Biosensing system for drug evaluation of amyotrophic lateral sclerosis based on muscle bundle and nano-biohybrid hydrogel composed of multiple motor neuron spheroids and carbon nanotubes

Abstract

Amyotrophic lateral sclerosis (ALS) results in muscle atrophy due to degeneration of the neuromuscular junctions (NMJs), the synapse responsible for the transmission of signals between motor neurons (MNs) and skeletal muscles. Even though NMJ models have been developed, in vitro drug evaluation models for ALS disease are still lacking due to the absence of a sensing system and immature NMJs in a model system. Moreover, a three-dimensional (3D) biosensing system that can observe drug-induced muscle recovery and movement still does not exist. In this study, we developed the first 3D NMJ biosensing system for drug evaluation of ALS based on muscle bundle and the 3D nano-biohybrid hydrogel composed of multiple motor neuron spheroids (multi-MNSs) and carboxylated carbon nanotubes (CNT-COOH). For the fabrication of biosensor-integrated NMJs, the use of multi-MNSs increased the number of neurites, which in combination with the muscle bundle, resulted in increased NMJ formation. In addition, the use of CNT-COOH in the 3D nano-biohybrid hydrogel induced neurogenesis of multi-MNSs, increased hydrogel conductivity, and formed more mature and functional NMJs. ALS-MNSs obtained from induced pluripotent stem cells (iPSCs) of sporadic ALS patients were added to the 3D nano-biohybrid hydrogel to examine the effects of bosutinib, one of the drug candidates for ALS treatment, leading to the recovery of contraction of the connected muscle bundle. The proposed 3D NMJ biosensing system using 3D nano-biohybrid hydrogel can be an extraordinary platform for drug screening of diverse neuromuscular diseases and toxicity assessment.