The migration and fusion events related to ROCK activity strongly influence the morphology of chicken embryo intestinal organoids.

The method of organoid culture has become a tool widely used in gastrointestinal research, but so far, the migration of organoids derived from gut epithelium and formed in 3D Matrigel matrix has not been reported and studied. The intestinal epithelial tissue derived from 19-day-old chicken embryo was cultured in Matrigel and the dynamic properties of the forming organoids were analyzed by time-lapse image analysis. It was observed that about one in ten organoids actively moved through the matrix, at a speed of 10-20 μm/h. Moreover, rotation was observed in the majority of organoids that did not migrate long distances. The fusion events took place between organoids, which collided during the movement or growth. In our previous paper, we showed that the presence of Toll-like receptor 4 ligand, Escherichia coli lipopolysaccharide (LPS, 1 μg/ml), increased the mean organoid diameter. Here, we confirm this result and demonstrate that the Rho-associated protein kinase (ROCK) inhibitor Y-27632 (10 μM) did not completely abolish organoid migration, but prevented the fusion events, in both LPS-treated and untreated cultures. In consequence, in the presence of Y-27632, the differences between cultures incubated with and without LPS were not visible. We conclude that migration and fusion of organoids may influence their morphology and suggest that these phenomena should be taken into account during the design of experimental settings.