WO2024090312A1 - Artificial organ and production method for same - Google Patents

Abstract

A production method for an artificial organ according to the present invention involves performing a decellularization treatment on a mammalian organ or a portion thereof to obtain a decellularized organ or a portion thereof and performing a cellularization treatment that grafts cells onto the decellularized organ or portion thereof to obtain an organ onto which the cells have been grafted. The cellularization treatment involves injecting an organoid that includes cells of the relevant organ or cells that can differentiate into cells of the relevant organ into the decellularized organ or portion thereof and infusing the blood vessels of the decellularized organ or portion thereof with cells of the relevant organ or cells that can differentiate into cells of the relevale organ. An artificial organ according to the present invention is obtained by means of the production method for an artificial organ.

Description

Artificial organs and their manufacturing methods

The present invention relates to an artificial organ and a method for manufacturing the same. This application claims priority based on Japanese Patent Application No. 2022-172278, filed on October 27, 2022, the contents of which are incorporated herein by reference.

　Recent developments in the field of regenerative medicine have accelerated research into the regeneration of thin tissues such as the skin, gastrointestinal mucosa, and cornea, as well as tissues with relatively simple structures and functions, such as bone and soft tissue. On the other hand, research and development into individual organs has lagged behind. This is because it is still difficult to understand and reproduce the extremely complex structures and functions of three-dimensional organs. The development of functional organ regeneration technology that could become a fundamental treatment for solid organ failure, especially for those with high societal needs, such as the liver, kidneys, and pancreas, is still only halfway there. To date, various technologies have been developed to realize the regeneration of three-dimensional organs.

To regenerate organ structures, 1) an appropriate extracellular matrix (ECM), 2) a continuous three-dimensional structure from microstructures to large blood vessels, and 3) a sufficient supply of cells are required. In order to realize the regeneration of such complex three-dimensional organs, Ott et al. reported in 2008 the world's first method of applying organ skeletons made by decellularizing solid organs themselves to regenerative medicine (see, for example, Patent Document 1). This method involves removing all cells from living tissues using various methods, and using the remaining ECM skeleton, which is a fibrous protein, for tissue regeneration. In fact, decellularized tissues obtained by a similar method using human skin (Alloderm (registered trademark)) and decellularized tissues using porcine heart valves (Hancock (registered trademark)) have already been commercialized and are being used clinically as medical materials.

The inventors have also reported that they have succeeded in producing an artificial liver with sufficient numbers of engrafted liver cells and vascular endothelial cells 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).

JP 2009-505752 A

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.

The method described in Non-Patent Document 1, etc., in which liver cells and vascular endothelial cells are injected from blood vessels into the inside of a decellularized liver, is excellent for reconstructing the vascular structure. However, the albumin production capacity is low, and there is room for improvement.

The present invention was made in consideration of the above circumstances, and provides an artificial organ with an excellent cell filling rate and in which the organ's functions are maintained, and a method for producing the same.

As a result of extensive research to achieve the above objective, the inventors discovered that by injecting cells (single cells) from the blood vessels into a decellularized organ and then directly injecting organoids, the cell filling rate can be improved and an artificial organ that maintains the organ's functions can be obtained, thus completing the present invention.

That is, the present invention includes the following aspects.
(1) A method for producing an artificial organ, comprising: performing a decellularization process on a mammalian organ or a part thereof to obtain a decellularized organ or a part thereof; and performing a cellularization process in which cells are engrafted onto the decellularized organ or a part thereof to obtain an organ engrafted with the cells, wherein the cellularization process comprises puncturing and injecting, into the decellularized organ or a part thereof, an organoid containing cells constituting the organ or cells capable of differentiating into said cells, and perfusing blood vessels of the decellularized organ or a part thereof with the cells constituting the organ or the cells capable of differentiating into said cells.
(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 cells of human origin.
(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).

The above-described artificial organ and manufacturing method thereof can provide an artificial organ and manufacturing method thereof that has an excellent cell filling rate and maintains the organ's functions.

