Method for creating human large organ with which vascular anastomosis can be performed by perfusion culture

Abstract

According to the present invention, a human large organ is created by perfusion culture through a large blood vessel by using an organoid attached to the large blood vessel. An artificial organ according to the present invention includes a large blood vessel and an organoid and can be transplanted by surgical vascular anastomosis, wherein the organoid is disposed adjacent to the large blood vessel, the large blood vessel has neovascularization ability, a blood vessel structure is formed in the organoid, and a blood vessel within the organoid and the large blood vessel are connected, enabling perfusion to the organoid via the large blood vessel.

Description

Creation of large human organs with vascular anastomosis capability by perfusion culture

[0001] The present invention relates to a technology for creating large human organs capable of vascular anastomosis by perfusion culture of vascularized organoids endowed with large blood vessels through the large blood vessels, thereby increasing the number of organ-constituting cells.

[0002] In recent years, three-dimensional cell culture methods using various epithelial cells, such as those from the intestine and liver, have been established for in vitro cell culture [Non-Patent Documents 1 and 2].

[0003] Following this technology, a technique for producing organoids with microvascular networks of 5-20 μm in diameter was developed by mixing organ precursor cells with vascular endothelial cells and mesenchymal cells and inducing autonomous cell aggregation on Matrigel [Non-Patent Document 3]. Furthermore, a technique for culturing large blood vessels with diameters of mm was developed [Patent Document 1].

[0004] Most tissues and organs in the body have a vascular system that supplies blood cells, oxygen, nutrients, etc., and removes waste products, etc. In an attempt to mimic this, perfusion culture using microfluidic devices has been actively researched, and its effectiveness in maintaining and growing cells has been demonstrated [Non-Patent Documents 4 and 5]. It has also been reported that perfusion is effective in maintaining the function of tissues removed from the body, and research into the preservation of human and porcine organs is also progressing [Non-Patent Documents 6 and 7].

[0005] Sato T et al., Nature 459, 262-265, 2009Huch M et al., Cell 160, 299-312, 2015Takebe T et al., Nature 499, 481-484, 2013Du Y et al., Lab Chip 17, 782-794, 2017Wang Y et al., Lab Chip 18, 3606-3616, 2018Nasralla D et al., Nature 557, 50-56, 2018Hozain AE et al., Nature, 2020

[0006] WO2019 / 087988

[0007] In the culture of organoids and cell aggregates, due to their requirement for nutrients and oxygen, they must be kept to a size of several hundred micrometers in diameter in order to avoid internal necrosis and enable long-term culture [Br. J. Cancer 53, 345-353, 1986]. Therefore, the introduction of perfusion culture is considered to be extremely useful for creating large organs.

[0008] On the other hand, microfluidic devices commonly used for perfusion culture are often closed systems, posing challenges in adapting to changes in organ size and in adapting enlarged organs for other applications after perfusion culture, such as transplantation.

[0009] The present invention aims to create large human organs by perfusion culture through large blood vessels using vascularized organoids endowed with large blood vessels.

[0010] The inventors constructed an open perfusion culture system, rather than a closed system, to accommodate changes in organ size, a problem posed by conventional microfluidic devices. Furthermore, to create transplantable replacement human organs in the future and ensure their immediate function after transplantation, organ transplantation via surgical vascular anastomosis is required as an application following perfusion culture. Therefore, they created large human organs containing human aortas that can be surgically anastomotically attached.

[0011] The gist of the present invention is as follows: (1) An artificial organ that can be transplanted by surgical vascular anastomosis, comprising a large blood vessel and an organoid, wherein the organoid is placed adjacent to the large blood vessel, the large blood vessel has angiogenic ability, the organoid has a vascular structure formed therein, and the blood vessels in the organoid are connected to the large blood vessel, allowing perfusion to the organoid via the large blood vessel. (2) A method for producing the artificial organ described in (1), comprising placing an organoid with angiogenic ability adjacent to the large blood vessel and then performing perfusion culture. (3) The method described in (2), comprising puncturing the large blood vessel before placing the organoid adjacent to the large blood vessel with angiogenic ability. (4) An artificial large blood vessel with angiogenic ability, comprising a tubular support (scaffold) made of a biocompatible material and seeded with smooth muscle cells and endothelial cells.

[0012] The present invention can increase the tissue mass and enhance the function of large vascularized organoids. Also, the present invention provides human large vascularized organoids that can be surgically anastomotically transplanted. This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2024-14943, which is the priority basis of this application.

[0013] This figure shows the scheme for generating fused liver buds with large blood vessels. The diagram shows the vascular structures formed in the liver buds at each stage of the scheme. Functional improvement of liver organoids through perfusion culture through large blood vessels. (A) Schematic of a new method for incorporating large blood vessels (BVs) into hiPSC liver organoids (LOs) under 1g conditions. Small hiPSC-LOs were fused on a cell culture insert, and the fused hiPSC-LOs were placed near a punctured rat femoral artery supported by a metal pipe. (B) Fluorescence image of fused hiPSC-LOs cultured for 2 days. Green: hepatic endoderm (HE). Red: endothelial cells (EC). (C) Fluorescence image of hiPSC-LOs with large BVs cultured for 5 days. Green: HE. Red: EC. (D) Hematoxylin and eosin (H&E) staining of fused hiPSC-LOs. (E) H&E staining of hiPSC-LOs with large BVs. (F) Immunofluorescence images of hiPSC-LOs containing large BVs. Green: GFP-positive cells derived from rat BVs. Red: ECs. White: human nuclei. (G) Human albumin secretion in fused LOs and hiPSC-LOs containing large BVs (n=4). (H) Schematic of the perfusion culture system (upper panel). Stereoscopic image of hiPSC-LOs containing large BVs after India ink perfusion (lower panel). (I) Fluorescence images of hiPSC-LOs containing large BVs before and after perfusion culture. Red: ECs. (J) Quantification of tissue size increase in hiPSC-LOs containing large BVs during perfusion culture (n=9-19). (K) Immunofluorescence images of hiPSC-LOs containing large BVs with and without perfusion. Green: hALBs. Red: HNF4a-positive cells (top), CK19-positive cells (middle), and CD31-positive cells (bottom). (L) Quantification of the area occupied by HNF4a-positive, hALB-positive, CK19-positive, and CD31-positive cells (n=4). (MO) Functional analysis of rat BV-loaded hiPSC-LOs in the perfusion (-) and perfusion (+) groups (n=5-20). Bar graphs represent mean ± SEM. Unless otherwise stated, the Mann-Whitney U test was used for statistical analysis. Enhancement of hepatic organoid function by the addition of hiPSC-derived large blood vessels. (A) Fluorescence images of hiPSC-BVs after 10 days of culture. Red: endothelial cells (ECs). Quantification of the length and number of newly formed blood vessels is shown in the graph (n=4).(B) Stereoscopic and fluorescent images of hiPSC-large BV-loaded hiPSC-LO. Red: EC. (C) Fluorescent images of hiPSC-large BV-loaded hiPSC-LO before and after perfusion culture. Red: EC. (D) Quantification of the size increase of hiPSC-large BV-loaded hiPSC-LO during perfusion culture (n=22). (E) Stereoscopic image of hiPSC-large BV-loaded hiPSC-LO after India ink perfusion (left) and histological images of hiPSC-LO blood vessels (middle and right). EC were stained with India ink (black arrows). (F) Immunohistochemical analysis of hiPSC-large BV-loaded hiPSC-LO with and without perfusion. Upper panel: CD31. Middle panel: hALB. Lower panel: CK19. (G) Quantification of the area occupied by CD31-positive, hALB-positive, and CK19-positive cells (n=6-10). (H-J) Functional analysis of hiPSC-BV-loaded hiPSC-LO in the perfusion (-) and perfusion (+) groups (n=7-22). (K) H&E staining (left) and immunofluorescence image (right) of the mesothelium of hiPSC-LO. Green: HNF4a. Red: podoplanin (PDPN). (L) Immunofluorescence image of blood vessels in hiPSC-LO in the perfusion (-) and perfusion (+) groups. Green: activated integrin β1. Red: CD31. (M) Quantification of the number of BVs in the perfusion (-) and perfusion (+) groups (n=4). (N) ELISA analysis of phospho-VEGFR3 in the perfusion (-) and perfusion (+) groups (n=5). (O) Quantitative RT-PCR analysis of HGF expression in the perfusion (-) and perfusion (+) groups (n=10-11). Bar graphs represent mean ± SEM. Unless otherwise stated, the Mann-Whitney U test was used for statistical analysis. Transplantation of large vessel-loaded hepatic organoids via vascular anastomosis. (A) Schematic diagram of transplantation of rat BV-loaded hiPSC-LO into the femoral artery of an immunodeficient rat (left). Stereomicroscope images of the host rat artery and rat BV-loaded hiPSC-LO before anastomosis (middle), and one side of the rat BV-loaded hiPSC-LO anastomosed to the host rat artery (right). (B) Stereomicroscope images of transplanted rat BV-loaded hiPSC-LO before (left) and after (middle) blood perfusion, and 2 weeks after transplantation (Tx) (right). (C) Immunofluorescence images of hepatocytes from hiPSC-LO before and after Tx. White: CK8 / 18. Red: hALB. (D) H&E staining of hiPSC-LO after Tx.(E) Immunofluorescence image of hiPSC-LO after transfection. Green: hALB. Red: HNF4a (left), CK19 (center), CD31 (right). (F) hALB secretion after transfection of rat BV-loaded hiPSC-LO. (G) Stereomicroscope images of transplanted hiPSC-BV-loaded hiPSC-LO before (left) and after (right) perfusion. (H) Magnified image of the surface of hiPSC-LO after perfusion. Black arrows indicate blood cells. (I) Stereomicroscope image of hiPSC-BV-loaded hiPSC-LO one week after transfection. (J) hALB and hfibrinogen secretion after transfection of hiPSC-BV-loaded hiPSC-LO. Unpaired t-test was used for statistical analysis (n=3). (K) H&E staining of hiPSC-LO after transfection (left). The graph shows quantification of blood cell localization (right). (LQ) Immunofluorescence images of hiPSC-LO after transfusion. Green: hALB. Red: CD31 (L), CK19 (M), hCYP3A4 (N), hA1AT (O), MRP2 (P), ZO-1 (Q). Bars represent mean ± SEM. Unless otherwise stated, the Mann-Whitney U test was used for statistical analysis. Perfusion culture of rat large blood vessel (BV)-embedded pluripotent stem cells (hiPSC)-liver organoids (LO). (A) Tissue size of large BV-embedded hiPSC-LO. (B) Schematic of the perfusion culture system. Four sets of rat large BV-embedded hiPSC-LO for perfusion culture and enlarged images of rat large BV-embedded hiPSC-LO on a 3D-printed perfusion culture jig are shown. (C) Quantitative RT-PCR analysis of rat large BV-containing hiPSC-LOs with and without perfusion. Characteristics of human pluripotent stem cell (hiPSC)-derived large blood vessels (BVs). (A) Phase-contrast images of hiPSCs, hiPSC-derived mesenchymal progenitor cells, and hiPSC-derived smooth muscle cells (SMCs). (B) Quantitative RT-PCR analysis during SMC differentiation. Human umbilical artery-derived SMCs (HUASMCs) were used as a positive control. (C) Schematic diagram of hiPSC-derived large BV generation and angiogenesis assay. hiPSC-SMCs and endothelial cells (ECs) were seeded into polyglycolic acid tubes (hiPSC-large BVs).The hiPSC-large BVs were embedded in a collagen / Matrigel (1:1) mixture and cultured for 10 days to observe angiogenesis. Characteristics of hiPSC-large vessel (BV)-loaded human pluripotent stem cell (hiPSC)-liver organoids (LO). (A) Electron microscopy images of hepatocytes (left), rough endoplasmic reticulum and mitochondria (center), and bile canaliculi formed between hepatocytes (right) within hiPSC-LO. (B) Gene ontology analysis of genes upregulated in the perfusion (+) group compared with the perfusion (-) group. (C) Quantitative RT-PCR analysis of hiPSC-LO loaded with hiPSC-BVs with and without perfusion. Schematic diagram of the mechanisms underlying the proliferation of human pluripotent stem cell (hiPSC)-liver organoids (LO) cells in response to perfusion culture through large vessels. VEGFR3 and integrin β1 expressed on endothelial cells (ECs) are activated by sensing the perfusion medium, inducing HGF expression, promoting hepatocyte proliferation and increasing angiogenesis. Effect of puncture treatment on large blood vessels. Fused human iPSC-derived liver buds were attached to mouse aortas (unpunctured group, top) and punctured group (bottom) and cultured for 7 days. Histological analysis images are shown on the right. Human vascular endothelial cells (red), mouse vascular endothelial cells (green), and nuclear staining (blue) are shown. Therapeutic effect of vascular anastomotic transplantation of large BV-containing hiPSC-LOs on acute liver failure. Large BV-containing hiPSC-LOs were surgically transplanted into the femoral artery of rats with acute liver failure due to thioacetamide administration (Tx group: large BV-containing hiPSC-LO transplant group). Sham transplantation groups received large BVs. Survival rates were analyzed using the log-rank test (p = 0.037; n = 8, log-rank test). Mechanism of cell proliferation promotion by perfusion culture. (A) Bulk RNA sequencing analysis confirmed an increase in HGF gene expression in the perfusion group. (B) Results of KEGG ontology enrichment analysis of genes whose expression was increased by 1.5-fold or more in the perfusion group in bulk RNA sequencing analysis. (C) Results of gene ontology enrichment analysis of genes whose expression was increased by 2-fold or more in vascular endothelial cells (ECs) from the perfusion group compared to ECs from the non-perfusion group in single-cell RNA sequencing analysis.

