Method for culturing tissue-derived cells and culture device comprising tissue-derived cells

Abstract

The purpose of the present invention is to provide a novel method for culturing tissue-derived cells. The method uses a cell culture method including a step for co-culturing cells derived from the skin and at least one type of cell selected from among cells derived from the intestine, cells derived from the heart, cells derived from the brain, cells derived from the stomach, cells derived from the embryonic membrane, cells derived from the kidney, and cells derived from the lung. And culturing the skin-derived cells.

Description

Technical Field

[0001] The present invention relates to a method for culturing cells from tissues and an apparatus for culturing cells containing cells from tissues. Background Technology

[0002] Previously, the standard culture medium for culturing cells from tissues was DMEM or RPMI-1640 containing 10%–20% serum (e.g., Scientific Reports volume 12, Article number: 827 (2022)). Summary of the Invention

[0003] The technical problem that the invention aims to solve The purpose of this invention is to provide a novel co-culture method for cells from multiple tissues and a novel method for culturing target cells using culture supernatants from cells from multiple tissues.

[0004] Technical solutions for solving technical problems [I] One embodiment of the present invention is a cell culture method, characterized in that it includes a step of co-culturing cells from the skin with one or more cells selected from cells from the intestine, heart, brain, stomach, embryonic membrane, kidney, and lung.

[0005] Another embodiment of the present invention is a cell culture method comprising culturing cells derived from skin in the presence of a culture supernatant obtained from culturing one or more cells selected from cells derived from the intestine, heart, brain, stomach, embryonic membrane, kidney, and lung. In this method, the culture supernatant may further comprise a culture supernatant obtained from culturing cells derived from skin. Furthermore, the culture may be a co-culture.

[0006] Another embodiment of the present invention is a culture system in which cells from the skin are co-cultured with one or more cells selected from cells from the intestine, heart, brain, stomach, embryonic membrane, kidney, and lung.

[0007] Another embodiment of the present invention is a culture system in which cells derived from skin are cultured in the presence of a culture supernatant obtained from culturing cells selected from one or more cells derived from the intestine, heart, brain, stomach, embryonic membrane, kidney, and lung. The culture supernatant may further include a culture supernatant obtained from culturing cells derived from skin. The culture can be a co-culture.

[0008] [II] One embodiment of the present invention is a cell culture method, characterized in that it includes a step of co-culturing cells from the intestine with one or more cells selected from cells from the skin, the heart, the brain, the stomach, the embryonic membrane, the kidney, and the lung.

[0009] Another embodiment of the present invention is a cell culture method comprising: culturing cells derived from the intestine in the presence of a culture supernatant obtained from culturing cells selected from one or more cells derived from skin, heart, brain, stomach, embryonic membrane, kidney, and lung. In this method, the culture supernatant may further comprise a culture supernatant obtained from culturing cells derived from the intestine. Furthermore, the culture may be a co-culture.

[0010] Another embodiment of the present invention is a culture system in which cells from the intestine are co-cultured with one or more cells selected from cells from the skin, heart, brain, stomach, embryonic membrane, kidney, and lung.

[0011] Another embodiment of the present invention is a culture system in which cells from the intestine are cultured in the presence of a culture supernatant obtained from culturing cells selected from one or more cells from the skin, heart, brain, stomach, embryonic membrane, kidney, and lung. The culture supernatant may further include a culture supernatant obtained from culturing cells from the intestine. The culture can be a co-culture.

[0012] [III] One embodiment of the present invention is a cell culture method, characterized in that it includes a step of co-culturing cells from the heart with one or more cells selected from cells from the skin, intestines, brain, stomach, embryonic membranes, kidneys, and lungs.

[0013] Another embodiment of the present invention is a cell culture method comprising: culturing cells derived from the heart in the presence of a culture supernatant obtained from culturing one or more cells selected from cells derived from skin, intestine, brain, stomach, embryonic membrane, kidney, and lung. In this method, the culture supernatant may further comprise a culture supernatant obtained from culturing cells derived from the heart. Furthermore, the culture may be a co-culture.

[0014] Another embodiment of the present invention is a culture system in which cells from the heart are co-cultured with one or more cells selected from cells from the skin, intestine, brain, stomach, embryonic membrane, kidney, and lung.

[0015] Another embodiment of the present invention is a culture system in which cells derived from the heart are cultured in the presence of culture supernatants obtained from culturing cells selected from one or more cells derived from skin, intestine, brain, stomach, embryonic membrane, kidney, and lung. The culture supernatants may further include culture supernatants obtained from culturing cells derived from the heart. The culture can be a co-culture.

[0016] [IV] One embodiment of the present invention is a cell culture method, characterized in that it includes a step of co-culturing cells from the brain with one or more cells selected from cells from the skin, intestines, heart, stomach, embryonic membranes, kidneys, and lungs.

[0017] Another embodiment of the present invention is a cell culture method comprising: culturing brain cells in the presence of a culture supernatant obtained from culturing one or more cells selected from skin cells, intestinal cells, heart cells, stomach cells, embryonic membrane cells, kidney cells, and lung cells. In this method, the culture supernatant may further comprise a culture supernatant obtained from culturing brain cells. Furthermore, the culture may be a co-culture.

