Optimized method for isolating highly purified and functional porcine aortic endothelial and smooth muscle cells
| Autor: | Marco A. Morales-Garza, Caitlin Winebrenner, Doris A. Taylor, Mitalben Patel, Eric Chau, Farideh Beigi, Luiz C. Sampaio, Andrea S. Gobin |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
CD31 Time Factors Physiology Clinical Biochemistry Cell Myocytes Smooth Muscle Sus scrofa Neovascularization Physiologic Cell Separation 030204 cardiovascular system & hematology Calcium in biology Muscle Smooth Vascular 03 medical and health sciences 0302 clinical medicine Von Willebrand factor medicine.artery Dispase Endopeptidases medicine Doubling time Animals Collagenases Aorta Cell Proliferation biology Endothelin-1 Chemistry Endothelial Cells Biological Transport Cell Biology Anatomy Flow Cytometry Cell biology Lipoproteins LDL 030104 developmental biology medicine.anatomical_structure Phenotype Microscopy Fluorescence Collagenase biology.protein Calcium Biomarkers medicine.drug |
| Zdroj: | Journal of cellular physiology. 232(11) |
| ISSN: | 1097-4652 |
| Popis: | Numerous protocols exist for isolating aortic endothelial and smooth muscle cells from small animals. However, establishing a protocol for isolating pure cell populations from large animal vessels that are more elastic has been challenging. We developed a simple sequential enzymatic approach to isolate highly purified populations of porcine aortic endothelial and smooth muscle cells. The lumen of a porcine aorta was filled with 25 U/ml dispase solution and incubated at 37°C to dissociate the endothelial cells. The smooth muscle cells were isolated by mincing the tunica media of the treated aorta and incubating the pieces in 0.2% and then 0.1% collagenase type I solution. The isolated endothelial cells stained positive for von Willebrand factor, and 97.2% of them expressed CD31. Early and late passage endothelial cells had a population doubling time of 38 hr and maintained a capacity to take up DiI-Ac-LDL and form tubes in Matrigel®. The isolated smooth muscle cells stained highly positive for alpha-smooth muscle actin, and an impurities assessment showed that only 1.8% were endothelial cells. Population doubling time for the smooth muscle cells was ∼70 hr at passages 3 and 7; and the cells positively responded to endothelin-1, as shown by a 66% increase in the intracellular calcium level. This simple protocol allows for the isolation of highly pure populations of endothelial and smooth muscle cells from porcine aorta that can survive continued passage in culture without losing functionality or becoming overgrown by fibroblasts. |
| Databáze: | OpenAIRE |
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