[0014] The present invention will be described in detail below.

[0015] The present invention provides an artificial organ that can be transplanted by surgical vascular anastomosis, comprising a large blood vessel and an organoid, wherein the organoid is positioned adjacent to the large blood vessel, the large blood vessel has angiogenic ability, a vascular structure is formed in the organoid, blood vessels within the organoid are connected to the large blood vessel, and perfusion to the organoid via the large blood vessel is possible.

[0016] In the present invention, the large blood vessels preferably have a diameter of 0.5 mm or more and a length of 3 mm or more, and preferably have a diameter of 1 mm or more and a length of 10 mm or more, with the upper limit of the diameter being approximately 12 mm and the upper limit of the length being approximately 100 mm. The thickness of the large blood vessels is preferably 0.1 mm to 2 mm, and preferably 0.15 mm to 0.5 mm.

[0017] In the present invention, the large blood vessels may be of the same origin as the organoids (homologous), or may be of a different origin from the organoids (heterologous).

[0018] The large blood vessels may have a tubular structure and may be derived from a living organism or may be artificial. The large blood vessels may take the form of a tubular structure. If the large blood vessels are artificial, they may be tubular supports (scaffolds) made from biocompatible materials. Examples of biocompatible materials include metals (e.g., stainless steel, cobalt alloys, titanium alloys, etc.), glass, ceramics, synthetic polymers (e.g., nylon, polypropylene, polydioxanone, polylactic acid, polyethylene terephthalate, Teflon (registered trademark), polyglycolic acid, etc.), and biomaterials (e.g., silk, collagen, gelatin, decellularized tissue). The large blood vessels are preferably blood vessels that can withstand the perfusion pressure of perfusion culture. The upper limit of the perfusion pressure for perfusion culture may be set based on the vascular blood pressure of the organ depending on the type of artificial organ to be produced. Human blood pressure varies significantly depending on whether the blood vessel is arterial or venous. It decreases rapidly with the size of the blood vessel, i.e., the aorta (80-120 mmHg in healthy individuals), arteries (80-120 mmHg to 60-100 mmHg as one approaches the arteriole), arterioles (60-100 mmHg to 15-35 mmHg as one approaches the capillaries), and capillaries (15-35 mmHg to 5-10 mmHg as one approaches the venules), and then gradually decreases from venules to veins and venae (several mmHg). At the start of perfusion culture, blood vessels within organoids and those extending from the large vessels to the organoids are elongating, forming a vascular network and developing. Therefore, perfusion pressure can be close to zero or even a few mmHg to prevent capillary breakdown. As the vascular network is constructed, the perfusion pressure may be gradually increased, increased in steps, or maintained constant at the same perfusion pressure as at the start of perfusion culture. To withstand the perfusion pressure of perfusion culture, large blood vessels are preferably made of synthetic polymers (e.g., polyglycolic acid). By making large blood vessels resistant to perfusion pressure, they can also withstand arterial pressure when transplanted into a living organism. Polyglycolic acid nonwoven fabric is a preferred material for fabricating artificial large blood vessels. The fibers constituting the nonwoven fabric should have a fiber diameter of 1 to 100 μm, preferably 2 to 10 μm, and more preferably 2-5 μm.The thickness of the nonwoven fabric is preferably 1 μm to 3 mm, more preferably 10 μm to 1 mm, and even more preferably 50 to 200 μm. The polyglycolic acid nonwoven fabric may be coated with gelatin nanofibers or polyglycolic acid nanofibers. The nanofibers should have a fiber diameter of 50 to 5,000 nm, preferably 150 to 1,000 nm, more preferably 150 to 500 nm, and even more preferably 150 to 400 nm.

[0019] In the present invention, it is preferable that the large blood vessels have angiogenic potential. Angiogenic potential can be measured by the angiogenesis assay described in the Examples below. Generally, large blood vessels derived from living organisms have angiogenic potential. However, when the large blood vessels are artificial, for example, they can be made to have angiogenic potential by seeding smooth muscle cells (SMCs) and endothelial cells (ECs) into the large blood vessels. The ratio of SMCs to ECs is preferably 1:1 to 3:1. The number of SMCs and ECs seeded into the large blood vessels is 2.5 to 7.5 x 10 per large blood vessel with a diameter of 1 mm, length of 10 mm, and thickness of 0.15 mm. 5 2.5 x 10 SMC 5 It is recommended to sow ECs.

[0020] The smooth muscle cells are preferably vascular smooth muscle cells, and the endothelial cells are preferably vascular endothelial cells.

[0021] SMC and EC have been previously reported (Y. Kamishibahara, S. Okamoto, T. Ohkuma, H. Taniguchi, Stabilized generation of human iPSC-derived liver organoids using a modified coating approach. Biol. Methods Protoc. 8, bpac034 (2023).; C. Patsch, L. Challet-Meylan, EC Thoma, E. Urich, T. Heckel, JF O'Sullivan, S.J. Grainger, FG Kapp, L. Sun, K. Christensen, Y. Iacone, CA SMCs can be prepared according to the method described in Cowan, "Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nat. Cell Biol. 17, 994-1003 (2015)," or by modifications of a previously published method. Briefly, SMCs are prepared by culturing 2-8 x 10 cells. 3 cells / cm 2iPSCs were seeded at a density of 3-5 x 10 cells / well and cultured for 4-6 days in StemFit medium supplemented with Y-27632 (10 μM). The medium was then replaced with DMEM / F12 medium supplemented with 1% Glutamax, 1% B27, CHIR99021 (8 μM), and BMP-4 (25 ng / ml). After 3 days of culture, the medium was replaced with DMEM / F12 medium supplemented with 1% Glutamax, 1% B27, Activin A (2 ng / ml), and PDGFBB (10 ng / ml). After 3 days, the cells were cultured at a density of 3-5 x 10 cells / well. 4 The cells were passaged at a density of 1000 cells / cm² and cultured in DMEM / F12 medium supplemented with heparin (2 ng / ml) and activin A (2 ng / ml) for three days. To prepare ECs, iPSCs were seeded and cultured for one day in StemFit medium supplemented with Y-27632 (10 μM). The next day, the medium was replaced with DMEM / F12 medium supplemented with 1% Glutamax, 1% B27, CHIR99021 (8 μM), and BMP-4 (25 ng / ml). After three days of culture, the medium was replaced with StemPro-34 SFM medium supplemented with VEGF (200 ng / ml) and forskolin (2 μM). Quality was assessed by FACS on day 7 after differentiation induction, confirming the expression of CD31 and CD144. The obtained vascular endothelial cells are passaged and expanded on fibronectin-coated culture dishes using Miracell medium.

[0022] To seed large blood vessels with SMCs and ECs, a suspension of mixed SMCs and ECs can be instilled into the large blood vessels.

[0023] The present invention also provides an angiogenic artificial vascular system, which consists of a tubular support (scaffold) made of a biocompatible material and seeded with smooth muscle cells and endothelial cells. After organoids are placed adjacent to the artificial vascular system, new blood vessels are generated from the artificial vascular system and connect to the blood vessels formed within the organoids, allowing perfusion to the organoids via the artificial vascular system. Perfusion culture of organoids through the artificial vascular system can promote the growth and maturation of organoid tissue. It can also enhance the formation of a vascular network within the organoids. The artificial vascular system may be punctured with a needle. This allows perfusion of culture medium from the artificial vascular system to the fused organoids. Alternatively, it can promote angiogenesis from the artificial vascular system to the fused organoids and perfusion of culture medium through the newly formed blood vessels. The puncture area may be the area where the organoids will be placed. The puncture interval should be 0.15 to 0.3 mm.

