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Engineered skin substitutes (ESS), composed of cultured autologous fibroblasts and keratinocytes inoculated on collagen-glycosaminoglycan (GAG) sponge, are effective adjuncts to split-thickness autograft in the treatment of burns exceeding 50% of total body surface area. The process of ESS fabrication is extremely materials and labor intensive, and these factors contribute to the high cost of the engineered tissue. The use of bioreactors to automate ESS fabrication process has the potential to decrease costs, increase reproducibility, and permit more efficient regulation of ESS anatomy and physiology. In these studies, two bioreactors were evaluated in vitro for their effects on ESS microanatomy, cell viability and proliferation, and barrier formation; and in vivo, by percent original wound area, and percent engraftment after transplantation to athymic mice. ESS fabricated with conventional culture techniques served as controls.The Kerator is a computer controlled bioreactor in which medium changes during keratinocyte culture are fully automated. The growth of keratinocytes on the culture surface of the Kerator, fluroethylene polymer (FEP) film, was compared with that of the polystyrene surface of tissue culture flasks by image analysis for percent confluence. Results indicated that although the confluence was higher in flasks if compared with the Kerator after four days of culture (28±2.3% vs. 18 0.93%), the confluence of keratinocytes in both conditions was similar after six days of culture. The subconfluent keratinocytes were harvested from the Kerator by modifying the method used in conventional keratinocyte culture to reduce trypsinization time. The results of clonal growth assays performed on the keratinocytes harvested from the Kerator demonstrated colony forming efficiencies and growth rate not statistically different from those harvested from flasks (p>0.05). ESS fabricated with keratinocytes harvested from the Kerator were not statistically different from control ESS in vitro and in vivo.A recirculating bioreactor system was developed for the perfusion culture of ESS. Medium flow rates of 5, 15 and 50ml/min were evaluated for their effects on ESS quality in vitro. Controls were ESS incubated in static conditions in Petri dishes. Results of the in vitro experiments demonstrated that ESS incubated at 5 ml/min flow rate had better microanatomy, higher cell viabilities after two and three weeks of culture, and comparable keratinocyte proliferation and epidermal barrier if compared to controls. Increasing the medium flow rate resulted in deterioration of ESS anatomy and physiology. In vivo, ESS incubated in 5 ml/min healed wounds as effectively as controls.The results of the experiments performed in these studies demonstrate that the Kerator and recirculating bioreactor system for ESS can introduce automation while maintaining, and in the instance of the latter bioreactor, increasing the quality assurance of ESS. By integrating these bioreactors together with bioreactors for fibroblast expansion and cell inoculation on collagen-GAG scaffolds, the majority of cell culture processes in ESS fabrication could be automated and regulated more efficiently and accurately. This will translate into greater availability of the product, reduced costs, and better treatment outcomes for patients.