Effect of Growth Factors and Defined Medium on Primary Hepatocyte Culture on Polyester Carriers with Varying Surface Treatment

Abstract

The growth and function of primary rat hepatocytes was characterized on commercial, electrostatically treated nonwoven polyester carriers (Fibra-Cel) designed for use in a packed-bed bioreactor. Such carriers are attractive in that they allow three-dimensional cellular interactions and afford shear protection. To initiate investigations of the potential of these carriers for hepatocyte support, novel hepatocyte media were evaluated along with a variety of carrier surface treatments in static cell culture. Different serum-free chemically defined media with recombinant growth factors known to be complete mitogens for hepatocytes were contrasted. Carrier treatments included unmodified surfaces contrasted with carriers coated with type I collagen or HepG2-derived extracellular matrix. Hepatocytes were also co-cultured with nonparenchymal cells. Cellular mitochondrial metabolism of 3-(4,5-dimethylthiozol- 2-yellow)-2,5 diphenyl tetrazolium bromide (MTT) was used to ascertain continued viability. Cell proliferation was assessed by total DNA measurements. Cell morphology was monitored by phase microscopy. 7-Ethoxycoumarin deethylation was used to indicate cytochrome p450 function. MTT metabolism initially declined, but stabilized at 8 days, and then was maintained on the treated carriers for at least 30 days with a novel hepatocyte media supplemented with hepatocyte growth factor and epidermal growth factor. Carrier treatment also had significant effects on MTT metabolism with HepG2 matrices < collagen < untreated carriers. Co-culture with nonparenchymal cells also appeared to improve MTT conversion by hepatocytes substantially over those cultured on carriers treated with collagen. Evenly distributed throughout the carriers early in the culture period, hepatocyte clustering became apparent over time. Cytochrome p450 activity was sustained for at least 18 days in hepatocytes maintained in static culture on carriers treated with collagen. The combination of this commercial carrier system with specifically designed hepatocyte media and modified carrier substrates holds promise as a new avenue for bioartificial liver design.