Epithelial restitution in 3D - Revealing biomechanical and physiochemical dynamics in intestinal organoids via fs laser nanosurgery

Sören Donath; Anna Elisabeth Seidler; Karlina Mundin; Johannes Wenzel; Jonas Scholz; Lara Gentemann; Julia Kalies; Jan Faix; Anaclet Ngezahayo; André Bleich; Alexander Heisterkamp; Manuela Buettner; Stefan Kalies

doi:10.1016/j.isci.2023.108139

Epithelial restitution in 3D - Revealing biomechanical and physiochemical dynamics in intestinal organoids via fs laser nanosurgery

iScience. 2023 Oct 5;26(11):108139. doi: 10.1016/j.isci.2023.108139. eCollection 2023 Nov 17.

Authors

Sören Donath^{1

2}, Anna Elisabeth Seidler^{1

2}, Karlina Mundin^{1

2}, Johannes Wenzel^{1

2}, Jonas Scholz³, Lara Gentemann^{1

2

4}, Julia Kalies^{1

2}, Jan Faix³, Anaclet Ngezahayo⁵, André Bleich^{2

4

6}, Alexander Heisterkamp^{1

2}, Manuela Buettner^{2

4

6}, Stefan Kalies^{1

2

4}

Affiliations

¹ Institute of Quantum Optics, Leibniz University Hannover, 30167 Hannover, Germany.
² Lower Saxony Center for Biomedical Engineering, Implant Research and Development (NIFE), 30625 Hannover, Germany.
³ Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany.
⁴ REBIRTH Research Center for Translational Regenerative Medicine, 30625 Hannover, Germany.
⁵ Institute of Biophysics, Leibniz University Hannover, 30167 Hannover, Germany.
⁶ Institute for Laboratory Animal Science, Hannover Medical School, 30625 Hannover, Germany.

Abstract

Intestinal organoids represent a three-dimensional cell culture system mimicking the mammalian intestine. The application of single-cell ablation for defined wounding via a femtosecond laser system within the crypt base allowed us to study cell dynamics during epithelial restitution. Neighboring cells formed a contractile actin ring encircling the damaged cell, changed the cellular aspect ratio, and immediately closed the barrier. Using traction force microscopy, we observed major forces at the ablation site and additional forces on the crypt sides. Inhibitors of the actomyosin-based mobility of the cells led to the failure of restoring the barrier. Close to the ablation site, high-frequency calcium flickering and propagation of calcium waves occured that synchronized with the contraction of the epithelial layer. We observed an increased signal and nuclear translocation of YAP-1. In conclusion, our approach enabled, for the first time, to unveil the intricacies of epithelial restitution beyond in vivo models by employing precise laser-induced damage in colonoids.

Keywords: Bioengineering; Cell biology; Molecular physiology; Optical imaging.