without FBS or LPS, while proliferation was significantly improved under Neosera and bPL + LPS conditions.

[0066] Figure 3 is a micrograph of the cells at the time of Figure 2. The jelly-like substance visible in (c) bPL + LPS is the residue of coagulated plasma components. Under the bPL + LPS conditions, the cells were numerous and compact, and in particular, compared to FBS, they exhibited characteristics of highly proliferative cells.

[0067] Figure 4 is a graph showing the evaluation of the proliferation of Neosera and hPL against bovine myoblasts. For reference, data using Neosera, a conventionally known PL composition, and human PL (hPL, AventaCell Biomedical Corp.) are shown. Neosera, derived from bovine cells, showed a concentration-dependent increase in proliferation rate. On the other hand, hPL, which shows high proliferation rate against human cells, actually decreased in proliferation rate against bovine cells. This suggests that while bovine serum is commonly used against human cells, the reverse is not possible. Furthermore, considering the results mentioned above, it was found that the highest proliferation rate was achieved when both the cultured cells and the culture medium composition were from bovine cells, suggesting that using cells and culture medium compositions derived from the same animal is effective.

[0068] [Test Example 3: Preparation of a culture medium composition with added β-glucan] A culture medium composition containing bovine PL was prepared in the same manner as in Test Example 1, except that β-glucan was used as the immunostimulant instead of LPS. The purified β-glucan used was a purified product of Aureobazidium culture medium (provided by Aureo Co., Ltd.). This purified product mainly consisted of β-1,3-1,6-glucan. When adding β-glucan, the purified β-glucan was added at an amount of 1 / 10000 of the amount of the recovered fraction. Bovine myoblasts were cultured using this culture medium composition under the same conditions as in Test Example 2.

[0069] Figure 5 is a photograph illustrating the effect of β-glucan-based culture medium compositions on bovine myoblasts. The cells were observed two days after seeding. The number of cells observed in bPL (bioblast culture) with β-glucan added was higher than in bPL without β-glucan. This effect was similarly observed in bPL with LPS (luper polyphosphate) added.

[0070] [Test Example 4: Preparation of Culture Medium Composition from Pig Blood] To verify the effectiveness of using a culture medium composition derived from pig blood for culturing pig cells, a culture medium composition was prepared from pig blood. Whole pig blood containing an anticoagulant, purchased from the aforementioned supplier, was used. The supernatant fraction containing platelets was collected by density gradient centrifugation of each whole blood sample with sucrose aqueous solution. When LPS was added to the collected fraction, it was added so that the concentration after addition was 1 μg / mL. Subsequently, CaCl２ By adding [a specific substance], coagulation components such as fibrin were coagulated and precipitated, and the coagulation components were removed. After removal, the sample was sterilized using a 0.22 μm filter to obtain a culture medium component composition derived from pig blood.

[0071] [Test Example 5: Culture of Pig Muscle Cells] The effect of the aforementioned porcine blood-derived culture medium composition on cell proliferation was evaluated when used for culturing porcine cells. Primary cells isolated from porcine muscle were used for evaluation. In this case, the additives (FBS, pPL) for each condition were added to DMEM containing antibiotics at a concentration of 10% and used in the culture medium. Culture was carried out in the same manner as for cattle, except that a nearly equivalent product derived from Gram-negative bacteria was used as LPS.

[0072] Figure 6 is a graph showing the evaluation of the growth potential of porcine cells in culture medium compositions derived from porcine blood. In the figure, LPS+ refers to the component incubated with LPS added to pPL, and LPS- refers to the component without the addition of LPS. The growth rate was measured on the fourth day after seeding. The culture medium composition with added LPS showed a growth rate comparable to that of the medium with added FBS.

[0073] The results above clearly show that culture medium compositions incubated with blood-derived components (including platelets derived from bovine or porcine blood) and immunostimulants (LPS, β-glucan) promote cell proliferation to an extent equivalent to or greater than that of serum components (FBS). In particular, when bovine cells were grown using a culture medium composition containing bovine blood-derived components, the proliferation effect was significantly superior to that of conventional techniques.

