Artificial organ and method for manufacturing the same

Abstract

To provide an artificial organ that has excellent cell filling factor, and in which a function of an organ is maintained, and a method for manufacturing the same.SOLUTION: A method for manufacturing an artificial organ includes: performing decellularizing treatment to a mammal organ or a part thereof to obtain a decellularized organ or a part thereof; and performing cellularizing treatment of engrafting a cell to the decellularized organ or a part thereof to obtain an organ to which the cell is engrafted, where the cellularizing treatment includes: injecting an organoid including cells constituting the organ or cells capable of differentiating into the cells into the decellularized organ or a part thereof by puncture; and perfusing cells constituting the organ or cells capable of differentiating into the cells into blood vessels of the decellularized organ or a part thereof. An artificial organ is obtained by the method for manufacturing the artificial organ.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an artificial organ and a method for producing the same. [Background technology]

[0002] Recent advances in the field of regenerative medicine have accelerated research into the regeneration of thin tissues, such as skin, gastrointestinal mucosa, and cornea, as well as tissues with relatively simple structures and functions, such as bone and soft tissue. Meanwhile, research and development into individual organs has lagged behind. This is due to the ongoing difficulty in understanding and reproducing the extremely complex structures and functions of three-dimensional organs. The development of functional organ regeneration technologies that could provide fundamental treatments for solid organ failure, particularly for those with high societal needs, such as the liver, kidney, and pancreas, is still in its infancy. Various technologies have been developed to realize the regeneration of three-dimensional organs.

[0003] Regenerating organ structures requires 1) an appropriate extracellular matrix (ECM), 2) a continuous three-dimensional structure extending from microstructures to large blood vessels, and 3) a sufficient supply of cells. To achieve the regeneration of such complex, three-dimensional organs, Ott et al. pioneered a method in 2008 for applying organ scaffolds, decellularized from solid organs, to regenerative medicine (see, for example, Patent Document 1). This method involves removing all cells from living tissue using various methods, and then using the remaining ECM scaffold, a fibrous protein, for tissue regeneration. In fact, decellularized tissues obtained using a similar method, such as human skin (Alloderm®) and porcine heart valve (Hancock®), have already been commercialized and are being used clinically as medical materials.

[0004] The inventors have also reported that they have successfully produced an artificial liver in which sufficient amounts of liver cells and vascular endothelial cells have taken root by decellularizing a pig liver and then injecting and filling the inside of the decellularized pig liver with pig liver cells and vascular endothelial cells through a blood vessel (see, for example, Non-Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2009-505752 [Non-patent literature]

[0006] [Non-Patent Document 1] Higashi H et al., “Transplantation of bioengineered liver capable of extended function in a preclinical liver failure model”, Am J Transplant., Vol. 22, Issue 3, pp. 731-744, 2022. Summary of the Invention [Problem to be solved by the invention]

[0007] The method of injecting liver cells and vascular endothelial cells into a decellularized liver via a blood vessel, as described in Non-Patent Document 1, etc., is excellent at reconstructing the vascular structure. However, the albumin production capacity is low, and there is room for improvement.

[0008] The present invention has been made in view of the above circumstances, and provides an artificial organ that has an excellent cell filling rate and maintains organ function, and a method for producing the same. [Means for solving the problem]

[0009] As a result of extensive research to achieve the above-mentioned objective, the inventors discovered that by injecting cells (single cells) into a decellularized organ via a blood vessel and then directly injecting organoids through a puncture, the cell filling rate can be improved and an artificial organ that maintains organ function can be obtained, leading to the completion of the present invention.

[0010] That is, the present invention includes the following aspects. (1) performing a decellularization process on a mammalian organ or a part thereof to obtain a decellularized organ or a part thereof; performing a cellularization treatment to engraft cells onto the decellularized organ or a part thereof, thereby obtaining an organ engrafted with the cells; Including, The cellularization treatment is Puncturing and injecting an organoid containing cells constituting the organ or cells that can differentiate into the decellularized organ or a part thereof; and perfusing cells constituting the organ or cells capable of differentiating into said cells into the blood vessels of the decellularized organ or part thereof; A method for producing an artificial organ, comprising: (2) The method for producing an artificial organ according to (1), wherein the mammal is a mammal other than a human. (3) The method for producing an artificial organ according to (1) or (2), wherein the cells are human-derived cells. (4) The method for producing an artificial organ according to any one of (1) to (3), wherein the organ is a solid organ. (5) The method for producing an artificial organ according to any one of (1) to (4), wherein the organ is a liver or a kidney. (6) An artificial organ obtained by the method for producing an artificial organ according to any one of (1) to (5). [Effects of the Invention]

