Polydimethylsilxane (PDMS) chambers were designed, casted and cut as previously described.[21a (link)] The PDMS NMJ chambers were incubated for 24 hours in 70% isopropanol to remove any unpolymerized monomers that could be toxic to the cells. The chambers were dipped in 100% ethanol and dried under sterile conditions for at least 2-hours prior to assembly. Glass coverslips (22 mm x 22 mm) were cleaned by plasma treatment for 2-minutes under oxygen pressure of 750 mTorr, sterilized with 70% ethanol and air-dried under sterile conditions before assembly. The sterile PDMS chambers adhered to the clean coverslips by applying gentle pressure around the edges and gently tapping on the tunnels. The SKM-side and MN-side of the chamber were coated with rat tail collagen I (ThermoFisher A1048301; 60 μg/mL) and laminin (ThermoFisher 23017015; 3 μg/mL), respectively. The collagen was removed after a 2-hour incubation period and rinsed twice with 1X phosphate buffer solution (PBS). The muscle proliferation medium (Adult Growth Medium, AGM) was added to the muscle-side before storing the systems at 4°C overnight. The laminin remained on the MN-side. After 24 hours, the laminin solution on the MN-side was removed and replaced with human motoneuron media (HMN). The NMJ systems were stored at 37°C and 5% CO2 1-hour before cell culture plating for equilibration.