Method for culturing liver-derived cells and culture system including liver-derived cells

Abstract

The purpose of the present invention is to provide a novel method for culturing liver-derived cells. Specifically, liver-derived cells are co-cultured with one or more cells selected from the group consisting of intestine-derived cells, lung-derived cells, and embryonic membrane-derived cells. Alternatively, the liver-derived cells may be cultured in the presence of a culture supernatant obtained by culturing one or more cells selected from the group consisting of intestine-derived cells, lung-derived cells, and embryonic membrane-derived cells.

Description

Method for culturing liver-derived cells and culture system containing liver-derived cells

[0001] The present invention relates to a method for culturing liver-derived cells and a culture system containing liver-derived cells.

[0002] Conventionally, hepatocytes have been cultured with feeder cells prepared from fibroblasts, endothelial cells, epithelial cells, or the like (JP 2009-183299 A; JP 2010-148386 A; JP 2007-516706 A), which has been known to cause hepatocytes to form spheroids (JP 2010-148386 A), and the feeder cells provide hepatocytes with an intracellular matrix and diffusible factors such as growth factors for proliferation and expansion of the hepatocytes (see, for example, Patent Document 3).

[0003] An object of the present invention is to provide a method for culturing liver-derived cells and a culture system containing liver-derived cells.

[0004] One embodiment of the present invention is a method for culturing liver-derived cells, which comprises co-culturing the cells with one or more cells selected from the group consisting of intestinal cells, lung cells, and fetal membrane cells. Head-derived cells and / or kidney-derived cells may also be co-cultured.

[0005] Another embodiment of the present invention is a method for culturing liver-derived cells, comprising culturing the cells in the presence of a culture supernatant obtained by culturing one or more cells selected from the group consisting of intestine-derived cells, lung-derived cells, and embryonic membrane-derived cells. The culture supernatant may further contain a culture supernatant obtained by culturing head-derived cells and / or kidney-derived cells. The culture to obtain the culture supernatant may be a co-culture.

[0006] A further embodiment of the present invention is a method for culturing cells derived from embryonic membranes, comprising co-culturing liver-derived cells with one or more cells selected from the group consisting of intestinal-derived cells, lung-derived cells, kidney-derived cells, and head-derived cells.

[0007] A further embodiment of the present invention is a method for culturing embryonic membrane-derived cells, comprising culturing the cells in the presence of a culture supernatant obtained by culturing liver-derived cells and a culture supernatant obtained by culturing one or more cells selected from the group consisting of intestinal-derived cells, lung-derived cells, kidney-derived cells, and head-derived cells. The culture supernatant may further comprise a culture supernatant obtained by culturing embryonic membrane-derived cells. The culture to obtain the culture supernatant may be a co-culture.

[0008] A further embodiment of the present invention is a method for culturing lung-derived cells, which comprises co-culturing liver-derived cells with one or more cells selected from the group consisting of intestinal-derived cells, fetal membrane-derived cells, kidney-derived cells, and head-derived cells.

[0009] A further embodiment of the present invention is a method for culturing lung-derived cells, comprising culturing the cells in the presence of a culture supernatant obtained by culturing liver-derived cells and a culture supernatant obtained by culturing one or more cells selected from the group consisting of intestine-derived cells, fetal membrane-derived cells, kidney-derived cells, and head-derived cells. The culture supernatant may further comprise a culture supernatant obtained by culturing lung-derived cells. The culture to obtain the culture supernatant may be a co-culture.

[0010] A further embodiment of the present invention is a method for culturing kidney-derived cells, which comprises co-culturing liver-derived cells with one or more cells selected from the group consisting of fetal membrane-derived cells and lung-derived cells.

[0011] A further embodiment of the present invention is a method for culturing kidney-derived cells, comprising culturing the cells in the presence of a culture supernatant obtained by culturing liver-derived cells and a culture supernatant obtained by culturing one or more cells selected from the group consisting of fetal membrane-derived cells and lung-derived cells. The culture supernatant may further comprise a culture supernatant obtained by culturing kidney-derived cells. The culture to obtain the culture supernatant may be a co-culture.

[0012] A further embodiment of the present invention is a method for culturing intestinal cells, wherein the cells are co-cultured with liver-derived cells, optionally with one or more cells selected from the group consisting of lung-derived cells, fetal membrane-derived cells, head-derived cells, and kidney-derived cells.

[0013] A further embodiment of the present invention is a method for culturing intestinal cells, comprising culturing the cells in the presence of a culture supernatant obtained by culturing liver-derived cells. The culture supernatant may further comprise a culture supernatant obtained by culturing one or more cells selected from the group consisting of lung-derived cells, fetal membrane-derived cells, head-derived cells, and kidney-derived cells. The culture supernatant may further comprise a culture supernatant obtained by culturing intestinal-derived cells. The culture to obtain the culture supernatant may be a co-culture.

[0014] A further embodiment of the present invention is a method for culturing cells derived from the head, wherein the cells are co-cultured with cells derived from the liver, and optionally with one or more cells selected from the group consisting of cells derived from the intestine, cells derived from the lung, and cells derived from an embryonic membrane.

