Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies

Abstract

Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs. However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates. Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development. In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations. Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry. Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed.

Characterization of hepatocyte-based in vitro systems for reliable toxicity testing

“…Upon dedifferentiation, hepatocytes adopt a fibroblast-like flattened phenotype. Hepatocytes either swell and become leaky, or shrink and produce apoptotic bodies when dying by necrosis or apoptosis, respectively (Fraczek et al 2013;Vinken et al 2014).…”

“…For this reason, a lot of efforts have been focused, and are still being focused, on the development of hepatocyte-based in vitro systems eligible for the safety evaluation of chemical compounds (Fraczek et al 2013;Hewitt et al 2007; Leist et al 2017). As a result, 2 main types of human hepatocyte-based in vitro systems are available today.…”

“…The first are liver-derived systems that are directly obtained from liver or liver tissue. They vary in complexity and longevity, and include whole isolated perfused livers, liver slices, primary hepatocytes and their diverse culture configurations, cell lines and subcellular liver fractions (Fraczek et al 2013;Godoy et al 2013). Among those, adherent cultures of primary human hepatocytes are still considered as the gold standard.…”

“…Among those, adherent cultures of primary human hepatocytes are still considered as the gold standard. Nevertheless, although a multitude of studies published in the past decades has demonstrated the potential of a variety of primary hepatocyte culture formats to predict different types of chemical-induced hepatotoxicity, they cope with progressive deterioration of their in vivo-like functional and morphological features (Fraczek et al 2013). This dedifferentiation process hampers the use of primary hepatocyte cultures for testing toxicity over longer terms (Godoy et al 2016).…”

Human stem cell-derived hepatocytes: breakthrough of an expedient tool for preclinical assessment of drug-induced liver injury?

“…Primary human hepatocyte cultures represent the gold standard, but their use is hampered by their low availability and their inability to proliferate in culture (Fraczek et al 2013). Due to intensive transplantation programs, hepatocytes can be seldom isolated from healthy human livers.…”