No abstract

Isolated rat primary hepatocytes were cultured between two layers of gelled collagen in a sandwich configuration that reinstates the cellular polarity necessary for long-term function in vitro. Maintenance of hepatocyte function, as measured by the secretion of albumin, was shown to be dependent on both the sandwich gel configuration and the continued presence of L-proline in the culture media. Cis-hydroxyproline, an analog of proline known to prevent the proper folding of triple helical collagen molecules, inhibited the response of sandwiched hepatocytes to proline in a dose-dependent and reversible manner. The addition of cis-hydroxyproline to cultures established for 7 days also resulted in the inhibition of hepatocyte function. These data support the hypothesis that continued collagen synthesis by hepatocytes is critical for hepatocyte function in the sandwich gel configuration.

Many potential applications of primary hepatocytes cultured on microcarriers, such as an artificial liver or hepatocyte transplantation, would benefit from having a large number of hepatocytes attached to each microcarrier. In addition, the supply of primary hepatocytes is usually limited, so the efficient utilization of hepatocytes during attachment to microcarriers is necessary. Several physical parameters involved in the attachment process have been investigated, and the number of cells attached per microcarrier and the fraction of hepatocytes which attach have been quantitatively monitored. Variation of the partial pressure of gas phase oxygen in the incubation flask produced significant effects on the attachment of hepatocytes to microcarriers, with higher partial pressures of oxygen found to be necessary for attachment. In addition, variation of fluid depth and cell number, both of which influence the partial pressure of oxygen at the cell surface, affected hepatocyte attachment. The partial pressure of oxygen at the cell surface as a function of the physical parameters was analyzed using a simple one-dimensional theoretical model. Variations in the cell-to-microcarrier ratio used for incubation indicate that a compromise must be made in terms of maximizing the number of cells per microcarrier and the fraction of total hepatocytes which attach. The maximum number of hepatocytes per microcarrier obtained in this work was approximately 100. The best attachment fraction, defined as the ratio of the number of hepatocytes attached to the total number added to the incubation, was approximately 90%.