Cell culture methods

Abstract

The present invention provides a cell culture method that enables organ organoids to be cultured and matured over a long period of time, and is suitable for producing three-dimensional organs. The cell culture method of the present invention is for culturing, while perfusing a culture solution, organ organoids immobilized in a chamber and / or cells constituting an organ. The culture solution is perfused so as to create a turbulent flow in the chamber.

Description

[Technical Field]

[0001] The present invention relates to a method for culturing organoids and / or cells constituting an organ, and more particularly to a method for culturing organoids and / or cells constituting an organ while perfusing a culture solution.

[0002] [Background of the invention] To develop drugs and realize regenerative medicine for various organ diseases, such as the liver and kidneys, research and development is underway to create organoids, or cellular structures that self-organize cultured cells and replicate the three-dimensional structure of each organ, as well as the complex structures of blood vessels, bile ducts, and other vascular systems. Advanced cell structures based on organoids have also been developed, including the creation of organ buds (e.g., hepatic buds), which form early in development, by culturing specific cell types, including undifferentiated cells. These "mini-organs," or organoids, offer significant value, as they can be used as a more in vivo evaluation system for drug efficacy and toxicity assessment than conventional, single-organ cultures. They can also be used for transplantation and plasma protein production.

[0003] Various organ organoids are generally produced by culture in a static environment (static culture) in which the culture medium (liquid medium) is stationary; however, culture in an environment where the culture medium is perfused (perfusion culture) has also been proposed. For example, Non-Patent Document 1 describes the development of glomerular blood vessels by perfusion culture of kidney organoids. Furthermore, Non-Patent Document 2 describes the use of a microfluidic device to place a biological tissue slice (the suprachiasmatic nucleus (SCN) of brain tissue) on a culture medium-permeable membrane, controlling the speed of the culture medium perfusion to maintain an appropriate amount of liquid covering the tissue, thereby maintaining a balance between nutrient supply and respiration while culturing for a long period of time. Non-Patent Document 3 describes the promotion of cell growth and differentiation by perfusion culture of lung epithelial cells at an air-liquid interface (ALI) compared to static culture. In perfusion culture of such organ organoids, tissues, etc., it has been common to perfuse the culture medium at a constant rate to create a "laminar flow" and to minimize shear stress on the organ organoids, tissues, etc. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Homan, KA et al., Flow-enhanced vascularization and maturation of kidney organoids in vitro. Nat Methods 16, 255-262 (2019) doi:10.1038 / s41592-019-0325-y [Non-patent document 2] Ota, N. et al., A Microfluidic Platform Based on Robust Gas and Liquid Exchange for Long term Culturing of Explanted Tissues. Analytical Sciences, Oct 2019, Vol.35 [Non-patent document 3] Long term Culturing of Explanted Tissues. Analytical Sciences, Oct 2019, Vol.35 Summary of the Invention [Problem to be solved by the invention]

[0005] It has been difficult to produce mature organoids, or even three-dimensional organs, through long-term culture using static culture, which has been the norm until now. For example, when liver organoids are cultured for long periods in static culture, it is difficult to maintain mature hepatocytes. After approximately two weeks of culture, cell death occurs, resulting in a decrease in cell density and the formation of gaps between cells.

[0006] An objective of the present invention is to provide a culture method that enables organoids to be cultured for a long period of time to mature and is suitable for producing three-dimensional organs. [Means for solving the problem]

[0007] The inventors discovered that by intermittently increasing the flow rate of the perfused culture medium (liquid medium), i.e., by intermittently increasing the flow rate of the culture medium in the chamber, and imposing a transient transitional state of "turbulence" on the cells that make up the organoids and / or organs, it is possible to produce denser organoids or three-dimensional organs composed of mature cells, and thus completed the present invention.

[0008] That is, in order to solve the above problems, the present invention provides the following [1] to

[15] . [1] A method for culturing organoids and / or cells constituting an organ immobilized in a chamber while perfusing a culture solution, comprising: A culture method, wherein the culture medium is perfused to generate turbulent flow within the chamber. [2] Item 1. The culture method according to Item 1, wherein the culture medium is perfused in the chamber so as to include a period during which the culture medium transitions from laminar flow to turbulent flow, a period during which turbulent flow is generated, and a period during which the culture medium transitions from turbulent flow to laminar flow. [3] Item 3. The culture method according to item 1 or 2, wherein the organoids and / or cells constituting the organ are immobilized in a matrix. [4] Item 4. The culture method according to any one of items 1 to 3, wherein the matrix in which the organoids and / or cells constituting the organ are embedded is immobilized in a chamber in a state suspended on a breathable membrane. [5] Item 5. The culture method according to any one of Items 1 to 4, wherein the organoid has a vascular structure, and / or the cells constituting the organ coexist with vascular cells. [6] Item 6. The culture method according to any one of items 1 to 5, wherein the organoid is a liver or kidney organoid, and / or the organ is a liver or kidney. [6a] Item 6. The culture method according to any one of Items 1 to 5, wherein the organoid is an organoid of the liver, kidney, pancreas, thyroid, parathyroid, lung, brain, or heart. [7] Item 7. A method for producing a three-dimensional organ, comprising a step of carrying out the culture method according to any one of Items 1 to 6. [8] Item 7. The culture method according to any one of Items 1 to 6, wherein the culture medium contains a drug. [9] Item 9. The culture method according to Item 8, wherein the drug is for treating a disease accompanied by ischemia. [9a] Item 9. The culture method according to Item 8, wherein the drug is for treating an ischemic disease, an infarct / necrotic disease, or a hemorrhagic disease. [9b] Item 9. The culture method according to Item 8, wherein the drug is for safety evaluation of side effects associated with ischemia or bleeding.

[10] Item 10. A method for evaluating a drug efficacy, comprising a step of carrying out the culture method according to Item 8 or 9. [10a] A drug evaluation method comprising carrying out the culture method according to paragraph 8, 9, 9a or 9b, and evaluating the efficacy and / or safety of the drug on the organ organoid.

[11] An organoid obtained by the culture method according to any one of items 1 to 6.

[12] Item 8. A three-dimensional organ obtained by the manufacturing method described in Item 7.

[13] Item 13. A method for evaluating drug efficacy, comprising the steps of adding a drug to a culture medium for the three-dimensional organ according to Item 12, and evaluating the efficacy of the drug on the three-dimensional organ. [13a] Item 13. A drug evaluation method comprising the steps of adding a drug to a culture medium for the three-dimensional organ according to Item 12, and evaluating the efficacy and / or safety of the drug on the three-dimensional organ.

[14] A chamber capable of immobilizing and accommodating organoids and / or cells constituting an organ, and having an inlet and an outlet for perfusing a culture solution over the organoids and / or cells constituting the organ; A culture system comprising a perfusion means for perfusing a culture solution, A culture system, wherein the perfusion means is controlled to generate turbulence in the culture medium within the chamber.

