Organoid for treating heart disease and method for producing same

Abstract

Provided is an organoid for treating heart disease. In the organoid, myocardial cells, mesenchymal stem cells, and optionally other cells are self-organized in a cooperative manner. Provided is a method for producing an organoid, the method comprising seeding and adhering individually dissociated myocardial cells 14 on a scaffold membrane 16 comprising molecules constituting a basement membrane, seeding individually dissociated mesenchymal stem cells 15 on the myocardial cells 14 adhered on the scaffold membrane 16, and culturing the myocardial cells 14 together with the mesenchymal stem cells 15 in a medium, wherein the number of the mesenchymal stem cells 15 is smaller than the number of the myocardial cells 14.

Description

Organoids for the treatment of heart disease and methods for producing same The present invention relates to organoids for the treatment of cardiac diseases and methods for their production. Paragraph of Patent Document 1

[0039] discloses the creation of cell sheets by mixing myoblasts and mesenchymal stem cells in a 4:1 ratio and seeding them on culture dishes coated with poly-N-isopropylacrylamide, a temperature-responsive polymer. Paragraph of Patent Document 2

[0058] discloses that a cell layer of skeletal muscle cells is formed on a Pluronic® hydrogel sheet, and a cell layer of skeletal muscle cells is formed on an alginate hydrogel sheet, so that the cell layers are in contact with each other. Paragraph of Patent Document 3

