title: Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology.
abstract: 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 4600Pa in both the presence and absence of liver ECM. Cell attachment, viability, and organization of the actin cytoskeleton improved with increased stiffness up to 4600Pa. 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 4600Pa 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 1200Pa stiffness, slightly below the stiffness of normal liver ECM at 3000Pa. 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.
authors.affiliation: Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, United States; Molecular Medicine and Translational Sciences, Wake Forest School of Medicine, Winston-Salem, NC, United States. Electronic address: ddeegan@wakehealth.edu.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Deegan
authors.affiliation: Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, United States.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Zimmerman
authors.affiliation: Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, United States.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Skardal
authors.affiliation: Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, United States.
authors.forename: Anthony
authors.initials: A
authors.lastname: Atala
authors.affiliation: Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, United States.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Shupe
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D017690:Cell Transplantation; D003593:Cytoplasm / Q000187:drug effects / Q000201:enzymology; D005109:Extracellular Matrix / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006025:Glycosaminoglycans / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry* / Q000494:pharmacology*; D020100:Hydrogels / Q000737:chemistry; D008099:Liver / Q000166:cytology*; D055595:Mechanical Phenomena; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D012995:Solubility; D023822:Tissue Engineering; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D001672:Biocompatible Materials; D015415:Biomarkers; D006025:Glycosaminoglycans; D020100:Hydrogels; D006820:Hyaluronic Acid; D011494:Protein Kinases; D020741:rho GTP-Binding Proteins
keywords: Decellularization; Hepatocytes; Hyaluronic acid; Hydrogels; Liver extracellular matrix; Stiffness