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A Large-Scale Automated Method for Hepatocyte Isolation: Effects on Proliferation in Culture

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Abstract:

Large-scale sterile methods for isolating hepatocytes are desirable for the development of bioartificial liver support systems. In this study the traditional centrifuge method was compared with the use of a Baylor Rapid Autologous Transfusion (BRAT) machine for isolating large quantities of porcine hepatocytes. After isolating hepatocytes, the methods were evaluated in terms of cell viability and yield per liver, proliferation over 7 days, and the effects on the cell cycle using the trypan blue exclusion test, conventional phase-contrast light microscopy, the lactate to pyruvate ratio, the leakage of lactate dehydrogenase (LD) and aspartate aminotransferase (AST), lidocaine clearance, albumin production, and flow cytometry. With the centrifuge method the mean cell viability was 92.5%, while with the BRAT method the viability was 95.9%. The minimal cell yields with the BRAT procedure were 7.3 × 109 for 250-ml centrifuge bowls and 2.8 × 109 for 165-ml bowls, which compares well with that found by other authors. Because the same initial procedures were employed in both methods the total hepatocyte yield per liver was comparable. Flow cytometry confirmed that the proliferation of hepatocytes was facilitated by oxygenation during the isolation procedure. The recovery of hepatocytes in culture following isolation was similar after either method. Daily microscopic investigation indicated that cytoplasmic vacuolization and granularities were present after either procedure and these disappeared following 3–4 days of culturing. Flow cytometry indicated that the hepatocyte cell cycle was similar after either method; at 7 days the profile indicated that the cells were still proliferating. Trends in the lactate to pyruvate ratio and the leakage of LD and AST indicated that the functional polarity of hepatocytes was regained after approximately 3 days. Lidocaine clearance at 4 days indicated that the cytochrome P450 system was active, while significant albumin production was apparent at day 5. The benefit of using BRAT technology in hepatocyte isolation lies in guaranteed sterility, convenience, speed, and the ability to oxygenate media and cell suspensions during the procedure.

Keywords: Bioartificial liver; Collagenase liver perfusion; Isolated hepatocytes; Liver cell isolation

Document Type: Review Article

DOI: http://dx.doi.org/10.3727/000000005783983007

Affiliations: 1: Department of Internal Medicine, University of Pretoria, Pretoria, South Africa, Department of Bioengineering, University of Pretoria, Pretoria, South Africa 2: Department of Internal Medicine, University of Pretoria, Pretoria, South Africa 3: Department of Immunology, National Health Laboratory Service, Tswane Academic Hospital, Pretoria, South Africa. 4: Department of Surgery, Pretoria Academic Hospital, University of Pretoria, Pretoria, South Africa 5: Department of Anaesthesiology, Onderstepoort Veterinary Hospital, University of Pretoria, Pretoria, South Africa 6: Department of Mechanical and Aeronautical Engineering, University of Pretoria, Pretoria, South Africa

Publication date: May 1, 2005

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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