Explanted Diseased Livers – A Possible Source of Metabolic Competent Primary Human Hepatocytes

Abstract

Being an integral part of basic, translational and clinical research, the demand for primary human hepatocytes (PHH) is continuously growing while the availability of tissue resection material for the isolation of metabolically competent PHH remains limited. To overcome current shortcomings, this study evaluated the use of explanted diseased organs from liver transplantation patients as a potential source of PHH. Therefore, PHH were isolated from resected surgical specimens (Rx-group; n = 60) and explanted diseased livers obtained from graft recipients with low labMELD-score (Ex-group; n = 5). Using established protocols PHH were subsequently cultured for a period of 7 days. The viability and metabolic competence of cultured PHH was assessed by the following parameters: morphology and cell count (CyQuant assay), albumin synthesis, urea production, AST-leakage, and phase I and II metabolism. Both groups were compared in terms of cell yield and metabolic function, and results were correlated with clinical parameters of tissue donors. Notably, cellular yields and viabilities were comparable between the Rx- and Ex-group and were 5.3±0.5 and 2.9±0.7×106 cells/g liver tissue with 84.3±1.3 and 76.0±8.6% viability, respectively. Moreover, PHH isolated from the Rx- or Ex-group did not differ in regards to loss of cell number in culture, albumin synthesis, urea production, AST-leakage, and phase I and II metabolism (measured by the 7-ethoxycoumarin-O-deethylase and uracil-5′-diphosphate-glucuronyltransferase activity). Likewise, basal transcript expressions of the CYP monooxygenases 1A1, 2C8 and 3A4 were comparable as was their induction when treated with a cocktail that consisted of 3-methylcholantren, rifampicin and phenobarbital, with increased expression of CYP 1A1 and 3A4 mRNA while transcript expression of CYP 2C8 was only marginally changed. In conclusion, the use of explanted diseased livers obtained from recipients with low labMELD-score might represent a valuable source of metabolically competent PHH which are comparable in viability and function to cells obtained from specimens following partial liver resection.

Cell sources forin vitrohuman liver cell culture models

“…Human liver cells isolated from explanted livers obtained from recipients with moderate alterations in blood parameters (low labMELD score) were comparable in viability and function to cells obtained from specimens following partial liver resection. 4 …”

Transcriptome analysis reveals a classical interferon signature induced by IFNλ4 in human primary cells

“…We established cultures of PHH 24 and primary HAE cells. 25 The two systems were chosen as the respective primary cells originate from tissues that are of biological relevance for type III IFN.…”

“…Cells were seeded in 6-well dishes on collagen directly after surgery. 24 Twenty-four hours post seeding, the PHHs were induced for 6 h with IFNs or PBS (mock control) diluted in hepatocyte culture medium (HCM, Lonza, Walkersville, MD, USA). The cells were then lysed in 1 ml Trizol reagent (Invitrogen, Karlsruhe, Germany) per well.…”

Inactivation of HCV and HIV by microwave: a novel approach for prevention of virus transmission among people who inject drugs

“…Primary human hepatocytes (PHH) were isolated from liver specimens obtained after partial hepatectomy, plated at a density of 1.3 × 10 6 on collagen in P6 dishes, and kept in hepatocyte culture medium (Lonza) as described47.…”