[0018] Another embodiment of the present invention is a culture system in which cells from the brain are co-cultured with one or more cells selected from cells from the skin, intestine, heart, stomach, embryonic membrane, kidney, and lung.

[0019] Another embodiment of the present invention is a culture system in which brain cells are cultured in the presence of culture supernatants obtained from culturing one or more cells selected from skin cells, intestinal cells, heart cells, stomach cells, embryonic membrane cells, kidney cells, and lung cells. The culture supernatants may further include culture supernatants obtained from culturing brain cells. The culture can be a co-culture.

[0020] [V] One embodiment of the present invention is a cell culture method, characterized in that it includes a step of co-culturing cells from the stomach with one or more cells selected from cells from the skin, intestines, heart, brain, embryonic membranes, kidneys, and lungs.

[0021] Another embodiment of the present invention is a cell culture method comprising: culturing cells from the stomach in the presence of a culture supernatant obtained from culturing cells selected from one or more cells from the skin, intestine, heart, brain, embryonic membrane, kidney, and lung. In this method, the culture supernatant may further include culture supernatant obtained from culturing cells from the stomach. Furthermore, the culture may be a co-culture.

[0022] Another embodiment of the present invention is a culture system in which cells from the stomach are co-cultured with one or more cells selected from cells from the skin, intestines, heart, brain, embryonic membranes, kidneys, and lungs.

[0023] Another embodiment of the present invention is a culture system in which cells from the stomach are cultured in the presence of a culture supernatant obtained from culturing cells selected from one or more cells from the skin, intestine, heart, brain, embryonic membrane, kidney, and lung. The culture supernatant may further include a culture supernatant obtained from culturing cells from the stomach. The culture can be a co-culture.

[0024] [VI] One embodiment of the present invention is a cell culture method, characterized in that it includes a step of co-culturing cells derived from the embryonic membrane with one or more cells selected from cells derived from the skin, intestine, heart, brain, stomach, kidney, and lung.

[0025] Another embodiment of the present invention is a cell culture method comprising: culturing cells derived from the embryonic membrane in the presence of a culture supernatant obtained from culturing one or more cells selected from cells derived from skin, intestine, heart, brain, stomach, kidney, and lung. In this method, the culture supernatant may further comprise a culture supernatant obtained from culturing cells derived from the embryonic membrane. Furthermore, the culture may be a co-culture.

[0026] Another embodiment of the present invention is a culture system in which cells derived from the embryonic membrane are co-cultured with one or more cells selected from cells derived from the skin, intestine, heart, brain, stomach, kidney, and lung.

[0027] Another embodiment of the present invention is a culture system in which cells derived from the embryonic membrane are cultured in the presence of a culture supernatant obtained from culturing cells selected from one or more cells derived from skin, intestine, heart, brain, stomach, kidney, and lung. The culture supernatant may further include a culture supernatant obtained from culturing cells derived from the embryonic membrane. The culture can be a co-culture.

[0028] [VII] One embodiment of the present invention is a cell culture method, characterized in that it includes a step of co-culturing cells from the kidney with one or more cells selected from cells from the skin, intestine, heart, brain, stomach, embryonic membrane and lung.

[0029] Another embodiment of the present invention is a cell culture method comprising: culturing kidney cells in the presence of a culture supernatant obtained from culturing one or more cells selected from skin cells, intestinal cells, heart cells, brain cells, stomach cells, embryonic membrane cells, and lung cells. In this method, the culture supernatant may further comprise a culture supernatant obtained from culturing kidney cells. Furthermore, the culture may be a co-culture.

[0030] Another embodiment of the present invention is a culture system in which cells from the kidney are co-cultured with one or more cells selected from cells from the skin, intestine, heart, brain, stomach, embryonic membrane and lung.

[0031] Another embodiment of the present invention is a culture system in which kidney cells are cultured in the presence of culture supernatants obtained from culturing one or more cells selected from skin cells, intestinal cells, heart cells, brain cells, stomach cells, embryonic membrane cells, and lung cells. The culture supernatants may further include culture supernatants obtained from culturing kidney cells. The culture can be a co-culture.

[0032] [VIII] One embodiment of the present invention is a cell culture method, characterized in that it includes a step of co-culturing cells from the lungs with one or more cells selected from cells from the skin, intestines, heart, brain, stomach, embryonic membranes and kidneys.

[0033] Another embodiment of the present invention is a cell culture method comprising: culturing lung cells in the presence of a culture supernatant obtained from culturing one or more cells selected from skin cells, intestinal cells, heart cells, brain cells, stomach cells, embryonic membrane cells, and kidney cells. In this method, the culture supernatant may further comprise a culture supernatant obtained from culturing lung cells. Furthermore, the culture may be a co-culture.

[0034] Another embodiment of the present invention is a culture system in which cells from the lungs are co-cultured with one or more cells selected from cells from the skin, intestines, heart, brain, stomach, embryonic membranes and kidneys.

