A new, automated and accurate in vitro method to quantify endothelial cells attached to vascular prostheses

Over a decade ago the idea of endothelial cell seeding was introduced in an attempt to improve the function of small caliber vascular prostheses. Although endothelial cell seeding is currently being applied clinically, several questions regarding the functional properties of the seeded endothelial cells remain. Evaluation of functional properties of endothelial cells on various types of vascular prostheses can be performed partly in vitro, but it is hampered by the fact that commonly used methods to quantify endothelial cells do not adequately apply to these cells on prosthetic materials. An accurate quantification method is described that is rapidly and easily applicable to endothelial cells attached to vascular prostheses. The method can also be used to quantify endothelial cells attached to culture dishes or microcarriers. Colorless, non-fluorescing, fluorescein-diacetate was used, which was taken up by the attached endothelial cells, and which was then intracellularly converted to yellow fluorescein, emitting green fluorescence. Subsequently, triton-X-100 was applicated to release fluorescein and levels of fluorescence were measured with the automated aperture-defined microvolume (ADM) method, using an inverted fluorescence microscope to which a photometer was connected. The measured level of fluorescence is linearly related to endothelial cell numbers attached to prostheses. The accuracy and the reproducibility of cell countings are high.