A novel bioengineering platform using functionalized self-assembly peptides to enhance CYP3A2 activity in modified rat hepatocyte sandwich cultures

Abstract

Isolated hepatocytes removed from their microenvironment soon lose their hepatospecific functions when cultured. Highly oxygen-demanding hepatocytes are commonly maintained under oxygen-deficient culture conditions, limited by culture medium thickness as well scaffold thickness. Thus, the cells are forced into anaerobic metabolic states that degenerate liver specific functions. Furthermore, cells separated from their extracellular matrix and disconnected from the synergistic interactions between other hepatic cells types further exacerbate hepatocellular function. This study aims to improve hepatospecific activity, especially CYP3A2 - a biomarker that is notoriously known to quickly lose expression in primary cultures, by creating a platform based on collagen sandwich cultures. The modified sandwich cultures are substituted with self-assembling peptide, RAD16-I, combined with integrin-binding sequence RGD or laminin receptor binding sequence YIGSR functional peptide motifs to create a cell-instructive peptide scaffold. To facilitate oxygen and nutrient diffusion and exchange, plasma modification technology is employed to control peptide layer dimension. We have successfully shown that plasma engineering can be used to optimize peptide thickness.

(cont.) Likewise, we have shown that the incorporation of the functional motifs enhanced hepatospecific activity. CYP3A2 expression from cultures on our platform improved over 256 times the levels found in collagen sandwich cultures, the current standard for hepatocyte cultures. This study demonstrates the capability of sandwich cultures with modified instructive self-assembling peptides and the importance of thinner cultures scaffolds to promote better oxygen and nutrient exchange. We believe that our novel bioengineered platform has the potential to greatly improve existing hepatocyte culture methods and be invaluable to future in vitro hepatocyte studies as well as toxicity tests.