Large fused cell mass, method for producing same, incubator, and support structure

Abstract

Provided is a large fused cell mass having a diameter of 3 cm or more.　A large fused cell mass according to the present invention can be produced by suppressing deflection of a cell culture insert film by means of the weight of a seeded cell mass, and preventing contraction of a fused cell mass that is being cultured. The present invention also provides: a method for producing a large fused cell mass having a diameter of 3 cm or more; an incubator; and a support structure.

Description

Large fused cell mass, its production method, culture vessel and support structure

[0001] The present invention relates to a large fused cell mass, a method for producing the same, a culture vessel and a support structure, and in particular to a large fused cell mass having a diameter of 3 cm or more, a method for producing the same, a culture vessel and a support structure.

[0002] Previously, a method for producing fused cell masses such as large organoids, which was independently developed by the present inventors, has been proposed (hereinafter referred to as the "conventional method"), which allows for the production of large organoids of 5 mm or more by fusing small cell masses of approximately 100-200 μm in diameter. In the conventional method, for example, by fusing a large number of small organoids produced by mixing hepatic endoderm cells, vascular endothelial cells, and mesenchymal cells, it is possible to produce large organoids containing not only hepatocytes but also advanced vascular and bile duct structures (see Patent Document 1).

[0003] Conventionally, large organoids with a diameter of 5 mm or more have been produced using incubators such as cell culture inserts. Cell culture inserts are incubators equipped with a porous membrane that is impermeable to cells but allows medium and oxygen to pass through. Cells are seeded on the membrane, which is placed higher than the bottom of the culture vessel. The cells are cultured with the membrane immersed in the medium, allowing the components of the medium and oxygen to reach the cells.

[0004] WO2019 / 189324

[0005] However, when using cell culture inserts to produce large organoids with a diameter of over 3 cm, the organoids frequently shrink. This is thought to be due to the membrane of the cell culture insert bending under the weight of the seeded cell clusters, causing the cell clusters to gather in the center. This can lead to defects such as different organoid shrinkage rates between production batches, making it difficult to produce large organoids with consistent size and cell function.

[0006] The present invention aims to produce large fused cell clusters by suppressing deflection of the cell culture insert membrane due to the weight of the seeded cell clusters and preventing contraction of the fused cell clusters during culture.

[0007] The present inventors have developed an improved cell culture insert that integrates a membrane support structure into the bottom surface of the membrane to prevent the membrane from bending under the weight of seeded cells, etc. Use of this improved cell culture insert can suppress membrane bending during the preparation of large organoids, preventing the large organoids from shrinking during culture. The present invention was completed based on these findings.

[0008] The gist of the present invention is as follows: (1) A large fused cell mass produced by fusing cell masses in vitro, the large fused cell mass having a diameter of 3 cm or more. (2) The large fused cell mass according to (1), wherein the cell mass is an organ bud. (3) The large fused cell mass according to (2), wherein the organ bud is formed from tissue or organ cells, mesenchymal cells, and vascular cells. (4) The large fused cell mass according to (3), wherein the ratio of tissue or organ cells, mesenchymal cells, and vascular cells is 10:0.1 to 10:0.1 to 10. (5) The large fused cell mass according to any of (2) to (4), wherein the organ bud is formed in a culture vessel having a non-cell adhesive surface. (6) The large fused cell mass according to any of (2) to (5), wherein the fused organ bud forms a vascular structure. (7) A method for producing large fused cell clusters according to (1), comprising seeding cell clusters on the front surface of a membrane permeable to medium components and oxygen, supplying medium from the back side of the membrane to carry out culture, and fusing the cell clusters together, wherein the back surface of the membrane is supported by a support structure that prevents the membrane from bending due to the weight of the seeded cell clusters. (8) The method according to (7), wherein the cell clusters are cultured with the front surface of the membrane exposed to the atmosphere. (9) The method according to (7) or (8), wherein the diameter of the membrane is 3 cm or more. (10) The method according to any one of (7) to (9), wherein the diameter of the cell clusters to be fused is 80 to 500 μm and the number of cells is 2,000 or more. (11) An incubator having a main body with a membrane at the bottom that is permeable to culture medium components and oxygen and on which a culture medium is seeded on the front surface, and in which culture medium is supplied into the main body from the back side of the membrane, characterized in that a support structure that prevents the membrane from sagging is attached to the bottom surface of the main body. (12) The incubator described in (11), characterized in that the support structure is in point contact, line contact, or surface contact with the back surface of the membrane, or a combination thereof. (13) The incubator described in (11), characterized in that the support structure consists of one or more frame members that contact the back surface of the membrane. (14) The incubator described in (13), characterized in that the frame members are formed so as to pass through the center of the back surface of the membrane and extend to the periphery.(15) The incubator according to (14), characterized in that the frame member divides the back surface of the membrane into two or more compartments, and each of the compartments is formed to include a part of the peripheral edge of the back surface of the membrane. (16) The incubator according to any of (11) to (15), characterized in that the main body is formed in a substantially cylindrical shape. (17) The incubator according to any of (11) to (16), characterized in that the support structure has legs at its bottom. (18) A support structure used in the incubator according to any of (11) to (17).

[0009] When using conventional cell culture inserts, which simply comprise a membrane that serves as a cell culture scaffold, to produce large organoids, the organoids frequently contract during culture, and the contraction rate varies from batch to batch, making it difficult to produce large organoids with stable size and cell function. The present invention suppresses membrane deflection during large organoid production, preventing large organoids from shrinking during culture, making it possible to produce large organoids with stable size. As a result, stabilization of cell function is also expected.

[0010] The present invention enables the production of large organoids with stable size. As a result, stabilization of cell function is expected. This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2024-13672, which is the priority basis of this application.

[0011] (A) is a perspective view of an incubator according to an embodiment of the present invention, (B) is a plan view thereof, (C) is a side view thereof, and (D) is a cross-sectional view along line II of (C). The incubator is used in a state where it is placed in an existing cell culture vessel such as a cell culture dish or a 6-well cell culture plate. (A) is a perspective view of the main body of an incubator according to an embodiment of the present invention, (B) is a plan view thereof, (C) is a side view thereof, and (D) is a cross-sectional view along line II-II of (C). (A) is a perspective view of a support structure according to an embodiment of the present invention, (B) is a plan view thereof, (C) is a side view thereof, and (D) is a cross-sectional view along line III-III of (C). (A) is a perspective view of a rim member of an incubator according to an embodiment of the present invention, (B) is a plan view thereof, (C) is a side view thereof, and (D) is a cross-sectional view along line IV-IV of (C). (A)-(G) are plan views showing modified examples of the support structure according to an embodiment of the present invention. Photographs show fused liver organoids (left) produced using cell culture inserts without a membrane support structure and those (right) produced using cell culture inserts with a membrane support structure. Histological analysis results of large fused liver organoids are shown. Macrophage migration activity evaluation results of large fused liver organoids are shown. Single RNA sequencing analysis results of fused liver organoids are shown. Single RNA sequencing data were obtained from large fused liver organoids (LfLB_1, 2) produced by the present invention and small fused liver organoids (SfLB_1, 2) produced by conventional methods, and UMAP analysis was performed using gene expression as an indicator. Each plot represents one cell. No bias in cell distribution was observed between samples produced by both production methods, suggesting no differences between the two production methods or variation between production lots. The results of clustering of constituent cells of fused liver organoids are shown. Using single RNA sequencing data from large and small fused hepatic organoids, cell populations were clustered using gene expression as an indicator, and the cell types that make up each cell cluster were identified by referencing marker genes specifically expressed in the constituent cell types of hepatic organoids. Because it was difficult to classify mesenchymal cells and hepatic stellate cells based on gene expression alone, they were included in the same cluster. The results of a comparison of the constituent cell ratios of fused hepatic organoids are shown below.The ratio of cells contained in each cell cluster (see Figure 9) was compared between large fused hepatic organoids (LfLB, n=2) and small fused hepatic organoids (SfLB, n=2). No clear difference was observed in the ratio of each constituent cell type between large and small fused hepatic organoids, suggesting the equivalence of the two.

