Development of a Closed Bioreactor System for Culture of Tissue-Engineered Skin at an Air–Liquid Interface

(This is Terry posting on the behalf of Elena, who's having blogger problems.)

Before autologous tissue-engineered skin providing a reasonable barrier can progress to the clinic, it is needed to design bioreactor that can construct cells at an air-liquid interface. This article investigates and compares the designs for continuous as well as batch-reaction bioreactors. A comparison between continuous perfusion with medium versus changing medium every few days was also used to determine which method would better support metabolic activity.

Four different scaffolds were used in this study: acellular deepidermized human dermis (DED), electrospun polystyrene (PS; 0.8–1.0 mm thick), a composite of electrospun poly-DL-lactide fiber and polystyrene (PDLA/PS) (0.8–1.0 mm thick), and a commercially available nonwoven scaffold (Azowipes). Normal human keratinocytes and fibroblasts were isolated and cultured and the viable cell density was assessed by MTT-ESTA (an assay based on the conversion of MTT to a colored formazan end product by intracellular dehydrogenase activity). Viability measurements by MTT indicated that fibroblast and endothelial single cultures preferred submerged culture conditions, whereas an air–liquid interface gave greater total cell viability for the single culture of keratinocytes and fibroblast–keratinocyte cocultures compared with submerged cultures. MTT measurements showed that total cell viability of fibroblast–keratinocyte cocultures was significantly higher in PS and PDLA/PS compared with DED.

Results also indicated that the cell viability of tissues cultured under continuous perfusion was significantly higher than that of tissues cultured under batch-fed culture (Fig. 5). It was noticeable that total cell viability was almost 2-fold higher for PS electrospun scaffolds with continuous feed versus DED with continuous feed. In the case of keratinocytes this may result in cells failing to proliferate or it may induce premature abnormal differentiation. Thus, continuously perfused medium is likely to be more effective than frequent changes of static culture medium every few days.

Overall, the bioreactor developed in this investigation shows several technical and operational advantages that will be of significance for skin tissue-engineering research. These advantages include: a completely closed system, a bioreactor that can be operated easily under sterile conditions, a design the potential for parallel connection of individual chambers, allowing for multiple experimental reactors to be stacked vertically and therefore minimizing culture space, and a bioreactor with multiple chambers to allow for simultaneous experiments.

I chose this article due to its relevance to cell-culture techniques used during our lab. The feeding technique use din lab consists of changing the medium every few days, however in this report, it is suggested that a continuous perfusion of medium resulted in an increased cell viability. In addition, this investigation focuses on an air-liquid interface cell culture, which would be needed in tissue-engineered skin to be produced for clinical use. This aspect relates to our project of tissue engineering dermis and can provide insight as to possible future work that could provide better results.