[0015] A further embodiment of the present invention is a method for culturing cells derived from the head of a mammal, comprising culturing the cells in the presence of a culture supernatant obtained by culturing cells derived from the liver. The culture supernatant may further contain a culture supernatant obtained by culturing one or more cells selected from the group consisting of cells derived from the intestine, cells derived from the lung, and cells derived from an embryonic membrane. The culture supernatant may further contain a culture supernatant obtained by culturing cells derived from the head of a mammal. The culture to obtain the culture supernatant may be a co-culture.

[0016] A further embodiment of the present invention is a cell culture system in which liver-derived cells and one or more cells selected from the group consisting of intestinal cells, lung cells, and fetal membrane-derived cells are co-cultured. Additionally, head-derived cells and / or kidney-derived cells may be co-cultured.

[0017] A further embodiment of the present invention is a cell culture system in which cells derived from embryonic membranes, cells derived from the liver, and one or more cells selected from the group consisting of cells derived from the intestine, cells derived from the lung, cells derived from the kidney, and cells derived from the head are co-cultured.

[0018] A further embodiment of the present invention is a cell culture system in which lung-derived cells, liver-derived cells, and one or more cells selected from the group consisting of intestinal-derived cells, fetal membrane-derived cells, kidney-derived cells, and head-derived cells are co-cultured.

[0019] A further embodiment of the present invention is a cell culture system in which one or more cells selected from the group consisting of kidney-derived cells, liver-derived cells, and fetal membrane-derived cells and lung-derived cells are co-cultured.

[0020] A further embodiment of the present invention is a cell culture system in which intestinal cells are co-cultured with liver-derived cells, and optionally one or more cells selected from the group consisting of lung-derived cells, fetal membrane-derived cells, head-derived cells, and kidney-derived cells.

[0021] A further embodiment of the present invention is a cell culture system in which head-derived cells and liver-derived cells are co-cultured, and one or more cells selected from the group consisting of intestinal-derived cells, lung-derived cells, and fetal membrane-derived cells may also be co-cultured.

[0022] A further embodiment of the present invention is a culture system for liver-derived cells, comprising a culture supernatant obtained by culturing one or more cells selected from the group consisting of intestinal-derived cells, lung-derived cells, and fetal membrane-derived cells. The culture supernatant may further comprise a culture supernatant obtained by culturing head-derived cells and / or kidney-derived cells. The culture supernatant may further comprise a culture supernatant obtained by culturing liver-derived cells. The culture to obtain the culture supernatant may be a co-culture.

[0023] A further embodiment of the present invention is a culture system for embryonic membrane-derived cells, comprising a culture supernatant obtained by culturing liver-derived cells and a culture supernatant obtained by culturing one or more cells selected from the group consisting of intestinal-derived cells, lung-derived cells, kidney-derived cells, and head-derived cells. The culture supernatant may further comprise a culture supernatant obtained by culturing embryonic membrane-derived cells. The culture to obtain the culture supernatant may be a co-culture.

[0024] A further embodiment of the present invention is a culture system for lung-derived cells, comprising a culture supernatant obtained by culturing liver-derived cells and a culture supernatant obtained by culturing one or more cells selected from the group consisting of intestine-derived cells, embryonic membrane-derived cells, kidney-derived cells, and head-derived cells. The culture supernatant may further comprise a culture supernatant obtained by culturing lung-derived cells. The culture to obtain the culture supernatant may be a co-culture.

[0025] A further embodiment of the present invention is a culture system for kidney-derived cells, comprising a culture supernatant obtained by culturing liver-derived cells and a culture supernatant obtained by culturing one or more cells selected from the group consisting of fetal membrane-derived cells and lung-derived cells. The culture supernatant may further comprise a culture supernatant obtained by culturing kidney-derived cells. The culture to obtain the culture supernatant may be a co-culture.

[0026] A further embodiment of the present invention is a culture system for intestinal cells, comprising a culture supernatant obtained by culturing liver-derived cells. The culture supernatant may further comprise a culture supernatant obtained by culturing one or more cells selected from the group consisting of lung-derived cells, fetal membrane-derived cells, head-derived cells, and kidney-derived cells. The culture supernatant may further comprise a culture supernatant obtained by culturing intestinal-derived cells. The culture to obtain the culture supernatant may be a co-culture.

[0027] A further embodiment of the present invention is a system for culturing cells, comprising a cell culture vessel for growing the cells and an apparatus for obtaining a culture supernatant contained in a medium for culturing the cells, the apparatus being an apparatus for co-culturing a plurality of cell types. The apparatus comprises a plurality of cell culture vessels, and some or all of the plurality of cell culture vessels may be connected so that the culture supernatant in the vessels circulates.

[0028] ==Cross-reference to related literature== This application claims priority based on Japanese Patent Application No. 2022-001828, filed on January 7, 2022, and the basic application is incorporated herein by reference.

[0029] FIG. 1 is a diagram showing one embodiment of a culture system for obtaining a culture supernatant for culturing target cells to be proliferated.

[0030] The objectives, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description in this specification, and those skilled in the art will be able to easily reproduce the present invention. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustrative or explanatory purposes, and do not limit the present invention thereto. It will be apparent to those skilled in the art that various changes and modifications can be made based on the description in this specification within the spirit and scope of the present invention disclosed in this specification.

