Mimicking acute airway tissue damage using femtosecond laser nanosurgery in airway organoids

Lara Gentemann; Sören Donath; Anna E Seidler; Lara Patyk; Manuela Buettner; Alexander Heisterkamp; Stefan Kalies

doi:10.3389/fcell.2023.1268621

Mimicking acute airway tissue damage using femtosecond laser nanosurgery in airway organoids

Front Cell Dev Biol. 2023 Sep 8:11:1268621. doi: 10.3389/fcell.2023.1268621. eCollection 2023.

Authors

Lara Gentemann^{1

2

3}, Sören Donath^{1

2}, Anna E Seidler^{1

2}, Lara Patyk^{1

2}, Manuela Buettner⁴, Alexander Heisterkamp^{1

2

3

5}, Stefan Kalies^{1

2

3

5}

Affiliations

¹ Institute of Quantum Optics, Leibniz University Hannover, Hannover, Germany.
² Lower Saxony Center for Biomedical Engineering, Implant Research and Development, Hannover, Germany.
³ REBIRTH Research Center for Translational Regenerative Medicine, Hannover, Germany.
⁴ Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany.
⁵ German Center for Lung Research (DZL), Gießen, Germany.

Abstract

Airway organoids derived from adult murine epithelial cells represent a complex 3D in vitro system mimicking the airway epithelial tissue's native cell composition and physiological properties. In combination with a precise damage induction via femtosecond laser-based nanosurgery, this model might allow for the examination of intra- and intercellular dynamics in the course of repair processes with a high spatio-temporal resolution, which can hardly be reached using in vivo approaches. For characterization of the organoids' response to single or multiple-cell ablation, we first analyzed overall organoid survival and found that airway organoids were capable of efficiently repairing damage induced by femtosecond laser-based ablation of a single to ten cells within 24 h. An EdU staining assay further revealed a steady proliferative potential of airway organoid cells. Especially in the case of ablation of five cells, proliferation was enhanced within the first 4 h upon damage induction, whereas ablation of ten cells was followed by a slight decrease in proliferation within this time frame. Analyzing individual trajectories of single cells within airway organoids, we found an increased migratory behavior in cells within close proximity to the ablation site following the ablation of ten, but not five cells. Bulk RNA sequencing and subsequent enrichment analysis revealed the differential expression of sets of genes involved in the regulation of epithelial repair, distinct signaling pathway activities such as Notch signaling, as well as cell migration after laser-based ablation. Together, our findings demonstrate that organoid repair upon ablation of ten cells involves key processes by which native airway epithelial wound healing is regulated. This marks the herein presented in vitro damage model suitable to study repair processes following localized airway injury, thereby posing a novel approach to gain insights into the mechanisms driving epithelial repair on a single-cell level.

Keywords: acute lung damage; airway organoids; epithelial repair; femtosecond laser; laser-based nanosurgery.

Grants and funding

The authors declare financial support was received for the research, authorship, and/or publication of this article. AH received funding by the biomedical research in endstage and obstructive lung disease Hannover (BREATH) from the German Center for Lung Research (DZL, 82DZL002B3). SK, MB, and AH received funding by the REBIRTH Research Center for Translational Regenerative Medicine (ZN3440, State of Lower Saxony Ministry of Science and Culture (Nieders. Vorab)). The publication of this article was funded by the Open Access Fund of Leibniz University Hannover.