Isolation of Primary Human Hepatocytes from Normal and Diseased Liver Tissue: A One Hundred Liver Experience

Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples

Fluid shear stress modulation of hepatocyte like cell function

Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However, primary hepatocyte scarcity, cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies, HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology, such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study, the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions, cytochrome P450 drug metabolism and serum protein secretion, in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug, primarily metabolised by Cyp2D6. In addition to metabolic capacity, fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker, alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype

Modeling human liver biology using stem cell-derived hepatocytes

Stem cell-derived hepatocytes represent promising models to study human liver biology and disease. This concise review discusses the recent progresses in the field, with a focus on human liver disease, drug metabolism and virus infection

A metabolomics cell-based approach for anticipating and investigating drug-induced liver injury

In preclinical stages of drug development, anticipating potential adverse drug effects such as toxicity is an important issue for both saving resources and preventing public health risks. Current in vitro cytotoxicity tests are restricted by their predictive potential and their ability to provide mechanistic information. This study aimed to develop a metabolomic mass spectrometry-based approach for the detection and classification of drug-induced hepatotoxicity. To this end, the metabolite profiles of human derived hepatic cells (i.e., HepG2) exposed to different well-known hepatotoxic compounds acting through different mechanisms (i.e., oxidative stress, steatosis, phospholipidosis, and controls) were compared by multivariate data analysis, thus allowing us to decipher both common and mechanism-specific altered biochemical pathways. Briefly, oxidative stress damage markers were found in the three mechanisms, mainly showing altered levels of metabolites associated with glutathione and γ-glutamyl cycle. Phospholipidosis was characterized by a decreased lysophospholipids to phospholipids ratio, suggestive of phospholipid degradation inhibition. Whereas, steatosis led to impaired fatty acids β-oxidation and a subsequent increase in triacylglycerides synthesis. The characteristic metabolomic profiles were used to develop a predictive model aimed not only to discriminate between non-toxic and hepatotoxic drugs, but also to propose potential drug toxicity mechanism(s)

Profiling the Impact of Medium Formulation on Morphology and Functionality of Primary Hepatocytes in vitro

The characterization of fully-defined in vitro hepatic culture systems requires testing of functional and morphological variables to obtain the optimal trophic support, particularly for cell therapeutics including bioartificial liver systems (BALs). Using serum-free fully-defined culture medium formulations, we measured synthetic, detoxification and metabolic variables of primary porcine hepatocytes (PPHs) - integrated these datasets using a defined scoring system and correlated this hepatocyte biological activity index (HBAI) with morphological parameters. Hepatic-specific functions exceeded those of both primary human hepatocytes (PHHs) and HepaRG cells, whilst retaining biotransformation potential and in vivo-like ultrastructural morphology, suggesting PPHs as a potential surrogate for PHHs in various biotech applications. The HBAI permits assessment of global functional capacity allowing the rational choice of optimal trophic support for a defined operational task (including BALs, hepatocellular transplantation, and cytochrome P450 (CYP450) drug metabolism studies), mitigates risk associated with sub-optimal culture systems, and reduces time and cost of research and therapeutic applications

Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile

© 2016 The Author(s)The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation. This phenomenon reduces many unique hepatocyte functions, limiting their use in drug disposition and toxicity assessment. The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells. We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation. Cells were cultured for 0, 24, 72 and 168 h as a monolayer in order to permit unrestricted hepatocyte dedifferentiation, so as to reveal the causative signalling pathways and factors in this process, by pathway analysis. A total of 3430 proteins were identified with a false detection rate of <1 %, of which 1117 were quantified at every time point. Increasing numbers of significantly differentially expressed proteins compared with the freshly isolated cells were observed at 24 h (40 proteins), 72 h (118 proteins) and 168 h (272 proteins) (p < 0.05). In particular, cytochromes P450 and mitochondrial proteins underwent major changes, confirmed by functional studies and investigated by pathway analysis. We report the key factors and pathways which underlie the loss of hepatic phenotype in vitro, particularly those driving the large-scale and selective remodelling of the mitochondrial and metabolic proteomes. In summary, these findings expand the current understanding of dedifferentiation should facilitate further development of simple and complex hepatic culture systems