Modulation of hepatocyte phenotype in vitro via chemomechanical tuning of polyelectrolyte multilayers

Abstract

It is increasingly appreciated that since cell and tissue functions are regulated by chemomechanical stimuli, precise control over such stimuli will improve the functionality of tissue models. However, due to the inherent difficulty in decoupling these cues as presented by extracellular materials, few studies have explored the independent modulation of biochemical and mechanical stimuli towards the manipulation of sustained cellular processes. Here, we demonstrate that both mechanical compliance and ligand presentation of synthetic, weak polyelectrolyte multilayers (PEMs) can be tuned independently to influence the adhesion and liver-specific functions of primary rat hepatocytes over extended in vitro culture (two weeks). These synthetic PEMs exhibited elastic moduli E ranging over 200 kPa -< E < 142 MPa, as much as one thousand-fold more compliant than tissue-culture polystyrene (E ∼ 2.5 GPa). The most compliant of these PEM substrata promoted hepatocyte adhesion and spheroidal morphology. Subsequent modification of PEMs with type I collagen and the proteoglycan decorin did not alter substrata compliance, but enhanced the retention of spheroids on surfaces and stabilized hepatic functions (albumin and urea secretion, CYP450 detoxification activity). Decorin exhibited unique compliance-mediated effects on hepatic functions, downregulating the hepatocyte phenotype when presented on highly compliant substrata while upregulating hepatocyte functions when presented on increasingly stiffer substrata. These results show that phenotypic functions of liver models can be modulated by leveraging synthetic polymers to study and optimize the interplay of biochemical and mechanical cues at the cell-material interface. More broadly, these results suggest an enabling approach for the systematic design of functional tissue models applied to drug screening, cell-based therapies and fundamental studies in development, physiology and disease.

Multiple Functionalities of Polyelectrolyte Multilayer Films: New Biomedical Applications

“…These cells are widely considered to be ideal for constructing liver tissue models but are known to rapidly lose their viability (within a few hours or days) and phenotype functions upon isolation from the native in vivo microenvironment of the liver. They found that, on unmodified PAH/PAA surfaces, hepatocyte attachment increased with PEM rigidity, [195] but this trend was canceled when the PEM substrata was modified with COL I or with COL I pre-mixed with the small proteoglycan decorin (Fig. 7).…”

“…7B). [195] As previously described in Section 4.4, Picart et al developed an alternate strategy for tuning film mechanical properties using a simple water-based chemical cross-linking method applicable to PEM films that contain amine and carboxylic groups. For PLL/HA films, fine-tuning the EDC cross-linker concentration in contact with the films made it possible to vary the film's stiffness over two orders of magnitude.…”

Polyelectrolyte Multilayers in Tissue Engineering

“…PAA/ PAH multilayers have been used to culture hepatocytes and these substrates appear to elicit increased urea and albumin production in comparison to hepatocytes cultured on a collagen surface. 102 Similarly, Wittmer et al have demonstrated that both PLL/ALG and PLL/PLGA PEMs facilitate adult rat hepatocyte adhesion and increased albumin production when compared to cells cultured on a collagen surface. 103 Further, in both reports, the adsorption of collagen on the terminal PE layer enhanced hepatocyte adhesion and phenotypic functions.…”

“…103 Further, in both reports, the adsorption of collagen on the terminal PE layer enhanced hepatocyte adhesion and phenotypic functions. 102,103 Hepatocytes are generally stable when cultured between two collagen gels (collagen sandwich). 104,105 When these cells are cocultured with nonparenchymal hepatic cells, their function can be further enhanced.…”

Three dimensional cultures of rat liver cells using a natural self‐assembling nanoscaffold in a clinically relevant bioreactor for bioartificial liver construction

“…Many biomaterials have been developed for liver cell culture (Janorkar et al, 2008;Khetani and Bhatia, 2008;Chen et al, 2009;Pavlica et al, 2009;Feng et al, 2010;Kim et al, 2010;Mehta et al, 2010), but these too are limited by short term culture (typically 5-15 days) so that cell culture also is still a long ways from clinical application for BAL purposes. A number of nanofabrication scaffolds have been used for liver cell culture (Feng et al, 2010), but the solvents used for nanofabrication can be a toxic concern.…”