Cultured meat complex and method for producing the same

Abstract

To provide a method to manufacture lump meat in a uniform shape by culturing animal cells efficiently.SOLUTION: A cultured edible meat complex includes hollow fiber membrane or resolvent or lysate thereof, and cultured edible meat containing animal cell group existing along the hollow fiber membrane or resolvent or lysate thereof. The cultured edible meat complex can be manufactured by feeding animal cell suspension to either of the hollow fiber inside space and the hollow fiber outside space of membrane module with hollow fiber membrane, and feeding culture fluid to the other to breed the animal cell contained in the animal cell suspension along the surface of the hollow fiber membrane.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to a cultured meat composite and a method for producing the same.

Background Art

[0002] Currently, as the global population increases and people's eating habits become more carnivorous, the demand for meat is increasing. Therefore, methods for improving supply capacity, such as improving livestock production efficiency and expanding mariculture, have been developed. However, the current situation is that supply has not been able to catch up with the increasing demand.

[0003] In addition, raising livestock requires a huge amount of time, vast land, feed, water, etc., and is inefficient. Furthermore, a large amount of methane gas generated from the manure of livestock raised for meat has an adverse impact on global warming, which is also a problem. Moreover, there is a risk that viruses dangerous to the human body infect livestock and that meat is contaminated by bacteria dangerous to the human body at the meat stage, posing problems in terms of hygiene.

[0004] As a method for solving the above problems, a method of industrially culturing cells collected from livestock or the like to produce meat is known. Non-Patent Document 1 describes a method of culturing cells in a culture dish. Patent Document 1 also describes a method of culturing cells while stirring in a tank filled with a culture solution (= tank stirring culture method).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

[0007] However, in the method of culturing cells in a culture dish, the bottom surface of the culture dish is used as a scaffold, and the cells proliferate while adhering to the bottom surface. Therefore, culturing large quantities of cells requires a large culture area, and the culture efficiency is low, which is a problem. In addition, while cells can proliferate while bonding with each other in a two-dimensional direction, it is difficult for them to proliferate while bonding with each other in a three-dimensional direction. For this reason, the above method can produce minced meat, but it cannot produce chunks of meat. On the other hand, in tank agitation culture, cells are cultured while suspended in a tank, allowing them to proliferate while binding to each other not only in two dimensions but also in three dimensions. However, controlling the size of the aggregates of the proliferated cells was difficult. Another problem was that cells were easily damaged by agitation. Furthermore, culturing large quantities required large culture tanks and large amounts of culture medium, which resulted in high costs. In other words, conventional culture methods made it difficult to efficiently culture cells and produce uniformly shaped chunks of meat.

[0008] Therefore, the object of this disclosure is to provide a method for efficiently culturing animal cells to produce a block of meat with a uniform shape. Another object of this disclosure is to provide a uniformly shaped block of meat produced by the method described above. [Means for solving the problem]

[0009] The inventors diligently studied to solve the above problems and obtained the following findings. That is, 1. Hollow fiber membranes have a very large specific surface area, and when used as a scaffolding material, even small modules can have a sufficient surface area for cell culture. 2. By culturing in a small module, it becomes possible to cultivate with a small amount of culture medium, thereby reducing the cost of the culture medium. 3. By culturing the cells with the hollow fiber membrane contained within the module, the aggregate size of the proliferated cells can be easily controlled. 4. The cultured cells and the culture medium exist in a space separated by a hollow fiber membrane within the membrane module. By adjusting the pore size of the hollow fiber membrane, components necessary for cell proliferation and differentiation can be supplied from the culture medium to the cell side, while components unnecessary for cell proliferation and differentiation can be removed from the cell side to the culture medium side. This allows for cell culture while maintaining an environment suitable for proliferation and differentiation. 5. If edible hollow fiber membranes, or hollow fiber membranes whose degradation products are edible, or hollow fiber membranes whose lysates are edible are used, the cultured cells can be removed from the module along with the hollow fiber membrane or its degradation products or lysates after culturing and consumed directly. This disclosure has been completed based on these findings.