[0035] Another embodiment of the present invention is a culture system in which lung cells are cultured in the presence of a culture supernatant obtained from culturing one or more cells selected from skin cells, intestinal cells, heart cells, brain cells, stomach cells, embryonic membrane cells, and kidney cells. The culture supernatant may further include culture supernatant obtained from culturing lung cells. The culture can be a co-culture.

[0036] ==Cross-citation with related literature== This application claims priority based on Japanese Patent Application 2023-111706, filed on July 6, 2023, which is incorporated herein by reference. Detailed Implementation

[0037] Based on the description in this specification, the objectives, features, advantages, and technical concepts of the present invention are clear to those skilled in the art, and can be readily reproduced by anyone skilled in the art. The embodiments and specific examples described below are intended to illustrate preferred embodiments of the invention and are for illustrative purposes only; the invention is not limited thereto. Those skilled in the art should understand that various changes and modifications can be made based on the description in this specification within the intent and scope of the invention disclosed herein.

[0038] [1] Cultivation method <Culturing methods for cells derived from the skin> One embodiment of the present invention provides a method for culturing skin-derived cells, comprising the step of co-culturing skin-derived cells with one or more cells selected from cells derived from the intestine, heart, brain, stomach, embryonic membrane, kidney, and lung. Particularly preferred is the co-culturing of skin-derived cells with cells selected from the following combinations: (1) Cells from the intestine and cells selected from cells from the heart, brain, stomach, kidney and lung; (2) Cells from the heart and cells selected from cells from the brain, stomach, embryonic membranes and lungs; (3) Cells from the brain and cells selected from cells from the stomach, cells from the embryonic membrane and cells from the lung; (4) Cells from the stomach and cells selected from cells from the kidneys, cells from the embryonic membranes and cells from the lungs; (5) Cells from the kidney and cells selected from cells from the embryonic membrane and cells from the lung; (6) Cells from the embryonic membrane and cells from the lung.

[0039] Another embodiment of the present invention is a cell culture method comprising: culturing cells derived from skin in the presence of a culture supernatant obtained from culturing one or more cells selected from cells derived from the intestine, heart, brain, stomach, embryonic membrane, kidney, and lung. Particularly preferred is the use of a culture supernatant obtained from co-culturing cells with cells selected from the following combinations: (1) Cells from the intestine and cells selected from cells from the heart, brain, stomach, kidney and lung; (2) Cells from the heart and cells selected from cells from the brain, stomach, embryonic membranes and lungs; (3) Cells from the brain and cells selected from cells from the stomach, cells from the embryonic membrane and cells from the lung; (4) Cells from the stomach and cells selected from cells from the kidneys, cells from the embryonic membranes and cells from the lungs; (5) Cells from the kidney and cells selected from cells from the embryonic membrane and cells from the lung; (6) Cells from the embryonic membrane and cells from the lung.

[0040] The culture supernatant may also include culture supernatant obtained from culturing cells derived from the skin. Additionally, the culture used to obtain the culture supernatant can be a co-culture.

[0041] <Culturing methods for cells derived from the gut> One embodiment of the present invention provides a method for culturing intestinal cells, comprising the step of co-culturing intestinal cells with one or more cells selected from cells derived from skin, heart, brain, stomach, embryonic membrane, kidney, and lung. Particularly preferred is co-culturing intestinal cells with cells selected from the following combinations: (1) Cells from the skin and cells selected from cells from the brain and cells from the kidneys; (2) Cells from the brain and cells from the stomach; (3) Cells from the stomach and cells selected from cells from the kidneys, cells from the embryonic membranes and cells from the lungs.

[0042] Another embodiment of the present invention is a cell culture method comprising: culturing cells from the intestine in the presence of a culture supernatant obtained from culturing one or more cells selected from cells from the skin, heart, brain, stomach, embryonic membrane, kidney, and lung. Particularly preferred is the use of a culture supernatant obtained by co-culturing cells with cells selected from the following combinations: (1) Cells from the skin and cells selected from cells from the brain and cells from the kidneys; (2) Cells from the brain and cells from the stomach; (3) Cells from the stomach and cells selected from cells from the kidneys, cells from the embryonic membranes and cells from the lungs.

[0043] The culture supernatant may also include culture supernatant obtained from culturing cells derived from the intestine. Additionally, the culture used to obtain the culture supernatant can be a co-culture.

[0044] <Culturing methods for cells derived from the heart> One embodiment of the present invention provides a method for culturing heart-derived cells, comprising the step of co-culturing heart-derived cells with one or more cells selected from cells derived from skin, intestines, brain, stomach, embryonic membranes, kidneys, and lungs. Particularly preferred is the co-culturing of heart-derived cells with cells selected from the following combinations: (1) Cells from the skin and cells selected from cells from the intestine and cells from the lung; (2) Cells from the intestine and cells from the embryonic membrane; (3) Cells from the brain and cells selected from cells from the stomach, cells from the embryonic membrane and cells from the lungs.

