Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Bioscience and Bioengineering  

Polarized epithelial cells are compartmentalized into apical and basement membranes with asymmetrically distributed proteins. This study aimed to establish a method for culturing epithelial cells at the fluorinated oil (Novec-7500) microdroplet surface for the formation of epithelial polarity, which is desirable for regenerative medicine and drug discovery research. Microdroplet surfaces treated with fibronectin, which regulates a variety of cell behaviors through direct interactions with cell surface integrin receptors, were prepared for culturing epithelial cells. The cells adhered rapidly to the fibronectin-coated fluorinated oil–medium interface and reached confluence. However, as the culture time progressed, the cells began to detach from the microdroplet surface. To promote adhesion to the microdroplet interface, the cells were exposed to the Rho-associated protein kinase inhibitor Y27632, which increased the frequency of microdroplets with cells adhering to the liquid–liquid interface by 1.63-fold. However, continuous exposure to Y27632 caused the cells to detach from the microdroplet surface. When the drug was switched from Y27632 to forskolin, which enhances cell–cell and cell–substrate adhesion, the cells remained as a monolayer on the microdroplet interface. Na+/K+-ATPase and zonula occludens-2 were localized to the apical and lateral membranes of the cells, respectively, while paxillin co-localized with fibronectin at the microdroplet interface. This suggests that the cells exhibited epithelial polarity. These findings indicate that the regulation of cell–cell and cell–substrate adhesion is crucial for establishing epithelial polarity in cells cultured on the microdroplet interface.

DOI： 10.1016/j.jbiosc.2024.12.009

Web of Science

Scopus

PubMed

Language：English   Publisher：Scientific Reports  

Epithelial tissue forms and maintains a critical barrier function in the body. A novel culture design aimed at promoting uniform maturation of epithelial cells using liquid materials is described. 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. To inhibit stress fiber formation and apoptosis, the exposure of cells on liquid–liquid interfaces to Y27632, a specific inhibitor of the Rho-associated protein kinase (ROCK), facilitated tight junction formation (frequency of ZO-2-positive cells, FZ = 0.73). 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 HLN, was 0.93 ± 0.01, indicated even cell distribution in the culture vessel at t = 72 h. The behavior of epithelial cells on liquid–liquid interfaces contributes to the promotion of their uniform maturation.

DOI： 10.1038/s41598-024-63115-7

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Pharmacology Research and Perspectives  

An understanding of the quantitative relationship between bile canaliculus (BC) dynamics and the disruption of tight junctions (TJs) during drug-induced intrahepatic cholestasis may lead to new strategies aimed at drug development and toxicity testing. To investigate the relationship between BC dynamics and TJ disruption, we retrospectively analyzed the extent of TJ disruption in response to changes in the dynamics of BCs cultured with entacapone (ENT). Three hours after adding ENT, the ZO-1-negative BC surface area ratio became significantly higher (4.1-fold) than those of ZO-1-positive BCs. Based on these data, we calculated slopes of surface area changes, m, of each ZO-1-positive and ZO-1-negative BC. BCs with m ≤ 15 that fell within the 95% confidence interval of ZO-1-positive BCs were defined as ZO-1-positive. To validate this method, we compared the frequency of ZO-1-positive BCs, FZ, with that of BCs with m ≤ 15, FT, in culture using drugs that regulate TJ, or induce intrahepatic cholestasis. FT values were correlated with FZ under all culture conditions (R2 =.99). Our results indicate that the magnitude of BC surface area changes is a factor affecting TJ disruption, suggesting that maintaining TJ integrity by slowing BC dilation inhibits cell death.

DOI： 10.1002/prp2.960

Scopus

Repository Public URL： https://hdl.handle.net/2324/7172695

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1242/bio.058606

Repository Public URL： https://hdl.handle.net/2324/7172697

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/adbi.202000198

Repository Public URL： https://hdl.handle.net/2324/7172698

Event date： 2025.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜   Country：Japan  

Event date： 2025.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜   Country：Japan  

Event date： 2024.10

Language：Japanese  

Venue：仙台   Country：Japan  

Language：Japanese  

Venue：仙台   Country：Japan  

Language：English  

Venue：Aachen   Country：Germany