# US 10,441,609 B2 · Decellularization and recellularization of solid organs

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- Number: US 10,441,609 B2
- Document ID: US10441609B2
- Status: Granted
- Kind: B2
- Application number: 13/725,030
- Filed: 2012-12-21
- Published: 2013-05-02
- Granted: 2019-10-15
- Earliest claimed: 2005-08-26
- Assignees: Miromatrix Medical Inc.
- Applicants: Miromatrix Medical Inc.
- Inventors: Harald Ott; Doris Taylor
- Web page: https://patentgrep.com/patent/US10441609B2

## CPC

- A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00)
- A61K 9/0024: {Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue (compositions for intravenous administration, normal injectable solutions or dispersions for, e.g. subcutaneous administration A61K9/0019; brain implants A61K9/0085; (coated) prostheses, catheters or stents A61L)}
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)}
- C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)}
- C12M 33/04: {by injection or suction, e.g. using pipettes, syringes, needles (pipettes in general B01L3/02)}
- C12N 5/0602: {Vertebrate cells}
- C12N 5/0657: {Cardiomyocytes; Heart cells}
- A61K 35/34: Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes (vascular smooth muscle A61K35/44)
- C12N 2506/00: Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention provides for methods and materials to decellularize a solid organ and to recellularize such a decellularized organ to thereby generate a solid organ.

## Claims

### Claim 1

A method, comprising
providing a decellularized extracellular matrix of a decellularized mammalian organ that retains the morphology of said extracellular matrix prior to decellularization, wherein said decellularized extracellular matrix of said decellularized organ comprises an intact exterior surface, wherein said decellularized extracellular matrix of said decellularized organ comprises the extracellular matrix components of a vascular tree comprising an intact vascular bed, and wherein said decellularized extracellular matrix retains a majority of fluid introduced to the decellularized extracellular matrix vascular tree; and
introducing a population of cells to the decellularized matrix, or a vessel, a duct, or a cavity of said decellularized extracellular matrix; and
perfusing a first solution into the vessel, the duct, or the cavity of the decellularized extracellular matrix, or injecting the first solution into the decellularized extracellular matrix, so that at least some of said cells distribute within and on said at least a portion of said decellularized extracellular matrix, thereby providing a recellularized extracellular matrix of the decellularized mammalian organ.

### Claim 2 (depends on 1)

The method of claim 1 wherein said organ is a heart, kidney, liver, spleen, pancreas, bladder, skeletal muscle, small bowel, large bowel, stomach, bone, brain, or a lung.

### Claim 3 (depends on 2)

The method of claim 2 wherein said heart is a pig heart, a human heart, a bovine heart, a sheep heart, or a canine heart.

### Claim 4 (depends on 2)

The method of claim 2 wherein said kidney comprises a substantially intact glomerular structure.

### Claim 5 (depends on 1)

The method of claim 1 wherein the cells invade the decellularized extracellular matrix within 72 hours of the introducing.

### Claim 6 (depends on 1)

The method of claim 1 wherein the decellularized mammalian organ is non-human.

### Claim 7 (depends on 1)

The method of claim 1 wherein said cells are cardiac fibroblasts, cardiac microvasculature endothelial cells, smooth muscle cells, aortic endothelial cells, or a combination thereof.

### Claim 8 (depends on 1)

The method of claim 1 wherein said cells are allogeneic or xenogeneic to said decellularized mammalian organ.

### Claim 9 (depends on 1)

The method of claim 1 further comprising introducing said recellularized extracellular matrix into a patient, wherein said cells are autologous to said patient.

### Claim 10 (depends on 1)

The method of claim 1 wherein said decellularized extracellular matrix is prepared by perfusing a cannulated mammalian organ with a first cellular disruption medium.

### Claim 11 (depends on 1)

The method of claim 1 wherein said recellularized extracellular matrix of said decellularized mammalian organ is exposed to a mechanical load or pressure.

### Claim 12 (depends on 1)

The method of claim 1 wherein said cells are multipotent adult progenitor cells (MAPC) or a subset thereof.

