WO2024090312A1 - Organe artificiel et son procédé de production - Google Patents

Organe artificiel et son procédé de production Download PDF

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WO2024090312A1
WO2024090312A1 PCT/JP2023/037746 JP2023037746W WO2024090312A1 WO 2024090312 A1 WO2024090312 A1 WO 2024090312A1 JP 2023037746 W JP2023037746 W JP 2023037746W WO 2024090312 A1 WO2024090312 A1 WO 2024090312A1
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organ
cells
image
liver
artificial
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PCT/JP2023/037746
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English (en)
Japanese (ja)
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洋 八木
倫範 土田
翔伍 長田
晃太郎 西
英樹 谷口
祥己 久世
理志 岡本
茉奈 大友
Original Assignee
慶應義塾
国立大学法人東京大学
公立大学法人横浜市立大学
ＭａｔｒｉＳｕｒｇｅ株式会社
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Publication of WO2024090312A1 publication Critical patent/WO2024090312A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

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  • 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.
  • 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.
  • Non-Patent Document 1 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).
  • Non-Patent Document 1 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • FIG. 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.
  • 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
  • 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.
  • 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 ⁇ -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.
  • 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 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").
  • an artificial organ 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.
  • 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.
  • a hollow organ refers to an organ that has a cavity inside.
  • a solid organ refers to an organ in which cells and extracellular matrix are tightly bound inside.
  • hollow organs include, but are not limited to, the esophagus, stomach, and intestines (duodenum, small intestine, large intestine, and colon).
  • 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.
  • liver or kidney are preferred as artificial organs.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ECM extracellular matrix
  • extracellular matrix 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.
  • 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.
  • 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.
  • 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.
  • organs 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
  • 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., ⁇ cells, ⁇ cells, ⁇ cells, ⁇ 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.
  • immune cells e.g., B
  • 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.
  • 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.
  • a mixed medium of endothelial cell growth medium KBM VEC-1, manufactured by Kohjin Bio
  • DMEM Dulbecco's Modified Eagle's Medium
  • 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.
  • 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).
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimum Essential Medium
  • 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).
  • 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.
  • 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.
  • the cells that constitute organs and cells that can differentiate into said cells 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.
  • the cell concentration can be, for example, 1 ⁇ 10 5 cells/mL or more and 1 ⁇ 10 7 cells/mL or less.
  • 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.
  • the perfusion pressure can be set 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.
  • 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.
  • the artificial organ of this embodiment is obtained by the above-mentioned method for producing an artificial organ.
  • the artificial organ of this embodiment 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.
  • 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 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.
  • 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.
  • 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.
  • 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
  • Patients and affected animals include animals similar to those that are the subject of treatment for the "artificial organs" mentioned above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a mixed medium of endothelial cell growth medium KBM VEC-1, Kohjin Bio Co., Ltd.
  • Dulbecco's modified Eagle medium DMEM, GIBCO Co., Ltd.
  • 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.
  • 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).
  • StemFit AK03N medium Alignment Co., Inc.
  • laminin 511 E8 fragment iMatrix-511, provided by Nippi.
  • Hepatic endoderm human iPSC-HE
  • iPSC-EC endothelial cells
  • iPSC-STM mesenchymal cells
  • 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.
  • KBM VEC-1 endothelial cell growth medium
  • DMEM Dulbecco's modified Eagle's medium
  • FBS final concentration 2.5 v/v%
  • Dexamethasone final concentration 50 nM, Sigma-Aldrich
  • Oncostatin M final concentration 10 ng/mL, R&D Systems
  • Y-27632 final concentration 5 ⁇ M, Fujifilm Wako Pure Chemical Industries
  • 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
  • mesenchymal cells iPSC-STM
  • hepatic endoderm human iPSC-HE
  • endothelial cells human iPSC-EC
  • mesenchymal cells human iPSC-STM
  • 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).
  • organoid suspension organoid concentration: 1.8 ⁇ 10 4 cells/mL.
  • 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.
  • the organoid was gently injected in several separate times (direct puncture injection method of organoid).
  • the amount of injection was adjusted to 20 ⁇ L or more and 100 ⁇ L 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.
  • 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.
  • 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.
  • 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).
  • hybrid loading group 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").
  • 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”
  • punctcture injection group a sample in which the liver organoid was only directly injected by puncture
  • the hybrid packing group had a higher cell packing rate of over 25% compared to the other sample groups.
  • 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
  • CD31 is an endothelial cell marker.
  • the liver tissue sections were nuclear stained using 4',6-diamidino-2-phenylindole (DAPI). The results are shown 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
  • the image at the bottom is a merged image of all these fluorescent images.
  • FIGS 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.
  • 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).
  • CHP 5-FAM-labeled Collagen Hybridizing Peptide
  • PROGEN PROGEN, #GP11
  • anti-collagen III antibody proteintech, 22734-1-AP
  • 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.
  • 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
  • the image at the bottom right is a merged image of all these fluorescent images.
  • 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.
  • 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.
  • 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 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
  • the first image from the right is a merged image of the fluorescent images of E-cadherin and CYP3A4.
  • 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
  • DPPIV is a type of protease known as a prolyl peptidase that dissociates proteins.
  • the liver tissue section was also stained with DAPI for nuclei. The results are shown 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
  • 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.
  • DPPIV-positive cells formed bile canaliculus-like structures in the gaps between ZO-1-positive hepatocytes.
  • liver organoids were densely packed into the extracellular matrix (ECM) framework surrounding the blood vessel-like structure covered with vascular endothelial cells, forming tissue.
  • ECM extracellular matrix
  • cell-cell interactions and ECM-cell interactions were confirmed in various cell populations.
  • 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.
  • 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
  • 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.
  • 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
  • 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).
  • Alexa Fluor registered trademark
  • the liver tissue sections were also stained for nuclei using DAPI. The results are shown in the lower part 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
  • the first image from the right is a merged image of these fluorescent images.
  • AFP ⁇ -fetoprotein
  • 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.
  • the graft was maintained in vivo.
  • the cells exhibited rapid human albumin production ability starting 7 days after transplantation.
  • the artificial organ and manufacturing method of this embodiment can produce an artificial organ with excellent cell filling rate and that maintains organ function.

