Cellulases and their promising use in tissue engineering and cell sheet technology

• Bacterial nanocellulose (BNC) was prepared in order to generate continuous scaffold-free sheets of human keratinocytes (HaCaT). • HaCaTs were cultured on four different BNC samples: air-dried (BNC_AD), lyophilized (BNC_L) and Ar + plasma-modified (PM) BNC, i.e. BNC_AD_PM and BNC_L_PM. • BNC was degraded by cellulase from Trichoderma reesei added into the culture medium (37.5 U/ml, pH 6.5). • The best results were obtained with the BNC_AD_PM, where continuous sheets of viable keratinocytes were separated after exposure to a cellulase solution for 3 h. Bacterial nanocellulose (BNC) was used to generate continuous scaffold-free sheets of human keratinocytes (HaCaT) after degradation by cellulase from Trichoderma reesei . First, we successfully grafted acidic cellulase from Trichoderma reesei onto a BNC nanostructure and prepared degradable BNC that dissolved in phosphate-buffered saline within one week. In addition, we selected a cellulase solution that degrades BNC within three hours under physiological conditions. HaCaTs were cultured on four different BNC samples: air-dried (BNC_AD), lyophilized (BNC_L) and plasma-modified (PM) BNC exposed to a direct current Ar + plasma discharge (BNC_AD_PM and BNC_L_PM). On days 1 and 3, the cells were stained with the LIVE/DEAD viability/cytotoxicity kit. The significantly highest cell number on day 3 was found on the BNC_AD_PM membrane (median 65,025 cells/cm 2 ), similar to the standard cell culture polystyrene (median 58,553 cells/cm 2 ). Cell viability on day 3 was greater than 90 % for all samples except BNC_L (62 %). Cell island formation was also preferentially observed on BNC_AD_PM samples. The significant positive effect of PM on cell growth was shown after BNC degradation on day 10 after cell seeding, when continuous sheets of viable keratinocytes were separated from the BNC samples by exposure to a cellulase solution (37.5 U/ml, pH 6.5) for 3 h. The number of cells obtained by cultivation on BNC_PM greatly exceeded the number of cells obtained from unmodified BNC. The approaches presented here may be useful for the preparation of (1) degradable BNC scaffolds for tissue engineering and (2) continuous scaffold-free cell sheets applicable in wound healing.