[0011] According to the above-described aspects of the artificial organ and the method for producing the same, it is possible to provide an artificial organ with an excellent cell filling rate and in which organ function is maintained, and a method for producing the same. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is an image showing the perfusion culture system in Example 1. [Figure 2] 1 is a graph comparing the cell loading rates of artificial liver tissues prepared by each loading method in Example 1. [Figure 3]1 shows images showing the localization of a single cell labeled with PKH26 in an artificial liver tissue prepared by the hybrid filling method in Example 1. The upper left image is a bright-field image, the upper right image is a fluorescent image, and the lower image is a merged image of the bright-field image and the fluorescent image. [Figure 4] Fluorescence image of a single cell labeled with PKH26 (top left), a fluorescence image of CK8 / 18 (top center), a fluorescence image of CD31 (top right), a fluorescence image of DAPI (top right corner of the fluorescence image of CD31) in an artificial liver tissue prepared by the hybrid filling method in Example 1, and a merged image of all these fluorescence images (bottom). [Figure 5] 1 shows a fluorescent image of 5-FAM-labeled Collagen Hybridizing Peptide (CHP) (upper left), a fluorescent image of DAPI (upper left corner of the CHP fluorescent image), a fluorescent image of CK8 / 18 (upper right), a fluorescent image of collagen III (lower left), and a merged image of all these fluorescent images (lower left) in the artificial liver tissue prepared by the hybrid filling method in Example 1. [Figure 6] The top row shows a fluorescent image of CK8 / 18 (first from the left), a fluorescent image of DAPI (upper right corner of the fluorescent image of CK8 / 18), a fluorescent image of albumin (second from the left), a fluorescent image of CK19 (second from the right), and a merged image of all these fluorescent images (first from the right) in the artificial liver tissue prepared by the hybrid filling method in Example 1. The bottom row is an enlarged image of the top row. [Figure 7] FIG. 1 shows a fluorescent image of CK8 / 18 (first from the left), a fluorescent image of DAPI (upper right corner of the fluorescent image of CK8 / 18), a fluorescent image of E-cadherin (second from the left), a fluorescent image of cytochrome P4503A4 (CYP3A4) (second from the right), and a merged image of the fluorescent images of E-cadherin and CYP3A4 (first from the right) in artificial liver tissue prepared by the hybrid filling method in Example 1. [Figure 8]FIG. 1 shows a fluorescent image of CK8 / 18 (first from the left), a fluorescent image of DAPI (upper right corner of the fluorescent image of CK8 / 18), a fluorescent image of ZO-1 (second from the left), a fluorescent image of dipeptidyl peptidase IV (DPPIV) (first from the left), and a merged image of the fluorescent images of ZO-1 and DPPIV (first from the right) in artificial liver tissue prepared by the hybrid filling method in Example 1. [Figure 9] 1 shows a scanning electron microscope (SEM) image of an artificial liver tissue prepared by the hybrid filling method in Example 1. [Figure 10] 1 shows a scanning electron microscope (SEM) image of an artificial liver tissue prepared by the hybrid filling method in Example 1. [Figure 11] 1 shows a fluorescent image of CK8 / 18 (first from the left in the top row), a fluorescent image of DAPI (upper right corner of the fluorescent image of CK8 / 18 in the top row), a fluorescent image of albumin (second from the left in the top row), a fluorescent image of collagen I (second from the right in the top row), and a merged image of all these fluorescent images (first from the right in the top row) of artificial liver tissue prepared by the hybrid filling method in Example 1. Also shown are a fluorescent image of CK8 / 18 (first from the left in the middle row), a fluorescent image of DAPI (upper right corner of the fluorescent image of CK8 / 18 in the middle row), a fluorescent image of E-cadherin (second from the left in the middle row), a fluorescent image of collagen IV (second from the right in the middle row), and a merged image of all these fluorescent images (first from the right in the middle row) of artificial liver tissue prepared by the hybrid filling method. Also shown are a fluorescent image of CK8 / 18 (first from the left in the bottom row), a fluorescent image of DAPI (top right corner of the fluorescent image of CK8 / 18 in the bottom row), a fluorescent image of CD31 (second from the left in the bottom row), a fluorescent image of laminin (second from the right in the bottom row), and a merged image of all these fluorescent images (first from the right in the bottom row). [Figure 12] 1 is a graph showing the time-dependent changes in the amounts of albumin, glucose-6-phosphate (G6P), and bile acid produced in the artificial liver tissue prepared by the hybrid loading method in Example 1. [Figure 13]1 is a graph showing the relative expression levels of the α-fetoprotein (AFP) gene and the albumin (ALB) gene in the artificial liver tissues prepared by each filling method in Example 1. [Figure 14] 1 is a graph showing the change over time in the production amounts of coagulation factors V, VII, and XI in the artificial liver tissue prepared by the hybrid loading method in Example 1. [Figure 15] 1 shows images illustrating the protocol for producing and transplanting an artificial liver graft in Example 1. [Figure 16] 1 shows a bright-field image (top) of an artificial liver graft on day 10 after transplantation in Example 1, and a hematoxylin-eosin (HE) stained image (bottom) of a section. [Figure 17] 1 is a graph showing the change over time in the amount of human albumin in the serum of NOG mice transplanted with an artificial liver graft in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0013] <Manufacturing method of artificial organs> The method for producing an artificial organ of this embodiment includes the following steps. performing a decellularization process on a mammalian organ or a part thereof to obtain a decellularized organ or a part thereof (hereinafter, sometimes referred to as the "decellularization process"); A cellularization treatment is carried out to engraft cells onto the decellularized organ or a part thereof, thereby obtaining an organ engrafted with the cells (hereinafter, this may be referred to as the "cellularization treatment step").

[0014] The cellularization treatment (cellularization treatment step) includes the following: Puncture injection of organoids containing cells constituting the organ or cells that can differentiate into the decellularized organ or a part thereof (hereinafter sometimes referred to as the "organoid puncture injection step"); and The cells that constitute the organ or cells that can differentiate into said cells are perfused into the blood vessels of the decellularized organ or part of it (hereinafter, this may be referred to as the "cell perfusion step").

[0015] Hereinafter, the organ engrafted with cells obtained by the method for producing an artificial organ of this embodiment may be referred to as an artificial organ.

[0016] In conventional methods of injecting and filling cells into the interior of a decellularized organ via blood vessels, there was a problem that the cell filling rate was lower in areas far from blood vessels compared to areas close to blood vessels, resulting in a bias in the distribution of engrafted cells. Furthermore, there was room for improvement in terms of improving organ function. From the perspective of improving organ function, it is conceivable to inject and fill organoids containing cells that constitute the organ or cells that can differentiate into such cells into the interior of the organ via blood vessels, but there is a risk that the organoids may cause vascular blockage, making this method not practical at all.

[0017] In contrast, the method for producing an artificial organ of this embodiment combines the method of injecting and filling cells into the interior of a decellularized organ via blood vessels with the method of directly injecting organoids, thereby dispersing cells throughout the organ and achieving an excellent cell filling rate. Furthermore, the method for producing an artificial organ of this embodiment can simultaneously impart stable functions derived from organoids and construct a vascular structure using cells injected via blood vessels, resulting in an artificial organ that maintains organ function.

[0018] The artificial organ produced in this embodiment may be a hollow organ, a parenchymal organ, or any other organ, but parenchymal organs are preferred because of the ease of puncture injection of organoids.

[0019] In this specification, a hollow organ refers to an organ having an internal cavity, while a solid organ refers to an organ in which cells and extracellular matrix are tightly bound together.

[0020] Examples of hollow organs include, but are not limited to, the esophagus, stomach, and intestines (duodenum, small intestine, large intestine, and colon).

[0021] Examples of solid organs include, but are not limited to, the liver, kidneys, spleen, adrenal glands, ovaries, pancreas, thymus, brain, and prostate gland.

[0022] Examples of organs other than hollow organs and solid organs include skin, muscle, bladder, lung, eyeball, uterus, testes, heart, blood vessels, and the like.

[0023] Among these, the liver or kidney is preferred as the artificial organ.

[0024] Next, each step of the method for producing an artificial organ according to this embodiment will be described in detail below.

[0025] <Decellularization process> In the decellularization treatment step, a mammalian organ or a part thereof is subjected to decellularization treatment to obtain a decellularized organ or a part thereof.

