Establishment of a simple one-step method for oligodendrocyte progenitor cell preparation from rodent brains.

Oligodendrocytes, which form myelin, enable rapid and efficient nerve conduction. Destruction of myelin causes demyelinating diseases such as multiple sclerosis. Primary oligodendrocyte progenitor cells (OPCs) from postnatal rodents have been utilized to elucidate the developmental mechanism of oligodendrocytes in vitro. However, this process is complicated and takes up to several weeks. We established a method to culture OPCs from neonatal rat brain in DMEM/F-12 with Stem-Pro, bFGF (10 ng/mL), and rhPDGF (30 ng/mL). The culture, without shaking or immunopanning, became OPC-enriched rather than a mixed glial culture. Immunofluorescent analysis using cell lineage markers suggested that these cells were initially glial progenitors, which gradually changed to OPCs with a few cells further differentiating into oligodendrocytes. Using compounds that promote OPC differentiation, we confirmed that these cells were compatible for high-throughput screening in a 96-well plate format. In co-culture with dorsal root ganglion neuron, OPCs showed myelin sheath-like morphologies. This method was also applicable to mouse OPCs. Although the purity of the OPCs was not comparable to that after immunopanning, most cells were of the oligodendrocyte lineage at 8 DIV, while less than 10% were astrocytes. This method requires mediums with only two growth factors without any specific equipment like antibodies or magnet and takes simple procedures. The simplicity and high yield of our method make it a good choice when working with oligodendrocytes/OPCs. We believe that this method is an affordable protocol for various biological applications without any special techniques or equipment.

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