人类脑器官样结构（COs）是由干细胞生成的，是了解脑部发育和神经退行性疾病的新兴动物替代品。COs的长期生长目前受到高效氧气渗透和持续营养供应的限制，导致器官样结构的核心区域出现普遍内部缺氧和细胞死亡。在这里，我们开发了一个具有动态液流干扰和供氧的三维（3D）微流体平台。我们证明，培养在3D微流体系统中的COs可以持续生长超过50天，核心区域没有细胞死亡。通过免疫荧光染色、蛋白质组学和代谢组学的验证，我们还观察到了细胞增殖的增加和细胞分化的增强。在第4天到第30天之间连续进行的时间点蛋白质组学鉴定发现了546个在COs生长过程中差异表达的蛋白质，主要与神经系统发育、子宫内胚胎发育、脑部发育和神经元迁移相关。我们的3D微流体平台为培养高同质性的人类器官样结构提供了潜在的应用价值。版权所有 © 2023 Elsevier B.V. 保留所有权利。

Human cerebral organoids (COs), generated from stem cells, are emerging animal alternatives for understanding brain development and neurodegeneration diseases. Long-term growth of COs is currently hindered by the limitation of efficient oxygen infiltration and continuous nutrient supply, leading to general inner hypoxia and cell death at the core region of the organoids. Here, we developed a three-dimensional (3D) microfluidic platform with dynamic fluidic perturbation and oxygen supply. We demonstrated COs cultured in the 3D microfluidic system grew continuously for over 50 days without cell death at the core region. Increased cell proliferation and enhanced cell differentiation were also observed and verified with immunofluorescence staining, proteomics and metabolomics. Time-lapse proteomics from 7 consecutive acquisitions between day 4 and day 30 identified 546 proteins differently expressed accompanying COs growth, which were mainly relevant to nervous system development, in utero embryonic development, brain development and neuron migration. Our 3D microfluidic platform provides potential utility for culturing high-homogeneous human organoids.Copyright © 2023 Elsevier B.V. All rights reserved.