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

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.

Quantitative understanding of HepaRG cells during drug-induced intrahepatic cholestasis through changes in bile canaliculi dynamics.

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.

Tight junction stabilization prevents HepaRG cell death in drug-induced intrahepatic cholestasis.

Entacapone (ENT), a catechol-O-methyltransferase inhibitor, causes liver injury by inducing bile canaliculi (BC) dilation through inhibition of the myosin light kinase pathway. Loss of tight junctions (TJs) induces hepatocyte depolarization, which causes bile secretory failure, leading to liver damage. To understand the influence of TJ structural changes as a consequence of BC dynamics, we compared the datasets of time-lapse and immunofluorescence images for TJ protein ZO-1 in hepatocytes cultured with ENT, forskolin (FOR), ENT/FOR, and those cultured without any drugs. Retrospective analysis revealed that the drastic change in BC behaviors caused TJ disruption and apoptosis in cells cultured with ENT. Exposure to FOR or sodium taurocholate facilitated TJ formation in the cells cultured with ENT and suppressed BC dynamic changes, leading to the inhibition of TJ disruption and apoptosis. Our findings clarify that hepatocyte TJ stabilization protects against cell death induced by BC disruption.

Switching of cell fate through the regulation of cell growth during drug-induced intrahepatic cholestasis.

Understanding the fundamental mechanisms that govern the fate of cells during drug-induced intrahepatic cholestasis provides strategies for the establishment of evaluation methods for drug screening. In the present study, the aggregates of a differentiated human hepatic cell line, HepaRG, were incubated in medium with Y27632 or bosentan to clarify the changes in the behavior of bile canaliculi (BC) with the growth of cells during drug-induced intrahepatic cholestasis. With elapsed exposure time, the aggregates in the culture with bosentan caused the dilation of BC, and the hepatocytes ultimately exhibited apoptotic death after the disruption of BC. Y27632 caused the disruption of BC in the aggregates after dilation. However, there was no change in the number of cells within the aggregates in the culture with Y27632, in spite of its cytotoxicity. After 144 h from the start of Y27632 exposure, the aggregates showed the rearrangement of BC. To inhibit cell division, the aggregates exposed to Y27632, which exhibited disruption of BC, were treated with mitomycin C for 2 h and continuously exposed to Y27632. The inhibition of cell division could not induce the rearrangement of BC within these aggregates, which was similar to the phenomenon observed in the aggregates exposed to bosentan. These findings indicate that growth is an important factor that influences the switching of cell fate toward survival or death in drug-induced intrahepatic cholestasis process. Thus, the autoregulation of growth is a major contributor to the rearrangement of BC within aggregates.

Phenotypic heterogeneity of human retinal pigment epithelial cells in passaged cell populations.

Human retinal pigment epithelial (RPE) cells at different population doublings (PDs) were cultured for 28 days to examine their phenotypic heterogeneity in a confluent state. In an early population (PD = 2.8), cells showed a cobblestone-like appearance (type I), which gradually became small and tight, and eventually exhibited dark pigmentation. Some cells showed a dome-like structure (type II), which detached from the culture surface during culture. With increasing PD, the cells showed active migration that caused a shift in phenotype from a single layer of large, flattened cells (type III) to a multiple cell layers (stratified) with flattened, irregularly shaped cells (type IV). Immunostaining of specific RPE markers, ZO-1 and Na+/K+-ATPase revealed that cells have markedly decreased expressions in a late population (PD = 10.1). RPE phenotypes were classified into four types by measuring the nuclear size and local density. The frequencies of type I cells decreased with increasing PD value, while the frequencies of type III and IV cells increased along with the decrease in type I. The frequencies of type IV cells at PD = 10.1 had increased by 10.3-fold compared with PD = 2.8. From these results, the nuclear size and local density were proposed as indicators for understanding phenotypic heterogeneity of RPE cells in the passaged cell population during cell expansion. It is concluded that the population doubling level is an important factor to affect the transition of RPE phenotype and thereby to modulate the quality of cultured cells.

Facilitation of uniform maturation of human retinal pigment epithelial cells through collective movement in culture.

Understanding of the fundamental mechanisms that govern tight junction formation of retinal pigment epithelial (RPE) cells provides surface design strategies for promoting their maturation in culture. RPE cells were cultured to investigate their migratory behavior and the expression of tight junction protein ZO-1 in the central and peripheral regions of a culture vessel. Regardless of locational differences in the culture vessel, the cells at day 1 were elongated in shape, did not form tight junctions, and migrated actively. As the culture progressed, the cells in the central region slowly moved with morphological change of a cobblestone-like shape via interaction between contact cells and exhibiting the shift from random migration to collective movement toward the center, accompanied by tight junction formation. On the other hand, the cells in the peripheral region maintained the random migration at day 5, meaning spatial heterogeneity in maturation in the vessel. At day 5, RPE cells were incubated in medium with Rac1 inhibitor and the exposure to the Rac1 inhibitor triggered the rapid conversion of migratory behavior from random migration to collective movement toward the center of the vessel, resulting in uniform maturation. These findings indicate that the change in migratory patterns is an important cues and the collective movement toward the center causes the facilitation of uniform maturation in the vessel.

Locational heterogeneity of maturation by changes in migratory behaviors of human retinal pigment epithelial cells in culture.

To better characterize human retinal pigment epithelial (RPE) cells, their maturation was studied by time-lapse observation and immunostaining of the tight junction protein ZO-1. During subconfluency with active migration, the cells had an elongated shape. During cell division to reach confluency, RPE cells became small and tight, exhibiting cobblestone-like morphology. In addition, RPE maturation at the peripheral region of the culture vessel was delayed when compared with the central region, demonstrating local heterogeneity during maturation. To correlate cellular migration and maturation, we compared frequencies of migration rate and number of ZO-1-positive cells at the central and peripheral regions. Cells having migration rates less than 5.0 µm/h in the central region were 1.4-fold higher than in the peripheral region at day 5. Regardless of locational differences in the culture vessel, the frequency of cells having migration rates less than 5.0 µm/h showed 90% agreement with the frequency of ZO-1-positive cells. To inhibit cell migration, RPE cells were exposed to medium containing 50 µg/ml Rac1 inhibitor at day 5. Frequencies of ZO-1-positive cells and cells having migration rates less than 5.0 µm/h at the peripheral region were similar to those at the central region. The results show that migration is an important factor affecting maturation, and demonstrate that location heterogeneity during maturation is caused by different migratory behaviors in the culture vessel.

Analysis of locality of early-stage maturation in confluent state of human retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells were cultured on the laminin-coated and plain surfaces. The measurement of local nucleus density in non-stratified region, which correlated with formation of tight junction, is the indicator of the maturation, and the parameters can be applied to the evaluation of the early-stage maturation of RPE cells in culture.