Sheikh Irshad A, Kiela Pawel R, Ghishan Fayez K
Department of Pediatrics, University of Arizona, Tucson, AZ, USA.
Bio Protoc. 2025 Apr 5;15(7):e5258. doi: 10.21769/BioProtoc.5258.
Matrix vesicles (MVs) represent a heterogeneous group of spherical membrane-bound extracellular vesicles in the range of 100-200 nm in diameter secreted by mineralizing osteoblasts. The initial synthesis of the amorphous calcium phosphate occurs within the confines of the intracellular MVs, which are capable of transporting P and Ca into the MV lumen. Thus, understanding the initial process of MV-mediated mineralization is critical in developing better therapeutic strategies for various bone-related disorders such as osteoporosis and addressing ectopic calcification of soft tissues. Although various techniques and commercially available kits are now available for isolating MVs, isolating a pure population of MVs is challenging mainly because of their variable size and lack of consensus protein markers. This ultracentrifugation-based protocol ensures high purity of isolated MVs by removing other contaminated extracellular vesicles and cellular debris through sequential centrifugation steps but also allows downstream functional mineralization assays of the isolated MVs. Key features • Simple and rapid high-quality isolation of MVs from in vitro culture of mineralizing osteoblasts by ultracentrifugation. • Use of isolated MVs for various functional assays such as mineralization efficacy. • Cell-free mineralization assay to determine intrinsic mineralization efficacy of the isolated MVs under desired experimental conditions.
基质小泡（MVs）是由矿化成骨细胞分泌的直径在100 - 200纳米范围内的一组异质性球形膜结合细胞外小泡。无定形磷酸钙的初始合成发生在细胞内MVs的范围内，MVs能够将磷和钙转运到MV内腔。因此，了解MV介导的矿化初始过程对于开发针对骨质疏松症等各种骨相关疾病的更好治疗策略以及解决软组织异位钙化至关重要。尽管现在有各种技术和市售试剂盒可用于分离MVs，但分离出纯的MVs群体具有挑战性，主要是因为它们大小可变且缺乏一致的蛋白质标志物。这种基于超速离心的方案通过连续离心步骤去除其他受污染的细胞外小泡和细胞碎片，确保了分离出的MVs的高纯度，同时也允许对分离出的MVs进行下游功能性矿化测定。关键特性 • 通过超速离心从矿化成骨细胞的体外培养物中简单快速地高质量分离MVs。 • 将分离出的MVs用于各种功能测定，如矿化功效。 • 无细胞矿化测定，以确定在所需实验条件下分离出的MVs的内在矿化功效。
