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Investigation of Colonic Regeneration via Precise Damage Application Using Femtosecond Laser-Based Nanosurgery
Sören Donath1,2, Leon Angerstein1,2, Lara Gentemann1,2
1Institute of Quantum Optics, Leibniz University Hannover, 30167 Hannover, Germany.
Abstract:
Organoids represent the cellular composition of natural tissue. So called colonoids, organoids derived from colon tissue, are a good model for understanding regeneration. However, next to the cellular composition, the surrounding matrix, the cell-cell interactions, and environmental factors have to be considered. This requires new approaches for the manipulation of a colonoid. Of key interest is the precise application of localized damage and the following cellular reaction. We have established multiphoton imaging in combination with femtosecond laser-based cellular nanosurgery in colonoids to ablate single cells in the colonoids' crypts, the proliferative zones, and the differentiated zones. We observed that half of the colonoids recovered within six hours after manipulation. An invagination of the damaged cell and closing of the structure was observed. In about a third of the cases of targeted crypt damage, it caused a stop in crypt proliferation. In the majority of colonoids ablated in the crypt, the damage led to an increase in Wnt signalling, indicated via a fluorescent lentiviral biosensor. qRT-PCR analysis showed increased expression of various proliferation and Wnt-associated genes in response to damage. Our new model of probing colonoid regeneration paves the way to better understand organoid dynamics on a single cell level.
Insights
Colonoids, or colon organoids, were precisely damaged using laser nanosurgery to study regeneration. This technique revealed rapid recovery, structural changes, and altered Wnt signaling, offering insights into organoid dynamics.
Area of Science:
- Regenerative Medicine
- Stem Cell Biology
- Gastroenterology
Background:
- Organoids, including colonoids, mimic natural tissue but require advanced manipulation techniques to study regeneration.
- Understanding colonoid regeneration necessitates considering cellular composition, matrix, cell-cell interactions, and environmental factors.
Purpose of the Study:
- To develop and apply a novel method for localized cellular damage in colonoids to investigate regeneration dynamics.
- To precisely ablate single cells within colonoid crypts and differentiated zones using femtosecond laser nanosurgery.
Main Methods:
- Established multiphoton imaging combined with femtosecond laser-based cellular nanosurgery for precise cell ablation in colonoids.
- Targeted single-cell ablation in colonoid crypts (proliferative zones) and differentiated zones.
Main Results:
- Approximately 50% of manipulated colonoids recovered within six hours, showing structural invagination and closure.
- Targeted crypt damage halted proliferation in about 33% of cases and increased Wnt signaling in the majority.
- Gene expression analysis (qRT-PCR) confirmed increased proliferation and Wnt-associated gene expression post-damage.
Conclusions:
- The developed laser-based nanosurgery model provides a powerful tool for studying colonoid regeneration at the single-cell level.
- Colonoid regeneration involves rapid structural repair and modulation of key signaling pathways like Wnt.
- This approach enhances our understanding of organoid dynamics and tissue repair mechanisms.
