The method for the formation of a histologically-complete skin substitute that is recovered from the culture by treatment with a protease comprising the steps of:
- (a) feeding basal keratinocyte stem cells a medium consisting essentially of N(2-OH-ethyl-)piperazine-N'-(2-ethane-sulfonic acid) in a concentration in the range of 14 mM to 22 mM, sodium chloride in a concentration in the range of 90 mM to 140 mM, calcium 2+ ion in a concentration in the range of 0.03 mM to 0.3 mM, histidine in a concentration in the range of 1.0.times.10.sup.-4 M to 2.5.times.10.sup.-4 M, isoleucine in a concentration in the range of 0.5.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, methionine in a concentration in the range of 1.0.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, phenylalanine in a concentration in the range of 1.0.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, tryptophan in a concentration in the range of 0.5.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, tyrosine in a concentration in the range of 1.0.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, hydrocortisone in a concentration in the range of 1.0.times.10.sup.-7 M to 5.0.times.10.sup.-7 M, phosphoethanolamine in a concentration in the range of 0.5.times.10.sup.-4 M to 2.0.times.10.sup.-4 M, ethanolamine in a concentration in the range of 0.5.times.10.sup.-4 M, to 2.0.times.10.sup.-4 M, epidermal growth factor in a concentration in the range of 1 ng/ml to 25 ng/ml, and insulin-like growth factor-1 in a concentration in the range of 0.3 ng/ml to 30 ng/ml;
- (b) replacing the preceding medium with a medium consisting essentially of N-(2-OH-ethyl)piperazine-N'-(2-ethane-sulfonic acid) in a concentration in the range of 14 mM to 22 mM, sodium chloride in a concentration in the range of 90 mM to 140 mM, calcium 2+ ion in a concentration in the range of 0.7 mM to 3.0 mM, histidine in a concentration in the range of 1.0.times.10.sup.-4 M to 2.5.times.10.sup.-4 M, isoleucine in a concentration in the range of 0.5.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, methionine in a concentration in he range of 1.0.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, phenylalanine in a concentration in the range of 1.0.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, tryptophan in a concentration in the range of 0.5.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, tyrosine in a concentration in the range of 1.0.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, hydrocortisone in a concentration in the range of 1.0.times.10.sup.-7 M to 10.0.times.10.sup.-7 M, phosphoethanolamine in a concentration in the range of 0.5.times.10.sup.-4 M to 2.0.times.10.sup.-4 M, ethanolamine in a concentration in the range of 0.5.times.10.sup.-4 M to 2.0.times.10.sup.-4 M, epidermal growth factor in a concentration in the range of 1 ng/ml to 5 ng/ml, insulin-like growth factor-1 in a concentration in the range of 0.3 ng/ml to 30 ng/ml, and Beta-transforming growth factor in a concentration in the range of 3.0 ng/ml to 30 ng/ml;
- and
- (c) replacing the preceding medium with a medium consisting essentially of N-(2-OHethyl)piperazine-N'-(2-ethane-sulfonic acid) in a concentration in the range of 14 mM to 22 mM, sodium chloride in a concentration in the range of 90 mM to 140 mM, calcium 2+ ion in a concentration in the range of 0.7 mM to 3.0 mM, histidine in a concentration in the range of 1.0.times.10.sup.-4 M to 2.5.times.10.sup.-4 M, isoleucine in a concentration in the range of 0.5.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, methionine in a concentration in the range of 1.0.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, phenylalanine in a concentration in the range of 1.0.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, tryptophan in a concentration in the range of 0.5.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, tyrosine in a concentration in the range of 1.0.times.10.sup.-4 M to 5.0.times.10.sup.-4 M, linoleic acid in a concentration in the range of 1 microgram/ml to 15 microgram/ml, hydrocortisone in a concentration in the range of 1.0.times.10.sup.-7 M to 10.0.times.10.sup.-7 M, phosphoethanolamine in a concentration in the range of 0.5.times.10.sup.-4 M to 2.0.times.10.sup.-4 M, and ethanolamine in a concentration in the range of 0.5.times.10.sup.-4 M to 2.0.times.10.sup.-4 M, with the replacements of said media preferably done by aspiration accomplished under sterile conditions.