[0031] [1] Culture Method <Culture Method of Liver-Derived Cells> A culture method of one embodiment of the present invention is a culture method in which liver-derived cells are co-cultured with one or more cells selected from the group consisting of intestine-derived cells, lung-derived cells, and embryonic membrane-derived cells. Furthermore, head-derived cells and / or kidney-derived cells may be co-cultured.

[0032] Another embodiment of the present invention relates to a method for culturing liver-derived cells in the presence of a culture supernatant obtained by culturing one or more cells selected from the group consisting of intestinal-derived cells, lung-derived cells, and embryonic membrane-derived cells. The culture supernatant may be a culture supernatant obtained by co-culturing head-derived cells and / or kidney-derived cells. In either case, the culture supernatant may be a culture supernatant obtained by co-culturing liver-derived cells.

[0033] <Method for culturing cells derived from the head> A method for culturing cells derived from the head in one embodiment of the present invention is a culture method in which cells derived from the head are co-cultured with liver-derived cells and fetal membrane-derived cells, or with liver-derived cells and lung-derived cells.

[0034] Another embodiment of the present invention relates to a method for culturing cells derived from the head of a mammal, comprising culturing the cells in the presence of a culture supernatant containing a culture supernatant from cells derived from the liver and cells derived from the embryonic membrane, or a culture supernatant obtained by co-culturing the cells with a culture supernatant from cells derived from the liver and a culture supernatant from cells derived from the lung. In either case, the culture supernatant may be a culture supernatant obtained by further co-culturing cells derived from the head of a mammal.

[0035] <Method for culturing intestinal-derived cells> A method for culturing intestinal-derived cells according to one embodiment of the present invention is a culture method in which the intestinal-derived cells are co-cultured with liver-derived cells. The intestinal-derived cells may further be co-cultured with one or more cells selected from the group consisting of lung-derived cells, fetal membrane-derived cells, head-derived cells, and kidney-derived cells.

[0036] Another embodiment of the present invention relates to a method for culturing intestinal cells, which comprises culturing the cells in the presence of a culture supernatant obtained by culturing liver-derived cells. The culture supernatant may further contain one or more culture supernatants selected from the group consisting of culture supernatants for lung-derived cells, fetal membrane-derived cells, head-derived cells, and kidney-derived cells. In either case, the culture supernatant may also be a culture supernatant obtained by co-culturing intestinal cells.

[0037] <Method for culturing kidney-derived cells> A method for culturing kidney-derived cells according to one embodiment of the present invention is a culture method in which kidney-derived cells are co-cultured with liver-derived cells and fetal membrane-derived cells, or with liver-derived cells and lung-derived cells.

[0038] In another embodiment of the present invention, the method for culturing kidney-derived cells comprises culturing the cells in the presence of a culture supernatant containing a culture supernatant of liver-derived cells and a culture supernatant of embryonic membrane-derived cells, or a culture supernatant containing a culture supernatant of liver-derived cells and a culture supernatant of lung-derived cells. In either case, the culture supernatant may be a culture supernatant obtained by further co-culturing kidney-derived cells.

[0039] <Method for culturing lung-derived cells> One embodiment of the present invention relates to a method for culturing lung-derived cells, which involves co-culturing the cells with liver-derived cells and one or more cells selected from the group consisting of head-derived cells, kidney-derived cells, lung-derived cells, and embryonic membrane-derived cells.

[0040] Another embodiment of the present invention relates to a method for culturing kidney-derived cells, which comprises culturing the cells in the presence of a culture supernatant containing one or more culture supernatants selected from the group consisting of a culture supernatant of liver-derived cells, and a culture supernatant of head-derived cells, a culture supernatant of kidney-derived cells, a culture supernatant of lung-derived cells, and a culture supernatant of embryonic membrane-derived cells. In any case, the culture supernatant may be a culture supernatant obtained by further co-culturing lung-derived cells.

[0041] <Method for culturing embryonic membrane-derived cells> One embodiment of the method for culturing embryonic membrane-derived cells is a culture method in which the cells are co-cultured with liver-derived cells and one or more cells selected from the group consisting of head-derived cells, kidney-derived cells, lung-derived cells, and intestine-derived cells.

[0042] Another embodiment of the present invention relates to a method for culturing embryonic membrane-derived cells, which comprises culturing the cells in the presence of a culture supernatant containing one or more culture supernatants selected from the group consisting of a culture supernatant of liver-derived cells, and a culture supernatant of head-derived cells, a culture supernatant of kidney-derived cells, a culture supernatant of lung-derived cells, and a culture supernatant of lung-derived cells. In either case, the culture supernatant may be a culture supernatant obtained by further co-culturing embryonic membrane-derived cells.

[0043] [2] Culture System <Culture System for Liver-Derived Cells> In one embodiment of the present invention, the culture system for liver-derived cells is a culture system in which liver-derived cells are co-cultured with one or more cells selected from the group consisting of intestine-derived cells, lung-derived cells, and fetal membrane-derived cells. Furthermore, head-derived cells and / or kidney-derived cells may be co-cultured.

