Department of Sensory and Motor Systems, ALS/Neuropathy Project (Laboratory of Peripheral Nerve Pathophysiology), Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506, Japan.
Histochem Cell Biol. 2012 Jun;137(6):829-39. doi: 10.1007/s00418-012-0934-3. Epub 2012 Feb 25.
Establishing stable coculture systems with neuronal and Schwann cell lines has been considered difficult, presumably because of their high proliferative activity and phenotypic differences from primary cultured cells. The present study is aimed at developing methods for myelin formation under coculture of the neural crest-derived pheochromocytoma cell line PC12 and the immortalized adult rat Schwann cell line IFRS1. Prior to coculture, PC12 cells were seeded at low density (3 × 10(2)/cm(2)) and maintained in serum-free medium with N2 supplement, ascorbic acid (50 μg/ml), and nerve growth factor (NGF) (50 ng/ml) for a week. Exposure to such a NGF-rich environment with minimum nutrients accelerated differentiation and neurite extension, but not proliferation, of PC12 cells. When IFRS1 cells were added to NGF-primed PC12 cells, the cell density ratio of PC12 cells to IFRS1 cells was adjusted from 1:50 to 1:100. The cocultured cells were then maintained in serum-free medium with B27 supplement, ascorbic acid (50 μg/ml), NGF (10 ng/ml), and recombinant soluble neuregulin-1 type III (25 ng/ml). Myelin formation was illustrated by light and electron microscopy performed at day 28 of coculture. The stable PC12-IFRS1 coculture system is free of technical and ethical problems arising from the primary culture and can be a valuable tool to study peripheral nerve degeneration and regeneration.
建立稳定的神经元和施万细胞系共培养体系一直被认为是困难的，这可能是因为它们具有较高的增殖活性，并且与原代培养细胞在表型上存在差异。本研究旨在开发神经嵴来源的嗜铬细胞瘤细胞系 PC12 和永生化成年大鼠施万细胞系 IFRS1 共培养条件下髓鞘形成的方法。在共培养之前，将 PC12 细胞以低细胞密度（3×102/cm2）接种，并在含 N2 补充物、抗坏血酸（50μg/ml）和神经生长因子（NGF）（50ng/ml）的无血清培养基中维持一周。在这种富含 NGF 且营养物质最少的环境中，PC12 细胞加速分化和轴突延伸，但不增殖。当 IFRS1 细胞被添加到 NGF 诱导的 PC12 细胞中时，将 PC12 细胞与 IFRS1 细胞的细胞密度比从 1:50 调整为 1:100。然后将共培养的细胞在含 B27 补充物、抗坏血酸（50μg/ml）、NGF（10ng/ml）和重组可溶性神经调节蛋白-1 型 III（25ng/ml）的无血清培养基中维持。在共培养 28 天时通过光镜和电镜观察到髓鞘形成。稳定的 PC12-IFRS1 共培养系统没有源于原代培养的技术和伦理问题，可以成为研究周围神经变性和再生的有价值的工具。
