Hydrogels with differential and patterned mechanics to study stiffness-mediated myofibroblastic differentiation of hepatic stellate cells

The differentiation of hepatic stellate cells (HSCs) into myofibroblasts is a key event in liver fibrosis. Due to the local stiffening of the extracellular matrix (ECM) during fibrosis, it is of great interest to develop mimics that can be used to investigate the cellular response to changes in mechanics. Here, we used a step-wise hydrogel crosslinking technique, where macromolecules are crosslinked using a sequence of addition then UV light-mediated radical crosslinldng, to generate hydrogels with tunable stiffness. Freshly isolated HSCs remained rounded with lipid droplets and high levels of PPAR gamma expression on soft substrates (E similar to 2 kPa); however, HSCs spread, lost their lipid droplets, and expressed high levels of a-smooth muscle actin (alpha-SMA) and type I collagen on stiff substrates (E similar to 24 kPa). Similarly, fully differentiated cells reverted to a quiescent state when plated on soft substrates. Stiffness-induced differentiation of HSCs was enhanced in the presence of exogenous TGF-beta 1, a dominant signal in fibrosis. When the UV-induced secondary crosslinking was restricted with a photomask to spatially control mechanics, HSCs responded based on the local hydrogel stiffness, although they remained quiescent on stiff substrates if the stiff feature size was not sufficient to allow cell spreading. This hydrogel system permits the investigation of HSC response to materials with diverse levels and spatially heterogeneous mechanical properties. (C) 2013 Elsevier Ltd. All rights reserved.