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Supplementary Material for: Gene Expression and Functional Analyses of Primary Rat Hepatocytes on Nanofiber Matrices

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posted on 01.06.2009, 00:00 by Brophy C.M., Luebke-Wheeler J.L., Amiot B.P., Remmel R.P., Rinaldo P., Nyberg S.L.
Long term culture of primary hepatocytes is valuable for diagnostic and therapeutic applications. However, standard monolayer culture of primary hepatocytes on tissue culture plastic (TCP) – either uncoated or coated with a biological material such as collagen or laminin – is problematic. Thus, novel support matrices are under development to better maintain gene expression and differentiated function of primary hepatocytes in vitro. In this study, a fabricated nanofiber matrix was compared to control conditions of uncoated and laminin-coated TCP. Gene expression and biochemical analyses were performed to compare functional abilities of the hepatocytes in the different conditions. Hepatocytes cultured on nanofibers maintained higher cytochrome P450 1A activity (0.49 ± 0.08 ng resorufin/ml/min) compared to hepatocytes on laminin (0.11 ± 0.05 ng resorufin/ml/min). In addition, albumin production of hepatocytes on nanofibers was greater than twice the production of hepatocytes on laminin (day 14, 34.4 ± 1.8 vs. 15.9 ± 4.5 µg albumin/ml/day). Hepatocytes demonstrated the ability to generate urea from ammonia in all conditions; however, hepatocytes performed ureagenesis more effectively on nanofibers than on laminin (0.55 ± 0.25 µM vs. 0.36 ± 0.24 µM urea, day 14). Gene expression of hepatocytes cultured on nanofiber and laminin conditions were similar on a per cell basis determined by analysis using a custom microarray of 250 genes expressed in hepatocytes. Similar cell attachment data between conditions and similar numbers of cells expressing the hepatocyte marker hepatocyte nuclear factor 4αindicates that hepatocytes grown on nanofibers only marginally display improved hepatic functions compared to laminin control conditions.

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