Biohybrid motor neuron spheroid composed of graphene/HUVEC/neural cell for 3D biosensing system to evaluate drug of amyotrophic lateral sclerosis

A 3D motor neuron (MN) spheroid has been developed to investigate neurodegenerative and neuromuscular junction (NMJ) disease. However, core necrosis and reduced neurogenesis, impairing neural network formation, were observed as the MN spheroid matured. In this study, to enhance neural network formation, a biohybrid MN spheroid composed of neural cells/reduced graphene oxide (rGO)/human umbilical vein endothelial cells (HUVECs) was generated for the first time and applied to 3D biosensing system for MNJ disease. By incorporating rGO and HUVECs at the onset of human neural stem cell (hNSC) culture, rGO and HUVECs were evenly distributed within MN spheroid generated by differentiation of hNSC, which improved oxygen- and nutrient- supply by reduction of core necrosis, and enhanced neurogenesis. The fabricated biohybrid MN spheroid improved neural network formation and electrophysiological signal. This method was also applied to generate biohybrid cerebral organoids from human induced pluripotent stem cells (hiPSCs), emphasizing its versatility for diverse 3D neural models. Then, a 3D NMJ biosensing system was fabricated by positioning the biohybrid MN spheroid with muscle bundles to evaluate its utility in neuromuscular disease modeling. Biohybrid MN spheroids generated from induced pluripotent stem cells of sporadic amyotrophic lateral sclerosis (ALS) patients were used to make NMJ. Reduced contraction of the connected muscle bundle due to ALS could be restored by upon treatment with the bosutinib, ALS drug, demonstrating the potential use for drug screening. The method to generate biohybrid spheroid can be applied to generation of various biohybrid brain organoids, and the proposed 3D NMJ biosensing system can be used to drug screening of diverse neuromuscular diseases.