[15] Item 15. The system according to Item 14, wherein the perfusion means is controlled to perfuse the culture solution so that there is a period of time in the chamber during which the flow transitions from laminar to turbulent, a period of time during which turbulent flow occurs, and a period of time during which the flow transitions from turbulent to laminar. [Effects of the Invention]

[0009] The culture method of the present invention makes it easier to produce organoids in which mature cells are distributed at a higher density than conventional organoids, and makes it easier to produce three-dimensional organs that are superior to conventional organoids. Furthermore, in organoids and three-dimensional organs obtained by the culture method of the present invention, or in artificial organs using three-dimensional organs, or in regenerative medicine in which three-dimensional organs are transplanted, it becomes possible to evaluate the efficacy and safety of various drugs under conditions that are considered to be more reproducible than conventional drug actions. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of the culture device (culture system of the present invention) constructed in Examples (common to Examples 1 and 2). [Figure 2] Figure 2 shows images of liquid medium in the culture chamber immediately before (top) and during (bottom) liquid flow under turbulent flow conditions (20 mL / min, intermittent) in the example (common to examples 1 and 2) and under conventional laminar flow conditions (4 mL / min, continuous) that do not produce turbulence, as described in Non-Patent Document 1 for reference. The liquid medium is stained with trypan blue dye. Under the liquid flow conditions of the reference example, the liquid medium flows into the chamber from the inlet on the left in a neat line (i.e., laminar flow). In contrast, under the liquid flow conditions of the example, the liquid medium flows into the chamber in a swirling manner (i.e., turbulent flow), and the liquid medium collides with the chamber wall, causing turbulence throughout the chamber. [Figure 3] Figure 3 shows optical microscope images (side and top), nuclear staining images, and fluorescent images of immunostained images (double staining images of ASGR1 and CD31, shown in red and green in the color image, respectively) of organoids (right) obtained by perfusion culture (the culture method of the present invention) in Example 1 and organoids (left) obtained by static culture in Comparative Example 1 as a control, as well as the ASGR1 positivity rate (mean ± standard deviation, mean value of n = 3) calculated from the area of ​​the fluorescent region in the fluorescent images. [Figure 4]Figure 4 shows an image of a matrix composition (right) containing organoids obtained by perfusion culture (the culture method of the present invention) in Example 2, and a matrix composition (left) containing organoids obtained by static culture in Comparative Example 2 as a control. [Figure 5] Figure 5 shows immunostained images (triple stained images of GS, E-cadherin, and DAPI, shown in red, green, and blue in the color image) of organoids (right) obtained by perfusion culture (the culture method of the present invention) in Example 2 and organoids (left) obtained by static culture in Comparative Example 2 as a control. The immunostained image (right) of Example 2 shows expression of the mature stem cell markers GS and E-cadherin, and is densely packed with cells. In contrast, the immunostained image (left) of Comparative Example 2 shows no expression of these markers, and cell loss is observed, particularly in the center of the organoid. DETAILED DESCRIPTION OF THE INVENTION

[0011] - Cultivation method for organoids, etc. - The culture method of the present invention is a method for culturing organ organoids and / or cells or tissues constituting an organ (collectively referred to in this specification as "organ organoids, etc.") immobilized in a chamber while perfusing a culture solution, in which the culture solution is perfused so as to generate turbulence within the chamber.

[0012] A "chamber" refers to a portion provided within a culture vessel (cell culture device) that is large enough to accommodate the organ organoids or the like to be cultured, and has a structure capable of immobilizing the organ organoids or the like. Generally, a chamber is equipped with an inlet and outlet for the culture medium to be perfused, and is configured as a portion that is larger in width and height than the culture medium flow path that communicates with the inlet and outlet. The culture medium that flows in from the inlet comes into contact with the immobilized organ organoids or the like, and then flows out from the outlet. General culture vessels for perfusion culture of cells are commercially available, and similar culture vessels can be used in the present invention.

[0013] <Turbulent / Laminar flow> In the present invention, "turbulent flow" of the culture medium in the chamber refers to the fact that the flow of the culture medium in the chamber is not uniform, and that vortices are generated in the culture medium by visual inspection, particularly around the fixed organ organoids or the matrix in which the organ organoids are embedded, or near the inlet or the wall of the chamber, preferably at a level higher than a predetermined standard (e.g., frequency per unit time, size, etc.). On the other hand, "laminar flow," which relates to a preferred embodiment of the present invention described below, refers to the fact that the flow of the culture medium in the chamber is nearly uniform, and that only vortices are generated by visual inspection, particularly around the fixed organ organoids or the matrix in which the organ organoids are embedded, or near the inlet or the wall of the chamber, at a level lower than a predetermined standard, preferably no or almost no vortices are generated in the culture medium. In addition, if it is possible to model the perfusion of culture medium in a chamber using piping flow, open channel flow, etc., and calculate the Reynolds number from the representative length (hydraulic diameter, for example, the diameter when a cross section perpendicular to the perfusion direction in piping flow is considered to be circular), representative flow velocity, density, and viscosity coefficient, it is also possible to compare the Reynolds number with a predetermined standard (2300, for example), and determine that if it is larger than that, it is turbulent flow, and if it is smaller, it is laminar flow.

[0014] The means for generating turbulence in the chamber is not particularly limited, but for example, turbulence can be generated in the chamber by increasing the flow rate (e.g., a value expressed in units such as "mL / min") or flow velocity (a value calculated by dividing the flow rate by the cross-sectional area of ​​the chamber) of the culture solution in the chamber above a predetermined level and causing the culture solution to collide relatively strongly with the wall of the chamber. Alternatively, a structure (e.g., protrusions) that generates turbulence in the chamber can be provided, and turbulence can be generated in the chamber by the culture solution colliding with the structure.

[0015] During the period in which organoids or the like immobilized in a chamber are cultured while being perfused with a culture medium, turbulence may be generated without changing the conditions, or may be generated while changing the conditions.

[0016] In a preferred embodiment of the present invention, the culture medium is perfused so as to include (i) a period during which the culture medium transitions from laminar to turbulent flow in the chamber, (ii) a period during which the culture medium generates turbulent flow, and (iii) a period during which the culture medium transitions from turbulent flow to laminar flow. In this embodiment, the culture medium in the chamber does not always generate turbulent flow, but rather alternates between (i) a state during which the culture medium is in the process of transitioning from laminar to turbulent flow, (ii) a state during which the culture medium is generating turbulent flow, (iii) a state during which the culture medium is in the process of transitioning from turbulent flow to laminar flow, and (iv) a state during which the culture medium is generating laminar flow (which may be stationary). The duration of each of the above states (i) to (iv) is not particularly limited, but can be adjusted, for example, by the flow rate or flow velocity of the perfused culture medium (hereinafter collectively referred to as "flow rate, etc."). That is, the duration of state (i) can be adjusted by the time it takes to increase the flow rate from a predetermined flow rate (hereinafter referred to as the "first flow rate") that generates laminar flow to a predetermined flow rate (hereinafter referred to as the "second flow rate") that generates turbulent flow, or by the time it takes for a pump or other fluid delivery device that can automatically change the flow rate, as described below, to switch from a first flow rate to a second flow rate, and the flow rate in the flow channel and chamber to change accordingly. Similarly, the duration of state (ii) can be adjusted by the time it takes to maintain the second flow rate, the duration of state (iii) can be adjusted by the time it takes to decrease from the second flow rate to the first flow rate, and state (iv) can be adjusted by the time it takes to maintain the first flow rate, or by the settings of the corresponding fluid delivery device. For example, in a culture vessel equipped with a certain chamber, a flow rate within a typical range that is typically set to generate "laminar flow" within the chamber can be treated as the "first flow rate," and a higher, non-typical range of flow rate can be treated as the "second flow rate."

[0017] <Organoids, etc.> "Organ organoid" is a tissue (three-dimensional structure) similar to artificially created organ or tissue. "Organ organoid" in the present invention is a general term for the organoid of various organs or tissues, and includes not only the organoid of organ or tissue that is in the mature stage, but also the structure that is called "organ bud" or "primordium" in the early stage of complexification.

[0018] The present invention can also be applied to "cancer organoids" (see, for example, JP 2018-110575 A) instead of "organ organoids." That is, according to the culture method of the present invention, when "cancer organoids" are immobilized in a chamber instead of "organ organoids" and the cancer organoids are cultured in a culture medium perfused to generate turbulence in the chamber, it is possible to produce cancer organoids in which cancer cells are distributed at a higher density than conventional cancer organoids. Therefore, the term "organ organoids" in this specification can be replaced with "cancer organoids" as necessary, or can be replaced with "organoids" as a general term for "organ organoids" and "cancer organoids." In addition, the "drug evaluation method" of the present invention can be carried out using "cancer organoids."

