D14 - Geometrically Controlled WNT Activation Drives Intestinal Morphogenesis

Introduction: : Digital Light Processing (DLP) 3D bioprinting offers a high fidelity and high-throughput platform to probe and decouple the mechanical cues involved in morphogenesis. Colorectal carcinoma (Caco-2) cells are robust and are a common cell type that can mimic the structure and function of the intestine, providing a strong cell type to study crypt-villi-like morphogenesis. While several investigations have shown that Caco-2 cells can undergo morphogenesis and form crypt-villi-like structures through environmental cues such as basolateral flow and co-culture conditions, WNT signaling has also been probed as a driving force in intestinal morphogenesis in a variety of studies. By integrating insights from the WNT signaling pathway with the influence of architectural cues, this work aims to systematically investigate how shaped microwell environments can direct epithelial morphogenesis. To this end, we hypothesized that engineered in concert with WNT signaling can enable spatially controlled intestinal tissue organization, providing a tunable platform for engineering physiologically relevant intestinal models.

Materials and

Methods: : Gelatin methacrylate (GelMA) was reconstituted with 0.25% LAP in PBS to create a 10% GelMA bioink for bioprinting. All bioprinting was performed with DLP via a BioNova-X printer from CellInk. Bioprinted constructs were allowed to swell in 0.1% LAP in PBS over 45 minutes followed by an additional 5 min of UV (405 nm) exposure to increase stiffness of the hydrogels. Caco-2 cells were grown and cultured within a variety of environmental conditions including human dermal fibroblast (HDF) conditioned media, WNT agonists, CHIR99021 dosed media, and geometrically controlled microwells. The HDF conditioned media components were confirmed via WNT signaling cytokine array analysis to have a variety of components relating to initiating WNT signaling. Crypt-villi-like structure analysis was performed using immunofluorescence staining of β-catenin and mucin-2 (MUC2), in addition to EdU proliferative staining for qualitative imaging. Functionality of the crypt-villi-like structures was analyzed via qRT-PCR of MUC2, MUC5AC, and AXIN2 genes.

Results, Conclusions, and Discussions:: The results of triggering WNT signaling with HDF conditioned media and small molecule CHIR99021 dosages lead to further confirmation of WNT signaling driving morphogenesis in crypt-villi-like structures. Under conditioned media with components corresponding to driving WNT signaling, there was a significant height increase from 20.45 µm to 52.6 µm and in CHIR dosages of 3 µM there is a further increase in height to 77.2 µm (Figure 1A). This height increase also led to an upregulation in MUC2 expression which is associated with a functional crypt-villi-like structure (Figure 1B and 1C). Further confirming the WNT signaling effects on driving morphogenesis, conditioned media exposed cultures were exposed to WNT signaling inhibitor dikkopf-1 (DKK1) lowering the MUC2 expression compared to the other cultures (Figure 1B). Furthermore, optimization of the DLP bioprinting and our stiffness tuning allowed for successful Caco-2 adherence to highly patterned geometric microwells. Using these optimized settings and a variety of shapes and patterns corresponding to a value deemed as ‘Edge Effect’ (equivalent to perimeter/area), we found a relationship to increase morphogenesis based solely on the architecture the Caco-2 cells grow within (Figure 1D). Testing the various geometries, MUC2 expression can be tuned following a trend corresponding to the Edge Effect in which WNT signaling is triggered and drives crypt-villi-like formation (Figure 1E). Further testing led to the creation of an abstract shape with a similar Edge Effect (Figure 1F) to the triangle microwell, resulting in similar MUC2 expression in both (Figure 1G). Lastly, a molecule to reduce cytoskeletal tension, blebbistatin, was used to determine if the shape truly drives the cell cultures' morphogenesis. When exposed to blebbistatin, the shapes express equal or less MUC2 than if the cells were cultured upon a flat surface (Figure 1H). In conclusion, WNT signaling can drive intestinal morphogenesis in crypt-villi-like formations and can be controlled with specifically patterned microenvironmental architecture.

Acknowledgements and/or References (Optional)::