[0026] The mammal from which the organ is derived is preferably a mammal other than a human, and particularly preferably a livestock mammal. Examples of livestock mammals include monkeys, marmosets, cattle, horses, camels, llamas, donkeys, yaks, sheep, pigs, goats, deer, alpacas, dogs, raccoon dogs, weasels, foxes, cats, rabbits, hamsters, guinea pigs, rats, mice, squirrels, and raccoons. Among these, pigs and rats are preferred because of their stable availability.

[0027] Decellularization is not particularly limited as long as it is a method that removes animal-derived cells, viruses, and bacteria. Examples of decellularization methods include surfactant treatment, enzyme treatment, osmotic pressure treatment, freeze-thaw treatment, and high hydrostatic pressure treatment, and can be selected appropriately depending on the type of mammal and organ. Among these, surfactant treatment or high hydrostatic pressure treatment is preferred. High hydrostatic pressure treatment is particularly preferred because it does not use agents that may have adverse effects on the human body, such as surfactants. The lower limit of the pressure applied in hydrostatic pressure treatment is generally 10 MPa, with 50 MPa or more being preferred and 150 MPa or more being more preferred. The upper limit is generally 1000 MPa, with 750 MPa or less being preferred and 500 MPa or less being more preferred. The pressurization step may be performed once, or pressurization and depressurization may be repeated multiple times.

[0028] The decellularization conditions can be appropriately selected depending on the type of mammal and organ, and specific examples include the conditions shown in the Examples below.

[0029] The decellularization step preferably includes a step of perfusing water into the organ (hereinafter, sometimes referred to as the "water perfusion step"). Perfusing water into the organ can be performed, for example, using a known perfusion device. The water perfused into the organ can contain a surfactant. Examples of surfactants include, but are not limited to, ionic surfactants and nonionic surfactants. These may be used alone or in combination of two or more. The water perfusion step may be performed alone or in combination with high hydrostatic pressure treatment. By performing the water perfusion step after high hydrostatic pressure treatment, decellularization can be performed efficiently.

[0030] Examples of ionic surfactants include sodium fatty acid, potassium fatty acid, sodium alpha-sulfofatty acid ester, sodium linear alkylbenzenesulfonate, sodium alkyl sulfate, sodium alkyl ether sulfate, sodium alpha-olefin sulfonate, and 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS). These surfactants may be used alone or in combination of two or more. Among these, sodium fatty acid or CHAPS is preferred, and sodium dodecyl sulfate (SDS) or CHAPS is more preferred.

[0031] Examples of nonionic surfactants include alkyl glycosides, alkyl polyoxyethylene ethers (Brij series, etc.), octylphenol ethoxylates (Triton X series, Igepal CA series, Nonidet P series, Nikkol OP series, etc.), polysorbates (Tween series such as Tween 20, etc.), sorbitan fatty acid esters, polyoxyethylene fatty acid esters, alkyl maltosides, sucrose fatty acid esters, glycoside fatty acid esters, glycerin fatty acid esters, propylene glycol fatty acid esters, fatty acid monoglycerides, etc. These may be used alone or in combination of two or more.

[0032] The decellularization step may include a step of perfusing water through the organ to wash it before decellularization (hereinafter, this step may be referred to as a "washing step"). Perfusing water through the organ can be performed, for example, using a known perfusion device.

[0033] The decellularization step can further include a step of washing the components. The method for washing the components can be appropriately selected depending on the type of decellularization method. Examples of washing methods include immersion in a washing solution and microwave irradiation.

[0034] The decellularized organ obtained in the decellularization process contains extracellular matrix (ECM) as its main component.

[0035] As used herein, "extracellular matrix (ECM)" refers to a substance found between cells in animal tissues and that functions as a structural element within the tissue. The ECM contains a mixture of proteins and polysaccharides secreted by cells. Specifically, the ECM is composed of collagen, laminin, fibronectin, glycosaminoglycans (GAGs), etc., and is particularly rich in collagen, but the types and proportions of the components contained vary depending on the type of organ from which it is derived.

[0036] <Cellification process> In the cellularization treatment step, a cellularization treatment is carried out to engraft cells onto the decellularized organ or a part thereof, thereby obtaining an organ engrafted with cells.

[0037] The cells used in the cellularization treatment step, i.e., the cells used in the organoid puncture injection step and the cell perfusion step, are preferably derived from the same species of mammal.

[0038] The mammal from which the cells are derived can be appropriately selected depending on the type of mammal to be transplanted. Examples of mammals include humans, monkeys, marmosets, cows, horses, sheep, pigs, goats, deer, alpacas, dogs, cats, rabbits, hamsters, guinea pigs, rats, and mice. Among these, humans are preferred.

[0039] The cellularization process includes an organoid puncture and injection process and a cell perfusion process. Either the organoid puncture and injection process or the cell perfusion process may be performed first, and the other may be performed later, or these processes may be performed simultaneously.

[0040] [Organoid puncture injection process] In the organoid puncture injection step, organoids containing cells that constitute the organ or cells that can differentiate into said cells are injected into a decellularized organ or a part thereof by puncture injection.

[0041] Organoids may have the functions of an organ, or may be aggregates of cells (spheroids).

[0042] The cells constituting an organ can be appropriately selected depending on the type of the target organ. Specific examples include, but are not limited to, cells collected from any organ, such as solid organs such as the liver, kidney, spleen, adrenal gland, ovary, pancreas, thymus, brain, and prostate; hollow organs such as the esophagus, stomach, and intestines (duodenum, small intestine, large intestine, and colon); and organs other than solid organs and hollow organs such as the skin, muscle, bladder, lung, eyeball, uterus, testis, heart, and blood vessels. More specific examples of somatic cells include, but are not limited to, fibroblasts, immune cells (e.g., B lymphocytes, T lymphocytes, neutrophils, macrophages, monocytes, etc.), erythrocytes, platelets, pericytes, dendritic cells, mesenchymal cells, epithelial cells, endothelial cells, vascular endothelial cells, lymphatic endothelial cells, hepatocytes, pancreatic islet cells (e.g., α cells, β cells, δ cells, ε cells, PP cells, etc.), cumulus cells, glial cells, nerve cells (neurons), oligodendrocytes, microglia, astrocytes, cardiac myocytes, squamous epithelial cells, mononuclear cells, basement membrane cells, keratinocytes, muscle cells, retinal pigment cells, astrocytes, bile duct epithelial cells, and the like.

[0043] Examples of cells that can differentiate into cells that constitute organs include, but are not limited to, stem cells and progenitor cells.

[0044] Stem cells are cells that have the ability to self-replicate and differentiate into cells of multiple lineages. Examples of stem cells include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic progenitor cells, pancreatic stem cells, germ stem cells, intestinal stem cells, and myoblasts.

