Summary
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-di-acetate 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 appli-cated 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.
