Phenotypic characterization of isolated valvular interstitial cell subpopulations.
BACKGROUND AND AIM OF THE STUDY: Valvular interstitial cells (VICs) demonstrate a heterogeneous range of phenotypes such as variable expression of smooth muscle alpha-actin (SMalphaA). Myofibroblast-like VICs, expressing high levels of SMalphaA, are thought to be involved in myxomatous degeneration of mitral valves. The inability to isolate specific cell types has restricted potential investigations of valvular disease mechanisms. Thus, investigations were conducted into methods of isolating different cell subpopulations from primary VICs as a preparatory step for cell type-specific evaluations of heart valve disease. METHODS: VICs were isolated from porcine valves, cultured to 80% confluency, and subdivided using differential detachment or adhesion. The subdivided cells were further cultured and analyzed phenotypically by immunocytochemistry and flow cytometry to characterize SMalphaA expression. Roundness and growth rates were also analyzed. RESULTS: VICs that were relatively sensitive to trypsinization expressed low and heterogeneous levels of SMalphaA (15-35%), whereas more-adherent VICs expressed higher and homogeneous levels (>98%) suggestive of a myofibroblast-like phenotype. The more-adherent cells also had lower growth potential and were less round than less-adhesive VICs. Separated cell subtypes were found to maintain their phenotype through several cell passages. CONCLUSION: VICs are a mixed population of cells, many of which express high levels of SMalphaA. Differential detachment and adhesion can effectively separate cell subpopulations from primary cultures of VICs. The ability to study valve cell subpopulations has substantial implications for future analyses of valvular biology, disease, and tissue engineering.