[0008] discloses repeatedly casting layer by layer of a mixture containing cardiomyocytes, non-cardiomyocytes, and collagen. Non-Patent Document 1 discloses a 3D prevascularized stem cell patch fabricated by stacking striped layers of bioinks made of cardiac progenitor cells and mesenchymal stem cells in a grid pattern. In Non-Patent Document 2, mesenchymal stem cells and cardiomyocytes are co-cultured on an amniotic membrane bilayer to prepare a cardiac patch. The entire contents of Patent Documents 1 to 3 and Non-Patent Documents 1 and 2 are incorporated herein by reference. International Publication No. 2011 / 058813 Special Publication No. 2021-531807 Special Publication No. 2023-546466 Jinah Jang, Hun-Jun Park, Seok-Won Kim, Heejin Kim, Ju Young Park, Soo Jin Na, Hyeon Ji Kim, Moon Nyeo Park, Seung Hyun Choi, Sun Hwa Park, Sung Won Kim, Sang-Mo Kwon, Pum-Joon Kim, Dong-Woo Cho, 3D printed complex tissue construct using stem cell-laden decellularized extracellular matrix bioinks for cardiac repair, Biomaterials, Volume 112, 2017, Pages 264-274. [retrieved on 2024-04-12]. Retrieved from <URL: https: / / doi.org / 10.1016 / j.biomaterials.2016.10.026> Sandora, N., Putra, M.A., Busro, P.W. et al. Preparation of Cell-Seeded Heart Patch In Vitro; Co-Culture of Adipose-Derived Mesenchymal Stem Cell and Cardiomyocytes in Amnion Bilayer Patch. Cardiovasc Eng Tech 13, 193-206 (2022). [retrieved on 2024-04-12]. Retrieved from <URL: https: / / doi.org / 10.1007 / s13239-021-00565-4> Discovery of the most effective mature stage of cardiomyocytes for transplantation: Improving the transplant efficacy of cardiomyocytes differentiated from human iPS cells | News | News & Events | CiRA | Center for iPS Cell Research and Application, Kyoto University (kyoto-u.ac.jp), 2016-01-08 [retrieved on 2024-08-02]. Retrieved from<URL: https: / / www.cira.kyoto-u.ac.jp / j / pressrelease / news / 160108-190000.html> Human stem cell culture supernatant | Column: The importance of cytokines and the "paracrine effect" (medrt.com), 2020-07-27 [retrieved on 2024-08-02].<URL: https: / / stemcell.medrt.com / human-stem-cell-culture-supernatant4 / > The present invention provides organoids for the treatment of cardiac diseases that can provide highly effective treatment. <1> Within the organoid, cardiomyocytes, mesenchymal stem cells, and optionally other cells cooperate and self-organize, Organoids. <2> The organoid described above having a heart wall-like morphology. <3> The organoid described above having a patch-like morphology. <4> Cardiomyocytes, mesenchymal stem cells, and optionally other cells are included, The number of cells contained in organoids is highest among cardiomyocytes. Organoids as described above. <5> The cardiomyocytes, the mesenchymal stem cells, and the other cells cooperate to self-organize, The organoid described above, wherein the other cells are at least one of fibroblasts, endothelial cells, and smooth muscle cells. <6> The method is produced by seeding cardiomyocytes, mesenchymal stem cells, and optionally other cells on a plate coated with laminin, Here, cardiomyocytes are seeded followed by mesenchymal stem cells. Organoids as described above. <7> The method is produced by seeding cardiomyocytes, mesenchymal stem cells, and optionally other cells on a plate coated with laminin, The cell number ratio of the seeded cardiomyocytes to mesenchymal stem cells is greater than 4:1 and less than 20:1; Organoids as described above. <8> Repeating contraction and expansion autonomously. Organoids as described above. <9> For the treatment of heart disease, Organoids as described above. <10> For transplantation into the heart, Organoids as described above. <11> For transplantation into areas of the heart that have lost blood flow, Organoids as described above. <12> A method for producing organoids, Cardiomyocytes dispersed in each other are seeded and attached onto a scaffold membrane made of molecules constituting a basement membrane, Seeding mesenchymal stem cells dispersed among each other on the cardiomyocytes adhered to the scaffold membrane; Cardiomyocytes are cultured in a medium together with mesenchymal stem cells, the number of said mesenchymal stem cells is less than the number of said cardiomyocytes, The organoid is for treating cardiac disease, and cardiomyocytes, mesenchymal stem cells, and optionally other cells cooperate and self-organize within the organoid. method. <13> The molecule constituting the basement membrane is laminin. The above method. <14> The molecule constituting the basement membrane is laminin, The unit area (cm) of the scaffold membrane ２ ) weight of 0.05 to 2 μg; The above method. <15> The molecule constituting the basement membrane is laminin, The unit area (cm) of the scaffold membrane ２) weighing between 0.05 and 2 μg; the concentration of serum in the medium is 1 to 20 (vol / vol%); The above method. <16> The mixing ratio of cardiomyocytes and mesenchymal stem cells at the time of seeding is greater than 4:1 and less than 20:1, The length of the culture period after seeding the mesenchymal stem cells does not exceed 48 hours. The above method. <17> The cell number ratio of cardiomyocytes to mesenchymal stem cells at the time of seeding is greater than 4:1 and less than 20:1, The length of the culture period after seeding the mesenchymal stem cells does not exceed 48 hours. The above method. The present invention provides organoids for the treatment of cardiac diseases that can provide highly effective treatment. Schematic diagram of cell sheet Schematic diagram of cell sheet production 1 Schematic diagram of cell sheet production 2 Stained image of cell sheet Cytokines produced by cell sheet Outline of Aorta ring assay Statistics of Aorta ring assay Periodic changes in contraction speed of cell sheet Maximum contraction speed and contraction synchronization of cell sheet (correlation) Survival rate of cell sheet transplanted into the heart Changes in ejection fraction (EF) value by echocardiography up to 8 weeks after transplantation <Organoids and the Paracrine Effect> One aspect of this embodiment is a three-dimensional structure (organoid) that mimics the heart and its tissues for the treatment of cardiac disease. Within the organoid, cardiomyocytes, mesenchymal stem cells, and optionally other cells cooperate to self-organize. In one aspect, the self-organization of these cells allows the organoid to reproduce some of the functions of the heart. Examples of some of the functions of the heart include contractility and vascular induction. The organoid may have a morphology resembling the heart wall (heart wall). This embodiment will be described below using an organoid in the form of a patch as an