Co-Culture of Lined Dorsal Root Ganglion Neurons ND7/23 and Schwann Cells IFRS1 as a Valuable Tool for Studying Myelination and Demyelination

Background/Objectives: Co-culture models of neurons and Schwann cells have been used to explore the mechanisms of myelination during development, axonal regeneration after injury, and the pathogenesis of various demyelinating neuropathies. A spontaneously immortalized Schwann cell line, IFRS1, established from the primary culture of adult Fischer rat peripheral nerves, can myelinate neurites in co-cultures with primary cultured dorsal root ganglion (DRG) neurons and neuronal cell lines, such as nerve growth factor (NGF)-primed PC12 cells and NSC-34 motor neuron-like cells. In this study, we aimed to establish a stable co-culture system using IFRS1 cells and the DRG neuron-like cell line ND7/23. Methods: ND7/23 cells were seeded at a low density (2 103/cm2) and maintained for 7 days in serum-containing medium supplemented with NGF (10 ng/mL) and the Rho kinase inhibitor Y27632 (5 μM) to promote neurite elongation. The cells were then treated with the anti-mitotic agent mitomycin C (1 μg/mL) for 12-16 h to suppress the proliferative activity. Following this, the cells were co-cultured with IFRS1 cells (2 x 104/cm2) and maintained in serum-containing medium supplemented with ascorbic acid (50 g/mL), NGF (10 ng/mL), and ciliary neurotrophic factor (10 ng/mL). Results: Double-immunofluorescence staining performed on day 21 of the co-culture revealed myelin protein 22- or myelin basic protein-immunoreactive IFRS1 cells surrounding βIII tubulin-immunoreactive neurites emerging from ND7/23 cells. Myelin formation was further confirmed by Sudan black B staining and electron microscopy. Conclusions: This co-culture system provides a valuable tool for studying the processes of myelination and demyelination in the peripheral nervous system, as well as the pathogenesis of various sensory neuropathies and potential novel therapeutic approaches for these conditions.