A GENERAL STRATEGY FOR ISOLATION OF ENDOTHELIAL-CELLS FROM MURINE TISSUES - CHARACTERIZATION OF 2 ENDOTHELIAL-CELL LINES FROM THE MURINE LUNG AND SUBCUTANEOUS SPONGE IMPLANTS

A rapid, reproducible method for the isolation of murine endothelial c ells (ECs) has been developed. Murine ECs were highly enriched by coll agenase digestion of mechanically minced lung and subcutaneous sponge implants followed by specific selection with rat anti-mouse CD31 (ie, PECAM-1) monoclonal antibody-coated magnetic beads (Dynabeads). Pure E C populations were isolated from primary cultures by a second cycle of immunomagnetic selection. The cells from the lung were then cloned by a limiting-dilution method to exclude the possibility of nonendotheli al cell contamination. Of the 300 cells plated, 29 clones (approximate to 10%) were obtained. The clones were positive for CD31 as measured by flow cytometry, and one clone from the lungs (1G11) and the cells f rom sponge implants (designated as SIECs) were then subjected to subse quent culture in vitro for 40 and 30 passages (up to 5 months), respec tively. Characterization was performed on cells between passage 3 and 10. Both cell types formed contact-inhibited monolayers on gelatin and capillary-like ''tubes'' on Matrigel. However, 1G11 cells exhibited a ''cobblestone'' morphology, whereas SIECs had a fibroblast-like appea rance at confluence. By flow cytometry and enzyme-linked immunosorbent assay, these cells constitutively expressed CD31, VE-cadherin (cadher in-5), CD34, ICAM-1, VCAM-1, and P-selectin. After stimulation with 30 ng/mL of tumor necrosis factor-alpha, the cells became positive for E -selectin (at 4 hours poststimulation) and the expression of ICAM-1, V CAM-1, and P-selectin was upregulated (after 24 hours of stimulation). The presence of VE-cadherin in 1G11 cells and SIECs was confirmed by fluorescence microscopy and Northern blot analysis. The phenotype and morphology of both cell types were stable during 5 months of culture, and there was no evidence of overgrowth by contaminating cells. Taken together, the approach outlined herein may provide a general strategy for the isolation and culture of ECs from a variety of murine tissues. The general strategy outlined here is simple, effective, and flexible , allowing the inclusion of further positive or negative selection ste ps.