[0044] Another embodiment of the liver-derived cell culture system of the present invention is a culture system in which liver-derived cells are cultured in the presence of a culture supernatant obtained by culturing one or more cells selected from the group consisting of intestine-derived cells, lung-derived cells, and embryonic membrane-derived cells. The culture supernatant may further contain a culture supernatant obtained by co-culturing head-derived cells and / or kidney-derived cells. The culture supernatant may further contain a culture supernatant obtained by co-culturing head-derived cells and / or kidney-derived cells. In either case, the culture supernatant may further contain a culture supernatant obtained by co-culturing liver-derived cells.

[0045] <Culture system for head-derived cells> The culture system for head-derived cells of one embodiment of the present invention is a culture system in which head-derived cells are co-cultured with liver-derived cells and fetal membrane-derived cells, or with liver-derived cells and lung-derived cells.

[0046] In another embodiment of the present invention, the culture system for head-derived cells is a culture system in which head-derived cells are cultured in the presence of a culture supernatant obtained by culturing liver-derived cells and embryonic membrane-derived cells, or by culturing liver-derived cells and lung-derived cells. In either case, the culture supernatant may further contain a culture supernatant obtained by co-culturing head-derived cells.

[0047] <Intestine-derived culture system> In one embodiment of the present invention, the intestine-derived cell culture system is a culture system in which intestine-derived cells are co-cultured with liver-derived cells, and may further be co-cultured with one or more cells selected from the group consisting of lung-derived cells, fetal membrane-derived cells, head-derived cells, and kidney-derived cells.

[0048] Another embodiment of the intestinal cell culture system of the present invention is a culture system in which intestinal cells are cultured in the presence of a culture supernatant obtained by culturing liver-derived cells. The culture supernatant may further contain a culture supernatant obtained by culturing one or more cells selected from the group consisting of lung-derived cells, fetal membrane-derived cells, head-derived cells, and kidney-derived cells. In either case, the culture supernatant may further contain a culture supernatant obtained by co-culturing intestinal cells.

[0049] <Culture system for kidney-derived cells> A culture system for kidney-derived cells in one embodiment of the present invention is a culture system in which kidney-derived cells are co-cultured with liver-derived cells and fetal membrane-derived cells, or with liver-derived cells and lung-derived cells.

[0050] In another embodiment of the present invention, the kidney-derived cell culture system is a culture system in which kidney-derived cells are cultured in the presence of a culture supernatant obtained by culturing liver-derived cells and fetal membrane-derived cells, or by culturing liver-derived cells and lung-derived cells. In either case, the culture supernatant may further contain a culture supernatant obtained by co-culturing kidney-derived cells.

[0051] <Lung-derived cell culture system> One embodiment of the present invention relates to a lung-derived cell culture system in which lung-derived cells are co-cultured with liver-derived cells and one or more cells selected from the group consisting of head-derived cells, kidney-derived cells, lung-derived cells, and embryonic membrane-derived cells.

[0052] In another embodiment of the present invention, the lung-derived cell culture system is a culture system in which lung-derived cells are cultured in the presence of a culture supernatant obtained by culturing liver-derived cells and one or more cells selected from the group consisting of head-derived cells, kidney-derived cells, lung-derived cells, and fetal membrane-derived cells. In either case, the culture supernatant may further contain a culture supernatant obtained by co-culturing lung-derived cells.

[0053] <Culture system for embryonic membrane-derived cells> The culture system for embryonic membrane-derived cells of one embodiment of the present invention is a culture system in which embryonic membrane-derived cells are co-cultured with liver-derived cells and one or more cells selected from the group consisting of head-derived cells, kidney-derived cells, lung-derived cells, and lung-derived cells.

[0054] In another embodiment of the present invention, the culture system for embryonic membrane-derived cells is a culture system in which embryonic membrane-derived cells are cultured in the presence of a culture supernatant obtained by co-culturing the embryonic membrane-derived cells with one or more cells selected from the group consisting of liver-derived cells, head-derived cells, kidney-derived cells, lung-derived cells, and lung-derived cells. In either case, the culture supernatant may further contain a culture supernatant obtained by co-culturing the embryonic membrane-derived cells.

[0055] <Specific Culture System> A culture system refers to a system in a culture state that includes a culture device including a cell culture vessel, a cell culture medium, and cultured cells. The culture device may be provided with a medium supply container for supplying fresh cell culture medium, a medium collection container for collecting cultured cell culture medium, and the like. The cell culture vessel is not particularly limited and may be, for example, a culture tank, a glass Petri dish, or a plastic dish. Those skilled in the art can select a cell culture medium that is suitable for the cells to be cultured. The medium may be a serum-containing medium, but a serum-free medium is preferable in that it is easier to control the substances contained therein.

[0056] When co-culturing, multiple types of cells may be cultured in a single culture vessel, or an independent culture vessel may be provided for each cell, and the cells may be cultured using a shared medium, allowing the cells to grow separately. In the latter case, the culture vessels may be connected to each other so that the medium is in communication between the culture vessels. The method of connecting the culture vessels can be determined appropriately depending on the purpose. For example, some or all of the culture vessels, including the culture vessel for the cells to be grown, may be connected in a ring shape. Alternatively, the culture vessel for the cells to be grown may be connected independently to each of the culture vessels for the cells to be co-cultured.