1 is an image showing a perfusion culture system in Example 1. 1 is a graph comparing the cell loading rates in artificial liver tissues prepared by each loading method in Example 1. 13 is an image showing the localization of a single cell labeled with PKH26 in an artificial liver tissue prepared by the hybrid loading 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. 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), and a merged image of all these fluorescence images (bottom). Fluorescence image of 5-FAM-labeled Collagen Hybridizing Peptide (CHP) (upper left), fluorescence image of DAPI (upper left corner of the CHP fluorescence image), fluorescence image of CK8/18 (upper right), fluorescence image of collagen III (lower left), and a merged image of all these fluorescence images (lower left) in the artificial liver tissue prepared by the hybrid loading method in Example 1. The upper 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 lower row shows an enlarged image of the upper row. 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 an artificial liver tissue prepared by the hybrid filling method in Example 1. 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 an artificial liver tissue prepared by the hybrid filling method in Example 1. 1 is a scanning electron microscope (SEM) image of an artificial liver tissue prepared by the hybrid filling method in Example 1. 1 is a scanning electron microscope (SEM) image of an artificial liver tissue prepared by the hybrid filling method in Example 1. 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) in the 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) in the 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 (upper 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), and a fluorescent image of laminin (second from the right in the bottom row), as well as a merged image of all these fluorescent images (first from the right in the bottom row). 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. 1 is a graph showing the relative expression levels of α-fetoprotein (AFP) gene and albumin (ALB) gene in artificial liver tissues prepared by each filling method in Example 1. 1 is a graph showing the change over time in the production amounts of coagulation factors V, VII, and XI in an artificial liver tissue prepared by the hybrid loading method in Example 1. 1 is an image showing the protocol for producing and transplanting an artificial liver graft in Example 1. 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. 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.

[Manufacturing method of artificial organs]
The method for producing an artificial organ of this embodiment includes the steps of performing a decellularization process on a mammalian organ or a part thereof to obtain a decellularized organ or a part thereof (hereinafter, this may be referred to as the "decellularization process step"), and performing a cellularization process to engraft cells onto the decellularized organ or a part thereof to obtain an organ engrafted with the cells (hereinafter, this may be referred to as the "cellularization process step"). The production method of this embodiment can be performed in vitro using an excised mammalian organ or a part thereof.

The cellularization treatment (cellularization treatment step) includes the following.
An organoid containing cells constituting the organ or cells capable of differentiating into said cells is injected into the decellularized organ or part thereof by puncture injection (hereinafter, this may be referred to as the "organoid puncture injection process"), and the cells constituting the organ or cells capable of differentiating into said cells are perfused into the blood vessels of the decellularized organ or part thereof (hereinafter, this may be referred to as the "cell perfusion process").

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.

　In the conventional method of injecting and filling cells into the interior of a decellularized organ via blood vessels, there was an issue that the cell filling rate was lower in areas far from blood vessels compared to areas close to blood vessels, resulting in a biased distribution of engrafted cells. There was also room for improvement in terms of improving organ function. From the perspective of improving organ function, it could be considered to inject and fill the interior of the organ via blood vessels with organoids containing cells that constitute the organ or cells that can differentiate into said cells, but there is a risk of the organoids causing vascular blockage, and this is not a practical method at all.

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, allowing cells to be dispersed throughout the organ and achieving an excellent cell filling rate. Furthermore, the method for producing an artificial organ of this embodiment can achieve both the imparting of stable functions derived from organoids and the construction of a vascular structure using cells injected from blood vessels, resulting in an artificial organ in which organ functions are maintained.

The artificial organ produced in this embodiment may be a hollow organ, a parenchymal organ, or another organ, but a parenchymal organ is preferable because of the ease of puncture injection of the organoid.

In this specification, a hollow organ refers to an organ that has a cavity inside. On the other hand, a solid organ refers to an organ in which cells and extracellular matrix are tightly bound inside.

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

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

Organs other than hollow organs and solid organs include, for example, skin, muscle, bladder, lungs, eyeball, uterus, testes, heart, blood vessels, etc.

Among these, liver or kidney are preferred as artificial organs.

Next, each step of the manufacturing method for the artificial organ of this embodiment will be described in detail below.

(Decellularization process)
In the decellularization process, a mammalian organ or a part thereof is subjected to a decellularization process to obtain a decellularized organ or a part thereof.

The mammal from which the organs are derived is preferably a mammal other than a human, and in particular a livestock mammal. Examples of livestock mammals include monkeys, marmosets, cows, 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. Of these, pigs and rats are preferred because of the stability of availability.

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

　Decellularization conditions can be appropriately selected depending on the type of mammal and organ. Specific examples include the conditions shown in the examples below.

The decellularization process preferably includes a process of perfusing water into the organ (hereinafter, may be referred to as the "water perfusion process"). Perfusion of 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 types. The water perfusion process may be performed alone or in combination with high hydrostatic pressure treatment. By performing the water perfusion process after the high hydrostatic pressure treatment, the decellularization process can be performed efficiently.

