Supplementary Material for: Isolation and Cultivation of Vascular Smooth Muscle Cells from the Mouse Circle of Willis

Introduction: Culturing cerebrovascular smooth muscle cells (CVSMCs) in vitro can provide a model for studying many cerebrovascular diseases. This study describes a convenient and efficient method to obtain mouse CVSMCs by enzyme digestion. Methods: Mouse circle of Willis was isolated, disgested, and cultured with platelet-derived growth factor-BB (PDGF-BB) to promote CVSMC growth, and CVSMCs were identified by morphology, immunofluorescence analysis, and flow cytometry. The effect of PDGF-BB on vascular smooth muscle cell (VSMC) proliferation was evaluated by cell counting kit (CCK)-8 assay, morphological observations, western blotting, and flow cytometry. Results: CVSMCs cultured in a PDGF-BB-free culture medium had a typical peak-to-valley growth pattern after approximately 14 days. Immunofluorescence staining and flow cytometry detected strong positive expression of the cell type-specific markers alpha-smooth muscle actin (α-SMA),mooth muscle myosin heavy chain 11(SMMHC), smooth muscle protein 22 (SM22), calponin, and desmin. In the CCK-8 assay and western blotting, cells incubated with PDGF-BB had significantly enhanced proliferation compared to those without PDGF-BB. Conclusion: We obtained highly purified VSMCs from the mouse circle of Willis using simple methods, providing experimental materials for studying the pathogenesis and treatment of neurovascular diseases in vitro. Moreover, the experimental efficiency improved with PDGF-BB, shortening the cell cultivation period.