Robust Generation of Quiescent Porcine Valvular Interstitial Cell Cultures

Abstract

Background Valvular interstitial cells ( VIC s) in the healthy aortic valve leaflet exhibit a quiescent phenotype, with <5% of VIC s exhibiting an activated phenotype. Yet, in vitro culture of VIC s on tissue culture polystyrene surfaces in standard growth medium results in rapid transformation to an activated phenotype in >90% of cells. The inability to preserve a healthy VIC phenotype during in vitro studies has hampered the elucidation of mechanisms involved in calcific aortic valve disease. This study describes the generation of quiescent populations of porcine VIC s in 2‐dimensional in vitro culture and their utility in studying valve pathobiology. Methods and Results Within 4 days of isolation from fresh porcine hearts, VIC s cultured in standard growth conditions were predominantly myofibroblastic (activated VIC s). This myofibroblastic phenotype was partially reversed within 4 days, and fully reversed within 9 days, following application of a combination of a fibroblast media formulation with culture on collagen coatings. Specifically, culture in this combination significantly reduced several markers of VIC activation, including proliferation, apoptosis, α‐smooth muscle actin expression, and matrix production, relative to standard growth conditions. Moreover, VIC s raised in a fibroblast media formulation with culture on collagen coatings exhibited dramatically increased sensitivity to treatment with transforming growth factor β1, a known pathological stimulus, compared with VIC s raised in either standard culture or medium with a fibroblast media formulation. Conclusions The approach using a fibroblast media formulation with culture on collagen coatings generates quiescent VIC s that more accurately mimic a healthy VIC population and thus has the potential to transform the study of the mechanisms of VIC activation and dysfunction involved in the early stages of calcific aortic valve disease.