[0045] Another embodiment of the present invention is a cell culture method comprising: culturing cells from the heart in the presence of a culture supernatant obtained from culturing one or more cells selected from cells from the skin, intestine, brain, stomach, embryonic membrane, kidney, and lung. Particularly preferred is the use of a culture supernatant obtained from co-culturing cells selected from the following combinations: (1) Cells from the skin and cells selected from cells from the intestine and cells from the lung; (2) Cells from the intestine and cells from the embryonic membrane; (3) Cells from the brain and cells selected from cells from the stomach, cells from the embryonic membrane and cells from the lungs.

[0046] The culture supernatant may also include culture supernatant obtained from culturing cells from the heart. Additionally, the culture used to obtain the culture supernatant can be a co-culture.

[0047] <Culturing methods for brain cells> One embodiment of the present invention provides a method for culturing brain-derived cells, comprising the step of co-culturing brain-derived cells with one or more cells selected from cells derived from skin, intestines, heart, stomach, embryonic membranes, kidneys, and lungs. Particularly preferred is co-culturing brain-derived cells with cells selected from the following combinations: (1) Cells from the skin and cells selected from cells from the intestine, stomach, kidney, embryonic membrane and lung; (2) Cells from the intestine and cells selected from cells from the heart, stomach, kidney and lung; (3) Cells from the heart and cells from the kidneys; (4) Cells from the stomach and cells from the lungs; (5) Cells from the kidneys and cells from the lungs; (6) Cells from the embryonic membrane and cells from the lung.

[0048] Another embodiment of the present invention is a cell culture method comprising: culturing brain cells in the presence of a culture supernatant obtained from culturing one or more cells selected from skin cells, intestinal cells, heart cells, stomach cells, embryonic membrane cells, kidney cells, and lung cells. Particularly preferred is the use of a culture supernatant obtained from co-culturing cells selected from the following combinations: (1) Cells derived from the skin and cells selected from cells derived from the intestine, stomach, kidney, embryonic membrane, and lung. (2) Cells from the intestine and cells selected from cells from the heart, stomach, kidneys and lungs. (3) Cells from the heart and cells from the kidneys, (4) Cells from the stomach and cells from the lungs (5) Cells from the kidneys and cells from the lungs, (6) Cells from the embryonic membrane and cells from the lung.

[0049] The culture supernatant may also include culture supernatant obtained from culturing cells from the brain. Additionally, the culture used to obtain the culture supernatant can be a co-culture.

[0050] <Culturing methods for cells from the stomach> One embodiment of the present invention provides a method for culturing stomach-derived cells, comprising the step of co-culturing stomach-derived cells with one or more cells selected from cells derived from skin, intestines, heart, brain, embryonic membranes, kidneys, and lungs. Particularly preferred is co-culturing stomach-derived cells with cells selected from the following combinations: (1) Cells derived from the skin and cells selected from cells derived from the intestine, the heart, the kidney, and the embryonic membrane; (2) Cells from the intestine and cells selected from cells from the brain and cells from the embryonic membrane; (3) Cells from the brain and cells from the embryonic membrane; (4) Cells from the kidney and cells from the embryonic membrane; (5) Cells from the embryonic membrane and cells from the lung.

[0051] Another embodiment of the present invention is a cell culture method comprising: culturing cells from the stomach in the presence of a culture supernatant obtained from culturing one or more cells selected from cells from the skin, intestine, heart, brain, embryonic membrane, kidney, and lung. Particularly preferred is the use of a culture supernatant obtained from co-culturing cells with cells selected from the following combinations: (1) Cells derived from the skin and cells selected from cells derived from the intestine, the heart, the kidney, and the embryonic membrane; (2) Cells from the intestine and cells selected from cells from the brain and cells from the embryonic membrane; (3) Cells from the brain and cells from the embryonic membrane; (4) Cells from the kidney and cells from the embryonic membrane; (5) Cells from the embryonic membrane and cells from the lung.

[0052] The culture supernatant may also include culture supernatant obtained from culturing cells from the stomach. Additionally, the culture used to obtain the culture supernatant can be a co-culture.

[0053] <Culturing methods for cells derived from the embryonic membrane> One embodiment of the present invention provides a method for culturing cells derived from the embryonic membrane, comprising the step of co-culturing cells derived from the embryonic membrane with one or more cells selected from cells derived from skin, intestine, heart, brain, stomach, kidney, and lung. Particularly preferred is co-culturing cells derived from the embryonic membrane with cells selected from the following combinations: (1) Cells from the skin and cells selected from cells from the brain, stomach and kidneys; (2) Cells from the intestine and cells selected from cells from the heart and kidneys; (3) Cells from the heart and cells selected from cells from the kidneys and lungs; (4) Cells from the brain and cells selected from cells from the kidneys and cells from the lungs; (5) Cells from the stomach and cells selected from cells from the kidneys and lungs.

