TY - JOUR

T1 - Mimicking acute airway tissue damage using femtosecond laser nanosurgery in airway organoids

AU - Gentemann, Lara

AU - Donath, Sören

AU - Seidler, Anna E.

AU - Patyk, Lara

AU - Buettner, Manuela

AU - Heisterkamp, Alexander

AU - Kalies, Stefan

N1 - Funding Information: 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.

PY - 2023/9/8

Y1 - 2023/9/8

N2 - 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.

AB - 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.

KW - acute lung damage

KW - airway organoids

KW - epithelial repair

KW - femtosecond laser

KW - laser-based nanosurgery

UR - http://www.scopus.com/inward/record.url?scp=85171886224&partnerID=8YFLogxK

U2 - 10.3389/fcell.2023.1268621

DO - 10.3389/fcell.2023.1268621

M3 - Article

AN - SCOPUS:85171886224

VL - 11

JO - Frontiers in Cell and Developmental Biology

JF - Frontiers in Cell and Developmental Biology

SN - 2296-634X

M1 - 1268621

ER -