[0010] In other words, this disclosure relates to hollow fiber membranes or their decomposition products or dissolved products, Cultured meat containing a population of animal cells present along the hollow fiber membrane or its decomposition products or lysates, The present invention provides a cultured meat complex containing the following:

[0011] The disclosure also provides the cultured meat complex wherein the hollow fiber membrane is a hollow fiber membrane or a degradation product or lysate thereof, mainly composed of at least one selected from proteins, lipids, carbohydrates, polyethers, and polyesters.

[0012] The disclosure also provides the cultured meat complex wherein the hollow fiber membrane or its degradation products or soluble products are edible hollow fiber membranes or their degradation products or soluble products.

[0013] The present disclosure also provides a method for producing a cultured meat composite, which supplies an animal cell suspension to one of the internal space and the external space of the hollow fiber of a membrane module equipped with hollow fibers, supplies a culture solution to the other, and grows the animal cells contained in the suspension along the surface of the hollow fiber membrane, thereby producing the cultured meat composite.

Advantages of the Invention

[0014] According to the method for producing a cultured meat composite of the present disclosure, animal cells can be efficiently cultured to produce a cultured meat composite having a uniform shape. In addition, in this method, a small module can be used to efficiently produce a cultured meat composite, without requiring a huge amount of time and a vast area as in the case of raising conventional livestock. It also does not generate greenhouse gases such as methane gas. And the cultured meat composite obtained by the method is hygienic. Furthermore, the cultured meat composite obtained by the method is in the form of a lump of meat rather than minced meat, and the texture and taste of the meat are well reproduced.

[0015] When the hollow fiber membrane of the cultured meat composite obtained by the method is non-edible, the product after removing the hollow fiber membrane from the cultured meat composite can be used for food. On the other hand, when the hollow fiber membrane, its decomposition products, and its dissolved substances are edible, the cultured meat composite can be eaten as it is. And by selecting the constituent material of the hollow fiber membrane, added value can be imparted to the cultured meat. For example, if an edible hollow fiber membrane formed of collagen is used, a cultured meat composite containing collagen can be obtained, and such a cultured meat composite can be expected to have a beauty effect and an effect of improving bone density.

Brief Description of the Drawings

[0016] [Figure 1] It is a schematic diagram (cross-sectional view) explaining the state where the cells introduced into the membrane module adhere to and grow on the surface of the hollow fiber membrane serving as a scaffold material. [Figure 2] It is a schematic diagram showing an example of a method for repeatedly producing a cultured meat composite using a membrane module. [Figure 3] It is a schematic diagram (cross-sectional view) showing an example of a membrane module. [Figure 4] It is a schematic diagram (cross-sectional view) showing another example of a membrane module. [Figure 5] It is a schematic diagram (cross-sectional view) showing another example of a membrane module.

Mode for Carrying Out the Invention

[0017] [Cultured meat composite] The cultured meat composite of the present disclosure includes a hollow fiber membrane or its decomposition product or dissolved product, and cultured meat containing a cell group of an animal existing along (or in the vicinity of) the hollow fiber membrane or its decomposition product or dissolved product.

[0018] In the cultured meat composite, a part or all of the hollow fiber membrane may be decomposed or dissolved. The hollow fiber membrane is preferably formed of a material having excellent affinity with cells, and particularly preferably formed of a material having excellent hydrophilicity. Alternatively, when it is formed of a material having poor hydrophilicity, it is preferably one whose surface has been hydrophilized.

[0019] The hollow fiber membrane or its decomposition product or dissolved product is preferably edible. Therefore, the hollow fiber membrane is preferably a hollow fiber membrane formed of an edible substance. The decomposition product is preferably a decomposition product of the hollow fiber membrane and is formed of an edible substance. The dissolved product is preferably a dissolved product of the hollow fiber membrane and is formed of an edible substance. Incidentally, if the decomposition product and the dissolved product are edible, the hollow fiber membrane before decomposition or dissolution may be non-edible, but it is particularly preferable that the hollow fiber membrane before decomposition or dissolution is also edible.

[0020] In this specification, "edible" means that a substance does not cause harm to humans when ingested, and does not necessarily have to be digestible. "Edible substances" are preferably, for example, substances approved for oral administration as pharmaceuticals or pharmaceutical additives, and / or substances approved as food or food additives.

