Collagen-I and fibronectin modified three-dimensional electrospun PLGA scaffolds for long-term in vitro maintenance of functional hepatocytes.

Extracellular matrix (ECM) proteins are important regulators of cellular behaviour in the native environment. It has been established that ECM proteins - collagen-I and fibronectin - are present in liver extracellular matrix and regulate specific functions of primary hepatocytes. While scaffolds grafted with the individual ECM protein have shown support for hepatocyte functional properties in vitro, the synergistic effects of both ECM proteins remain to be explored. Such studies are even more limited when three-dimensional (3D) scaffolds are involved. In the current work, the fabrication of a series of highly porous poly(lactic-co-glycolic acid) (PLGA) 3D electrospun scaffolds, simultaneously modified with both collagen-I and fibronectin, has been demonstrated. Different ratios of collagen-I to fibronectin were optimized to study the synergistic effects of the proteins in supporting the viability and functional properties of Huh-7.5 cells. The ratio of collagen-I to fibronectin at 3:1 was found to provide the most efficient chemisorption on the 3D scaffolds. At this ratio, the total protein content that can be grafted on the scaffolds was the highest and the most homogeous. This led to remarkable enhancement of cell seeding efficiency as well as proliferation. Most importantly, liver specific genes such as albumin and cytochrome P450 enzymes i.e. CYP3A4 and CYP3A7 were significantly upregulated by ~12.5, 7 and 4.5 fold respectively, as compared to unmodified PLGA scaffolds after 28 days of culture. Compared to single-protein modified scaffolds, scaffolds modified with 3:1 collagen to fibronectin result in a rise of the albumin gene expression of cultured cells by ~8 to 10 fold, whereas CYP3A4 gene expression improved by ~5 to 7 fold and CYP3A7 gene expression improved by ~4 to 4.5 fold after a long culture period of 28 days. Albumin secretion was improved by ~4 fold compared to unmodified PLGA scaffolds, ~3 fold compared to collagen-I modified culture groups and ~2 fold compared to fibronectin modified culture groups. The multi-protein modified scaffolds, at the optimum ratio, were able to significantly enhance functional properties of the liver cells. This simple yet highly functioning platform would be useful for in vitro culture of liver cells for both drug screening as well as translational purposes.