Open AccessA high‐yield isolation and enrichment strategy for human lung microvascular endothelial cells
Author(s) -
Gaskill Christa,
Majka Susan M.
Publication year - 2017
Publication title -
pulmonary circulation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.791
H-Index - 40
ISSN - 2045-8940
DOI - 10.1177/2045893217702346
Subject(s) - medicine , mesenchymal stem cell , lung , pathology , mesenchyme , angiogenesis , phenotype , fibrosis , endothelial stem cell , cancer research , immunology , in vitro , biology , biochemistry , gene
Vasculopathies, characterized by the formation of fragile and abnormal microvessels, are associated with the severity of many chronic lung diseases, including pulmonary fibrosis, emphysema/chronic obstructive pulmonary disease, systemic sclerosis, and hypertension. However, the study of human lung vasculature has been limited by the ability to isolate generous quantities of microvascular endothelial cells (MVEC) free from mesenchymal contamination. Expansion and passaging of primary human MVEC in vitro typically results in loss of a traditional phenotype in favor of an intermediate mesenchymal one, as early as passage five. Here we provide a detailed protocol for the selection of large quantities of enriched primary human lung MVEC based upon differential adherence from mesenchyme and simple magnetic separation, which decreases the need for excessive passaging, in order to obtain sufficient cell numbers to successfully freeze stock cultures. Additional protocols are provided for Ac‐di‐LDL selection, characterization, and a sandwich angiogenesis method of functional tube formation. The complete protocol including cell isolation and characterization takes approximately six weeks to complete.