example. Another aspect of this embodiment relates to manipulating cardiomyocytes and mesenchymal stem cells so that these cells cooperate to self-organize. Organoids according to one aspect of this embodiment comprise cardiomyocytes, mesenchymal stem cells, and optionally other cells. The other cells may be at least one of fibroblasts, endothelial cells, and smooth muscle cells. In one aspect, cardiomyocytes are the most abundant cells in the organoids. In one aspect, mesenchymal stem cells are the second most abundant cells in the organoids. In another aspect, mesenchymal stem cells are the second most abundant cells after other cells, such as smooth muscle cells. In one aspect, the other cells are present in fewer amounts than the cardiomyocytes and mesenchymal stem cells. One aspect of this embodiment is to create organoids, which have traditionally been used as experimental models in drug discovery and the like, for therapeutic use using the paracrine effect. One interpretation of the paracrine effect in this embodiment is that factors secreted by other cells have an effect on target cells. For example, this applies when some substance secreted by cells transplanted into a transplant recipient affects the recipient's original cardiomyocytes and restores their function. In one aspect of this embodiment, a high therapeutic effect is achieved by using organoids composed of multiple cells, such as a mix of cardiomyocytes and mesenchymal stem cells. This high therapeutic effect is brought about by the cytokine production ability of organoids, which leads to the paracrine effect. While the paracrine effect itself is a phenomenon, organoids composed of multiple cell types such as those described above can be used as transplant materials or devices, i.e., as objects themselves. Furthermore, the contractile properties, angiogenic ability, and other functions of organoids further enhance the therapeutic effect. The therapeutic effect of organoids is higher than that of transplant materials produced from a single cell type, such as cardiomyocytes. In one aspect of this embodiment, the characteristics of both cardiomyocytes and mesenchymal stem cells are expressed without compromising each other. Furthermore, by mixing cardiomyocytes and mesenchymal stem cells with each other, these cells are tightly organized within the organoid. Therefore, the organoid of this embodiment provides a paracrine effect. The organoids in this embodiment may be interpreted as artificially produced. The organoids in this embodiment have physiological functions similar to those of tissues extracted from a living body. However, the organoids in this embodiment are artificial structures produced according to the production method exemplified below, for example. The organoids may not yet have blood vessels formed, or blood vessels may already have been formed. One aspect of this embodiment is a transplantation material or device containing organoids for the treatment of cardiac disease that provide a high therapeutic effect. Another aspect of this embodiment is the production of organoids for the treatment of cardiac disease that provide a high therapeutic effect. In one example, cardiomyocytes and mesenchymal stem cells are seeded at a specific ratio onto a scaffold membrane formed by applying laminin to a plate. Organoids formed by this method contain a mixture of areas rich in cardiomyocytes or high in cardiomyocyte density and areas rich in mesenchymal stem cells or high in mesenchymal stem cell density. At least one of these areas may contain other types of cells. In one aspect of this embodiment, the organoids produced by the production method of the above example may have a patch-like shape. However, the shape of the organoids is not limited to a patch-like shape. Furthermore, the method for producing organoids of this embodiment is not limited to the above example. The types of cells that can be combined in the production of organoids of this embodiment are not limited to cardiomyocytes and mesenchymal stem cells. Cardiomyocytes and mesenchymal stem cells may be prepared by induction from pluripotent stem cells such as iPS cells and ES cells. Furthermore, any of fibroblasts, endothelial cells, and smooth muscle cells may be mixed into the population of cells prepared as cardiomyocytes or mesenchymal stem cells. Below, as a non-limiting example of organoids for treating cardiac disease in this embodiment, a patch of organoids provided as a cell sheet will be described. <Cell sheet> FIG. 1 shows an artificial cell sheet 10 according to an example of the form of an organoid patch of this embodiment. The cell sheet 10 has a first layer 11 and a second layer 12. The first layer 11 is laminated on the second layer 12. The first layer 11 may be in direct contact with the second layer 12. The first layer 11 is composed of cardiomyocytes 14. In one aspect, the first layer 11 contains cardiomyocytes but does not contain mesenchymal stem cells. The second layer 12 is composed of mesenchymal stem cells 15. In one aspect, the second layer 12 contains mesenchymal stem cells but does not contain cardiomyocytes. 1 shows that the number of mesenchymal stem cells 15 contained in the second layer 12 is smaller than the number of cardiomyocytes 14 contained in the first layer 11. Even when mesenchymal stem cells 15 are contained in the first layer 11 or when cardiomyocytes 14 are contained in the second layer 12, the number of mesenchymal stem cells 15 contained in the first layer 11 and the second layer 12 is smaller than the number of cardiomyocytes 14 contained in the first layer 11 and the second layer 12. In the total number of cardiomyocytes 14 and mesenchymal stem cells 15 in the first layer 11 and the second layer 12, the ratio of the number of cardiomyocytes 14 to the number of mesenchymal stem cells 15 is preferably greater than 4:1 and less than 20:1. In the cell sheet 10 shown in FIG. 1 , it is preferable that the cardiomyocytes 14 and mesenchymal stem cells 15 are in direct contact with each other. It is preferable that the cardiomyocytes 14 and mesenchymal stem cells 15 are not separated at the boundary between the first layer 11 and the second layer 12. From this perspective, the first layer 11 and the second layer 12 may be partially reversed. There may be areas where the cardiomyocytes 14 and mesenchymal stem cells 15 are well mixed between the first layer 11 and the second layer 12, with no difference in density between them. Furthermore, at least one of the first layer 11 and the second layer 12 may be partially missing. Furthermore, at least one of the first layer 11 and the second layer 12 may be partially thickened. In this way, the cell sheet 10 may have some imperfections. The cell sheet 10 shown in FIG. 1 may contain cell types other than cardiomyocytes 14 and mesenchymal stem cells 15. The first layer 11 may also contain mesenchymal stem cells or other cell types. However, the most abundant cells in the first layer 11 are cardiomyocytes 14. The second layer 12 may also contain cardiomyocytes or other cell types. However, the most abundant cells in the second layer 12 are mesenchymal stem cells 15. An example of the above cell types is endothelial cells, such as vascular endothelial cells. Another example is fibroblasts, such as myofibroblasts. Another example is smooth muscle cells, such as myofibroblasts. The other cell types may be mixed in a population of cardiomyocytes 14 or mesenchymal stem cells 15 when these cells are prepared in advance. 