# US 12,599,670 B2 · Composition comprising three-dimensional astrocyte bundles of bi-polar and aligned astrocyte processes and a method of making thereof

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- Number: US 12,599,670 B2
- Document ID: US12599670B2
- Status: Granted
- Kind: B2
- Application number: 15/534,934
- Filed: 2015-12-11
- Published: 2017-12-28
- Granted: 2026-04-14
- Earliest claimed: 2014-12-12
- Assignees: The Trustees of the University of Pennsylvania
- Applicants: The Trustees of the University of Pennsylvania
- Inventors: D. Kacy Cullen
- Web page: https://patentgrep.com/patent/US12599670B2

## CPC

- A61K 47/36: Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- 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)}
- 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
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 5/06: {Animal cells or tissues; Human cells or tissues (preservation of excised living parts A01N1/10)}
- C12N 5/0618: {Cells of the nervous system}
- C12N 5/0619: {Neurons}
- C12N 5/0622: {Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells}
- 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
- 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 includes a composition comprising an astrocyte comprising aligned and elongated astrocyte processes resulting from ex vivo machine-driven, physical stretching of the astrocyte maintained in culture, as well as a method of making it. The invention also includes a composition comprising an astrocyte comprising aligned bi-polar processes resulting from growth within hydrogel micro-columns in culture, as well as a method of making it. The invention also provides methods of treatment of nervous system injury or degeneration by implanting the compositions of the invention.

## Claims

### Claim 1

A composition comprising three-dimensional astrocyte bundles of bi-polar and aligned astrocyte processes, wherein the astrocyte bundles are enclosed in a micro-column comprising a biocompatible matrix on the inner surface of the micro-column.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the micro-column has an inner diameter ranging from about 0.1 mm to about 0.9 mm, or about 0.3 mm to about 0.35 mm.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the inner diameter of the micro-column is about 0.18 mm.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the biocompatible matrix is a hydrogel selected from the group consisting of extracellular matrix, collagen, agarose, methylcellulose, biodegradable polyurethanes, and cross-linked hyaluronic acid.

### Claim 5 (depends on 4)

The composition of claim 4, wherein the biocompatible matrix comprises collagen and the micro-column comprises agarose.

### Claim 6 (depends on 1)

The composition according to claim 1, further comprising astrocytes comprising aligned and elongated astrocyte processes having a length of at least 0.2 millimeters.

### Claim 7

A composition comprising at least one three-dimensional bundle of bi-polar and aligned astrocyte processes made by a method comprising seeding a micro-column comprising a biocompatible matrix and an inner diameter in a range of about 0.1 mm to about 0.9 mm, or about 0.3 mm to about 0.35 mm with a plurality of astrocytes at a density in a range of 9-12×105 cells/ml; and culturing the astrocytes in the micro-column.

### Claim 8

A method of producing three-dimensional astrocyte bundles of bi-polar aligned astrocyte processes, comprising (i) seeding a micro-column comprising a biocompatible matrix with a plurality of astrocytes at a density in a range of 9-12×105 cells/ml.; and (ii) culturing the micro-column so seeded of (i) in a culture medium, wherein the culture medium comprises Neurobasal® (A neuronal culture medium for cultivating neuronal cells from hippocampus, cortex and other regions of the brain and containing no L-glutamine, L-glutamic acid, or aspartic acid), B-27® supplement (a neuronal culture supplement comprising insulin, transferrin, progesterone, putrescine, selenium, thyroid hormone T3, fatty acids, and antioxidants including vitamin E and glutathione), G-5 supplement (a chemically defined serum free supplement based on Bottenstein's G-5 formulation for growth and expression of glial cells and astrocytes) and L-glutamine.

### Claim 9 (depends on 8)

The method of claim 8, wherein the micro-column has an inner diameter ranging from about 0.1 mm to about 0.9 mm, or about 0.3 mm to about 0.35 mm.

### Claim 10 (depends on 9)

The method of claim 9, wherein the inner diameter of the micro-column is about 0.18 mm.

### Claim 11 (depends on 8)

The method of claim 8, wherein the biocompatible matrix is a hydrogel selected from the group consisting of extracellular matrix, collagen, agarose, methylcellulose, biodegradable polyurethanes, and cross-linked hyaluronic acid.

### Claim 12 (depends on 11)

The method of claim 11, wherein the biocompatible matrix is collagen and the micro-column comprised agarose.

