Nakayama Yoshiaki, Wada Ayumi, Inoue Rei, Terasawa Kazuya, Kimura Ikuo, Nakamura Naosuke, Kurosaka Akira
Laboratory of Neuroglycobiology, Department of Molecular Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto 603-8555, Japan.
Center for Genomics Medicine, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan.
J Neurosci Methods. 2014 Apr 30;227:100-6. doi: 10.1016/j.jneumeth.2014.02.011. Epub 2014 Feb 28.
P19 mouse embryonic carcinoma cells are conventionally induced to differentiate into neural cells by suspension culture in the presence of retinoic acid to form cell aggregates, followed by adhesion culture in a poly-l-lysine-coated dish. Drawbacks of this procedure include it taking more than 10 days to obtain mature neurons, and non-neuronal proliferating cells occupying the majority of the cell population with time.
Here, we show a novel method for the rapid and efficient neurogenesis of P19 cells, without aggregate formation in a suspension culture. The new approach is based on an adherent serum-free culture in a laminin-coated dish in the presence of FGF8, a γ-secretase inhibitor, and cytosine arabinoside.
The new method efficiently induced P19 cells to differentiate into neurons within 4 days, and subsequently into mature neurons that were responsive to several neurotransmitters, giving spontaneous neuronal network activity within 6 days.
The novel method accelerated neuritogenesis and enhanced population of neuron selectively compared to the conventional method. Proliferating non-neuronal cells were eliminated by adding cytosine arabinoside during neuronal maturation.
The method is useful for studying neuronal differentiation or activities.
传统上，P19小鼠胚胎癌细胞在视黄酸存在的情况下通过悬浮培养形成细胞聚集体，从而诱导其分化为神经细胞，随后在聚-L-赖氨酸包被的培养皿中进行贴壁培养。该方法的缺点包括获得成熟神经元需要超过10天的时间，并且随着时间的推移，非神经元增殖细胞占据了细胞群体的大部分。
在此，我们展示了一种P19细胞快速高效神经发生的新方法，在悬浮培养中不形成聚集体。新方法基于在层粘连蛋白包被的培养皿中，在FGF8、γ-分泌酶抑制剂和阿糖胞苷存在的情况下进行无血清贴壁培养。
新方法在4天内有效地诱导P19细胞分化为神经元，随后分化为对多种神经递质有反应的成熟神经元，在6天内产生自发的神经元网络活动。
与传统方法相比，新方法加速了神经突的形成，并选择性地增加了神经元的数量。在神经元成熟过程中添加阿糖胞苷可消除增殖的非神经元细胞。
该方法对于研究神经元分化或活动很有用。
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