Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology

Tissue engineering and cell based liver therapies have utilized primary hepatocytes with limited success due to the failure of hepatocytes to maintain their phenotype in vitro. In order to overcome this challenge, hyaluronic acid (HA) cell culture substrates were formulated to closely mimic the composition and stiffness of the normal liver cellular microenvironment. The stiffness of the substrate was modulated by adjusting HA hydrogel crosslinking. Additionally, the repertoire of bioactive molecules within the HA substrate was bolstered by supplementation with normal liver extracellular matrix (ECM). Primary human hepatocyte viability and phenotype were determined over a narrow physiologically relevant range of substrate stiffnesses from 600 to 4600 Pa in both the presence and absence of liver ECM. Cell attachment, viability, and organization of the actin cytoskeleton improved with increased stiffness up to 4600 Pa. These differences were not evident in earlier time points or substrates containing only HA. However, gene expression for the hepatocyte markers hepatocyte nuclear factor 4 alpha (HNF4α) and albumin significantly decreased on the 4600 Pa stiffness at day 7 indicating that cells may not have maintained their phenotype long-term at this stiffness. Function, as measured by albumin secretion, varied with both stiffness and time in culture and peaked at day 7 at the 1200 Pa stiffness, slightly below the stiffness of normal liver ECM at 3000 Pa. Overall, gel stiffness affected primary human hepatocyte cell adhesion, functional marker expression, and morphological characteristics dependent on both the presence of liver ECM in gel substrates and time in culture.