# US 2019/0376044 A1 · Stable three-dimensional blood vessels and methods for forming the same

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- Number: US 2019/0376044 A1
- Document ID: US20190376044A1
- Status: Published application
- Kind: A1
- Application number: 16/483,095
- Filed: 2018-02-02
- Published: 2019-12-12
- Earliest claimed: 2017-02-03
- Assignees: Cornell University
- Applicants: Cornell University
- Inventors: Shahin RAFII; Brisa PALIKUQI
- Web page: https://patentgrep.com/patent/US20190376044A1

## CPC

- C12N 5/0691: {Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels}
- 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 35/44: Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
- A61L 27/38: {containing added animal cells (organs or tissue containing native cells A61L27/36)}
- A61L 27/3808: {Endothelial cells}
- A61L 27/507: {for artificial blood vessels (apparatus for applying cells on a blood vessel prosthesis A61F2/062)}
- C12N 15/09: Recombinant DNA-technology
- A61L 2400/06: Flowable or injectable implant compositions
- A61L 2430/22: for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
- A61L 2430/40: Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
- C12N 2500/02: Atmosphere, e.g. low oxygen conditions
- C12N 2510/00: Genetically modified cells
- C12N 2533/50: Proteins
- C12N 2533/52: Fibronectin; Laminin
- C12N 2533/54: Collagen; Gelatin
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)

## Abstract

The present disclosure provides methods for forming stable three-dimensional vascular structures, such as blood vessels and uses thereof. More specifically, the present disclosure provides methods for culturing differentiated endothelial cells that include an exogenous nucleic acid encoding ETV2 transcription factor on a matrix under conditions that express exogenous ETV2 protein in the endothelial cell to form stable three-dimensional artificial blood vessels without the use of a scaffold, pericytes or perfusion. The present disclosure also provides stable three-dimensional blood vessels that are capable of autonomously forming a functional three-dimensional vascular network, and uses thereof. In addition, the present disclosure includes methods for vascularizing an organoid and a decellularized organ by culturing the organoid or decellularized organ with endothelial cells that include an exogenous nucleic acid encoding ETV2 transcription factor under conditions that express exogenous ETV2 protein in the endothelial cell to vascularize the organoid or decellularized organ.

## Claims

### Claim 1

A method for producing a stable three-dimensional blood vessel comprising:
a) culturing an endothelial cell comprising an exogenous nucleic acid encoding an ETV2 transcription factor on a matrix under conditions wherein the endothelial cell expresses the ETV2 transcription factor for at least 3-4 weeks to induce vessel formation; and
b) isolating said stable three-dimensional blood vessel.

### Claim 2 (depends on 1)

The method of claim 1, further comprising culturing the cells of step a) for an additional period of time, wherein said further culturing occurs under conditions wherein the cells do not express the ETV2 transcription factor

### Claim 3 (depends on 2)

The method of claim 2, wherein said further culturing occurs for at least one week.

### Claim 4 (depends on 1)

The method of claim 1, wherein said matrix comprises laminin, entactin, collagen, or a combination thereof.

### Claim 5 (depends on 4)

The method of claim 4, wherein said matrix comprises:
laminin and entactin at a combined concentration of 5.25 mg/mL; and
0.2 mg/mL collagen IV.

### Claim 6 (depends on 1)

The method of claim 1, wherein said matrix is Matrigel™.

### Claim 7 (depends on 1)

The method of claim 1, wherein said endothelial cell comprises a human umbilical vein endothelial cell (HUVEC), an adipose derived endothelial cell or an organ-specific endothelial cell.

### Claim 8 (depends on 7)

The method of claim 7, wherein said organ-specific endothelial cell is selected from the group consisting of heart, muscle, kidney, testis, ovary, lymphoid, liver, pancreas, brain, lungs, bone marrow, spleen, large intestine, and small intestine.

### Claim 9 (depends on 1)

The method of claim 1, wherein said 3-4 week culturing step comprises culturing said endothelial cells in serum free media for at least 7 days.

