Method for manufacturing cardiac organoids

Abstract

To provide a simple and stable method for producing large quantities of cardiac organoids. [Solution] A method for producing cardiac organoids is provided, which includes culturing a mixture of pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells in a porous vessel.

Description

Technical Field

[0006] , , , ,

[0001] The present invention relates to a method for producing myocardial organoids.

Background Art

[0002] An organoid is a three-dimensional structure composed of a plurality of tissue- or organ-specific cells. Since an organoid can mimic the environment of a living tissue or organ, it is used in various fields such as the construction of disease models, drug discovery, and research on regenerative medicine. To date, attempts have been made to produce organoids of various tissues or organs by differentiating pluripotent stem cells.

[0003] In Non-Patent Document 1, the production of cardiomyocyte spheroids derived from human induced pluripotent stem cells (hiPSCs) by three-dimensional culture using a microwell plate was attempted. However, there is still a demand for the large-scale and stable production of uniform myocardial organoids.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a method for producing uniform bead-shaped myocardial organoids in large quantities and stably in order to produce highly functional myocardial organoids.

Means for Solving the Problems

[0006] As a result of diligent research, the inventors have surprisingly discovered that a large quantity of uniform, bead-shaped, highly functional cardiac organoids can be produced stably by a simple method of culturing a mixture of pluripotent stem cell-derived cardiomyocytes and pluripotent stem cell-derived mesenchymal stem cells in a porous vessel. Thus, the present invention has been completed.

[0007] This application provides the following embodiments. [1] A method for producing cardiac organoids, comprising culturing a mixture of pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells in a porous vessel. [2] The method according to [1], wherein cardiomyocytes derived from pluripotent stem cells are pre-cultured. [3] The method according to [1] or [2], wherein the cardiomyocytes derived from pluripotent stem cells are cardiomyocytes derived from induced pluripotent stem cells or embryonic stem cells. [4] The method according to any one of [1] to [3], wherein the mesenchymal stem cells are mesenchymal stem cells derived from pluripotent stem cells. [5] The method according to any one of [1] to [4], wherein the porous container is a plate, a bag, or a dish. [6] The method according to any one of [1] to [5], wherein the mixture comprises cardiomyocytes derived from pluripotent stem cells and mesenchymal stem cells in a ratio of 1:1 to 20:1. [7] A method for producing cardiomyocytes, comprising pre-culturing cardiomyocytes derived from pluripotent stem cells, and culturing a mixture of the pre-culturised cardiomyocytes derived from pluripotent stem cells and mesenchymal stem cells in a porous vessel. [8] The method according to [7], wherein the cardiomyocytes derived from pluripotent stem cells are cardiomyocytes derived from induced pluripotent stem cells or embryonic stem cells. [9] The method according to [7] or [8], wherein the mesenchymal stem cells are mesenchymal stem cells derived from pluripotent stem cells.

[10] The method according to any one of [7] to [9], wherein the porous container is a plate, a bag, or a dish.

[11] The method according to any one of [7] to

[10] , wherein the mixture comprises cardiomyocytes derived from pluripotent stem cells and mesenchymal stem cells in a ratio of 1:1 to 20:1.

[12] Cardiac organoids containing cardiomyocytes and mesenchymal stem cells derived from pluripotent stem cells.

[13] A cardiomyocyte organoid as described in

[12] , comprising pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells in a ratio of 6:4 to 20:1.

[14] A cardiac organoid as described in

[12] or

[13] , having a diameter or maximum diameter of 80 μm to 350 μm.

[15] A method for producing a cardiac organoid comprising the following steps, wherein a bead-shaped cardiac organoid having a diameter or maximum diameter of 80 μm to 350 μm is produced: 1. A step in which pluripotent stem cell-derived cardiomyocytes are pre-cultured at 25-37°C. 2. A step of mixing the pre-cultured cardiomyocytes and mesenchymal stem cells in a ratio of 5:1 to 20:1. 3. The step of placing the cell mixture into each pore of a porous container so that the number of cells is approximately 200 to 500. 4. A step in which cells placed in a porous vessel are cultured at 25-37°C for 2-14 days. [Effects of the Invention]