[0024] In the present invention, "organoids (organ buds)" refer to structures that can differentiate into organs upon maturation. As an example, WO2013 / 047639 discloses a method for producing organ buds from three types of cells: tissue or organ cells, endothelial cells (e.g., vascular endothelial cells), and mesenchymal cells (e.g., undifferentiated mesenchymal cells such as mesenchymal stem 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 has differentiated 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 described above (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), an organ bud that differentiates into the pancreas (pancreatic bud), or an organ bud that differentiates into the intestinal tract, 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 (an organ bud can be determined if one or more of the marker proteins 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 intestinal tract 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)).

[0025] 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 (if one or more of the marker proteins 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, SCA1, TER119, 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) or 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.

[0026] Endothelial cells can be isolated from vascular tissue such as umbilical vein, but are not limited to cells isolated from vascular tissue. They may also be differentiated from totipotent or pluripotent cells such as iPS cells or ES cells. Vascular endothelial cells are preferred as endothelial cells, and umbilical vein-derived endothelial cells are commercially available and easily available. In the present invention, "vascular endothelial cells" refers 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 such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3, and CD41 (expression of one or more of these marker proteins indicates that the cell is a vascular endothelial cell). 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. 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.

[0027] As used herein, 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, such as Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, and Nestin (expression of one or more of these marker proteins 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.

[0028] A method for producing all three types of cells (tissue or organ cells, endothelial cells, and mesenchymal cells) from iPS cells and 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.

[0029] Briefly, definitive endoderm (DE) cells (tissue or organ cells) can be derived from iPS cells as follows: Cell culture dishes or cell culture plates (Falcon) are filled with iMatrix-511 (Nippi, 0.4-0.6 μg / cm 2 ) at 37°C for 1 hour and then washed with PBS. Human iPSCs were cultured for one week in a 10 cm cell culture dish. After washing with PBS, 2 ml of Accutase was added and treated at 37°C for 5 to 10 minutes to detach the cells. After cell collection and centrifugation, the cell supernatant was removed and the cells were suspended in RPMI medium supplemented with penicillin / streptomycin (1%), B27 (2%), Wnt3a (50 ng / ml), Activin A (100 ng / ml), and Y-27632 (10 μM). 5-10 x 10 cells were plated onto a laminin-coated dish. 4 cells / cm 2The cells were seeded at a density of 1000 x 1000 mm. On days 1 and 3 of culture, the medium was replaced with RPMI medium supplemented with penicillin / streptomycin (1%), B27 (2%), Wnt3a (50 ng / ml), Activin A (100 ng / ml), and sodium butyrate (0.5 mM). On day 4 of culture, the medium was replaced with RPMI medium supplemented with penicillin / streptomycin (1%), B27 (2%), Wnt3a (50 ng / ml), and Activin A (100 ng / ml). On day 6 of culture, the cells were designated as definitive endoderm (DE) cells. On days 6 and 8 of culture, the medium was replaced with RPMI medium supplemented with penicillin / streptomycin (1%), B27 (2%), basic FGF (10 ng / ml), and BMP-4 (20 ng / ml). The cells on day 10 of culture were designated as hepatic endoderm (HE) cells.

[0030] Mesenchymal cells (MCs) can be derived from iPS cells as follows: 2-8 x 10 3 cells / cm 2 iPSCs were seeded at a density of 1.5-2.5 x 10 cells / well and cultured for 4-6 days in StemFit medium supplemented with Y-27632 (10 μM). The medium was replaced with DMEM / F12 medium supplemented with 1% Glutamax, 1% B27, CHIR99021 (8 μM), and BMP-4 (25 ng / ml). After 3 days of culture, the medium was replaced with DMEM / F12 medium supplemented with 1% Glutamax, 1% B27, Activin A (2 ng / ml), and PDGFBB (10 ng / ml). After 3 days, the cells reached a density of 1.5-2.5 x 10 cells / well. 4 cells / cm 2 The cells are subcultured at a density of 1000×1000×1000, and then the medium is replaced with DMEM / F12 medium supplemented with FGF2 (10 ng / ml) and PDGFBB (10 ng / ml), followed by culturing for 3 days.

[0031] The method for inducing endothelial cells (ECs) from iPS cells has been described above.

[0032] The culture ratio of the three types of cells in co-culture is not particularly limited as long as it is within a range that allows the formation of an organ bud, but a suitable cell number ratio is tissue or organ cells:endothelial cells:mesenchymal cells=10:0.1-10:0.1-10. If the number of endothelial cells and mesenchymal cells is low, blood vessel formation becomes difficult. If the number of mesenchymal cells is high, the adhesion between cells becomes stronger than adhesion to surfaces that can be cell-adhered, which often leads to contraction and makes it difficult to fuse cell clusters. If the number of endothelial cells is high, the amount of mesenchymal cells necessary for blood vessel construction becomes insufficient, inhibiting blood vessel formation.

[0033] The ratio (numerical ratio) of mesenchymal cells to endothelial cells is preferably 1:0.01-100, more preferably 1:0.1-10, and even more preferably 1:0.5-2. Co-culturing approximately 230,000 tissue or organ cells, approximately 160,000 endothelial cells, and approximately 160,000 mesenchymal cells can form organ buds measuring approximately 3,500-4,500 micrometers. The higher the proportion of mesenchymal cells, the finer the vascular network structure formed in the organ bud. The numerical ratio of cells suitable for forming a vascular network structure is tissue or organ cells:endothelial cells:mesenchymal cells = 10:1-10:1-10, more preferably 10:2-7:2-7.

[0034] The medium used for co-culturing cells to form organ buds may be any medium that allows organ bud formation, but is preferably a medium for vascular endothelial cell culture, a medium for tissue or organ cell culture, a mixture of the two media, etc. Any medium may be used for vascular endothelial cell culture, but it is preferable to use one that contains 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 (Lonza), EGM Bullet Kit (Lonza), VascuLife EnGS Comp Kit (LCT), Human Endothelial-SFM Basal Growth Medium (Invitrogen), and Human Microvascular Endothelial Cell Growth Medium (TOYOBO). Any media can be used for tissue or organ cell culture; however, 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. Examples of media that can be used for hepatocyte culture include HCM Bullet Kit (Lonza) minus hEGF (recombinant human epidermal growth factor), and RPMI 1640 (Sigma-Aldrich) supplemented with 1% B27 Supplements (GIBCO) and 10 ng / mL hHGF (Sigma-Aldrich). Regarding the formation of human liver buds, it has been found that adding dexamethasone, oncostatin M, and HGF to a 1:1 mixture of EGM BulletKit (manufactured by Lonza) and HCM BulletKit (manufactured by Lonza) minus hEGF (recombinant human epidermal growth factor) is effective in promoting liver bud maturation.

[0035] The temperature for culturing cells to form organ buds is preferably 30 to 40°C, more preferably 37°C.

[0036] The cell culture period for organ bud formation is preferably half a day to seven days, and more preferably one to two days. The organ bud does not need to have a vascular network (Figure 1B). In the present invention, the organ bud may be produced without using a cell-adherent surface such as a matrix (matrix-free), for example, in a culture vessel having a cell-nonadherent surface.

[0037] The cultureware having a non-cell-adhesive surface is preferably one that has been subjected to a low-adsorption surface treatment, for example, one in which the culture surface is coated with a non-cell-adhesive polymer. Examples of non-cell-adhesive polymers include, but are not limited to, phospholipids, phospholipid-polymer complexes, poly(2-hydroxyethyl methacrylate) (PHEMA), polyvinyl alcohol, agarose, chitosan, polyethylene glycol, albumin, and photocrosslinked superhydrophilic polymers.

[0038] The bottom of the culture vessel may have a large number of hemispherical or truncated cone-shaped depressions. For example, the bottom of the culture vessel may have a large number of hemispherical or truncated cone-shaped depressions (the volume of the depression is 0.068 mm 3 A 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 RB 500 400 NA (Kuraray) and 96-well U-bottom or V-bottom plates (Sumitomo Bakelite), and are suitable for use in the present invention.

[0039] In the present invention, the organoids (organ buds) placed adjacent to the large blood vessels are preferably the fusion of two or more organoids (fused organoids). Increasing the number of organoids allows for the production of larger fused organoids (see WO2019 / 18924). To fuse organoids, the organoids are seeded on a cell-adhesive surface so that the organoids occupy 40-100% of the seeding surface. The organoids preferably occupy 60-100%, more preferably 80-100% of the seeding surface. The organoids' area ratio to the seeding surface can be determined by measuring the projected shadow area of ​​the organoids and calculating the ratio to the area of ​​the seeding surface. The projected shadow area of ​​the organoids can be measured using the following method. The projected shadow area of ​​the organoids is calculated using image analysis software such as FIJI, ImageJ, or Photoshop.

[0040] In the organoid fusion method, organoids can be seeded at high density on a cell-adherent surface, e.g., 1 cm spacing for 150 μm diameter organoids. 3 The number of organoids present per 1000 cells was 9.5 x 10 4 ~3.8 x 10 5 1.9 x 10 5 ~3.8 x 10 5 and more preferably 2.9 x 10 5 ~3.8 x 10 5 There are individuals.

[0041] The size of the organoid is suitably 80 to 500 μm, preferably 100 to 250 μm.

[0042] Any medium suitable for culturing organoids may be used. For example, if the organoids are liver buds, preferred media include a 1:1 mixture of EGM BulletKit (Lonza) and HCM BulletKit (Lonza) minus hEGF (recombinant human epidermal growth factor), to which dexamethasone, oncostatin M, and HGF have been added; a 1:1 mixture of EGM BulletKit (Lonza) and VascuLife EnGS Comp Kit (LCT); and a 1:1 mixture of EGM BulletKit (Lonza) and Endothelial Cell Growth Medium MV (LCT).

[0043] "Organoid fusion" refers to the formation of a continuous structure from multiple organoids, and the fused organoids (fused organoids) can self-organize internally to form connected vascular structures. The fusion of organoids not only increases the size of the organoids, but also allows them to form and further develop vascular networks, and may improve the function of the organoids.

[0044] In producing fused organoids, organoids are seeded on a surface capable of cell adhesion, and cultured while supplying medium from the front and back sides of the seeded surface, thereby fusing the organoids.