[0012] 1: Incubator 2: Membrane 3: Main body 4: Support structure 4a: Frame member 4b: Legs 5: Rim member

[0013] The present invention provides large fused cell masses produced by fusing cell masses in vitro, the large fused cell mass having a diameter of 3 cm or more. In the present invention, the diameter (φ) of the large fused cell mass may be 3 cm or more, 5 cm or more, 6 cm or more, or 11 cm or more. The large fused cell masses of the present invention can be produced by a method for producing fused cell masses, in which cell masses are seeded on the front side of a membrane permeable to medium components and oxygen, cultured by supplying medium from the back side of the membrane, and fusing the cell masses together, characterized in that the back side of the membrane is supported by a support structure that prevents the membrane from bending due to the weight of the seeded cell masses. The present invention also provides a method for producing the large fused cell masses described above.

[0014] The cell mass may be any cell aggregate, such as an organ bud (organoid) or a spheroid, but preferably contains vascular endothelial cells. An "organ bud" is a structure that can differentiate into an organ upon maturation. For example, WO2013 / 047639 discloses a method for producing organ buds from three types of cells: tissue or organ cells, vascular endothelial cells, and undifferentiated mesenchymal cells or cells differentiated therefrom. Organ buds produced by this method can be suitably used in the present invention. Whether a structure is an organ bud can be confirmed, for example, by transplanting the structure into a living organism and examining whether it can differentiate into the desired organ (if it differentiates into the desired organ, it can be determined to be an organ bud), and / or by examining whether the structure contains all three types of cells (if it contains all three types of cells, it can be determined to be an organ bud). The organ bud may be, for example, an organ bud that differentiates into an organ such as the kidney, heart, lung, spleen, esophagus, stomach, thyroid, parathyroid, thymus, gonads, brain, or spinal cord. However, an organ bud that differentiates into an endodermal organ, such as an organ bud that differentiates into the liver (hepatobud), pancreas (pancreatic bud), or intestine, is preferred. Whether a structure is an organ bud that differentiates into an endodermal organ can be confirmed by examining the expression of marker proteins or corresponding genes (an organ bud can be determined if one or more of the marker proteins or corresponding genes described below are expressed). For example, markers for liver buds include HHEX, SOX2, HNF4A, AFP, and ALB; markers for pancreatic buds include PDX1, SOX17, and SOX9; and markers for organ buds that differentiate into the intestine include CDX2 and SOX9.Among terms used by those skilled in the art, the organ buds of the present invention include liver bud, liver diverticula, liver organoid, pancreatic (dorsal or ventral) buds, pancreatic diverticula, pancreatic organoid, intestinal bud, intestinal diverticula, and intestinal organoid (K. Matsumoto, et al. Science. 19; 294 (5542): 559-63. (2001)).

[0015] In the present invention, the term "tissue or organ cells" refers to functional cells that constitute a tissue or organ, or undifferentiated cells that differentiate into functional cells. Examples of "undifferentiated cells that differentiate into functional cells" include cells that can differentiate into functional cells that constitute organs such as the kidney, heart, lung, spleen, esophagus, stomach, thyroid gland, parathyroid gland, thymus, gonads, brain, and spinal cord. Examples include cells that can differentiate into functional cells that constitute ectodermal organs such as the brain, spinal cord, adrenal medulla, epidermis, hair, nails, and skin glands, sensory organs, peripheral nerves, and lens; cells that can differentiate into functional cells that constitute 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 functional cells that constitute endodermal organs such as the liver, pancreas, intestine, lung, thyroid gland, parathyroid gland, and urinary tract. Whether a cell is capable of differentiating into functional cells that make up ectodermal, mesodermal, or endodermal organs can be confirmed by examining the expression of marker proteins or corresponding genes (if one or more of the marker proteins or corresponding genes are expressed, it can be determined that the cell is capable of differentiating into functional cells that make up endodermal organs). For example, markers for cells that can differentiate into functional cells that make up the liver include HHEX, SOX2, HNF4A, AFP, and ALB; markers for cells that can differentiate into functional cells that make up the pancreas include PDX1, SOX17, and SOX9; markers for cells that can differentiate into functional cells that make up the intestinal tract include CDX2 and SOX9; markers for cells that can differentiate into functional cells that make up the kidney include SIX2 and SALL1; markers for cells that can differentiate into functional cells that make up the heart include NKX2-5, MYH6, ACTN2, MYL7, and HPPA; markers for cells that can differentiate into functional cells that make up the blood include C-KIT (the corresponding gene is KIT), SCA1 (the corresponding gene is ATXN1), TER119 (the corresponding gene is Ly76), and HOXB4; and markers for cells that can differentiate into functional cells that make up the brain and spinal cord include HNK1, AP2, and NESTIN.Among the terms used in the art are hepatoblast, hepatic progenitor cells, pancreatoblast, hepatic precursor cells, pancreatoblast, pancreatic progenitors, pancreatic progenitor cells, pancreatic precursor cells, endocrine precursors, intestinal progenitor cells, intestinal precursor cells, intermediate mesoderm, metanephric mesenchymal precursor cells, multipotent nephron progenitor, renal progenitor cell, cardiac mesoderm, cardiovascular progenitor cells, cardiac progenitor cells, (JR. Spence, et al. Nature.;470(7332):105-9.(2011), Self, et al. EMBO J.; 25(21): 5214-5228.(2006), J. Zhang, et al. Circulation Research.; 104: e30-e41 (2009), G. Lee, et al. Nature Biotechnology 25, 1468-1475 (2007)) are included in the undifferentiated tissue or organ cells of the present invention. Undifferentiated cells that differentiate into functional cells can be collected from tissues or organs, or can be produced from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods. Furthermore, undifferentiated cells that differentiate into functional cells may be cells that are in the middle stage of differentiation from pluripotent stem cells such as iPS cells into tissues or organs, such as primitive gut endoderm cells (PGECs) (Japanese Patent No. 5777127).PGECs have the advantage of being able to differentiate into hepatocytes, pancreatic cells, and intestinal cells (high differentiation function), not expressing markers related to cancer malignancy (high safety), and being prepared by inducing differentiation from iPS cells in a feeder-free environment, making them suitable for clinical applications. Furthermore, PGECs can be prepared in large quantities. PGECs can be prepared by the method described in Japanese Patent No. 5777127. Alternatively, PGECs can be induced into CXCR4- and E-cadherin-positive endodermal cells by culturing pluripotent stem cells such as iPS cells in a serum-free medium with the addition of activin, or by further culturing the resulting endodermal cells with the addition of BMP4 and FGF2 for two days to obtain a CXCR4-negative, HNF4α-positive hepatic endoderm population. In addition, for example, organ cells that can be differentiated into the liver can be prepared according to K. Si-Taiyeb, et al. Hepatology, 51 (1): 297-305 (2010), T. Touboul, et al. Hepatology. 51 (5): 1754-65. (2010), organ cells that can be differentiated into the pancreas can be prepared according to D. Zhang, et al. Cell Res.; 19 (4): 429-38. (2009), organ cells that can be differentiated into the intestinal tract can be prepared according to J. Cai, et al. J Mol Cell Biol.; 2 (1): 50-60 (2010), R. Spence, et al. Nature.; 470 (7332): 105-9. (2011), and organ cells that can be differentiated into the heart can be prepared according to J. Zhang, et al. Circulation Research.; 104: e30-e41 (2009). Cells that can differentiate into the brain or spinal cord can be produced according to G. Lee, et al. Nature Biotechnology 25, 1468-1475 (2007). Examples of "differentiated tissue or organ cells" include pancreatic endocrine cells, pancreatic duct epithelial cells, liver hepatocytes, intestinal epithelial cells, renal tubular epithelial cells, renal glomerular epithelial cells, cardiac myocytes, blood lymphocytes, granulocytes, and erythrocytes, brain neurons and glial cells, and spinal cord neurons and Schwann cells.Tissue or organ cells are primarily derived from humans, but tissue or organ cells derived from animals other than humans (e.g., animals used as laboratory animals, pet animals, working animals, racehorses, fighting dogs, etc., specifically, mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.) may also be used.