[0008] According to the present invention, uniform, bead-shaped cardiac organoids can be produced in large quantities and stably by a simple method of culturing a mixture of pluripotent stem cell-derived cardiomyocytes and pluripotent stem cell-derived mesenchymal stem cells in a porous container. Furthermore, cardiac organoids produced by the present invention exhibit suppression of the heart rate and improvement of the contraction speed associated with cardiomyocyte maturation, thus enabling the production of highly functional cardiac organoids. Moreover, using the present invention, cardiac organoids can be produced in a short culture period of approximately 2 days, or approximately 4 days including pre-culture. Furthermore, according to the present invention, one organoid can be produced for each pore of the porous container, making it easy to collect the produced cardiac organoids. In addition, cardiac organoids produced by the present invention can be transported together with the porous container, making them easy to handle. [Brief explanation of the drawing]

[0009] [Figure 1] Shows the diameters of the organoids due to differences in the mixing ratios of iPS-CM and iPS-MSC. [Figure 2] Shows the sphericity of the organoids due to differences in the mixing ratios of iPS-CM and iPS-MSC. [Figure 3] Shows the contraction characteristics of the organoids due to differences in the mixing ratios of iPS-CM and iPS-MSC. [Figure 4-1] Shows the changes in myocardial maturity of iPS-CM within the organoids due to differences in the mixing ratios of iPS-CM and iPS-MSC. [Figure 4-2] Shows the changes in myocardial maturity of iPS-CM within the organoids due to differences in the mixing ratios of iPS-CM and iPS-MSC. [Figure 5] Micrographs of organoids produced with different numbers of added cells. [Figure 6] Graph showing the relationship between the size of the organoids and the number of added cells. [Figure 7] Shows the diameters of the organoids produced with mixtures of iPS-CM and iPS-MSC in wells of different diameters and depths. [Figure 8] Shows the sphericity of the organoids produced with mixtures of iPS-CM and iPS-MSC in wells of different diameters and depths. [Figure 9] Shows the size distribution of the organoids produced by culturing mixtures of iPS-CM and iPS-MSC in well bags S500 and well bag S350. [Figure 10] Shows the size distribution of the organoids produced by culturing mixtures of iPS-CM and iPS-MSC in well bags S350 and well bag L350. [Figure 11] Shows micrographs and contraction rates of the organoids produced by culturing mixtures of iPS-CM and iPS-MSC in well bags S350 and well bag L350. [Figure 12] Micrographs of organoids produced with or without pre-culture of iPS-CM. [Figure 13] Shows the contraction speed and contraction characteristics of organoids produced with or without pre-culture of iPS-CMs. [Figure 14] Shows the cell distribution of myocardial organoids.

Mode for Carrying Out the Invention

[0010] 1. Cardiac organoids In the present specification, "myocardial organoid" refers to an artificial tissue structure that mimics the function of myocardium, and can mimic the myocardium of various organisms depending on the origin of the cells constituting the organoid.

[0011] The organism is not particularly limited and may be any organism, but is preferably a mammal, such as primates such as humans, monkeys, gorillas, chimpanzees, orangutans, rodents such as mice, rats, hamsters, guinea pigs, and dogs, cats, rabbits, cows, horses, sheep, goats, pigs, etc.

[0012] The myocardial organoid of the present application is essentially composed of cardiomyocytes derived from pluripotent stem cells and mesenchymal stem cells. Here, "essentially" means that although the majority of the cells constituting the myocardial organoid are either cardiomyocytes derived from pluripotent stem cells or mesenchymal stem cells, it cannot be excluded that the myocardial organoid contains a small number, that is, a negligible number of other cells. Examples of such other cells include cells differentiated from mesenchymal stem cells, heart-derived cells mixed during production, smooth muscle cells, fibroblasts, endothelial cells, etc., but are not limited thereto.

[0013] The origin of the mesenchymal stem cells constituting the myocardial organoid of the present application is not limited. The mesenchymal stem cells may be, for example, derived from pluripotent stem cells or cells (primary cells) collected from any living tissue. Examples of living tissue include, but are not limited to, fat, bone marrow, umbilical cord, dental pulp, etc.