[0045] Organoid fusion is preferably performed on a cell-adhesive surface. For example, if the cell-adhesive surface has a porous membrane structure, it is considered advantageous for post-fusion culture in terms of the ability to supply nutrients and oxygen to fused organoids from above and below. Examples of cell-adhesive surfaces include those that have been negatively charged and made hydrophilic by atmospheric corona discharge or vacuum gas plasma polymerization treatment (cell adhesion surface treatment), those with gelatinized surfaces, those coated with extracellular matrix (collagen, laminin, fibronectin, etc.) or mucopolysaccharides (heparin sulfate, hyaluronic acid, chondroitin sulfate, etc.), those coated with basic synthetic polymers (poly-D-lysine, etc.), those with synthetic nanofiber surfaces, those with hydrophilic and neutral hydrogel layer surfaces, and collagen membranes (Koken). When the cell-adhesive surface has a porous membrane structure, the pore size should be 0.4-8 μm. Suitable cultureware with a cell-adherent surface include the Falcon Cell Culture Plate (Corning), Falcon Multi-Cell Culture Plate (Corning), and Falcon Cell Culture Insert (Corning). Culture may be performed by batch culture, semi-batch culture (fed-batch culture), or continuous culture (perfusion culture). Static culture, aerobic culture, agitation culture, shaking culture, or rotary culture may also be used, with static culture being preferred.

[0046] The culture temperature for organoid fusion is preferably 25 to 37°C, more preferably 37°C.

[0047] The culture period for organoid fusion is preferably 0.5 to 7 days, more preferably 2 to 3 days. The fused organoids located adjacent to large blood vessels are in the middle stage of vascular network formation (Figure 1B).

[0048] The above method allows for the formation of large organoids without the use of a support such as Matrigel.

[0049] By the above method, fusion organoids of φ100 μm or more, φ1 mm or more, φ2 mm or more, φ2.5 mm or more, φ4 mm or more, φ6 mm or more, and φ8 mm or more can be produced. Fusion organoids of φ100 μm or more, φ1 mm or more, φ2 mm or more, φ2.5 mm or more, φ4 mm or more, φ6 mm or more, and φ8 mm or more can be produced from organoids of about 80 to 150 μm in size, 2 to 4, 150 to 200, 300 to 400, 350 to 500, 600 to 800, 1200 to 1600, and 2400 to 2800, respectively.

[0050] The fused organoids may have improved function compared to the unfused organoids. For example, if the organoids are liver buds, the fused liver buds may have higher gene expression levels of hepatic differentiation markers (e.g., FoxA2, ​​AFP, CYP3A7, CYP7A1) than the unfused liver buds. Furthermore, the liver function may be improved compared to the unfused liver buds. For example, the fused liver buds may have higher gene expression levels of hepatic differentiation markers (e.g., ALB, OTC, CYP3A7, GLUT2), albumin production, transferrin production, and ammonia metabolism compared to the unfused liver buds.

[0051] In the present invention, organoids are placed adjacent to large blood vessels with angiogenic potential, and then perfusion culture is performed to produce artificial organs that can be transplanted by surgical vascular anastomosis.

[0052] The organoids placed adjacent to the large blood vessels may be organoids before fusing organoids together, or may be fused organoids formed by fusing organoids together, but fused organoids are preferred. Before placing the organoids adjacent to the large blood vessels with angiogenic capacity, the large blood vessels may be punctured with a needle. Puncturing the large blood vessels allows perfusion of culture medium from the large blood vessels to the fused organoids. Alternatively, angiogenesis from the large blood vessels to the fused organoids and perfusion of culture medium through the newly formed blood vessels are promoted. The punctured area may be the area where the organoids will be placed. The puncture interval may be 0.15 to 0.3 mm. Any medium suitable for culturing organoids may be used; media suitable for culturing organoids have been described above.

[0053] For example, 400 to 1200 organoid cells can be placed in a large blood vessel with a diameter of 1 mm and a length of 10 mm, preferably as a single fused organoid. If the diameter and length of the large blood vessel are different, the number of cells in the organoid can be determined accordingly. The thickness of the organoid (preferably, the fused organoid) is, for example, initially 80 to 200 μm (when the organoid is placed adjacent to the large blood vessel), and gradually thickens with culture, eventually reaching 250 to 1000 μm.

[0054] Organoids (preferably fused organoids) are placed adjacent to large blood vessels and incubated in an incubator for approximately 1-2 hours, preferably 1.5 hours, allowing the organoids to attach to the large blood vessels and integrate with each other (hereinafter, sometimes referred to as "large blood vessel-bearing organoids"). Culture medium is then added and the cells are allowed to stand for approximately 1-7 days, preferably 2-4 days. A vascular network with a luminal structure can be formed within the statically cultured large blood vessel-bearing organoids (Figure 1B). Any culture medium suitable for organoid culture may be used; suitable culture media for organoid culture are described above.

[0055] Furthermore, when large blood vessel-bearing organoids are perfused, the large blood vessels are connected to the blood vessels within the organoids (preferably fused organoids), allowing perfusion of culture medium into the organoids via the large blood vessels, thereby enhancing interactions between organoids and between the organoids and the large blood vessels and potentially improving the function of the organoids.

[0056] The perfusion culture period should be between 2 hours and 14 days, with 3 to 7 days being preferable. For example, when fused organoids attached to large blood vessels were statically cultured for 4 days and then perfusion cultured for 2 hours, a response to perfusion stimulation was observed, suggesting a connection between the large blood vessels and the vascular network within the fused organoids (Figure 3L, M, N, O). Perfusion culture should be performed so that the medium passes through the inside of the large blood vessels. In the examples described below, metal pipes were attached to both ends of the large blood vessels of the large blood vessel-bearing organoids, connected to a pump via tubing, and placed in a culture vessel containing medium to circulate the medium. Open-system perfusion culture is recommended when the connection between the blood vessels and large blood vessels within the organoids is weak (e.g., 2 to 4 days after static culture). Closed-system perfusion culture is recommended once the connection between the blood vessels and large blood vessels within the organoids has strengthened (e.g., 5 to 7 days after static culture). Open-system perfusion culture is perfusion culture in which the medium flows with a portion of the medium flow path open, while closed-system perfusion culture is perfusion culture in which the medium flows without opening the medium flow path. In the perfusion culture system (Figure 5B) used in the examples described below, two tubes connected to a pump constitute the medium flow path, with one tube connected to one of two metal pipes attached to both ends of a large blood vessel (the metal pipe not connected to the tube is open to the medium in the culture vessel), and the other tube is inserted into the medium in the culture vessel, and the two tubes are not connected (open-system perfusion culture). To perform closed-system perfusion culture, each end of the large blood vessel is connected to a tube via a metal pipe, and the medium is circulated through the tube.

[0057] The perfusion may be performed at a flow rate of 10 microliters / minute to 250 milliliters / minute, preferably 100 microliters / minute to 1 milliliter / minute. The medium may be any suitable medium for culturing organoids, and suitable media for culturing organoids have been described above.

[0058] Figure 5B shows the perfusion culture system used in the Examples described below. In the perfusion culture system shown in Figure 5B, metal pipes are attached to both ends of the large blood vessels of the large blood vessel-bearing liver buds, which are fixed in the medium in the culture vessel with a jig. Two tubes connected to the pump function as the medium flow path; one end of the tube is inserted into the medium in the culture vessel, and the other end is connected to one of the metal pipes attached to both ends of the large blood vessels. The other end of the metal pipe attached to both ends of the large blood vessels is open to the medium in the culture vessel. The medium flow rate is controlled by increasing or decreasing the pump rotation speed using a pump controller receiving commands from a computer. In the system shown in Figure 5B, the culture vessel also serves as a drainage reservoir, eliminating the need for a drainage reservoir.

[0059] In the perfusion culture system shown in Figure 5B, the culture medium flows in through the inflow channel, flows out through the outflow channel, and the outflowed culture medium flows in again through the inflow channel repeatedly. Large blood vessels with organoids arranged on their outer surfaces are immersed in a liquid bath of culture medium, and the upstream end of the outflow channel is opened into the liquid bath to perfusion culture the extraluminal regions of the large blood vessels. One end of the large blood vessels is connected to the downstream end of the inflow channel, and the other end of the large blood vessel is opened into the liquid bath. The culture medium is allowed to flow from the inflow channel into the intraluminal regions of the large blood vessels, and the culture medium that has flowed through the intraluminal regions of the large blood vessels is allowed to flow out into the liquid bath to perfusion culture the intraluminal regions of the large blood vessels, thereby perfusion culture the organoids arranged on the outer surfaces of the large blood vessels (open system perfusion culture).

[0060] The perfusion culture system shown in Figure 5B can be implemented using a perfusion culture device that perfusion cultures organoids arranged on the outer surface of a large blood vessel. This device has a culture vessel that contains a liquid bath of culture medium, an inlet channel that is connected to one end of the large blood vessel at its downstream end and allows the culture medium to flow into the culture vessel, an outlet channel that opens at its upstream end into the liquid bath and allows the culture medium to flow out of the culture vessel, and a culture medium circulation device.

[0061] When changing the above-mentioned open system perfusion culture to a closed system perfusion culture, in a perfusion culture system in which the medium flowing in from the inflow channel flows out from the outflow channel, and the outflowed medium flows in again from the inflow channel, the organoids arranged on the outer surface of the large blood vessels can be perfusion cultured by immersing the large blood vessels on their outer surface in a liquid medium bath to culture the extraluminal region of the large blood vessels, connecting one end of the large blood vessels to the downstream end of the inflow channel and the other end of the large blood vessels to the upstream end of the outflow channel, allowing the medium to flow from the inflow channel into the intraluminal region of the large blood vessels, and allowing the medium that has flowed through the intraluminal region of the large blood vessels to flow out into the outflow channel, thereby perfusion culturing the intraluminal region of the large blood vessels.

[0062] The above-mentioned closed system perfusion culture can be performed using a perfusion culture device that perfusion cultures organoids arranged on the outer surface of large blood vessels. This device has a culture vessel that contains a liquid bath of culture medium, one end of the large blood vessel connected to a downstream end portion and the other end of the large blood vessel connected to an upstream end portion, an inlet channel for introducing culture medium into the large blood vessel, an outlet channel for discharging the culture medium from the large blood vessel, and a culture medium circulation device.

[0063] In both the open system perfusion apparatus and the closed system perfusion apparatus, the medium circulation device preferably comprises a pump, a pump controller, and a personal computer.