[0021] The cultured meat contains at least a population of animal cells. The cell population may form tissues. That is, the cultured meat may contain animal muscle tissue, and may further contain blood vessels, adipose tissue, etc.

[0022] When the cultured meat complex contains hollow fiber membranes that have not decomposed or dissolved, it is preferable that the cultured meat exists in a state of being adhered to the surface of the hollow fiber membranes.

[0023] In the total amount of the cultured meat complex, the proportion of the sum of [hollow fiber membrane or its degradation products or lysates] and [cultured meat containing animal cell populations] is, for example, 50% by weight or more, preferably 60% by weight or more, particularly preferably 70% by weight or more, most preferably 80% by weight or more, and especially preferably 90% by weight or more.

[0024] In the cultured meat complex, the ratio (by weight) of [hollow fiber membrane or its degradation or lysate] to [cultured meat containing animal cells] is, for example, 3 / 97 to 50 / 50, preferably 5 / 95 to 40 / 65, and particularly preferably 10 / 90 to 30 / 70.

[0025] [Method for producing cultured meat complex] The cultured meat complex can be produced, for example, by supplying an animal cell suspension to one of the intracapillary spaces (ICS) and extracapillary spaces (ECS) of a membrane module equipped with a hollow fiber membrane, and supplying a culture medium to the other, thereby allowing the animal cells contained in the suspension to utilize the hollow fiber membrane as a scaffold and growing the animal cells along the surface of the hollow fiber membrane.

[0026] In the above method, an animal cell suspension is supplied to one of the ICS and ECS, and a culture medium is supplied to the other. This separates the space where cells proliferate from the space where the culture medium circulates using a hollow fiber membrane with semipermeable properties, allowing the supply of components necessary for cell proliferation and differentiation from the space where the culture medium circulates to the space where cells proliferate. Therefore, the space where cells proliferate can be easily prepared to create an environment conducive to cell differentiation and proliferation.

[0027] In the above method, it is preferable to supply an animal cell suspension to the ECS and culture the cells in the ECS.

[0028] Furthermore, it is preferable to circulate the culture medium through the ICS. Note that the culture medium can be circulated by either collecting the culture medium that has passed through the ICS and passing it through the ICS again, or by discarding the culture medium that has passed through the ICS and passing it through a new culture medium. Additionally, if the culture medium contains components unnecessary for cell proliferation and differentiation, the pore size of the hollow fiber membrane can be adjusted to prevent these unnecessary components from permeating the membrane, thus eliminating the need to purify the culture medium beforehand.

[0029] As shown in Figure 1, when an animal cell suspension is supplied to the ECS6, the animal cells 13 in the animal cell suspension use the surface of the hollow fiber membrane 2 as a scaffold and adhere to it. The animal cells 13 that adhere to the surface of the hollow fiber membrane 2 then proliferate and form a cell population (or aggregate). The cells that make up the cell population then combine with each other to form a tissue.

[0030] When an animal cell suspension containing muscle cells is cultured, muscle cell populations are formed, and muscle tissue is created. For example, when an animal cell suspension containing vascular endothelial cells is cultured, blood vessels are formed, and when an animal cell suspension containing adipocytes is cultured, adipose tissue is formed. Therefore, by selecting the type of cells in the animal cell suspension, it is possible to produce cultured meat with desired properties (marbling, lean meat, etc.).

[0031] The culture temperature for animal cells is preferably 10-50°C, and particularly preferably 30-40°C, as this promotes active cell proliferation and differentiation.

[0032] After the animal cell culture is complete, a maturation process may be included. More specifically, the cells may be matured at a temperature of 0-60°C for approximately 1 to 336 hours.

[0033] Furthermore, collagen is a compound that has the property of becoming gelatinized when subjected to heat treatment. Therefore, a cultured meat complex obtained using a hollow fiber membrane composed of collagen may be subjected to heat treatment to gelatinize the collagen. According to this method, a cultured meat complex containing gelatin is obtained, but since gelatin has a lower viscosity than collagen, it can be easily removed from the cultured meat complex, and cultured meat that does not contain components derived from the hollow fiber membrane can be produced.