[0019] Various types of "organ organoids" are already known, including organoids of the liver, pancreas, kidney, heart, lung, spleen, esophagus, stomach, thyroid, parathyroid, thymus, gonads, brain, spinal cord, skin, and inner ear (see, for example, https: / / www.nejm.org / doi / pdf / 10.1056 / NEJMra1806175, https: / / www.nature.com / articles / s41568-018-0007-6, and http: / / www.amsbio.com / brochures / organoid-culture-handbook.pdf). Various organ organoids can be used depending on the purpose of implementing the culture method of the present invention. For example, organoids of the liver, kidney, pancreas, thyroid, parathyroid, lung, brain, and heart are preferred organ organoids in the present invention. These organ organoids can be particularly used to implement a method for evaluating the efficacy of drugs for treating ischemic diseases, etc., as described below.

[0020] The basic embodiments of the method for producing various organ organoids, such as the required cells and culture conditions, are known, and the embodiment of the culture method of the present invention can also be basically made to conform to the known method for producing organ organoids.For example, the method described in WO2013 / 047639, WO2015 / 129822, etc., in which (A) organ-constituting cells, (B) vascular endothelial cells, and (C) mesenchymal cells (preferably mesenchymal stem cells) are co-cultured, is also preferred as a method for producing organ organoids in the culture method of the present invention.

[0021] In the present invention, "cells or tissues constituting an organ" refers to cells necessary for constituting at least a portion of an organ organoid, or tissues containing such cells, used to prepare organ organoids (along with other cells such as vascular endothelial cells and mesenchymal cells, if necessary). "Cells or tissues constituting an organ" may be in the stage of a simple cell aggregate (spheroid) that does not have the structure or characteristics of organ organoids, or may be cells or tissues isolated from an individual (e.g., a patient-derived biopsy sample). Various "cells or tissues constituting an organ" are known, and cells can be selected according to the type of organ organoid. Note that the "cells constituting an organ" immobilized in the chamber can be interpreted as "cells or tissues for preparing organ organoids, including at least cells or tissues constituting an organ," if necessary.

[0022] "Cells that constitute organs" include (A1) parenchymal cells that constitute organs and tissues and (A2) non-parenchymal cells that constitute organs and tissues. Furthermore, (A1) parenchymal cells and (A2) non-parenchymal cells each include (m) differentiated and mature cells or cells that have reached terminal differentiation and have the specified functionality of parenchymal or non-parenchymal cells (simply referred to herein as "differentiated cells"). Also included are (n) cells that have the potential to differentiate into parenchymal or non-parenchymal cells or are destined (committed) to differentiate, but are undifferentiated or at the stem or progenitor cell stage and do not yet fully possess the specified functionality of parenchymal or non-parenchymal cells (simply referred to herein as "undifferentiated cells").

[0023] The "cells that constitute an organ" can be at least one type of cell selected from the group consisting of differentiated parenchymal cells, undifferentiated parenchymal cells, differentiated non-parenchymal cells, and undifferentiated non-parenchymal cells, preferably a combination of two or more types of cells that can form organ organoids (preferably organ buds).

[0024] Examples of differentiated "parenchymal cells" include hepatocytes in the liver, endocrine cells (e.g., α cells, β cells, δ cells, ε cells, PP cells) and pancreatic duct epithelial cells, tubular epithelial cells and glomerular epithelial cells in the kidney, alveolar epithelial cells in the lung, cardiac myocytes, intestinal epithelial cells, neurons and glial cells in the brain, and neurons and Schwann cells in the spinal cord.

[0025] Examples of differentiated "non-parenchymal cells" include sinusoidal endothelial cells, hepatic stellate cells, and Kupffer cells in the liver, pancreatic stellate cells and pancreatic microvascular endothelial cells in the pancreas, renal glomerular endothelial cells in the kidney, pulmonary artery endothelial cells and pulmonary fibroblasts in the lung, cardiac microvascular endothelial cells, aortic endothelial cells, coronary artery endothelial cells, and cardiac fibroblasts in the heart, intestinal microvascular endothelial cells in the intestine, cerebral microvascular endothelial cells, vascular pericytes, choroid plexus endothelial cells, and cerebrovascular adventitia fibroblasts in the brain, etc.

[0026] Examples of "undifferentiated cells" that have the ability to differentiate into parenchymal or non-parenchymal cells include cells that can differentiate into ectodermal organs such as the brain, spinal cord, adrenal medulla, epidermis, hair, nails, skin glands, sensory organs, peripheral nerves, and lens; cells that can differentiate into mesodermal organs such as the kidney, ureter, heart, blood, gonads, adrenal cortex, muscle, skeleton, dermis, connective tissue, and mesothelium; and cells that can differentiate into endodermal organs such as the liver, pancreas, intestine, lung, thyroid gland, parathyroid gland, and urinary tract.

[0027] The cells that constitute the organ may be primary cultured cells, subcultured cells (established cell lines), or cells obtained by differentiating ES cells, iPS cells, or the like.

[0028] Organ organoids and / or cells constituting organs (organ organoids, etc.) may be derived from humans or from non-human animals, such as mammals such as mice, rats, dogs, pigs, and monkeys. That is, organ organoids, etc. may be derived from humans or from non-human animals. From the viewpoint of detecting drugs that cause drug addiction that are difficult to discover in conventional animal experiments or human cell tests in the development of human medicines, organoids are preferably derived from humans. The same applies to other cells (vascular endothelial cells, mesenchymal cells, etc.) that are used together with cells constituting organs as needed to produce organ organoids.

[0029] The type of organ or tissue that the organoid of the present invention is used for culturing method can be selected according to its purpose and is not particularly limited.Hepatic organoid and kidney organoid are exemplified below, but the present invention can also be applied to the organoid of pancreas, thyroid, parathyroid, lung, brain, heart and other organs.

[0030] In a preferred embodiment of the present invention, organ organoid is liver organoid (preferably liver bud), and the cells that constitute organ are the cells that constitute liver (liver parenchymal cells and / or non-parenchymal cells).Liver organoid (liver bud) can be produced by co-culturing liver endoderm cells (corresponding to the undifferentiated cells of liver parenchymal cells), vascular endothelial cells and mesenchymal cells (preferably mesenchymal stem cells), for example, according to the method described in WO2013 / 047639, WO2015 / 129822 etc.

[0031] Among the "cells constituting the liver," liver parenchymal cells (hepatocytes) encompass both differentiated hepatocytes (differentiated hepatocytes) and cells that have committed to hepatocyte differentiation but have not yet differentiated into hepatocytes (undifferentiated hepatocytes), so-called hepatic progenitor cells (e.g., hepatic endoderm cells). Differentiated hepatocytes may be cells collected from a living organism (isolated from the liver in the living organism), or may be cells obtained by differentiating pluripotent stem cells such as ES cells and iPS cells, hepatic progenitor cells, or other cells capable of differentiating into hepatocytes. Undifferentiated hepatocytes may be collected from a living organism, or may be obtained by differentiating pluripotent stem cells such as ES cells and iPS cells, or other stem or progenitor cells. Cells capable of differentiating into hepatocytes can be produced, for example, according to K. Si-Taiyeb, et al. Hepatology, 51(1):297-305 (2010) and T. Touboul, et al. Hepatology, 51(5):1754-65 (2010). Methods for differentiating pluripotent stem cells such as ES cells and iPS cells, hepatic progenitor cells, and other cells capable of differentiating into hepatocytes into hepatocytes are known. For example, differentiation of iPS cells can be performed in accordance with the methods described in Hepatology, 2010; 51(1):297-305, Cell Rep. 2017; 21(10):2661-2670, etc. For both cell populations collected from a living body and cell populations prepared by inducing differentiation of ES cells, iPS cells, etc. (especially the latter), a cell population with a high purity of differentiated hepatocytes or a cell population with a high purity of undifferentiated hepatocytes may be used, or a cell mixture containing differentiated hepatocytes and undifferentiated hepatocytes in any ratio may be used.