[0064] Perfusion culture of vascularized organoids allows the connection of intracellular blood vessels with the large blood vessels, allowing perfusion of culture medium through the large blood vessels, resulting in proliferation of the organoid's constituent cells, increased organoid size, and improved organoid function. When organoids are hepatoblasts, increased secretion of albumin, ammonia metabolism, urea production, fibrinogen, C3 (β1C / A globulin), and α1-antitrypsin (hA1AT) may occur. Furthermore, increased expression of genes encoding hepatic metabolic enzymes, such as alcohol dehydrogenase 4 (ADH4), aldehyde dehydrogenase 8 family member A1 (ALDH8A1), Gc globulin (GC), and CYP3A7, may also occur. Furthermore, perfusion culture of large-vessel organoids can increase the expression of genes related to angiogenesis (angiopoietin-like 3 (ANGPTL3), angiomotin-like 2 (AMOTL2), CXC motif chemokine ligand 8 (CXCL8), interleukin-18 (IL18)), cell proliferation (EPH receptor A7 (EPHA7), fibroblast growth factor receptor 2 (FGFR2), and glial cell line-derived neurotrophic factor (GDNF)), and apoptosis suppression (Dickkopf-1 (DKK1)). The area of ​​CD31-positive blood vessels can also be increased. A vascular network with a luminal structure can be observed within the organoids (Figure 1B).

[0065] By perfusion culturing large blood vessel-bearing organoids, it is possible to prepare artificial organs that can be transplanted by surgical vascular anastomosis. In this artificial organ, the blood vessels within the organoids are connected to the large blood vessels, allowing perfusion to the organoids via the large blood vessels. Therefore, when this artificial organ is transplanted into a human or non-human animal by surgical vascular anastomosis, blood perfusion begins between the host and the artificial organ in vivo, and tissues and organs with highly ordered tissue structures can be created. Animals that can be transplanted include humans, as well as animals used for laboratory animals, pets, working animals, racehorses, fighting dogs, etc., specifically, non-human animals such as mice, rats, rabbits, pigs, dogs, monkeys, cows, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, and crabs. Furthermore, non-human animals are preferably immunocompromised to avoid immune rejection. The implantation site of the prosthetic organ may be any site that allows implantation by surgical vascular anastomosis, and examples include the portal vein, central vein, hepatic artery, renal artery, renal vein, omentum, etc. By implanting the prosthetic organ into a living body (human, non-human animal, etc.), tissues or organs that have lost or decreased function can be regenerated, and further, if human tissues or organs can be produced in non-human animals, they can be used for drug discovery screening.

[0066] Alternatively, the artificial organs 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.

[0067] The present invention will be described in more detail below with reference to examples.

[0068] The scheme for generating fused liver buds with macrovascularization in this example is shown in Figure 1A. Figure 1B also shows the vascular structures formed in the liver buds at each stage of the scheme. Explanation of Terms: Liver bud: In this example, this refers to the stage of the material used to generate fused liver buds after one (or two) days of culture. Endothelial cells are present inside the organoids, and no vascular network has formed (Figure 1B). Fused liver bud: A culture in which liver buds after one (or two) days of culture are densely arranged on a cell culture insert and cultured. After one day (two days in total, including liver bud generation), the liver buds fuse together and form a single mass. Endothelial cells migrate within the fused liver buds, begin to connect with each other, and initiate the formation of a vascular network. Endothelial cell migration is observed within the fused liver buds by three days of culture (four days in total), and a vascular network with a luminal structure forms between days four and seven (five to eight days in total) of culture (Figure 1B). In this example, the fused liver buds attached to the large blood vessels were in the intermediate stage of vascular network formation (see Figure 1B). Large blood vessel-attached liver buds (static culture): After 2–3 days of culture (3–4 days in total), the fused liver buds were removed from the cell culture insert and attached to the large blood vessels. After 4 days of culture (8 days in total, including liver bud formation), a vascular network with a luminal structure was observed within the hiPSC liver buds. In some cases, the culture was continued as a control for perfusion culture. After 4 days of culture (8 days in total, including liver bud formation), a 2-hour perfusion culture was performed, and responses to perfusion stimuli, such as an increase in the diameter of blood vessels within the hiPSC liver buds and activation of VEGFR3 expressed in vascular endothelial cells, were observed, suggesting the connection between the large blood vessels and the vascular network within the hiPSC liver buds (Figure 3L, M, N, O). - Liver buds with large vessels (perfusion culture): Liver buds with large vessels were cultured after 4 days (8 days in total, including liver bud formation) and placed in a perfusion device and cultured for 7 days. Compared to liver buds with large vessels (static culture), tissue-constituting cells proliferated and tissue size increased (Fig. 3C, D, H, I, J). A vascular network with a luminal structure was observed within the hiPSC liver buds (Fig. 3F, G, K).

[0069] Example 1: Creation of human iPSC-derived organs capable of immediate blood perfusion. Abstract: Human induced pluripotent stem cell (hiPSC)-derived organoids are a promising alternative to organ transplantation to address the shortage of donor organs. However, the lack of large blood vessels (BVs) suitable for anastomosis and immediate blood perfusion presents obstacles after transplantation. We developed a culture technique that incorporates large BVs into hiPSC liver organoids (LOs) by promoting tissue fusion. When perfused through large BVs, hiPSC-LOs respond to shear stress, resulting in increased tissue size and enhanced metabolic function. Finally, individual large BV-loaded hiPSC-LOs were transplanted into the femoral artery of immunodeficient rats via vascular anastomosis, where they functioned for 1 week. In summary, we successfully created hiPSC-derived organs suitable for surgical vascular anastomosis, potentially impacting future organ transplantation efforts.

[0070] Significant advances have been made in organoid culture technology using pluripotent stem cells. These advances have led to the creation of disease models, provided insights into the mechanisms of human organ formation, and raised hopes of generating human organs to alleviate the shortage of donor organs for transplantation. Currently, in regenerative medicine, single cells or small tissues such as sheets of retinal pigment epithelium derived from human induced pluripotent stem cells (hiPSCs) are used for transplantation, particularly for patients with exudative age-related macular degeneration (AMD) (1).

[0071] However, generating larger human organs and effectively utilizing them in regenerative medicine remains a challenge. Beyond a certain size (approximately 300 μm in diameter or larger), tissue viability declines due to oxygen and nutrient depletion, making blood perfusion through the vascular system necessary to maintain the viability of large organs in vivo (2, 3). Along with the delivery of oxygen and nutrients and the removal of waste products, mechanical forces generated by blood flow, such as shear stress and hydrostatic pressure, have recently attracted attention for their beneficial role in tissue development and regeneration. During organ development, blood flow to the liver begins at embryonic day 10.5 (E10.5) in mice. Integrin β1 and VEGFR3 on endothelial cells (ECs) sense these mechanical forces and induce hepatocyte proliferation via the secretion of hepatocyte growth factor (HGF) (4). During liver regeneration after partial hepatectomy, a transient increase in blood flow to the remaining liver tissue induces hepatocyte proliferation through a decrease in TGF-β1 production from ECs and an increase in HGF secretion from stellate cells (5).

[0072] Early access to blood flow also contributes significantly to functional recovery after transplantation. We have successfully generated vascularized liver organoids (LO) from hiPSCs (hiPSC-LO), which improved survival in a mouse model of subacute liver injury (6, 7). Furthermore, vascularized pancreatic islets have shown improved efficacy in treating type 1 diabetes (8). However, while transplanted donor organs have immediate access to a blood supply through vascular anastomoses, organoids, even when vascularized, do not undergo blood perfusion for several days. Overcoming this hurdle requires the development of organoids with large blood vessels (BVs) for surgical anastomosis. The introduction of large BVs into organoids is crucial for upgrading organoid cultures to a platform capable of generating transplantable human organs. Recent advances in this field include the construction of complex tissues by fusing heterologous organoids, which serve as a model for human organ development. For example, the fusion of human iPSC-derived foregut and midgut organoids resulted in the formation of visceral tissues with adjacent liver, pancreas, and bile duct structures (9). Similarly, the fusion of medial basal ganglia primitive organoids with cortical organoids reflects the functional integration of different brain regions observed during human brain development (10, 11). Furthermore, a pathological model of Hirschsprung's disease was created by adding neural progenitor cells to human intestinal organoids (12). Fusion techniques facilitate the incorporation of large BVs into organoids by bringing the tissues into close proximity. To achieve this, we utilized a microgravity environment, providing static conditions without media convection, thereby promoting cell aggregation and 3D culture (13-16).

[0073] In this study, we formed large hiPSC-LOs by fusing small organoids and fusing them into large BVs placed in close proximity. To increase tissue size and improve liver function, perfusion culture of hiPSC-LOs through large BVs was established. Furthermore, we developed large BVs from hiPSCs that resemble embryonic BVs with angiogenic potential and observed further enhancement of liver function after perfusion culture. Finally, we used in vivo surgical anastomotic transplantation of BV-loaded hiPSC-LOs to investigate immediate blood perfusion and flow into intraorgan vasculature and evaluate graft function. Overall, we believe that large BV-loaded hiPSC-LOs capable of perfusion culture and transplantation via vascular anastomosis could overcome major technical obstacles in the construction of artificial human organs and potentially address the shortage of donor organs for transplantation.

