Fluorescence activated cell sorting: A reliable method in tissue engineering of a bioprosthetic heart valve

Abstract

Background. Techniques of tissue engineering are used to seed human autologous cells in vitro on degradable mesh to create new functional tissue like a bioprosthetic heart valve. A precondition is subsequent seeding of native-valve–analogous pure endothelial and myofibroblast cell lines. The aim of this study is to find a safe method of isolating viable cell lines out of tissues from the operating room. Methods. Mixed cells from ascending aorta obtained from the operating room were incubated with an endothelial-specific fluorescent marker. The labeled cells were activated and sorted by flow cytometry. Isolated cell lines were cultured and thereafter square sheets of polymeric scaffold were seeded with myofibroblasts, followed by endothelial cells. The created tissue was stained with hematoxylin and eosin, van Gieson stain, and stains for factor VIII and CD34. Results. Control culture samples (n = 25) revealed vital uncontaminated endothelial and myofibroblast cell lines. Microscopy of the seeded meshes (n = 16) demonstrated a tissue-like structure. Van Gieson stain showed production of collagen. Endothelial cells formed a superficial monolayer, demonstrated by factor VIII and CD34; no invasive formation of capillaries was detectable. Conclusions. These results demonstrate that fluorescence activated cell sorting is a reliable and safe method to gain pure vital autologous cell lines out of human mixed cells for subsequent seeding on degradable mesh and that those cells are active to form new tissue.