Examples of ionic surfactants include sodium fatty acid, potassium fatty acid, sodium alpha sulfo fatty acid ester, sodium linear alkylbenzene sulfonate, sodium alkyl sulfate, sodium alkyl ether sulfate, sodium alpha olefin sulfonate, 3-[(3-cholamidopropyl)dimethylammonium]propanesulfonate (CHAPS), etc. These 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.

Nonionic surfactants include, for example, 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), 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.

The decellularization process may include a process of perfusing water into the organ to wash it before decellularization (hereinafter, may be referred to as the "washing process"). Perfusing water into the organ may be performed, for example, using a known perfusion device.

The decellularization process may further include a step of washing the components. The method of washing the components may be appropriately selected depending on the type of decellularization method. Examples of washing methods include immersing the components in a washing solution and irradiating them with microwaves.

The decellularized organ obtained through the decellularization process contains extracellular matrix (ECM) as its main component.

As used herein, "extracellular matrix (ECM)" refers to the material found between the cells of animal tissues and that functions as a structural element within the tissue. ECM contains a mixture of proteins and polysaccharides secreted by cells. Specifically, 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.

(Cellulization process)
In the cellularization treatment step, a cellularization treatment is carried out to allow cells to engraft on the decellularized organ or a part thereof, thereby obtaining an organ with engrafted cells.

The cells used in the cellularization process, i.e., the cells used in the organoid puncture injection process and the cell perfusion process, are preferably derived from the same mammalian species.

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. Of these, humans are preferred.

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

(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.

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

The cells constituting the 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, prostate, etc.; hollow organs such as the esophagus, stomach, intestine (duodenum, small intestine, large intestine, colon), etc.; hollow organs such as the skin, muscle, bladder, lung, eyeball, uterus, testes, heart, blood vessels, etc., and organs other than solid organs. More specifically, somatic cells include, but are not limited to, for example, fibroblasts, immune cells (e.g., B lymphocytes, T lymphocytes, neutrophils, macrophages, monocytes, etc.), red blood cells, platelets, pericytes, dendritic cells, mesenchymal cells, epithelial cells, endothelial cells, vascular endothelial cells, lymphatic endothelial cells, hepatic cells, 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, etc.

Cells that can differentiate into cells that make up organs include, but are not limited to, stem cells and progenitor cells.

Stem cells are cells that have the ability to replicate themselves 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.

　Progenitor cells are cells that are in the middle of differentiating from the stem cells into specific somatic cells or germ cells.

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

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 x 10 ⁶ human iPS cell-derived hepatic endoderm cells (culture day 10), 7 x 10 ⁵ endothelial cells, and 1 x 10 ⁵ human mesenchymal cells are suspended in a medium. As the medium, a mixed medium of endothelial cell growth medium (KBM VEC-1, manufactured by Kohjin Bio) and Dulbecco's Modified Eagle's Medium (DMEM) containing 2.5 v/v% FBS, dexamethasone (50 nM), and oncostatin M (10 ng/mL) can be used. The cell suspension is seeded on a 6-well Elplasia round-bottom plate (manufactured by Corning). Liver organoids can be obtained by culturing in the above medium for about 3 to 21 days.

Organoids are used in the form of a suspension in a medium or buffer solution. The medium or buffer solution can be selected appropriately depending on the type of organoid.

Specifically, the medium may be a basal culture medium that contains components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, etc.), such as Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Examples include, but are not limited to, 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).

In the suspension, the concentration of the organoids can be, for example, 3.0 x 10 ³ cells/mL or more and 2.0 x 10 ⁴ cells/mL or less.

After the organoid suspension is loaded into a syringe or a microinjector or other device for puncture injection, the organoid suspension is directly injected into the decellularized organ or part thereof. The injection site for the organoid suspension in the decellularized organ or part thereof may be a site that avoids blood vessels, and is injected into one or more sites according to the size of the organ or part thereof.

(Cell perfusion process)
In the cell perfusion step, cells constituting the organ or cells capable of differentiating into said cells are perfused into the blood vessels of the decellularized organ or part of it.

As cells that constitute organs and cells that can differentiate into said cells, the cells exemplified in the "organoid puncture injection process" above can be used.

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 depending on the type of cells. Specifically, the medium exemplified in the "Organoid puncture injection process" above can be used.

In the cell suspension, the cell concentration can be, for example, 1×10 ⁵ cells/mL or more and 1×10 ⁷ cells/mL or less.

