Assessment of an automated bioreactor to propagate and harvest keratinocytes for fabrication of engineered skin substitutes.
Engineered skin substitutes (ESS) composed of autologous fibroblasts and keratinocytes attached to collagen-glycosaminoglycan (GAG) scaffolds are effective adjuncts in the treatment of massive burns. The Kerator, an automated bioreactor for keratinocyte culture, could hypothetically reduce labor and material requirements, and increase availability of ESS. Human keratinocytes were cultured in the Kerator and also in tissue-culture flasks. It was found that keratinocyte confluence increased exponentially with time in both the Kerator (r2=0.99) and the flasks (r2=0.96). Confluence (mean+/-SEM) of keratinocytes in the flasks (28+/-2.3%) was significantly higher than in the Kerator (18+/-0.93%) at day 4. However, there was no difference in confluence at harvest. The colony forming efficiency (CFE) and population doublings (PD) per day of keratinocytes harvested from the Kerator were 67+/-4.7% and 0.80+/-0.06, respectively, and were not different from the corresponding values for keratinocytes from flasks. ESS fabricated with keratinocytes from the Kerator or from the flasks were comparable in vitro in terms of histological anatomy, cellular viability, and surface hydration. These findings show that there are no differences between keratinocytes from the Kerator and those from the flasks regarding (a) growth to confluence, (b) CFE and growth rate (PD/day), or (c) quality of ESS in vitro, suggesting that the Kerator can automate fabrication of ESS and increase its availability for treatment of skin wounds.
Kalyanaraman
Balaji
B
Department of Biomedical Engineering, University of Cincinnati, and Department of Research, Shriners Burn Hospital, Cincinnati, Ohio 45229, USA.
Boyce
Steven
S
D021522:Bioartificial Organs; D001705:Bioprosthesis; D019149:Bioreactors; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D017768:Skin, Artificial; D023822:Tissue Engineering; D014947:Wounds and Injuries / Q000628:therapy