Type of endothelial cells affects HepaRG cell acetaminophen metabolism in both 2D and 3D porous scaffold cultures.

Abstract

Recent advances in developing in vitro tissue models show that function of hepatocytes is altered in when cultured in 3D configuration and co-culturing with various non-parenchymal cells. However, tissue source for such non-parenchymal cells on viability and metabolic products of hepatocytes have not been explored. In this study, we evaluated the effect of 2D and 3D cultures either with HepaRG cells alone or in combination with liver sinusoidal endothelial cells (LSECs) and human umbilical vein ECs (HUVECs). For 3D cultures, we used chitosan-gelatin porous structures formed by freeze-drying. We cultured cells for 8days before challenging with 1mm acetaminophen (APAP) and assessed APAP, APAP-sulfate and APAP-glucuronide for 24hours at 6-hour time intervals using high-performance liquid chromatography. We used multiple methods (phase contrast, confocal and scanning electron microscopy and histology via hematoxylin and eosin staining) to ensure cell distribution. We also measured total protein content and albumin secretion and viability. HUVEC 3D co-cultures showed the lowest HepaRG cell viability, while both 2D and 3D LSEC co-cultures had highest HepaRG cell viability. In addition, 3D cultures had significantly higher EC viability relative to 2D cultures. Further, HUVEC co-cultures showed reduced total protein content and albumin expression as early as day 4. However, urea production on a total protein content basis did not change. In addition, LSEC 3D co-cultures had the highest APAP conversion with reduced APAP-sulfate and APAP-glucuronide formation. CYP3A4 was higher in co-culture with HUVEC for 2D and 3D cultures. In conclusion, HepaRG cells with EC co-cultures demonstrated sensitivity to the EC line used.