[0034] According to the method for producing cultured meat complexes of this disclosure, for example, as shown in Figure 2, an animal cell suspension and culture medium are supplied to a membrane module 1, which contains a hollow fiber membrane 2 in a housing 3, to allow the animal cells to proliferate and differentiate, and then, if necessary, mature, a cultured meat complex 12 is formed in the housing 3. After removing the formed cultured meat complex 12 from the housing 3, a new hollow fiber membrane 2 is stored in the housing 3, and the cultured meat complex 2 can be repeatedly produced by supplying the animal cell suspension and culture medium.

[0035] Furthermore, according to the above manufacturing method, since cells are cultured within the membrane module 1, a cultured meat complex 12 with a uniform shape can be produced.

[0036] According to the method for producing cultured meat complexes of this disclosure, by selecting materials that constitute the hollow fiber membrane, it is possible to produce a cultured meat complex containing a lysate of the hollow fiber membrane (= a cultured meat complex containing the hollow fiber membrane in a lysate state), or a cultured meat complex containing a degradation product of the hollow fiber membrane (= a cultured meat complex containing the hollow fiber membrane in a degraded state). For example, if a hollow fiber membrane is used that is made of a material with sufficient water solubility to maintain its shape during cell culture, a cultured meat complex can be obtained in which some or all of the contained hollow fiber membrane dissolves in water. For example, by using a hollow fiber membrane made of a material that can be converted from insoluble to soluble by applying a specific stimulus, cells can be cultured while the hollow fiber membrane remains insoluble, and after the cell culture is complete, the hollow fiber membrane can be converted from insoluble to soluble, thereby obtaining a cultured meat complex in which some or all of the contained hollow fiber membrane is dissolved in water. For example, by using a hollow fiber membrane made of a material that decomposes when a specific stimulus is applied, cells can be cultured while maintaining the shape of the hollow fiber membrane, and after the cell culture is complete, the hollow fiber membrane can be decomposed to obtain a cultured meat complex in which some or all of the hollow fiber membrane it contains has been decomposed. Examples of the specific stimulation methods include treatment with degrading enzymes (e.g., hydrolytic enzyme treatment), ultrasonic treatment, pressurization treatment, and heat treatment. A method for degrading the hollow fiber membrane by the aforementioned degrading enzyme treatment is to supply the degrading enzyme to the ICS.

[0037] (Membrane module) The aforementioned membrane module comprises a hollow fiber membrane that cells use as a scaffold.

[0038] The membrane module preferably has a configuration in which a hollow fiber membrane is housed in a housing. The shape of the housing is not particularly limited as long as it is a shape that can fill the ECS with liquid (e.g., animal cell suspension or culture medium) when the ECS is supplied with the liquid, but is cylindrical for example.

[0039] As shown in Figure 3, the membrane module 1 (Embodiment 1) has a configuration in which multiple hollow fiber membranes 2, each approximately the same length as the housing 3, are bundled together and housed in the housing 3.

[0040] The side of the housing 3 is provided with an inlet 4 and an outlet 5. The inlet and outlet are continuous via an ECS 6.

[0041] A bundle of hollow fiber membranes 2 is inserted into the housing 3, and the housing 3 is provided with caps 7 having openings at both ends. One of the openings is referred to as the first opening 8, and the other as the second opening 9.

[0042] At both ends of the bundle of hollow fiber membranes 2, the spaces between adjacent hollow fiber membranes 2 and the spaces between the bundle of hollow fiber membranes 2 and the sides of the housing 3 are sealed with a sealing material 10, and the first opening 8 and the second opening 9 are continuous via the ICS 11.

[0043] As shown in Figure 4, the membrane module 1' (Embodiment 2) has a configuration in which a hollow fiber membrane 2, which is longer than the length of the housing 3', is stored in the housing 3' in a folded state.

[0044] As shown in Figure 5, the membrane module 1" (Embodiment 3) has a configuration in which a hollow fiber membrane 2, which is longer than the length of the housing 3", is stored in the housing 3" in a folded state.