When using a cell suspension containing multiple types of cells, the total cell concentration should be adjusted to be within the above range.

Perfusion of the cell suspension can be performed using a known perfusion device, for example, the perfusion culture system shown in Figure 1.

The perfusion pressure in the cell perfusion process 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 to be equal to or more than the lower limit, the cells can be more thoroughly distributed throughout the organ or the entire part of it. On the other hand, by setting the perfusion pressure to be less than the upper limit or equal to or less than the upper limit, cell death due to shear stress can be further suppressed.

The perfusion flow rate in the cell perfusion process may be any rate that results in a perfusion pressure in the above range, and may be, for example, from 0.5 mL/min to 10.0 mL/min, and preferably from 1.0 mL/min to 5.0 mL/min.

The culture conditions in the cellularization process, i.e., the organoid puncture injection process and the cell perfusion process, are usually at a temperature of 30° C. or higher and 40° C. or lower, preferably 37° C. Other culture conditions are usually performed under an atmosphere with a _CO2 concentration of about 5% by volume.

The number of days for which the organoids are cultured after the organoids are punctured and injected in the organoid puncture injection process should be long enough for the cells in the organoids to be sufficiently attached to the skeleton of the organ, and can be, for example, from 2 to 21 days, and preferably from 4 to 10 days.

The number of days for culturing in the cell perfusion process should be long enough for the cells to be sufficiently attached to the organ skeleton, and can be, for example, from 2 to 21 days, preferably from 4 to 10 days.

The cellularization process can further include a process of shredding the organ to which the cells have been 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 device.

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

Compared to artificial organs obtained by other manufacturing methods, the artificial organ of this embodiment has improved organ function, as shown in the examples described below. However, in order to identify such differences and identify the artificial organ of this embodiment based on gene expression patterns, etc., a significant amount of trial and error would be required, which is practically 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.

The artificial organ of this embodiment can be preferably used as a transplant organ for patients or animals with various organ-related diseases.

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

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

Diseases include various diseases that require organ transplants.

Liver diseases are not particularly limited as long as they involve liver deficiency due to disease or liver deficiency 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.

In addition, 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, renal angiomyolipoma, etc.

Patients and affected animals include animals similar to those that are the subject of treatment for the "artificial organs" mentioned above.

Examples of areas to be treated include areas where part of an organ has been lost due to surgical treatment, or areas where an organ has been damaged due to an organ disease.

The present invention will be explained below using examples, but the present invention is not limited to the following examples.

[Example 1]
(1) Preparation of miniature porcine decellularized liver graft (1-1) Harvesting and preservation of porcine liver After intravenous injection of heparin (5000 IU) into a pig (Gottingen miniature pig), the liver was mobilized and the gallbladder was removed. Next, the bile duct, hepatic artery, and inferior hepatic vena cava were ligated, and the liver was removed. The portal vein and superior hepatic inferior vena cava were cannulated, and perfused with saline from the portal vein until blood was no longer discharged. After perfusion, the liver was frozen and stored at -80°C while immersed in saline.

(1-2) Decellularization by Perfusion The frozen liver was slowly thawed at 4°C. It was completely thawed in about 3 days. Next, the thawed liver was perfused with phosphate buffered saline (PBS) from the portal vein for 1 day until the waste liquid became transparent (flow rate of 70 mL/min to 100 mL/min). Next, 0.5 w/v% sodium dodecyl sulfate (SDS) aqueous solution was perfused from the portal vein for 1 day. The flow rate was started at 70 mL/min, and the flow rate was adjusted to 70 mL/min to 320 mL/min depending on the degree of cell removal. In many cases, about 60 L to 80 L of 0.5 mass% SDS aqueous solution was perfused at 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 amphoteric surfactant (CHAPS) (final concentration 2 mM) were dissolved in 20 L of PBS. The prepared solution was perfused from the portal vein at a flow rate of 150 mL/min. After perfusion of the first 10 L, the remaining 10 L was circulated (about 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 this order to obtain a decellularized pig liver.

