Abstract
It is well known that a three-dimensional (3D) culture environment and the presence of extracellular matrix (ECM) proteins facilitate hepatocyte viability and maintenance of the liver-specific phenotype in vitro. However, it is not clear whether specific ECM components such as collagen or fibronectin differentially regulate such processes, especially in 3D scaffolds. In this study, a series of ECM-functionalized inverted colloidal crystal (ICC) microporous scaffolds were fabricated and their influence on Huh-7.5 cell proliferation, morphology, hepatic-specific functions, and patterns of gene expression were compared. Both collagen and fibronectin promoted albumin production and liver-specific gene expression of Huh-7.5 cells, compared with the bare ICC scaffold. Interestingly, cells in the fibronectin-functionalized scaffold exhibited different aggregation patterns to those in the collagen-functionalized scaffold, a variation that could be related to the distinct mRNA expression levels of cell adhesion-related genes. Based on these results, we can conclude that different ECM proteins, such as fibronectin and collagen, indeed play distinct roles in the phenotypic regulation of cells cultured in a 3D environment.
Highlights
It is well known that a three-dimensional (3D) culture environment and the presence of extracellular matrix (ECM) proteins facilitate hepatocyte viability and maintenance of the liver-specific phenotype in vitro
As polystyrene beads with a 139 ± 2.9 μm diameter were used as the templates, a similar pore size was obtained in the Poly(ethylene glycol) diacrylate (PEG-DA) inverted colloidal crystal (ICC) scaffolds
The differential effect of specific ECM components on hepatocyte behavior has been studied in monolayer cell culture[33,34,35,36,37], but is not well understood in 3D culture
Summary
It is well known that a three-dimensional (3D) culture environment and the presence of extracellular matrix (ECM) proteins facilitate hepatocyte viability and maintenance of the liver-specific phenotype in vitro. Cells in the fibronectin-functionalized scaffold exhibited different aggregation patterns to those in the collagenfunctionalized scaffold, a variation that could be related to the distinct mRNA expression levels of cell adhesion-related genes Based on these results, we can conclude that different ECM proteins, such as fibronectin and collagen, play distinct roles in the phenotypic regulation of cells cultured in a 3D environment. They cultured the cells between two layers of collagen, the major ECM protein, and were able to reconstruct the hepatocyte polarity found in in vivo physiology and to maintain hepatic function for as long as 6 weeks Kotov and his team reported the controlled formation of uniformly-sized liver tumor cell spheroids in inverted colloidal crystal (ICC) scaffolds with uniform porosity[11,12,13,14,15], as well as cells displaying morphological similarities to liver tissue such as bile canaliculi[14]. Mooney et al suggested that hepatocytes maintained high levels of albumin gene expression and liver-specific protein excretion regardless of the type of ECM molecule used for cell attachment[37]
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