# US 7,407,805 B2 · Skin substitutes with improved barrier function

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- Number: US 7,407,805 B2
- Document ID: US7407805B2
- Status: Granted
- Kind: B2
- Application number: 11/235,814
- Filed: 2005-09-27
- Published: 2006-06-08
- Granted: 2008-08-05
- Earliest claimed: 2001-03-02
- Inventors: Allen Comer; Lynn Allen-Hoffmann; Michael Hoffmann
- Web page: https://patentgrep.com/patent/US7407805B2

## CPC

- C12N 5/0698: {Skin equivalents}
- C12N 2500/36: Lipids
- C12N 2500/38: Vitamins
- 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 2501/13: Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
- C12N 2501/385: of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
- C12N 2501/39: Steroid hormones
- C12N 2501/60: Transcription factors
- C12N 2502/094: keratinocytes
- C12N 2502/1323: Adult fibroblasts
- C12N 2510/00: Genetically modified cells
- C12N 2533/54: Collagen; Gelatin
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to in vitro cultured skin substitutes, and in particular to in vitro cultured skin substitutes that have improved barrier function. In some embodiments, improved barrier function is a result of improved culture conditions, while in other embodiments, improved barrier function results from genetic modification of keratinocytes. Improved culture conditions to improve barrier function include organotypic culture in the presence of linoleic acid and/or linoleic acid at about 75% humidity. Suitable genetic modifications for improving barrier function includes transfection with a DNA construct capable of expressing GKLF.

## Claims

### Claim 1

A method of making a skin equivalent having improved barrier function, the method comprising:
providing (a) a composition comprising near-diploid immortalized keratinocyte (NIKS) cells and (b) a culture medium comprising ascorbic acid, linoleic acid, isoproterenol, carnitine, serine, oleic acid, arachidonic acid, α-tocopherol, and epidermal growth factor in amounts effective to improve barrier function; and
culturing said composition (a) in said culture medium (b) under conditions wherein said culturing provides a skin equivalent having a surface electrical capacitance of 40 to 240 pF measured over a 10 second interval in vitro.

### Claim 2 (depends on 1)

The method of claim 1, wherein said ascorbic acid is at a concentration of about 10 to 100 micrograms/ml.

### Claim 3 (depends on 1)

The method of claim 1, wherein said ascorbic acid is at a concentration of about 0.05 mg/ml.

### Claim 4 (depends on 1)

The method of claim 1, wherein said linoleic acid is at a concentration of about 5 to 80 micromolar.

### Claim 5 (depends on 1)

The method of claim 1, wherein in said skin equivalent the content of ceramides 5, 6, and 7is about 20 to about 50% of the total ceramide content.

### Claim 6 (depends on 1)

The method of claim 1, wherein in said skin equivalent the content of ceramide 2 is about 10 to about 40% of the total ceramide content.

### Claim 7 (depends on 1)

The skin equivalent produced by the method of claim 1.