1 shows that the cell sheet 10 may have a scaffold membrane 16. The scaffold membrane 16 is located on the second layer 12. The scaffold membrane 16 is located on one of both surfaces of the second layer 12, facing the first layer 11. The scaffold membrane 16 may be located on the first layer 11. The scaffold membrane 16 may be located on one of both surfaces of the first layer 11, facing the second layer 12. In one aspect, the scaffold membrane 16 is made of molecules that constitute a basement membrane, such as laminin. 1 shows that a cell sheet 10 autonomously repeats contraction and extension. As shown in the lower panel, the cell sheet 10 contracts along the direction of extension of the cell sheet 10. As shown in the upper panel, the cell sheet 10 extends along the direction of extension of the cell sheet 10. The contraction and extension of the cell sheet 10 may be temporarily stopped by any chemical treatment. 1 shows an embodiment in which blood vessels have not yet formed within the cell sheet 10. In other embodiments, blood vessels may be further formed within the cell sheet 10. In FIG. 1 , regardless of whether or not contraction and expansion occur, or whether or not blood vessels are formed, the cell sheet 10 can be suitably used as an artificial myocardial patch for transplantation into the heart. In this embodiment, the myocardial patch may be simply referred to as a patch. When organoids in the form of a patch are transplanted into the heart, the cell sheet 10 is transplanted into the heart with the first layer 11 or the second layer 12 facing the heart. The cell sheet 10 may be transplanted into a site in the heart that has lost blood flow. New blood vessels may be generated on the transplanted cell sheet 10. 1, regardless of the presence or absence of contraction and expansion, or the presence or absence of blood vessel formation, the effect of a drug on the myocardium can be evaluated by contacting the cell sheet 10 with the drug. The cell sheet 10 is useful for developing drugs to prevent and / or treat myocardial diseases, such as myocardial infarction. The above is an example of the use of the cell sheet 10 shown in Figure 1. The use of the cell sheet is not limited. <Production of cell sheets> Figure 2 shows an example of a method for producing a cell sheet. The upper part of the figure shows seeding of cardiomyocytes 14 dispersed among one another onto a scaffold membrane 16. The scaffold membrane 16 is applied to the bottom of a culture vessel 19. In one embodiment, the scaffold membrane 16 is made of molecules that constitute basement membranes, such as laminin. The seeded cardiomyocytes 14 settle in a culture medium 17. In the culture medium 17, the cardiomyocytes 14 adhere to the scaffold membrane 16. This forms a first layer 11 composed of cardiomyocytes 14, as shown in the middle part. In FIG. 2, the unit area (cm ２ The weight per 1000 mg / kg body weight may be 0.01 to 10 μg, and may be any of 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 μg. 2 shows that mesenchymal stem cells 15 are further seeded dispersedly on the scaffold membrane 16 to which the cardiomyocytes 14 are attached. After seeding, the cardiomyocytes 14 are cultured together with the mesenchymal stem cells 15 in a medium 18. The mesenchymal stem cells 15 migrate through the gaps between the cardiomyocytes 14 in the first layer 11. 2 shows that a second layer 12 made of mesenchymal stem cells 15 is formed on the scaffold membrane 16. In this way, the second layer 12 is formed between the scaffold membrane 16 and the first layer 11. As a result, the first layer 11 is laminated on the second layer 12. Figure 3 shows another example of a method for producing a cell sheet. In the upper part of Figure 3, cardiomyocytes 14 are seeded dispersedly on a scaffold membrane 16, as in the upper part of Figure 2. In the middle part of Figure 3, mesenchymal stem cells 15 are further seeded dispersedly on the scaffold membrane 16 to which the cardiomyocytes 14 have adhered. After seeding, the cardiomyocytes 14 are cultured together with the mesenchymal stem cells 15 in a medium 18. The mesenchymal stem cells 15 settle on the first layer 11. In one example, the mesenchymal stem cells 15 do not migrate through the gaps between the cardiomyocytes 14. The lower part of Fig. 3 shows that a second layer 12 made of mesenchymal stem cells 15 is formed on the first layer 11 on the scaffold membrane 16. In this way, a structure is formed in which the first layer 11 is sandwiched between the scaffold membrane 16 and the second layer 12. As a result, the second layer 12 is laminated on the first layer 11. 2 and 3, the serum concentration (volume fraction) in medium 18 may be 0.1 to 50 (volume / volume %), or may be any of 0.2, 0.5, 1, 5, and 20 (volume / volume %). Note that in the upper part of Fig. 2, medium 17 may have the same composition as medium 18. 2 and 3, the number of seeded mesenchymal stem cells 15 is smaller than the number of seeded cardiomyocytes 14. The cell number ratio between seeded cardiomyocytes 14 and seeded mesenchymal stem cells 15 may be greater than 1:1 and less than 100:1. The cell number ratio may be any of 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, 10:1, 20:1, 30:1, 40:1, 50:1, and 60:1. 2 and 3, the length of the culture period after seeding of the mesenchymal stem cells 15 is preferably not more than 2 days, and more preferably not more than 1 day. The length of the culture period is longer than 0 hours and shorter than 48 hours. The length of the culture period may be any of 3, 6, 9, 12, 18, 24, and 36 hours. The length of the culture period may be extended or shortened depending on the condition of the cardiomyocytes 14 and mesenchymal stem cells 15 before seeding. 