Mason P W, Attema B L, DeVries G H
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0614.
J Neurosci Res. 1992 Apr;31(4):731-44. doi: 10.1002/jnr.490310417.
Much of our knowledge about the development and maintenance of the peripheral nervous system has been learned through studying the interaction of neurons, or their isolated membranes, with Schwann cells (SC), in tissue culture. Numerous approaches have been employed to obtain an adequate quantity of SC, but all have been limited by either the uncertainty of obtaining a sufficient amount of starting material, the time and expertise required to isolate the SC, or by the limited number of SC that can be generated. We have developed a procedure to isolate SC from cryopreserved sciatic nerves. This procedure allows for sciatic nerves to be pooled until adequate numbers of nerves are obtained, yet still produces cells that retain the functional abilities of SC isolated from fresh nerves. Sciatic nerves were isolated from 2 day old rat pups, placed in either DME media and used fresh or placed in a freezing solution containing DME media (25%), DMSO (25%), fetal calf serum (50%), frozen at -70 degrees C and stored in liquid nitrogen. The frozen nerves were rapidly thawed to 37 degrees C and single cells were prepared from both fresh and frozen nerves using enzymatic and mechanical disruption as previously described (Brockes et al., Brain Res 165: 105-118, 1979). Comparable cell yields were obtained for SC isolated from both frozen and fresh nerves. Immunohistochemical staining of both fresh and frozen SC produced similar staining patterns with antibodies to GFAP, laminin, CNPase, S100, MBP, and P0 protein. Addition of axolemmal enriched membrane fractions to both the frozen and fresh SC gave a similar dose response curve of 3H-thymidine incorporation, with SC from frozen sciatic nerves responding even better than fresh sciatic nerves at higher doses (50 micrograms and 100 micrograms of protein/ml). As demonstrated by the cell yield, immunohistochemical staining and responses to axolemmal mitogens, this procedure produces SC from frozen sciatic nerves with similar characteristics to those isolated from fresh nerves. This procedure will allow the production and utilization of a large number of SC, which will be critical in further studies on the development and maintenance of the peripheral nervous system.
我们关于周围神经系统发育和维持的许多知识，都是通过在组织培养中研究神经元或其分离的细胞膜与雪旺细胞（SC）之间的相互作用而获得的。人们采用了多种方法来获取足够数量的雪旺细胞，但所有方法都受到以下限制：要么获取足够起始材料存在不确定性，要么分离雪旺细胞所需的时间和专业知识，要么是能够产生的雪旺细胞数量有限。我们已经开发出一种从冷冻保存的坐骨神经中分离雪旺细胞的方法。该方法允许将坐骨神经汇集起来，直到获得足够数量的神经，同时仍然能产生保留从新鲜神经中分离出的雪旺细胞功能能力的细胞。从2日龄大鼠幼崽中分离出坐骨神经，将其置于DME培养基中，新鲜使用或置于含有DME培养基（25%）、二甲基亚砜（25%）、胎牛血清（50%）的冷冻溶液中，在-70℃下冷冻并储存在液氮中。将冷冻的神经迅速解冻至37℃，并按照先前描述的方法（Brockes等人，《脑研究》165：105 - 118，1979），通过酶解和机械破碎从新鲜和冷冻的神经中制备单细胞。从冷冻和新鲜神经中分离出的雪旺细胞获得了相当的细胞产量。用针对GFAP、层粘连蛋白、CNPase、S100、髓磷脂碱性蛋白（MBP）和P0蛋白的抗体对新鲜和冷冻的雪旺细胞进行免疫组织化学染色，产生了相似的染色模式。向冷冻和新鲜的雪旺细胞中添加富含轴膜的膜组分，得到了类似的3H - 胸腺嘧啶核苷掺入剂量反应曲线，在较高剂量（50微克和100微克蛋白质/毫升）下，来自冷冻坐骨神经的雪旺细胞的反应甚至比新鲜坐骨神经的雪旺细胞更好。正如细胞产量、免疫组织化学染色以及对轴膜有丝分裂原的反应所表明的那样，该方法从冷冻坐骨神经中产生的雪旺细胞具有与从新鲜神经中分离出的雪旺细胞相似的特征。这种方法将允许大量雪旺细胞的产生和利用，这对于进一步研究周围神经系统的发育和维持至关重要。
