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.

引言：体外培养脑血管平滑肌细胞（cerebrovascular smooth muscle cells, CVSMCs）可为研究多种脑血管疾病提供实验模型。本研究介绍了一种通过酶解法获取小鼠脑血管平滑肌细胞的便捷高效方法。
方法：分离小鼠大脑动脉环（circle of Willis），经酶解后使用血小板衍生生长因子-BB（platelet-derived growth factor-BB, PDGF-BB）培养以促进CVSMCs增殖，并通过形态学观察、免疫荧光分析及流式细胞术对CVSMCs进行鉴定。本研究采用细胞计数试剂盒-8（cell counting kit-8, CCK-8）检测法、形态学观察、蛋白质印迹法及流式细胞术，评估PDGF-BB对血管平滑肌细胞（vascular smooth muscle cell, VSMC）增殖的影响。
结果：在不含PDGF-BB的培养基中培养的CVSMCs，在约14天后呈现典型的峰谷状生长模式。免疫荧光染色及流式细胞术检测显示，细胞特异性标志物α-平滑肌肌动蛋白（alpha-smooth muscle actin, α-SMA）、平滑肌肌球蛋白重链11（smooth muscle myosin heavy chain 11, SMMHC）、平滑肌蛋白22（smooth muscle protein 22, SM22）、钙蛋白（calponin）及结蛋白（desmin）均呈强阳性表达。在CCK-8检测及蛋白质印迹实验中，与未添加PDGF-BB的细胞相比，经PDGF-BB孵育的细胞增殖能力显著增强。
结论：本研究通过简便方法从小鼠大脑动脉环中获取了高纯度的VSMCs，为体外研究神经血管疾病的发病机制及治疗方案提供了实验材料。此外，添加PDGF-BB可提升实验效率，缩短细胞培养周期。