[0074] Results: Establishment of an LO perfusion culture system with large BVs. To generate large BV-loaded hiPSC-LOs, green fluorescent protein (GFP)-labeled hepatic endoderm cells (HE cells), Kusabira orange (KO)-labeled ECs, and diaphragm mesenchymal cells were differentiated from hiPSCs. These cell types were seeded on micropatterned plates to generate small hiPSC-LOs. Large hiPSC-LOs were generated by first fusing smaller hiPSC-LOs and placing them adjacent to a large BV (punctured rat femoral artery) (Figure 2A and B). By day 5 of coculture, the large hiPSC-LOs had fused with the large BVs, revealing well-developed vascular structures within the hiPSC-LOs (Figure 2C–F). Notably, BV-loaded hiPSC-LOs exhibited a 1.53-fold increase in hALB secretion compared to unmodified hiPSC-LOs, highlighting the impact of large BVs on hiPSC-LO function (Figure 2G; p=0.0286). Enhanced tissue formation was observed in BV-loaded hiPSC-LO (Figure 5A). Furthermore, perfusion of India ink into large BVs demonstrated successful medium perfusion within the hiPSC-LO vasculature (Figure 2H, Figure 5B). The size of hiPSC-LO increased 1.5-fold in the perfused group after 1 and 2 weeks compared with the control group (Figure 2I and J; p=0.01 and p=0.004, respectively). Expression of liver genes (OTC, RBP4, TTR, glucose-6-phosphatase catalytic subunit [G6PC], arginase 1 [ARG1], and glycogen synthase 2 [GYS2]) tended to increase in the perfused group compared with the control group (Figure 5C). Compared with the control group, significant increases in the area of ​​HNF4a-positive hALB-positive hepatocytes (4.18-fold) and CD31-positive vascular areas (4.91-fold) were detected in the large BV-loaded hiPSC-LO in the perfusion group (Figure 2K and L; p=0.0286 and p=0.0286, respectively). Comparable CK19-positive bile duct areas were observed in the perfusion and control groups (Figure 2K and L). Furthermore, a significant increase in hALB secretion (2.81-fold and 2.48-fold at 1 and 2 weeks, respectively) depending on the perfusion period was evident in the perfusion group compared with the control group (Figure 2M; p<0.0001 and p<0.0001, respectively).Notably, the secretion of human fibrinogen (hFibrinogen), hC3, and human α1-antitrypsin (hA1AT) significantly increased by 1.5-fold, 1.4-fold, and 1.68-fold, respectively, compared to the control group (Figure 2M; p=0.0011, p=0.0104, and p=0.007, respectively). A trend toward increased ammonia metabolism and a significant 1.5-fold increase in urea production were observed in the perfusion group compared to the control group (Figure 2N and O; p=0.3272 and p=0.0104, respectively). These results highlight the successful establishment of a method for generating large BV-loaded hiPSC-LOs and a perfusion system that promotes their tissue expansion and functional enhancement.

[0075] Enhanced LO Function by Incorporating Large hiPSC-Derived BVs. Liver tissue is composed of various large BVs (e.g., portal vein [PV] and central vein [CV]) that secrete vascular signals and induce tissue polarization known as "liver zonation" (a location-dependent change in liver function and oxygen gradient within the liver). Hepatocytes in the PV region exhibit distinct liver enzyme profiles compared with those in the CV region. Furthermore, bile duct formation is initiated by interactions between SMCs and ECs in the PV, indicating the potential impact of large BVs on hiPSC-LO function (17-19). Therefore, to enhance hiPSC-LO function, we attempted to generate large hiPSC-derived BVs. We generated large BVs with angiogenic potential by seeding hiPSC-derived vascular smooth muscle cells (SMCs) and ECs onto PGA tubes (Figure 6A and B) (20). Large BVs with a 2:1 SMC-to-EC ratio were optimal for generating large BV-loaded hiPSC-LOs due to their enhanced angiogenic potential compared to large BVs with a 5:1 SMC-to-EC ratio (Figure 3A; Figure 6C). Placing hiPSC-LOs near these hiPSC-derived large BVs generated hiPSC-LOs with hiPSC-BVs (Figure 3B). Perfusion culture through these large BVs resulted in a 1.24-fold increase in tissue size compared to the control group (Figure 3C and D; p=0.0003). Addition of India ink demonstrated perfusion of fluid into the blood vessels formed within the hiPSC-LOs (Figure 3E). Histological analysis revealed a 1.7-fold increase in CD31-positive vasculature, a 2.58-fold increase in hALB-positive hepatocyte area, and a 2.39-fold increase in CK19-positive bile duct area in the perfused group compared with the control group (Figure 3F and G; p = 0.0027, p < 0.0001, and p = 0.0002, respectively). The perfused group also showed a significant increase in the secretion of hALB (1.76-fold; p < 0.0001), hfibrinogen (4.5-fold; p = 0.0006), hC3 (3.38-fold; p = 0.0006), and hA1AT (4.04-fold; p = 0.0006) compared with the control group (Figure 3H).Furthermore, ammonia metabolism (13.3-fold; p = 0.0025) and urea production (1.39-fold; p = 0.0037) were significantly increased in the perfused group compared with the control group ( Fig. 3I and J ).

[0076] Electron microscopy revealed that hepatocytes within hiPSC-LO contained abundant ER structures and mitochondria, and bile canaliculi were formed between hepatocytes (Figure 7A). Furthermore, a mesothelium formed on the outside of the hiPSC-LO, preventing medium leakage from the hiPSC-LO (Figure 3K). Comprehensive RNA-seq-based GO analysis revealed upregulation of genes related to retinoic acid metabolism, xenobiotic metabolism, vitamin transport, and complement activity in the perfusion group compared with the control group (Figure 7B). In particular, the expression of genes related to angiogenesis (angiopoietin-like 3 (ANGPTL3), angiomotin-like 2 (AMOTL2), C-X-C motif chemokine ligand 8 (CXCL8), interleukin-18 (IL18)), cell proliferation (EPH receptor A7 (EPHA7), fibroblast growth factor receptor 2 (FGFR2), and glial cell line-derived neurotrophic factor (GDNF)), and apoptosis suppression (Dickkopf-1 (DKK1)) was increased in the perfusion group (Fig. 7C). This is consistent with the histological findings (Fig. 3F). The expression of genes encoding hepatic metabolism-related enzymes, such as alcohol dehydrogenase 4 (ADH4), aldehyde dehydrogenase 8 family member A1 (ALDH8A1), Gc globulin (GC), and CYP3A7, was also increased in the perfusion group compared with the control group (Fig. 7C).

[0077] Finally, we investigated the activation of VEGFR3 and integrin β1, which are involved in sensing blood perfusion stimuli and the resulting upregulation of HGF and hepatocyte proliferation in developing and regenerating liver tissue (4). VEGFR3 is also essential for hepatic sinusoid development (21). As a result, 2 hours after medium perfusion, large BV-loaded hiPSC-LO cells showed increased VEGFR3 phosphorylation, integrin β1 activation, and HGF expression (Figure 3L-O).

[0078] These results highlight that large BV-loaded hiPSC-LOs have the ability to respond to mechanostress induced by in vitro perfusion, promoting angiogenesis and increasing hepatocyte mass through activation of signaling pathways, similar to the responses observed in vivo (Figure 8).

[0079] Transplantation of Large BV-Loaded LOs via Vascular Anastomosis Next, we verified in vivo blood perfusion into these organoids by establishing a vascular anastomosis with the large BVs. Therefore, we transplanted large BV-loaded hiPSC-LOs into the femoral artery of immunodeficient rats via surgical vascular anastomosis (Figure 4A). Considering the possibility of immune rejection, evaluation was performed 2 weeks after transplantation. Upon resumption of blood flow after transplantation, blood perfusion within the hiPSC-LOs occurred immediately, confirming the presence of the graft 2 weeks after transplantation (Figure 4B). Histological analysis revealed weak hALB staining in CK8 / 18-positive hepatocytes before transplantation, but strong hALB staining was observed after transplantation, indicating maturation of the liver tissue (Figure 4C). The presence of numerous blood cells within the vasculature of the graft confirmed blood perfusion into the hiPSC-loaded liver tissue (Figure 4D). Further analysis demonstrated the presence of hALB-positive / HNF4a-positive hepatocytes, CK19-positive bile ducts, and CD31-positive vasculature in the transplanted tissue, demonstrating graft functionality (Figure 4E). Secretion of hALB in the rat blood 2 weeks after transplantation provided further evidence of graft functionality (Figure 4F).

[0080] Finally, we transplanted large BV-loaded hiPSC-LOs into rat femoral arteries via surgical vascular anastomosis. Evaluation was performed 1 week after transplantation. Upon resumption of blood flow, the human iPSC-derived large BVs withstood blood pressure and promoted immediate blood perfusion within the LO (Figure 4G and H). One week after transplantation, the functionality of the grafts was confirmed by the secretion of hALB and hfibrinogen detected in the rat blood (Figure 4I and J). Histological analysis revealed the presence of many blood cells within the graft vasculature, but few outside the vascular area, indicating successful blood perfusion of the hiPSC-liver tissue (Figure 4K). Furthermore, histological examination of the transplanted tissue revealed the presence of hALB-positive hepatocytes, CD31-positive vascular structures, and CK19-positive bile duct structures (Figure 4L and M). Expression of hCYP3A4 and hA1AT was observed in hepatocytes 1 week after transplantation, suggesting hepatocyte maturation (Figure 4N and O). Furthermore, the expression of bile canaliculi-associated multidrug resistance protein 2 (MRP2) and ZO-1 suggests the formation of highly complex liver structures within the grafts ( Fig. 4P and Q ).

[0081] Effect of puncture treatment on large blood vessels We tested the effectiveness of puncture treatment on large blood vessels using mouse large blood vessels. The puncture treatment resulted in the extension of mouse-derived vascular endothelial cells (stained with a mouse CD31-specific antibody) from the lumen of the mouse large blood vessels into the interior of the hiPSC-LO (Figure 9). In contrast, no extension of mouse-derived vascular endothelial cells into the interior of the hiPSC-LO was observed in the group that did not undergo puncture treatment (Figure 9). These results suggest that puncture treatment of large blood vessels promotes the connection between the large blood vessels and the internal vascular structure of the hiPSC-LO.

[0082] Therapeutic Effect of Vascular Anastomotic Transplantation of Large-Vessel-Loaded hiPSC-LOs on Liver Failure We created an acute liver failure model in immunodeficient rats by administering thioacetamide. Subsequently, large-sized BVs (sham group) or large-sized BV-loaded hiPSC-LOs (transplantation group (Tx group)) were transplanted into the rat femoral artery via surgical vascular anastomosis to verify the therapeutic effect. The results showed a significant improvement in survival rate in the transplantation group compared with the sham group (Figure 10) (p = 0.037, Log-rank test, n = 8).

[0083] We used gene expression analysis to elucidate the mechanism by which perfusion stimulates cell proliferation. First, we extracted RNA from the perfused and non-perfused groups, which underwent 2-hour perfusion, and compared the two groups by sequence analysis. Similar to the quantitative PCR analysis shown in Figure 3O, we observed increased HGF gene expression (Figure 11A). Next, we extracted genes whose expression levels were 1.5-fold higher in the perfused group compared to the non-perfused group in the RNA sequence data. KEGG ontology enrichment analysis revealed that the expression of cell proliferation-related genes, including the MAPK signaling pathway, TGF-beta signaling pathway, PI3K-Akt signaling pathway, ErbB signaling pathway, and Hippo signaling pathway, was increased in response to perfusion stimulation (Figure 11B). Furthermore, single-cell RNA sequencing analysis identified genes that showed a two-fold increase in expression in perfused ECs compared to non-perfused ECs. Gene ontology enrichment analysis revealed not only promotion of angiogenesis but also an increase in genes related to cell signaling pathways (Figure 11C).

