# US 10,501,725 B2 · Method for producing therapeutic corneal endothelial substitute cell sphere

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- Number: US 10,501,725 B2
- Document ID: US10501725B2
- Status: Granted
- Kind: B2
- Application number: 15/535,032
- Filed: 2015-12-11
- Published: 2017-11-30
- Granted: 2019-12-10
- Earliest claimed: 2014-12-11
- Assignees: Keio University
- Applicants: Keio University
- Inventors: Shigeto Shimmura; Shin Hatou; Kazuo Tsubota
- Web page: https://patentgrep.com/patent/US10501725B2

## CPC

- C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)}
- A61K 31/203: Retinoic acids {; Salts thereof}
- 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
- A61K 38/22: Hormones (derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin A61K38/33, e.g. corticotropin A61K38/35)
- A61K 38/28: Insulins
- A61K 47/36: Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K 48/0091: {Purification or manufacturing processes for gene therapy compositions}
- A61P 27/02: Ophthalmic agents
- C12N 5/10: Cells modified by introduction of foreign genetic material
- A61F 9/0017: {implantable in, or in contact with, the eye, e.g. ocular inserts}
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61F: FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS (dental prosthetics A61C)

## Abstract

The problem of the present invention is to provide a method of efficiently producing therapeutic alternative corneal endothelial cells, particularly, a method capable of stably producing them in a condition suitable for intraocular transplantation (in the anterior chamber) in a large amount. Furthermore, the present invention aims to provide a composition for transplantation, which is preferable for intraocular administration, particularly, into the anterior chamber. A therapeutic alternative corneal endothelial cell sphere can be produced by culturing stem cells in suspension in a differentiation induction medium containing a GSK3 inhibitor, retinoic acid and a ROCK inhibitor. Addition of a viscoelastic substance during intraocular (into the anterior chamber) transplantation of the sphere or cultured corneal endothelial cells dispersed into single cells can increase the number of adherent cells after transplantation.

## Claims

### Claim 1

A pharmaceutical composition for transplantation comprising a therapeutic corneal endothelial cell sphere suspension having a cell density of 1-5×106 cells/ml, and a viscoelastic substance, wherein the viscoelastic substance is 5-40 mg/ml hyaluronic acid and/or 5-50 mg/ml chondroitin sulfate, and wherein the ratio of viscoelastic substance:cell sphere suspension is 1:1 to 1:5.

### Claim 2

A pharmaceutical composition for transplantation comprising a cultured corneal endothelial cell suspension having a cell density of 1-5 ×106 cells/ml, and a viscoelastic substance, wherein the cultured corneal endothelial cells are dispersed as single cells or in the form of a cell aggregate, wherein the viscoelastic substance is 5-40 mg/ml hyaluronic acid and/or 5-50 mg/ml chondroitin sulfate, and wherein the ratio of viscoelastic substance:cell suspension is 1:1 to 1:5.

### Claim 3 (depends on 1)

The pharmaceutical composition according to claim 1, which is for transplantation into anterior chamber.

### Claim 4 (depends on 1)

The pharmaceutical composition according to claim 1, wherein the sphere of therapeutic corneal endothelial cells is obtained by a production method comprising culturing stem cells in suspension in a differentiation induction medium, wherein the differentiation induction medium comprises a GSK3inhibitor, retinoic acid and a ROCK inhibitor.

### Claim 5 (depends on 2)

The pharmaceutical composition according to claim 2, wherein the cultured corneal endothelial cells in the suspension are dispersed into single cells.

### Claim 6 (depends on 1)

The pharmaceutical composition according to claim 1, further comprising retinoic acid and a ROCK inhibitor.

### Claim 7 (depends on 6)

The pharmaceutical composition according to claim 6, further comprising insulin, EGF and bFGF.

### Claim 8 (depends on 2)

The pharmaceutical composition according to claim 2, which is for transplantation into anterior chamber.

### Claim 9 (depends on 2)

The pharmaceutical composition according to claim 2, further comprising retinoic acid and a ROCK inhibitor.

### Claim 10 (depends on 9)

The pharmaceutical composition according to claim 9, further comprising insulin, EGF and bFGF.

### Claim 11

A method for improving cell engraftment during transplantation, comprising transplanting a corneal endothelial cell composition comprising (a) a cultured corneal endothelial cell suspension having a cell density of 1-5×106 cells/ml and (b) a viscoelastic substance into an anterior chamber of an eye, wherein the viscoelastic substance is 5-40 mg/ml hyaluronic acid and/or 5-50 mg/ml chondroitin sulfate, wherein the cells are dispersed into single cells or used in the form of a cell aggregate, wherein the ratio of viscoelastic substance:cell suspension is 1:1 to 1:5.