(1-3) Preparation of grafts In a clean bench, the decellularized porcine liver was immersed in PBS containing an antibiotic (Pen-Strep; a mixture of penicillin and streptomycin), and separated into each liver lobe (left lobe, right lobe, left middle lobe, and right middle lobe) using an automatic suture device or the like. PBS was injected into the vasculature of the separated liver lobe to inflate it. At that time, care was taken not to let air (air bubbles) enter the vasculature. A single blade of scissors was inserted into the central vein of the separated liver lobe, and an incision was made up to about 5 cm from the outer edge. The incised central vein and vascular system were observed, and a catheter with a 14G Surflo indwelling needle was inserted into the appropriate vasculature, and PBS or culture medium reddened with phenol red was injected to confirm the area in the decellularized liver where fluid delivery and perfusion were possible. It was confirmed that this perfusion-enabled area was within 4 cm square, including the outer edge, and cut out with scissors. The tip of a 14G Surflo indwelling needle catheter was cut obliquely to approximately 2 cm, and the tip was trimmed to make it blunt. The catheter was then inserted approximately 5 mm into the central vein of the excised decellularized liver and secured by suturing.

Next, PBS or culture medium containing phenol red was injected through the central vein, and the site of the portal vein stump was explored using leakage of the delivered fluid as an indicator. Using the same method as for catheter intubation into the central vein, an 18G Surflo indwelling needle was inserted into the portal vein of the Glisson's capsule, the thickest of the portal vein stump sites, and secured by suturing. PBS or culture medium containing phenol red was alternately injected through the inserted catheter, and it was confirmed that there was no leakage. If leakage was found from the amputation site or parenchymal site, the leakage site was ligated with sutures.

Then, the inside of the decellularized liver was washed by perfusing PBS containing colistatin and PBS containing gentamicin, after which a cap was attached to the catheter hub. It was then placed in a container filled with PBS containing gentamicin, tightly sealed, and sterilized with gamma rays (25 kGy). After gamma sterilization, it was frozen and stored at -30°C.

(2) Formation of liver organoids derived 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.), liver organoids derived from human iPS cells were prepared. Specifically, 1 × 10 ⁶ human iPS cell-derived hepatic endoderm cells (culture day 10), 7 × 10 ⁵ endothelial cells, and 1 × 10 ⁵ human mesenchymal cells were suspended in the medium. As the medium, a mixed medium of endothelial cell growth medium (KBM VEC-1, Kohjin Bio Co., Ltd.) and Dulbecco's modified Eagle medium (DMEM, GIBCO Co., Ltd.) containing 2.5 v / v % FBS, dexamethasone (50 nM), and oncostatin M (10 ng / mL) was used. The cell suspension was seeded on a 6-well Elplasia round-bottom plate (manufactured by Corning). The cells were cultured in the above medium for 3 to 21 days to obtain liver organoids.

Specifically, first, a human iPS cell line (QHJI01s04) was maintained and cultured in StemFit AK03N medium (Ajinomoto Co., Inc.) on a dish 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 induced to differentiate using the method described in Reference 1 above.

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) in 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 in a 6-well Elplasia round-bottom plate (Corning) to form human iPSC liver organoids in vitro. The human iPSC liver organoids were maintained for 7 to 8 days by replacing half of the culture supernatant with the above mixed medium without Y-27632 every 24 hours.

(3) Preparation of cells (single cells) Human iPS cell line (Ff-I01s04) was maintained and cultured on a dish coated with laminin 511 E8 fragment (iMatrix-511, provided by Nippi) in StemFit AK03N medium (Ajinomoto Co., Inc.). Hepatic endoderm (human iPSC-HE), endothelial cells (iPSC-EC) and mesenchymal cells (iPSC-STM) were induced to differentiate by the method described in Reference 1 above. Before injection into the decellularized liver of a miniature pig, cells were stained using PKH26 red fluorescent cell linker kit (Sigma-Aldrich).

(4) Preparation of an artificial partial liver by the hybrid filling method The frozen decellularized pig liver was transferred to 4°C and thawed the day before use. All handling of the decellularized pig liver was performed using sterile gloves and sterilized instruments. In a clean bench, the decellularized pig liver was placed on a transmitted light device so that the vascular structure in the decellularized pig liver could be visually observed. The catheter on the central vein side was connected to a syringe containing a medium so that the decellularized pig liver could be appropriately inflated. A 1 mL syringe with an injection needle (27G or 29G, with an inner diameter of at least 200 μm and a needle length of 10 mm to 40 mm) or a Myjector was prepared.