2 and 3, the length of the culture period from seeding of the cardiomyocytes 14 to seeding of the mesenchymal stem cells 15 is longer than 0 hours and shorter than 48 hours. The length of the culture period may be any of 3, 6, 9, 12, 18, 24, and 36 hours. The length of the culture period may be extended or shortened depending on the condition of the cardiomyocytes 14 before seeding. 2 and 3, a cell sheet 10 consisting of a first layer 11 and a second layer 12 is peeled off from a culture vessel 19. At this time, the scaffold membrane 16 may remain attached to the second layer 12. The cell sheet 10 may be made to easily peel off from the bottom of the culture vessel 19 by repeatedly contracting and extending. The bottom of the culture vessel 19 may be made to be less likely to expand and contract in the extension direction so that the cell sheet 10 can easily peel off from the bottom of the culture vessel 19. For example, the axial rigidity of the bottom of the culture vessel 19 in the extension direction may be higher than the axial rigidity of the cell sheet 10. 2 and 3 show that mesenchymal stem cells 15 are seeded on a layer of cardiomyocytes 14, ultimately forming their respective layers. It is acceptable for some of the cells to mix during the seeding and culturing process. For example, the second layer 12 of the cell sheet 10 may contain a small number of cardiomyocytes. Even when the cells are mixed in this manner, the number of mesenchymal stem cells 15 in the second layer 12 is greater than the number of cardiomyocytes. The first layer 11 may also contain a small number of mesenchymal stem cells. Even when the cells are mixed in this manner, the number of cardiomyocytes 14 in the first layer 11 is greater than the number of mesenchymal stem cells. In either case, the total number of mesenchymal stem cells in the second layer 12 is less than the total number of cardiomyocytes in the first layer 11 and the second layer 12. The ratio of the total number of cardiomyocytes to the total number of mesenchymal stem cells in the first layer 11 and the second layer 12 may be greater than 1:1 or less than 100:1. The ratio of cell numbers may be any of 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, and 50:1. 2 and 3 show that the actively proliferating mesenchymal stem cells 15 are seeded after the slowly proliferating cardiomyocytes 14. By adopting this seeding order, a cell sheet consisting of cardiomyocytes and mesenchymal stem cells can be obtained. The ratio of the number of these cells in the cell sheet does not change significantly from the time of cell seeding, so there is little variation in the quality of the cell sheet. 2 and 3, the ratio of the number of cardiomyocytes 14 to the number of mesenchymal stem cells 15 is 4:1 to 20:1, preferably 10:1. Here, the serum concentration (volume fraction) in the medium 18 is 5 to 10 (volume / volume %), and laminin is used as the scaffold membrane 16 in a volume per unit area (cm ２ It is preferable to use 0.5 to 2 μg of laminin per unit area (cm ２ It is particularly preferred to use 1 to 2 μg per 1000 mg of PEG. 2 and 3, the total number of cardiomyocytes 14 and mesenchymal stem cells 15 seeded per cell sheet may be in the following range: For example, a total of 5.5×10 5 Larger than 8.8 x 10 6 Seed fewer than 1.1 x 10 cells, preferably a total of 1.1 x 10 6 pcs or more, 4.4×10 6 Seed the cells in a number not exceeding 1000 cells / well. 2 and 3, the number of cardiomyocytes 14 seeded per cell sheet may be in the following range: For example, 5.0×10 5 Larger than 8.0 x 10 6 Seed fewer than 1.0 x 10 cells, preferably 1.0 x 10 6 pcs or more, 4.0×10 6 Seed the cells in a number not exceeding 1000 cells / well. 2 and 3, the area of ​​the bottom of the culture vessel 19 and the total number of the cardiomyocytes 14 and mesenchymal stem cells 15 to be seeded may be within the following ranges: For example, a total of 1.1 × 10 cells per well of a 24-well plate with a diameter of 1.547 cm. 6 Larger than 8.8 x 10 6 Seed fewer than 10 cells, preferably a total of 2.2 x 10 cells per well. 6 pcs or more, 4.4×106 Or, the bottom area of ​​the culture vessel 19 is 1 cm. 2 5.9 x 10 per 5 Larger than 4.6 x 10 6 Fewer than 1.1 x 10 cells are seeded, preferably 1.1 x 10 6 pcs or more, 2.4×10 6 Seed the cells in a number not exceeding 1000 cells / well. 2 and 3, the area of ​​the bottom of the culture vessel 19 and the number of cardiomyocytes 14 to be seeded may be in the following ranges: For example, 1×10 for one well of a 24-well plate with a diameter of 1.547 cm. 6 Larger than 8 x 10 6 Fewer than 10 cardiomyocytes 14 are seeded per well, preferably a total of 2 x 10 6 pcs or more, 4×10 6 The number of cardiomyocytes 14 is seeded in the culture vessel 19 within the area of ​​the bottom of 1 cm. 2 5.4 x 10 per 5 Larger than 4.2 x 10 6 The number of cardiomyocytes 14 seeded is less than 1.0×10 6 pcs or more, 2.2×10 6 Cardiomyocytes 14 are seeded in a number of cells equal to or less than 100 cells / well. <Order of cell seeding> In this example, cell sheets were prepared as organoids in the form of patches. First, differences were observed between seeding cardiomyocytes (CM cells, hereafter sometimes referred to as CM) and mesenchymal stem cells (MSCs) simultaneously and seeding CM followed by MSCs. Cardiomyocytes induced from iPS cells (iPS-CMs) were used as CM. Mesenchymal stem cells induced from iPS cells (iPS-MSCs) were used as MSCs. Hereinafter, unless otherwise specified, both CM and MSCs refer to cells induced from iPS cells. We previously identified other cells present in the iPS-CM cell population. Analysis of individual cells using next-generation sequencing (RNA-seq) detected fibroblasts and vascular endothelial cells. Furthermore, cells in myocardial sheets created solely from iPS-CMs were dispersed and analyzed by flow cytometry. Not only were cells positive for cTnT (cardiac Troponin T), a cardiomyocyte marker, but also for Vimentin, a mesenchymal cell marker for fibroblasts and other cells, aSMA (smooth muscle Actin alpha), a maker of smooth muscle-like cells and motile cells, and CD31 (Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1)), a marker for vascular endothelial cells and peripheral blood, were detected. Each well of a 24-well plate or 48-well plate was used as the culture vessel. The bottom of the culture vessel was coated with laminin (LN) as a scaffold membrane. LN allows for the efficient collection of a strong cell sheet from the bottom of the culture vessel. DMEM was used as the culture medium. Four cell sheets were prepared for each experiment. Immunostaining was performed targeting cTnT to identify the amount of CMs present. Immunostaining was performed targeting CD90 (Cluster of Differentiation 90) to identify the amount of MSCs present. Figure 4 shows a cross-section of the stained cell