The main findings and results obtained during the course of the project are:
1, In the first phase of the project, I successfully developed different 3D hydrogel scaffolds mimicking the 3D microarchitecture of intestine tissue. These include PAA hydrogel, PEG hydrogel, and Matrigel scaffolds. I also developed 3D microtubes and microfibers to study 3D epithelial dynamics on curved surfaces.
2, I then aimed to study 3D epithelial dynamics with a specific focus on the role of Epithelial Cell Adhesion Molecule (EpCAM) in modulating intestinal epithelial homeostasis and morphogenesis. I have successfully grown Caco2 and mouse primary intestinal cells on the aforementioned 3D hydrogel scaffolds. I recorded 3D epithelial dynamics on curved surfaces using high-resolution 3D fluorescent confocal microscopy. Additionally, I analyzed the 3D cell dynamics with PIV. The EpCAM’s role in 3D epithelial dynamics is currently under careful investigation. Our current observations show that the substrate curvatures showed a strong impact on 3D collective cell behaviours, resulting in collective 3D rotation. This is a novel behaviour mimicking many in vivo and in vitro morphogenesis processes.
3, After I carefully investigated the dynamics and organization of actomyosin networks and adherens junctions (AJs) in various 3D contexts. I grew primary mouse intestinal cells and other epithelial cells on 3D hydrogel scaffolds with varying curvatures. I observed that actin cytoskeleton organization within epithelial tissues changes radically according to the curvature of the scaffolds. Besides the topographic influence, I also observed a significant impact of substrate rigidity on the cell shape and epithelial organization. While the complicated interaction between EpCAM and actomyosin networks/AJs in different 3D contexts is still under investigation, my work led to a discovery of a novel, tissue-level cytoskeleton organization that we believe to stabilize spatial epithelial integration and homeostasis. This discovery is currently being summarized into a research article for submission to high-quality peer-reviewed journals.
4, Finally, I focused on the implementation of a microfluidic-based in vitro intestine model. I developed a permeable chip to be integrated into a two-layer microfluidic system (co-developed with our collaborators at ENS, Paris) for the long-term culture of primary mouse intestinal epithelia on 2D and 3D hydrogel scaffolds. The primary cells could be cultured on these novel scaffolds for more than 3 months. Furthermore, our culture methods allow IE cells to sustain for the long-term, pack into high density, develop columnar shape with improved apical-basal polarity and differentiation marker expression, a phenotype reminiscent of features in in vivo mouse IE.
Final results overview: the novel approach developed in this project enables, for the first time, the construction of biomimetic 2D/3D intestinal tissues for long-term culture and various studies. Furthermore, it allows convenient analysis of cell dynamics and tissue homeostasis during epithelial remodeling in response to different 3D cues.
Dissemination of the results: The work in point 1 has been presented via a poster "Ex vivo biomimetic intestinal model for developmental studies" in Euromech Colloquium 6060 - Mechanics in and for cell migration, October 2019, Paris. In addition, the outcome of this action will lead to at least 3 high-level research articles: two (from point 2 &3) in preparation for high-quality open-assess journals and 1 (from point 4) has been submitted and is currently under reviewed.
Outreach activities: My plan of attending multiple international conferences for communication and dissemination was significantly impeded by Covid-19 pandemic. Despite the difficulties, I communicated regularly with my collaborators and many international researchers via zoom meetings and emails. I also disseminated our discoveries via online academic seminar presentations and to general audients such as visiting high school students in Paris and other researchers at the University of Paris. To foster collaboration and transfer knowledge, I visited research groups in Paris, Lille, Amsterdam and Asia whenever it was possible. Moreover, I was invited to write a review for the journal Biology of the Cell on the topic of bioengineered organoids. I guided two Ph.D. students to write, correct, and edit the review article, which was accepted in September 2021.