[0014] Examples of pluripotent stem cells include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). In this application, the cardiomyocytes and mesenchymal stem cells derived from pluripotent stem cells may be derived from any type of pluripotent stem cell, preferably iPS cells or ES cells. When the cardiomyocyte organoid of this application includes cardiomyocytes and mesenchymal stem cells derived from pluripotent stem cells, the cardiomyocytes and mesenchymal stem cells derived from pluripotent stem cells may be derived from different types of pluripotent stem cells, or they may be derived from the same type of pluripotent stem cell.

[0015] The organism from which the pluripotent stem cells originate is not particularly limited and is appropriately selected according to the desired cardiac organoid. Examples of such organisms are as described above. Preferably, mammalian-derived pluripotent stem cells are used, more preferably human-derived pluripotent stem cells.

[0016] Pluripotent stem cell-derived cardiomyocytes may be obtained by differentiating pluripotent stem cells into cardiomyocytes, or by using established cell lines. Differentiation induction from pluripotent stem cells into cardiomyocytes may be carried out by methods known in the field, such as the method described in Biochemical and Biophysical Research Communications, 425 (2012) 321-327, or the method described in Proc Natl Acad Sci USA, 2013 Aug 27;110(35):E3281-90, but are not limited to these. Alternatively, commercially available pluripotent stem cell-derived cardiomyocytes may be used, such as iCell cardiomyocytes from FUJIFILM Cellular Dynamics.

[0017] Pluripotent stem cell-derived mesenchymal stem cells may be obtained by differentiating pluripotent stem cells into mesenchymal stem cells, or by using established cell lines. Differentiation induction from pluripotent stem cells into mesenchymal stem cells may be carried out by methods known in the field, for example, the method described in World J Stem Cells, 2021 Aug 26; 13(8): 1094-1111, etc., can be used, but is not limited to these. Alternatively, commercially available pluripotent stem cell-derived mesenchymal stem cells may be used, for example, iCell mesenchymal stem cells from FUJIFILM Cellular Dynamics.

[0018] The ratio of pluripotent stem cell-derived cardiomyocytes to mesenchymal stem cells contained in the cardiomyocyte organoid of the present invention is, for example, in the range of 1:2 to 20:1, preferably in the range of 2:1 to 10:1, and more preferably in the range of 6:4 to 10:1.

[0019] The shape of the myocardial organoids of this application is bead-like, preferably spherical or substantially spherical. The size of the myocardial organoids of this application may vary depending on the number of cells constituting the organoid, the size of the pores of the porous container used in the method for manufacturing the myocardial organoids described later, and / or the number of cells placed into the pores. The size of the myocardial organoids of this application is not limited, but examples include a diameter or maximum diameter of 80 μm to 350 μm, preferably 100 μm to 300 μm. In this specification, "maximum diameter" means the longest distance between two opposing points on the contour of the organoid when they are connected by a straight line, as observed visually or under a microscope.

[0020] The myocardial organoids of this invention exhibit high viability, with even the cells in their central region remaining viable. Viability can be measured by conventional methods; for example, after dispersing the organoids until they become single cells, it can be calculated by staining. The myocardial organoids of this invention possess the function of beating and can synchronize within myocardial tissue. The myocardial organoids of this invention have a suppressed beating rate and are mature as cardiomyocytes. Therefore, the myocardial organoids of this invention exhibit reduced variability in size and / or shape, making them stable and useful for drug discovery research.

[0021] 2. Method for manufacturing cardiac organoids The cardiomyocyte organoid of the present invention can be produced by a method (hereinafter also referred to as "the method of the present invention") which includes culturing a mixture of cardiomyocytes derived from pluripotent stem cells and mesenchymal stem cells in a porous vessel. The cardiomyocytes derived from pluripotent stem cells and mesenchymal stem cells are as described above.

[0022] Cardiomyocytes derived from pluripotent stem cells and mesenchymal stem cells may be cryopreserved cells. When using cryopreserved cardiomyocytes and mesenchymal stem cells derived from pluripotent stem cells, they should be thawed by an appropriate method before use. The thawing method may be any method known in the art, for example, by using an incubator or water bath at approximately 37°C until the ice particles become small.

