(368f) Biomanufacturing of Neuroactive Biologics Using Engineered Neuromuscular Junction (NMJ) in Vitro Model

Skeletal muscles are well known for enabling voluntary movement, yet they also crucially stabilize internal homeostasis and the functions of organs throughout the body. Skeletal muscles secrete a variety of peptides and cytokines, named myokines, which affect cells through autocrine, paracrine, and endocrine actions. Moreover, muscles produce extracellular vesicles such as exosomes, another class of biological factors. These vesicles, ranging from 40 to 150 nm, transport various biological molecules such as proteins, lipids, and nucleic acids to target cells, thereby facilitating intercellular communication. Muscle-derived exosomes have demonstrated effects on neuronal survival, neurite extension, and nerve regeneration.

Physical exercise is an important regulator of muscle secretion, with different types of activities boosting myokine and exosome secretion, implying the significant role of muscle contraction in releasing muscle-derived biological factors that maintain body homeostasis and even brain health.

To control and enhance the production of muscle-secreted neurotrophic factors, we propose that the connections and activity of neurons are crucial in determining the release and functional impact of myokines and exosomes secreted by muscles by using an in vitro neuromuscular junction (NMJ) model. We engineered a two-dimensional muscle construct on both flat and micro-grooved polymer substrates to manipulate the extent of neural connections and muscular efficacy. Neural stem cells were then differentiated into motor neurons directly atop the muscle layer, prompting the formation of neuromuscular junctions. We evaluated how such neural interaction influences muscle contractions, the expression of genes linked to myokines, the secretion of myokines, and the release of exosomes. Furthermore, we cataloged the miRNAs within exosomes sourced from both neuron-absent and neuron-innervated muscle tissue, exploring their impact on neural growth. Additionally, we explored how these muscle-derived factors promote the development, intercellular vesicle transport, and firing activity of cultured primary hippocampal neurons. Summarily, our investigation highlights the potential of our in vitro NMJ model as a biomanufacturing platform for generating neuro-supportive myokines and exosomes with significant ramifications for in vitro nervous system cultures.