### Claim 13 (depends on 8)

The method of claim 8, wherein the B27® supplement (a neuronal culture supplement comprising insulin, transferrin, progesterone, putrescine, selenium, thyroid hormone T3, fatty acids, and antioxidants including vitamin E and glutathione) in the culture medium is at a concentration ranging from 1% to 5%.

### Claim 14 (depends on 8)

The method of claim 8, wherein the G-5 supplement (a chemically defined serum free supplement based on Bottenstein's G-5 formulation for growth and expression of glial cells and astrocytes) in the culture medium is at a concentration ranging from 0.1% to 3%.

### Claim 15 (depends on 8)

The method of claim 8, wherein the of L-glutamine in the culture medium is at a concentration ranging from 0.1% to 1.0%.

### Claim 16 (depends on 8)

The method of claim 8, wherein the micro-column is co-seeded with neurons.

### Claim 17 (depends on 8)

The method of claim 8, wherein the astrocytes so cultured are extracted from the micro-column.

### Claim 18

A method of producing aligned and elongated astrocyte processes from an astrocyte comprising:
a) culturing an astrocyte in a culture medium, wherein the culture medium comprises Neurobasal® (a neuronal culture medium for cultivating neuronal cells from hippocampus, cortex and other regions of the brain and containing no L-glutamine, L-glutamic acid or aspartic acid), B-27® supplement (a neuronal culture supplement comprising insulin, transferrin, progesterone, putrescine, selenium, thyroid hormone T3, fatty acids, and antioxidants including vitamin E and glutathione), G-5 supplement (a chemically defined serum free supplement based on Bottenstein's G-5 formulation for growth and expression of glial cells and astrocytes) and L-glutamine;
b) plating the cultured astrocyte onto an overlying membrane and an underlying membrane so that the cultured astrocyte adheres to both membranes, wherein the overlying membrane or the underlying membrane is selected from polystyrene, ACLAR® film (a clear, poly-chloro-trifluoroethylene (PCTFE) film), collagen coated ACLAR® film (a clear, poly-chloro-trifluoroethylene (PCTFE) film) or MATRIGEL® (a gelatinous protein mixture) coated ACLAR® film (a clear, poly-chloro-trifluoroethylene (PCTFE) film), and wherein the overlying membrane is a towing membrane and the underlying membrane is a stationary membrane, or wherein the underlying membrane is the towing membrane and the overlying membrane is the stationary membrane; and
c) moving one of the two membranes—the overlying membrane and the underlying membrane-across the other under an ex vivo force at a stretching rate of 0.1-0.5 millimeter/day so that astrocyte processes from the astrocyte are stretched and aligned along the ex vivo force.

### Claim 19 (depends on 18)

The method of claim 18, wherein the B-27® supplement (a neuronal culture supplement comprising insulin, transferrin, progesterone, putrescine, selenium, thyroid hormone T3, fatty acids, and antioxidants including vitamin E and glutathione) in the culture medium is at a concentration ranging from 1% to 5%.

### Claim 20 (depends on 18)

The method of claim 18, wherein the G-5 supplement (a chemically defined serum free supplement based on Bottenstein's G-5 supplement (a chemically defined serum free supplement based on Bottenstein's G-5 formulation for growth and expression of glial cells and astrocytes) formulation for growth and expression of glial cells and astrocytes) in the culture medium is at a concentration ranging from 0.1% to 3%.

### Claim 21 (depends on 18)

The method of claim 18, wherein the L-glutamine in the culture medium is at a concentration ranging from 0.1% to 1.0%.

### Claim 22 (depends on 18)

The method of claim 18, wherein the ex vivo force is a mechanical force.

### Claim 23 (depends on 22)

The method of claim 22, wherein the mechanical force is generated from a device comprising structures of a stepper motor, an adaptor and a mechano-bioreactor, and wherein the mechano-bioreactor consists of a towing block attached to elongator rods that are connected to a motorized stepper that gradually pulls the towing blocks backward as shown in FIG. 8B-8D.

### Claim 24 (depends on 18)

The method of claim 18, wherein the astrocyte processes are elongated to greater than 1 millimeter in length, or to greater than 2 millimeters in length.

### Claim 25 (depends on 18)

The method of claim 18, wherein the towing membrane and/or the stationary membrane are selected from collagen coated ACLAR® film (a clear, poly-chloro-trifluoroethylene (PCTFE) film) or MATRIGEL® (a gelatinous protein mixture) coated ACLAR® film (a clear, poly-chloro-trifluoroethylene (PCTFE) film).