Isolation and long-term expansion of functional, myelinating oligodendrocyte progenitor cells from neonatal rat brain

Oligodendrocytes are the myelinating cells of the central nervous system (CNS). The isolation of purified oligodendrocyte progenitor cells (OPCs) in large numbers has been sought after as a source of cells for repair following CNS-demyelinating diseases and injuries, such as multiple sclerosis (MS) and spinal cord injury (SCI). Methods for isolation of OPCs from rodent neonatal brains are well established and have formed the basis for research in myelin repair within the CNS for many years. However, long-term maintenance of OPCs has been a challenge owing to small cellular yields per animal and spontaneous differentiation within a short period of time. Much effort has been devoted to achieving long-term culture and maintenance of OPCs, but little progress has been made. Here, protocols are presented for preparation of highly enriched rat OPC populations and for their long-term maintenance as oligospheres using mixed-glial-conditioned medium. Functional myelinating oligodendrocytes can be achieved from such protocols, when co-cultured with primary neurons. This approach is an extension of our normal shaking method for isolating OPCs, and incorporates some adaptations from previous OPC culture methods.