A large-scale automated method for hepatocyte isolation: effects on proliferation in culture.
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 x 10(9) for 250-ml centrifuge bowls and 2.8 x 10(9) 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.
Nieuwoudt
M J
MJ
Department of Internal Medicine, University of Pretoria, Pretoria, South Africa. martin@postino.up.ac.za
Kreft
E
E
Olivier
B
B
Malfeld
S
S
Vosloo
J
J
Stegman
F
F
Kunneke
R
R
Van Wyk
A J
AJ
Van der Merwe
S W
SW
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D048708:Cell Growth Processes / Q000502:physiology; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002498:Centrifugation; D005260:Female; D005434:Flow Cytometry; D022781:Hepatocytes / Q000166:cytology* / Q000637:transplantation; D019164:Liver, Artificial; D008858:Microscopy, Phase-Contrast; D013552:Swine