[0045] A precursor cell is a cell that is at an intermediate stage in the differentiation of the stem cell into a specific somatic cell or germ cell.

[0046] Organoids can be prepared appropriately using known methods depending on the type of target organ.

[0047] For example, liver organoids can be prepared from human iPS cells using the method described in Reference 1 (Sekine K et al., "Generation of human induced pluripotent stem cell-derived liver buds with chemically defined and animal origin-free media," Scientific Reports, Vol. 10, Article number 17937, 2020). Specifically, 1 × 10 6 7 x 10 human iPS cell-derived hepatic endoderm cells (culture day 10) 5 endothelial cells, and 1 x 10 5 Human mesenchymal cells were suspended in a medium containing 2.5% (v / v) FBS, dexamethasone (50 nM), and oncostatin M (10 ng / mL), and a mixture of endothelial cell growth medium (KBM VEC-1, Kohjin Bio) and Dulbecco's Modified Eagle's Medium (DMEM). The cell suspension was seeded onto a 6-well Elplasia round-bottom plate (Corning). Liver organoids were obtained by culturing the cells in the medium for 3 to 21 days.

[0048] The organoids are used in the form of a suspension in a medium, a buffer solution, etc. The medium or buffer solution can be appropriately selected depending on the type of organoid.

[0049] Specifically, the medium may be a basal culture medium containing components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins), etc., and examples thereof include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), RPMI-1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM / F-12), and Glasgow Minimum Essential Medium (Glasgow MEM).

[0050] In suspension, the concentration of organoids is, for example, 3.0 x 10 3 pcs / mL or more 2.0×10 4 It can be made less than 1 / mL.

[0051] The organoid suspension is loaded into a syringe, microinjector, or other puncture injection device, and then the organoid suspension is directly injected into a decellularized organ or a portion thereof. The injection site of the organoid suspension into the decellularized organ or portion thereof may be one or more sites that avoid blood vessels, depending on the size of the organ or portion.

[0052] [Cell perfusion process] In the cell perfusion step, cells that constitute the organ or cells that can differentiate into said cells are perfused into the blood vessels of the decellularized organ or part of it.

[0053] As cells that constitute organs and cells that can differentiate into such cells, the cells exemplified in the above "organoid puncture and injection step" can be used.

[0054] These cells are used in the form of a cell suspension suspended in a medium or buffer solution. The medium or buffer solution can be appropriately selected and used depending on the type of cell. Specifically, the medium exemplified in the above "organoid puncture injection process" can be used.

[0055] In the cell suspension, the cell concentration is, for example, 1 × 10 5 cells / mL or more 1×10 7 cells / mL or less.

[0056] When a cell suspension containing a mixture of multiple types of cells is used, the total cell concentration may be adjusted to fall within the above range.

[0057] The cell suspension can be perfused using a known perfusion device, for example, the perfusion culture system shown in FIG.

[0058] The perfusion pressure in the cell perfusion step can be 0.1 kPa or more and less than 10.0 kPa, and preferably 0.5 kPa or more and less than 5.0 kPa. By setting the perfusion pressure at or above the lower limit, cells can be more thoroughly distributed throughout the organ or a part thereof. On the other hand, by setting the perfusion pressure below the upper limit or equal to or less than the upper limit, cell death due to shear stress can be further suppressed.

[0059] The perfusion flow rate in the cell perfusion step may be set so that the perfusion pressure falls within the above range, and may be, for example, 0.5 mL / min or more and 10.0 mL / min or less, and preferably 1.0 mL / min or more and 5.0 mL / min or less.

[0060] The culture conditions for the cellularization process, i.e., the organoid puncture injection process and the cell perfusion process, are typically a temperature of 30° C. to 40° C., preferably 37° C. Other culture conditions include typically an atmosphere with a CO2 concentration of about 5% by volume.

[0061] The number of days for culturing the organoids after puncture and injection in the organoid puncture and injection process should be long enough to allow the cells in the organoids to fully attach to the organ skeleton, and can be, for example, from 2 to 21 days, preferably from 4 to 10 days.

[0062] The number of days for culturing in the cell perfusion step should be long enough for the cells to be sufficiently engrafted onto the organ skeleton, and can be, for example, from 2 to 21 days, preferably from 4 to 10 days.

[0063] The cellularization process can further include a step of shredding the organ to which the cells have engrafted after cellularization (hereinafter, sometimes referred to as the "shredding process"). This allows the size of the artificial organ to be adjusted to fit the transplant site. The shredding of the organ can be performed, for example, using a known automatic suturing instrument.

[0064] <Artificial organs> The artificial organ of this embodiment is obtained by the above-described method for producing an artificial organ.

[0065] The artificial organ of this embodiment has improved organ function compared to artificial organs obtained by other manufacturing methods, as shown in the examples described below. However, identifying such differences and identifying the artificial organ of this embodiment based on gene expression patterns, etc., would require a significant amount of trial and error, making it virtually impossible. Therefore, it can be said that it is practical to identify the artificial organ of this embodiment by the fact that it was produced by the above-mentioned manufacturing method.

[0066] The artificial organ of this embodiment can be preferably used as a transplant organ for patients or animals suffering from various organ-related diseases.

[0067] The animals to be treated using the artificial organ of this embodiment are preferably mammals, such as humans, monkeys, marmosets, cows, horses, sheep, pigs, goats, deer, alpacas, dogs, cats, rabbits, hamsters, guinea pigs, rats, and mice. Of these, humans are preferred.

[0068] Other Embodiments In one embodiment, the present invention provides a method for transplanting an organ, in which an artificial organ produced by the production method is transplanted into a target site for treatment in a patient or animal suffering from an organ disease. In one embodiment, the present invention provides a method for treating an organ disease, which comprises transplanting an artificial organ produced by the production method into a target site of treatment in a patient or animal suffering from an organ disease.

[0069] The diseases include various diseases that require organ transplantation.

[0070] The liver disease is not particularly limited as long as it is a liver disease accompanied by liver loss due to disease or liver loss due to surgical treatment, and examples include liver cancer, liver cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, fulminant hepatic failure, Wilson's disease, cystic liver disease, hereditary ATTR amyloidosis (FAP), cholangiocarcinoma, metastatic liver cancer, hepatoblastoma, etc.

