Nicolicht-Amorim Priscila, Delgado-Garcia Lina M, Nakamura Thabatta Karollynne Estevam, Courbassier Natália Rodrigues, Mosini Amanda Cristina, Porcionatto Marimelia A
Laboratory of Molecular Neurobiology, Department of Biochemistry, Universidade Federal de São Paulo, São Paulo, Brazil.
Laboratory of Neurobiology, Department of Physiology, Universidade Federal de São Paulo, São Paulo, Brazil.
Front Cell Neurosci. 2022 Oct 13;16:949412. doi: 10.3389/fncel.2022.949412. eCollection 2022.
The neurovascular unit (NVU) is a multicellular structure comprising of neurons, glial cells, and non-neural cells, and it is supported by a specialized extracellular matrix, the basal lamina. Astrocytes, brain microvascular endothelial cells (BMECs), pericytes, and smooth muscle cells constitute the blood-brain barrier (BBB). BMECs have a mesodermal origin and invade the nervous system early in neural tube development, forming the BBB anatomical core. BMECs are connected by adherent junction complexes composed of integral membrane and cytoplasmic proteins. and studies have shown that, given the proximity and relationship with neural cells, BMECs acquire a unique gene expression profile, proteome, and specific mechanical and physical properties compared to endothelial cells from the general vasculature. BMECs are fundamental in maintaining brain homeostasis by regulating transcellular and paracellular transport of fluids, molecules, and cells. Therefore, it is essential to gain in-depth knowledge of the dynamic cellular structure of the cells in the NVU and their interactions with health and disease. Here we describe a significantly improved and simplified protocol using C57BL/6 newborn mice at postnatal day 1 (PND1) to isolate, purify, and culture BMECs monolayers in two different substrates (glass coverslips and transwell culture inserts). characterization and validation of the BMEC primary culture monolayers seeded on glass or insert included light microscopy, immunolabeling, and gene expression profile. Transendothelial electrical resistance (TEER) measurement and diffusion test were used as functional assays for adherent junction complexes and integrity and permeability of BMECs monolayers. The protocol presented here for the isolation and culture of BMECs is more straightforward than previously published protocols and yields a high number of purified cells. Finally, we tested BMECs function using the oxygen-glucose deprivation (OGD) model of hypoxia. This protocol may be suitable as a bioscaffold for secondary cell seeding allowing the study and better understanding of the NVU.
神经血管单元（NVU）是一种多细胞结构，由神经元、神经胶质细胞和非神经细胞组成，并由一种特殊的细胞外基质——基膜提供支持。星形胶质细胞、脑微血管内皮细胞（BMECs）、周细胞和平滑肌细胞构成血脑屏障（BBB）。BMECs起源于中胚层，在神经管发育早期侵入神经系统，形成血脑屏障的解剖学核心。BMECs通过由整合膜蛋白和细胞质蛋白组成的黏附连接复合体相连。 研究表明，鉴于与神经细胞的接近程度和关系，与来自一般脉管系统的内皮细胞相比，BMECs具有独特的基因表达谱、蛋白质组以及特定的机械和物理特性。BMECs通过调节液体、分子和细胞的跨细胞和细胞旁运输，在维持脑内环境稳定方面起着至关重要的作用。因此，深入了解NVU中细胞的动态细胞结构及其与健康和疾病的相互作用至关重要。在此，我们描述了一种显著改进和简化的方案，使用出生后第1天（PND1）的C57BL/6新生小鼠，在两种不同的基质（玻璃盖玻片和Transwell培养小室）中分离、纯化和培养BMECs单层。接种在玻璃或小室上的BMEC原代培养单层的表征和验证包括光学显微镜检查、免疫标记和基因表达谱分析。跨内皮电阻（TEER）测量和扩散试验用作BMECs单层黏附连接复合体以及完整性和通透性的功能测定。此处介绍的BMECs分离和培养方案比以前发表的方案更简单，且能产生大量纯化细胞。最后，我们使用缺氧的氧糖剥夺（OGD）模型测试了BMECs的功能。该方案可能适合作为二次细胞接种的生物支架，有助于研究和更好地理解NVU。
