Skin substitutes with improved purity

1. A method for forming a skin equivalent for application to a human subject comprising:

a) providing human product cells, wherein said product cells are near-diploid human keratinocyte (NIKS) cells, providing feeder layer cells, and an agent that prevents cellular replication;

b) treating said feeder layer cells with said agent that prevents cellular replication to provide replication inactive feeder layer cells;

c) assaying said replication inactive feeder layer cells for replication, wherein said assay comprises plating said replication inactive feeder layer cells at a density of from about 7,810 to about 15,630 cells/cm 2 ;

d) cryopreserving said replication inactive feeder layer cells;

e) thawing said replication inactive feeder layer cells to provide thawed replication inactive feeder layer cells;

f) culturing said product cells on said thawed replication inactive feeder layer cells;

g) separating said product cells and said thawed replication inactive feeder layer cells;

h) assaying a representative portion said product cells for the presence of feeder layer cell DNA;

i) selecting product cells harvested from a feeder layer for which said representative portion of said product cells contain less than 0.015% feeder cell DNA equivalents within the total cell population; and

j) forming a skin equivalent from said product cells, and k) assaying said skin equivalent for feeder cell DNA and discarding skin equivalents having more than 1.45×10 4 murine cell DNA equivalents per NIKS cell dose, wherein a NIKS cell dose is 44.2 cm 2 of the finished skin equivalent.

2. The method of claim 1 , wherein said product cells are capable of stratifying into squamous epithelia.

3. The method of claim 1 , wherein said feeder layer provides growth factors and extracellular matrix molecules.

4. The method of claim 1 , wherein the feeder layer is murine fibroblast cells.

5. The method of claim 4 , wherein the murine fibroblast cells are 3T3 cells.

6. The method of claim 4 , wherein said assaying said product cells for the presence of feeder layer cell DNA is accomplished with a mouse DNA PCR assay.

7. The method of claim 1 , wherein said agent is mitomycin-C.

8. The method of claim 1 , wherein said separating said product cells and said feeder layer cells is accomplished with EDTA.

9. The method of claim 1 , wherein said assaying said feeder layer cells for replication is accomplished with a proliferation assay.

10. The method of claim 9 , wherein said feeder layer cells are substantially free of replicating mouse cells.

11. The method of claim 1 , wherein said feeder layer cells demonstrating cell replication are discarded.

12. The method of claim 1 , wherein said assaying said replication inactive feeder layer cells for replication further comprises assaying about 7×10 6 replication inactive feeder layer cells.