[0084] In summary, we successfully constructed large BV-loaded human iPSC-derived liver tissues. These tissues tolerated arterial pressure and facilitated immediate in vivo blood perfusion via surgical vascular anastomosis. Transplantation of large BV-loaded human iPSC-derived liver tissues into an acute liver failure model via surgical vascular anastomosis demonstrated improved survival.

[0085] Discussion: In this example, we describe a technique for generating large human tissues by incorporating large, millimeter-sized BVs into hiPSC-derived LOs, enabling subsequent transplantation via surgical anastomosis. Following perfusion of fluid through the large BVs, tissue expansion and enhanced hiPSC-LO function were observed. Furthermore, anastomosed transplantation of large BVs promoted immediate in vivo blood perfusion and maturation of hiPSC-LOs. Furthermore, vascular anastomotic transplantation in an acute liver failure model demonstrated a statistically significant improvement in survival rate.

[0086] We established a basic technique for in vitro perfusion culture to increase tissue size and mature organoids, which is promoted by the addition of large BVs. We elucidated the underlying mechanism by which hiPSC-LOs with large BVs detect mechanical stress from perfusion stimuli by activating VEGFR3 and integrin β1 on ECs and increasing HGF expression, leading to enhanced vascular structure and tissue volume (4).

[0087] Compared to rat BVs, the use of human hiPSC-derived BVs significantly enhanced liver functions, including functional protein production and ammonia metabolic capacity, after perfusion culture. This highlights the important role of signals derived from large BVs in regulating tissue function. Previous studies have documented the involvement of signals from the PV and CV in the formation and maintenance of the liver zonation (17-19). Therefore, optimizing large BVs improves liver function and the generation of liver zonation-specific hiPSC liver tissue. This may be valuable for pathological analysis of metabolic dysfunction associated with fatty liver disease models that progress from the CV region (28).

[0088] A major limitation in constructing large human artificial organs is that hiPSC-BVs are not suitable for long-term perfusion culture due to hydrolysis of PGA within the scaffold. Pulse perfusion promotes vascular smooth muscle differentiation in large BVs engineered with PGA, generating durable BVs capable of withstanding abdominal aortic blood pressure (20). Our RNA-seq analysis confirmed this trend by demonstrating increased expression of vascular smooth muscle differentiation-related genes in the perfusion culture group. Therefore, optimizing perfusion conditions may be useful for developing large artificial human organs with hiPSC-derived BVs suitable for long-term perfusion culture.

[0089] The acute liver failure model used in this study is a very severe model, with 87.5% of rats dying within two days in the sham transplant group. Under these conditions, the statistically significant improvement in survival rate observed by vascular anastomosis of hiPSC-BV-loaded hiPSC-LO to the rat femoral artery is a major step forward for regenerative medicine. This suggests that the developed vascularly anastomotic hiPSC-BV-loaded hiPSC-LO immediately accessed the bloodstream after transplantation, thereby exerting its therapeutic effect. Further enhancement of therapeutic efficacy is expected by generating and transplanting larger hiPSC-BV-loaded hiPSC-LO in the future.

[0090] In conclusion, we have reported a novel technique that advances organoid culture for the generation of human organs. The remarkable achievement of incorporating large BVs into organoids allows for surgical vascular anastomosis, thereby improving survival in a model of acute liver failure requiring immediate therapeutic effects. This technological innovation holds promise as a solution to the global shortage of organs for transplantation.

[0091] Materials and Methods Animals Eight-week-old male Fischer 344 / NSlc rats and eight-week-old female C57BL / 6J mice were purchased from Japan SLC Corp. Inc. (Shizuoka, Japan). Eight-week-old male Fischer 344 / / DuCrlCrlj and Fischer 344 / / NJcl-rnu / rnu rats were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). 12- to 15-week-old male immunodeficient rats, F344-Il2rg, were used. em1Iexas were purchased from CLEA Japan (Tokyo, Japan). Rats were maintained in accordance with the Yokohama City University Institutional Guidelines for the Use of Laboratory Animals (approval numbers: FA-19-016 and FA-22-062).

[0092] Culture of hiPSCs and differentiation into hepatic endoderm (HE). hiPSCs (Ff-I01s04) were provided by the Center for iPS Cell Research and Application, Kyoto University. hiPSCs were cultured in StemFit AK02N medium (Ajinomoto Co., Tokyo, Japan) containing laminin (iMatrix-511). TM The hiPSCs were maintained on dishes coated with α-heptane (Nippi Inc., Tokyo, Japan). The medium was changed every other day. HE differentiation was performed as previously described with minor modifications (29). Briefly, hiPSCs were seeded on laminin-coated dishes containing RPMI-1640 (Fujifilm Wako Pure Chemical Industries, Ltd.) containing StemFit for differentiation (Ajinomoto), human activin A (100 ng / mL, Ajinomoto), 2 μM CHIR99021 (Cayman Chemical, Ann Arbor, MI, USA), and Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.). The next day, the medium was changed to RPMI-1640 containing StemFit for differentiation, 100 ng / mL human activin A, 2 μM CHIR99021, and 500 μM sodium butyrate (Sigma-Aldrich, St. Louis, MO, USA). CHIR99021 and sodium butyrate were removed on days 3 and 4 of culture, respectively. Definitive endoderm cells (day 6) were cultured using a 20% knockout system. TMKnockOut containing Serum Replacement (KSR, Thermo Fischer Scientific), 2 mM L-glutamine (Thermo Fischer Scientific), 1% NEAA (Thermo Fischer Scientific), 100 μM β-mercaptoethanol (Thermo Fischer Scientific), and 1% DMSO (Nacalai Tesque, Kyoto, Japan). TM HE cells were obtained by treatment with DMEM (Thermo Fischer Scientific, Waltham, MA, USA) for 4 days.

[0093] Differentiation of endothelial cells (EC), mesenchymal cells (MC), and smooth muscle cells (SMC) from human induced pluripotent stem cells (hiPSCs). Differentiation of ECs, MCs, and SMCs was performed as previously described with minor modifications (29, 30). hiPSCs were seeded onto laminin-coated dishes in StemFit® AK02N medium containing 10 μM Y-27632. Starting on day 1, cells were treated with DMEM / F12 (Thermo Fischer Scientific) containing 1% GlutaMAX, differentiation-grade StemFit, 8 μM CHIR99021, and 25 ng / mL bone morphogenetic protein 4 (BMP4) for 3 days. Lateral mesoderm cells were then differentiated into endothelial lineage cells by treatment with StemPro-34 SFM medium (Thermo Fischer Scientific) containing 200 ng / mL VEGF (Fujifilm Wako) and 2 μM forskolin (Sigma-Aldrich) for 5 days. ECs were expanded by passage in MiraCell® EC culture medium (Takara Bio, Shiga Prefecture, Japan) for 7 days. For transverse septum MCs, lateral mesoderm cells were treated with DMEM / F12 containing 1% GlutaMAX, differentiation-grade StemFit, 2 ng / mL activin A, and 10 ng / mL PDGF-BB for 2 days. Mesenchymal progenitor cells were passaged and treated with DMEM / F12 containing 1% GlutaMAX, differentiation-grade StemFit, 10 ng / mL basic fibroblast growth factor, and 12 ng / mL BMP4 for 3 days. For SMCs, mesenchymal precursors were passaged and treated with DMEM / F12 containing 1% GlutaMAX, StemFit for differentiation, 2 ng / mL activin A, and 1 U / mL heparin (Mochida Pharmaceutical Co., Ltd., Tokyo, Japan).

[0094] Three-dimensional culture and fusion of liver organoids (LO) hiPSC-derived HE cells, ECs, and MCs were harvested using trypsin and seeded at a ratio of 10:2:2 (5 × 10 cells) into 24-well Elplasia plates (Corning, Inc., Corning, NY, USA) supplemented with LO medium (a 1:1 ratio of DMEM [Thermo Fischer Scientific] and KBM VEC1 [Kohjin Bio Co., Ltd., Saitama, Japan]) containing 2.5% FBS, 2.5 μg / mL insulin, 0.25 μg / mL hydrocortisone, 5 μg / mL holotransferrin, 10 ng / mL oncostatin M, and 25 nM dexamethasone, respectively. 5 The next day, 120 organoids from two wells were collected, resuspended in 100 μL of LO medium, and transferred to an adhesive silicone frame (Culture-Inserts, ibidi GmbH, Gräfelfing, Germany) set on a cell culture insert (0.4 μm pore, Corning). Medium was added to the underside of the cell culture insert and replaced every other day. Organoids were observed using a BZ-X710 fluorescence microscope (Keyence, Japan) and a confocal microscope SP5 (Leica Microsystems GmbH, Wetzlar, Germany).

[0095] Generation of fused hiPSC-LO cells containing large BVs. After isoflurane anesthesia, mouse or rat BVs were isolated from the femoral artery under a stereomicroscope (Leica Microsystems) and cultured in LO medium supplemented with 5 μg / mL voriconazole as an antibiotic for 1 day. To prepare hiPSC-derived BVs, a 1:1 mixture of hiPSC-derived SMCs and ECs was placed in a PGA tube (0.5, 1, or 3 mm diameter, Gunze Medical Co., Ltd.) at a concentration of 4.55 × 10 cells. 4 cells / mm 2 The hiPSC-LO cells were seeded in a 34-gauge needle (Terumo Corporation, Tokyo, Japan) and cultured in LO medium for 3 days. The BV was punctured with a 34G needle (Terumo Corporation, Tokyo, Japan), and the fused hiPSC-LO cells were placed in the punctured area of ​​the BV at 37°C for 20 minutes. The large BV-loaded hiPSC-LO cells were cultured for 5 days and then subjected to perfusion culture or transplantation.

[0096] Perfusion culture of large BV-loaded hiPSC-LOs. For perfusion culture, large BV-loaded hiPSC-LOs were attached to a 0.6 mm (rat BV) or 1 mm (hiPSC-B) diameter stainless steel pipe. The pipe-mounted hiPSC-LOs with hiPSC-BVs were connected to a mini peristaltic pump system (icomes Lab Co. Ltd., Iwate, Japan) via PTFE tubing (TUF-100, Chukoh Chemical Industry Co., Ltd., Tokyo, Japan). The hiPSC-BV-loaded human iPSC-LOs were placed on a culture device printed by Guider2s (Flashforge Corp., Zhejiang, China), and the culture vessel was covered with a Breathe-Easy® sealing membrane (Sigma-Aldrich). Medium was perfused through the large BV at 100 μL / min, and the medium was changed weekly for 2 weeks. The culture medium was analyzed by enzyme-linked immunosorbent assay (ELISA) to quantify secreted proteins. LO samples were studied by histological analysis and functional assays, including evaluation of ammonia metabolic capacity and urea production.

