Optogenetic-enabled biomanufacturing of long-projecting motor tracts for neural reconstruction

Tissue-engineered spinal tracts (TE-STs), a subclass of micro-tissue-engineered neural networks, are designed to replace long-range axonal pathways but are limited by the slow intrinsic growth of many neuronal subtypes, particularly spinal motor neurons. Optogenetic stimulation provides cell-type-specific, precise control of activity that may accelerate neurite extension during biofabrication. Accordingly, this study evaluates whether integrating optogenetic stimulation into the biomanufacturing process enhances motor axonal outgrowth within tailored hydrogel micro-columnsin vitro.Motor neuron aggregates from embryonic rat spinal cord were generated by forced aggregation and encased within agarose micro-columns (701µm OD, ∼300µm ID; 3-12 mm length) to create TE-STs. Neurons were transduced to express the red-shifted channelrhodopsin ChrimsonR. Stimulation parameters were first optimized in planar cultures, where a 1-hour red-light train at 10 Hz (625 nm) maximized neurite outgrowth without significant heating. TE-STs were then stimulated at 10 Hz for 1 h at 7 din vitro(DIV) and then fixed at 21 DIV. Groups included transduced and non-transduced neurons, each with and without light stimulation. Axonal extension was quantified within the micro-column lumen, and two-way analysis of variance was used for analysis. TE-STs exhibited robust axonal growth with organized tract architecture, preserved neuronal health, and stable motor neuron phenotype. Transduced neurons with and without light stimulation extended axons faster than non-transduced controls, whereas no differences were observed between non-transduced neurons with or without light. Notably, transduced neurons exposed to a single round of optogenetic stimulation at 7 DIV reached ∼5.5 mm of growth by 21 DIV, representing a ∼2.5-fold increase over baseline controls. Brief, targeted optogenetic activation is sufficient to accelerate axonal growth of engineered spinal motor tracts duringin vitrobiomanufacturing. These findings establish optogenetic stimulation as a programmable biomanufacturing input that accelerates axonal assembly through activity-dependent mechanisms while remaining compatible with scalable three-dimensional tissue fabrication.