TY  - JOUR
TI  - Isolation of In Vitro Osteoblastic-Derived Matrix Vesicles by Ultracentrifugation and Cell-Free Mineralization Assay
AU  - Sheikh, Irshad A.
AU  - Kiela, Pawel R.
AU  - Ghishan, Fayez K.
VL  - 15
IS  - 7
PY  - 2025
DA  - 2025/04/05
SP  - e5258
C1  - Bio-protocol 2025;15:e5258
DO  - 10.21769/BioProtoc.5258
UR  - https://doi.org/10.21769/BioProtoc.5258
AB  - 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 Pi and Ca2+ 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.
KW  - Extracellular vesicles
KW  - Matrix vesicles
KW  - MC3T3-E1 subclone 4 preosteoblast cell line
KW  - Ultracentrifugation
KW  - Mineralization
KW  - Nanoparticle tracking analysis
KW  - Energy dispersive spectroscopy (EDS) analysis in scanning electron microscopy (SEM)
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False