### Claim 13 (depends on 1)

The method of claim 1 wherein said cells are capable of differentiation.

### Claim 14 (depends on 1)

The method of claim 1 wherein said decellularized mammalian organ is a heart, kidney, liver, or lung.

### Claim 15 (depends on 1)

The method of claim 1 wherein said cells are introduced to the vessel of the decellularized extracellular matrix.

### Claim 16 (depends on 1)

The method of claim 1 wherein said cells are infused into said decellularized extracellular matrix.

### Claim 17 (depends on 1)

The method of claim 1 wherein the first solution is perfused through the vessel.

### Claim 18 (depends on 1)

The method of claim 1 wherein the decellularized mammalian organ is a liver, lung or kidney.

### Claim 19 (depends on 1)

The method of claim 1 wherein the cells are introduced and the first solution is perfused into the vessel.

### Claim 20 (depends on 19)

The method of claim 19 further comprising perfusing a second solution into the vessel after the cells are introduced and the first solution is perfused.

### Claim 21 (depends on 19)

The method of claim 19 further comprising perfusing a second solution into the vessel before the cells are introduced and the first solution is perfused.

### Claim 22 (depends on 21)

The method of claim 21 further comprising perfusing a third solution into the vessel after the introducing.

### Claim 23 (depends on 1)

The method of claim 1 wherein the cells and the first solution are concurrently perfused into the vessel, the duct, or the cavity, or are concurrently injected into the decellularized extracellular matrix.

### Claim 24 (depends on 1)

The method of claim 1 wherein a system is employed to perform said introducing and said perfusing.

### Claim 25 (depends on 24)

The method of claim 24 wherein the system includes a cannulation device and an apparatus comprising a pump, and provides for exchange of gases.

### Claim 26

An ex vivo method of recellularization of a decellularized mammalian organ comprising
providing a decellularized extracellular matrix of a decellularized mammalian organ, wherein said decellularized extracellular matrix of said decellularized organ comprises an intact exterior surface, wherein said decellularized organ comprises the extracellular matrix components of a vascular tree comprising an intact vascular bed, and wherein said decellularized extracellular matrix retains a majority of fluid introduced to the decellularized extracellular matrix vascular tree;
perfusing or injecting said decellularized organ prior to recellularization with a solution comprising one or more compounds thereby generating a perfused decellularized organ; and
introducing a population of cells to said perfused decellularized organ, wherein the one or more compounds stimulate at least some of the cells as compared to a comparable population of cells that undergo said introducing but are absent said perfusing.

### Claim 27 (depends on 26)

The method of claim 26 wherein said one or more compounds comprise a growth factor.

### Claim 28 (depends on 26)

The method of claim 26 wherein said one or more compounds comprise a factor that modifies the coagulation cascade or is an immune modulating agent.

### Claim 29 (depends on 26)

The method of claim 26 wherein said decellularized mammalian organ is a heart, kidney, liver, spleen, pancreas, bladder, skeletal muscle, small bowel, large bowel, stomach, bone, brain, or a lung.

### Claim 30 (depends on 26)

The method of claim 26 wherein the cells are perfused to a vessel, a duct, or a cavity of said decellularized extracellular matrix, or the cells are injected into the decellularized extracellular matrix.

### Claim 31 (depends on 30)

The method of claim 30 wherein the cells invade the decellularized extracellular matrix within 72 hours of said introducing.

### Claim 32 (depends on 30)

The method of claim 30 wherein the cells are perfused to the vessel, the duct, or the cavity before the one or more compounds are perfused, or the cells are injected into the decellularized extracellular matrix before the one or more compounds are injected.