[0054] Another embodiment of the present invention is a cell culture method comprising: culturing cells derived from the embryonic membrane in the presence of a culture supernatant obtained from culturing one or more cells selected from cells derived from skin, intestine, heart, brain, stomach, kidney, and lung. Particularly preferred is the use of a culture supernatant obtained from co-culturing cells with cells selected from the following combinations: (1) Cells from the skin and cells selected from cells from the brain, stomach and kidneys; (2) Cells from the intestine and cells selected from cells from the heart and kidneys; (3) Cells from the heart and cells selected from cells from the kidneys and lungs; (4) Cells from the brain and cells selected from cells from the kidneys and cells from the lungs; (5) Cells from the stomach and cells selected from cells from the kidneys and lungs.

[0055] The culture supernatant may also include culture supernatant obtained from culturing cells derived from the embryonic membrane. Additionally, the culture used to obtain the culture supernatant can be a co-culture.

[0056] <Culturing methods for cells derived from the kidney> One embodiment of the present invention provides a method for culturing kidney-derived cells, comprising the step of co-culturing kidney-derived cells with one or more cells selected from cells derived from skin, intestines, heart, brain, stomach, embryonic membranes, and lungs. Particularly preferred is co-culturing kidney-derived cells with cells selected from the following combinations: (1) Cells from the skin and cells selected from cells from the intestine, heart, brain, stomach and embryonic membrane; (2) Cells from the intestine and cells selected from cells from the heart, stomach and lungs; (3) Cells from the heart and cells from the lungs; (4) Cells from the stomach and cells from the embryonic membrane.

[0057] Another embodiment of the present invention is a cell culture method comprising: culturing kidney cells in the presence of a culture supernatant obtained from culturing one or more cells selected from skin cells, intestinal cells, heart cells, brain cells, stomach cells, embryonic membrane cells, and lung cells. Particularly preferred is the use of a culture supernatant obtained from co-culturing cells with a combination of the following: (1) Cells from the skin and cells selected from cells from the intestine, heart, brain, stomach and embryonic membrane; (2) Cells from the intestine and cells selected from cells from the heart, stomach and lungs; (3) Cells from the heart and cells from the lungs; (4) Cells from the stomach and cells from the embryonic membrane.

[0058] The culture supernatant may also include culture supernatant obtained from culturing cells from the kidney. Additionally, the culture used to obtain the culture supernatant can be a co-culture.

[0059] <Culturing methods for lung cells> One embodiment of the present invention provides a method for culturing lung cells, comprising the step of co-culturing lung cells with one or more cells selected from cells derived from skin, intestines, heart, brain, stomach, embryonic membranes, and kidneys. Particularly preferred is co-culturing lung cells with cells selected from the following combinations: (1) Cells from the skin and cells selected from cells from the intestine, heart, brain, stomach and embryonic membrane; (2) Cells from the intestine and cells selected from cells from the brain and cells from the kidney; (3) Cells from the heart and cells from the stomach; (4) Cells derived from the brain and cells selected from cells derived from the kidneys and cells derived from the embryonic membrane; (5) Cells from the stomach and cells from the kidneys; (6) Cells from the kidney and cells from the embryonic membrane.

[0060] Another embodiment of the present invention is a cell culture method comprising: culturing lung cells in the presence of a culture supernatant obtained from culturing one or more cells selected from skin cells, intestinal cells, heart cells, brain cells, stomach cells, embryonic membrane cells, and kidney cells. Particularly preferred is the use of a culture supernatant obtained from co-culturing cells selected from the following combinations: (1) Cells from the skin and cells selected from cells from the intestine, heart, brain, stomach and embryonic membrane; (2) Cells from the intestine and cells selected from cells from the brain and cells from the kidney; (3) Cells from the heart and cells from the stomach; (4) Cells derived from the brain and cells selected from cells derived from the kidneys and cells derived from the embryonic membrane; (5) Cells from the stomach and cells from the kidneys; (6) Cells from the kidney and cells from the embryonic membrane.

[0061] The culture supernatant may also include culture supernatant obtained from culturing cells derived from the lungs. Additionally, the culture used to obtain the culture supernatant can be a co-culture.

[0062] [2] Cultivation system A culture system refers to a system in a culture state, including a culture apparatus containing cell culture containers, a culture medium for cell culture, and cells in culture. The culture apparatus may include a culture medium supply container for supplying fresh cell culture medium and a culture medium recovery container for recovering the cultured cell culture medium. The cell culture container is not particularly limited; for example, it can be a culture tank, a glass shale, or a plastic dish. Regarding the cell culture medium, those skilled in the art can select a culture medium suitable for culturing the cells. The culture medium can be a serum-containing medium, but from the perspective of ease of control over the substances contained, a serum-free medium is preferred.

[0063] In co-culture, multiple cell types can be cultured in a single culture vessel, or separate culture vessels can be set up for each cell type, sharing the same culture medium to allow the cells to proliferate separately. In the latter case, the culture vessels can be connected by linking the culture media between them. The method of connecting the culture vessels can be appropriately determined according to the purpose. For example, the culture vessels can be connected by a connecting tube, and a switch control device such as a valve can be installed in the connecting tube to control the flow of the culture medium. Alternatively, a pump can be installed in the connecting tube to allow the culture medium to flow in any direction.

