A novel strategy to facilitate uniform epithelial cell maturation on liquid-liquid interfaces

Abstract Background: Liquid-liquid interfaces, such as lacrimal fluid of the eye, are pervasive in living organism. While culturing cells in an environment that closely mimics in vivo conditions enhances our understanding of cell characteristics and unveils underlying mechanisms, practical considerations have led to predominant use of solid surfaces, such as polystyrene or glass, for cell culture. In this study, we introduce a novel culture design aimed at promoting uniform maturation of epithelial cells using liquid materials. Results: Culturing Madin–Darby canine kidney (MDCK) cells at the liquid-liquid interface yielded reduced migration and stimulated active cell growth. Similar to solid-liquid interfaces, cells cultured on a fibronectin-coated liquid-liquid interface exhibited active migration and growth, ultimately reaching a confluent state. These cells exhibited reduced stress fiber formation and adopted a cobblestone-like shape, which led to their even distribution in the culture vessel. Analysis of staining images of the tight junction (TJ) protein ZO-2 revealed that the frequency of ZO-2-positive cells, F Z , in cultures on the fibronectin-coated liquid-liquid interface was 2.2-fold higher than that on the non-coated liquid-liquid interface at t = 72 h. To inhibit stress fiber formation and apoptosis, the exposure of cells to Y27632, a specific inhibitor of the Rho-associated protein kinase (ROCK), facilitated uniform maturation ( F Z = 0.73). Additionally, a similar trend was observed in cells cultured on the fibronectin-coated liquid-liquid interface ( F Z = 0.80) at t = 72 h. In Y27632-exposed cells on the liquid-liquid interface, the value obtained by subtracting the standard deviation of the ratio of nucleus densities in each region that compartmentalized a culture vessel from 1, denoted as H LN , was 0.93 0.01, indicating even cell distribution in the culture vessel at t = 72 h. Across all culture conditions, the H LN and F Z values exhibited a close correlation ( R 2 = 0.93), suggesting that cell distribution in the culture vessel is related to the formation of TJs in the late phase of maturation. Conclusions: Our findings suggest that the behaviors of epithelial cells on liquid-liquid interfaces contribute to the promotion of their uniform maturation.