Correlation between valvular interstitial cell morphology and phenotypes: A novel way to detect activation

Valvular interstitial cells (VICs) constitute the major cell population in heart valves. Quiescent fibroblastic VICs are seen in adult healthy valves. They become activated myofibroblastic VICs during development, in diseased valves and in vitro. 2D substrate stiffness within a 5-15 kPa range along with high passage numbers promote VIC activation in vitro. In this study, we characterize VIC quiescence and activation across a 1-21 kPa range of substrate stiffness and passages. We define a cell morphology characterization system for VICs as they transform. We hypothesize that VICs show distinct morphological characteristics in different activation states and the morphology distribution varies with substrate stiffness and passage number. Four VIC morphologies - tailed, spindle, rhomboid and triangle - account for the majority of VIC in this study. Using α-smooth muscle actin (α-SMA), non-muscle myosin heavy chain B (SMemb) and transforming growth factor β (TGF-β) as activation markers for validation, we developed a system where we categorize morphology distribution of VIC cultures, to be potentially used as a non-destructive detection method of activation state. We also show that this system can be used to force stiffness-induced deactivation. The reversibility in VIC activation has important implications in in vitro research and tissue engineering.