Characterization of spontaneous cell death in monolayer cultures of primary hepatocytes

Abstract

Monolayer cultures of primary hepatocytes, isolated from freshly removed livers, represent widely used in vitro tools in the area of liver physiology and pathology, pharmacology and toxicology. However, a major shortcoming of these systems is that they cope with dedifferentiation, which is accompanied by spontaneous cell death. The goal of the present study was to elucidate the mechanisms that drive the process of self-generated cell demise in primary hepatocyte cultures. For this purpose, isolated rat hepatocytes were cultivated under conventional conditions, and the occurrence of apoptosis and necrosis was monitored during 4 days by performing a set of acknowledged cell death assays. These included examination of cell morphology by light microscopy, quantification of apoptotic and necrotic cell populations by Hoechst 33342 and propidium iodide in situ staining, assessment of apoptotic and necrotic activities by measuring caspase 3-like activity and extracellular leakage of lactate dehydrogenase, and studying the expression of apoptosis regulators through immunoblot analysis. In essence, two cell death peaks were observed, namely shortly after cell seeding and in the final stages of the cultivation period, both involving apoptotic and necrotic actions. The outcome of this study not only sheds new light onto the molecular processes that underlie spontaneous cell death in primary hepatocyte cultures, but also opens perspectives for the establishment of strategies to increase cell survival in these popular in vitro systems.

Inhibition of cytochrome P450s enhances (+)‐usnic acid cytotoxicity in primary cultured rat hepatocytes

“…We were not able to consistently observe a decrease of UA cytotoxicity in hepatocytes pre‐treated with CYP inducers for 24 h, although the cytotoxicity of leflunomide was significantly decreased using a 24‐h pre‐treatment with CYP inducers, as reported (Shi et al ., ). It has been well recognized that monolayer cultures of primary hepatocytes tend to lose many normal phenotypes 24 h after isolation (Vinken et al ., ). This might be the reason that hepatocytes treated with CYP inducers for 24 h did not respond to UA challenge as expected.…”

Hepatocytes maintain greater fluorescent bile acid accumulation and greater sensitivity to drug-induced cell death in three-dimensional matrix culture

“…For instance, hepatocytes appear to show less spreading when cultured in the presence of serum and on substrates other than collagen (Vinken et al. ; Godoy et al. ).…”

Overexpression of Hematopoietically Expressed Homeoprotein Induces Nonapoptotic Cell Death in Mouse Prechondrogenic ATDC5 Cells

“…35) Noy and colleagues also indicated that knockdown of Hex gene increased cell proliferation, and the increase was enhanced by stimulation with VEGF in K562 cells. 32) However, the precise molecular mechanism and the executioner of the cell death by Hex were not described in the report.…”