The hepatic endoderm (human iPSC-HE), endothelial cells (human iPSC-EC) and mesenchymal cells (human iPSC-STM) used for injection, as well as organoids, were collected, resuspended in 500 μL of medium and placed on ice. The syringe or Myjector was filled with a cell suspension (total cell concentration of three types of cells: 5.4 × 10 ⁶ cells/mL; human iPSC-HE concentration: 3.0 × 10 ⁶ cells/mL, human iPSC-EC concentration: 2.1 × 10 ⁶ cells/mL, human iPSC-STM concentration: 3.0 × 10 ⁵ cells/mL) or an organoid suspension (organoid concentration: 1.8 × 10 ⁴ cells/mL). The vascular structure inside the miniature pig decellularized liver was confirmed using a transmitted light device, and the injection needle was punctured into the parenchymal area while avoiding the main vasculature. At that time, the end of the decellularized liver expanded by sending liquid from the central venous catheter was held with tweezers so that the injection needle could be accurately punctured into the target site. While withdrawing the injection needle, the organoid was gently injected in several separate times (direct puncture injection method of organoid). At that time, the amount of injection was adjusted to 20 μL or more and 100 μL or less per injection site, and the diameter of the cell aggregate expected to be formed at the injection site was adjusted to be less than 1 mm. In addition, if the organoid precipitated in the syringe, the inside of the syringe was appropriately stirred. Before completely withdrawing the injection needle, the angle was changed and the needle was punctured deeply again and injected. This puncture injection was performed at multiple sites of the miniature pig decellularized liver. After the puncture injection, the miniature pig decellularized liver was inflated with a culture medium to check whether the injected organoid was excessively discharged from the puncture site. When excessive discharge was observed, the leaked organoid was collected and puncture injected again in the same manner as the above method.

Next, the small pig decellularized liver into which the organoids had been injected by puncture injection was placed in a perfusion culture device (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 device circuit closest to the central venous catheter of the installed small pig decellularized liver. Next, the cell suspension was injected into the small pig decellularized liver via the central vein, taking care not to introduce 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 to 2 mmHg or more from before injection, the injection of the cell suspension was interrupted until the pressure decreased. An artificial partial liver was produced by perfusion culture at a flow rate (approximately 3 mL/min) that did not exceed a pressure of 1.5 kPa (approximately 11 mmHg).

(5) Evaluation of cell loading rate On the 7th to 8th day after the start of perfusion culture, the prepared artificial partial liver was taken out (hereinafter, sometimes referred to as the "hybrid loading group"). As controls, a sample in which the cell suspension was only injected into the decellularized liver of a small pig via the central vein (hereinafter, sometimes referred to as the "single cell injection group"), a sample in which the liver organoid was only injected into the decellularized liver of a small pig via the central vein (hereinafter, sometimes referred to as the "IVC injection group"), and a sample in which the liver organoid was only directly injected by puncture (hereinafter, sometimes referred to as the "puncture injection group") were also prepared.

Then, after sectioning, the tissue was immunostained and an image of the entire section was obtained using an all-in-one fluorescence microscope BZ-X800 (Keyence), after which the cell filling rate within the liver tissue of each sample was calculated using an analysis application (Keyence, BZ-H4C/Hybrid Cell Count). The results are shown in Figure 2.

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

(6) Localization of single cells and organoids in liver tissue Next, the localization of single cells labeled with PKH26 was confirmed for liver tissue sections of the hybrid-loaded group using an all-in-one fluorescence microscope BZ-X800 (manufactured by KEYENCE). Bright-field images (upper left in FIG. 3), fluorescent images (upper right in FIG. 3), and merged images of the bright-field and fluorescent images (lower in FIG. 3) are shown in FIG. 3.

In addition, liver tissue sections from the hybrid-loaded group were immunostained using anti-CK8/18 antibody (PROGEN, #GP11), anti-CD31 antibody (Dako, M0823), and secondary antibodies Alexa Fluor (registered trademark) 488 (ThermoFisher SCIENTIFIC, A-11073) and Alexa Fluor (registered trademark) 568 (ThermoFisher SCIENTIFIC, A-11004) corresponding to each primary antibody. CK8/18 is a hepatocyte marker, and CD31 is an endothelial cell marker. In addition, the liver tissue sections were nuclear stained using 4',6-diamidino-2-phenylindole (DAPI). The results are shown in Figure 4. In Figure 4, the image at the top left is a fluorescent image of PKH26, the image at the top center is a fluorescent image of CK8/18, the image at the top right is a fluorescent image of CD31, the image at the top 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.

