Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Nat Protoc. 2022 Jan;17(1):95-128. doi: 10.1038/s41596-021-00635-w. Epub 2022 Jan 7.
The blood-brain barrier (BBB) greatly restricts the entry of biological and engineered therapeutic molecules into the brain. Due to challenges in translating results from animal models to the clinic, relevant in vitro human BBB models are needed to assess pathophysiological molecular transport mechanisms and enable the design of targeted therapies for neurological disorders. This protocol describes an in vitro model of the human BBB self-assembled within microfluidic devices from stem-cell-derived or primary brain endothelial cells, and primary brain pericytes and astrocytes. This protocol requires 1.5 d for device fabrication, 7 d for device culture and up to 5 d for downstream imaging, protein and gene expression analyses. Methodologies to measure the permeability of any molecule in the BBB model, which take 30 min per device, are also included. Compared with standard 2D assays, the BBB model features relevant cellular organization and morphological characteristics, as well as values of molecular permeability within the range expected in vivo. These properties, coupled with a functional brain endothelial expression profile and the capability to easily test several repeats with low reagent consumption, make this BBB model highly suitable for widespread use in academic and industrial laboratories.
血脑屏障（BBB）极大地限制了生物和工程治疗分子进入大脑。由于将动物模型的结果转化为临床应用存在挑战，因此需要相关的体外人 BBB 模型来评估病理生理学分子转运机制，并为神经疾病设计靶向治疗方法。本方案描述了一种在微流控装置中自组装的源自干细胞的或原代脑内皮细胞、原代脑周细胞和星形胶质细胞的体外人 BBB 模型。该方案需要 1.5 天用于设备制造，7 天用于设备培养，最多 5 天用于下游成像、蛋白质和基因表达分析。还包括测量 BBB 模型中任何分子通透性的方法，每个设备需要 30 分钟。与标准的 2D 测定相比，BBB 模型具有相关的细胞组织和形态特征，以及在体内预期范围内的分子通透性值。这些特性，加上功能性脑内皮表达谱和能够以低试剂消耗轻松测试多个重复的能力，使该 BBB 模型非常适合在学术和工业实验室中广泛使用。
