Isolation, Culture, and Cryopreservation of Adult Rodent Schwann Cells Derived from Immediately Dissociated Teased Fibers
Isolation, Culture, and Cryopreservation of Adult Rodent Schwann Cells Derived from Immediately Dissociated Teased Fibers
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DOI：
10.1007/978-1-4939-7649-2_4
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发表时间：
2018-01-01
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影响因子：
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通讯作者：
Monje, Paula V.
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文献类型：
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作者：
Andersen, Natalia D.;Monje, Paula V.
成年许旺细胞（SC）培养物通常来源于经过漫长预变性步骤的神经，以促进酶消化和活细胞的恢复。为了克服预变性的需要，我们开发了一种方法，该方法允许在组织收获后立即分离成年大鼠坐骨神经SC。这种方法结合了实现神经纤维的快速酶促解离和细胞与髓鞘的直接分离的优点，从而提高了细胞产量和活力。基本上，该方法包括（1）在去除神经外膜层和广泛梳理神经纤维后立即用胶原酶和分散酶进行急性解离，（2）通过选择性地将细胞附着到高度粘附的聚-L-赖氨酸/层粘连蛋白底物上来去除髓鞘碎片，（3）在含有化学有丝分裂原的培养基中扩增初始SC群，和（4）准备用于转移或延迟实验的低温储备。该方案允许在神经组织解剖后3-4天获得去除髓鞘和成纤维细胞生长的均质SC培养物。SC培养物可以原样用于实验或在使用、纯化或冷冻保存之前进行连续轮扩增。
Adult Schwann cell (SC) cultures are usually derived from nerves subjected to a lengthy step of predegeneration to facilitate enzymatic digestion and recovery of viable cells. To overcome the need for predegeneration, we developed a method that allows the isolation of adult rat sciatic nerve SCs immediately after tissue harvesting. This method combines the advantages of implementing a rapid enzymatic dissociation of the nerve fibers and a straightforward separation of cells versus myelin that improves both cell yield and viability. Essentially, the method consists of (1) acute dissociation with collagenase and dispase immediately after removal of the epineurium layer and extensive teasing of the nerve fibers, (2) removal of myelin debris by selective attachment of the cells to a highly adhesive poly-L-lysine/laminin substrate, (3) expansion of the initial SC population in medium containing chemical mitogens, and (4) preparation of cryogenic stocks for transfer or delayed experimentation. This protocol allows for the procurement of homogeneous SC cultures deprived of myelin and fibroblast growth as soon as 3-4 days after nerve tissue dissection. SC cultures can be used as such for experimentation or subjected to consecutive rounds of expansion prior to use, purification, or cryopreservation.