Development of hydrogel cavities of tuneable stiffness for the growth of epithelial crypts

Abstract

Depending on their function, the epithelial cell monolayers that line the inner surfaces of organs adopt a variety of three-dimensional shapes. Traditional studies in vitro have been using mainly flat cell culture dishes, overseeing the impact of these in vivo shapes in tissue function. Recent research has begun to address this issue, by trying to mimic the 3D structures found in tissues. However, those novel culture platforms still have some limitations, especially in cases where the architecture must correspond with the original tissue’s stiffness. Tissues have a quite low physiological rigidity, and most of the microfabrication techniques used nowadays need quite firm materials to achieve the desired 3D structures without issue. A type of structure difficult to fabricate using soft materials are invaginations, which can be found in vivo in kidneys, lungs, and the small intestine. Low rigidity substrates are typically characterized by high deformability and lack of structural support, which can result in unprecise final features due to distortions of the material during the microfabricating process. In this project, 3D cavities have been fabricated into polyacrylamide (PA), a material which allows the tuneability of its rigidity by changing the proportion of acrylamides during the synthesis of the prepolymer solution. Replica moulding has been employed to acquire these structures. The invaginations successfully recreated key aspects of the in vivo environment, both with their shape and stiffness, and multiple copies were created easily, enabling precise characterization. Finally, after the assessment of the mechanical properties and the architectural features of the microcavities, the functionalization of the samples was successful, confirming the suitability of the resulting scaffold as a model to study epithelial growth, morphology, and conformity in these inward bended structures.