[0023] The present invention provides a method for producing cardiac organoids from a mixture of pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells. Conventionally, attempts to produce cardiac organoids using only cardiomyocytes have not yielded cell aggregates of sufficient size. However, the present invention, by using mesenchymal stem cells in addition to cardiomyocytes, has succeeded in producing cardiac organoids of sufficient size. The mixing ratio of pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells is, for example, in the range of 1:1 to 20:1, preferably in the range of 5:1 to 20:1, and more preferably about 10:1. The higher the proportion of mesenchymal stem cells in the mixture, the more cell aggregation is promoted, and the less variation there is in the diameter and sphericity of the resulting organoids. However, the above mixing ratio is appropriate from the viewpoint of the organoid's ability to mimic cardiac muscle.

[0024] The mixing of pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells may be performed in each pore of the porous vessel used for culture, or the pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells may be mixed first and then the mixture placed in the porous vessel.

[0025] Pluripotent stem cell-derived cardiomyocytes and / or mesenchymal stem cells may be pre-cultured before mixing. Preferably, pluripotent stem cell-derived cardiomyocytes are pre-cultured. In particular, when using cryopreserved cells, it is preferable to pre-culture the frozen pluripotent stem cell-derived cardiomyocytes after thawing. For example, pluripotent stem cell-derived cardiomyocytes that have been pre-cultured after thawing may be mixed with mesenchymal stem cells that have been thawed as is (without pre-culture).

[0026] The culture medium used for pre-culture is not particularly limited, as long as it contains the components necessary for cell proliferation and / or maintenance. For example, basal media used in animal cell culture may be used, and examples of such basal media include, but are not limited to, BME (Basal medium Eagle's) medium, IMDM (Iscove's Modified Dulbecco's) medium, Medium 199 medium, Eagle MEM medium, α MEM medium, DMEM (Dulbecco's Modified Eagle's) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, etc., and mixed media of these. Substances such as fetal bovine serum, fetal calf serum, human platelet lysate, or human serum albumin may be added to the medium. The medium may be either serum-containing or serum-free. In addition, liquid or solid culture media are typically used.

[0027] The pre-culture period should be at least two days, for example, 2 to 7 days, preferably 2 to 5 days. The culture temperature should be, for example, about 25 to 37°C. The above and other culture conditions can be set as appropriate by those skilled in the art. Pre-culture may be performed by suspension culture or static culture.

[0028] In this specification, "porous container" means a culture container having multiple pores (also called wells). The shape of the container is not particularly limited and may be any shape that can be used for cell culture, such as plates, bags, dishes, or petri dishes. The shape of the pores is not particularly limited, but examples of top surface shapes include circular, elliptical, square, and rectangular, and examples of bottom shapes include U-bottom, V-bottom, or flat bottom. Among these, U-bottom or V-bottom pores are preferred. The surface of the pores is preferably coated with a cell-non-adherent coating agent, such as a polymer such as hydrogel or lipid, but may also be coated with a coating agent such as collagen, fibronectin, or laminin, which are cell adhesion substances.

[0029] The number and size of pores in the container are not particularly limited and can be appropriately selected according to the desired quantity and size of organoids. In the method of this invention, one organoid is produced per pore, so the size of the organoid can be adjusted by the number of cells introduced into each pore. That is, to produce larger organoids, a porous container with larger pores should be used. The size of the pore is determined by the diameter or maximum diameter of the pore surface (hereinafter also referred to as "pore diameter"), the depth of the pore, the shape of the pore bottom, etc. However, when the same number of cells are introduced into pores of the same depth and bottom shape, differences in pore diameter do not significantly affect the size of the resulting organoids. On the other hand, if the pores are shallow, cells or organoids may move between pores during culture (for example, entering adjacent pores), and organoids of uniform size and shape cannot be obtained. Therefore, although it depends on the number of cells introduced (i.e., the desired size of the organoids), pores with a depth of at least about 100 μm are preferable. The pore size can be appropriately selected depending on the number of cells to be introduced, but examples include approximately 200 μm to approximately 500 μm. Various commercially available porous containers can be used in the method of this application. For example, although not limited to these, U-bottom well plates (e.g., 96 U-bottom well plates), V-bottom well plates, microwell plates, well bags, or well dishes, such as EZspehre®, Elplasia®, CELLSOLUT®, etc. may be used.