[0032] Whether a cell is a differentiated hepatocyte can be determined by the positive expression of one or more mature hepatocyte markers, such as asialoglycoprotein receptor 1 (ASGR1), glutamine synthase (GS), E-cadherin, the immature hepatocyte marker (early hepatic differentiation marker) alpha-fetoprotein (AFP), and the early hepatic differentiation markers albumin (ALB), retinol-binding protein (RBP4), transthyretin (TTR), and glucose-6-phosphatase (G6PC). On the other hand, whether a cell is an undifferentiated hepatocyte can be determined by the positive expression of one or more cell markers, such as HHEX, SOX2, HNF4α, AFP, and ALB (weak positive expression for ALB).

[0033] The term "vascular endothelial cells" encompasses both hemogenic endothelial cells (HECs) and non-hemogenic endothelial cells (non-HECs). HECs are endothelial cells capable of producing hematopoietic stem cells (having hematopoietic potential) and are also known as blood cell-producing endothelial cells. On the other hand, non-HECs are endothelial cells that do not possess such hematopoietic potential. The use of endothelial cells can provide organoids with a fine vascular network.

[0034] The vascular endothelial cells may be a highly purified cell population of vascular endothelial cells collected from a living body (e.g., microvessel endothelial cells (MVEC), liver sinusoidal endothelial cells (LSEC), umbilical-vein endothelial cells (UVEC)), etc.), or a highly purified cell population of vascular endothelial cells obtained by differentiating pluripotent stem cells such as ES cells and iPS cells, or other cells capable of differentiating into vascular endothelial cells.

[0035] Methods for differentiating pluripotent stem cells such as ES cells and iPS cells, and other cells capable of differentiating into vascular endothelial cells, into hematopoietic endothelial cells (HECs) are known. For example, methods for differentiating iPS cells can be performed in accordance with the methods described in PLoS One, 2013; 8(4): e59243, Nat Biotechnol. 2014; 32(6): 554-61, Sci Rep. 2016; 6: 35680, etc.

[0036] Whether a cell is a vascular endothelial cell can be determined by whether it is positive for one or more vascular endothelial cell markers, such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3, and CD41 (if positive, it is a vascular endothelial cell). Furthermore, whether a cell is a differentiated vascular endothelial cell can be determined by whether it is positive for one or more markers, such as CD31 and CD144 (if positive, it is a differentiated vascular endothelial cell).

[0037] "Mesenchymal cells" refers to connective tissue cells that exist primarily in connective tissue derived from the mesoderm and form a support structure for cells that function in tissues. The term encompasses both differentiated cells (differentiated mesenchymal cells) and cells whose differentiation fate into mesenchymal cells has been determined but which have not yet differentiated into mesenchymal cells (undifferentiated mesenchymal cells), so-called mesenchymal stem cells. However, although "vascular endothelial cells" are a type of cell that differentiates from undifferentiated mesenchymal cells, they are excluded from the definition of "mesenchymal cells" in this specification.

[0038] Whether a cell is an undifferentiated mesenchymal cell or a differentiated mesenchymal cell can be determined by whether it is positive for one or more of the markers for undifferentiated mesenchymal cells, such as Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, and Nestin (if positive, it is an undifferentiated mesenchymal cell; if negative, it is a differentiated mesenchymal cell).

[0039] Mesenchymal cells may also express cell markers specific to a particular organ (tissue) depending on the organoid of interest or the cells constituting the organ or tissue used in combination with them. Examples of such cell markers include FOXF1, COL4A, and ALCAM, which are cell markers for septum transversum mesenchyme (STM).

[0040] In a preferred embodiment of the present invention, the organoid is a renal organoid (preferably a kidney primordium), and the cells that constitute the organ are the cells that constitute the kidney (kidney parenchymal cells and / or non-parenchymal cells). Renal organoids and kidney primordium can be produced by co-culturing kidney or metanephric cells, vascular endothelial cells and mesenchymal cells (preferably mesenchymal stem cells), for example, according to the method described in WO2013 / 047639, WO2015 / 129822, etc. The vascular endothelial cells and mesenchymal cells used to produce renal organoids (kidney primordium) are the same as those used to produce the liver organoids (liver buds) described above.

[0041] Among the "cells that constitute the kidney," kidney parenchymal cells (renal glomerular endothelial cells, glomerular epithelial cells, proximal tubule cells, loop of Henle cells, and distal tubule cells) encompass both cells that have differentiated into parenchymal cells (differentiated kidney cells) and cells whose differentiation fate into parenchymal cells has been determined but that have not yet differentiated (undifferentiated kidney cells), so-called renal progenitor cells. Differentiated kidney cells may be cells collected from a living organism (isolated from the kidney or other part of the body), or may be cells obtained by differentiating pluripotent stem cells such as ES cells and iPS cells, renal progenitor cells, or other cells that have the ability to differentiate into kidney parenchymal cells. Undifferentiated kidney cells may be collected from a living organism, or may be cells obtained by differentiating pluripotent stem cells such as ES cells and iPS cells, or other stem or progenitor cells. Cells that can be differentiated into kidney cells can be produced, for example, according to Morizane et al., Nat Protoc 12:195-207 (2017), Taguchi et al., Cell Stem Cell 14(1):53-67 (2014), Narayanan et al., Kidney Int. 83(4):593-603 (2013), etc. Methods for differentiating pluripotent stem cells such as ES cells and iPS cells, renal progenitor cells, and other cells capable of differentiating into renal cells into renal cells are known. For example, iPS cells can be differentiated into renal cells as described above, as well as in Morizane et al., Nat Protoc 12:195-207 (2017), Taguchi et al., Cell Stem Cell 14(1):53-67 (2014), Narayanan et al., Kidney Int. 83(4):593-603 (2013), etc. For both cell populations collected from a living body and cell populations prepared by inducing the differentiation of ES cells, iPS cells, etc. (especially the latter), a highly purified cell population of differentiated renal cells or a highly purified cell population of undifferentiated renal cells may be used, or a cell mixture containing differentiated renal cells and undifferentiated renal cells in any ratio may be used.

[0042] Whether a cell is a differentiated renal cell can be determined by whether it is positive for the expression of one or more mature renal cell markers, such as PODXL, LTL, and CDH1, whereas whether a cell is an undifferentiated renal cell can be determined by whether it is positive for the expression of one or more cell markers, such as SIX2 and SALL1.

[0043] In a preferred embodiment of the present invention, organoid has vascular structure, and / or the cells that constitute the organ coexist with vascular cells." has vascular structure " specifically refers to that in the preparation of organoid, the cells that constitute organ and vascular cells coexist (co-culture), so that organoid forms microvascular structure.

[0044] " Vascular cell " can be exemplified by vascular endothelial cells, vascular smooth muscle cells and pericytes.For example, by co-culturing hepatic endoderm cells (corresponding to the undifferentiated cells of liver parenchymal cells), vascular endothelial cells and mesenchymal cells (preferably mesenchymal stem cells), can produce hepatic organoids with vasculature.In addition, by co-culturing kidney or metanephric cells, vascular endothelial cells and mesenchymal cells (preferably mesenchymal stem cells), can produce renal organoids with vasculature.

[0045] Whether organ organoids have been obtained from cells constituting an organ and other cells used as needed can be determined, for example, by confirming the formation of a three-dimensional structure (three-dimensional structure) by macroscopic or microscopic observation. Furthermore, in addition to the formation of such a three-dimensional structure, the creation of organ organoids can also be determined by whether certain cell markers, particularly markers for organ parenchymal cells, are positive (e.g., whether the ratio of positive cells exceeds a predetermined standard), and, if necessary, by whether the proteins of these markers are secreted into the culture supernatant (e.g., whether the secretion amount exceeds a predetermined standard). For example, in the case of hepatic organoids or liver buds, positive expression of markers such as HHEX, SOX2, AFP, ALB, and HNF4α and / or secretion of albumin (ALB) into the culture supernatant can be used for determination. In the case of kidney organoids or kidney primordia, positive expression of markers such as SIX2, SALL1, PODXL, LTL, and CDH1 can be used for determination.

