The effects of immunosuppressive agents on the function of human hepatocytes in vitro

T Serrano, R R Mitry, C Terry, S C Lehec, A Dhawan, R D Hughes

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    12 Citations (Scopus)

    Abstract

    Calcineurin inhibitors (tacrolimus) and steroids continue to be an important component of hepatocyte transplantation protocols, despite reports of hepatotoxicity and inhibitory effects of steroids on cell proliferation. The aim of the study was to investigate whether isolated human hepatocytes were more vulnerable to the toxicity of these agents and also to investigate their effects on hepatocyte VEGF secretion, a vascular permeability factor suggested to be involved in the cell engraftment process. Human hepatocytes were isolated from donor livers/segments rejected or unused for orthotopic liver transplantation using a collagenase perfusion technique. Hepatocytes were plated for cell function tests and to determine VEGF production. Tacrolimus (0-50 ng/ml) and methylprednisolone (0-500 ng/ml) were added to the culture media and cells incubated for 24 h. Cell metabolic activity was assessed using. the MTT assay, cell number using the SRB assay, and cell attachment from hepatocyte total protein content and protein synthesis using [C-14]leucine incorporation. VEGF in culture supernatants was measured by ELISA. Tacrolimus and methylprednisolone had no statistically significant inhibitory effects on metabolic activity or protein synthesis compared to controls at all concentrations of the agents tested when added after plating. There were also no significant effects on cell attachment when tacrolimus or methylprednisolone was added at the time of cell plating. There were no differences in the responses obtained when either fresh or cryopreserved hepatocytes were used. The amount of VEGF secreted by untreated hepatocytes was highly variable (0-1400 pg/10(6) cells/24 h). VEGF levels in the culture supernatant from hepatocytes isolated from
    Original languageEnglish
    Pages (from-to)777 - 783
    Number of pages7
    JournalCell Transplantation
    Volume15
    Issue number8-9
    Publication statusPublished - 2006

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