A method to isolate human dermal microvascular pericytes without the use of magnetic beads sorting in vitro

Abstract

Human dermal microvascular pericytes (HDMPCs) are a critical component of the skin flap microvasculature and play a role in regulating flap blood flow and integrity. Pericytes were isolated mostly via magnetic bead sorting in the published literature. In this study, we discuss in detail how to separate and concentrate pericytes from human facial flaps using enzyme digestion and differential adherence instead of magnetic bead sorting. Cultured HDMPCs were seen to have well-spread irregular edges, with most cells having two longitudinal pericytic processes. The phalloidin staining revealed that HDMPCs had prominent stress fibers, and the nucleus deviated to the side that interacted with the neighboring pericytic processes. Flow cytometry analysis showed that the positive rates of NG2 in the first and second passages were 91.2% ± 0.7% and 98.2% ± 0.1% separately. And the immunofluorescence and western blot results demonstrated a positive expression of α smooth muscle actin (αSMA), platelet-derived growth factor receptor β (PDGFRβ), and NG-2, while the endothelial cell marker CD31 was negatively expressed. In summary, we established a straightforward methodology for selectively isolating and identifying HDMPCs as well as generating high-purity cell cultures in vitro without the use of magnetic bead sorting.