The pathways that control protein transport across the blood–brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

中文翻译：

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使用血脑屏障类器官阵列研究受体介导的抗体转胞吞作用

控制蛋白质跨血脑屏障 (BBB) 转运的途径仍然缺乏特征。尽管在体外概括人类 BBB 方面取得了很大进展，但目前的模型不适合系统分析抗体转运的分子机制。我们对抗体转胞吞作用的机制理解方面的差距阻碍了新的治疗递送策略的发展。我们应用了一种新的生物工程方法，通过星形胶质细胞、周细胞和脑内皮细胞的自组装产生人类 BBB 类器官，使用微图案水凝胶具有前所未有的通量和可重复性。我们设计了一种半自动化和可扩展的成像测定来测量受体介导的抗体转胞吞作用。最后，我们开发了一个工作流程，在 BBB 类器官阵列中使用 CRISPR/Cas9 基因编辑来敲除脑内皮细胞中的内吞调节因子，以剖析受体介导的转胞吞作用的分子机制。BBB 有机体阵列允许以高度可重复的方式在每个单独的实验中同时生长 3000 多个同质有机体。BBB 类器官阵列显示出对大分子的低渗透性并阻止了人类非靶向抗体的转运。相比之下，针对人转铁蛋白受体的单价抗体在类器官中经历了剂量和时间依赖性的胞吞作用。在 BBB 类器官阵列中使用 CRISPR/Cas9 基因编辑，我们发现网格蛋白而非小窝蛋白是转铁蛋白受体依赖性转胞吞作用所必需的。人 BBB 类器官阵列是一个强大的高通量平台，可用于发现受体介导的抗体转胞吞作用的新机制。在药物发现的早期阶段实施该平台可以加速新的大脑递送技术的开发。