[0071] Furthermore, kidney diseases are not particularly limited as long as they involve kidney loss due to disease or kidney loss due to surgical treatment, and examples include polycystic kidney disease, nephritis, renal parenchymal tumor (renal cell carcinoma), renal pelvic tumor (renal pelvic cancer), diabetic nephropathy, chronic kidney disease, collagen disease-related nephropathy, nephrosclerosis, pyelitis, renal abscess, pyonephrosis, perinephritis, perinephric abscess, metastatic renal carcinoma, and renal angiomyolipoma.

[0072] Examples of patients and animal patients include animals similar to those that are the subject of treatment for the above-mentioned "artificial organs."

[0073] Examples of the treatment target site include a site where a part of an organ has been lost due to surgical treatment, and a site where an organ has been damaged due to an organ disease. [Example]

[0074] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0075] [Example 1] 1. Preparation of Miniature Pig Decellularized Liver Grafts (1) Collection and preservation of pig liver After intravenous injection of heparin (5000 IU) into a pig (Göttingen minipig), the liver was mobilized and the gallbladder was removed. The bile duct, hepatic artery, and inferior hepatic vena cava were then ligated, and the liver was removed. The portal vein and superior hepatic inferior vena cava were cannulated, and saline was perfused through the portal vein until no more blood was discharged. After perfusion, the liver was stored frozen at -80°C while immersed in saline.

[0076] (2) Decellularization by perfusion Cryopreserved livers were slowly thawed at 4°C. Complete thawing took approximately 3 days. The thawed livers were then perfused with phosphate-buffered saline (PBS) via the portal vein for 1 day (flow rate: 70 mL / min to 100 mL / min) until the effluent became transparent. Next, a 0.5 w / v% aqueous solution of sodium dodecyl sulfate (SDS) was perfused via the portal vein for 1 day. The flow rate was initially 70 mL / min, and was adjusted to 70 mL / min to 320 mL / min depending on the degree of cell removal. In most cases, approximately 60 L to 80 L of 0.5 wt% SDS aqueous solution was perfused at a rate of 70 mL / min to 120 mL / min. Next, 100 mL of Triton-X100 (final concentration: 0.5% v / v), 10 g of glycol ether diamine tetraacetic acid (EGTA) (final concentration: 0.05% w / v), 10 g of sodium azide (final concentration: 0.05% w / v), and 24 g of zwitterionic surfactant (CHAPS) (final concentration: 2 mM) were dissolved in 20 L of PBS. The resulting solution was perfused through the portal vein at a flow rate of 150 mL / min. After the first 10 L was perfused, the remaining 10 L was circulated (approximately 6 hours). Next, 500 mL of PBS containing 0.3 mg / mL colistin and 500 mL of PBS containing 0.2 mg / mL gentamicin were perfused in that order to obtain a decellularized porcine liver.

[0077] (3) Preparation of grafts In a clean bench, decellularized porcine livers were immersed in PBS containing an antibiotic (Pen-Strep; a mixture of penicillin and streptomycin) and separated into individual liver lobes (left lobe, right lobe, left median lobe, and right median lobe) using an automatic stapler. PBS was injected into the vasculature of each separated liver lobe to inflate it. Care was taken to avoid introducing air bubbles into the vasculature. A single blade of scissors was inserted into the central vein of each separated liver lobe and an incision was made approximately 5 cm from the outer edge. The central vein and vasculature were observed, and a 14G Surflo catheter was inserted into the appropriate vasculature. PBS or culture medium dyed with phenol red was injected to confirm the area within the decellularized liver where fluid delivery and perfusion were possible. The perfusion area was confirmed to be within a 4 cm square, including the outer edge, and then excised with scissors. The tip of a 14G Surflo indwelling needle catheter was cut diagonally to approximately 2 cm, and the tip was trimmed to make it blunt. It was then inserted approximately 5 mm into the central vein of the excised decellularized liver and secured by suturing.

[0078] Next, PBS or culture medium containing phenol red was infused through the central vein, and the portal vein stump site was explored using fluid leakage as an indicator. Using the same method as central vein catheter intubation, an 18G Surflo indwelling needle was inserted into the portal vein at the Glisson's capsule, the thickest of the portal vein stump sites, and secured with sutures. PBS or culture medium containing phenol red was alternately injected through the inserted catheter to confirm the absence of leakage. If leakage was found from the amputation site or parenchyma, the leakage site was ligated with sutures.

[0079] The decellularized liver was then perfused with PBS containing colistatin and PBS containing gentamicin to wash the interior of the liver. The catheter hub was then capped and placed in a container filled with PBS containing gentamicin, tightly sealed, and sterilized with gamma radiation (25 kGy). After gamma sterilization, the liver was frozen and stored at -30°C.

[0080] 2. Formation of liver organoids derived from human iPS cells Human iPS cell-derived liver organoids were generated using the method described in Reference 1 (Sekine K et al., “Generation of human induced pluripotent stem cell-derived liver buds with chemically defined and animal origin-free media”, Scientific Reports, Vol. 10, Article number 17937, 2020.). Specifically, 1 × 10 6 7 x 10 human iPS cell-derived hepatic endoderm cells (culture day 10) 5 endothelial cells, and 1 x 10 5 Human mesenchymal cells were suspended in a medium containing 2.5% (v / v) FBS, dexamethasone (50 nM), and oncostatin M (10 ng / mL), a mixture of endothelial cell growth medium (KBM VEC-1, Kohjin Bio) and Dulbecco's modified Eagle's medium (DMEM, GIBCO). The cell suspension was seeded onto a 6-well Elplasia round-bottom plate (Corning). Liver organoids were obtained by culturing the cells in the medium for 3 to 21 days.

[0081] Specifically, a human iPS cell line (QHJI01s04) was maintained and cultured in StemFit AK03N medium (Ajinomoto Co.) on dishes coated with laminin-511 E8 fragment (iMatrix-511, provided by Nippi). Hepatic endoderm (human iPSC-HE), endothelial cells (iPSC-EC), and mesenchymal cells (iPSC-STM) were differentiated using the method described in Reference 1.

[0082] Next, a total of 900 cells (human iPSC-HE / iPSC-EC / iPSC-STM) per spot were resuspended in a mixture of endothelial cell growth medium (KBM VEC-1, Kohjin Bio) and Dulbecco's modified Eagle's medium (DMEM, GIBCO) at a ratio of 10:7:1. Furthermore, FBS (final concentration 2.5% v / v), dexamethasone (final concentration 50 nM, Sigma-Aldrich), oncostatin M (final concentration 10 ng / mL, R&D Systems), and Y-27632 (final concentration 5 μM, Fujifilm Wako Pure Chemical Industries) were added to the medium, and the cell suspension was seeded into 6-well Elplasia round-bottom plates (Corning) to form human iPSC liver organoids in vitro. Human iPSC liver organoids were maintained for 7 to 8 days by replacing half of the culture supernatant with the above-mentioned mixed medium without Y-27632 every 24 hours.