sheet. Sarcomere structures were observed both with and without the addition of MSCs. Furthermore, the more MSCs present, the thicker the cell sheet. Table 1 shows the results of the test. In the group in which CM and MSC were seeded simultaneously, the pre-seeding CM / MSC cell ratio was not maintained after two days of culture. As shown in the table, the CM abundance ratio decreased at all cell ratios, as did the MSC abundance ratio. This is thought to be because MSCs adhered to the LN before CM, allowing for efficient proliferation. In contrast, in the group in which MSCs were seeded the day after CM seeding, the pre-seeding CM / MSC cell ratio was largely maintained after two days of culture from CM seeding. <Serum volume and coating volume> Next, we investigated the amount of LN to coat the bottom of the culture vessel. Table 2 shows the cell sheets prepared by varying the volume fraction (%) of FBS in the medium and the amount of LN per area. Culture was carried out independently in three batches, Lot 1, Lot 2, and Lot 3, in that order. MSCs were seeded the day after CM seeding. The seeded cell ratio was CM:MSC = 10:1. The volume fractions of serum and FBS in the medium were kept constant during both the period when only CM was cultured and the period when only MSCs were cultured. The results are shown in Table 2 below. For example, in Lot 1 of 2.5% FBS DMEM, LN was 0.15 μg / cm 2 Under these conditions, the percentage of cTnT-positive cells was 73.8%, as shown on the left. The percentage of CD90-positive cells was 12.7%, as shown on the right. Please refer to these figures for other culture conditions as well. The higher the FBS content in the medium and the lower the amount of LN coating, the easier the cell sheet detached from the bottom of the culture vessel. Detachment of the cell sheet is thought to occur due to repeated contraction of the cell sheet. The culture conditions that resulted in detachment in the tests for Lot 1 and Lot 2 were not used in the culture test for Lot 3. When the FBS content in the medium was low and the amount of LN coating was high, the cell sheet did not detach in Lot 3. The serum volume fraction is 1-10% and the LN concentration is 0.05-2 μg / cm 2 By seeding CM and MSCs in sequence, a sheet was obtained after 2 days of culture and could be artificially detached. In this case, the serum volume fraction was higher than 1% and the LN concentration was 0.5 μg / cm. 2 When the serum volume fraction was lower than 2.5%, the cell sheet was easily artificially detached from the culture vessel. When the serum volume fraction was higher than 2.5%, a strong cell sheet tended to be obtained. Cell sheets were prepared with CM to MSC cell number ratios of 20:1, 10:1, and 4:1. The serum volume fraction and LN coating amount were as shown in Table 3. The serum volume fraction was 5% and the LN coating amount was 2 μg / cm.2 When the serum volume fraction was increased to 10%, a cell sheet could be formed on the bottom surface of the culture vessel at any cell number ratio. The same was true when the serum volume fraction was increased to 10%. Furthermore, when the LN coating amount was 1 μg / cm 2 The same was true when the In the following tests, the cell number ratio of CM to MSC was 20:1, 10:1, or 4:1, the serum volume fraction was 5 to 10%, and the amount of LN coated on the culture vessel was 1 to 2 μg / cm. 2 CM were seeded and cultured for one day, and then MSCs were seeded and cultured for another day. The total culture period until the cell sheet was obtained was two days. <Angiogenic potential of cell sheets> Cell sheets were prepared using CM to MSC ratios of 20:1, 10:1, and 4:1. For comparison, cell sheets were also prepared from CM alone. As shown in Figure 5, when the amounts of cytokines secreted by the cell sheets into the culture supernatant were measured using an antibody array, the secretion levels of cytokines involved in angiogenesis, such as HGF and MMP-9, were increased in both cell sheets containing MSCs compared to cell sheets not containing MSCs (CM:MSC = 1:0). These are cytokines that are also frequently expressed by mesenchymal stem cells. Furthermore, the secretion levels of cytokines involved in angiogenesis, such as Angiopoetin-1, 2, VEGF, and SDF-1a, were maintained in both cell sheets containing MSCs. These are also cytokines that are naturally frequently expressed by cardiomyocytes. Based on the above, we investigated the effect of cell sheet secretions on angiogenesis. As shown in Figure 6, an Aorta ring assay was performed using the culture supernatant generated during the preparation of cell sheets. Mouse aortas were sliced ​​into rings. The aortic rings were then cultured in Matrigel. The cell supernatant from the cell sheet was then added to the culture medium for the aorta. Endothelial cells in the aorta were observed to form capillaries from the cut surface of the aorta toward the Matrigel. Whether the cell sheet contained MSCs or not, the culture supernatant promoted angiogenesis from the cut surface of the aorta, similar to the positive control VEGF. Figure 7 shows the results of statistical analysis of the measured values ​​in the Aorta ring assay. The total area, total number, and total length of newly formed blood vessels increased, particularly at a CM:MSC ratio of 10:1. A t-test was performed on each measured value between the VEGF (positive control group) and culture supernatant-exposed group and the negative control group, and a significant difference was observed at a significance level of 5%. It was found that the cell sheet strongly promotes angiogenesis. As described above, the cell sheet of this example possesses excellent vascular induction ability. Thus, the cell sheet of this example is able to reproduce some of the functions of the heart. This indicates that cardiomyocytes and mesenchymal stem cells work together to self-organize. As described above, the organoids constituting the cell sheet of this example actively secrete cytokines. Furthermore, the organoids of this example affect surrounding cells through cytokine secretion. Therefore, the paracrine effect of the organoids of this example is evident from the above results. However, the paracrine effect of the organoids of this example is not limited to the scope of angiogenesis. <Cell sheet contractility> Figure 8 is a graph showing the periodic changes in the contraction speed of the cell sheet. The upper panel of Figure 9 shows the maximum contraction speed of the cell sheet. The lower panel shows the contraction synchronization (correlation). Compared to cell sheets without MSCs, cell sheets containing MSCs at a CM:MSC ratio of 10:1 or 4:1 exhibited higher contractile force. The contraction