[0016] In the present invention, "vascular endothelial cells" refer to cells that constitute the vascular endothelium or cells that can differentiate into such cells. Whether a cell is a vascular endothelial cell can be confirmed by examining the expression of marker proteins or corresponding genes, such as TIE2, VEGFR-1 (corresponding gene is FLT1), VEGFR-2 (corresponding gene is KDR), VEGFR-3 (corresponding gene is FLT4), and CD31 (corresponding gene is PECAM1). (The expression of one or more of the marker proteins or corresponding genes indicates that the cell is a vascular endothelial cell.) The vascular endothelial cells used in the present invention may be differentiated or undifferentiated. Whether a vascular endothelial cell is a differentiated cell can be confirmed by examining CD31 and CD144 (corresponding gene is CDH5). Among terms used by those skilled in the art, endothelial cells, umbilical vein endothelial cells, endothelial progenitor cells, endothelial precursor cells, vasculogenic progenitors, and hemangioblasts (HJ. Joo, et al., Blood. 25;118(8):2094-104. (2011)) are included in the vascular endothelial cells of the present invention. Preferred vascular endothelial cells are umbilical vein-derived vascular endothelial cells. Vascular endothelial cells can be collected from blood vessels or prepared from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods. Vascular endothelial cells used are mainly derived from humans, but vascular endothelial cells derived from animals other than humans (e.g., animals used for laboratory experiments, pets, working animals, racehorses, fighting dogs, etc., specifically, mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.) may also be used.

[0017] In the present invention, the term "mesenchymal cells" refers to connective tissue cells that are primarily present in connective tissue derived from the mesoderm and form a support structure for cells that function in tissues. However, the term also encompasses cells whose differentiation fate into mesenchymal cells has been determined but that have not yet differentiated into mesenchymal cells. The mesenchymal cells used in the present invention may be differentiated or undifferentiated. Whether a cell is an undifferentiated mesenchymal cell can be confirmed by examining the expression of marker proteins or corresponding genes, such as Stro-1, CD29 (corresponding gene is ITGB1), CD44, CD73 (corresponding gene is NT5E), CD90 (corresponding gene is THY1), CD105 (corresponding gene is ENG), CD133 (corresponding gene is PROM1), CD271 (corresponding gene is NGFR), and nestin. (Expression of one or more of these marker proteins or corresponding genes indicates that the cell is an undifferentiated mesenchymal cell.) Furthermore, mesenchymal cells that do not express any of the markers listed above can be determined to be differentiated mesenchymal cells. Among terms used by those skilled in the art, the mesenchymal cells of the present invention include mesenchymal stem cells, mesenchymal progenitor cells, and mesenchymal cells (R. Peters, et al. PLoS One. 30;5(12):e15689.(2010)). Preferred mesenchymal cells are bone marrow-derived mesenchymal cells (particularly mesenchymal stem cells). Mesenchymal cells can be collected from tissues such as bone marrow, adipose tissue, placental tissue, umbilical cord tissue, and dental pulp, or can be produced from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods. Mesenchymal cells are mainly derived from humans, but undifferentiated mesenchymal cells derived from animals other than humans (for example, animals used as laboratory animals, pets, working animals, racehorses, fighting dogs, etc., specifically mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.) may also be used.

