A rapid and versatile method for the isolation, purification and cryogenic storage of Schwann cells from adult rodent nerves.
A rapid and versatile method for the isolation, purification and cryogenic storage of Schwann cells from adult rodent nerves.
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DOI：
10.1038/srep31781
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发表时间：
2016-08-23
影响因子：
4.6
通讯作者：
Monje PV
中科院分区：
文献类型：
--
作者：
Andersen ND;Srinivas S;Piñero G;Monje PV
我们在此开发了一种用于快速获取成人神经源性许旺细胞（SC）的方案，该方案被优化以实施新鲜神经组织的立即酶促解离，同时保持高细胞活力，提高产量并最大限度地减少成纤维细胞和髓鞘污染。本方案介绍：（1）在去除神经外膜和广泛梳理神经纤维后立即进行酶促细胞释放的有效方法;（2）用于选择性细胞附着、去除髓鞘碎片和在化学成分确定的培养基中扩增初始SC群体的适应性滴板法;（3）用于快速和有效消除成纤维细胞的磁激活细胞分选纯化方案;和（4）任选的冷冻保存步骤，用于储存过量的细胞。在神经处理的三天内获得了缺乏髓鞘和成纤维细胞生长的高度增殖的SC培养物。对初始、扩增和冻存细胞产物的表征证实了整个过程中SC鉴别、活力和生长速率的维持。最重要的是，即使在冷冻保存后，SC仍保持其对有丝分裂原的敏感性和分化潜力。总之，这种易于实施和临床相关的方案允许制备可扩展的均质SC培养物，同时最大限度地减少时间、细胞操作和暴露于培养变量。
We herein developed a protocol for the rapid procurement of adult nerve-derived Schwann cells (SCs) that was optimized to implement an immediate enzymatic dissociation of fresh nerve tissue while maintaining high cell viability, improving yields and minimizing fibroblast and myelin contamination. This protocol introduces: (1) an efficient method for enzymatic cell release immediately after removal of the epineurium and extensive teasing of the nerve fibers; (2) an adaptable drop-plating method for selective cell attachment, removal of myelin debris, and expansion of the initial SC population in chemically defined medium; (3) a magnetic-activated cell sorting purification protocol for rapid and effective fibroblast elimination; and (4) an optional step of cryopreservation for the storage of the excess of cells. Highly proliferative SC cultures devoid of myelin and fibroblast growth were obtained within three days of nerve processing. Characterization of the initial, expanded, and cryopreserved cell products confirmed maintenance of SC identity, viability and growth rates throughout the process. Most importantly, SCs retained their sensitivity to mitogens and potential for differentiation even after cryopreservation. To conclude, this easy-to-implement and clinically relevant protocol allows for the preparation of expandable homogeneous SC cultures while minimizing time, manipulation of the cells, and exposure to culture variables.