The effects of 3D-culture and stimulation with 5-Azacytidine and Vitamin C on HepG2 cells in culture

Abstract

Primary human hepatocytes are the gold standard for in vitro toxicity testing, however, in classical 2D culture, they dedifferentiate rapidly, necessitating research into new culture modalities as well as alternative cell lines. In this study we investigated the effects of sandwich and cryogel 3D culture as well as stimulation with 5-azacytidine in combination with vitamin C on the metabolic activity of HepG2 cells, an immortalized hepatoma cell that has been shown to maintain residual hepatocyte-like function. We created a scaffold from pHEMA, bis-acrylamide, collagen, cold fish gelatin, cross-linked by APS and TEMED. We were able to show regular pore formation and established a protocol for cell seeding and cell culture. We were able to see an increase in the activity of several key enzymes in 3D and sandwich culture and we could also demonstrate an increase in gene expression as a result of epigenetic modification with AzaC as well as sandwich cultivation. Despite seeing higher activity, we could not detect a significant difference when comparing AzaC 3D culture with unstimulated 3D culture. 3D culturing of primary human hepatocytes offers promising avenues of research, but first, scaffolds need to be thoroughly optimized to mimic in vivo conditions as closely as possible.