The Effects of Immunosuppressive Agents on the Function of Human Hepatocytes In Vitro

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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 [14C]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/106 cells/24 h). VEGF levels in the culture supernatant from hepatocytes isolated from ≤20-year-old donors (687 ± 59 pg/106 cells/24 h) was significantly greater than from older donors (61 ± 7 pg/106 cells/24 h; p = 0.003). Tacrolimus and methylprednisolone did not significantly affect VEGF secretion by hepatocytes. Tacrolimus and methylprednisolone did not have detrimental effects on the metabolic function of human hepatocytes, cell attachment, or VEGF secretion after cell isolation.

Keywords: Hepatocytes; Methylprednisolone; Tacrolimus; Transplantation; Vascular endothelial growth factor

Document Type: Research Article

DOI: http://dx.doi.org/10.3727/000000006783981530

Affiliations: King's College London School of Medicine at King's College Hospital, Institute of Liver Studies, London, UK

Publication date: August 1, 2006

More about this publication?
  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.
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