[0046] <Immobilization matrix> In the culture method of the present invention, organ organoids and / or cells constituting an organ (organ organoids, etc.) are immobilized in a chamber. The means for "immobilization" are not particularly limited, as long as the culture medium perfused in the chamber to generate turbulence prevents organ organoids, etc. from being washed away from the chamber. For example, the entire or part of the bottom of the chamber can be covered with a matrix (similar to that used to embed organ organoids, details of which will be described later) or a similar scaffolding material, and organ organoids, etc. (prescribed cells for producing organ organoids) can be seeded on the covering and cultured on a plate, thereby immobilizing organ organoids, etc. in the chamber. In addition, as in a preferred embodiment of the present invention described below, embedding organ organoids, etc. in a matrix, for example, suspending the matrix in which organ organoids, etc. are embedded on a breathable membrane, is also a suitable means for immobilizing organ organoids, etc. in a chamber.

[0047] In a preferred embodiment of the present invention, organ organoids and / or cells constituting an organ (organ organoids, etc.) are immobilized in a state embedded in a matrix.

[0048] The "matrix" used to embed organoids and the like can be a typical extracellular matrix that is solid at room temperature or above and is used in cell culture, particularly three-dimensional cell culture. Extracellular matrices are primarily composed of fibrous proteins and proteoglycans, including elastin, entactin, osteonectin, collagen (e.g., type IV collagen), tenascin, thrombospondin, perlecan, vitronectin, fibrillin, fibronectin, heparin (sulfate), and laminin. For example, a basement membrane matrix known under the trade name "Matrigel" (Corning), which contains laminin, collagen (type IV collagen), and entactin, as well as growth factors such as EGF, IGF-1, PDGF, and TGF-β, is a preferred example of a matrix for the present invention. Self-assembling peptides known as hydrogels, such as hydrogels containing arginine, glycine, and asparagine, can also be used as matrices for the present invention. The matrix may be any one of the above, or two or more of them may be used in combination or not (so as to form different layers when solidified).

[0049] Those skilled in the art can adjust the composition and concentration of the matrix (either undiluted or diluted) as needed to ensure that the matrix (hereinafter referred to as the "matrigel composition") in which organoids or the like are embedded has an appropriate hardness. For example, the matrix (e.g., Matrigel) can be mixed with a culture medium as needed to improve handling and ensure appropriate hardness when solidified. In such an embodiment, the culture medium to be mixed with the matrix can be the same as the culture medium used to culture organoids or the like perfused in the culture method of the present invention.

[0050] A matrix composition can generally be prepared by adding an appropriate amount of matrix containing appropriate components to a culture medium containing organ organoids or the like, and then solidifying the matrix (adding an ionic solution or ionic molecules to solidify the hydrogel, if necessary). For example, a matrix (e.g., Matrigel) that is liquid at 4°C or below and organ organoids or the like are stirred and mixed, and then allowed to stand at 37°C or above to solidify the matrix, thereby obtaining a matrix composition. When adding a matrix to a relatively large amount of medium in which organ organoids or the like are suspended, it is appropriate to add a sufficient amount of matrix to ensure that the matrix is ​​sufficiently solid so that the organ organoids or the like are retained in the matrix without settling to the bottom.

[0051] The matrix composition may be, for example, any one of the following (i) to (iii). (i) Organoids are produced from selected cells, then isolated and mixed with a matrix. (ii) Organoids are produced from selected cells by three-dimensional culture, and then the organoids are mixed with a matrix. (iii) Organoids are obtained by adding a matrix to a cell population containing specific cells (cells that make up an organ) to be used to create organoids, solidifying the matrix, and then culturing the cell population embedded in the matrix to create organ organoids.

[0052] The means for immobilizing organ organoids and / or cells constituting an organ (organ organoids, etc.) embedded in a matrix, i.e., the matrix composition, within the chamber is not particularly limited. The matrix composition may be placed in an appropriate location within the chamber so that the perfused culture medium can contact the matrix in a turbulent state with the organ organoids, etc. embedded in the chamber. In the present invention, for example, the organ organoids, etc. (embedded in the matrix) can be immobilized within the chamber by dropping a mixture of the organ organoids, etc. and the matrix before solidification into an appropriate location within the chamber and solidifying the matrix.

[0053] In a preferred embodiment of the present invention, a matrix (matrix composition) in which organoids and / or cells constituting an organ are embedded is immobilized in a chamber while suspended from a breathable membrane.

[0054] A "breathable membrane" is a membrane that is at least oxygen permeable and, if necessary, also carbon dioxide or other desired gas permeability. Various breathable membranes are known, including membranes made from fibers such as polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), fluorocarbon, polytetrafluoroethylene (PTFE), and polyurethane. If necessary, the breathable membrane may be surface-treated to enhance or reduce cell adhesion, for example, by coating with an extracellular matrix (ECM) such as collagen. Furthermore, if necessary, the breathable membrane may be laminated with a porous membrane (mesh) made from fibers different from the breathable membrane (hybrid membrane).

[0055] A matrix composition "suspended on a breathable membrane" can be produced by dropping the matrix composition onto a breathable membrane before solidification, allowing it to solidify, and then inverting the membrane so that the solidified matrix composition attached to the breathable membrane faces downward (convex downward). By fixing the breathable membrane with the suspended matrix composition to a chamber (e.g., the ceiling) using an appropriate member such as a holder, the matrix composition can be immersed in a perfused culture medium, while the breathable membrane can be placed in the atmosphere (or a desired culture atmosphere) without being immersed. This allows organoids embedded in the matrix to be cultured while ensuring gas exchange through the breathable membrane.

[0056] <Culture solution> In the present invention, the perfused culture medium (liquid medium) can be selected appropriately depending on the type of organ organoid and / or organ-constituting cells (organ organoid, etc.). Culture media for organ organoids and the like are known, and generally, a mixture of media for culturing the organ-constituting cells (usually a cell population of a mixture of multiple types of cells) used to produce organ organoids can be used. For example, when culturing hepatic organoids and / or liver-constituting cells, a mixed medium of a hepatic cell medium and a vascular endothelial cell medium can be used, and when culturing renal organoids and / or kidney-constituting cells, a mixed medium of a renal cell medium and a vascular endothelial cell medium can be used.

[0057] Examples of basal media for vascular endothelial cells include DMEM / F-12 (Gibco), Stempro-34 SFM (Gibco), Essential 6 Medium (Gibco), Essential 8 Medium (Gibco), EGM (Lonza), BulletKit (Lonza), EGM-2 (Lonza), BulletKit (Lonza), EGM-2 MV (Lonza), VascuLife EnGS Comp Kit (LCT), Human Endothelial-SFM Basal Growth Medium (Invitrogen), and human microvascular endothelial cell growth medium (TOYOBO). Examples of additives for vascular endothelial cells include B27 Supplements (GIBCO), BMP4 (bone morphogenetic protein 4), GSKβ inhibitors (e.g., CHIR99021), VEGF (vascular endothelial growth factor), FGF2 (fibroblast growth factor (also known as bFGF (basic fibroblast growth factor))), folskolin, SCF (stem cell factor), TGFβ receptor inhibitors (e.g., SB431542), Flt-3L (Fms-related tyrosine kinase 3 ligand), IL-3 (interleukin 3), and IL-6 (interleukin 6), TPO (thrombopoietin), hEGF (recombinant human epidermal growth factor), hydrocortisone, ascorbic acid, IGF1, FBS (fetal bovine serum), antibiotics (e.g., gentamicin, amphotericin B), heparin, L-glutamine, phenol red, and BBE.