[0057] When culturing cells to be grown by adding a culture supernatant, the culture vessel for the cells to be grown may be independent of the cell vessel for the cells from which the culture supernatant is obtained. The number of cell vessels for the cells from which the culture supernatant is obtained may be one or more, regardless of the number of cell types used, but it is preferable to have one or more for each cell type. They may be independent or may be connected depending on the purpose. It is preferable that the cells from which the culture supernatant is obtained include cells of the same type as the cells to be grown.

[0058] The tubes connecting the culture vessels may be provided with an on-off control device such as an on-off valve, and the communication of the culture medium may be controlled by controlling the on-off. Also, a pump may be provided in the tubes between the culture vessels to allow the culture medium to flow in either direction.

[0059] For example, when there are two culture vessels, they may be connected by one tube, or by two or more tubes. Connecting the culture vessels with multiple tubes allows for the circulation of the medium. When there are three or more culture vessels, they may be connected in series or in parallel, and an appropriate culture system can be formed by connecting the culture vessels appropriately according to the purpose.

[0060] The number of culture vessels for each cell may be one or more. In either case, appropriate culture vessels can be combined to form an appropriate culture system according to the purpose.

[0061] Specific methods for preparing and culturing the cells are described below.

[0062] [3] Specific Cell Preparation and Culturing Methods <Cell Origin> The animal from which each cell is obtained is not particularly limited, but is preferably a vertebrate such as a mammal, and more preferably an animal that lays eggs with eggshells, such as a bird or reptile. In particular, the biological species from which liver-derived cells, head-derived cells, kidney-derived cells, lung-derived cells, and lung-derived cells are derived are not particularly limited, but are preferably a vertebrate, and may be any of mammals, reptiles, birds, and amphibians, such as humans, cows, pigs, rabbits, chickens, sheep, and goats. Furthermore, the biological species from which embryonic membrane-derived cells are derived is not particularly limited as long as they are oviparous, and may be birds, reptiles, or chickens. In the case of chickens, embryos from 0-day to 21-day embryos are acceptable, but may also be 2-day to 19-day embryos, 4-day to 17-day embryos, or 6-day to 15-day embryos. Cells from each tissue may be derived from the same animal or different animals.

[0063] <Cell Types and Preparation Methods> Liver-derived cells, head-derived cells, intestine-derived cells, kidney-derived cells, and lung-derived cells can be obtained by removing the liver, head (above the neck, which may or may not include the neck), intestine, kidney, and lung from an animal embryo, and isolating the cells by chemical treatment with enzymes such as collagenase, dispase, or trypsin, or by physical treatment such as cutting and crushing with a scalpel or razor, or pipetting using a microchip. Specific methods are not particularly limited, as long as they are already established as common technical knowledge and allow for the recovery of live cells.

[0064] Liver-derived cells include one or more cells selected from the group consisting of parenchymal cells (i.e., hepatocytes), non-parenchymal cells (e.g., sinusoidal wall cells, myofibroblasts, bile duct epithelial cells, connective tissue cells, hepatic stellate cells, etc.), and their progenitor or stem cells.

[0065] Cells derived from the head include one or more cells selected from the group consisting of retinal cells, nerve cells, glial cells, pituitary cells, muscle cells, connective tissue cells, fibroblasts, vascular endothelial cells, vascular endothelial cells, and their progenitor or stem cells.

[0066] The intestine-derived cells include one or more cells selected from the group consisting of intestinal epithelial cells, CBC cells, Paneth cells, enteroendocrine cells, goblet cells, absorptive epithelial cells, M cells, intestinal wall cells, connective tissue cells, muscle cells, fibroblasts, vascular endothelial cells, vascular epithelial cells, and their precursor cells or stem cells. The intestine may be the duodenum, small intestine, large intestine, or colon, or may be a mixture of two or more of these.

[0067] Kidney-derived cells include one or more cells selected from the group consisting of epithelial cells that make up the renal glomeruli, endothelial cells, mesangial cells, visceral epithelial cells, parietal visceral cells, endothelial cells that make up the renal tubules, epithelial cells, connective tissue cells that fill other tissues, fibroblasts, vascular endothelial cells, vascular perithelial cells, and their precursor cells or stem cells.

[0068] Lung-derived cells include one or more cells selected from the group consisting of alveolar macrophages, type II alveolar epithelial cells, type I alveolar epithelial cells, basal cells, ciliated cells, goblet cells, mucus cells, serous cells, connective tissue cells, muscle cells, fibroblasts, vascular endothelial / epithelial cells, and their progenitor or stem cells.

[0069] Cells derived from embryonic membranes include one or more cells selected from the group consisting of amnion, chorion, allantois, and yolk sac.

[0070] The cells may be primary cultured cells or established cell lines, and may be cultured in the form of tissues or organs, in the form of cell clumps, or as individually isolated cells.

[0071] The culture may be suspension culture or adherent culture, or may be plate culture or three-dimensional culture. The culture to be selected depending on the cell type can be easily and appropriately determined by those skilled in the art.