[0097] Histological Analysis and Image Quantification. Samples were fixed overnight in 4% PFA / PBS. After embedding in paraffin, samples were sectioned at 5–7 μm thickness using a microtome (Leica Biosystems, Wetzlar, Germany). For frozen sections, samples were embedded in Tissue-Tek® OCT Compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan) and sectioned at 12 μm thickness using a cryostat (Leica Biosystems). Sections were stained with hematoxylin and eosin. For immunohistochemistry, after antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) at 121°C for 20 minutes (paraffin sections), sections were blocked with protein block (Agilent Technologies, Inc., Santa Clara, CA, USA) and stained with primary and secondary antibodies (Table 1). For immunofluorescence staining, nuclei were stained with DAPI (Dojindo Laboratories, Kumamoto, Japan), and images were acquired using a BZ-X710 fluorescence microscope (Keyence Corporation, Osaka, Japan) or a confocal microscope SP5 (Leica Microsystems).

[0098] HRP-conjugated secondary antibodies were reacted with 3,3′-diaminobenzidine (Agilent Technologies, Santa Clara, CA, USA) and counterstained with hematoxylin. Images were quantified using Fiji software (version 2.14.0 / 1.54f; https: / / fiji.sc / ).

[0099] ELISA analysis was performed as previously described (6). Briefly, human albumin (ALB), human C3, A1AT, and fibrinogen in the culture medium, as well as human ALB and fibrinogen in rat serum, were measured using a human albumin ELISA quantification kit (Bethyl Laboratories, Montgomery, TX, USA), a human complement C3 ELISA kit (Abcam, Cambridge, UK), a human SerpinA1 ELISA kit (Abcam), and a high-sensitivity human fibrinogen ELISA kit (Abcam) according to the manufacturer's instructions. Phosphorylated VEGFR3 was measured using a Human Phospho-VEGFR3 / Flt4 DuoSet IC ELISA (R&D Systems Inc., MN, USA) according to the manufacturer's instructions.

[0100] Measurement of ammonia metabolic capacity and urea production in hiPSC-LO. To evaluate the ammonia metabolic capacity of hiPSC-LO, ammonium chloride solution was added to the culture medium at a final concentration of 2 μM. The medium was collected at 0 and 24 hours, and the ammonia concentration was measured using an Ammonia Test Kit II (ARKRAY, Inc., Japan). The urea concentration in the medium was measured using a Quantichrom Urea Assay Kit (BioAssay Systems, CA, USA) according to the manufacturer's instructions.

[0101] Quantitative RT-PCR analysis: Total RNA was extracted using the PureLink™ RNA Mini Kit (Thermo Fisher Scientific), and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative RT-PCR analysis using the TaqMan Probe System (Thermo Fisher Scientific) or THUNDERBIRD® SYBR qPCR Mix (Takara Bio Inc.) was performed using a LightCycle® 480 (Roche Diagnostics KK, Tokyo, Japan). The primer / probe sets used here are listed in Table 2.

[0102] Angiogenesis assay. The angiogenesis assay was performed as previously described (31) with minor modifications. Briefly, hiPSC-large BVs were embedded in a 1:1 mixture of rat collagen I (Cultrex, 1 mg / mL) and Matrigel and allowed to solidify for 20 min at 37°C in a CO2 incubator. GlutaMAX TM Opti-MEM containing Supplement (51985034, Thermo Fisher Scientific) TM Angiogenesis medium consisting of reduced serum medium, 1% penicillin / streptomycin, 1% FBS, and 30 ng / mL human VEGF (Fujifilm Wako) was added to the solidified gel and cultured for 10 days.

[0103] Transmission electron microscopy analysis. Samples were fixed overnight at 4°C with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (PB). After three washes with PB, the samples were post-fixed with 2% osmium tetroxide in 0.1 M PB for 2 hours at 4°C, dehydrated through graded ethanol solutions, treated twice with propylene oxide (PO) for 30 minutes each, and then transferred to a PO / Quetol-812 (Nisshin EM Co., Tokyo, Japan) mixture (70:30) for 1 hour. Next, the samples were immersed in 100% Quetol-812 overnight and polymerized at 60°C for 48 hours. Ultrathin sections (70 nm, Ultracut UCT, Leica Microsystems) were stained with 2% uranyl acetate for 15 minutes at room temperature. After washing with distilled water, the samples were stained with lead stain (Sigma-Aldrich) for 3 minutes at room temperature. The samples were observed using a transmission electron microscope (JEM-1400Plus, JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 100 kV. Digital images were acquired with a CCD camera (EM-14830RUBY2, JEOL Ltd.).

[0104] cDNA Library Construction and Sequencing. Total RNA was isolated from two 1g group samples, three μg group samples, four control (perfusion-negative) group samples, and four perfusion group samples of hiPSC-BV-transduced hiPSC-LO. The quality and quantity of total RNA were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and an Agilent RNA 6000 Nano Kit (Agilent Technologies). After mRNA enrichment using the NEBNext rRNA Depletion Kit v2 (human / mouse / rat, New England Biolabs, Ipswich, MA, USA), cDNA libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions.

[0105] Libraries were sequenced using 121-bp single-read sequencing on an Illumina NextSeq1000 (Illumina, San Diego, CA, USA). Reads were generated in FASTQ format using bcl2fastq2 Conversion Software V2.20 (Illumina). Read data were submitted to the DDBJ Read Archive (accession number DRA017179).

[0106] RNA-seq Data Analysis. Gene expression was analyzed using CLC Genomics Workbench 22 (Qiagen, Hilden, Germany). First, raw read data were cleaned using the following parameters: quality limit = 0.001, ambiguity limit = 2, number of 5'-terminal nucleotides = 15, number of 3'-terminal nucleotides = 6, and minimum number of nucleotides in reads = 50. The cleaned reads were mapped to the Homo sapiens (human) reference genome, GRCh38.p14, retrieved from the NCBI Genome Database (https: / / www.ncbi.nlm.nih.gov / genome / ). The mapping parameters were as follows: mismatch cost = 2, insertion cost = 3, deletion cost = 3, length ratio = 0.8, and similarity ratio = 0.8. After statistical analysis based on a generalized linear model, differentially expressed genes with a difference > |2|-fold or |1.5|-fold and a false discovery rate (FDR)-adjusted p-value < 0.05 were selected and enriched using the public database DAVID (https: / / david.ncifcrf.gov / ). Results were visualized in the form of volcano plots using R (version 3.5.2).

[0107] Single-cell gene expression analysis was performed using the 10xGenomics FLEX system according to the manufacturer's instructions. After enzymatic dissociation of fixed samples, libraries were prepared using Chromium X and the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit, the Chromium Fixed RNA Kit, Human Transcriptome, 4rxns × 4 BC, and the Chromium Next GEM Chip Q Single Cell Kit (10xGenomics). Sequencing was performed using a NovaSeq 6000. Sequencing data were preprocessed using Cell Ranger, and integrated analysis was performed using Seurat (version 5) in R software to define individual cell clusters. Pseudo-bulk analysis of gene expression in endothelial cells was then performed to compare gene expression between the non-perfused and perfused groups. Differentially expressed genes with a difference of >2-fold were selected and enriched using the public database DAVID (https: / / david.ncifcrf.gov / ). Results were visualized as bar graphs using GraphPad Prism software.

[0108] Transplantation of large BV-loaded hiPSC-LOs via vascular anastomosis. After anesthesia with isoflurane, the femoral artery of an F344 nude rat was carefully exposed under a stereomicroscope (Leica Microsystems), and blood flow was stopped using a microvascular clip (Bear Medic Corp., Tokyo, Japan). After cutting the artery, the large BV-loaded hiPSC-LOs were anastomosed using 10-0 sutures with a 3 mm microsurgery needle (Bear Medic). Blood perfusion was resumed by removing the microvascular clip. If negligible blood leakage was observed, the skin was sutured to close the surgical area, and the animals were maintained for two weeks for rat BV-loaded hiPSC-LOs and one week for hiPSC-BV-loaded hiPSC-LOs.

[0109] Therapeutic effect of transplantation of large BV-loaded hiPSC-LO in a rat model of acute liver failure. Immunodeficient rat F344-Il2rg em1Iexas The day before transplantation, mice (CLEA Japan, Tokyo, Japan) were administered 300 mg / kg of thioacetamide (TAA, Sigma-Aldrich) and transplanted with large BV-loaded hiPSC-LO the following day, as described in

[0105] . A sham transplantation group was also administered with large BVs not loaded with hiPSC-LO. After transplantation, the animals were maintained with 300 mg / kg of TAA.

[0110] Statistical Analysis: Groups were compared using the Mann-Whitney U test with GraphPad Prism software (version 10.0.2, alpha level set at 0.05). Data are presented as mean ± standard error of the mean (SEM). Post-transplant survival data were compared using the log-rank test with GraphPad Prism software.

[0111] Availability of data and materials: The RNA-seq data used in this study have been deposited with DDBJ under accession number DRA017179. PGA tubes were provided by Gunze Medical Co., Ltd. under an MTA contract.

[0112] (Table 1)

[0113] (Table 1) (continued)

[0114] (Table 2)

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[0116] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0117] Organoids with large blood vessels can be perfused through large blood vessels to promote their proliferation and differentiation, and the presence of large blood vessels also allows for vascular anastomosis transplantation.

Claims

1. An artificial organ that can be transplanted by surgical vascular anastomosis, comprising a large blood vessel and an organoid, wherein the organoid is positioned adjacent to the large blood vessel, the large blood vessel has angiogenic ability, a vascular structure is formed in the organoid, blood vessels within the organoid are connected to the large blood vessel, and perfusion to the organoid via the large blood vessel is possible.

2. A method for producing the artificial organ described in claim 1, comprising placing organoids adjacent to large blood vessels having angiogenic capacity, followed by perfusion culture.

3. The method of claim 2, further comprising puncturing a large blood vessel having angiogenic potential before placing the organoid adjacent to the large blood vessel.

4. An artificial large blood vessel with angiogenic properties, consisting of a tubular support (scaffold) made from biocompatible materials seeded with smooth muscle cells and endothelial cells.

Images