[0058] Basal media for hepatocytes include, for example, RPMI (Fujifilm) and HCM (Lonza). Additives for hepatocytes include, for example, one or more selected from the group consisting of Wnt3a, activin A, BMP4, FGF2, FBS, HGF (hepatocyte growth factor), oncostatin M (OSM), and dexamethasone (Dex). If necessary, the hepatocyte culture medium can be supplemented with at least one selected from ascorbic acid, BSA-FAF, insulin, hydrocortisone, and GA-1000. More specifically, hepatocyte culture media include, for example, HCM BulletKit (Lonza) without hEGF (recombinant human epidermal growth factor); and RPMI1640 (Sigma-Aldrich) supplemented with 1% B27 Supplements (GIBCO) and 10 ng / mL hHGF (Sigma-Aldrich). In particular, when preparing liver buds, a medium can be used that is a 1:1 mixture of EGM Bullet Kit (Lonza) and HCM Bullet Kit (Lonza) minus hEGF (recombinant human epidermal growth factor), to which dexamethasone, oncostatin M, and HGF have been added.

[0059] Examples of basal media for renal cells include ReproFF2 (REPROCELL) and StemFit Basic (Ajinomoto). Examples of additives for renal cells include FGF9. If necessary, the renal cell medium can be supplemented with one or more members selected from the group consisting of CHIR and heparin. More specifically, examples of media for renal cells include ReproFF2 supplemented with 10 ng / mL FGF9 (R&D). In particular, when preparing kidney primordia, a medium supplemented with 3 μM CHIR and 10 ng / mL FGF9 (R&D) can also be used.

[0060] The viscosity of the culture solution (liquid medium) is not particularly limited as long as it allows perfusion, but from the viewpoint of facilitating turbulence in the chamber, it is preferable that the viscosity be, for example, 0.7 to 0.8 mPa·s at the temperature under culture conditions (e.g., approximately 37°C).

[0061] <Culture conditions, etc.> Other conditions for the culture method of the present invention, such as atmosphere, temperature, period, etc., can be adjusted as appropriate depending on the purpose of carrying out the culture method of the present invention and the embodiment of the organ organoid, etc. For example, to form a three-dimensional structure of an organ organoid such as a liver or kidney or its organ bud (primordium) from cells constituting the organ and other cells used as needed, culture can be carried out at 5% CO2, 30 to 40°C (preferably about 37°C) for 1 to 10 days (1 to 3 days for organ bud (primordium)).

[0062] <Drugs> In a preferred embodiment of the present invention, the culture medium contains a drug. The "drug" is not particularly limited as long as it acts on organ organoids or is used to analyze whether it acts, and can be selected according to the purpose. The "drug" is not particularly limited as long as it can be used as an active ingredient in a pharmaceutical, such as a small molecule drug, an antibody drug, a peptide drug, or a nucleic acid drug. Furthermore, substances used to embolize blood vessels in hepatic arterial chemoembolization (e.g., gelatin sponge, porous gelatin granules) can be included in the culture medium, particularly in the culture medium in the vasculature of organ organoids, or as a drug in a drug evaluation method using a three-dimensional organ (which may be transplanted into a non-human animal), as described below. The "drug" is not limited to drugs that are actually commercially available as pharmaceuticals, but may also be drugs used in clinical trials or non-clinical trials, or drugs in the early stages of development (candidate active ingredients for pharmaceuticals).

[0063] For example, drugs for treating ischemic diseases (diseases accompanied by ischemia), infarction / necrotic diseases, or hemorrhagic diseases (collectively referred to herein as "ischemic diseases, etc.") in each organ are preferred drugs in the present invention. A therapeutic drug for ischemic diseases, etc. in each organ can be added to a culture medium for organoids, etc. corresponding to the organ, and cultured. Examples of ischemic diseases, etc. in the liver include ischemic liver damage (ischemic hepatitis), ischemic bile duct damage, and hepatic rupture (secondary bleeding due to cancer, trauma, infection, etc.). Examples of ischemic diseases, etc. in the kidney include ischemic renal damage and idiopathic renal hemorrhage. An example of a therapeutic drug for ischemic renal damage approved in Japan, etc., is eculizumab. Examples of ischemic diseases, etc. in the pancreas include ischemic acute pancreatitis and ischemic chronic pancreatitis. Examples of ischemic diseases, etc. (reduced blood flow, etc.) in the thyroid or parathyroid gland include subacute thyroiditis and painless thyroiditis. Examples of ischemic diseases in the heart include ischemic heart disease. Examples of therapeutic drugs for ischemic heart disease approved in Japan include isosorbide dinitrate. Examples of rejection diseases in the brain include cerebral ischemia.

[0064] From another perspective, drugs that may cause side effects involving ischemia or bleeding in various organs are also preferred drugs for safety evaluation in the present invention. Examples of drugs that may cause ischemia as a side effect include anticancer drugs, antiviral drugs, and acetaminophen, which can cause serious drug-induced liver injury in the liver, as well as antibiotics (which affect multiple organs), cardiotonic drugs, steroids (for the heart), diuretics, renin-angiotensin system (RAS) inhibitors, contrast agents, and SGLT2 inhibitors (for the kidneys). Examples of drugs that may cause bleeding as a side effect include antiplatelet drugs and thrombolytic drugs.

[0065] -Drug evaluation method- The culture method of the present invention can be applied to a method for evaluating a drug by using a culture medium containing the drug. The viewpoint from which the drug is evaluated is not particularly limited, but for example, if the drug is used to treat ischemic diseases in each organ, the effectiveness of the treatment can be evaluated. Furthermore, if the drug may cause side effects involving ischemia or bleeding in each organ, the safety of the drug can be evaluated.

[0066] That is, the drug evaluation method according to the present invention using organ organoids, etc., includes a step of carrying out the culture method of the present invention using a culture medium containing the drug as described above (hereinafter referred to as the "drug-addition culture step"), and further includes a step of evaluating the effectiveness and / or safety of the drug on the organ organoids.

[0067] More specifically, examples of the method for evaluating the efficacy of a drug according to the present invention (sometimes referred to herein as a "drug efficacy evaluation method") include a drug evaluation method comprising a drug-addition culture step carried out using a culture medium containing a drug for treating ischemic diseases in the liver, kidneys, etc. Furthermore, examples of the method for evaluating the safety of a drug according to the present invention include a drug efficacy evaluation method comprising a drug-addition step carried out using a drug that may cause side effects involving ischemia or bleeding in each organ.

[0068] The culture conditions (atmosphere, temperature, period, etc.) in the drug-addition culture step can basically be the same as those in the culture method of the present invention, and can be appropriately adjusted as needed, for example, to a culture period sufficient for evaluation depending on the drug.

[0069] The drug evaluation method of the present invention may optionally include a step other than the drug-added culture step.For example, for the cells contained in the organoids etc. that have undergone the drug-added culture step, (i) use a reagent (for example, propidium iodide; PI) to detect dead cells, and measure the ratio of dead cells in cells (cell death rate), and / or (ii) measure the production amount of a biomarker that reflects the symptoms of the disease to be treated (for example, the concentration of a specific substance in the culture supernatant), and compare it with a control, to evaluate the efficacy and / or safety of the drug.

[0070] In the drug evaluation method of the present invention, organ organoids and the like are preferably derived from patients (humans) who have developed a disease that is the target of drug treatment or other subjects. In this embodiment, the cells that constitute the organ and other cells that are used as needed may be cells (primary cultured cells) collected from such patients or subjects, or may be iPS cells or subcultured cells (established cell lines) prepared using them. For example, iPS cell lines prepared using cells collected from patients with ischemic diseases, etc., are suitable for standardizing the drug evaluation method of the present invention to prepare organ organoids, that is, for inducing differentiation into cells that constitute the organ for preparing organ organoids and other cells that are used as needed.

