Specific determination of endothelial cell viability in the whole cell fraction from cryopreserved canine femoral veins using flow-cytometry.

Abstract

An efficient method for specifically determining the viability of endothelial cells (EC) from cells dissociated from the human saphenous vein was investigated. Three different methods, trypan blue staining assay, [3H]-proline incorporation assay, and flow cytometry (FCM), combined with the fluorescein isothiocyanate conjugated with Griffonia simplicifolia agglutins (GS1-FITC)/propidium iodide (PI) double staining, were used. Both trypan blue staining and [3H] proline incorporation assays demonstrated less sensitivity to determine viability of EC differentially from the other cells. FITC-GS1 showed prominent binding to the vascular EC and could be counted by FCM including PI on dead cells. Following the cryopreservation process, the GS1-FITC/PI FCM analytical method was adopted to test simultaneously the viability of whole cells and EC from the same tissue, human saphenous veins, and mongrel dogs' femoral veins after harvesting, antibiotic solution treatment, and thawing. The viability of the whole cells from veins decreased with a significant difference (p < 0.05) from that of EC after thawing.