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.