A fluid-walled microfluidic platform for human neuronal microcircuits and axotomy

<strong>ABSTRACT</strong> Recapitulation of human neuronal circuits <em>in vitro</em> will significantly facilitate investigation of human neuronal physiology and pathophysiology. This paper demonstrates and characterises a novel system for culturing neuronal circuits within fluid-walled microenvironments. Fluid-walled technology leverages the immiscibility between aqueous cell media and hydrophobic bio-inert fluorocarbon (FC40) to create miniaturised cell cultures inside standard Petri dishes. Here, we demonstrate the capacity of fluid-walled technology in recreating the human corticostriatal microcircuit. Induced pluripotent stem cell-derived cortical and striatal neurons were co-cultured in fluid-walled dumbbells which, employing intrinsic pressures, allows i) separation of different neuronal subtypes, ii) compartmentalisation of cortical somas and dendrites from cortical axons, and iii) unidirectional projection of cortical axons toward their postsynaptic striatal partner. Exploiting unique features of fluid walls, we also describe a contactless method to perform localised cortical axotomy at pre-defined location along the dumbbells using a micro-jet of cell media. Axotomy of all cortical axonal tracts on the same dish is systematically obtained within 90 seconds. Subsequent axonal regeneration was observed and significantly promoted in the presence of postsynaptic striatal neurons. The ability to control compartmentalisation and directionality of neuronal connections based on fluid dynamics offers an efficient and highly reproducible system for long-term culturing of human neuronal circuits over conventional monoculture of cortical neurons. Combined with reproducible axotomy, we envision this system to be adaptable to large scale screening of regenerative therapeutic candidates in a highly physiological manner. <strong>FILE DESCRIPTIONS</strong> Source Data.xlsx: Tabular datasets plotted on main figures 2 and 5. Key Resources Table.xlsx: Table containing key resources (primary and secondary antibodies, cell lines and software) used in this study.**摘要** 体外重建人类神经元环路，将显著推动人类神经元生理学与病理生理学研究。本文展示并表征了一种可在流体壁微环境中培养神经元环路的新型系统。流体壁技术利用水性细胞培养基与疏水生物惰性全氟碳化合物（FC40）之间的不混溶性，在标准培养皿中构建微型细胞培养体系。本文证实了流体壁技术可用于重建人类皮质纹状体微环路：将诱导多能干细胞（induced pluripotent stem cell）分化而来的皮质神经元与纹状体神经元共培养于流体壁哑铃型微腔中；借助体系内的固有压力，该系统可实现：① 不同神经元亚型的分离；② 皮质神经元胞体与树突同皮质轴突的区域化分隔；③ 皮质轴突向突触后纹状体神经元的单向投射。此外，依托流体壁的独特特性，本文还报道了一种非接触式方法：利用细胞培养基微射流，沿哑铃型微腔的预设位置对皮质轴突实施局部轴突切断术。同一培养皿内的所有皮质轴突束可在90秒内完成系统性轴突切断。后续观察发现，在突触后纹状体神经元存在的情况下，轴突再生现象显著增强。相较于传统的皮质神经元单培养体系，该系统可通过流体动力学精准调控神经元连接的区域化与方向性，为人类神经元环路的长期培养提供了一种高效且可高度重复的方案。结合可重复的轴突切断术，我们预期该系统可适配大规模再生治疗候选药物的筛选，且能高度模拟生理环境。 **文件说明** Source Data.xlsx：主图2与主图5对应的表格数据集。 Key Resources Table.xlsx：本研究使用的关键资源汇总表，涵盖一抗、二抗、细胞系及相关软件。