University of Burgundy, INSERM U866, 21000 Dijon, France.
J Neurosci Methods. 2010 Mar 30;187(2):243-53. doi: 10.1016/j.jneumeth.2010.01.017. Epub 2010 Jan 25.
As the major immuno-competent cells of the brain, microglia are highly implicated in neuro-protection as well as in neurodegeneration. Therefore, they are of key interest for research on numerous CNS diseases. Currently, to model inflammation in the brain, microglial cell lines or primary microglia prepared from embryonic or neo-natal rodents are widely used. However, these in vitro microglial models are not suitable for research in the field of neuro-degenerative diseases where aging is a crucial parameter. Only a few in vitro studies on aged microglia have been published so far, most of which use ex vivo microglia which cannot be kept in culture for prolonged periods of time. In the present study, we provide a new approach which allows the isolation and culture of an almost pure population of microglia from adult mouse brains. The isolation is based on a procedure which combines density separation and a subsequent culture selection process. After these steps, microglia form a non-adherent floating cell layer that can be easily and repeatedly harvested and replated. This method is simple and allows for a comparatively high yield and purity of adult microglial cells. The collected primary adult microglia proliferate and can be kept in culture for extended periods of time. We compared the primary adult microglia to primary microglia from neo-natal mice as well as to the C8-B4 microglial cell line. We found that adult microglia have similar, but not identical, immuno-phenotypic, functional and electrophysiological characteristics to the other in vitro models.
作为大脑中的主要免疫活性细胞，小胶质细胞在神经保护和神经退行性变中都有重要作用。因此，它们是研究多种中枢神经系统疾病的关键。目前，为了模拟大脑中的炎症，广泛使用从小鼠胚胎或新生期制备的小胶质细胞系或原代小胶质细胞。然而，这些体外小胶质细胞模型并不适合研究与衰老有关的神经退行性疾病，因为衰老是一个关键参数。迄今为止，只有少数关于衰老小胶质细胞的体外研究已经发表，其中大多数使用的是离体小胶质细胞，离体小胶质细胞不能在培养中长期保存。在本研究中，我们提供了一种新的方法，可以从成年小鼠大脑中分离和培养几乎纯的小胶质细胞群体。该分离方法基于一种结合密度分离和后续培养选择过程的程序。经过这些步骤，小胶质细胞形成了一个不易贴壁的悬浮细胞层，可以轻松且反复地收获和再铺板。这种方法简单，可获得相对较高的产量和纯度的成年小胶质细胞。收集的原代成年小胶质细胞增殖，并可在培养中长期保存。我们将原代成年小胶质细胞与新生小鼠的原代小胶质细胞以及 C8-B4 小胶质细胞系进行了比较。我们发现成年小胶质细胞具有与其他体外模型相似但不完全相同的免疫表型、功能和电生理特征。
