Preparation of cultured skin for transplantation using insulin-like growth factor I in conjunction with insulin-like growth factor binding protein 5, epidermal growth factor, and vitronectin.
BACKGROUND
Cultured skin for transplantation is routinely prepared by growing patient keratinocytes in the presence of semidefined sources of growth factors including serum and feeder cells, but these materials require substantial risk remediation and can contribute to transplant rejection.


METHODS
We have therefore investigated the potential of a novel combination of recombinant and purified growth factors to replace serum and feeder cells in cultures of human keratinocytes suitable for clinical application. Our technique was investigated with respect to culture establishment, serial propagation, colony-forming efficiency, immunocytochemistry, epidermal reconstruction, and suitability to support transplantation by aerosolization.


RESULTS
We demonstrate that insulin-like growth factor (IGF)-I--used in conjunction with epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP)-5 and vitronectin--supports growth in the absence of serum. Moreover, a threefold greater number of cells are generated within 7 days compared to those grown under current best practice conditions using serum (P<0.05). The resulting test cultures are suitable for epidermal reconstruction and support the option for delivery in the form of an aerosolized cell suspension. Serial propagation, with the view to producing confluent sheets for extensive injuries, was achieved but with less consistency and this result correlated with a significant decline in colony-forming efficiency compared to controls.


CONCLUSIONS
IGF-I used in conjunction with IGFBP-5, EGF, and vitronectin provides a superior alternative to serum for the rapid expansion and transplantation of cultured keratinocytes within the first week of treatment. Nevertheless, further optimization is required with respect to elimination of feeder cells and serial expansion of cultures for treatment of extensive injuries.
Dawson
Rebecca A
RA
Tissue Repair and Regeneration Domain, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia. r2.dawson@qut.edu.au
Upton
Zee
Z
Malda
Jos
J
Harkin
Damien G
DG
D000818:Animals; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004817:Epidermis / Q000187:drug effects; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D015603:Keratinocytes / Q000187:drug effects*; D051379:Mice; D010641:Phenotype; D016038:Skin Transplantation; D046509:Tissue Culture Techniques / Q000379:methods*; D019096:Vitronectin / Q000494:pharmacology*
D016895:Culture Media, Serum-Free; D018975:Insulin-Like Growth Factor Binding Protein 5; D019096:Vitronectin; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I