[0083] 3. Cell (Single Cell) Preparation Human iPS cell line (Ff-I01s04) was maintained and cultured in StemFit AK03N medium (Ajinomoto Co.) on dishes coated with laminin-511 E8 fragment (iMatrix-511, provided by Nippi). Hepatic endoderm (human iPSC-HE), endothelial cells (iPSC-EC), and mesenchymal cells (iPSC-STM) were differentiated as described in Reference 1. Cells were stained using the PKH26 red fluorescent cell linker kit (Sigma-Aldrich) before injection into decellularized miniature pig livers.

[0084] 4. Fabrication of an artificial partial liver using the hybrid filling method The cryopreserved decellularized miniature pig liver was transferred to 4°C and thawed the day before use. All handling of the decellularized miniature pig liver was performed using sterile gloves and sterile instruments. The decellularized miniature pig liver was placed on a transmitted light device in a clean bench so that the vascular structure within the decellularized miniature pig liver could be visualized. A syringe containing medium was connected to the central venous catheter, allowing the decellularized miniature pig liver to be inflated as needed. A 1 mL syringe with an injection needle (27G or 29G, inner diameter of at least 200 μm, needle length of 10 mm to 40 mm) or a Myjector was prepared.

[0085] The hepatic endoderm (human iPSC-HE), endothelial cells (human iPSC-EC), mesenchymal cells (human iPSC-STM), and organoids to be injected were collected, resuspended in 500 μL of medium, and placed on ice. The cell suspension (total cell concentration of the three types of cells: 5.4 × 10) was added to the syringe or microinjector. 6 cells / mL; concentration of human iPSC-HE: 3.0 × 10 6 cells / mL, human iPSC-EC concentration: 2.1 x 10 6 cells / mL, human iPSC-STM concentration: 3.0 × 10 5 cells / mL) or organoid suspension (organoid concentration: 1.8 × 10 4The organoids were injected into the syringe at a concentration of 1000 μL / mL. The vascular structure inside the miniature decellularized pig liver was confirmed using a transmitted light device, and the injection needle was inserted into the parenchyma, avoiding major vascular structures. The end of the decellularized liver, which had been expanded by fluid delivery from the central venous catheter, was held with tweezers to ensure accurate insertion of the injection needle into the target area. The organoids were slowly injected in multiple doses while the injection needle was withdrawn (direct organoid injection method). The volume was adjusted to between 20 μL and 100 μL per injection site, and the diameter of the cell aggregates expected to form at the injection site was less than 1 mm. If the organoids settled within the syringe, the syringe was stirred as needed. Before completely withdrawing the injection needle, the angle was changed and the needle was inserted deeper again for injection. This injection procedure was performed at multiple sites within the miniature decellularized pig liver. After the injection, the miniature pig decellularized liver was inflated with culture medium to check for excessive leakage of the injected organoids from the puncture site. If excessive leakage was observed, the leaked organoids were collected and injected again using the same method as above.

[0086] Next, the decellularized miniature pig liver into which the organoids had been injected via puncture injection was placed in a perfusion culture apparatus (see Figure 1). An extension tube and a syringe containing the cell suspension were connected to the three-way stopcock built into the perfusion culture apparatus circuit closest to the central venous catheter of the installed decellularized miniature pig liver. Next, the cell suspension was injected into the decellularized miniature pig liver via the central vein, taking care to avoid the introduction of air (air bubbles). The cell suspension was injected at a rate of 1 mL / min while stirring to prevent precipitation (single-cell injection method). If the pressure rose above 2 mmHg from before injection, the injection of the cell suspension was suspended until the pressure decreased. An artificial partial liver was created by perfusion culture at a flow rate (approximately 3 mL / min) that did not exceed 1.5 kPa (approximately 11 mmHg).

[0087] 5. Cell Packing Rate Assessment On the 7th or 8th day after the start of perfusion culture, the artificial partial livers were removed (hereinafter, sometimes referred to as the "hybrid-filled group"). As controls, a sample in which the cell suspension was simply injected into the decellularized liver of a miniature pig via the central vein (hereinafter, sometimes referred to as the "single-cell injection group"), a sample in which liver organoids were injected into the decellularized liver of a miniature pig via the central vein (hereinafter, sometimes referred to as the "IVC injection group"), and a sample in which liver organoids were directly injected (hereinafter, sometimes referred to as the "puncture injection group") were also prepared.

[0088] The sections were then sectioned and immunostained. Images of the entire sections were captured using an all-in-one fluorescence microscope (BZ-X800, manufactured by KEYENCE Corporation). Cell density within the liver tissue of each sample was then calculated using an analytical application (BZ-H4C / Hybrid Cell Count, manufactured by KEYENCE Corporation). The results are shown in Figure 2.

[0089] As shown in Figure 2, the hybrid packed group had a higher cell packing rate of over 25% compared to the other sample groups.

[0090] 6. Localization of single cells and organoids within liver tissue Next, the localization of PKH26-labeled single cells in liver tissue sections from the hybrid-loaded group was confirmed using an all-in-one fluorescence microscope, BZ-X800 (Keyence Corporation). Figure 3 shows a bright-field image (upper left in Figure 3), a fluorescent image (upper right in Figure 3), and a merged image of the bright-field and fluorescent images (lower right in Figure 3).

[0091] In addition, liver tissue sections from the hybrid-loaded group were immunostained using anti-CK8 / 18 antibody (PROGEN, #GP11) and anti-CD31 antibody (Dako, M0823), and the corresponding secondary antibodies Alexa Fluor® 488 (ThermoFisher Scientific, A-11073) and Alexa Fluor® 568 (ThermoFisher Scientific, A-11004). CK8 / 18 is a hepatocyte marker, and CD31 is an endothelial cell marker. Furthermore, nuclear staining of these liver tissue sections was performed using 4',6-diamidino-2-phenylindole (DAPI). The results are shown in Figure 4. In Figure 4, the image in the upper left is a fluorescent image of PKH26, the image in the upper center is a fluorescent image of CK8 / 18, the image in the upper right is a fluorescent image of CD31, the image in the upper right corner of the fluorescent image of CD31 is a fluorescent image of DAPI, and the image at the bottom is a merged image of all these fluorescent images.

[0092] Figures 3 and 4 reveal that organoids (PKH26-labeled) and single cells (PKH26-labeled) coexist and are in close proximity to each other, contributing to tissue formation.

