Skin substitutes with improved barrier function

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.

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

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

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

5. 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.

6. 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.

7. The skin equivalent produced by the method of claim 1 .