Periodic Reporting for period 1 - TOPOGRAPHYSENSING (Effects of 3D topographies on mechanosensing in intestine epithelial architecture and dynamics)
Período documentado: 2019-09-01 hasta 2021-08-31
The intestine epithelium (IE) consists of spatially segregated cells that organize into groups of various functions at different locations of the 3-dimensional (3D) curved epithelial monolayer. How geometric cues contribute to the maintenance of the sophisticated epithelial architecture and dynamics in 3D remains unknown until now. Thus, a systematic investigation on the effect of curvature on IE architecture, organization, and dynamics in the context of 3D mechanosensing is highly needed to significantly improve our understanding of normal IE structure maintenance and homeostasis, etc.
In this project, I developed a novel 3D intestinal model based on biomimetic hydrogels that allow the long-term culture of primary intestinal cells. Substrate elasticity was designed to match the mechanical properties of the physiological extracellular matrix (ECM). By applying two-step lithography and soft molding methods, I produced 3D scaffolds reflecting in vivo intestinal 3D architectures. I then grew primary intestinal stem cells on these scaffolds to realize in vivo-like compartmentalization and tissue structure. With this, I systematically studied 3D cellular dynamics and cytoskeleton organization on out-of-plane curvature. Furthermore, I could conveniently integrate the cell-laden scaffolds into a two-layer microfluidic system, which offers a practical approach for monitoring epithelial remodeling processes in 3D contexts.
This new platform can possibly be translated into a disease model for studying some rare intestinal diseases, such as Congenital Tufting Enteropathy (CTE). The outcome of this action then has the potential to influence fields in fundamental cell development research and therapeutic studies. It could also become a successful paradigm for other tissue engineering studies.
In this project, I developed a novel 3D intestinal model based on biomimetic hydrogels that allow the long-term culture of primary intestinal cells. Substrate elasticity was designed to match the mechanical properties of the physiological extracellular matrix (ECM). By applying two-step lithography and soft molding methods, I produced 3D scaffolds reflecting in vivo intestinal 3D architectures. I then grew primary intestinal stem cells on these scaffolds to realize in vivo-like compartmentalization and tissue structure. With this, I systematically studied 3D cellular dynamics and cytoskeleton organization on out-of-plane curvature. Furthermore, I could conveniently integrate the cell-laden scaffolds into a two-layer microfluidic system, which offers a practical approach for monitoring epithelial remodeling processes in 3D contexts.
This new platform can possibly be translated into a disease model for studying some rare intestinal diseases, such as Congenital Tufting Enteropathy (CTE). The outcome of this action then has the potential to influence fields in fundamental cell development research and therapeutic studies. It could also become a successful paradigm for other tissue engineering studies.
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.
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.
We have achieved the major goals of the proposed project, i.e. the establishment of 3D in vitro intestinal models and the characterization of the intestinal mechanosensing in 3D microenvironment, at the end of the action. The results are of great scientific value to unveil the relationship between mechanosensing proteins/mechanical tension distribution and topography. The mapping of epithelial cell behaviors in 3D is also significantly informative and can provide guidelines for future biophysical research on out-of-plane surfaces. For instance, the epithelial dynamics acquired in 3D contexts will directly link to in vivo intestinal renewal and homeostasis. Furthermore, the achievement in microfabrication protocol is highly helpful for the design of novel biomaterials. Taken together, all of the knowledge obtained via this proposed research is of keen interest to multidisciplinary scientific communities. Finally, the long-lasting questions including the mechanisms through which epithelial cells can modulate their organization and terminal differentiation according to external physical and chemical properties are adequately addressed during the project.