Optimizing large-scale autologous human keratinocyte sheets for major burns-Toward an animal-free production and a more accessible clinical application

Autologous keratinocyte sheets constitute an important component of the burn wound treatment toolbox available to a surgeon and can be considered a life-saving procedure for patients with severe burns over 50% of their total body surface area. Large-scale keratinocyte sheet cultivation still fundamentally relies on the use of animal components such as inactivated murine 3T3 fibroblasts as feeders, animal-derived enzymes such as trypsin, as well as media components such as fetal bovine serum (FBS). This study was therefore aimed to optimize autologous keratinocyte sheets by comparing various alternatives to critical components in their production.

Autologous keratinocyte sheets constitute an important component of the burn wound treatment toolbox available to a surgeon and can be considered a life-saving procedure for patients with severe burns over 50% of their total body surface area. Large-scale keratinocyte sheet cultivation still fundamentally relies on the use of animal components such as inactivated murine 3T3 fibroblasts as feeders, animal-derived enzymes such as trypsin, as well as media components such as fetal bovine serum (FBS). This study was therefore aimed to optimize autologous keratinocyte sheets by comparing various alternatives to critical components in their production.

This study demonstrated that animal-free components could be used successfully to reduce the risk profile of large-scale autologous keratinocyte sheet production, and thereby increase clinical accessibility.

Human skin samples were retrieved from remnant operative tissues. Cell isolation efficiency and viability were investigated by comparing the efficacy of porcine-derived trypsin and animal-free enzymes (Accutase and TrypLESelect). The subsequent expansion of the cells and the keratinocyte sheet formation was analyzed, comparing various cell culture substrates (inactivated murine 3T3 fibroblasts, inactivated human fibroblasts, Collagen I or plain tissue culture plastic), as well as media containing serum or chemically defined animal-free media.

The cell isolation step showed clear cell yield advantages when using porcine-derived trypsin, compared to animal-free alternatives. The keratinocyte sheets produced using animal-free serum were similar to those produced using 3T3 feeder layer and FBS-containing medium, particularly in mechanical integrity as all grafts were liftable. In addition, sheets grown on collagen in an animal-free medium showed indications of advantages in homogeneity, speed, reduced variability, and differentiation status compared to the other growth conditions investigated. Most importantly, the procedure was compatible with the up-scaling requirements of major burn wound treatments.