In vitro co-culture of human skin keratinocytes and fibroblasts on a biocompatible and biodegradable scaffold.
Extensive full-thickness burns require replacement of both epidermis and dermis. In designing skin replacements, the goal has been to re-create this model and make a product which has both essential components.



In the present study, we developed procedures for establishing confluent, stratified layers of cultured human keratinocytes on the surface of modified collagen-chitosan scaffold that contains fibroblasts. The culture methods for propagation of keratinocytes and fibroblasts isolated from human neonatal foreskin were developed. The growth and proliferation of normal human keratinocytes were evaluated in serum-free (keratinocyte growth medium) and our modified medium. Characterization of human keratinocytes was determined by using pan-keratin and anti-involucrin monoclonal antibodies. For fabrication of relevant biodegradable and biocompatible collagen-chitosan porous scaffold with improved biostability, modified method of freeze-gelation was used. In generating organotypic co-cultures, epidermal keratinocytes were plated onto the upper surface of scaffold containing embedded fibroblasts.



The results showed that the growth of isolated human skin fibroblasts and keratinocytes in our modified medium was more than that in the serum-free medium. The different evaluations of collagen-chitosan scaffold showed that it is relevant to growth of cells (fibroblast and keratinocyte) and has a good flexibility in manipulation of the living skin equivalents.



These findings indicate that the integration of collagen-chitosan scaffold with co-cultured keratinocyte and fibroblast in vitro provides a potential source of living skin for grafting in vivo.
Pajoum Shariati
Seyed Ramin
SR
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran.|Biomedical Group, Dept. of Chemical Engineering and Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, Iran.
Shokrgozar
Mohammad Ali
MA
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran.
Vossoughi
Manouchehr
M
Biomedical Group, Dept. of Chemical Engineering and Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, Iran.
Eslamifar
Ali
A
Dept. of Clinical Research, Pasteur Institute of Iran, Tehran, Iran.
D016475:3T3 Cells; D020341:Absorbable Implants; D000818:Animals; D001672:Biocompatible Materials / Q000138:chemical synthesis / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D048271:Chitosan / Q000494:pharmacology; D018920:Coculture Techniques / Q000295:instrumentation / Q000379:methods; D003094:Collagen / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D008422:Materials Testing; D051379:Mice; D009924:Organ Culture Techniques; D012867:Skin / Q000166:cytology*; D054457:Tissue Scaffolds / Q000737:chemistry
D001672:Biocompatible Materials; D003094:Collagen; D048271:Chitosan