[0018] A method for generating all three types of cells (tissue or organ cells, vascular endothelial cells, and mesenchymal cells) from iPS cells and then creating organ buds from these cells is described in Takebe T et al., 2017, Cell Reports 21, 2661-2670, and these cells and organ buds may be used in the present invention. This method may be improved or modified as described in Kamishibahara Y et al., "Stabilized generation of human iPSC-derived liver organoids using a modified coating approach." Biology Methods & Protocols, 8(1):bpac034, 2023. Briefly, to induce definitive endoderm (DE) cells (tissue or organ cells), hiPSCs are seeded onto a culture dish using DE induction medium containing 10 mM Y-27632 using either the Standard method, Direct method, or Uncoating method (Kamishibahara Y et al., supra). DE induction medium was prepared by adding 20% ​​StemFit for Differentiation (Ajinomoto) to RPMI-1640 (Thermo Fisher Scientific) and containing 33 ng / ml Activin A (Ajinomoto) and 2 mM CHIR99021 (Cayman). The following day, 500 mM sodium butyrate (Sigma) was added to the medium. The following day, CHIR99021 and sodium butyrate were removed from the medium. To induce HE differentiation, DE cells were cultured for 8 days in hepatic endoderm (HE) induction medium containing Basic03 (Ajinomoto), 1 mM L-glutamine (Gibco), 1% non-essential amino acids (Gibco), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), and 1% dimethyl sulfoxide (DMSO) (Nacalai Tesque). The medium was refreshed daily during the HE differentiation period.To differentiate into mature hepatocytes (MH), HE cells were cultured for 8 days in MH induction medium consisting of Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific) supplemented with 5% fetal bovine serum (FBS) (MP Biomedicals), 100 nM dexamethasone (Dex) (Sigma), and 20 ng / ml oncostatin M (OSM) (R&D). The medium was changed every 3 days. The differentiation of endothelial cells (EC) and mesenchymal cells (MC) from iPS cells was performed as previously described, with modifications [Takebe T, Sekine K, Kimura M et al. Massive and reproducible production of liver buds entirely from human pluripotent stem cells. Cell Rep 2017;21:2661-70.] For EC differentiation, hiPSCs were dissociated with Accutase and plated onto StemFit AK02N culture dishes containing 10 mM Y-27632 and iMatrix-511. The next day, the medium was replaced with mesoderm induction medium consisting of DMEM / F12 (Thermo Fisher Scientific) supplemented with 20% StemFit for Differentiation, 1% GlutaMAX, 25 ng / ml BMP4 (R&D), and 8 mM CHIR99021. After an additional 3 days, the mesoderm induction medium was replaced with EC induction medium consisting of StemPro-34 SFM medium (Thermo Fisher Scientific) supplemented with 200 ng / ml vascular endothelial growth factor (VEGF) (Fujifilm Wako) and 2 mM forskolin (Cayman). The induction medium was refreshed daily. On day 10 of differentiation, ECs were replicated in EC maintenance medium supplemented with iMatrix-511 without Y-27632. EC maintenance medium is StemPro-34 SFM medium supplemented with 50 ng / ml VEGF. The following day, this medium was replaced with EC expansion medium. EC expansion medium was replaced every 3 days.For MC differentiation, hiPSCs were dissociated with Accutase and plated onto culture dishes in StemFit AK02N containing 10 mM Y-27632 and iMatrix-511. The following day, the medium was replaced with mesoderm induction medium. After 3 days of culture, the cells were exposed to 10 ng / ml platelet-derived growth factor (PDGF) BB (PeproTech) and 0.66 ng / ml activin A for 2 days. On day 6 of differentiation, the medium was replaced with MC induction medium containing DMEM / F12 supplemented with 20% StemFit for Differentiation, 1% Glutamax, 10 ng / ml bFGF (Fujifilm Wako), and 12 ng / ml BMP4. The MC induction medium was refreshed daily.

[0019] The culture ratio of the three types of cells in the co-culture is not particularly limited as long as it allows the formation of an organ bud. A preferred cell ratio is tissue or organ cells:vascular endothelial cells:mesenchymal cells = 10:1 to 10:1 to 10. Co-culture of approximately 230,000 tissue or organ cells, approximately 160,000 vascular endothelial cells, and approximately 160,000 mesenchymal cells can form organ buds measuring approximately 3,500 to 4,500 micrometers. The higher the proportion of mesenchymal cells, the finer the vascular network structure formed in the organ bud. A preferred cell ratio for forming a vascular network structure is tissue or organ cells:vascular endothelial cells:mesenchymal cells = 10:1 to 10:1 to 10, more preferably 10:4 to 7:4 to 7. Any medium may be used when co-culturing cells to form organ buds, as long as it allows the formation of organ buds. However, it is preferable to use a medium for vascular endothelial cell culture, a medium for tissue or organ cell culture, or a mixture of the two media. Any medium may be used for vascular endothelial cell culture, but it is preferable to use one containing at least one of hEGF (recombinant human epidermal growth factor), VEGF (vascular endothelial growth factor), hydrocortisone, bFGF, ascorbic acid, IGF1, FBS, antibiotics (e.g., gentamicin, amphotericin B, etc.), heparin, L-glutamine, phenolred, and BBE. Examples of media that can be used for vascular endothelial cell culture include EGM-2 Bullet Kit (manufactured by Lonza), EGM Bullet Kit (manufactured by Lonza), VascuLife EnGS Comp Kit (manufactured by LCT), Human Endothelial-SFM Basal Growth Medium (manufactured by Invitrogen), and human microvascular endothelial cell growth medium (manufactured by TOYOBO). Any medium may be used for culturing tissue or organ cells, but when the organ cells are hepatocytes, it is preferable to use a medium containing at least one of ascorbic acid, BSA-FAF, insulin, hydrocortisone, and GA-1000.Media for hepatocyte culture include HCM BulletKit (Lonza) minus hEGF (recombinant human epidermal growth factor) and RPMI1640 (Sigma-Aldrich) supplemented with 1% B27 Supplements (GIBCO) and 10 ng / mL hHGF (Sigma-Aldrich). Regarding human liver bud formation, a 1:1 mixture of EGM BulletKit (Lonza) and HCM BulletKit (Lonza) minus hEGF (recombinant human epidermal growth factor) supplemented with dexamethasone, oncostatin M, and HGF has been shown to promote liver bud maturation. The culture temperature for organ bud formation is preferably 30-40°C, more preferably 37°C. The culture period for organ bud formation is preferably 0.5 to 10 days, more preferably 1 to 7 days.

[0020] In the present invention, the cell mass (e.g., organ bud) may be prepared without using a cell adhesive surface such as a matrix (matrix-free), and may be formed, for example, in a culture vessel having a non-cell adhesive surface. The culture vessel having a non-cell adhesive surface may be subjected to a low-adsorption surface treatment, and for example, may have a culture surface coated with a non-cell adhesive polymer. Examples of non-cell adhesive polymers include phospholipids, phospholipid-polymer complexes, poly(2-hydroxyethyl methacrylate) (PHEMA), polyvinyl alcohol, agarose, chitosan, polyethylene glycol, albumin, and photocrosslinked superhydrophilic polymers. The bottom of the culture vessel may have a large number of hemispherical or truncated conical depressions. For example, hemispherical or truncated conical depressions (with a volume of 0.068 mm) may be formed. 3A 24-well culture vessel containing 600 nuclei per well is used, and a total of 200,000 to 3,000,000 cells are cultured per well to form cell clusters. The cell clusters can be 80 to 500 micrometers in size. The above culture vessel and culture conditions are described in WO2015 / 182159, which is incorporated herein by reference. Examples of culture vessels with non-cell-adhesive surfaces include Elplasia plates (Corning) and 96-well U-bottom or V-bottom plates (Sumitomo Bakelite), and are suitable for use in the present invention.

