Cryopreservation of Bulk-Produced Primary Rat Oligodendrocyte Progenitor Cells

Primary oligodendrocyte cultures are a crucial driving force for in vitro research on oligodendrocytes (OLs) and myelin. Various methods are available to obtain oligodendrocyte lineage cells, primarily from neonatal rodent brains or human induced pluripotent stem cells (iPSCs). In this protocol, we describe a step-by-step procedure for detaching and cryopreserving primary rat oligodendrocyte progenitor cells (OPCs), followed by the thawing, proliferation, and differentiation of the cryopreserved OPCs. After freezing in a serum-free cryopreservation medium, the OPCs can be preserved at -80 °C for up to two months without notable changes in viability, proliferation, or differentiation into mature OLs. Cryopreserved OPCs can be differentiated into mature OLs with robust myelin processes and the capacity to wrap around neuron-mimicking structures. Combined with the author's method for primary OL culture, which allows for bulk production of OPCs, OPC cryopreservation may substantially improve the efficiency of in vitro OL research. Key features • This protocol recommends the use of a specific culture method that enables the simple, bulk production of primary rat OPCs. • Through this protocol, researchers may obtain large numbers of cryopreserved OPCs, which can be reserved for up to two months. • This protocol facilitates the planning of in vitro experiments and reduces the effort required to maintain adequate numbers of primary OPCs for large-scale experiments.