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.
Highlights
Despite considerable improvements achieved in the cryopreservation and hypothermic storage of primary human hepatocytes (PHH) [1,2,3,4] the availability of PHH for cell biology use and hepatocyte transplantation (HCTx) remains insufficient due to the limited number of adequate donors.the demand for PHH has continuously increased due to tightened standards in drug testing [5,6] and an expanded use of primary cells in basic and clinical research [7,8]
General donor characteristics Due to the severity of the underlying diseases that eventually necessitated LTx, the macroscopic appearance of the explanted organs was grossly altered as compared to the liver specimens obtained from partial hepatectomy (Figures 1A–E)
No statistically significant differences in liver function tests were observed between the Rx- and Explant group (Ex)-group and likewise the labMELD-score did not differ (Table 1)
Summary
Despite considerable improvements achieved in the cryopreservation and hypothermic storage of primary human hepatocytes (PHH) [1,2,3,4] the availability of PHH for cell biology use and hepatocyte transplantation (HCTx) remains insufficient due to the limited number of adequate donors. The demand for PHH has continuously increased due to tightened standards in drug testing [5,6] and an expanded use of primary cells in basic and clinical research [7,8]. Large specimens from liver resections are less frequently available since extended liver surgery is less readily performed as a result of advancement in surgical techniques. The existing gap between supply and demand of PHH will widen in the near future
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