An automated platform for simultaneous, longitudinal analysis of engineered neuromuscular tissues for applications in neurotoxin potency testing

Animal models of the neuromuscular junction (NMJ) have been widely studied but exhibit critical differences from human biology limiting utility in drug and disease modelling. Challenges with scarcity, scalability, throughput, and ethical considerations further limit the suitability of animal models for preclinical screening. Engineered models have emerged as alternatives for studying NMJ functionality in response to genetic and/or pharmacological challenge. However, these models have faced challenges associated with their poorly scalable creation, sourcing suitable cells, and the extraction of reliable, quantifiable metrics. We present a turnkey iPSC-based model of the NMJ employing channelrhodopsin-2 expression within the motor neuron (MN) population driving muscle contraction in response to blue light. MNs co-cultured with engineered skeletal muscle tissues produced twitch forces of 34.7 ± 22.7 µN in response to blue light, with a response fidelity > 92 %. Histological analysis revealed characteristic punctate acetylcholine receptor staining co-localized with the presynaptic marker synaptic vesicle protein-2. Dose-response studies using botulinum neurotoxin showed loss of function in a dose- and time-dependent manner (EC₅₀ − 0.11 ± 0.015 µg). Variability of the EC₅₀ values between 2 different iPSC differentiations of both cell types and 2 users was less than 2 %. Further testing with the acute neurotoxins acetylcholine mustard and d-tubocurarine validated the biological relevance of the postsynaptic machinery of the model. This model marks a meaningful progression of 3D engineered models of the NMJ, providing engineered tissues at a throughput relevant to potency and screening applications with an abundant iPSC cell source and standardized hardware-software ecosystem allowing technology transfer across laboratories.