Abstract
While conventional cell culture methodologies have relied on flat, two-dimensional cell monolayers, three-dimensional engineered tissues are becoming increasingly popular. Often, engineered tissues can mimic the complex architecture of native tissues, leading to advancements in reproducing physiological functional properties. In particular, engineered intestinal tissues often use hydrogels to mimic villi structures. These finger-like protrusions of a few hundred microns in height have a well-defined topography and curvature. Here, we examined the cell morphological response to these villus-like microstructures at single-cell resolution using a novel embedding method that allows for the histological processing of these delicate hydrogel structures. We demonstrated that by using photopolymerisable poly(ethylene) glycol as an embedding medium, the villus-like microstructures were successfully preserved after sectioning with vibratome or cryotome. Moreover, high-resolution imaging of these sections revealed that cell morphology, nuclei orientation, and the expression of epithelial polarization markers were spatially encoded along the vertical axis of the villus-like microstructures and that this cell morphological response was dramatically affected by the substrate curvature. These findings, which are in good agreement with the data reported for in vivo experiments on the native tissue, are likely to be the origin of more physiologically relevant barrier properties of engineered intestinal tissues when compared with standard monolayer cultures. By showcasing this example, we anticipate that the novel histological embedding procedure will have a positive impact on the study of epithelial cell behavior on three-dimensional substrates in both physiological and pathological situations.
Highlights
Many epithelial tissues exhibit complex morphologies that are mostly dominated by curved surfaces such as those found in lung alveoli and intestinal villi (Torras et al, 2018; Baptista et al, 2019)
A prepolymer solution containing 6.5% w/v 6 kDa polyethylene glycol diacrylate (PEGDA), 0.3% w/v acrylic acid (AA), and 1% w/v Irgacure D-2959 photoinitiator in phosphate-buffered saline (PBS) was flown into a chip fabricated with a 1 mm thick polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning) stencil containing an array of pools of 6.5 mm diameter
The villus-like microstructured hydrogels were embedded in 10% w/v low molecular weight PEGDA, P575, by UV-initiated crosslinking (Figure 2A)
Summary
Many epithelial tissues exhibit complex morphologies that are mostly dominated by curved surfaces such as those found in lung alveoli and intestinal villi (Torras et al, 2018; Baptista et al, 2019). Hydrogels are widely used as scaffold materials for tissue engineering and 3D cell culture applications (Slaughter et al, 2009; Caliari and Burdick, 2016) Due to their porous network structure and soft mechanical properties, hydrogels mimic important elements of the native extracellular matrix. Did these scaffolds support the growth and differentiation of intestinal epithelial Caco-2 cells along the villi but they induced improved cell polarization and tissue barrier properties compared to the standard monolayer cell cultures These benefits were attributed to the more physiologically realistic environment provided by the 3D model
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
