Isolation of microvascular endothelial cells from tissues using counterflow elutriation

Abstract

The purpose of this study was to optimize the isolation of pure and viable microvascular endothelial cell (EC) populations from a variety of murine tissues. While several methods are reported in the literature, many of these are lengthy and result in low yields. Although counterflow elutriation has been used to isolate a variety of cell types, we are unaware of its use for the isolation of ECs from tissues other than from the liver. Therefore, we proposed to evaluate the conditions necessary for separation of ECs from various tissues. Previously described procedures for isolating liver sinusoidal ECs via counterflow elutriation were optimized using our equipment. Additionally, methods for isolating ECs from aortae and kidney were developed and optimized. All cells were isolated from mice overexpressing endothelial‐specific GFP (Tg(TIE2GFP)287Sato/J). Flow cytometry was used to determine purity of isolated EC populations using staining of GFP, CD31 and DiI(3,3′‐dioctadecylindocarbocyanine)‐labeled acetylated LDL. The methods described herein result in EC populations of at least 98% purity. These cells have been successfully used for downstream applications, including molecular biological assays and cell culture. Counterflow elutriation can be employed to obtain high‐yield, high‐purity EC populations from a variety of murine tissues.This work was funded, in part, by NHLBI T32 HL090610.