### Claim 33

A method for recellularizing an extracellular matrix of a decellularized mammalian organ, comprising
providing a decellularized extracellular matrix of a decellularized mammalian organ that retains the morphology of said extracellular matrix prior to decellularization, wherein said decellularized extracellular matrix of said decellularized organ comprises an intact exterior surface, wherein said decellularized extracellular matrix of said decellularized organ comprises the extracellular matrix components of a vascular tree comprising an intact vascular bed, and wherein said decellularized extracellular matrix retains a majority of fluid introduced to the decellularized extracellular matrix vascular tree; and
introducing a population of stem cells to the decellularized extracellular matrix, or a vessel, a duct, or a cavity, of said decellularized extracellular matrix; and
perfusing a solution into the vessel, the duct, or the cavity of the decellularized extracellular matrix, or injecting the solution into the decellularized extracellular matrix, so that at least some of said stem cells distribute within and on at least a portion of said decellularized extracellular matrix, thereby providing a recellularized extracellular matrix of the decellularized mammalian organ.

### Claim 34 (depends on 33)

The method of claim 33 wherein said stem cells comprise embryonic stem cells, umbilical cord cells or a subset thereof, bone marrow cells or a subset thereof, peripheral blood cells or a subset thereof, adult-derived stem cells or a subset thereof, tissue-derived stem cells or a subset thereof, mesenchymal stem cells (MSC) or a subset thereof.

### Claim 35

A method for recellularizing an extracellular matrix of a decellularized mammalian liver, comprising
providing a decellularized extracellular matrix of a decellularized mammalian liver that retains the morphology of said extracellular matrix prior to decellularization, wherein said decellularized extracellular matrix of said decellularized liver comprises an intact exterior surface, wherein said decellularized extracellular matrix of said decellularized liver comprises the extracellular matrix components of a vascular tree comprising an intact vascular bed, and wherein said decellularized extracellular matrix retains a majority of fluid introduced to the decellularized extracellular matrix vascular tree; and
perfusing a population of endothelial cells or liver-specific cells and a first solution into a vessel, a duct or a cavity of said decellularized extracellular matrix so that said endothelial cells distribute and engraft in the vascular tree or said liver-specific cells distribute and engraft in the cavity, and optionally multiply, wherein the endothelial cells re-endothelialize the vascular tree and the liver-specific cells have an organ-specific activity of corresponding cells present in the liver prior to decellularization.

### Claim 36 (depends on 35)

The method of claim 35 wherein the mammal is a human or a pig.

### Claim 37 (depends on 35)

The method of claim 35 wherein the cells are allogeneic or xenogeneic to the liver.

### Claim 38 (depends on 35)

The method of claim 35 wherein the population of endothelial cells or liver-specific cells are perfused into the vessel or the duct.

### Claim 39 (depends on 38)

The method of claim 38 wherein the population of endothelial cells is perfused into the vessel.

### Claim 40 (depends on 38)

The method of claim 38 wherein the population of liver-specific cells is perfused into the vessel.

### Claim 41 (depends on 38)

The method of claim 38 further comprising perfusing a second solution into the vessel after the population of endothelial cells or liver-specific cells are perfused.

### Claim 42 (depends on 35)

The method of claim 35 further comprising perfusing a second solution into the vessel after the population of endothelial cells or liver-specific cells and the first solution are perfused.

### Claim 43 (depends on 35)

The method of claim 35 further comprising perfusing a second solution into the vessel before the population of endothelial cells or liver-specific cells and the first solution are perfused.

### Claim 44 (depends on 43)

The method of claim 43 further comprising perfusing a third solution into the vessel after the population of endothelial cells or liver-specific cells are perfused.

### Claim 45 (depends on 35)

The method of claim 35 wherein a system is employed to perfuse the population of endothelial cells or liver-specific cells and the first solution.

### Claim 46 (depends on 45)

The method of claim 45 wherein the system includes a cannulation device and an apparatus comprising a pump, and provides for exchange of gases.

### Claim 47 (depends on 35)

The method of claim 35 further comprising perfusing a second solution comprising one or more compounds in an amount effective to enhance recellularization of the population of endothelial cells or liver-specific cells.

### Claim 48 (depends on 35)

The method of claim 35 wherein the endothelial cells or the liver-specific cells are autologous.