[0064] For example, when there are two culture containers, they can be connected by one connecting tube, or by two or more connecting tubes. By connecting the culture containers with multiple connecting tubes, the culture medium can be refluxed. When there are three or more culture containers, they can be connected in series or in parallel, and the culture containers can be appropriately combined according to the purpose to form a suitable culture system.

[0065] Each cell culture vessel can be used in one or more containers. In any case, culture containers can be appropriately combined to form a suitable culture system, depending on the purpose.

[0066] The specific methods for cell preparation and culture are described below.

[0067] [3] Specific cell preparation and culture methods <The Origin of Cells> The animals used to obtain tissues such as skin, intestines, heart, brain, stomach, kidneys, and lungs are not particularly limited, but vertebrates such as mammals, birds, reptiles, and amphibians are preferred. The source organisms for cells from the skin, intestines, heart, brain, stomach, kidneys, and lungs are not particularly limited, but vertebrates are preferred; they can be any of mammals, reptiles, birds, or amphibians, such as humans, cattle, pigs, rabbits, and chickens. Furthermore, the animals used to obtain the embryonic membranes are not particularly limited, as long as they are oviparous, but birds or reptiles are preferred. The source organisms for cells from the embryonic membranes are not particularly limited, as long as they are oviparous; they can be birds or reptiles, such as chickens. In the case of chickens, embryos aged 0 to 21 days are acceptable, including embryos aged 2 to 19 days, 4 to 17 days, and 6 to 15 days. Moreover, the cells for each tissue can come from the same animal or different animals.

[0068] <Types and preparation methods of cells> Cells from the skin, intestines, heart, brain, stomach, embryonic membranes, kidneys, and lungs can be obtained by taking the skin, intestines, heart, brain, stomach, embryonic membranes, kidneys, and lungs from an animal embryo, respectively, or by using the entire embryo and separating the cells through chemical treatment with enzymes such as collagenase, dispersinase, and trypsin, or through physical treatment such as cutting or breaking with a scalpel or razor, or blowing with a micropipette tip. Specific methods are established as common technical knowledge; any method that can recover living cells is acceptable and there are no particular limitations.

[0069] Cells derived from the skin include epithelial cells and dermal cells.

[0070] Cells derived from the intestine include one or more cells selected from intestinal epithelial cells, CBC cells, Paneth cells, enteroendocrine cells, goblet cells, absorptive epithelial cells, M cells, intestinal wall cells, connective tissue cells, muscle tissue, fibroblasts, vascular endothelial cells, vascular perithelial cells, and their precursor cells or stem cells. The intestine can be any one of the duodenum, small intestine, large intestine, or colon, or a mixture of any two or more types.

[0071] Cells derived from the heart include one or more cells selected from cardiomyocytes, nerve cells, connective tissue cells, fibroblasts, vascular endothelial cells, vascular perithelial cells, blood cells, and their precursor cells or stem cells.

[0072] Cells derived from the brain include one or more cells selected from nerve cells, glial cells, pituitary cells, vascular endothelial cells, vascular periothelial cells, and their precursor cells or stem cells.

[0073] Cells from the stomach include one or more of the following: chief cells, parietal cells, neck mucous cells, and their precursor cells or stem cells.

[0074] Cells derived from the embryonic membrane include one or more of the following: amnion, chorion, allantois, and yolk sac.

[0075] Cells from the kidney include one or more cells selected from the following: epithelial cells, endothelial cells, mesangial cells, visceral epithelial cells, parietal epithelial cells, endothelial cells, epithelial cells, and connective tissue cells, fibroblasts, vascular endothelial cells, vascular perithelial cells, and their precursor cells or stem cells that constitute the glomerulus.

[0076] Cells derived from the lungs include one or more types of cells selected from endothelial cells, alveolar epithelial cells, and their precursor cells or stem cells.

[0077] The cells can be primary cultured cells or constructed cell lines. Furthermore, they can be cultured in the form of tissues or organs, in cell clusters, or as isolated individual cells.

[0078] The culture can be a suspension culture or an adherent culture. Furthermore, it can be a planar culture or a three-dimensional culture. Those skilled in the art can easily and appropriately determine which culture method to choose based on the cell type.

[0079] Regarding specific cell culture methods, those skilled in the art can use the above-described culture system to select a cell culture method suitable for the culture purpose.

[0080] When using culture supernatants obtained from culturing other cell species in culture, the culture supernatant can, for example, allow cells to be cultured at a concentration of 1 × 10⁻⁶. 4 pcs / ~1×10 7Cells are seeded at a density of cells / mL in cell containers and cultured for approximately 1–30 days before being recovered. Afterward, centrifugation can be performed to remove precipitates such as cell debris. The resulting culture supernatant can be used directly (i.e., 100% cell supernatant) to culture the target cells for proliferation, or a culture medium supplemented with 10%, 30%, 50%, or 70% or more fresh culture medium can be used. When adding culture supernatants of two or more cell types to the culture medium of the cells to be proliferated, the two or more cell types used to obtain the culture supernatant can be cultured separately, their supernatants mixed, or a portion or all of the cells can be co-cultured. During co-culture, a portion or all of the cells can be cultured in a single culture container, or the culture medium can be shared by using separate culture containers for each cell type, allowing the cells to proliferate separately. In the latter case, the culture containers can be connected by connecting the culture medium between the containers. The method of connecting the culture containers can be appropriately determined according to the purpose. For example, a portion or all of the culture containers can be combined into a ring, which may include culture containers containing the same cell type as the cells to be proliferated.