[0030] The number of cells to be placed in each pore of a porous vessel can be appropriately selected depending on the desired organoid size and is not particularly limited. For example, although not limited, approximately 50 to 3000 cells may be placed in each pore as the total number of pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells. Preferably, approximately 100 to 2000, 100 to 1500, or 100 to 1000 cells may be placed in each pore, and more preferably, approximately 200 to 500 cells may be placed.

[0031] Culture of a mixture of pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells in a porous vessel should be performed by static culture to prevent migration of cells or organoids between pores.

[0032] The culture medium used for culturing a mixture of pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells is not particularly limited, as long as it contains the components necessary for cell proliferation and / or maintenance. For example, a basal medium used for animal cell culture may be used, and examples of such basal media include, but are not limited to, BME (Basal medium Eagle's) medium, IMDM (Iscove's Modified Dulbecco's) medium, Medium 199 medium, Eagle MEM medium, α MEM medium, DMEM (Dulbecco's Modified Eagle's) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, etc., and mixed media of these. Substances such as fetal bovine serum, fetal calf serum, human platelet lysate, or human serum albumin may be added to the medium. The culture medium may be either a serum-containing medium or a serum-free medium. Preferably, a liquid medium is used.

[0033] The culture period is not particularly limited and can be any period necessary for organoid formation. Organoid formation can be confirmed by observing, either visually or under a microscope, the formation of a single cell aggregate in each pore that is proportional in size to the number of cells added, and the clear outline of the cell aggregate. In the method of this application, organoids are usually formed after culturing for at least two days. Furthermore, culturing may be continued even after organoid formation. Therefore, examples of culture periods include two days or more, for example, 2 to 14 days, preferably 2 to 7 days. The culture temperature is, for example, about 25 to 37°C. The above and other culture conditions can be appropriately set by those skilled in the art.

[0034] Thus, by the method of the present invention, multiple myocardial organoids having uniform size and uniform properties (e.g., pulsating function) can be manufactured. The manufactured myocardial organoids can be stored at low temperatures until use. Examples of storage temperatures include approximately 4°C to 37°C.

[0035] Furthermore, terms used in this specification should be interpreted in the sense commonly used in the art. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. In this specification, the term “about” is to be understood by those skilled in the art and is subject to some degree of variation depending on the context in which it is used. “About” typically means a number within a range of ±10%, more typically ±5%, more typically ±4%, more typically ±3%, more typically ±2%, and even more typically ±1% of the number to which the term is attached. [Examples]

[0036] Example 1: Production of cardiac organoids at different mixing ratios Frozen stocks of iPS cell-derived cardiomyocytes (hereinafter referred to as iPS-CM), obtained by differentiation induction from iPS cells, were thawed in a 37°C water bath and suspended in DMEM medium containing 10% FBS. Next, frozen iPS cell-derived mesenchymal stem cells (hereinafter referred to as iPS-MSC), obtained by differentiation induction from iPS cells, were thawed in a 37°C water bath and suspended in DMEM medium containing 10% FBS. Then, the iPS-CM:iPS-MSC mixtures were seeded into EZsphere (6-well plates; AGC Technoglass Co., Ltd.) in mixed ratios of iPS-CM:iPS-MSC = 1:0, 20:1, 10:1, 5:1, 2:1, and 1:1, with a total cell count of 200, and cultured at 37°C for 2 days. After 2 days of static culture, the formation of spherical organoids in each well was confirmed, and the diameter and sphericity of the formed organoids were measured using Cell3iMager duos (SCREEN Co., Ltd.), and the average values ​​were calculated.

[0037] The results are shown in Table 1. Furthermore, graphs created from the obtained results are shown in Figures 1 and 2, respectively. From these results, it was found that organoid size is larger and variation is reduced when iPS-CM is mixed with iPS-MSC, and sphericity is also improved at mixing ratios up to 2:1. Note that a sphericity value closer to 1 indicates that the organoid is closer to a sphere.