[0072] <Specific Cell Culture Method> A specific cell culture method can be selected by those skilled in the art using the above-mentioned culture system, and is suitable for the cells to be cultured.

[0073] In the case of co-culture, multiple types of cells may be cultured together, or the cells may be grown separately by dividing the culture vessel into compartments for each cell and culturing them in a shared medium. Alternatively, individual containers may be prepared for each cell, and the containers may be connected to each other so that the medium is communicated between the containers and a uniform medium is obtained throughout. Alternatively, individual containers may be prepared for each cell, and the containers may be connected to each other so that the medium is communicated between the containers, but the flow of the medium may be controlled so that there are periods when the medium is communicated between the containers and periods when the medium remains in each container without communication.

[0074] When using a culture supernatant from culture of other cell types, the culture supernatant may be used to culture, for example, 1×10 cells. ４ pieces / ~1×10 ７ The cells can be seeded into a cell container at a density of 100 cells / mL and harvested after approximately 1 to 30 days of culture. Cell debris and other precipitates can then be removed by centrifugation. The resulting culture supernatant can be used as is (i.e., 100% cell supernatant) to culture the target cells for growth, or a medium containing 10% or more, 30% or more, 50% or more, or 70% or more fresh medium can be added. When adding the culture supernatant of two or more types of cells to a medium for growing cells, the two or more types of cells used to obtain the culture supernatant can be cultured separately and the supernatants mixed, or some or all of the cells can be co-cultured. When co-culturing, some or all of the cells to be co-cultured can be cultured in a single culture vessel, or independent culture vessels can be provided for each cell type and cultured sharing the same medium, allowing the cells to grow separately. In the latter case, the culture vessels can be connected to each other so that the medium is connected between the culture vessels. The method of connecting the culture vessels can be determined appropriately depending on the purpose. For example, some or all of the culture vessels may be connected in a ring shape, and a culture vessel for cells of the same type as the cells to be grown may be included among them.

[0075] <Culture system for obtaining culture supernatant> An example of a culture system for obtaining a culture supernatant, which uses a culture device including culture vessels and tubes connecting the vessels, is shown below with reference to FIG. 1 ; however, the embodiment of the culture system is not limited to this, and a person skilled in the art would be able to easily make various improvements and modifications.

[0076] This culture system includes a scaffold 20 for cell culture, multiple culture vessels 101-103 containing cultured cells 41-43, and culture supernatants 51-53. The cultured cells 41-43 cultured in each culture vessel 101-103 may be the same or different types, and may contain a cell species to be propagated using the resulting supernatant. The culture vessels 101-103 are connected by a communicating tube 10 to circulate the culture supernatants 51-53, and three-way valves 61-63 are provided between each vessel 101-103. Recovery pipes 31-33 are provided so that the supernatant can be recovered from this medium circulation system into a recovery container 90 by controlling the three-way valves 61-63. A medium container 400 containing fresh medium 55 is connected to this medium circulation system using a three-way valve 70. A liquid delivery pump 80 is provided to the communicating tube 10 and the recovery tube 30. The culture vessels 101 to 103 and the medium vessel 400 are provided with tubes that are open to the outside air and have on-off valves 71 to 73 and 74 attached, and the tubes are provided with filters to prevent the intrusion of microorganisms and minute objects from the outside air.

[0077] By using such a device, it is possible to recover a supernatant that meets the purpose. For example, by opening valve 61 only in the direction from culture vessel 101 to collection vessel 90 and opening valves 63 and 70 only in the direction from medium vessel 400 to culture vessel 101, a culture supernatant obtained by culturing only cultured cells 41 can be obtained. Alternatively, by opening valves 61 to 63 and valve 70 only in the direction that circulates the medium, a homogenized culture supernatant obtained by co-culturing cultured cells 41 to 43 circulates through the circulation system. Then, by opening valve 71 and opening valve 63 only in the direction from vessel 103 to collection vessel 90, the homogenized culture supernatant can be recovered. Then, by closing valve 71, opening valves 73 and 74, opening valves 61 to 63 only in the direction that circulates the medium, and opening valve 70 only in the direction from medium vessel 400 to vessel 101, fresh medium is supplied to culture vessels 101 to 103. Alternatively, by appropriately combining the opening and closing of each valve, the amount of culture medium in each culture vessel 101 to 103 and the combination of culture medium to be collected can be freely controlled.

[0078] In this way, a supernatant suitable for the purpose can be collected in collection container 90, and the collected supernatant can be used to culture the cells to be grown. Note that the collection container 90 may be connected to a culture container for the cells to be grown, and the supernatant collected in collection container 90 may be automatically supplied to the culture container for the cells to be grown. In this case, the amount, frequency, and number of times of supply of the supernatant may be controlled by a computer.

[0079] Example 1 Cell Preparation Method Cells were prepared from each tissue as follows: First, fertilized chicken eggs were incubated, and a mixture of embryonic membrane, urinary membrane, and vitelline membrane (referred to as 3Mix in this specification), head, liver, kidney, lung, and intestine were collected from 12-day-old embryos. The tissues were then dispersed into single cells by chemical treatment with collagenase or physical treatment using a mesh (100 μm).