[0081] Example [1] Cell preparation methods The following procedures are used to prepare cells from various tissues (skin, stomach, heart, brain, intestine, embryo, kidney, lung).

[0082] First, the fertilized chicken eggs are incubated, and the tissues from the 12-day embryos are collected separately. The tissues are dispersed into single cells and collected by physical processing using a sieve (100μm).

[0083] [2] Cell culture methods The recovered cells were seeded with cell scaffolds in three cell culture plates connected by connecting tubes, and cultured in YE (DMEM containing 0.01% yeast extract) at 37°C with 5% CO2. After 6 days of culture, the medium was replaced with 50% medium, and after 12 days, the cells were recovered and counted. The relative cell counts were calculated, taking the cell count obtained separately from the cells in plates #1-#3 cultured under the same conditions with a single cell species as 1. The experiment was repeated eight times, and the mean ± standard error is recorded in Table 1.

[0084] [3] Evaluation For each combination, a value greater than 1 for a specific cell (#1 > 1) is considered to promote cell proliferation of that specific cell. Such cells are labeled 0 (Evaluation 1). Next, a value greater than 1 for all three cell types used in the combination (All > 1) is considered to promote cell proliferation of all three cell types used in the combination. Such cells are labeled 0 (Evaluation 2). These evaluations are described in the following tables.

[0085] [4] Results [Table 1] As shown in Table 1, co-culturing skin cells with various cell species can promote proliferation. In particular, co-culturing skin cells with cell combinations selected from the following groups significantly promotes cell proliferation.

[0086] (1) Cells from the intestine and cells selected from cells from the heart, brain, stomach, kidney and lung; (2) Cells from the heart and cells selected from cells from the brain, stomach, embryonic membranes and lungs; (3) Cells from the brain and cells selected from cells from the stomach, cells from the embryonic membrane and cells from the lung; (4) Cells from the stomach and cells selected from cells from the kidneys, cells from the embryonic membranes and cells from the lungs; (5) Cells from the kidney and cells selected from cells from the embryonic membrane and cells from the lung; (6) Cells from the embryonic membrane and cells from the lung.

[0087] [Table 2] As shown in Table 2, co-culturing intestinal cells with various cell species can promote proliferation. In particular, co-culturing intestinal cells with cell types selected from the following combinations significantly promotes cell proliferation.

[0088] (1) Cells from the skin and cells selected from cells from the brain and cells from the kidneys; (2) Cells from the brain and cells from the stomach; (3) Cells from the stomach and cells selected from cells from the kidneys, cells from the embryonic membranes and cells from the lungs.

[0089] [Table 3] As shown in Table 3, co-culturing heart-derived cells with various cell species can promote proliferation. In particular, co-culturing heart-derived cells with cell types selected from the following combinations significantly promotes cell proliferation.

[0090] (1) Cells from the skin and cells selected from cells from the intestine and cells from the lung; (2) Cells from the intestine and cells from the embryonic membrane; (3) Cells from the brain and cells selected from cells from the stomach, cells from the embryonic membrane and cells from the lungs.

[0091] [Table 4] As shown in Table 4, co-culturing brain-derived cells with various cell species can promote proliferation. In particular, co-culturing brain-derived cells with cell types selected from the following combinations significantly promotes cell proliferation.

[0092] (1) Cells from the skin and cells selected from cells from the intestine, stomach, kidney, embryonic membrane and lung; (2) Cells from the intestine and cells selected from cells from the heart, stomach, kidney and lung; (3) Cells from the heart and cells from the kidneys; (4) Cells from the stomach and cells from the lungs; (5) Cells from the kidneys and cells from the lungs; (6) Cells from the embryonic membrane and cells from the lung.

[0093] [Table 5] As shown in Table 5, co-culturing stomach cells with various cell species can promote proliferation. In particular, co-culturing stomach cells with cell types selected from the following combinations significantly promotes cell proliferation.

[0094] (1) Cells derived from the skin and cells selected from cells derived from the intestine, the heart, the kidney, and the embryonic membrane; (2) Cells from the intestine and cells selected from cells from the brain and cells from the embryonic membrane; (3) Cells from the brain and cells from the embryonic membrane; (4) Cells from the kidney and cells from the embryonic membrane; (5) Cells from the embryonic membrane and cells from the lung.

[0095] [Table 6] As shown in Table 6, co-culturing kidney-derived cells with various cell species can promote proliferation. In particular, co-culturing kidney-derived cells with cell types selected from the following combinations significantly promotes cell proliferation.

