Abstract
This study reports the first hydrogel based compartmentalized in vitro platform for neuromuscular junction (NMJ) formation. The study was performed in three parts including the fabrication of a biomimetic substrate adhesive to both cell types involved in NMJ formation, optimizing the media formulation for these cells, and designing and testing a compartmentalized hydrogel system. A laminin coated gelatin (GEL-LN) hydrogel was fabricated and used to assess C2C12 mouse skeletal muscle cells, primary rat Schwann cells, and human induced pluripotent stem cell derived spinal spheroid (SpS). The GEL- LN substrate increased maturation and improved morphology when used to culture C2C12 cells and SpS. Additionally, GEL-LN improved adhesion of SpS and Schwann cells compared to gelatin hydrogels. Next, a media formulation ideal for culturing both cell types was explored. The neuronal base media containing DMEM/F12, D-glucose, L-ascorbic acid, N2 supplement, and B27 supplement with no additional supplements resulted in controlled skeletal muscle differentiation leading to longer culture times, which is optimal for NMJ formation. There was no statistical difference for SpS cultured in base neuron media compared to the full neuron media with supplements, therefore all subsequent experiments used the neuron base media formulation. A compartmentalized hydrogel was designed including: (1) isotropic area with added laser engraved well for SpS adherence, (2) microchannels to guide axonal growth, (3) engineered anisotropic skeletal muscle. The compartmentalized design was micromolded onto GEL-LN hydrogels, after which C2C12 cells and SpS were seeded in the respective compartments. When compared to traditional co-culture methods, the compartmentalized system had increased axonal growth and larger acetylcholine receptor cluster sizes signifying enhanced NMJ formation. Both pre- and post- synaptic markers were present in compartmentalized cultures indicating NMJ formation.