Primary culture of capillary endothelial cells from the spiral ligament and stria vascularis of bovine inner ear

Abstract

Methods for isolation and culture of microvascular endothelial cells of the inner ear were devised to provide an in-vitro system for studying endothelial functions in this tissue. Capillaries from the stria vascularis and spiral ligament were treated enzymatically to free them from surrounding tissue. Contamination by extraneous tissue was minimized by banding capillary segments in Percoll gradients and culture in plasma-derived serum on a fibronectin-coated substrate. Although only small amounts of inner ear tissue were available, tritiated thymidine autoradiography demonstrated that considerable growth in culture was possible. Addition of heparin and endothelial cell growth supplement to the medium enhanced proliferation. The endothelial origin of the cultured cells was confirmed by immunofluorescent demonstration of the presence of Factor VIII-related antigen and angiotensin-converting enzyme. In addition, tight junctions between cells were observed in both thin sections and platinum replicas obtained by freeze-fracture techniques. Endothelial cells from neither the stria vascularis nor the spiral ligament allowed passage of horseradish peroxidase across the monolayer during a 5-min period. However, endothelial cells from the stria vascularis exhibited a greater amount of pinocytotic activity than those of the spiral ligament, a difference that is also observed in vivo. Methods for expanding a small population of endothelial cells with retention of specialized properties into one of sufficient size for morphologic and biochemical studies have been demonstrated for the inner ear.

Functional interaction between mesenchymal stem cells and spiral ligament fibrocytes

“…Inner ear cell culture has been successfully reported in epithelial (Rarey and Patterson,1989; Achouche et al,1991; Melichar and Gitter,1992) and endothelial (Bowman et al,1985) cells. Recently, cell culture of SLFs has also been established and characterized for type I SLFs in gerbils (Gratton et al,1996), mice (Suko et al,2000), and rats (Yian et al,2006).…”