Live imaging of human airway epithelial repair in precision-cut lung slices after targeted cell damage.

Precision-cut lung slices (PCLS) are a complex three-dimensional ex vivo model system comprised of all resident cell types of the lung, thus closely mimicking the in vivo situation in regards to structural composition and function. The herein described application of a precise airway epithelial lesion via femtosecond laser-based nanosurgery and subsequent longitudinal imaging via two-photon or confocal microscopy enables the examination of the tissue's repair responses on a single-cell level. Allowing for live observation of intercellular cross-talk, this study demonstrates an endogenous repair program is induced in human PCLS upon damage induction. As early reaction to a small epithelial lesion, physiological stress responses, including transient airway constriction and increased mucus secretion, occur, followed by epithelial restitution within 24 h. Automated cell detection and subsequent cell track analysis reveal a more linearly confined cellular movement in the course of repair. Further, non-stationary, motile cells directly interact with cell debris, thereby contributing to final resolution of the lesion. Together, these findings emphasize the suitability of PCLS, combined with localized laser-based damage induction and state-of-the-art microscopy techniques, as a model system to study complex intercellular interactions in the course of endogenous repair processes.