[0074] [Test Example 6: Cell Growth Based on Reaction Temperature] In the incubation reaction of blood-related components and immunostimulants, we investigated whether it was possible to maintain cell growth performance by reacting in both a low-temperature environment preferred for food hygiene and a high-temperature environment preferred for reaction efficiency. LPS from E. coli, Serotype EH100 (Ra) (TLRGRADE(R)) (Ready-to-Use) ALX-581-010 (Enzo Biochem Inc.) was added to the supernatant containing bovine blood-derived platelets to a concentration of 1 μg / mL, and the reaction was carried out overnight in environments of 37°C and 4°C. After sterilization by gamma irradiation, the cells were used to culture bovine myoblasts, and cell growth was measured by staining and counting the number of cell nuclei each day of culture. Other conditions were the same as in Test Example 2.

[0075] Figure 7 is a graph showing cell proliferation under different temperature conditions for the activation of the culture medium component composition. The figure shows the mean ± standard deviation of the number of cell nuclei for three samples. As shown in the figure, nearly the same number of cell nuclei was observed whether the activation incubation was performed at 4°C or 37°C, meaning that nearly the same cell proliferation rate was observed. From these results, it was shown that the culture medium component composition can be manufactured not only at 37°C, where the reaction is generally likely to occur, but also at low temperatures of 10°C or below, as stipulated by food hygiene regulations, without any performance issues.

[0076] While preferred embodiments of the present invention have been described and illustrated above, it should be understood that these are illustrative and not limiting. Additions, omissions, substitutions, and other modifications can be made without departing from the spirit or scope of the invention. Therefore, the present invention is not limited by the foregoing description but is limited only by the scope of the appended claims.

[0077] According to the present invention, a culture medium component composition with high functionality and suitable for cell culture, a method for producing the culture medium component composition, a method for cell proliferation, and a food product can be obtained.

Claims

1. A culture medium component composition comprising blood-related components, including blood-derived components or processed products thereof, and immunostimulants that induce or promote an immune response.

2. The culture medium component composition according to claim 1, for use in cell culture for food manufacturing, cosmetics, pharmaceuticals, or research and development.

3. The culture medium component composition according to claim 1 or 2, wherein the immunostimulant is one or more selected from the group consisting of microbial components, nucleic acids, lipids, glycans, peptides, polyphenols, fermentation products, or extracts thereof.

4. The culture medium component composition according to claim 1 or 2, wherein the immunostimulant is one or more selected from the group consisting of lipopolysaccharide, β-glucan, peptidoglycan, vitamins, minerals, lactic acid bacteria, bifidobacteria, and plant-derived components.

5. The culture medium component composition according to claim 1 or 2, comprising 1 pg / g or more, 1 pg / mL or more, or 0.01 EU / mL or more of the immunostimulant.

6. The culture medium component composition according to claim 1 or 2, wherein the blood-related component is derived from an adult animal other than a human.

7. The culture medium component composition according to claim 1 or 2, wherein the blood-related component is derived from birds, fish, or mammals.

8. The culture medium component composition according to claim 1 or 2, wherein the blood-related component is bovine or porcine.

9. A method for producing a culture medium component composition according to claim 1 or 2, comprising the step of activating the blood-related component by adding the immunostimulator to the blood-related component and incubating it to obtain the culture medium component composition.

10. A method for producing a culture medium component composition according to claim 9, comprising the step of dissolving the platelets by freezing and thawing the activated composition, wherein the blood-related component includes platelets, to obtain a blood-related component containing platelet lysates, and thereby obtaining a culture medium component composition.

11. The method for producing the culture medium component composition according to claim 9, wherein the activation step is carried out in an environment of 10°C or lower.

12. A method for producing a culture medium component composition according to claim 9, further comprising a step of reducing or removing the immunostimulant after the activation step.

13. A method for cell proliferation, comprising culturing and growing animal cells using the culture medium component composition described in claim 1 or 2.

14. The cell proliferation method according to claim 13 for producing food using the animal cells.

15. The cell proliferation method according to claim 13, wherein the blood-related components contained in the culture medium component composition and the animal cells are derived from the same species of animal.

16. The cell proliferation method according to claim 15, wherein the blood-related component is derived from a bovine and the animal cell is a bovine myoblast, or the blood-related component is derived from a pig and the animal cell is a pig muscle tissue-derived cell.

17. The cell proliferation method according to claim 13, wherein the cultivation and proliferation of the animal cells is carried out at a temperature of 10°C or lower.

18. A food product comprising a cellular product obtained by growing animal cells using the culture medium component composition described in claim 1 or 2.

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