### Claim 10 (depends on 9)

The method of claim 9, wherein said culturing in serum free media is carried out at less than atmospheric (20%) oxygen tension.

### Claim 11 (depends on 10)

The method of claim 10, wherein said oxygen tension is 5%.

### Claim 12 (depends on 1)

The method of claim 1, wherein said culturing step a) occurs in the absence of a scaffold or perivascular support.

### Claim 13 (depends on 1)

A stable three-dimensional blood vessel produced by the method of claim 1.

### Claim 14

A stable three-dimensional blood vessel capable of autonomously forming a three-dimensional vascular network.

### Claim 15 (depends on 14)

The stable three-dimensional blood vessel of claim 14, wherein said stable three-dimensional blood vessel comprises a tubular structure having at least one contiguous layer of reprogrammed endothelial cells.

### Claim 16 (depends on 15)

The stable three-dimensional blood vessel of claim 15, wherein said reprogrammed endothelial cells comprise an exogenous nucleic acid sequence encoding an ETV2.

### Claim 17 (depends on 14)

The stable three-dimensional blood vessel of claim 14, wherein said stable three-dimensional blood vessel is functional for at least 2 months.

### Claim 18 (depends on 13)

A method for promoting vascularization in a subject comprising administering to the subject the stable three-dimensional blood vessel of claim 13.

### Claim 19 (depends on 18)

The method of claim 18, wherein said administration comprises implanting said stable three-dimensional blood vessel in said subject to form a functional three-dimensional vascular network in said subject.

### Claim 20 (depends on 19)

The method of claim 19, wherein said functional three-dimensional vascular network comprises a capillary, an arteriole, a venule or a lymphatic vessel.

### Claim 21

A method for vascularizing an organoid comprising culturing said organoid with an endothelial cell comprising an exogenous nucleic acid encoding an ETV2 transcription factor on a matrix under conditions wherein the endothelial cell expresses the ETV2 transcription factor.

### Claim 22 (depends on 21)

The method of claim 21, wherein the matrix comprises laminin, entactin, collagen, or a combination thereof.

### Claim 23 (depends on 21)

The method of claim 21, wherein the organoid is selected from the group consisting of a small intestine organoid, colon organoid, kidney organoid, heart organoid and a tumor organoid.

### Claim 24 (depends on 21)

The method of claim 21, wherein said culturing comprises culturing in media comprising basic FGF (FGF-2) and heparin for at least 7 days.

### Claim 25 (depends on 21)

The method of claim 21, further comprising isolating said vascularized organoid.

### Claim 26 (depends on 25)

The method of claim 25, wherein said isolated vascularized organoid is administered to a subject.

### Claim 27 (depends on 25)

The method of claim 25, wherein said isolated vascularized organoid is administered to the subject by surgical implantation.

### Claim 28 (depends on 25)

The method of claim 25, wherein said isolated vascularized organoid is contacted with a therapeutic agent, and the efficacy of said therapeutic agent is determined.

### Claim 29

A method for revascularizing a decellularized organ comprising, culturing said decellularized organ with an endothelial cell comprising an exogenous nucleic acid encoding an ETV2 transcription factor under conditions wherein the endothelial cell expresses the ETV2 transcription factor, wherein said culturing vascularizes said decellularized organ.

### Claim 30 (depends on 29)

The method of claim 29, wherein said decellularized organ is selected from the group consisting of heart, kidney, testis, ovary, bone, lymph, liver, pancreas, brain, lungs, bone marrow, spleen, large intestine and small intestine.

### Claim 31 (depends on 29)

The method of claim 29, wherein said vascularization comprises the formation of an artery, a vein, a capillary, an arteriole, a venule or a combination thereof.

### Claim 32 (depends on 29)

The method of claim 29, wherein said culturing occurs in a bioreactor.

### Claim 33 (depends on 29)

The method of claim 29, further comprising isolating said vascularized decellularized organ.

### Claim 34 (depends on 33)

The method of claim 33, further comprising administering said vascularized decellularized organ to a subject.