# US 11,439,730 B2 · Cell sheet construct for neurovascular reconstruction and manufacture thereof

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- Number: US 11,439,730 B2
- Document ID: US11439730B2
- Status: Granted
- Kind: B2
- Application number: 16/705,314
- Filed: 2019-12-06
- Published: 2020-04-09
- Granted: 2022-09-13
- Earliest claimed: 2014-08-15
- Assignees: National Defense Medical Center
- Applicants: National Defense Medical Center
- Inventors: Chung-Hsing Chou; Dueng-Yuan Hueng; Tsung-Neng Tsai
- Web page: https://patentgrep.com/patent/US11439730B2

## CPC

- A61L 27/3891: {as distinct cell layers}
- A61K 35/30: Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
- A61K 35/44: Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
- A61L 27/24: Collagen
- A61L 27/3633: {Extracellular matrix [ECM]}
- A61L 27/3675: {Nerve tissue, e.g. brain, spinal cord, nerves, dura mater}
- A61L 27/3808: {Endothelial cells}
- A61L 27/383: {Nerve cells, e.g. dendritic cells, Schwann cells}
- A61L 27/3878: {Nerve tissue, brain, spinal cord, nerves, dura mater}
- C12N 5/0618: {Cells of the nervous system}
- C12N 5/0697: {Artificial constructs associating cells of different lineages, e.g. tissue equivalents (blood vessels C12N5/0691)}
- A61L 2430/32: for nerve reconstruction
- C12N 2500/25: Insulin-transferrin; Insulin-transferrin-selenium
- C12N 2500/46: Amines, e.g. putrescine
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/392: Sexual steroids
- C12N 2502/088: neural stem cells
- C12N 2502/28: Vascular endothelial cells
- C12N 2533/54: Collagen; Gelatin
- 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)
- 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)
- 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 relates to a cell sheet construct for neurovascular reconstruction. The cell sheet construct has a vascular endothelial cell layer and a neural stem cell layer, and the two layers are physically in direct contact with each other, where the vascular endothelial cell layer forms branching vasculatures, and the neural stem cell layer differentiates into neurons. The invention also relates to a method for manufacturing the cell sheet construct, having the following steps: culturing vascular endothelial cells on a substrate to form a vascular endothelial cell layer, seeding neural stem cells on the vascular endothelial cell layer to make the neural stem cells be physically in direct contact with the vascular endothelial cell layer, and culturing the neural stem cells and the vascular endothelial cell layer to differentiate into neurons and branching vasculatures to form a cell sheet construct.

## Claims

### Claim 1

A method for manufacturing a cell sheet constructfor neurovascular reconstruction, consisting of:
step 1: culturing vascular endothelial cells on a cell culture dish with a vascular endothelial cell medium to form a vascular endothelial cell layer;
step 2: seeding neural stem cells on the vascular endothelial cell layer to ensure the neural stem cells being physically in direct contact with the vascular endothelial cell layer;
step 3: co-culturing the neural stem cells and the vascular endothelial cell layer to form a cell sheet construct of neural stem cells-vascular endothelial cells (NSC-EC);
step 4: detaching the cell sheet construct of NSC-EC from the culture dish and transferring the same to a cell culture surface; and
step 5: culturing the cell sheet construct of NSC-EC obtained from step 4 with an NSC-EC co-culture medium to provide a cell sheet construct for neurovascular reconstruction.

### Claim 2 (depends on 1)

The method of claim 1, wherein the neural stem cells are human cerebral neural stem cell line, SCC007, Millipore, Merck.

### Claim 3 (depends on 1)

The method of claim 1, wherein in step 1, the cell culture dish has a temperature-responsive cell culture surface.

### Claim 4 (depends on 1)

The method of claim 1, wherein in step 1, the cell density of the vascular endothelial cell layer is 100,000 cells/cm2 to 300,000 cells/cm2.

### Claim 5 (depends on 1)

The method of claim 1, wherein in step 2, the neural stem cells are seeded with a density of 10,000 cells/cm2 to 100,000 cells/cm2.

### Claim 6 (depends on 1)

The method of claim 1, wherein the vascular endothelial cell medium is an endothelial cell growth-2 (EGM™-2, Lonza) medium comprising 1-10% (v/v) fetal bovine serum (FBS), 0.1-5% (v/v) 10,000 U/mL penicillin and 0.1-5% (v/v) 10,000 μg/mL streptomycin.

### Claim 7 (depends on 1)

The method of claim 1, wherein in step 3, the neural stem cells and the endothelial cell layer are co-cultured in a neural stem cell/endothelial cell co-culture medium.

### Claim 8 (depends on 7)

The method of claim 7, wherein the neural stem cell/endothelial cell co-culture medium comprises 50% (v/v) DMEM/F-12 medium and 50% (v/v) EGM™-2 medium and 0.001-0.03% (v/v) human albumin solution, 100-5000 μg/ml transferrin, 0.1-100 μg/ml putrescine DiHCl, 0.1-10 μg/ml insulin, 1-100 ng/ml progesterone, 0.1-10 mM L-glutamine, and 1-100 ng/ml sodium selenite.