[0021] In the method for producing fused cell clusters of the present invention, cell clusters are seeded on the front side of a membrane that is permeable to medium components and oxygen, and culture is performed by supplying medium from the back side of the membrane, resulting in fusion of the cell clusters. The size of the cell clusters is preferably 80 to 500 μm in diameter, preferably 80 to 250 μm in diameter, and more preferably 100 to 200 μm in diameter. In the method of the present invention, cell clusters are preferably fused on a cell adhesion surface where cell clusters are arranged at high density. "High density" refers to, for example, a density of 18,000 ± 6,000 cell clusters per ml of space (18,000 ± 6,000 cells / ml) when the size of the cell clusters is approximately 100 to 200 μm. The number of cell clusters should be two or more; increasing the number of cell clusters allows for the production of larger fused cell clusters. According to the method of the present invention, it is possible to fuse 2,000 or more cell clusters, and even 10,000 or more, 20,000 or more, 50,000 or more, 100,000 or more, 120,000 or more, and 500,000 or more cell clusters. The upper limit of the number of cell clusters to be fused is thought to be about 1,000,000. Therefore, according to the method of the present invention, it is possible to fuse 2,000 to 4,000 cell clusters, 10,000 to 20,000 cell clusters, 20,000 to 40,000 cell clusters, 50,000 to 100,000 cell clusters, 100,000 to 200,000 cell clusters, 120,000 to 240,000 cell clusters, and 500,000 to 1,000,000 cell clusters. The medium may be any medium suitable for culturing cell clusters. For example, when the cell clusters are hepatoblasts, preferred media include a medium prepared by mixing EGM BulletKit (manufactured by Lonza) and HCM BulletKit (manufactured by Lonza) in a 1:1 ratio, with the exception of hEGF (recombinant human epidermal growth factor), and adding Dexamethasone, Oncostatin M, and HGF; a medium prepared by mixing EGM BulletKit (manufactured by Lonza) and VascuLife EnGS Comp Kit (manufactured by LCT) in a 1:1 ratio; and a medium prepared by mixing EGM BulletKit (manufactured by Lonza) and Endothelial Cell Growth Medium MV (manufactured by LCT) in a 1:1 ratio."Fusion of cell clusters" refers to the formation of a continuous structure by multiple cell clusters, and the fused cell clusters can self-organize internally to form connected vascular structures. The fusion of cell clusters not only increases the size of the cell clusters, but also allows a vascular network structure to form in the cell cluster, further develops the vascular network structure, and can improve the function of the cell cluster.

[0022] Fusion of cell clusters is carried out on a membrane permeable to medium components and oxygen. A porous membrane structure is believed to be advantageous for post-fusion culture, allowing nutrients and oxygen to be supplied to the fused cell clusters from above and below. Examples of membranes permeable to medium components and oxygen include those negatively charged and hydrophilic by atmospheric corona discharge or vacuum gas plasma polymerization (cell adhesion surface treatment), those with gelatinized surfaces, those coated with extracellular matrix (e.g., collagen, laminin, fibronectin, etc.) or mucopolysaccharides (e.g., heparin sulfate, hyaluronic acid, chondroitin sulfate, etc.), those coated with basic synthetic polymers (e.g., poly-D-lysine), those with synthetic nanofiber surfaces, those with a hydrophilic, neutral hydrogel layer surface, and collagen membranes (Koken). When the membrane permeable to medium components and oxygen has a porous membrane structure, the pore size should be 0.4 to 8 μm. Examples of cultureware with a membrane that allows the permeability of medium components and oxygen include Falcon cell culture inserts (Corning), Millicell cell culture inserts (Merck), cell culture inserts (Thermo Fisher Scientific), and FibColl highly permeable atelocollagen inserts (Koken). The membrane diameter (φ) should be 3 cm or more, 5 cm or more, 6 cm or more, or 11 cm or more.

[0023] In the method of the present invention, cell aggregates are preferably cultured with the front surface of a membrane permeable to medium components and oxygen exposed to the atmosphere (air-liquid interface culture). Air-liquid interface (ALI) culture originated in research on the thymus and has since been used extensively in culturing tissues exposed to air, such as the skin and lungs. Developmental biology 1960 Jun;2:271-84. Morphogenetic interactions in the development of the mouse thymus gland R AUERBACH PMID: 13795076, DOI: 10.1016 / 0012-1606(60)90009-9 cultured epithelial tissue isolated from the mouse thymus using ALI (Fig. 2). The Journal of investigative dermatology. 1983 Jul;81(1 Suppl):28s-33s. Methods for cultivation of keratinocytes with an air-liquid interface M Prunieras, M Regnier, D Woodley PMID: 6190962, DOI: 10.1111 / 1523-1747.ep12540324 is a review discussing ALI culture of skin epidermal cells (keratinocytes). Transplantation 1984 May;37(5):499-503. Survival of human fetal pancreatic explants in organ culture as reflected in insulin secretion and oxygen consumption A Andersson, N Christensen, CG Groth, C Hellerstrom, B Petersson, S Sandler PMID: 6375004, DOI: 10.1097 / 00007890-198405000-00015. However, no positive results have been obtained with ALI culture.A paper has been published in recent years stating that hypoxia is better for the pancreas. The culture temperature for cell cluster fusion is preferably 25 to 37°C, and more preferably 37°C. The culture period for cell cluster fusion is preferably 1 to 10 days, and more preferably 1 to 8 days.

[0024] According to the present invention, large cell masses can be formed without using a support such as Matrigel. The method of the present invention allows the production of large cell masses with diameters (φ) of 3 cm or more, 6 cm or more, or 11 cm or more. For example, in the case of hepatic organoids (size (diameter) approximately 100-200 μm) formed from hepatic endoderm cells (HE), vascular endothelial cells (EC), and mesenchymal cells (MC) (ratio of 10:4:4), a large fused hepatic organoid with a diameter of 3 cm can be produced in a volume of 2.4 × 10 4 (The number of HEs at the time of preparation was 2.0 × 10 7 Large fused liver organoids with a diameter of 6 cm can be generated from 1.2 × 10 5 (The number of HEs at the time of preparation was 1.0 × 10 8 Large fused liver organoids with a diameter of 11 cm can be generated from 5.0 × 10 5 (The number of HEs at the time of preparation was 4.2 × 10 8Hepatic organoids can be produced from hepatic organoids (cells). The thickness of large cell clusters produced by the methods of the present invention can be approximately 100-1000 μm. When the large cell clusters are large fused hepatic organoids, their thickness can be 200-400 μm. Large organoids with a diameter of 5 mm or more can be produced using culture devices such as cell culture inserts. However, when producing large organoids with a diameter of more than 5 cm using cell culture inserts, organoid contraction frequently occurs. This is thought to be due to the cell culture insert membrane bending due to the weight of the seeded cell clusters, causing the cell clusters to aggregate in the center. In the method for producing large cell clusters of the present invention, the back surface of the membrane, which is permeable to medium components and oxygen, is supported by a support structure, thereby suppressing membrane bending due to the weight of the seeded cell clusters and the resulting accumulation of cell clusters in the center of the membrane. When using cell culture inserts, it is recommended to use improved cell culture inserts in which a support structure is added to or integrated with the bottom surface of the membrane to prevent the cell culture insert membrane from bending under the weight of seeded cells, etc. Use of such improved cell culture inserts suppresses bending of the membrane during the preparation of large cell clusters and prevents shrinkage of large cell clusters during culture. By supporting the back surface of the membrane, which is permeable to medium components and oxygen, on a support structure, the shrinkage rate of the cell clusters can be suppressed to 30% or less, preferably 5% or less, and more preferably 0% or less. The shrinkage rate of the cell clusters can be measured as follows: (Shrinkage rate) = (area of ​​large cell clusters viewed from directly above) ÷ (culturable membrane area) x 100. If the cell clusters shrink, cells in the shrinking areas may die due to a lack of oxygen and nutrients, resulting in functional impairment. Furthermore, shrinkage may cause the large cell clusters to become unusable due to an inappropriate shape.