[0045] The hollow fiber membrane is a straw-shaped separation membrane made of a material having semipermeable membrane properties. It can pass through some components of the dispersion medium and dispersed phosphate contained in animal cell suspensions and culture media, but not animal cells. It is preferable to select the components that can pass through depending on the type of animal cells being cultured. The components that can pass through can be controlled by adjusting the material constituting the hollow fiber membrane and the pore size of the hollow fiber membrane.

[0046] The inner diameter of the hollow fiber membrane is, for example, 100 to 1000 μm, preferably 150 to 500 μm. The film thickness of the hollow fiber membrane is, for example, 10 to 150 μm, preferably 20 to 100 μm. The pore size of the hollow fiber membrane is, for example, 0.001 to 0.5 μm.

[0047] The membrane permeability of the hollow fiber membrane is, for example, 10 to 10,000 L / (m³). 2 (hr·0.1MPa), preferably 100-7000 L / (m 2The values ​​are (h, 0.1 MPa). The membrane permeability is measured at room temperature (25°C) and normal pressure, with one end of the hollow fiber membrane closed and pure water supplied from the other end at 0.1 MPa. The volume of pure water that permeates the hollow fiber membrane in a certain time is measured using the sampling time (h) and the membrane surface area (m²) of the inner surface of the hollow fiber membrane. 2 It can be found by dividing by ).

[0048] Since the hollow fiber membrane functions as a biological scaffold, it is preferable that it be made of a material with excellent affinity for animal cells, and in particular, a material with excellent hydrophilicity. If the hollow fiber membrane is made of a material with poor hydrophilicity, it is preferable that its surface be treated to make it hydrophilic.

[0049] The hollow fiber membrane is preferably an edible hollow fiber membrane, that is, a hollow fiber membrane made of an edible material. This is because if an edible hollow fiber membrane is used, the resulting cultured meat complex can be consumed as is without removing the hollow fiber membrane.

[0050] The material constituting the hollow fiber membrane is, for example, at least one selected from proteins, lipids, carbohydrates, polyethers, and polyesters.

[0051] Examples of the aforementioned lipids include paraffin, shellac, beeswax, carnauba wax, lanolin, and candelilla wax.

[0052] Examples of the polyether include polyethylene glycol.

[0053] Examples of the aforementioned polyester include polyglycolic acid and glycolic acid copolymer.

[0054] Among the materials constituting the edible hollow fiber membrane, at least one selected from fibrous proteins, polysaccharides, complex carbohydrates, and derivatives thereof is preferred.

[0055] Examples of the fibrous protein or its derivatives include collagen, fibronectin, keratin, myosin, elastin, gelatin, and gluten.

[0056] Examples of the polysaccharides or their derivatives include cellulose, cellulose derivatives (e.g., cellulose acetate, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), methylcellulose, carboxymethylcellulose, etc.), bacterial cellulose, starch, glycogen, chitin, chitosan, agar, glucomannan, xylose, oligosaccharides, fucoidan, pullulan, guar gum, xanthan gum, carrageenan, locust bean gum, alginic acid, alginates such as sodium alginate, and dextrin.

[0057] Examples of the aforementioned complex carbohydrates or their derivatives include hyaluronic acid, chondroitin sulfate, heparin, proteoglycans, and pectin.

[0058] Furthermore, the edible hollow fiber membrane is composed of a material that becomes an edible decomposition product or lysate after the completion of cell culture, and within the cultured meat complex, some or all of the edible hollow fiber membrane may change into an edible decomposition product or lysate, and it is not necessary for the hollow fiber membrane to retain its shape.

[0059] Materials for forming edible hollow fiber membranes in cultured meat complexes, in which at least a portion becomes edible decomposed or dissolved products, include water-soluble materials, materials that can be converted from insoluble to water-soluble by applying specific stimuli, or materials that decompose by applying specific stimuli.

[0060] For example, the solubility of cellulose acetate in water changes depending on the total degree of acetyl substitution, and cellulose acetate with a total degree of acetyl substitution of about 0.5 to 1.5 has moderate water solubility. When cells are cultured using an edible hollow fiber membrane formed from such cellulose acetate, the hollow fiber membrane dissolves in water over time, resulting in a cultured meat complex in which some or all of the contained hollow fiber membrane is dissolved.

