Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration

Abstract

Rat primary hepatocytes were cultured under different extracellular matrix configurations and evaluated for the acquisition and maintenance of structural and functional cell polarity. De novo repolarization of the plasma membrane was variable in rate and extent in hepatocyte cultures maintained on a conventional single layer of either gelled or ungelled collagen. However, cultures maintained in a collagen-sandwich configuration initiated uniform formation of a contiguous anastomosing network of bile canaliculi throughout the entire culture. Localization of apical membrane markers demonstrated normal distribution at the canalicular membrane. A marked rearrangement of the intracellular microfilaments to the cell periphery was observed and coincided with the development of the bile canaliculi. Acquisition of normal bile canalicular function and integrity was observed within 3-4 days postoverlay as indicated by the concentration and retention of carboxyfluorescein within the canalicular network. These results demonstrate that cultures of hepatocytes maintained in a sandwich configuration may serve as a more reliable and representative model in which to study the physiology of hepatic function as well as the morphogenesis of polarized membrane domains in vitro.

Bile acid stimulates hepatocyte polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway

“…Parallel to the morphological change, bile acid synthesis accelerates (16), suggesting that bile acids may influence hepatocyte polarization. Because of difficulties in conducting in vivo developmental studies in fetal liver, we took advantage of a novel in vitro collagen sandwich culture of rat primary hepatocytes, which mimics the in vivo system morphologically and functionally (31,32). After perfusion of rat liver with collagenase, isolated hepatocytes are not polarized; however, in collagen sandwich culture, they sequentially develop into a fully polarized canalicular network, which is accelerated by taurocholate.…”

“…Although bile acids had no effect on cAMP production in suspension culture of hamster hepatocytes (33), taurocholate rapidly increased cAMP in sandwich cultures and adenylate cyclase inhibitor prevented taurocholatemediated increased cAMP. In contrast to suspension cultures, collagen sandwich culture morphologically and functionally mimics events in vivo (31,32). The culture medium contains insulin (0.02 mg/mL) and glucagon (0.0284 μg/mL), which increase cAMP (34,35); however, taurocholate and forskolin significantly increased cAMP compared with control cells.…”

Delamanid Does Not Inhibit or Induce Cytochrome P450 Enzymes <i>in Vitro</i>

“…[20][21][22][23][24][25] Three lots of human fresh hepatocytes were used for the assessment of CYP1A2, CYP2C9, and CYP3A4 activities and the relative mRNA levels. Hepatocytes were seeded at approximately 1.3×10 6 viable cells/mL on collagencoated 60 mm culture dishes and were placed in a humidified culture chamber (37°C, at 95% relative humidity, 95/5% air/ carbon dioxide).…”

Shared apical sorting of anion exchanger isoforms AE2a, AE2b1, and AE2b2 in primary hepatocytes

“…To analyze the sorting of each AE2 variant in polarized liver cells we used the system of collagen-sandwiched primary rat hepatocytes [21,26]. This has been considered an appropriate model to investigate the characteristics of differentiated hepatocytes [27], and their dynamics of repolarization [28].…”