[0025] The present invention also provides an incubator having a main body with a membrane at the bottom, permeable to medium components and oxygen, on the front surface of which a culture medium is seeded, and in which culture medium is supplied into the main body from the back side of the membrane, characterized in that a support structure is attached to the bottom surface of the main body to prevent the membrane from bending. Examples of culture medium include cells, cell clusters (spheroids, organoids, etc.), tissues, organs, and individual animals (fetuses and adults). The present invention also provides a support structure for use in the incubator. The support structure may be in point contact, line contact, or surface contact with the back surface of the membrane, or a combination thereof. The support structure may comprise one or more frame members that contact the back surface of the membrane, which is permeable to medium components and oxygen. The frame members may be formed to extend through the center of the back surface of the membrane, which is permeable to medium components and oxygen, to the periphery. The center refers to the center point of the membrane and the area nearby. The peripheral portion refers to the outer periphery of the membrane and the area nearby. The frame member preferably divides the rear surface of the membrane permeable to medium components and oxygen into two or more compartments, with each compartment preferably including a portion of the peripheral portion of the rear surface of the membrane. The frame may have any shape as long as each area divided by the frame includes a portion of a circumferential arc. The member supporting the membrane permeable to medium components and oxygen may be a point instead of a frame. When supported by a point, the point may be located anywhere as long as the membrane remains flat. A very short line may be used instead of a point. The member supporting the membrane permeable to medium components and oxygen may be a combination of a frame and a point. The body of the incubator of the present invention is preferably formed in an approximately cylindrical shape. The incubator may have legs at the bottom of the support structure. Figure 1(A) is a perspective view of an incubator according to an embodiment of the present invention, (B) is a plan view thereof, (C) is a side view thereof, and (D) is a cross-sectional view along line I-I of (C). 2A is a perspective view showing the main body of an incubator according to an embodiment of the present invention, (B) is a plan view thereof, (C) is a side view thereof, and (D) is a cross-sectional view taken along line II-II of (C).Figure 3-1 (A) is a perspective view of a support structure according to an embodiment of the present invention, (B) is a plan view thereof, (C) is a side view thereof, and (D) is a cross-sectional view along line III-III of (C). Figure 3-2 (A) is a perspective view of a rim member of an incubator according to an embodiment of the present invention, (B) is a plan view thereof, (C) is a side view thereof, and (D) is a cross-sectional view along line IV-IV of (C). As shown in Figures 1(A)-(D), an incubator 1 according to an embodiment of the present invention has a main body 3 with a membrane 2 at the bottom that is permeable to medium components and oxygen and on which the culture target (e.g., cell clumps) is seeded on the front surface. A support structure 4 is attached to the bottom of the main body 3 to prevent the membrane 2 from sagging. The incubator is used in an existing cell culture vessel, such as a cell culture dish or a 6-well cell culture plate. A rim member 5 may be attached to the lower inside of the main body 3, for example, by adhesive or the like. After attaching the rim member 5 to the main body 3, the membrane 2 can be attached to the bottom of the main body 3 and the rim member 5. The membrane 2 can be fixed to the main body with adhesive or the like. As shown in Figures 3-2(A)-(D), the rim member 5 can be ring-shaped with a downward inward slope. Attaching a rim member has the following advantages: 1. It prevents large fused cell masses (e.g., large organoids) from spontaneously detaching from the edge of the membrane during culture. In other words, it reduces the risk of shrinkage. 2. It makes it easier to detach large fused cell masses (e.g., large organoids) from the membrane when retrieving them. In other words, it reduces the risk of breakage. 3. It has the effect of enhancing membrane adhesion and fixation. Regarding 2, when retrieving large fused cell masses (e.g., large organoids), a spatula or the like is used to make it easier to physically detach the large fused cell masses (e.g., large organoids) from the membrane. However, since the large fused cell masses (e.g., large organoids) only weakly adhere to the rim member, it is easy to insert a spatula or the like between the rim member and the large fused cell masses (e.g., large organoids). The body, rim member, and membrane are preferably all made of polycarbonate. The polycarbonate membrane allows organoids to adhere and allows observation under a phase-contrast microscope. Even if the membrane and rim member are made of the same polycarbonate, differences in the strength of organoid adhesion are thought to be due to differences in surface smoothness and electrical charge.Typically, the surfaces of the main body, rim member, and support structure are not specially treated, and therefore are not considered to be as smooth as cell culture membranes. On the other hand, the cell culture surfaces of cell culture dishes and cell culture membranes may be altered by plasma discharge treatment or corona discharge treatment to promote cell adhesion and proliferation. However, the materials used for the main body, rim member, and membrane are not necessarily limited to polycarbonate. The incubator 1 performs culture by supplying culture medium into the main body 3 from the back side of the membrane 2. If the culture object is a cell cluster, the cell clusters may fuse together during culture. As shown in Figures 2(A)-(D), the main body 3 is formed in an approximately cylindrical shape. The main body 3 and support structure 4 may or may not be fixed with adhesive or the like. The support structure 4 is configured in point contact, line contact, surface contact, or a combination of these with the back surface of the membrane 2. The frame members of the support structure should be designed to allow for the removal of air bubbles generated under the membrane in the incubator. To achieve this, it is recommended that the outer periphery of the frame members of the support structure not be below the underside of the membrane. Taking the support structure shown in Figure 3-1 as an example, the membrane rests on the radial beams of the frame members, with the outer periphery of the frame members being flush with the membrane. The support structure is made of polycarbonate (PC), for example, but may also be made of other synthetic resins such as polystyrene (PS), polypropylene (PP), and polyethylene (PE). The support structure 4, as shown in Figures 3-1(A)-(D), consists of one or more frame members 4a (four in this embodiment) that make surface or line contact with the back surface of the membrane 2. The frame members 4a are formed to pass through the center of the back surface of the membrane 2 and extend to the periphery. Alternatively, the frame members 4a may divide the back surface of the membrane 2 into two or more compartments, each of which may include a portion of the periphery of the back surface of the membrane 2. The bottom of the support structure 4 may have legs 4b to facilitate the supply of culture medium to the culture object (e.g., cell mass). The culture vessel and the support structure are preferably integrated, and the main body 3 may be adhesively fixed in the order of the rim member 5, membrane 2, and support structure 4. Figures 4(A)-(G) are plan views showing modified examples of the support structure according to the embodiment of the present invention.To prevent the membrane 2 from sagging, the support structure 4 may be configured to be in line or surface contact with the membrane 2, as shown in Figures 4(A)-(D), or in point contact with the membrane 2, as shown in Figures 4(E) and 4(F), or may be configured in a combination of point, line, and surface contact with the membrane 2, as shown in Figure 4(G). According to the incubator 1 of the embodiment of the present invention, the support structure 4 that prevents the membrane 2 from sagging is attached to the bottom surface of the main body 3, so the membrane does not sag due to the weight of the seeded cell clusters, and the cell clusters do not gather in the center. As a result, fused cell clusters with stable size and cell function can be produced.