[0061] Furthermore, the solubility of collagen changes depending on the pH of the aqueous solution. Collagen is insoluble in aqueous solutions with a pH of 7 or higher (neutral or alkaline aqueous solutions) and soluble in aqueous solutions with a pH of less than 7 (acidic aqueous solutions). Therefore, by using a hollow fiber membrane composed of collagen, maintaining the shape of the hollow fiber membrane by keeping the pH of the aqueous solution supplied to the membrane module at 7 or higher during cell culture, and then changing the pH of the aqueous solution supplied to the membrane module to less than 7 after the completion of cell culture, a cultured meat complex can be obtained in which some or all of the collagen constituting the hollow fiber membrane is dissolved in the aqueous solution.

[0062] Furthermore, hollow fiber membranes composed of polysaccharides or their derivatives (e.g., cellulose, cellulose derivatives, chitin, collagen, etc.) can be degraded by treatment with degrading enzymes (e.g., hydrolytic enzymes such as cellulase, hemicellulase, chitinase, collagenase, etc.). Therefore, by performing cell culture using the aforementioned hollow fiber membranes composed of polysaccharides or their derivatives, and then supplying degrading enzymes (e.g., hydrolytic enzymes) to the ICS after the cell culture is completed to degrade the hollow fiber membranes, a cultured meat complex can be obtained in which some or all of the contained hollow fiber membranes have been degraded.

[0063] (Animal cell suspension) The animal cell suspension is a suspension of animal cells in a dispersion medium. Examples of animal cells include muscle cells such as muscle satellite cells and myoblasts, mesenchymal stem cells, adipocytes, fibroblasts, and vascular endothelial cells from animals. These can be included individually or in combination of two or more types. The animal cells can be appropriately selected according to the desired properties (marbling, lean meat, etc.) of the cultured meat complex.

[0064] The animals mentioned above include terrestrial animals and aquatic animals. However, humans are not included in the animals mentioned above. The aquatic animals mentioned above include marine animals and freshwater animals. Examples of terrestrial animals include livestock (including poultry) such as cattle, pigs, and chickens. Examples of aquatic animals include fish, crustaceans, mollusks, shellfish, etc.

[0065] (Culture solution) The culture medium is not particularly limited as long as it contains components necessary for the growth of the animal cells to be cultured. The components necessary for the growth of the cells include various organic and inorganic substances, and dissolved gases such as oxygen. As the culture medium, for example, a general-purpose liquid culture medium such as Dulbecco's MEM, RPMI1640, or HAM F12 can be used, to which serum, various growth factors, differentiation-inducing factors, etc., have been added.

[0066] The configurations and combinations thereof described above are examples only, and additions, omissions, substitutions, and modifications to the configurations may be made as appropriate, without departing from the spirit of this disclosure. [Explanation of symbols]

[0067] 1. Membrane module 1' Membrane Module 1” membrane module 2 Hollow fiber membrane 3 Housing 3' Housing 3" Housing 4 ECS inlet 5 ECS outlet 6 ECS 7 Housing cap 8. First opening 9. Second opening 10. Sealing material 11 ICS 12 Cultured meat complex 13. Animal cells to be cultured

Claims

1. A hollow fiber membrane that may be partially decomposed or dissolved, The cultured meat includes a population of animal cells present along the outer surface of the hollow fiber membrane, The hollow fiber membrane is a hollow fiber membrane mainly composed of cellulose acetate, A cultured meat complex in which the proportion of the hollow fiber membrane or its decomposition product or lysate to the cultured meat (by weight ratio) is 5 / 95 to 40 / 65, the inner diameter of the hollow fiber membrane is 150 to 500 μm, and the cellulose acetate is cellulose acetate with a total acetyl substitution degree of 0.5 to 1.

5.

2. A method for producing a cultured meat complex according to claim 1, comprising supplying an animal cell suspension to one of the hollow fiber internal space and the hollow fiber external space of a membrane module equipped with a hollow fiber membrane, and supplying a culture medium to the other, thereby causing the animal cells contained in the suspension to grow along the surface of the hollow fiber membrane.

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