Takaku Shizuka, Sango Kazunori
Diabetic Neuropathy Project, Department of Diseases and Infection, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
Neurol Int. 2025 Sep 1;17(9):138. doi: 10.3390/neurolint17090138.
: 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 Fischer rat Schwann cell line 1 (IFRS1), established from the primary culture of adult Fischer344 rat peripheral nerves, can myelinate neurites in co-cultures with primary cultured dorsal root ganglion 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 ND7/23 sensory neuron-like cells. : ND7/23 cells were seeded at a low density (2 × 10/cm) 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 proliferative activity. Following this, the cells were co-cultured with IFRS1 cells (2 × 10/cm) and maintained at 37 °C in serum-containing medium supplemented with ascorbic acid (50 μg/mL), NGF (10 ng/mL), and ciliary neurotrophic factor (10 ng/mL). : 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 via Sudan Black B staining and electron microscopy. : This co-culture system may provide a valuable tool for studying the processes of myelination in the peripheral nervous system, as well as the pathogenesis of various sensory neuropathies and potential novel therapeutic approaches for these conditions.
神经元与雪旺细胞的共培养模型已被用于探索发育过程中的髓鞘形成机制、损伤后的轴突再生以及各种脱髓鞘性神经病的发病机制。从成年Fischer344大鼠外周神经原代培养物中建立的自发永生化Fischer大鼠雪旺细胞系1（IFRS1），可在与原代培养的背根神经节神经元和神经元细胞系（如神经生长因子（NGF）预处理的PC12细胞和NSC-34运动神经元样细胞）的共培养中使神经突形成髓鞘。在本研究中，我们旨在使用IFRS1细胞和ND7/23感觉神经元样细胞建立一个稳定的共培养系统。：将ND7/23细胞以低密度（2×10/cm）接种，并在补充有NGF（10 ng/mL）和Rho激酶抑制剂Y27632（5 μM）的含血清培养基中培养7天，以促进神经突伸长。然后用抗有丝分裂剂丝裂霉素C（1 μg/mL）处理细胞12 - 16小时，以抑制增殖活性。此后，将细胞与IFRS1细胞（2×10/cm）共培养，并在补充有抗坏血酸（50 μg/mL）、NGF（10 ng/mL）和睫状神经营养因子（10 ng/mL）的含血清培养基中于37°C下培养。：在共培养第21天进行的双重免疫荧光染色显示，髓磷脂蛋白22或髓磷脂碱性蛋白免疫反应性IFRS1细胞围绕着从ND7/23细胞发出的βIII微管蛋白免疫反应性神经突。通过苏丹黑B染色和电子显微镜进一步证实了髓鞘形成。：这种共培养系统可能为研究外周神经系统中的髓鞘形成过程、各种感觉神经病的发病机制以及这些病症的潜在新治疗方法提供一个有价值的工具。