[0038] [Table 1]

[0039] Furthermore, the contraction velocity and heart rate of the obtained organoids were measured using the SI8000 cell motion imaging system (Sony Corporation). The measurement results are shown in Figure 3. As is clear from Figure 3, when iPS-CM and iPS-MSC were mixed, the contraction velocity of the myocardial organoids increased and the heart rate decreased, indicating the maturation of myocardial cells. When the mixing ratio of MSCs was 1:1, the heart rate decreased further, while the contraction velocity remained unchanged, suggesting that MSCs became dominant and hindered contraction.

[0040] Organoids prepared using different mixing ratios of iPS-CM and iPS-MSC were further subjected to RNA extraction using the RNeasy Mini Kit (QIAGEN), followed by cDNA synthesis. Real-time PCR was then performed using the QuantStudio 5 System (Thermo Fisher Scientific) to measure MYH6, MYH7, TNNI1, and TNNI3, which are markers of myocardial maturation. The results are shown in Figure 4. First, regarding the expression of immature myocardial MYH6 and mature myocardial MYH7, compared with organoids prepared using only iPS-CM, it was confirmed that the relative expression level of mature myocardial MYH7 was increased in organoids prepared with iPS-CM:iPS-MSC mixing ratios of 20:1, 10:1, 5:1, or 2:1. Similarly, regarding the expression of immature myocardial TNNI1 and mature myocardial TNNI3, mixing with iPS-MSC increased the relative expression level of mature myocardial TNNI3. Therefore, the maturation of iPS-CMs, which are a mixture of iPS-MSCs, was also confirmed by the expression of marker genes.

[0041] Example 2: Production of cardiac organoids with different cell counts Frozen stocks of iPS-CM and iPS-MSC were thawed in a 37°C water bath and suspended in 10% FBS-containing DMEM medium. Next, these were added to each well of a 96-well U-bottom plate and an EZsphere (35mm dish, approximately 2700 wells; AGC Technoglass Co., Ltd.) in a cell number ratio of iPS-CM:iPS-MSC = 10:1, resulting in total cell counts of 100, 200, 500, 1000, or 2000. Each well contained 100 μL of 10% FBS-containing DMEM medium. The well plates and dishes were incubated at 37°C. After 2 days of static culture, the formation of spherical organoids in each well was confirmed. The size (diameter or maximum diameter) of the formed organoids was then measured under a microscope, and the average value was calculated.

[0042] The results are shown in Table 2. Furthermore, micrographs of the obtained organoids and the relationship between organoid size and the number of added cells are shown in Figures 5 and 6, respectively. These results show that the size of the produced organoids can be adjusted by changing the number of cells used.

[0043] [Table 2]

[0044] Example 3: Production of myocardial organoids in vessels with different diameters and depths EZsphere (custom-made 6-well plate, AGC Technoglass Co., Ltd.) was used as a porous vessel with different bore sizes. Frozen iPS-CM and iPS-MSCs were added to each well of the EZsphere in a cell number ratio of 10:1, resulting in a total of 200 cells. Each well contained 2 mL of 10% FBS-containing DMEM medium. After static incubation at 37°C for 2 days, the formation of spherical organoids in each well was confirmed. Next, the diameter and sphericity of the formed organoids were measured using Cell3iMager duos (SCREEN Corporation), and the average values ​​were calculated.

[0045] The results are shown in Table 3. Furthermore, graphs created from the obtained results are shown in Figures 7 and 8. From these results, it was found that in wells with a diameter exceeding 1000 μm for a total cell count of 200, there was a large variability, and stable organoid production was not possible.

[0046] [Table 3]

[0047] Example 4: Production of organoids using porous vessels with different pore sizes Wellbag S500 (500 μm well diameter, approximately 18,000 wells; manufactured by Toyo Seikan Co., Ltd.) and Wellbag S350 (350 μm well diameter, approximately 32,000 wells; manufactured by Toyo Seikan Co., Ltd.) were used as porous containers. Frozen iPS-CMs were thawed at 37°C. Frozen iPS-MSCs were thawed at 37°C. Then, iPS-CMs and iPS-MSCs were added to each well of the Wellbag in a cell number ratio of iPS-CM:iPS-MSC = 10:1, with a total cell count of 200 cells. Each Wellbag contained 20 mL of 10% FBS-containing DMEM medium. After static incubation at 37°C for 2 days, the formation of spherical organoids in each well was confirmed. Then, the size (diameter or maximum diameter) of the formed organoids was measured under a microscope, and the average value was calculated.

