Dexamethasone Effects on Rat Hepatocyte Spheroid Formation and Function

Abstract

Hepatocytes cultured on moderately adhesive surfaces or in spinner flasks spontaneously self-assemble into spherical tissue-like aggregates (spheroids). These spheroids have smooth surfaces and tissue-like polarized cell morphology, including bile canalicular-like channels, and maintain high viability and liver-specific functions for extended culture periods. Dexamethasone (DEX), a synthetic glucocorticoid, is known to elicit various responses in gene expression, and is often added to hepatocyte culture medium. The morphology and liver-specific protein production of hepatocyte spheroids were assessed under DEX concentrations ranging from 50 nM to 10 microM. DEX altered the kinetics of spheroid formation in a concentration-dependent fashion, with increasing concentrations inhibiting aggregation and promoting aggregate disassembly on culture dishes. DEX addition to spinner cultures resulted in smaller, more irregularly shaped spheroids and a higher incidence of aggregate clumping. Albumin and urea production were also higher in DEX cultures, but this effect was not as sensitive to concentration and occurred irrespective of the state of aggregation. RTPCR was utilized to assess the mRNA levels of extracellular matrix proteins, E-cadherin, and cytochrome P-450 enzymes. Results indicated a slight increase in fibronectin and collagen III mRNA early in the cultures, possibly contributing to the changes in morphology observed.

Three-Dimensional Primary Hepatocyte Culture in Synthetic Self-Assembling Peptide Hydrogel

“…Although the underlying mechanism is unclear, this result is consistent with one report using suspension cultures in spinner flasks. 25 This prior publication also reported a higher albumin secretion rate with Dex, which agrees with our data showing that albumin production was increased in the presence of Dex for all culture configurations. The albumin gene promoter region is known to have a glucocorticoid response element, which may mediate this observed increase.…”

“…26 Another possible explanation may be due to the antiinflammatory properties of Dex, which may reverse albumin suppression induced by a proinflammatory or acute phase response triggered by the isolation procedure. 27 On the contrary, we did not observe more urea production in the presence of Dex, unlike what was reported by Abu-Absi et al 25 Interestingly, Dex had no effect on the CYP450 activity in PuraMatrix cultures, but inhibited the recovery of CYP450 function beyond day 4 in the two culture configurations using type I collagen. Clearly, the effects of Dex on hepatic functions in hepatocyte in vitro culture remain to be elucidated; however, we propose that including 0.1 mM Dex in the hepatocyte culture medium for the first 3 to 4 days after seeding is an efficient way to control the size of the spheroids on PuraMatrix and help cells recover from isolation injury.…”

Glucocorticoid receptor signalling activates YAP in breast cancer

“…On the basis of our results, we hypothesized that the endocrine control exerted by GCs on YAP might have an impact in sustaining the self-renewal potential of breast CSCs35. To assess whether the GR–YAP axis plays a causal role in defining CSC traits, we tested the effect of BM on the self-renewal potential of mammary epithelial cells from a panel of different basal breast cancer cell lines by testing their capacity to form and propagate mammospheres in vitro 363738.…”

A method for the effective formation of hepatocyte spheroids using a biodegradable polymer nanosphere

“…Hepatocyte spheroids ranging from 50 to 100 μm were formed between 2 and 3 days in the conventional method. 18, 20 In the present study, hepatocyte spheroids in the nanosphere method formed between 24 and 36 h. The higher cell viability in the nanosphere‐added cultures may be due to the increased speed at which the spheroids formed and the higher efficiency of hepatocyte spheroid formation, since single hepatocytes in suspension die rapidly 18, 24. Our previous studies have demonstrated that the formation of cell spheroids using nanospheres enhanced the rate of cell aggregation, subsequent growth of the aggregated cells, and the production of a recombinant protein more than the cell spheroid formations without nanospheres 22–24.…”