Epithelial monolayer development and tight junction assembly on nanopillar arrays

Nanostructured materials provide an outstanding opportunity to both stimulate and measure cellular processes. In the context of tight junctions, it was previously reported that transient application of a nanotopographic surface over the apical brush border membrane of epithelial monolayers triggers redistribution of ZO-1, claudins, and F-actin that increases paracellular macromolecular flux. In excitable tissues, nanomaterials have been used to apply and measure electrical signals, such action potentials. As a first step towards translating these technologies for use in analysis of epithelial function, we sought to culture monolayers composed of transporting epithelia over nanopillar arrays without perturbing cellular structure or function. Madin-Darby Canine kidney I (MDCK I) cells were cultured on collagen-coated silicon chips with [~]1 m diameter nanopillar arrays. Fluorescence and scanning electron microscopy were used to assess the impact of height on nanopillar-epithelial interactions. Monolayers formed over and were largely unaffected by short nanopillars. These nanopillars were located beneath basal epithelial surfaces and were not preferentially located within lateral intercellular spaces or beneath ZO-1-containing junctions. In contrast, tall nanopillars that exceeded cell height disrupted MDCK I monolayer growth. Cells interacted with, encircled, and extended cytoplasm over the top of tall nanopillars, and dense ZO-1 and F-actin accumulations occasionally surrounded apical membranes adjacent to nanopillars. Finally, when grown over arrays composed of nanopillars 1 - 2 m shorter than cells, MDCK I frequently grew between nanopillars. As a result, nanopillars were more commonly present within lateral intercellular spaces beneath junctions. Apical complex structure was intact, as assessed by fluorescence microscopy of ZO-1, occludin, claudin-2, F-actin, and E-cadherin. Apical microvilli were also unaffected. We therefore show that conditions can be defined to allow growth of mature, correctly assembled epithelial monolayers with nanopillars localized to lateral intercellular spaces. This sets the stage for application of nanotechnologies for perturbation and analysis of epithelial biology.