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Cytotoxicity and apoptosis are common problems in the isolation and storage of human hepatocytes. In vitro environments of hepatocytes during cell infusion may be critical to reducing cellular damage and enhancing cell viability. We examined the effects of donor liver histology (40–50% steatosis vs. normal), incubation time, temperature, and three solutions for infusion on banked primary human hepatocytes, by studying: trypan blue exclusion, AST release, LDH release, MTT assay, detection of DNA ladder, and a hepatocyte proliferation assay. In addition, the microstructure functions of the endoplasmic reticulum and mitochondria of the intact hepatocytes were determined by measuring correlates of UGT 1A1 and cytochrome P-450 3A (CYP3A4) activity. In general, hepatocyte viability decreased significantly within 60 min after thawing. Cells suspended in 5% dextrose lactated Ringers solution (D5LR) maintained greater cell viability. Hepatocytes from normal liver donors showed less AST and LDH enzyme leak in comparison with cells from fatty liver donors. Mild hypothermic temperature (32°C) inhibited cellular damage that otherwise significantly increased at 60 min. Hepatocytes did not proliferate until 12 h from thaw, regardless of supernatant or conditions of suspension. CYP3A4 activity and a marker for UGT 1A1 activity in hepatocytes from normal donor livers were higher than those from steatotic donor livers. These findings suggest that hepatocytes suspended for infusion after isolation from normal liver donors have normal biological functions and less cellular damage/necrosis in contrast with those isolated from fatty liver donors. These damages are inhibited significantly by maintaining hepatocytes at a mild hypothermic temperature (32°C). D5LR alone maintained the best cell viability for up to 60 min. Media of D5LR + adenosine and HMM were able to partially inhibit hepatocyte apoptosis in hepatocytes from steatotic livers.
*Department of Surgery, Virginia Commonwealth University Medical Center, Richmond, VA 23298
Publication date: January 1, 2004
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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.