Ruck Tobias, Bittner Stefan, Epping Lisa, Herrmann Alexander M, Meuth Sven G
Department of Neurology, University of Münster;
Department of Neurology, University of Münster; Interdisciplinary Center for Clinical Research (IZKF) Münster.
J Vis Exp. 2014 Nov 14(93):e52204. doi: 10.3791/52204.
The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies. This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.
血脑屏障在超微结构上由单层脑微血管内皮细胞（BMEC）组装而成，这些细胞通过紧密连接和黏附连接的连接复合体相互连接。它们与其他细胞类型，如星形胶质细胞或周细胞一起，形成神经血管单元（NVU），该单元专门调节外周血与中枢神经系统之间的液体、分子和细胞交换。通过这个复杂而动态的系统，BMEC参与维持中枢神经系统稳态的各种过程。血脑屏障功能障碍被认为是许多严重中枢神经系统疾病发病机制中的关键步骤。然而，由于其潜在机制仍远未被理解，特异性和靶向性治疗非常有限。动物模型和体外模型已被广泛用于深入了解复杂的生理和病理生理过程。通过简化和还原，可以将研究重点聚焦于感兴趣的主题，并排除各种混杂因素。然而，模型系统的可比性和可转移性也会降低，在评估时必须考虑到这一点。最常见的动物模型以小鼠为基础，主要原因之一是方法学的可能性不断增加。对分离的小鼠BMEC进行体外研究可能有助于深入分析其特性以及生理和病理生理条件下的血脑屏障。对血脑屏障复杂机制的进一步深入了解可能为新的治疗策略提供基础。本方案描述了一种通过一系列物理和化学纯化步骤分离原代小鼠微血管内皮细胞的方法。讨论了MBMEC纯度和培养的特殊注意事项以及质量控制、潜在应用和局限性。