[0080] The collected cells were placed in a 500 ml culture bottle (BelloCell: CESCO) at 1 × 10 ８ The cells were seeded in DMEM containing 10% FBS (fetal bovine serum) and cultured for two weeks.

[0081] <Cell culture method> (1) Preparation of culture supernatant for each cell combination Using the above medium, cells were cultured in a 500 ml culture bottle at 1 × 10 ８ At least 100 cells were seeded and incubated at 37°C, 5% CO using BioNOC II matrix (CESCO Bioengineering). ２ Two weeks after the start of culture, each culture supernatant was collected and centrifuged to remove precipitates such as cell debris, and the resulting culture supernatant was used for the following culture.

[0082] (2) Culture The cells to be examined for proliferation were cultured in a plastic cell culture dish at 5 × 10 ５ The cells were seeded at a density of 100 cells / ml or more and incubated at 37°C, 5% CO for 10 days. ２The cells were cultured in the presence of α-glucan. A food basal medium was used at the start of culture, to which 50% of the culture supernatant of the other cell types obtained in (1) was added. From days 0 to 6 of culture, 50% of the medium was replaced every two days. From days 6 to 10 of culture, 50% of the medium was replaced daily. The above medium without the culture supernatant was used for medium replacement. After 10 days of culture, the cells were recovered, the cell count was measured, and the numerical value was calculated assuming the number of cells at the time of seeding as 1. The results are shown in Result I of Table 1. Each experiment was performed using three petri dishes, and the average values ​​are shown in Table 1. Furthermore, the proliferation rate was calculated assuming the proliferation rate when cultured alone as 1, and the results are shown in Result II of Table 1.

[0083]

[0084] <Result Analysis> When the culture supernatant of other cells is added to the medium compared to when each cell is cultured alone, if proliferation is promoted, the value in Result II will be greater than 1.0. For liver cells, 3Mix, lung, or intestinal cell supernatant, and a mixture of head cell supernatant and kidney cell supernatant were used; for 3Mix cells, a mixture of liver cell supernatant and lung, intestinal, kidney, or head cell supernatant were used; for lung cells, a mixture of liver cell supernatant and 3Mix, intestinal, kidney, or head cell supernatant were used; for kidney cells, a mixture of liver cell supernatant and 3Mix or lung cell supernatant were used; for intestinal cells, liver cell supernatant was used; and for head cells, liver cell supernatant, preferably a mixture of liver cell supernatant and 3Mix, lung, or intestinal cell supernatant, was used. [Example 2]

[0085] <Cell Preparation Method> Cells were prepared from each tissue as follows: First, fertilized chicken eggs were incubated, and 3Mix, liver, and lung were collected from 12-day-old embryos, and the tissues were dispersed by physical treatment using scissors or a mesh (100 μm).

[0086] <Perfusion culture method> (1) Preparation of culture supernatant for each cell combination The perfusion culture was performed using a diaphragm-type constant-volume pump (SIMDOS) as the center, and three 500 ml culture bottles containing one 500 ml medium bottle and BioNOC II matrix (CESCO Bioengineering) were connected in a ring shape with tubing. Using the same basal food medium as in Example 1, 3Mix, liver, and lung cells were cultured at 1 x 10 in separate culture bottles. ８ The cells were seeded at 1000 x g and incubated at 37°C in 5% CO ２ After 12 days of culture, the culture supernatant was collected and centrifuged to remove precipitates such as cell debris, and the resulting culture supernatant was used in the following experiments.

[0087] (2) Culturing The cells to be examined for proliferation (duck liver-derived cells) were cultured in a plastic cell culture plate at 2 × 10 ４ The cells were seeded at a density of 100 cells / ml and cultured for 2 days at 37°C in the presence of 5% CO2. At the start of culture, a food-grade basal medium was used, supplemented with 50% of the culture supernatant obtained in (1). After 2 days of culture, ATP levels were measured as an indicator of cell number using a CellTiter-Glo 2.0 Cell viability assay kit (Promega). The value was calculated by setting the number of cells cultured in food-grade medium alone at 1. Each experiment was performed using three wells, and the average values ​​ranged from 5.0 to 24.4. Thus, co-culture of multiple cells to obtain supernatant and use of the resulting supernatant were also effective in proliferating liver-derived cells.

[0088] The present invention provides a method for culturing liver-derived cells and a culture system containing liver-derived cells.

Claims

1. A method for culturing liver-derived cells, comprising: A culture method in which the cells are co-cultured with one or more cells selected from the group consisting of intestinal-derived cells, lung-derived cells, and fetal membrane-derived cells.

2. The culture method according to claim 1, further comprising co-culturing cells derived from the head and / or cells derived from the kidney.

3. A method for culturing liver-derived cells, comprising: A culture method comprising culturing one or more cells selected from the group consisting of intestinal-derived cells, lung-derived cells, and embryonic membrane-derived cells in the presence of a culture supernatant obtained by culturing the cells.

4. The culture method according to claim 3 , wherein the culture supernatant further contains a culture supernatant obtained by culturing head-derived cells and / or kidney-derived cells.

5. The culture method according to claim 3 , wherein the culture supernatant further contains a culture supernatant obtained by culturing liver-derived cells.