[0026] When the cell clusters contain vascular endothelial cells, the large cell clusters produced by the method of the present invention can form a vascular network structure. The vascular network structure may be formed before the cell clusters are fused together, or may be formed after the cell clusters are fused together. While the cell clusters are fused together, angiogenesis may further progress. The large cell clusters produced by the method of the present invention can have improved functionality compared to the cell clusters before fusion. For example, when the cell clusters are liver buds (liver organoids), the large fused liver buds can secrete albumin. Furthermore, when the large cell clusters produced by the method of the present invention are liver buds, the large fused liver buds can have macrophage migration ability.

[0027] By transplanting large cell masses into humans or non-human animals, for example, a vascular network is established in the transplanted tissue or organ, blood perfusion begins, and tissues or organs with a highly ordered tissue structure can be created. Thus, large cell masses can be transplanted into non-human animals to produce tissues or organs. Furthermore, large cell masses can be transplanted into humans or non-human animals to regenerate or restore the function of tissues or organs. Examples of animals that can be transplanted include humans, as well as animals used as laboratory animals, pets, working animals, racehorses, fighting dogs, etc., specifically, non-human animals such as mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, and crabs. Furthermore, it is preferable that the non-human animals be immunodeficient to avoid immune rejection reactions. The transplantation site for large cell clusters may be any site as long as it is transplantable, and examples include blood vessels, intracranial space, mesentery, liver, spleen, kidney, subrenal capsule, supraportal vein, etc. By transplanting large cell clusters into a living organism (human, non-human animal, etc.), it is possible to regenerate tissues or organs that have lost or reduced function, and furthermore, if human tissues or organs can be produced in non-human animals, they can be used in drug discovery screening.

[0028] Alternatively, the large cell masses may be cultured in vitro to further improve their function and used as organ analogs or organs in human biology, regenerative medicine, drug discovery screening, and the like.

[0029] By transplanting and engrafting large cell masses into non-human animals, non-human chimeric animals can be produced. Non-human animals (e.g., mice) transplanted with large cell masses can mimic the physiological functions of the species (e.g., humans) from which the cell masses originated.

[0030] Furthermore, drugs can be evaluated using at least one selected from the group consisting of large cell clusters, tissues and organs produced from large cell clusters, and non-human chimeric animals transplanted with large cell clusters. Drug evaluations can include, for example, evaluation of drug metabolism (e.g., prediction of drug metabolic profiles), efficacy evaluation (e.g., screening for drugs effective as pharmaceuticals), toxicity evaluation, drug interaction evaluation, etc.

[0031] Drug metabolism can be evaluated by administering a drug candidate compound to large cell masses obtained by fusing cell masses produced from cells derived from humans or non-human animals, tissues and organs produced from large cell masses, or non-human chimeric animals transplanted with large cell masses, and then collecting and analyzing biological samples to obtain a human-type drug metabolism profile. This makes it possible to predict the distribution, metabolism, and excretion processes of drugs in humans, which was extremely difficult to achieve with conventional technology, and is expected to dramatically accelerate the development of safe and effective drugs.

[0032] Screening for drugs effective as pharmaceuticals can be performed by administering novel drug candidate compounds to large cell masses obtained by fusing cell masses produced from cells derived from humans or non-human animals, tissues and organs produced from large cell masses, or non-human chimeric animals transplanted with large cell masses. This is expected to significantly improve the accuracy of predicting drug efficacy when administered to actual humans, which was previously insufficient in conventional in vitro tests.

[0033] Toxicity assessment can improve the accuracy of damage prediction by administering a test substance to large cell clusters obtained by fusing cell clusters prepared from cells derived from humans or non-human animals, tissues and organs prepared from large cell clusters, or non-human chimeric animals transplanted with large cell clusters, and then measuring tissue damage markers, etc.

[0034] Drug interaction evaluation can be performed by administering multiple drugs to a large cell mass obtained by fusing cell masses prepared from cells derived from humans or non-human animals, to tissues and organs prepared from large cell masses, or to a non-human chimeric animal into which a large cell mass has been transplanted, and then evaluating the pharmacokinetics, toxicity, and efficacy of each drug, including the distribution, metabolism, and excretion processes of each drug.

[0035] Large cell masses produced by the method of the present invention can be used as active ingredients in compositions for regenerative medicine.

[0036] The regenerative medicine composition can be transplanted into the body of a human or non-human animal to produce tissue or organs, and can also be transplanted into the body to regenerate or restore the function of tissue or organs.

[0037] After the regenerative medicine composition is transplanted into a human or non-human animal body, the large cell masses can differentiate into tissues or organs with a vascular network. Blood perfusion can occur in the vascular network. It is believed that the generation of blood perfusion in the vascular network makes it possible to create tissues and organs with a highly ordered tissue structure equivalent to or close to that of adult tissue.

[0038] The regenerative medicine composition may contain tissue vascularization promoters such as FGF2, HGF, and VEGF, gelatin sponge (trade name: Spongel, Astellas Inc.) for hemostasis during transplantation, and tissue adhesives such as Bolheal (Teijin Pharma Limited), Veriplast (CSL Behring Co., Ltd.), and Tachoseal (CSL Behring Co., Ltd.) used to fix transplanted tissue, collagen, and Matrigel.