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

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), as well as secondary antibodies corresponding to each primary antibody, Alexa Fluor (registered trademark) 594 (ThermoFisher SCIENTIFIC, A-11076) and Alexa Fluor (registered trademark) 647 (abcam, ab150063). F-CHP is a probe that specifically binds to denatured collagen chains and forms a triple helix structure, and was used primarily to detect the skeleton of organoids and decellularized liver tissue. Anti-collagen III antibody was used to detect the center of the organoid. Furthermore, the liver tissue section was also stained nuclearly using DAPI. The results are shown in Figure 5. In Figure 5, the image at the top left is a fluorescent image of F-CHP, the image at the top left corner of the fluorescent image of F-CHP is a fluorescent image of DAPI, the image at the bottom left is a fluorescent image of collagen III, and the image at the bottom right is a merged image of all these fluorescent images.

As shown in Figure 5, single cells were localized surrounding the organoid.

(7) Confirmation of hepatocyte function The liver tissue sections of the obtained hybrid-loaded group were subjected to immunofluorescence staining using anti-CK8/18 antibody (PROGEN, #GP11), anti-albumin (ALB) antibody (Sigma-Aldrich, A6684), and anti-cytokeratin 19 (CK19) antibody (Dako, M0888), and secondary antibodies corresponding to each primary antibody, Alexa Fluor (registered trademark) 488 (ThermoFisher SCIENTIFIC, A-11073), Alexa Fluor (registered trademark) 555 (ThermoFisher SCIENTIFIC, A-21137), and Alexa Fluor (registered trademark) 647 (ThermoFisher SCIENTIFIC, A-21137). Immunostaining was performed using a fluorescent staining agent (A-21240, manufactured by SCIENTIFIC). Furthermore, nuclear staining was also performed on this liver tissue section using DAPI. The results are shown in FIG. 6. In the upper part of FIG. 6, 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 ALB, the second image from the right is a fluorescent image of CK19, and the first image from the right is a merge image of all these fluorescent images. The images in the lower part are enlarged images of the images in the upper part. The arrowheads indicate ALB-positive and CK19-positive cells.

As shown in Figure 6, albumin-highly expressing cells were localized in the gaps between and around the organoids, and albumin- and CK19-positive hepatic progenitor/hepatocyte-like cells were also present.

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), as well as secondary antibodies corresponding to each primary antibody, Alexa Fluor (registered trademark) 488 (ThermoFisher SCIENTIFIC, A-11073), Alexa Fluor (registered trademark) 568 (ThermoFisher SCIENTIFIC, A-11004), and Alexa Fluor (registered trademark) 647 (abcam, ab150063). Furthermore, the liver tissue sections were stained with DAPI for nuclei. 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.

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

Liver tissue sections from the hybrid-loaded group were immunostained using anti-CK8/18 antibody (PROGEN, #GP11), anti-ZO-1 antibody (ThermoFisher SCIENTIFIC, 33-9100), and anti-dipeptidyl peptidase IV (DPPIV, CST, 40134S), as well as secondary antibodies corresponding to each primary antibody: Alexa Fluor (registered trademark) 488 (ThermoFisher SCIENTIFIC, A-11073), Alexa Fluor (registered trademark) 568 (ThermoFisher SCIENTIFIC, A-11004), and Alexa Fluor (registered trademark) 647 (abcam, ab150063). ZO-1 is a protein that links cell membrane proteins to the actin cytoskeleton, and DPPIV is a type of protease known as a prolyl peptidase that dissociates proteins. Furthermore, the liver tissue section was also stained with DAPI for nuclei. The results are shown in FIG. 8. In FIG. 8, 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 ZO-1, the second image from the right is a fluorescent image of DPPIV, and the first image from the right is a merged image of the fluorescent images of ZO-1 and DPPIV. The arrowheads indicate the areas where ZO-1-positive cells and DPPIV-positive cells are adjacent.

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

(8) Microstructural Analysis The liver tissue sections of the hybrid-loaded group were observed with a scanning electron microscope (SEM). The results are shown in Figures 9 and 10.

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

As shown in Figure 10, cell-cell interactions and ECM-cell interactions were confirmed in various cell populations.

In addition, 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), as well as secondary antibodies corresponding to each primary antibody, Alexa Fluor (registered trademark) 488 (ThermoFisher SCIENTIFIC, A-11073), Alexa Fluor (registered trademark) 568 (ThermoFisher SCIENTIFIC, A-11004), and Alexa Fluor (registered trademark) 647 (abcam, ab150063). Furthermore, the liver tissue sections were stained with DAPI for nuclei. The results are shown in the top row 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.