[0093] In addition, liver tissue sections from the hybrid-loaded group were immunostained using 5-FAM-labeled Collagen Hybridizing Peptide (CHP) (F-CHP, 3-Helix, Red60), anti-CK8 / 18 antibody (PROGEN, #GP11), and anti-collagen III antibody (Proteintech, 22734-1-AP), along with the corresponding secondary antibodies Alexa Fluor® 594 (ThermoFisher SCIENTIFIC, A-11076) and Alexa Fluor® 647 (Abcam, ab150063). F-CHP specifically binds to denatured collagen chains, forming a triple helix structure, and was primarily used to detect the skeleton of organoids and decellularized liver tissue. Anti-collagen III antibody was used to detect the center of organoids. Furthermore, nuclear staining of these liver tissue sections was also performed using DAPI. The results are shown in Figure 5. In Figure 5, the image in the upper left corner is a fluorescent image of F-CHP, the image in the upper left corner of the fluorescent image of F-CHP is a fluorescent image of DAPI, the image in the lower left is a fluorescent image of collagen III, and the image in the lower right is a merged image of all these fluorescent images.

[0094] As shown in Figure 5, single cells were localized around the organoid.

[0095] 7. Confirmation of hepatocyte function Liver tissue sections from the hybrid-loaded group were immunostained using anti-CK8 / 18 antibody (PROGEN, #GP11), anti-albumin (ALB) antibody (Sigma-Aldrich, A6684), and anti-cytokeratin 19 (CK19) antibody (Dako, M0888), along with the corresponding secondary antibodies Alexa Fluor® 488 (ThermoFisher SCIENTIFIC, A-11073), Alexa Fluor® 555 (ThermoFisher SCIENTIFIC, A-21137), and Alexa Fluor® 647 (ThermoFisher SCIENTIFIC, A-21240). Furthermore, nuclear staining was also performed on these liver tissue sections using DAPI. The results are shown in Figure 6. In the top row of Figure 6, the first image from the left is a CK8 / 18 fluorescent image, the image in the upper right corner of the CK8 / 18 fluorescent image is a DAPI fluorescent image, the second image from the left is an ALB fluorescent image, the second image from the right is a CK19 fluorescent image, and the first image from the right is a merged image of all these fluorescent images. The bottom image is an enlarged version of the top image. The arrowheads indicate ALB- and CK19-positive cells.

[0096] As shown in Figure 6, cells with high albumin expression were localized in the gaps between and around the organoids, and albumin-positive and CK19-positive liver progenitor / hepatocyte-like cells were also present.

[0097] In addition, liver tissue sections from the hybrid-loaded group were immunostained using anti-CK8 / 18 antibody (PROGEN, #GP11), anti-E-cadherin antibody (Abcam, ab76055), and anti-cytochrome P4503A4 (CYP3A4) antibody (Abcam, ab231816), along with the corresponding secondary antibodies Alexa Fluor® 488 (ThermoFisher Scientific, A-11073), Alexa Fluor® 568 (ThermoFisher Scientific, A-11004), and Alexa Fluor® 647 (Abcam, ab150063). Furthermore, nuclear staining was also performed on these liver tissue sections using DAPI. The results are shown in Figure 7. In Figure 7, the first image from the left is a fluorescent image of CK8 / 18, the image in the upper right corner of the fluorescent image of CK8 / 18 is a fluorescent image of DAPI, the second image from the left is a fluorescent image of E-cadherin, the second image from the right is a fluorescent image of CYP3A4, and the first image from the right is a merged image of the fluorescent images of E-cadherin and CYP3A4.

[0098] As shown in Figure 7, CYP3A4 expression was confirmed in organized regions with cell-cell interactions mediated by E-cadherin.

[0099] Liver tissue sections from the hybrid-loaded group were immunostained with anti-CK8 / 18 antibody (PROGEN, #GP11), anti-ZO-1 antibody (ThermoFisher SCIENTIFIC, 33-9100), and anti-dipeptidyl peptidase IV (DPPIV, CST, 40134S), along with the corresponding secondary antibodies Alexa Fluor® 488 (ThermoFisher SCIENTIFIC, A-11073), Alexa Fluor® 568 (ThermoFisher SCIENTIFIC, A-11004), and Alexa Fluor® 647 (Abcam, ab150063). ZO-1 is a protein that connects cell membrane proteins to the actin cytoskeleton, and DPPIV is a type of protease known as a prolyl peptidase that dissociates proteins. In addition, nuclear staining of these liver tissue sections was also performed using DAPI. The results are shown in Figure 8. In Figure 8, the first image from the left is a CK8 / 18 fluorescent image, the image in the upper right corner of the CK8 / 18 fluorescent image is a DAPI fluorescent image, the second image from the left is a ZO-1 fluorescent image, the second image from the right is a DPPIV fluorescent image, and the first image from the right is a merged image of the ZO-1 and DPPIV fluorescent images. The arrowheads indicate the area where ZO-1-positive cells and DPPIV-positive cells are adjacent.

[0100] As shown in Figure 8, it was confirmed that DPPIV-positive cells formed bile canaliculus-like structures in the spaces between ZO-1-positive hepatocytes.

[0101] 8. Microstructural analysis The liver tissue sections of the hybrid-loaded group were observed using a scanning electron microscope (SEM), and the results are shown in Figures 9 and 10.

[0102] As shown in Figure 9, it was confirmed that liver organoids were densely packed within the extracellular matrix (ECM) framework surrounding the blood vessel-like structures covered with vascular endothelial cells, forming tissue.

[0103] As shown in FIG. 10, the formation of cell-cell interactions and ECM-cell interactions was confirmed in various cell populations.

[0104] Liver tissue sections from the hybrid-loaded group were immunostained using anti-CK8 / 18 antibody (PROGEN, #GP11), anti-albumin (ALB) antibody (Sigma-Aldrich, A6684), and anti-collagen I (COL.1) antibody (Abcam, ab34710), along with the corresponding secondary antibodies Alexa Fluor® 488 (ThermoFisher SCIENTIFIC, A-11073), Alexa Fluor® 568 (ThermoFisher SCIENTIFIC, A-11004), and Alexa Fluor® 647 (Abcam, ab150063). Nuclear staining was also performed on these liver tissue sections using DAPI. The results are shown in the upper panel of Figure 11. In the top row of Figure 11, the first image from the left is a fluorescent image of CK8 / 18, the image in the upper left corner of the fluorescent image of CK8 / 18 is a fluorescent image of DAPI, the second image from the left is a fluorescent image of ALB, the second image from the right is a fluorescent image of COL.1, and the first image from the right is a merged image of these fluorescent images.

