# US 9,040,299 B2 · Generating a mucin-producing cell from an umbilical cord amniotic membrane epithelial stem cell

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- Number: US 9,040,299 B2
- Document ID: US9040299B2
- Status: Granted
- Kind: B2
- Application number: 13/652,371
- Filed: 2012-10-15
- Published: 2013-02-14
- Granted: 2015-05-26
- Earliest claimed: 2005-10-21
- Assignees: Cellresearch Corporation Pte. Ltd.
- Applicants: Cellresearch Corporation Pte Ltd
- Inventors: Toan-Thang Phan
- Web page: https://patentgrep.com/patent/US9040299B2

## CPC

- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- A61P 11/00: Drugs for disorders of the respiratory system
- B01F 33/30: Micromixers
- B01F 35/81: Forming mixtures with changing ratios or gradients
- B01L 3/5027: {by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip}
- B01L 3/502784: {specially adapted for droplet or plug flow, e.g. digital microfluidics}
- C12N 5/0605: {Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly}
- G01N 27/44743: {Introducing samples}
- B01L 2200/0605: Metering of fluids
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/0867: Multiple inlets and one sample wells, e.g. mixing, dilution
- B01L 2400/0418: electro-osmotic flow [EOF]
- B01L 2400/0421: electrophoretic flow
- B01L 2400/0487: fluid pressure, pneumatics
- C12N 5/063: {Kereatinocyte stem cells; Keratinocyte progenitors}
- C12N 2500/25: Insulin-transferrin; Insulin-transferrin-selenium
- C12N 2501/105: Insulin-like growth factors [IGF]
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/117: Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
- C12N 2501/119: Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
- C12N 2501/135: Platelet-derived growth factor [PDGF]
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2502/094: keratinocytes
- C12N 2506/025: from extra-embryonic cells, e.g. trophoblast, placenta
- Y10T 436/11: Automated chemical analysis
- Y10T 436/25: including sample preparation
- Y10T 436/25375: Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
- Y10T 436/255: including use of a solid sorbent, semipermeable membrane, or liquid extraction
- Y10T 436/25625: Dilution
- Y10T 436/2575: Volumetric liquid transfer
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

The present invention relates to the generation of a mucin-producing cell using stem/progenitor cells obtained from the amniotic membrane of umbilical cord and therapeutic uses of such mucin-producing cells.

## Claims

### Claim 1

A method of generating a mucin-producing cell comprising:
placing umbilical cord amniotic membrane epithelial stem cells (UCEC) in a container;
culturing the UCEC in the container in a growth medium adapted for the cultivation of a mucin-producing cell through the addition of a growth factor selected from the group consisting of epidermal growth factor (EGF), insulin-like growth factor-1, platelet-derived growth factor-BB (PDGFb), transforming growth factor-β, keratinocyte growth factor (KGF), TGF-αand amphiregulin and combinations thereof; and
permitting the UCEC to differentiate into mucin producing cells as indicated by observing mucin in the culture medium.

### Claim 2 (depends on 1)

A method according to claim 1, wherein said growth medium adapted for the cultivation of mucin-producing cells further comprises insulin, transferrin, and selenous acid.

### Claim 3 (depends on 2)

A method according to claim 2, wherein said growth medium adapted for the cultivation of mucin-producing cells comprises a medium selected from the group consisting of KGM®-Keratinocyte Medium (Cambrex), MEGM™-Mammary Epithelial Cell Medium (Cambrex), EpiLife® medium (Cascade Biologics), Green's Medium, CMRL1066 (Mediatech, Inc.), M171 (Cascade Biologics), L-15 medium, Dulbecco's Modified Eagle Medium (DMEM), DMEM-F12 and RPMI-1640 media.

### Claim 4 (depends on 1)

A method according to claim 1, wherein said medium adapted for the cultivation of a mucin-producing cell comprises a growth medium for the cultivation of mucin-producing cells, epidermal growth factor (EGF), insulin, transferrin and selenous acid.

### Claim 5 (depends on 4)

A method according to claim 4, wherein said medium adapted for the cultivation of a mucin-producing cell comprises about 98,8 to about 99,4% (v/v) of a growth medium for the cultivation of mucin-producing cells, about 0,2 to about 0,4% (v/v) insulin, about 0,2 to about 0,4% (v/v) transferrin, about 0,2 to about 0,4% (v/v) selenous acid and about 10 ng/ml epidermal growth factor (EGF).

### Claim 6 (depends on 5)

A method according to claim 5, wherein said medium adapted for the cultivation of a mucin-producing cell comprises about 98,8 to about 99,4% (v/v) CMRL1066 (Mediatech, Inc.), about 0,2 to about 0,4% (v/v) insulin, about 0,2 to about 0,4% (v/v) transferrin, about 0,2 to about 0,4% (v/v) selenous acid and about 10 ng/ml epidermal growth factor (EGF).

### Claim 7 (depends on 6)

A method according to claim 6, wherein said medium adapted for the cultivation of a mucin-producing cell comprises about 98,8to about 99,4% (v/v) M171 (Cascade Biologics), about 0,2 to about 0,4% (v/v) insulin, about 0,2 to about 0,4% (v/v) transferrin, about 0,2 to about 0,4% (v/v) selenous acid and about 10 ng/ml epidermal growth factor (EGF).