[0039] The present invention will be described in more detail below with reference to the following examples. (Example 1) Human iPS cells (QHJI01s04) were induced to differentiate into hepatic endoderm cells, vascular endothelial cells, and mesenchymal cells according to the method of Kamishibahara et al. (1). The resulting hepatic endoderm cells, vascular endothelial cells, and mesenchymal cells were mixed in a culture medium containing DMEM (GIBCO) with 50% KBM VEC-1 (Kohjin Bio), 2.5% fetal bovine serum (BioWest), 50 nM dexamethasone (Sigma), 20 ng / ml oncostatin M (R&D), and 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries) at a ratio of 10:4:4, and 4.5 x 10 6Cells / well were seeded into 40 wells of an Elplasia 6-well plate (Corning). The next day, the formed cell clusters (liver organoids) were collected and seeded onto collagen I-coated cell culture inserts (Fig. 1, membrane diameter 6 cm). From the next day, the medium was replaced every other day with a culture medium containing DMEM (GIBCO) mixed with 50% KBM VEC-1 (Kohjin Bio), 2.5% fetal bovine serum (BioWest), 50 nM dexamethasone (Sigma), and 20 ng / ml oncostatin M (R&D). The cells were cultured at the air-liquid interface for 8 days to form large fused liver organoids (analysis of photographed images showed an area of ​​approximately 20 cm). 2 ) were obtained. To verify the effectiveness of cell culture inserts with membrane supports, we compared them with cell culture inserts of the same culture area without membrane supports (Figure 5). When cell culture inserts without membrane supports were used, shrinkage of 89.9 ± 4.7% (mean ± SEM, n = 3) was observed, while when cell culture inserts with membrane supports were used, shrinkage was reduced to 4.9 ± 1.6% (mean ± SEM, n = 4) (Mann-Whitney U test, * p < 0.05). These results demonstrate that cell culture inserts with membrane supports reduce shrinkage during culture and enable the generation of stable, large-scale organoids. (1) Kamishibahara Y et al. Stabilized generation of human iPSC-derived liver organoids using a modified coating approach. Biology Methods & Protocols, 8(1):bpac034, 2023

[0040] (Example 2) Histological analysis of large fused hepatic organoids was performed. The large fused hepatic organoids obtained by the method of Example 1 were fixed with 4% paraformaldehyde, embedded in paraffin, and then thinly sliced ​​with a microtome and stained with hematoxylin-eosin. Numerous hepatocyte-like cell clusters were observed. Furthermore, staining with anti-human CD31 antibody revealed numerous human CD31-positive luminal structures, confirming the formation of vascular structures (Figure 6). Furthermore, the human albumin level in the culture supernatant of large fused hepatic organoids cultured on cell culture inserts for 8 days was measured using an ELISA kit (Bethyl), and was found to be 5998±2133 ng / 10 6 Human albumin secretion into the supernatant was observed at a rate of 100 cells / 24 hours (mean ± SEM, n=4). These results demonstrate that large fused hepatic organoids generated using cell culture inserts with membrane-supported structures are capable of forming mature liver tissue.

[0041] Example 3: Macrophage migration ability of large fused hepatic organoids was evaluated. First, large fused hepatic organoids were excised using a Φ8mm trephine (KAI). Using a 6-well plate cell culture insert system, large fused hepatic organoid pieces were placed in the lower layer, and THP-1 cells (GFP-expressing strain) induced to differentiate into macrophages were seeded, and the migration ability of THP-1 cells was evaluated by co-culture. As a result, compared to the control (assay using THP-1 cells only, without co-culture with large fused organoids), co-culture with large fused hepatic organoid pieces increased the migration ability of THP-1 cells (Mann-Whitney U test, *p<0.05) (Figure 7). This indicates that large fused hepatic organoids prepared using the cell culture insert of the present invention have macrophage migration ability.

[0042] Example 4: To confirm the equivalence of large fused hepatic organoids and small fused hepatic organoids produced by conventional methods, single-cell RNA sequencing analysis was performed on both. Small fused hepatic organoids produced by conventional methods were produced according to a previously reported method (Tadokoro T, Murata S, Kato M. et al., Sci Transl Med 2024; 16:eadg0338.). Specifically, hepatic endoderm cells, vascular endothelial cells, and mesenchymal cells induced from human iPS cells (QHJI01s04) were allowed to form cell clusters on Elplasia plates as in Example 1, and then seeded into culture insert wells (ibidi) placed on the membrane of a 6-well cell culture insert (Falcon). The cell cluster seeding volume per culture insert well was 1 / 100 of the volume of large hepatic organoids. Other culture conditions, such as the medium used and culture period, were the same for both. Single-cell RNA sequencing data obtained from large fused liver organoids (LfLB) and small fused liver organoids (SfLB) were processed using CellRanger, followed by UMAP analysis and cell type clustering using gene expression as an indicator using Seurat (Figures 8 and 9). No bias was observed between samples in the UMAP plot (Figure 8). Furthermore, comparison of the cell content between each cell cluster in large and small fused liver organoids revealed no clear differences between the two (Figure 10). These results suggest that the large fused liver organoids generated by the method of the present invention are comparable to small fused liver organoids generated by conventional methods. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

[0043] The present invention can be used in human biology, regenerative medicine, drug discovery screening, and the like.

Claims

1. A large fused cell mass produced by fusing cell masses in vitro, with a diameter of 3 cm or more.

2. The large fused cell cluster according to claim 1, wherein the cell cluster is an organ bud.

3. The large fused cell mass according to claim 2, wherein the organ bud is formed from tissue or organ cells, mesenchymal cells and vascular cells.

4. The large fused cell mass according to claim 3, wherein the ratio of tissue or organ cells, mesenchymal cells and vascular cells is 10:0.1-10:0.1-10.

5. The large fused cell mass according to any one of claims 2 to 4, wherein the organ bud is formed in a culture vessel having a non-cell adhesive surface.

6. The large fused cell mass according to any one of claims 2 to 5, wherein the fused organ bud forms a vascular structure.

7. A method for producing large fused cell clusters according to claim 1, comprising seeding cell clusters on the front surface of a membrane that is permeable to culture medium components and oxygen, supplying culture medium from the back side of the membrane to carry out cultivation, and fusing the cell clusters together, wherein the back surface of the membrane is supported by a support structure that prevents the membrane from bending due to the weight of the seeded cell clusters.

8. The method according to claim 7, wherein the cell mass is cultured with the front surface of the membrane exposed to the atmosphere.

9. The method according to claim 7 or 8, wherein the membrane has a diameter of 3 cm or more.

10. The method according to any one of claims 7 to 9, wherein the cell clusters to be fused have a diameter of 80 to 500 µm and number of 2000 or more.

11. An incubator having a body with a membrane at the bottom that is permeable to the components of the culture medium and oxygen and on which the culture subject is seeded on the front surface, in which culture medium is supplied into the body from the back side of the membrane to perform culture, characterized in that a support structure that prevents the membrane from bending is attached to the bottom surface of the body.

12. The incubator according to claim 11, wherein the support structure is in point contact, line contact, or surface contact with the back surface of the membrane, or a combination thereof.

13. The incubator of claim 11, wherein the support structure comprises one or more frame members that contact the back surface of the membrane.

14. The incubator according to claim 13, wherein the frame member is formed so as to pass through the center of the back surface of the membrane and extend to the periphery.

15. The incubator according to claim 14, wherein the frame member divides the back surface of the membrane into two or more compartments, each of the compartments being formed to include a portion of the periphery of the back surface of the membrane.

16. The incubator according to any one of claims 11 to 15, characterized in that the main body is formed in a substantially cylindrical shape.

17. The incubator according to any one of claims 11 to 16, characterized in that the support structure has legs at its bottom.

18. A support structure for use in the incubator according to any one of claims 11 to 17.

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