Establishment of an efficient and economical method for primary oligodendrocyte progenitor cell culture from neonatal mouse brain

The primary culture of oligodendrocyte progenitor cells (OPCs) provides an indispensable tool for characterizing their biological properties and myelin repair potential. However, the current OPC preparation methods are mainly limited to rat tissues, and it remains a substantial challenge for replicating the primary culture from mouse tissues to generate large quantities of high-quality OPCs. Here, we describe a protocol to successfully establish highly enriched OPC cultures from the cerebral cortex of mice at the age of neonatal 3 days. OPCs were isolated and purified from the bed layer of astrocytes by shaking for 6 h at 250 rpm. Using this protocol, mouse OPCs can be easily produced in bulk and economically without the need for specific cell-surface antibodies and equipment. These mouse OPC cultures were identified by immunocytochemical, immunobloting and RNA-seq analysis. Furthermore, they could be expanded in vitro and differentiate into mature oligodendrocytes. We propose this method as a viable and affordable protocol to obtain mouse OPC culture, which should significantly facilitate studies on OPC lineage progression and their application in myelin-related disease modeling and regenerative medicine.