[0096] (1) Cells from the skin and cells selected from cells from the intestine, heart, brain, stomach and embryonic membrane; (2) Cells from the intestine and cells selected from cells from the heart, stomach and lungs; (3) Cells from the heart and cells from the lungs; (4) Cells from the stomach and cells from the embryonic membrane.

[0097] [Table 7] As shown in Table 7, co-culturing lung cells with various cell species can promote proliferation. In particular, co-culturing lung cells with cell types selected from the following combinations significantly promotes proliferation.

[0098] (1) Cells from the skin and cells selected from cells from the intestine, heart, brain, stomach and embryonic membrane; (2) Cells from the intestine and cells selected from cells from the brain and cells from the kidney; (3) Cells from the heart and cells from the stomach; (4) Cells derived from the brain and cells selected from cells derived from the kidneys and cells derived from the embryonic membrane; (5) Cells from the stomach and cells from the kidneys; (6) Cells from the kidney and cells from the embryonic membrane.

[0099] [Table 8] As shown in Table 7, co-culturing cells derived from the embryonic membrane with various cell species can promote proliferation. In particular, co-culturing cells derived from the embryonic membrane with cell types selected from the following combinations significantly promotes cell proliferation. (1) Cells from the skin and cells selected from cells from the brain, stomach and kidneys; (2) Cells from the intestine and cells selected from cells from the heart and kidneys; (3) Cells from the heart and cells selected from cells from the kidneys and lungs; (4) Cells from the brain and cells selected from cells from the kidneys and cells from the lungs; (5) Cells from the stomach and cells selected from cells from the kidneys and lungs.

[0100] Industrial availability The present invention provides a method for culturing cells from tissues and a device for culturing cells containing cells from tissues.

Claims

1. A cell culture method, characterized in that, include: The process of co-culturing cells from the skin with one or more cells selected from the intestine, heart, brain, stomach, embryonic membrane, kidney, and lung.

2. A cell culture method, characterized in that, include: The process of culturing cells from the skin in the presence of a culture supernatant obtained from one or more cells selected from the intestine, heart, brain, stomach, embryonic membrane, kidney, and lung.

3. The cell culture method as described in claim 2, characterized in that: The culture supernatant also includes culture supernatant obtained from culturing cells from the skin.

4. The cultivation method as described in claim 2 or 3, characterized in that: The culture described is a co-culture.

5. The cell culture method according to any one of claims 1 to 4, characterized in that: The cells derived from the skin include epithelial cells and / or dermal cells.

6. The cell culture method according to any one of claims 1 to 4, characterized in that: The cells derived from the intestine include one or more cells selected from intestinal epithelial cells, CBC cells, Paneth cells, enteroendocrine cells, goblet cells, absorptive epithelial cells, M cells, intestinal wall cells, connective tissue cells, muscle tissue, fibroblasts, vascular endothelial cells, vascular perithelial cells, and their precursor cells or stem cells.

7. The cell culture method according to any one of claims 1 to 4, characterized in that: The cells derived from the heart include one or more cells selected from cardiomyocytes, nerve cells, connective tissue cells, fibroblasts, vascular endothelial cells, vascular perithelial cells, blood cells, and their precursor cells or stem cells.

8. The cell culture method according to any one of claims 1 to 4, characterized in that: The brain-derived cells include one or more cells selected from nerve cells, glial cells, pituitary cells, vascular endothelial cells, vascular perithelial cells, and their precursor cells or stem cells.

9. The cell culture method according to any one of claims 1 to 4, characterized in that: The cells derived from the stomach include one or more of the following: autosomes, parietal cells, neck mucous cells, and their precursor cells or stem cells.

10. The cell culture method according to any one of claims 1 to 4, characterized in that: The cells derived from the embryonic membrane include cells selected from one or more of the amnion, chorion, allantois, and yolk sac.

11. The cell culture method according to any one of claims 1 to 4, characterized in that: The cells derived from the kidney include one or more cells selected from epithelial cells, endothelial cells, mesangial cells, visceral epithelial cells, parietal epithelial cells, connective tissue cells, fibroblasts, vascular endothelial cells, vascular perithelial cells, and their precursor cells or stem cells.

12. The cell culture method according to any one of claims 1 to 4, characterized in that: Cells derived from the lung include one or more types of cells selected from endothelial cells, alveolar epithelial cells and their precursor cells or stem cells.

13. The cell culture method according to any one of claims 1 to 12, characterized in that: Cultured using serum-free medium.

14. A culture system, characterized in that: Cells from the skin are co-cultured with one or more cells selected from the following sources: intestinal cells, heart cells, brain cells, stomach cells, embryonic membrane cells, kidney cells, and lung cells.

15. A culture system, characterized in that: Cells from the skin were cultured in the presence of culture supernatants obtained from one or more cells selected from the intestine, heart, brain, stomach, embryonic membrane, kidney, and lung.

16. The cultivation method as described in claim 15, characterized in that: The culture supernatant also includes culture supernatant obtained from culturing cells from the skin.

17. The culture system as described in claim 15 or 16, characterized in that: The culture described is a co-culture.

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