TY - JOUR
T1 - Epithelial restitution in 3D
T2 - Revealing biomechanical and physiochemical dynamics in intestinal organoids via fs laser nanosurgery
AU - Donath, Sören
AU - Seidler, Anna Elisabeth
AU - Mundin, Karlina
AU - Wenzel, Johannes
AU - Scholz, Jonas
AU - Gentemann, Lara
AU - Kalies, Julia
AU - Faix, Jan
AU - Ngezahayo, Anaclet
AU - Bleich, André
AU - Heisterkamp, Alexander
AU - Buettner, Manuela
AU - Kalies, Stefan
N1 - Funding Information:
We acknowledge Puja Pandey, Maria Mellin, Anja Siebert (all Hannover Medical School) for providing colonoid culture medium. We thank Prof. Jan Faix (Hannover Medical School) for primary anti-VASP-antibody and help with antibody stainings. Furthermore, we thank Despina Kiriazi (Hannover Medical School) for actin and myosin 2 inhibitors. This study was funded by the REBIRTH Research Center for Translational Regenerative Medicine ( ZN3440 , State of Lower Saxony Ministry of Science and Culture (Nieders. Vorab)). J.K. and A.H. were supported by the biomedical research in endstage and obstructive lung disease Hannover (BREATH) from the german lung center (DZL). M.B. and A.B. were funded by R2N , Federal State of Lower Saxony . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.
Funding Information:
We acknowledge Puja Pandey, Maria Mellin, Anja Siebert (all Hannover Medical School) for providing colonoid culture medium. We thank Prof. Jan Faix (Hannover Medical School) for primary anti-VASP-antibody and help with antibody stainings. Furthermore, we thank Despina Kiriazi (Hannover Medical School) for actin and myosin 2 inhibitors. This study was funded by the REBIRTH Research Center for Translational Regenerative Medicine (ZN3440, State of Lower Saxony Ministry of Science and Culture (Nieders. Vorab)). J.K. and A.H. were supported by the biomedical research in endstage and obstructive lung disease Hannover (BREATH) from the german lung center (DZL). M.B. and A.B. were funded by R2N, Federal State of Lower Saxony. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article. Conceptualization, S.D. and S.K; methodology, S.K.; software, J.W. K.M. and S.K.; validation, S.D. A.E.S. L.G. J.W. and S.K.; formal analysis, S.D. and S.K.; investigation, S.D. A.E.S. J.W. K.M. J.S. J.K. J.F. and S.K.; resources, J.K. M.B. and S.K.; data curation, S.D. J.W. K.M. A.N. M.B. and S.K.; writing—original draft preparation, S.D. and S.K.; writing—review and editing, all authors; visualization, S.D. and S.K.; supervision, S.K.; project administration, A.B. A.N. A.H. M.B. and S.K.; funding acquisition, A.B. A.H. and S.K. All authors have read and agreed to the published version of the article. The authors declare no competing interest.
PY - 2023/11/17
Y1 - 2023/11/17
N2 - 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.
AB - 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.
KW - Bioengineering
KW - Cell biology
KW - Molecular physiology
KW - Optical imaging
UR - https://www.scopus.com/pages/publications/85173812140
U2 - 10.1016/j.isci.2023.108139
DO - 10.1016/j.isci.2023.108139
M3 - Article
SN - 2589-0042
VL - 26
JO - iScience
JF - iScience
IS - 11
M1 - 108139
ER -