synchronization (correlation) was not impaired in these cell sheets. Furthermore, no significant differences were observed between groups LN1 and LN2, which differed in the amount of LN. As described above, the cell sheet of this example possesses excellent contractile ability. Thus, the cell sheet of this example is able to partially reproduce cardiac function. This indicates that cardiomyocytes and mesenchymal stem cells cooperate to self-organize. <Transplantation into animal hearts> As described above, the cell sheet of this example was confirmed to be an organoid in the form of a patch. This cell sheet was transplanted onto the surface of the heart of a nude rat in which ischemic cardiomyopathy had been artificially induced. The schedule for this in vivo test is as follows: At -2 weeks (-2W), 7-week-old female immunodeficient nude rats (F344 / NJcl-rnu / rnu) underwent ligation of the left anterior descending artery (LAD) in the heart, resulting in ischemic cardiomyopathy (ICM). The test began at week 0 (0W). Rats that had undergone surgery two weeks prior and whose ejection fraction (EF) had decreased to 40±5% were recruited for subsequent testing. The rats were divided into groups A to E as follows. The following cell sheets were transplanted into each rat. Group A: Sham operation Group B: CM:MSC=1:0, number of seeded cells 4.0x10 6 , 15 to 16 mm diameter cell sheets Group C: CM:MSC=10:1, number of seeded cells 4.4x10 6 , 15 to 16 mm diameter cell sheets Group D: CM:MSC=10:1, number of seeded cells 2.2x106 , 15 to 16 mm diameter cell sheets Group E: CM:MSC=10:1, number of seeded cells 1.1x10 6 , 8 to 9 mm diameter cell sheets Both CMs and MSCs were derived from human iPS cells. Cardiac function of each rat was assessed weekly by echocardiography from week 0 to week 8 (8W). Heart specimens were collected from one rat at week 4 (4W). Heart specimens were collected from three rats at week 8 (8W). Figure 10 and Table 4 show the state of the remaining cell sheet up to 4 and 8 weeks after transplantation. The sheet survival rate is the number of individuals in which the cell sheet was found to remain on the surface of the heart as a result of pathological analysis, divided by the number of individuals from which heart specimens were harvested each time. At 4 weeks after sheet transplantation, survival of the transplanted cell sheet was confirmed in groups B to E. At 8 weeks after sheet transplantation, survival of the transplanted cell sheet was not confirmed in group B. In contrast, survival of the transplanted cell sheet was confirmed in groups C to E. As such, the cell sheet of this embodiment is more likely to survive than a cell sheet that does not contain MSCs. Furthermore, the cause of the loss of the cell sheet is expected to be rejection of the cell sheet by the immune system of the recipient. The inventors considered this result to suggest that the cell sheet of this embodiment is more tolerant to immune rejection than a cell sheet that does not contain MSCs. Figure 11 shows the results of the therapeutic efficacy verification by echocardiography up to 8 weeks. The EF values ​​of each group were compared by analysis of variance (ANOVA). At 4 weeks, Group B and each of Groups C to E tended to have higher EF values ​​than Group A. The P value between Group A and Group C was below 0.01. The P value between Group A and Group D was below 0.05. At 8 weeks, Group B and each of Groups C to E continued to have higher EF values ​​than Group A. At 4 weeks and 8 weeks, the EF value increased with increasing number of cells in the cell sheet for each of Groups C to E, indicating a tendency for the cell sheet to be highly effective in recovering from myocardial infarction. Furthermore, at 4 weeks and 8 weeks, Group C and Group B, which contained the same number of cardiomyocytes, tended to have a higher EF value than Group B. This confirmed that the cell sheet of this embodiment tends to have a greater therapeutic effect than a cell sheet consisting of CM alone. Although not shown, cardiac specimens at 4W and 8W were immunostained with antibodies against human cytoplasm-specific markers STEM121 and h-TnT. At 4W, expression of STEM121 and h-TnT was observed in groups B to E. At 4W, it was confirmed that cell sheets remained regardless of the presence or absence of MSCs. At 8W, expression of STEM121 and h-TnT was observed in groups C to E, but not in group B. At 8W, it was confirmed that the cell sheet of this embodiment remained better than a cell sheet consisting of CM alone. At 4W and 8W, cardiac specimens were stained with Sirius Red. Sirius Red detects areas of fibrosis due to the progression of cardiomyopathy. At 4W, cell sheets not stained with Sirius Red were observed surrounded by areas stained with Sirius Red in groups B to E. This confirmed that cell sheets survived at 4W regardless of the presence or absence of MSCs. Furthermore, the area stained with Sirius Red was largest in group A, followed by group B. It was small in groups C to E. Therefore, it was confirmed that the cell sheet of this embodiment tends to have a higher therapeutic effect than a cell sheet consisting of CM alone. At 8W, cell sheets not stained with Sirius Red were observed surrounded by areas stained with Sirius Red. It was not observed in group B. This confirmed that cell sheets of this embodiment survived better at 8W than a cell sheet consisting of CM alone. Furthermore, the area stained with Sirius Red was largest in group A, followed by group B. It was small in groups C to E. The observation results at 4W and 8W confirmed that the cell sheet of this embodiment tended to have a higher therapeutic effect than a cell sheet consisting of CM only. As mentioned above, the organoids that make up the cell sheet of this example actively secrete cytokines. Furthermore, they release a different combination of cytokines from those released by cardiomyocytes and mesenchymal stem cells. Furthermore, the organoids of this example affect surrounding cells through cytokine secretion. Therefore, the above results confirm that the therapeutic effect is enhanced by the paracrine effect of the organoids of this example. This application is based on Japanese Patent Application No. 2024-148694, entitled "Method for producing cell aggregates, aggregate populations, and cell culture methods," filed on August 30, 2024, and claims the benefit of priority from this Japanese patent application. The entire contents of this Japanese patent application are incorporated herein by reference. 10: Cell sheet, 11: First layer, 12: Second layer, 14: Cardiomyocytes, 15: Mesenchymal stem cells, 16: Scaffold membrane, 17: Culture medium, 18: Culture medium, 19: Culture vessel