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Abstract

Un procédé de production d'un organe artificiel selon la présente invention implique la réalisation d'un traitement de décellularisation sur un organe de mammifère ou une partie de celui-ci pour obtenir un organe décellularisé ou une partie de celui-ci et la réalisation d'un traitement de cellularisation qui greffe des cellules sur l'organe décellularisé ou une partie de celui-ci pour obtenir un organe sur lequel les cellules ont été greffées. Le traitement de cellularisation comprend l'injection d'un organoïde qui comprend des cellules de l'organe ou des cellules pertinents qui peuvent se différencier en cellules de l'organe pertinent dans l'organe décellularisé ou une partie de celui-ci et l'infusion des vaisseaux sanguins de l'organe décellularisé ou d'une partie de celui-ci avec des cellules de l'organe ou des cellules pertinents qui peuvent se différencier en cellules de l'organe pertinent. Un organe artificiel selon la présente invention est obtenu au moyen du procédé de production d'un organe artificiel.
PCT/JP2023/037746 2022-10-27 2023-10-18 Organe artificiel et son procédé de production WO2024090312A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019526255A (ja) * 2016-08-26 2019-09-19 ザ ユニバーシティ オブ クィーンズランド 心筋細胞の成熟
WO2021113747A1 (fr) * 2019-12-04 2021-06-10 Miromatrix Medical Inc. Procédés de décellularisation et de recellularisation d'organes et de parties d'organes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019526255A (ja) * 2016-08-26 2019-09-19 ザ ユニバーシティ オブ クィーンズランド 心筋細胞の成熟
WO2021113747A1 (fr) * 2019-12-04 2021-06-10 Miromatrix Medical Inc. Procédés de décellularisation et de recellularisation d'organes et de parties d'organes

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Title
HISANOBU HIGASHI: "Transplantation of bioengineered liver capable of extended function in a preclinical liver failure model", AMERICAN JOURNAL OF TRANSPLANTATION, BLACKWELL MUNKSGAARD, DK, vol. 22, no. 3, 1 March 2022 (2022-03-01), DK , pages 731 - 744, XP093162737, ISSN: 1600-6135, DOI: 10.1111/ajt.16928 *

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