# US 7,611,895 B2 · Method for growth of human conjunctival tissue equivalents for research, clinical ocular surface transplantation and tissue engineering

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 7,611,895 B2
- Document ID: US7611895B2
- Status: Granted
- Kind: B2
- Application number: 11/398,557
- Filed: 2006-04-06
- Published: 2006-08-10
- Granted: 2009-11-03
- Earliest claimed: 2002-03-29
- Assignees: Singapore Eye Research Institute
- Inventors: Donald Tan; Leonard Ang; Roger Beuerman
- Web page: https://patentgrep.com/patent/US7611895B2

## CPC

- C12N 5/0623: {Stem cells}
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)}
- 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)
- C12N 2500/14: Calcium; Ca chelators; Calcitonin
- C12N 2500/84: from mammals
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2501/01: Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/33: Insulin
- C12N 2501/39: Steroid hormones
- C12N 2533/92: Amnion; Decellularised dermis or mucosa
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The invention relates to materials and methods for producing equivalents of tissues of the ocular surface, especially conjunctival tissue equivalents. The method of the invention involves biopsy of an appropriate tissue, primary culture in a certain medium, proliferative culture in a second medium, and differentiative culture in a third medium. The tissue equivalents are typically grown upon a substrate, typically amniotic membrane, which provides ease of handling as one advantage.

## Claims

### Claim 1

A method for producing conjunctival tissue equivalents comprising:
i) culturing cells of a tissue comprising conjunctival stem cells in a medium I comprising
a) Dulbecco's Modified Essential Medium and Ham's F-12 medium in a ratio of 1:1;
b) Fetal Bovine Serum or Human Serum Albumin;
c) human Epidermal Growth Factor
d) insulin;
e) cholera toxin;
f) hydrocortisone;
g) an antibiotic or mixture of antibiotics;
ii) further culturing the cells in a medium II comprising:
a) Keratinocyte Growth Medium;
b) human Epidermal Growth Factor;
c) insulin;
d) hydrocortisone;
e) pituitary extract;
f) an antibiotic or mixture of antibiotics; and
iii) further culturing the cells to obtain conjunctival tissue equivalents in a medium III comprising:
a) Dulbecco's Modified Essential Medium and Ham's F-12 medium in a ratio of 3:1;
b) Fetal Bovine Serum or Human Serum Albumin;
c) human Epidermal Growth Factor
d) insulin;
e) cholera toxin;
f) hydrocortisone;
g) an antibiotic or mixture of antibiotics;
said culturing steps (i), (ii), (iii) being performed on the surface of a basement membrane side of an amniotic membrane; thereby obtaining a conjunctival tissue equivalent.

### Claim 2

A method for producing equivalents of an ocular tissue comprising:
i) culturing explants of tissue from the ocular surface in a medium I comprising
a) Dulbecco's Modified Essential Medium and Ham's F-12 medium in a ratio of 1:1;
b) Fetal Bovine Serum or Human Serum Albumin;
c) human Epidermal Growth Factor
d) insulin;
e) cholera toxin;
f) hydrocortisone;
g) an antibiotic or mixture of antibiotics;
ii) culturing cells from the explant in a medium II comprising:
a) Keratinocyte Growth Medium;
b) human Epidermal Growth Factor;
c) insulin;
d) hydrocortisone;
e) pituitary extract;
f) an antibiotic or mixture of antibiotics;
iii) culturing the cells to obtain conjunctival tissue equivalents in a medium III comprising:
a) Dulbecco's Modified Essential Medium and Ham's F-12 medium in a ratio of 3:1;
b) Fetal Bovine Serum or Human Serum Albumin;
c) human Epidermal Growth Factor
d) insulin;
e) cholera toxin;
f) hydrocortisone;
g) an antibiotic or mixture of antibiotics;
thereby obtaining an equivalent of said ocular tissue.

### Claim 3 (depends on 2)

The method of claim 2, wherein said tissue comprises corneal epithelial cells or limbal stem cells.

### Claim 4 (depends on 2)

The method of claim 2, in which the tissue from an ocular surface comprising conjunctival stem cells is obtained by biopsy of tissue distal from the cornea in a manner that the cornea remains intact.

### Claim 5

A method for producing equivalents of an ocular tissue comprising:
culturing explants of tissue comprising conjunctival stem cells in a medium I comprising
a) Dulbecco's Modified Essential Medium and Ham's F-12 medium in a ratio of 1:1;
b) Fetal Bovine Serum or Human Serum Albumin;
c) human Epidermal Growth Factor
d) insulin;
e) cholera toxin;
f) hydrocortisone;
g) an antibiotic or mixture of antibiotics;
ii) culturing cells from the explant in a medium II comprising:
a) Keratinocyte Growth Medium;
b) human Epidermal Growth Factor;
c) insulin;
d) hydrocortisone;
e) pituitary extract;
f) an antibiotic or mixture of antibiotics; and
iii) culturing the cells to obtain ocular tissue equivalents in a medium III comprising:
a) Dulbecco's Modified Essential Medium and Ham's F-12 medium in a ratio of 3:1;
b) Fetal Bovine Serum or Human Serum Albumin;
c) human Epidermal Growth Factor
d) insulin;
e) cholera toxin;
f) hydrocortisone;
g) an antibiotic or mixture of antibiotics;
thereby obtaining an equivalent of said ocular tissue.

### Claim 6 (depends on 5)

The method of claim 5, in which the tissue comprising conjunctival stem cells is obtained by biopsy of tissue distal from the cornea in a manner that the cornea remains intact.