# US 12,006,515 B2 · Composition and method for preventing or treating eye disorder

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- Number: US 12,006,515 B2
- Document ID: US12006515B2
- Status: Granted
- Kind: B2
- Application number: 17/136,080
- Filed: 2020-12-29
- Published: 2021-07-01
- Granted: 2024-06-11
- Earliest claimed: 2019-12-30
- Assignees: Buddhist Tzu Chi Medical Foundation
- Applicants: Buddhist Tzu Chi Medical Foundation
- Inventors: Yuan-Chieh Lee; Li-Yi Sun
- Web page: https://patentgrep.com/patent/US12006515B2

## CPC

- C12N 5/0668: {Mesenchymal stem cells from other natural sources}
- A61P 27/02: Ophthalmic agents
- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)}
- C12N 5/0667: {Adipose-derived stem cells [ADSC]; Adipose stromal stem cells}
- C12N 2500/32: Amino acids
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Provided is a method for preparing mesenchymal stem cell-conditioned medium, including isolating mesenchymal stem cells from a subject, maintaining the mesenchymal stem cells in a mesenchymal stem cell maintenance medium, collecting the mesenchymal stem cells at passages 2 to 5, culturing the mesenchymal stem cells in a medium supplemented with fetal bovine serum and mesenchymal stem cell culture adjuvant for hours to obtain a mesenchymal stem cell-conditioned medium, harvesting the medium and centrifuging followed by filtering. Also provided is a method for preventing or treating epithelial tissue disorder of eyes such as dry eye syndrome, including applying the medium to an eye of a subject. Further provided is a medical composition including the medium.

## Claims

### Claim 1

A method for preventing or treating environmentally-induced evaporative dry eye syndrome in a subject in need thereof, comprising administering to the eye of said subject a therapeutically effective amount of a formulation comprising a composition comprising a fraction with molecular weights less than 30 kDa prepared by:
obtaining adipose-derived stem cells (ADSCs) from adipose tissue of the subject, wherein the subject is human;
culturing the ADSCs in a medium supplemented with serum and mesenchymal stem cell culture adjuvant (MCA) for 2 to 5 passages,
wherein the medium is selected from the group consisting of alpha minimum essential medium (alpha-MEM), Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute (RPM!) medium, improved minimum essential medium (IMEM), and Iscove's Modified Dulbecco's Medium (IMDM),
wherein the serum is fetal bovine serum or human serum having a concentration in a range of from 5% to 15% in the medium,
wherein the MCA comprises at least one of fibroblast growth factor 2 (FGF2), N-acetyl-L-cysteine (NAC) and L-ascorbic acid-2-phosphate (AsA2P);
harvesting the medium from the culture of 2 to 5 ADSC passages;
centrifuging the harvested medium followed by filtering; and
obtaining the fraction with molecular weights less than 30 kDa from the filtered medium.

### Claim 2 (depends on 1)

The method of claim 1, wherein the fibroblast growth factor 2 (FGF2) has a concentration in a range of from 5 ng/mL to 15 ng/mL in the mesenchymal stem cell culture adjuvant (MCA).

### Claim 3 (depends on 1)

The method of claim 1, wherein the N-acetyl-L-cysteine (NAC) has a concentration in a range of from 1 mM to 5 mM in the mesenchymal stem cell culture adjuvant (MCA).

### Claim 4 (depends on 1)

The method of claim 1, wherein the L-ascorbic acid-2-phosphate (AsA2P) has a concentration in a range of from 0.1 mM to 0.5 mM in the mesenchymal stem cell culture adjuvant (MCA).

### Claim 5 (depends on 1)

The method of claim 1, wherein the fraction with molecular weights less than 30 kDa comprises a fraction with molecular weight less than 10 kDa.

### Claim 6 (depends on 5)

The method of claim 5, wherein the fraction with molecular weights less than 10 kDa comprises a fraction with molecular weight less than 3 kDa.

### Claim 7 (depends on 6)

The method of claim 6, wherein the fraction with molecular weights less than 3 kDa comprises a fraction with molecular weight less than 1 kDa.

### Claim 8 (depends on 1)

A composition comprising the fraction with molecular weights less than 30 kDa according to claim 1.