6. The culture method according to any one of claims 3 to 5, wherein the culture is a co-culture.

7. The culture method according to any one of claims 1 to 5, wherein the liver-derived cells are one or more cells selected from the group consisting of parenchymal cells (hepatocytes), non-parenchymal cells (sinusoidal wall cells, myofibroblasts, bile duct epithelial cells, connective tissue cells, and hepatic stellate cells), and their progenitor cells or stem cells.

8. The culture method according to any one of claims 1 to 5, wherein the intestine-derived cells are one or more cells selected from the group consisting of intestinal epithelial cells, CBC cells, Paneth cells, enteroendocrine cells, goblet cells, absorptive epithelial cells, M cells, intestinal wall cells, connective tissue cells, muscle cells, fibroblasts, vascular endothelial / epithelial cells, and their precursor cells or stem cells.

9. The culture method according to any one of claims 1 to 5, wherein the lung-derived cells are one or more cells selected from the group consisting of alveolar macrophages, type II alveolar epithelial cells, type I alveolar epithelial cells, basal cells, ciliated cells, goblet cells, mucus cells, serous cells, connective tissue cells, muscle cells, fibroblasts, vascular endothelial / epithelial cells, and their progenitor cells or stem cells.

10. The culture method according to any one of claims 1 to 5, wherein the embryonic membrane-derived cells are one or more cells selected from the group consisting of amniotic membrane, chorion, allantois, and yolk sac.

11. The culture method according to claim 2 or 4, wherein the cells derived from the head are one or more cells selected from the group consisting of retinal cells, nerve cells, glial cells, pituitary cells, muscle cells, connective tissue cells, fibroblasts, vascular endothelial / epithelial cells, and their progenitor cells or stem cells.

12. The culture method according to claim 2 or 4, wherein the kidney-derived cells are one or more cells selected from the group consisting of epithelial cells, endothelial cells, mesangial cells, visceral epithelial cells, parietal visceral cells, endothelial cells that constitute the renal tubules, epithelial cells, connective tissue cells that fill other tissues, fibroblasts, vascular endothelial / epithelial cells, and their precursor cells or stem cells.

13. The culture method according to any one of claims 1 to 5, wherein the culture is carried out using a serum-free medium.

14. A cell culture system comprising: Liver-derived cells, and one or more cells selected from the group consisting of intestinal-derived cells, lung-derived cells, and fetal membrane-derived cells; A culture system in which the two are co-cultured.

15. The culture system according to claim 14, further comprising co-cultured cells derived from the head and / or cells derived from the kidney.

16. A liver-derived cell culture system, comprising: A culture system comprising a culture supernatant obtained by culturing one or more cells selected from the group consisting of intestinal-derived cells, lung-derived cells, and embryonic membrane-derived cells.

17. The culture system according to claim 16 , wherein the culture supernatant further comprises a culture supernatant obtained by culturing head-derived cells and / or kidney-derived cells.

18. The culture system according to claim 16 , wherein the culture supernatant further comprises a culture supernatant obtained by culturing liver-derived cells.

19. The culture system according to any one of claims 16 to 18, wherein the culture is a co-culture.

20. The culture system according to any one of claims 14 to 18, wherein the liver-derived cells are one or more cells selected from the group consisting of parenchymal cells (hepatocytes), non-parenchymal cells (sinusoidal wall cells, myofibroblasts, bile duct epithelial cells, connective tissue cells, and hepatic stellate cells), and their progenitor or stem cells.

21. The culture system according to any one of claims 14 to 18, wherein the intestine-derived cells are one or more cells selected from the group consisting of intestinal epithelial cells, CBC cells, Paneth cells, enteroendocrine cells, goblet cells, absorptive epithelial cells, M cells, intestinal wall cells, connective tissue cells, muscle cells, fibroblasts, vascular endothelial / epithelial cells, and their precursor cells or stem cells.

22. The culture system according to any one of claims 14 to 18, wherein the lung-derived cells are one or more cells selected from the group consisting of alveolar macrophages, type II alveolar epithelial cells, type I alveolar epithelial cells, basal cells, ciliated cells, goblet cells, mucus cells, serous cells, connective tissue cells, muscle cells, fibroblasts, vascular endothelial / epithelial cells, and their progenitor cells or stem cells.

23. The culture system according to any one of claims 14 to 18, wherein the cells derived from the embryonic membrane are one or more cells selected from the group consisting of amnion, chorion, allantois, and yolk sac.

24. The culture system of claim 17, wherein the cells derived from the head are one or more cells selected from the group consisting of retinal cells, nerve cells, glial cells, pituitary cells, muscle cells, connective tissue cells, fibroblasts, vascular endothelial / epithelial cells, and their progenitor or stem cells.

25. The culture system of claim 17, wherein the kidney-derived cells are one or more cells selected from the group consisting of epithelial cells constituting the renal glomeruli, endothelial cells, mesangial cells, visceral epithelial cells, parietal visceral cells, endothelial cells constituting the renal tubules, epithelial cells and connective tissue cells filling other tissues, fibroblasts, vascular endothelial / epithelial cells, and their progenitor cells or stem cells.

26. The culture system according to any one of claims 14 to 18, comprising a serum-free medium.