Claims

1. An organoid in which cardiomyocytes, mesenchymal stem cells, and optionally other cells cooperate and self-organize within the organoid.

2. The organoid of claim 1, having a heart wall-like morphology.

3. The organoid of claim 1 having a patch-like morphology.

4. The organoid of claim 1, comprising cardiomyocytes, mesenchymal stem cells, and optionally other cells, wherein the number of cells contained within the organoid is greatest among cardiomyocytes.

5. The organoid described in claim 1, wherein the cardiomyocytes, the mesenchymal stem cells, and the other cells cooperate to self-organize, and the other cells are at least one of fibroblasts, endothelial cells, and smooth muscle cells.

6. The organoid of claim 1, produced by seeding cardiomyocytes, mesenchymal stem cells, and optionally other cells onto a laminin-coated plate, wherein the cardiomyocytes are seeded followed by the mesenchymal stem cells.

7. The organoid of claim 1, which is produced by seeding cardiomyocytes, mesenchymal stem cells, and optionally other cells on a plate coated with laminin, wherein the cell number ratio of the seeded cardiomyocytes to mesenchymal stem cells is greater than 4:1 and less than 20:

1.

8. The organoid of claim 1, which autonomously repeats contraction and extension.

9. The organoid of claim 1 for use in treating heart disease.

10. The organoid of claim 1 for transplantation into the heart.

11. The organoid of claim 1, for transplantation into a site of the heart that has lost blood flow.

12. A method for producing organoids, comprising: seeding and adhering cardiomyocytes dispersed among themselves onto a scaffold membrane made of molecules constituting a basement membrane; seeding mesenchymal stem cells dispersed among themselves onto the cardiomyocytes adhered onto the scaffold membrane; culturing the cardiomyocytes together with the mesenchymal stem cells in a culture medium, wherein the number of the mesenchymal stem cells is less than the number of the cardiomyocytes; and the organoids are for treating heart disease, and the cardiomyocytes, mesenchymal stem cells, and optionally other cells cooperate and self-organize within the organoids.

13. The method according to claim 12, wherein the molecule constituting the basement membrane is laminin.

14. The molecule constituting the basement membrane is laminin, and the unit area (cm ２ 13. The method of claim 12, wherein the weight per 1000 mg / ml of the sachet is 0.05 to 2 μg.

15. The molecule constituting the basement membrane is laminin, and the unit area (cm ２ 13. The method of claim 12, wherein the weight of the serum in the medium is 0.05 to 2 μg per 1000 μg of the culture medium, and the concentration of serum in the medium is 1 to 20% (volume / volume).

16. The method according to claim 12, wherein the mixing ratio of cardiomyocytes to mesenchymal stem cells at the time of seeding is greater than 4:1 and less than 20:1, and the length of the culture period after seeding the mesenchymal stem cells does not exceed 48 hours.

17. The method according to claim 12, wherein the cell number ratio of cardiomyocytes to mesenchymal stem cells at the time of seeding is greater than 4:1 and less than 20:1, and the length of the culture period after seeding the mesenchymal stem cells does not exceed 48 hours.

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