[0071] The drug evaluation method of the present invention can also be carried out in the same way when using a three-dimensional organ obtained by the manufacturing method described below instead of organ organoids etc. (organ organoids and / or cells that constitute organs).For example, in a preferred embodiment of the present invention, the drug evaluation method of the present invention using a three-dimensional organ can include the steps of adding a drug to the culture medium of the three-dimensional organ, and evaluating the effectiveness (drug efficacy) and / or safety of the drug on the three-dimensional organ.

[0072] Furthermore, the present invention also provides a drug evaluation method comprising the steps of administering a drug to a non-human animal transplanted with a three-dimensional organ, and evaluating the efficacy and / or safety of the drug on the three-dimensional organ.

[0073] -Method of manufacturing three-dimensional organs- The method for producing a three-dimensional organ of the present invention includes carrying out the method for culturing organoids of the present invention as described above. A "three-dimensional organ" is a structure that includes a cell population or structure that is more mature than an organ organoid, and can also be called a mature organ organoid.

[0074] The culture conditions (atmosphere, temperature, duration, etc.) in the method for producing a three-dimensional organ of the present invention can be basically the same as those in the culture method of the present invention, and can be adjusted appropriately as necessary, for example, a culture period sufficient for the formation of a three-dimensional organ can be set. For example, when producing three-dimensional organs such as liver or kidney, culture can be performed for 20 to 30 days from the day when the culture of the specified cells for producing each organ organoid is started, or culture for an additional 10 to 20 days from the day when the formation of a three-dimensional structure as the organ organoid is confirmed for maturation.

[0075] Whether a three-dimensional organ has been obtained from an organoid can be determined from one or more perspectives, such as the density of cells in the structure (whether it exceeds a predetermined standard, etc.), the three-dimensional shape of the structure (whether it is three-dimensional beyond a certain level, etc.), function or trait (whether a predetermined function or trait, such as metabolic function, has been acquired, etc.), cell marker (whether the expression of the gene or protein of the cell marker is positive, whether the density of positive cells exceeds a predetermined standard, whether the amount of marker protein secreted into the culture supernatant exceeds a predetermined standard, etc.). The above-mentioned cell density, three-dimensional shape, function or trait, cell marker, etc. can be appropriately set depending on the organ organoid and three-dimensional organ, but for example, whether a level comparable to or similar to that of an organ in vivo has been achieved can be used as the basis for such determination.

[0076] For example, when preparing a liver as a three-dimensional organ, the acquisition of metabolic functions such as glutamine and citrate production and gluconeogenesis, the positive expression of markers such as ASGR1 and drug-metabolizing enzymes (CYP3A4, CYP7A1), for example, whether ASGR1-positive cells are present at a density of 70% or higher, can be used to determine this.When preparing a kidney as a three-dimensional organ, the positive expression of some or all of markers such as PODXL, LTL, and CDH1, ciliary proteins (PKD1, PKD2, NPHP1, NPHP6, PKHD1), transport proteins (SLC34A1, ATP1A1, SLC6A19, SLC9A3, SLC2A2, ABCB1, LRP2), etc. can be used to determine this.

[0077] By connecting the three-dimensional organ to an extracorporeal circulation system, an artificial organ can be produced. Such an artificial organ can be used as an organ failure model or to evaluate organ function. For such uses, see, for example, WO2013 / 047720.

[0078] -Cultivation system- The culture system of the present invention is a culture system that can immobilize and accommodate organ organoids and / or cells that constitute organs (organ organoids, etc.), and is equipped with a "chamber" equipped with an inlet and outlet for perfusing culture medium into the organ organoids, etc., and a "perfusion means" for perfusing the culture medium, and the perfusion means is controlled to generate turbulence in the culture medium within the chamber.

[0079] The chamber is as described above in relation to the "method for culturing organoids, etc." of the present invention, and the culture system of the present invention uses such a chamber or a culture vessel (cell culture device) equipped with such a chamber.

[0080] The perfusion means in the culture system of the present invention can be any common or known means used for perfusion culture of organoids or other tissues, cells, etc. The perfusion means typically includes, as its basic components, a pump (such as a peristaltic pump) for delivering the culture medium or other fluid delivery means, a reservoir for storing the culture medium, and a reservoir and a pump, etc., a pump and an inlet of the chamber, an outlet of the chamber and the reservoir, and channels (such as tubes) connecting them.

[0081] The perfusion means, particularly a fluid delivery means such as a pump, is preferably controlled so that the flow rate and other parameters can be automatically changed at any (preset) timing. For example, in a preferred embodiment of the present invention, the perfusion means is controlled to perfuse the culture solution so that it includes (i) a time for transition from laminar flow to turbulent flow in the chamber, (ii) a time for turbulent flow, and (iii) a time for transition from turbulent flow to laminar flow. In this embodiment, the above periods (i) to (iii) can be added with (iv) a time for laminar flow (which may be stationary), and these periods can be controlled so that they are continuously repeated. For example, one cycle can consist of delivery of fluid at a first flow rate or the like for a predetermined time, or no delivery of fluid for a predetermined time (corresponding to a case where the first flow rate or the like is zero), followed by delivery of fluid at a second flow rate or the like for a predetermined time, and this cycle can be repeated a predetermined number of times (for a predetermined time). Note that the control program for the perfusion means, particularly a fluid delivery means such as a pump, can also constitute a part of the culture system of the present invention.

[0082] The culture system of the present invention may, if necessary, be equipped with other means, configurations, etc. that are used in general or known culture systems for perfusion culture, such as means for maintaining the culture environment at an appropriate temperature and atmosphere (temperature control device / mechanism, atmosphere control device / mechanism, etc.). [Example]

[0083] [Example 1] [1] Creation of human non-hematopoietic vascular endothelial cells Human iPS cells (1383D2; Kyoto University iPS Cell Research Institute) were cultured in 10 mL of DMEM / F-12 (Gibco) supplemented with 1% B-27 Supplements (Gibco), BMP4 (25 ng / mL), and CHIR99021 (8 μM) at 37°C with 5% CO2 for 3 days. The resulting mesodermal cells were then cultured in 10 mL of Stempro-34 SFM (Gibco) supplemented with VEGF (200 ng / mL) and folskolin (2 μM) at 37°C with 5% CO2 for 7 days to generate a CD31-, CD73-, and CD144-positive human non-hematopoietic endothelial cell population.

[0084] [2] Preparation of human hepatic endoderm cells Human iPS cells (1383D2; Kyoto University iPS Cell Research Institute) were cultured for 5 days at 5% CO2 and 37°C in 2 ml of basal RPMI (Fujifilm) medium supplemented with Wnt3a (50 ng / mL) and activin A (100 ng / mL). Endodermal cells were then induced. The resulting endodermal cells were cultured for an additional 5 days at 37°C and 5% CO2 in the same basal medium supplemented with 1% B27 Supplements (GIBCO) and FGF2 (10 ng / mL). AFP-, ALB-, and HNF4α-positive human liver endoderm cell populations were obtained.

[0085] [3] Preparation of human mesenchymal cells Human iPS cells (1383D2; Kyoto University iPS Cell Research Institute) were cultured for 3 days at 5% CO2 and 37°C in 10 mL of DMEM / F-12 (Gibco) medium supplemented with 1% B-27 Supplements (GIBCO), BMP4 (25 ng / mL), and CHIR99021 (8 μM). The resulting mesodermal progenitor cells were cultured for an additional 3 days at 5% CO2 and 37°C in the same medium supplemented with PDGFBB and activin A. The cultured cells were then harvested and replated onto new gelatin-coated plates. The basal medium was supplemented with bFGF and BMP4 and cultured for an additional 4 days at 5% CO2 and 37°C to obtain a human mesenchymal cell population positive for FOXF1, COL4A, ALCAM, and CD73.