[0105] Liver tissue sections from the hybrid-loaded group were immunostained using anti-CK8 / 18 antibody (PROGEN, #GP11), anti-E-cadherin (ECAD) antibody (Abcam, ab76055), and conjugated anti-collagen IV (COL.4) antibody (Abcam, ab6586), along with the corresponding secondary antibodies Alexa Fluor® 488 (ThermoFisher Scientific, A-11073), Alexa Fluor® 568 (ThermoFisher Scientific, A-11004), and Alexa Fluor® 647 (Abcam, ab150063). Furthermore, nuclear staining was also performed on these liver tissue sections using DAPI. The results are shown in the middle panel of Figure 11. In the middle of Figure 11, the first image from the left is a fluorescent image of CK8 / 18, the image in the upper left corner of the fluorescent image of CK8 / 18 is a fluorescent image of DAPI, the second image from the left is a fluorescent image of ECAD, the second image from the right is a fluorescent image of COL.4, and the first image from the right is a merged image of these fluorescent images.

[0106] Liver tissue sections from the hybrid-loaded group were immunostained using anti-CK8 / 18 antibody (PROGEN, #GP11), anti-CD31 antibody (Dako, M0823), and anti-laminin antibody (Abcam, ab11575), along with the corresponding secondary antibodies Alexa Fluor® 488 (ThermoFisher Scientific, A-11073), Alexa Fluor® 568 (ThermoFisher Scientific, A-11004), and Alexa Fluor® 647 (Abcam, ab150063). Furthermore, nuclear staining was also performed on these liver tissue sections using DAPI. The results are shown in the bottom panel of Figure 11. In the bottom row of Figure 11, the first image from the left is a fluorescent image of CK8 / 18, the image in the upper left corner of the fluorescent image of CK8 / 18 is a fluorescent image of DAPI, the second image from the left is a fluorescent image of CD31, the second image from the right is a fluorescent image of laminin, and the first image from the right is a merged image of these fluorescent images.

[0107] As shown in Figure 11, self-organization was promoted by the ECM scaffold and the ECM produced by the filled cells.

[0108] 9. Evaluation of Hepatocyte Differentiation The production of albumin (ELISA kit: Bethyl Laboratories, E88-129), glucose-6-phosphate (G6P, ELISA kit: BioAssay Systems, EG6P-100), bile acids (ELISA kit: BioAssay Systems, EFBA-100), and factors V (ELISA kit: Abcam, ab137976), VII (ELISA kit: Abcam, ab190810), and IX (ELISA kit: Abcam, ab188393) in the liver tissue of the hybrid-loaded group was measured daily from days 0 to 8 of culture. The results are shown in Figure 12 (albumin, G6P, and bile acids) and Figure 14 (factors V, VII, and IX).

[0109] To evaluate the degree of hepatocyte differentiation, RNA was collected from the liver tissue of the hybrid-loaded group, and the expression of the alpha-fetoprotein (AFP) gene and albumin gene was measured by RT-PCR. The primer sequences used are shown in the table below. The results are shown in Figure 13.

[0110] [Table 1]

[0111] As shown in Figure 12, the hybrid-loaded group showed a significant increase in albumin production from the early stage of culture compared to the single-cell-injected group. The production of G6P and bile acids also increased over time. Furthermore, as shown in FIG. 14, the production amount of each coagulation factor increased over time. Furthermore, as shown in Figure 13, the hybrid-filled group showed increased expression of the ALB gene, which is an indicator of maturation, and decreased expression of the AFP gene, which is an indicator of immaturity, compared to the other groups.

[0112] These findings suggest that the differentiation and maturation of hepatocytes was promoted in the hybrid-loaded group.

[0113] 10. In Vivo Functional Assessment On day 8 of perfusion culture, liver tissue from the hybrid-loaded group was collected from the perfusion apparatus and minced to prepare grafts. The grafts were then transplanted between the liver lobes of NOG mice (see Figure 15). On day 10 after transplantation, the grafts were collected, sectioned, and stained with hematoxylin and eosin (HE). The results are shown in Figure 16.

[0114] In addition, blood was collected from the NOG mice on days 0, 3, 5, 7, and 10 after transplantation, and the amount of human albumin in the serum was measured using an ELISA kit (Bethyl Laboratories, E88-129). The results are shown in Figure 17.

[0115] As shown in Figure 16, the grafts were retained in vivo.

[0116] As shown in FIG. 17, a rapid human albumin production ability was demonstrated from 7 days after transplantation.

[0117] These results suggest that the maturation of transplanted hepatocytes was promoted in vivo.

[0118] From the above results, the following was observed in the liver tissue of the hybrid-loaded group. 1. Improvement in cell filling rate and mutual communication between single cells and organoids were observed. 2. Single cells and organoids were engrafted uniformly and densely throughout the entire graft area. 3. Single cells were present in the interstitial spaces of the organoids, forming heterogeneous ALB-positive cell populations, suggesting the possibility of liver organization, including bile canalicular differentiation. 4. Dense cell engraftment was observed especially around blood vessels, suggesting improved ALB synthesis and angiogenesis mediated by cell-ECM adhesion. 5. Measurement of the production levels of ALB, G6P, bile acids, and multiple coagulation factors, as well as gene expression analysis, confirmed that hepatocyte function was maintained. 6. It was suggested that the maturation of cells within the graft may be promoted in vivo. [Industrial Applicability]

[0119] According to the artificial organ and the method for producing the same of this embodiment, an artificial organ with an excellent cell filling rate and in which organ function is maintained can be obtained.

Claims

1. performing a decellularization treatment on a mammalian organ or a part thereof to obtain a decellularized organ or a part thereof; performing a cellularization treatment to engraft cells onto the decellularized organ or a part thereof, thereby obtaining an organ engrafted with the cells; Including, The cellularization treatment is Puncturing and injecting an organoid containing cells constituting the organ or cells that can differentiate into the decellularized organ or a part thereof; and perfusing cells constituting the organ or cells capable of differentiating into said cells into the blood vessels of the decellularized organ or part thereof; A method for producing an artificial organ, comprising:

2. The method for producing an artificial organ according to claim 1 , wherein the mammal is a mammal other than a human.

3. The method for producing an artificial organ according to claim 1 or 2, wherein the cells are human-derived cells.

4. The method for producing an artificial organ according to claim 1 or 2, wherein the organ is a solid organ.

5. 3. The method for producing an artificial organ according to claim 1, wherein the organ is a liver or a kidney.

6. An artificial organ obtained by the method for producing an artificial organ according to claim 1 or 2.

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