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 conjugate anti-collagen IV (COL.4) antibody (abcam, ab6586), as well as secondary antibodies corresponding to each primary antibody, Alexa Fluor (registered trademark) 488 (ThermoFisher SCIENTIFIC, A-11073), Alexa Fluor (registered trademark) 568 (ThermoFisher SCIENTIFIC, A-11004), and Alexa Fluor (registered trademark) 647 (abcam, ab150063). Furthermore, the liver tissue sections were stained with DAPI for nuclei. The results are shown in the middle 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.

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), as well as secondary antibodies corresponding to each primary antibody, Alexa Fluor (registered trademark) 488 (ThermoFisher SCIENTIFIC, A-11073), Alexa Fluor (registered trademark) 568 (ThermoFisher SCIENTIFIC, A-11004), and Alexa Fluor (registered trademark) 647 (abcam, ab150063). In addition, the liver tissue sections were also stained for nuclei using DAPI. The results are shown in the lower part 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.

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

(9) Evaluation of hepatocyte differentiation Albumin (ELISA kit: Bethyl Laboratories, E88-129), glucose-6-phosphate (G6P, ELISA kit: BioAssay Systems, EG6P-100), bile acid (ELISA kit: BioAssay Systems, EFBA-100), and factor V (ELISA kit: abcam, ab137976), factor VII (ELISA kit: abcam, ab190810) and factor IX (ELISA kit: abcam, ab188393) production in the liver tissue of the hybrid-loaded group were measured using an ELISA kit every day from the start of culture to day 8. The results are shown in Figure 12 (albumin, G6P and bile acids) and Figure 14 (factors V, VII and IX).

In addition, to evaluate the degree of differentiation of hepatocytes, RNA was extracted from the liver tissue of the hybrid-loaded group, and the expression of the α-fetoprotein (AFP) gene and albumin gene was measured by RT-PCR. The sequences of the primers used are shown in the table below. The results are shown in Figure 13.

12, in the hybrid-loaded group, a significant increase in the amount of albumin produced was observed from the early stage of culture compared to the single-cell-injected group. The production amounts 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 FIG. 13, the hybrid-loaded 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.

These findings suggest that the differentiation and maturation of liver cells is promoted in the hybrid packed group.

(10) In vivo functional evaluation Liver tissue from the hybrid-loaded group on the 8th day of perfusion culture was collected from the perfusion apparatus and cut into pieces to prepare grafts. The grafts were then transplanted between the liver lobes of NOG mice (see FIG. 15). The grafts were collected on the 10th day after transplantation, and sections were prepared and stained with hematoxylin-eosin (HE). The results are shown in FIG. 16.

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.

As shown in Figure 16, the graft was maintained in vivo.

As shown in Figure 17, the cells exhibited rapid human albumin production ability starting 7 days after transplantation.

These results suggest that maturation of transplanted hepatocytes was promoted in vivo.

From the above results, the following was observed in the liver tissue of the hybrid loading group.
(1) Improved cell loading rate and mutual communication between single cells and organoids were observed.
(2) Single cells and organoids were engrafted uniformly and distributed throughout the entire graft area, with high density localization.
(3) Single cells were present in the gaps between the organoids, forming a heterogeneous population of ALB-positive cells, suggesting the possibility of liver organization, including bile canalicular differentiation.
(4) Dense cell adhesion was observed especially around blood vessels, suggesting improved ALB synthesis ability and angiogenesis mediated by cell-ECM adhesion.
(5) Maintenance of hepatocyte function was confirmed by measuring the production of ALB, G6P, bile acids, and multiple coagulation factors, as well as by gene expression analysis.
(6) It was suggested that the maturation of cells within the graft may be promoted in vivo.

The artificial organ and manufacturing method of this embodiment can produce an artificial organ with excellent cell filling rate and that maintains organ function.

Claims (6)

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 for engrafting cells onto the decellularized organ or a part thereof to obtain an organ engrafted with the cells;
Including,
The cellularization treatment includes:
Injecting an organoid containing cells constituting the organ or cells capable of differentiating into the decellularized organ or a part thereof by puncturing the organ,
Perfusing cells constituting the organ or cells capable of differentiating into said cells into the blood vessels of the decellularized organ or a part thereof;
A method for producing an artificial organ, comprising: The method for producing an artificial organ according to claim 1, wherein the mammal is a mammal other than a human. The method for producing an artificial organ according to claim 1 or 2, wherein the cells are human-derived cells. The method for producing an artificial organ according to claim 1 or 2, wherein the organ is a solid organ. The method for producing an artificial organ according to claim 1 or 2, wherein the organ is a liver or a kidney. An artificial organ obtained by the method for producing an artificial organ according to claim 1 or 2.

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