Fluorescent activated cell sorting to isolate canine microvascular endothelial cells from adipose tissue

Abstract

Adipose tissue offers an abundant source for isolation of microvascular endothelial cells (MVECs). Several cell types result from the enzymatic digestion of adipose tissue, including MVECs, mesothelial cells and fibroblasts. Pure populations of MVECs must be isolated from the mixed cultures or fibroblasts overgrow the population. Canine subcutaneous or mesenteric fat is obtained during elective surgical procedures and digested with collagenase. Microvascular endothelial cells are separated from capillary fragments by Percoll gradient centrifugation and plated in culture. At confluence, microscopic identification of most cultured cells indicate the typical cobblestone morphology of ECs, while other spindle shaped cells resemble fibroblasts. Micro- vascular endothelial cells are identified by their uptake of acetylated-low density lipoprotein (Ac-LDL). Mesothelial cells, which closely resemble MVECs in morphology, and fibroblasts are removed from cultures of MVECs using a fluorescent activated cell sorter (FACS). Acetylated-low density lipoprotein tagged with a fluorescent probe, 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl- indocarbocyanine perchlorate (DiI), is incubated with the mixed cultures and the cells are sorted using a FACS. The pure populations of MVECs that result are tested immunocytochemically and are identified by their positive staining with antibodies against factor VIII related antigen. Mesothelial cells are identified by positive staining for cytokeratin 18 antibody. Contaminating fibroblasts show negative staining for smooth muscle alpha actin, cytokeratin 18 and factor VIII related antigen antibodies. This study examines the efficiency of the fluorescent activated cell sorter to obtain pure populations of MVECs harvested from adipose tissue.