OPTIMIZATION OF USE OF UEA-1 MAGNETIC BEADS FOR ENDOTHELIAL-CELL ISOLATION

Most endothelial cells (EC) in the body belong to the microvasculature . Isolation and subsequent culture of these microvessel EC contributes greatly to our understanding of the heterogeneity and vascular specif icity that exist between one organ site and another. However, a major obstacle is the overgrowth of contaminating cells (fibroblasts, pericy tes, smooth-muscle cells) in cultures. Since 1990 the use of magnetic beads in combination with either a lectin, Ulex europaeus agglutinin-1 (UEA-1) or a monoclonal antibody has represented a powerful tool for the isolation/purification of microvessel EC. In the former case, oper ative conditions remain to be optimized to obtain pure cultures of EC. We have performed studies to optimize conditions of use for magnetic beads coated with UEA-1. Incubating beads with cells, the influences a re studied of time, temperature, cell concentration, and number of bea ds per target cell for two cell types, human umbilical vein EC (HUVEC) and skin fibroblasts HSF) either isolated or mixed. The effect of the last parameter was also checked on the behavior of cells undergoing p roliferation after isolation. Results, expressed as isolation efficien cy (from 40% to 90%) allowed us to select a 15-min incubation time at 4 degrees C with rotary agitation, an optimal concentration of 4 x 10( 5) cells/ml, and an optimal cell:bead ratio of 1:3. From a mixed cell population and in these conditions, even very low HUVEC:HSF proportion s of 2.5:97.5 allowed us to obtain a pure HUVEC population in subseque nt culture.