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The Optimal Hepatocyte Density for a Hollow-Fiber Bioartificial Liver

Address correspondence to Qingxiang Xu, M.D., Hepatobiliary Surgical Department, Affiliated Drum Tower Hospital of Nanjing University Medical College, Zhongshan Road 321, Nanjing, PR China 210008; tel 86 25 3304616 11601; fax: 86 25 3317016; e-mail: xqx008{at}hotmail.com.

A bioartificial liver (BAL) based on viable porcine hepatocytes can serve as a bridge to liver transplantation in patients with acute liver failure (ALF). To support liver functions, an adequate mass of hepatocytes is needed, which depends upon the cell density in the BAL device. This study evaluated the optimal density of hepatocytes within BAL devices that were constructed by perfusing porcine hepatocyte suspensions mixed with cytodex-3 into polysulfon hollow-fibers. The BAL devices were prepared with 6 different cell densities. The mass of hepatocytes in each device was evaluated for (a) cell viability, (b) ability to degrade diazepam, (c) ability to synthesize urea, (d) incorporation of [³H]-leucine into protein, (e) glucose-6-phosphatase activity, (f) total RNA content, and (g) p53 gene expression. Hepatocyte viability was about 90% in each device. With increasing hepatocyte density, the diazepam concentration in the medium decreased from 9.26±0.96 mg/L at 1 x 10⁵ cells/ml to a minimum of 5.25±1.02 mg/L at 5 x10⁶ cells/ml and thereafter remained at low levels. Urea production and [³H]-leucine incorporation into protein increased progressively until the cell density reached 5 x 10⁶/ml and thereafter remained at high levels. Glucose-6-phosphatase activity and total RNA content stayed at high levels until the cell density reached 5 x 10⁶/ml and then progressively decreased. p53 gene expression differed from the other parameters, since it increased only when the cell density reached 5 x 10⁷/ml. In conclusion, the density of 5 x 10⁶ cells/ml is a critical inflection point for most of the functional parameters, although p53 gene expression is not elevated at this cell density. These findings suggest that 5 x 10⁶ cells/ml is the optimal hepatocyte density in the hollow-fiber BAL device.

Keywords: bioartificial liver, acute liver failure, hepatocytes