Poster BB8 - Engineering High-Throughput Human Neuromuscular Microphysiological System for Phenotypic Drug Testing

Introduction: In vitro neuromuscular junctions (NMJ) modeling with functional synaptic connections are considered to be challenging due to the structural complexity seen in NMJs in living organisms. The existing in vitro systems for assessing NMJ function typically involved low-throughput methods, limiting the ability to simultaneously analyze various factors influencing maturation and making drug dose-response studies less practical. To overcome these limitations, we have developed a compartmentalized microfluidic chamber and an in situ functional assessment method based on muscle contractility that enables high-throughput, real-time functional measurements as well as compartmentalized molecular phenotypic studies.

Materials and

Methods: We developed a novel approach enables the simultaneous evaluation of human-induced pluripotent stem cell (hiPSC)-derived nerve-muscle co-cultures, accommodating 24-, 48-, and 96-well plate formats. This NMJ model involves the co-culturing of engineered muscle tissue (EMT) with optogenetically active hiPSC-derived motor neurons. The system employs polydimethylsiloxane (PDMS) posts and embedded permanent magnets to measure the contractile behavior of EMTs when stimulated optically by motor neurons. Within four days of fabrication and co-culture, the EMTs respond to various electrical stimuli, exhibiting both twitch and tetanic forces. Notably, as the stimulation frequency increases to 20 Hz, single twitch forces transition into sustained tetanic forces. For functional measurement, 96 anisotropic magneto-resistance sensors are strategically aligned with the magnets to monitor muscle contractile function. These sensors measure parameters such as muscle twitch force, speed, and power in real-time by detecting changes in magnetic flux resulting from the displacement of the magnets during muscle contractions. We characterized the development of neuromuscular tissue over time to demonstrate its physiological relevance and potential utility in pharmaceutical contexts using human-sourced neurons and skeletal muscle myocytes. This longitudinal analysis ensures that the tissue models accurately mimic human neuromuscular physiology, which is critical for their application in drug testing and disease modeling.

Results, Conclusions, and Discussions: Our fabrication process demonstrated the neuromuscular junction tissue formation success over 95% with scalability to automate the production of 96 tissues. The magnetic-based functional measurements demonstrated a signal-to-noise ratio over 60 dBf. Sensor readouts, calibrated against the golden standards with optical tracking of tissue contractions, show a linear correlation that facilitates direct translation between these two measurement modalities. Immunofluorescence analysis reveals a tubular, striated structure of contractile protein sarcomeric alpha-actinin, along with the presence of acetylcholine receptors and Pax7-positive satellite cells, highlighting the intricate microstructure of EMTs that closely mimics the native skeletal muscle niche. Also, pre-synaptic markers of motor neurons such as Hb9, a transcription factor critical for the development of motor neurons and ChAT, an enzyme responsible for synthesizing the neurotransmitter is confirmed. This system effectively produces three-dimensional NMJ tissues from human induced pluripotent stem cells (hiPSCs) and human primary cells, exhibiting robust physiological functions and gene expression profiles. Over four weeks, Human primary skeletal muscles respond to electrical stimuli with twitch forces stabilizing from day 7 to 28 post-biofabrication, with forces increasing from 1 Hz single twitches at 18.04 ± 10.47 μN (n = 5) to 30 Hz sustained tetanic forces at 93.27 ± 51.87 μN (n = 5). Stable expressions of myosin heavy chain and ion-regulating proteins of skeletal muscle tissues suggest maturation of both muscle types at the transcriptional level. We virally transduced hiPSC-derived motor neurons with Lenti-EF1A-hChR2 for optogenetic control of neuron firing that in turn stimulates muscle. We demonstrated the muscle contraction with varying frequency of neuron firing from 1Hz to 10Hz to confirm functional connection in neuromuscular junction.

Acknowledgements (Optional): The described system significantly enhances the throughput of NMJ tissue fabrication and functional evaluation, reducing costs, time, and labor required for large-scale production. This advancement offers a promising approach to advance the use of NMJ chip devices in large-scale drug screening and disease modeling.