Abstract：
The blood-brain barrier (BBB) constitutes a crucial protective anatomical layer with a microenvironment that tightly controls material transit. Constructing an in vitro BBB model to replicate in vivo features requires the sequential layering of constituent cell types. Maintaining heightened integrity in the observed tight junctions during both the establishment and post-experiment phases is crucial to the success of these models. We have developed an in vitro BBB model that replicates the cellular composition and spatial orientation of in vivo BBB observed in humans. The experiment includes comprehensive procedures and steps aimed at enhancing the integration of the four-cell model. Departing from conventional in vitro BBB models, our methodology eliminates the necessity for pre-coated plates to facilitate cell adhesion, thereby improving cell visualization throughout the procedure. An in-house coating strategy and a simple yet effective approach significantly reduce costs and provides superior imaging of cells and corresponding tight junction protein expression. Also, our BBB model includes all four primary cell types that are structural parts of the human BBB. With its innovative and user-friendly features, our in-house optimized in vitro four-cell-based BBB model showcases novel methodology and provides a promising experimental platform for drug screening processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Coating and culture system Basic Protocol 2: Cell seeding and Transwell insert handling Basic Protocol 3: Assessment of model functionality.
摘要：
血脑屏障（BBB）构成了至关重要的保护性解剖层，具有严格控制物质运输的微环境。构建体外BBB模型以复制体内特征需要组成细胞类型的顺序分层。在建立阶段和实验后阶段，在观察到的紧密连接中保持高度的完整性对于这些模型的成功至关重要。我们已经开发了一种体外BBB模型，该模型复制了在人体中观察到的体内BBB的细胞组成和空间方向。该实验包括旨在增强四细胞模型的集成的全面程序和步骤。与常规体外BBB模型不同，我们的方法消除了预涂层板促进细胞粘附的必要性，从而改善整个过程中的细胞可视化。内部涂层策略和简单而有效的方法显着降低成本，并提供细胞和相应的紧密连接蛋白表达的优异成像。此外，我们的BBB模型包括作为人BBB结构部分的所有四种原代细胞类型。凭借其创新和用户友好的功能，我们内部优化的体外四细胞BBB模型展示了新的方法学,并为药物筛选过程提供了一个有前景的实验平台.©2024作者WileyPeriodicalsLLC出版的当前协议。基本方案1:涂覆和培养系统基本方案2:细胞接种和Transwell插入物处理基本方案3:模型功能的评估。