[0048] The results are shown in Table 4. Furthermore, Figure 9 shows the size distribution of organoids produced using Wellbag S500 and Wellbag S350. As is clear from Figure 9, the difference in well diameter between 350 μm and 500 μm did not result in a significant difference in the size of the resulting organoids.

[0049] [Table 4]

[0050] Example 5: Organoid production using a scaled-up porous vessel Two types of well bags of different sizes were used as porous containers: Well Bag S350 (350 μm well diameter, approximately 32,000 wells; manufactured by Toyo Seikan Co., Ltd.) and Well Bag L350 (350 μm well diameter, approximately 680,000 wells; manufactured by Toyo Seikan Co., Ltd.). Frozen iPS-CMs were thawed at 37°C. Frozen iPS-MSCs were thawed at 37°C. Then, iPS-CMs and iPS-MSCs were added to each well of the well bag in a cell number ratio of iPS-CM:iPS-MSC = 10:1, so that the total number of cells was 200. S350 contained 20 mL of 10% FBS-containing DMEM medium per well bag, and L350 contained 400 mL of 10% FBS-containing DMEM medium per well bag. After static incubation at 37°C for 2 days, it was confirmed that spherical organoids had formed in each well. Next, the size (diameter or maximum diameter) of the formed organoids was measured under a microscope, and the average value was calculated. Then, the organoid contraction rate was measured using the Cell Motion Imaging System SI8000 (manufactured by Sony). Note that the overall size of the Wellbag L350 container is approximately 20 times larger than that of the Wellbag S350.

[0051] The results are shown in Table 5. Furthermore, Figure 10 shows the size distribution of organoids cultured and produced in Wellbag S350 and Wellbag L350. In addition, Figure 11 shows micrographs of the organoids and their shrinkage rates. As is clear from these results, there was no significant difference in organoid size even when the size of the porous container was different.

[0052] [Table 5]

[0053] Example 6: Comparison of organoids with and without pre-culture Differentiation-induced iPS-CMs and iPS-MSCs were used. Well bags (approximately 18,000 wells; manufactured by Toyo Seikan Co., Ltd.) were used as porous vessels. Frozen iPS-CMs were thawed at 37°C and then pre-cultured in 20% FBS-containing DMEM medium at 37°C for 2 days, or used without pre-culture. Next, frozen iPS-MSCs were thawed at 37°C and added to each well of the well bag along with the iPS-CMs, in a cell number ratio of iPS-CM:iPS-MSC = 10:1, resulting in a total cell count of 3.6E+06. Each well contained 15 mL of 10% FBS-containing DMEM medium. After static culture at 37°C for 2 days, the formation of spherical organoids in each well was confirmed.

[0054] As a result, micrographs of the fabricated organoids are shown in Figure 12. As is clear from Figure 12, the size of the organoids obtained when iPS-CM was pre-cultured was larger than when iPS-CM was used without pre-culture. Furthermore, when iPS-CM was pre-cultured, the outlines of the obtained organoids were clearer than when iPS-CM was used without pre-culture, indicating that the cells had sufficiently aggregated to form organoids.

[0055] Example 7: Comparison of organoid performance with and without pre-culture and with different cell mixing ratios Differentiation-induced iPS-CMs and iPS-MSCs were used. Wellbags (approximately 18,000 wells; manufactured by Toyo Seikan Co., Ltd.) were used as porous vessels. Frozen iPS-CMs were thawed at 37°C and then pre-cultured in 20% FBS-containing DMEM medium at 37°C for 2 days, or used without pre-culture. Next, frozen iPS-MSCs were thawed at 37°C and added to each well of the wellbag along with the iPS-CMs in cell number ratios of iPS-CM:iPS-MSC = 10:1, 20:1, and 40:1, so that the total number of cells was 3.6E+06. Each well contained 15 mL of 10% FBS-containing DMEM medium. After static culture at 37°C for 2 days, the formation of spherical organoids in each well was confirmed. Subsequently, the contraction rate and pulsation rate of the organoids were measured using the SI8000 Cell Motion Imaging System (manufactured by Sony Corporation).