### Claim 49

A method for recellularizing an extracellular matrix of a decellularized human or pig liver, comprising:
providing a decellularized extracellular matrix of a decellularized human or pig liver that retains the morphology of said extracellular matrix prior to decellularization, wherein said decellularized extracellular matrix of said decellularized human or pig liver comprises an intact exterior surface, wherein said decellularized extracellular matrix of said decellularized human or pig liver comprises the extracellular matrix components of a vascular tree comprising an intact vascular bed, and wherein said decellularized extracellular matrix retains a majority of fluid introduced to the decellularized extracellular matrix vascular tree;
selecting an amount of human or pig endothelial cells and hepatocytes for recellularization; and
introducing the human and pig endothelial cells or hepatocytes to a vessel or a duct of said decellularized extracellular matrix and perfusing a solution into the vessel or duct of the decellularized extracellular matrix so that said endothelial cells and hepatocytes distribute and engraft, and optionally multiply, within and on said decellularized extracellular matrix, thereby providing a recellularized human or pig liver decellularized extracellular matrix having engrafted human or pig hepatocytes with an activity of cells of the human or pig liver prior to decellularization and having engrafted human or pig endothelial cells that re-endothelialize the vascular tree.

### Claim 50

A method for recellularizing an extracellular matrix of a decellularized human or pig liver, comprising
providing in a bioreactor a decellularized extracellular matrix of a decellularized human or pig liver that retains the morphology of said extracellular matrix prior to decellularization, wherein said decellularized extracellular matrix of said decellularized human or pig liver comprises an intact exterior surface, wherein said decellularized extracellular matrix of said decellularized human or pig liver comprises the extracellular matrix components of a vascular tree comprising an intact vascular bed, and wherein said decellularized extracellular matrix retains a majority of fluid introduced to the decellularized extracellular matrix vascular tree;
perfusing a population of human or pig endothelial cells or liver-specific cells in a first solution into a vessel or a duct of said decellularized extracellular matrix so that said endothelial cells distribute and engraft and optionally multiply in the vascular tree or said liver-specific cells distribute and engraft and optionally multiply, wherein the engrafted endothelial cells re-endothelialize the vascular tree and the engrafted liver-specific cells have an activity of corresponding cells present in the liver prior to decellularization; and
perfusing a second solution into the re-endothelialized vessel to maintain the viability of the cells in the recellularized liver.

### Claim 51

A method for recellularizing an extracellular matrix of a decellularized human or pig liver, comprising
providing a decellularized extracellular matrix of a decellularized human or pig liver that retains the morphology of said extracellular matrix prior to decellularization, wherein said decellularized extracellular matrix of said decellularized human or pig liver comprises an intact exterior surface, wherein said decellularized extracellular matrix of said decellularized human or pig liver comprises the extracellular matrix components of a vascular tree comprising an intact vascular bed, and wherein said decellularized extracellular matrix retains a majority of fluid introduced to the decellularized extracellular matrix vascular tree;
selecting an amount of human or pig endothelial cells or liver-specific cells for recellularization;
perfusing a first solution through the extracellular matrix components of the vascular tree; and
perfusing the human or pig endothelial cells or liver-specific cells in a second solution into a vessel or a duct of said decellularized extracellular matrix so that said endothelial cells distribute and engraft and optionally multiply in the vascular tree or said liver-specific cells distribute and engraft in the cavity and optionally multiply, wherein the engrafted endothelial cells re-endothelialize the vascular tree or the engrafted liver-specific cells have an activity of corresponding cells present in the liver prior to decellularization.

### Claim 52

An at least partially recellularized organ comprising a perfusion decellularized extracellular matrix and at least about 10,000,000 regenerative cells engrafted thereon; wherein prior to the recellularization, the perfusion decellularized organ included an intact exterior surface, an intact, non-vasculature decellularized extracellular matrix and an intact, vasculature decellularized extracellular matrix, wherein said vasculature decellularized extracellular matrix retains a majority of fluid introduced to said vasculature decellularized extracellular matrix, wherein said vascular decellularized extracellular matrix comprises human cells, and wherein said decellularized extracellular matrix is from a kidney, lung, liver, pancreas, or spleen.