[0086] [4] Embedding cells into extracellular matrix and preparation of matrix composition A hepatic induction medium (HCM, Lonza) containing FBS, HGF, OSM, and Dex was mixed with EGM (Lonza) at a volume ratio of 1:1 to prepare a medium (referred to herein as "organoid medium"). Furthermore, a matrix (referred to herein as "organoid medium / Matrigel mixed matrix") was prepared by mixing the organoid medium with Matrigel (BD Pharmingen) at a volume ratio of 1:1.

[0087] 5 μL of the organoid medium / Matrigel mixed matrix was dropped onto an inverted hanging drop insert (Millipore) and solidified by raising the temperature to 37°C to form a "third matrix layer."

[0088] The human hepatic endoderm cells, human non-hemopoietic endothelial cells, and human mesenchymal cells prepared as described above were mixed in a 10:7:1 ratio in an organoid medium / Matrigel mixed matrix at 4°C. 3 μL of the resulting mixture was dropped onto the third matrix layer formed as described above, and the mixture was solidified by raising the temperature to 37°C to form a "second matrix layer."

[0089] Furthermore, 8 μL of the organoid culture medium / Matrigel mixture was dropped onto the second matrix layer formed as described above, and the mixture was solidified by raising the temperature to 37°C to form a "first matrix layer."

[0090] 600 μL of organoid medium supplemented with a Rock (Rho-associated coiled-coil forming kinase) inhibitor (10 ng / mL) was added to a low-adsorption 24-well plate. The matrix composition consisting of the first to third matrix layers formed on the hanging drop insert as described above was inserted into the well, facing the medium. After 24 hours, half of the medium was replaced with organoid medium without the Rock inhibitor (10 ng / mL). Thereafter, medium replacement was performed daily, with half of the medium replaced, until the third day after the start of culture.

[0091] [5] Perfusion culture On day 3 after the initiation of culture, the hanging drop inserts with the matrix composition containing organoids were removed, and perfusion culture was initiated using a QuasiVivo QV600 Barrier System Starter Kit (Kirkstall), which included a culture chamber and a supply medium reservoir. A Watson-Marlow tubing pump (530SN / 313D) was used to deliver the culture medium, and Tygon LMT-55 tubing (8 mm inner diameter x 11 mm outer diameter) was connected to the pump tubing. Intermittent perfusion was achieved by delivering the culture medium at 20 mL / min every 30 seconds for 30 seconds. A schematic diagram of the apparatus is shown in Figure 1. During perfusion, the generation of turbulent vortices within the culture chamber was visually confirmed (see Figure 2). Culture was performed in organoid medium at 5% CO2 and 37°C for 11 days, with medium changes performed by replacing the organoid medium in the supply medium reservoir every 3 days.

[0092] On day 11 after the start of perfusion culture, the hanging drop inserts with the organoid-containing matrix composition attached were removed, and the organoids were detached using a cell scraper. The organoids were then placed in a 2 mL tube containing 4% paraformaldehyde / PBS fixative solution and fixed.

[0093] [6] Static culture (control) As a control, the same organoids were cultured in a matrix composition and organoid medium containing the same organoids as above using conventional static culture, i.e., without perfusion culture from the third day after the start of culture, in a low-adsorption 24-well plate for the same period as above, and then fixed in the same manner.

[0094] [7] Immunostaining (ASGR1 etc.) After removing the fixative, the sections were washed twice with PBS for 15 minutes each and then permeabilized with 20% DMSO / ethanol, 80% ethanol, and 50% ethanol for 15 minutes each. Immunostaining was performed using anti-ASGR1 antibody (R&D, MAB4394), anti-CD31 antibody (Abcam, AB28364), and fluorescently labeled secondary antibodies (Novex Donkey Anti-Mouse IgG (H+L) Secondary Antibody, Novex Donkey Anti-Rabbit IgG (H+L) Secondary Antibody (Invitrogen)) and a nuclear stain (DAPI). The sections were then cleared using Visikol HISTO-M (Visikol Inc.). Fluorescence images of the stained sections were observed using an LSM880 confocal laser microscope (Zeiss). The ASGR1 fluorescence area was binarized from the fluorescence image and divided by the binarized DAPI fluorescence area to calculate the ASGR1 positivity rate. The results are shown in Figure 3.

[0095] [Example 2] Human hepatic endoderm cells, human non-hemopoietic endothelial cells, and human mesenchymal cells prepared in the same manner as in [1] to [3] of Example 1 were used to obtain organoids by perfusion culture of the matrix composition in the same manner as in [4] and [5] of Example 1, and then fixed. As a control, organoids were obtained by static culture of the matrix composition in the same manner as in [6] of Example 1, and then fixed. Observation images (three-dimensional structures) of the matrix composition containing organoids obtained by perfusion culture and static culture are shown in Figure 4.

[0096] [8] Immunostaining (GS, E-cadherin, etc.) After removing the fixative, the sections were washed twice with PBS for 15 minutes each and then permeabilized with 20% DMSO / ethanol, 80% ethanol, and 50% ethanol for 15 minutes each. Immunostaining was performed using anti-glutamine synthase (GS) antibody (Abcam, ab73593), anti-E-cadherin antibody (Abcam, ab76055), fluorescently labeled secondary antibodies (Novex Donkey anti-Mouse IgG (H+L) Secondary Antibody, Novex Donkey anti-Rabbit IgG (H+L) Secondary Antibody (Invitrogen)) and a nuclear stain (DAPI). The sections were then cleared using Visikol HISTO-M (Visikol Inc.). Fluorescence images of the stained sections were obtained using an LSM880 confocal laser scanning microscope (Zeiss). The results are shown in Figure 5.

Claims

1. A method for culturing organoids having a vascular structure and / or cells constituting an organ coexisting with vascular cells immobilized in a chamber while perfusing a culture solution, comprising: A culture method in which the culture medium is perfused in the chamber so that it includes a period during which it transitions from laminar flow to turbulent flow, a period during which turbulent flow is generated, and a period during which it transitions from turbulent flow to laminar flow.

2. The culture method according to claim 1, wherein the organoids and / or cells constituting the organ are immobilized in a matrix.

3. The culture method according to claim 1, wherein the matrix in which the organoids and / or cells constituting the organ are embedded is immobilized in a chamber while suspended on a breathable membrane.

4. The culture method according to claim 1, wherein the organ organoid is a liver or kidney organoid, and / or the organ is a liver or kidney.

5. A method for producing a three-dimensional organ, comprising a step of carrying out the culture method according to any one of claims 1 to 4.

6. The culture method according to any one of claims 1 to 4, wherein the culture medium contains a drug.

7. The culture method according to claim 6 , wherein the drug is for treating a disease accompanied by ischemia.

8. A method for evaluating a drug efficacy, comprising a step of carrying out the culture method according to claim 6 or 7.

9. An organoid obtained by the culture method of any one of claims 1 to 4.

10. A three-dimensional organ obtained by the method of claim 5.

11. A drug efficacy evaluation method comprising the steps of adding a drug to a culture medium for the three-dimensional organ according to claim 10, and evaluating the drug efficacy of the drug on the three-dimensional organ.

12. A chamber capable of immobilizing and accommodating organoids having a vascular structure and / or cells constituting an organ that coexist with vascular cells, the chamber having an inlet and an outlet for perfusing a culture medium over the organoids and / or cells constituting the organ; A culture system comprising a perfusion means for perfusing a culture solution, A culture system wherein the perfusion means is controlled to perfuse the culture solution so that there are periods in the chamber during which the flow transitions from laminar to turbulent, periods during which turbulent flow is generated, and periods during which the flow transitions from turbulent to laminar.

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