Elucidating Extracellular Matrix and Stiffness Control of Primary Human Hepatocyte Phenotype via Cell Microarrays

How the liver's extracellular matrix (ECM) protein composition and stiffness cooperatively regulate primary human hepatocyte (PHH) phenotype is unelucidated. Here, protein microarrays and high‐content imaging with single‐cell resolution are utilized to assess PHH attachment/functions on 10 major liver ECM proteins in single‐ and two‐way combinations robotically spotted onto polyacrylamide gels of 1 or 25 kPa stiffness. Albumin, cytochrome‐P450 3A4 (CYP3A4), and hepatocyte nuclear factor alpha (HNF4α) positively correlate with each other and cell density on both stiffnesses. The 25 kPa stiffness supports higher average albumin and HNF4α expression after 14 d, while ECM protein composition significantly modulates PHH functions across both stiffnesses. Unlike previous rodent data, PHH functions are highest only when collagen‐IV or fibronectin are mixed with specific proteins, whereas noncollagenous proteins without mixed collagens downregulate functions. Combination of collagen‐IV and hyaluronic acid retains high CYP3A4 on 1 kPa, whereas collagens‐IV and ‐V better retain HNF4α on 25 kPa over 14 d. Adapting ECM conditions to 96‐well plates containing conjugated hydrogels reveals novel regulation of other functions (urea, CYP1A2/2A6/2C9) and drug‐mediated CYP induction by the ECM protein composition/stiffness. This high‐throughput pipeline can be adapted to elucidate ECM's role in liver diseases and facilitate optimization of engineered tissues. High‐throughput cell microarrays and high‐content imaging with single‐cell resolution elucidate synergistic and novel effects of extracellular matrix (ECM) protein composition and stiffness on diverse functions of primary human hepatocytes (PHHs), which is useful for drug screening, disease modeling, and regenerative medicine. Select ECM conditions adapted to multiwell plates improve PHH functions and drug responsiveness for 2 weeks in culture.