Investigation of Colonic Regeneration via Precise Damage Application Using Femtosecond Laser-Based Nanosurgery

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.

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

“…Single-cell ablation in the crypt area is indicated by a strong increase in autofluorescence of the destroyed cell. 12 Upon the application of 100 μM blebbistatin, an inhibitor of actomyosin-based cell mobility, the spatial extent of autofluorescence remained identical over time ( Figure 5 A) and the damaged cell mass was not pulled into the lumen of the colonoid, which was observed in untreated controls ( Figure S2 ). While the crypt survived the lesion in the epithelium for 120 min, a total loss of structural integrity of the crypt and whole colonoid occurred after approximately 270 min.…”

“…We could previously show that single-cell ablation in the crypt area leads to increased Wnt signaling. 12 Via Wnt activation at the frizzled receptor, a short calcium pulse occurs. 81 Based on this interaction between Wnt and calcium and the direct interplay between Wnt and the primary sensor of the cellular microenvironment YAP/TAZ, 47 , 82 Hippo YAP/TAZ signaling was verified to be activated after ablation via YAP1 immunostaining.…”

“…The setup was described previously. 12 , 13 , 89 The laser beam was focused into the sample via a 40x/1.2 water immersion objective. A wavelength of 730 nm, a pulse energy of 0.9 nJ, and a dissection speed of 15 μm/s were used for single-cell ablation.…”

“…To understand the precise initiation and progression of restitution events, we have recently established a colonoid wounding model, which involves the targeted laser-based ablation of individual or multiple cells in the colonoid crypt. 12 Such precise manipulation can hardly be achieved by ionizing radiation or treatment with various chemical components. Instead, we employ a femtosecond laser system leading to non-linear optical interactions with the investigated tissue and allowing for precise removal of (sub-)cellular structures.…”

“…Instead, we employ a femtosecond laser system leading to non-linear optical interactions with the investigated tissue and allowing for precise removal of (sub-)cellular structures. 12 , 13 This process, called femtosecond laser nanosurgery, is based on the creation of a low-density plasma of free electrons, produced mainly by multiphoton ionization. 14 , 15 As the process involves no significant heating, no thermal or mechanical energy is released in neighboring cells.…”