[0056] The results are shown in Figure 13. As is clear from Figure 13, when pre-cultured iPS-CMs were used, the contraction speed of the myocardial organoids increased and the heart rate decreased, indicating maturation. On the other hand, when the mixing ratio of MSCs decreased, the heart rate increased, indicating that the organoids became more immature.

[0057] Example 8: Cell distribution of the fabricated organoids Wellbag S350 (350 μm well diameter, approximately 32,000 wells; manufactured by Toyo Seikan Co., Ltd.) was used as a porous container. Frozen iPS-CMs were thawed at 37°C. Frozen iPS-MSCs were thawed at 37°C. Then, iPS-CMs and iPS-MSCs were added to each well of the wellbag in a cell number ratio of iPS-CM:iPS-MSC = 1:0 or 10:1, so that the total number of cells was 200. Each wellbag contained 20 mL of 10% FBS-containing DMEM medium. After static culture at 37°C for 2 days, the harvested organoids were separated into single cells by enzymatic dispersion with collagenase, and then fluorescently stained with cTnT (cardiac troponin), a cardiomyocyte marker, and CD73, a mesenchymal stem cell or fibroblast marker, and then evaluated by FACS (fluorescence-activated cell sorting). The obtained cell distribution results are shown in Figure 14.

[0058] In cardiac spheres produced using iPS-CM:iPS-MSC=1:0, 91.8% of cells were cTnT-positive. In contrast, in cardiac organoids produced using iPS-CM:iPS-MSC=10:1, 75.4% of cells were cTnT-positive. CD73-positive cells were present in 2.55% of cardiac spheres and 18.2% of cardiac organoids. From this, it was confirmed that the cardiac organoids of this invention contained approximately 15% MSCs that had proliferated by 2 days of culture, and also contained a small amount of fibroblasts. In addition, approximately 5% of the cells were double-negative, meaning they did not react to either marker.

[0059] Example 9: Manufacturing efficiency of cardiac organoids Myocardial organoids were prepared using the same method as in Example 5. However, a mixing ratio of iPS-CM:iPS-MSC = 10:1 was used, and the total number of cells used for organoid preparation was 1.28E+07. The prepared myocardial organoids were collected, and the number of organoids was calculated by measuring a portion of the collected organoids under a microscope. Next, the collected organoids were separated into single cells by enzymatic dispersion with collagenase, and the number of cells per organoid and cell viability were calculated by measuring the cells. Furthermore, the number of recovered cells was calculated from the number of recovered organoids and the number of cells per organoid, and the recovery rate was calculated. The results are shown in Table 6.

[0060] [Table 6]

[0061] It has been found that cardiac organoids can be efficiently produced according to the method of the present invention.

Claims

1. A method for producing cardiac organoids, comprising culturing a mixture containing cardiomyocytes derived from induced pluripotent stem cells and mesenchymal stem cells derived from induced pluripotent stem cells in a ratio of 2:1 to 20:1 in a porous vessel.

2. The method according to claim 1, wherein the cardiomyocytes derived from induced pluripotent stem cells are cells that have been thawed and pre-cultured at 25-37°C for two days or more after the cryopreservation of induced pluripotent stem cell-derived cardiomyocytes.

3. The method according to claim 1, wherein the porous container is a plate, a bag, or a dish.

4. A method for producing a cardiac organoid comprising the following steps, wherein a bead-shaped cardiac organoid having a diameter or maximum diameter of 80 μm to 350 μm is produced:

1. A step of pre-culturing induced pluripotent stem cell-derived cardiomyocytes at 25-37°C.

2. A step of mixing the pre-cultured cardiomyocytes and mesenchymal stem cells derived from induced pluripotent stem cells in a ratio of 5:1 to 20:

1.

3. The step of placing the cell mixture into each pore of a porous container so that the number of cells is between 200 and 500.

4. A step in which cells placed in a porous vessel are cultured at 25-37°C for 2-14 days.

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