Integrated culture and purification of rat Schwann cells from freshly isolated adult tissue
Integrated culture and purification of rat Schwann cells from freshly isolated adult tissue
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DOI：
10.1038/nprot.2012.118
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发表时间：
2012-11-01
期刊：
影响因子：
14.8
通讯作者：
Haycock, John W.
中科院分区：
文献类型：
--
作者：
Kaewkhaw, Rossukon;Scutt, Andy M.;Haycock, John W.
我们描述了一种简单，快速和高选择性的协议，原代培养雪旺细胞在体外新鲜分离的成年大鼠神经。该方案是基于包含有丝分裂原（毛喉素和任选的N2补充剂加上牛垂体提取物）的选择性培养基，以刺激许旺细胞的生长，加上抑制性底物以同时限制成纤维细胞过度生长（D-缬氨酸），其包含在DMEM中。该方案与其他可用方法的不同之处在于，它使用了施万细胞代谢D-缬氨酸的优先能力，因为施万细胞和成纤维细胞之间D-氨基酸氧化酶（DAAO）酶的表达存在差异，加上存在选择性促分裂原以刺激施万细胞的生长。这允许直接从新鲜的成人神经中获得高纯度的许旺细胞。在19天后可以达到97%的平均许旺细胞纯度，而无需预变性、纯化或抗有丝分裂步骤。
We describe a simple, rapid and highly selective protocol for the primary culture of Schwann cells in vitro from freshly dissociated adult rat nerve. The protocol is based on a selective culture medium comprising both mitogens (forskolin and optionally N2 supplement plus bovine pituitary extract), to stimulate growth of Schwann cells, plus an inhibitory substrate to simultaneously restrict fibroblast overgrowth (D-valine), contained in DMEM. This protocol differs from other available methods in that it uses the preferential capacity of Schwann cells to metabolize D-valine because of the difference in expression of a D-amino acid oxidase (DAAO) enzyme between Schwann cells and fibroblasts plus the presence of a selective mitogen to stimulate growth of Schwann cells. This permits derivation of highly pure Schwann cells directly from fresh adult nerve. Average Schwann cell purities of 97% can be achieved after 19 d without pre-degeneration, purification or antimitotic steps.