6 well adherent plates

Human Schwann cells (SCs) derived from human embryonic spinal nerve (Cat. No.10HU-188, Lot no. 200240 ) at passage 1 were obtained from iXCells Biotechnologies, USA and expanded to passage 4 in 1:1 Dulbecco's Modified Eagles Medium/Nutrient mixture F-12 (D8437, Sigma-Aldrich, UK) supplemented with 10% FBS (Gibco, UK), 2% penicillin/streptomycin (Sigma-Aldrich, UK) and 1% Amphotericin B (Gibco, UK). The SCs were seeded at a density of 5 × 10 4 cells per well in 6 well-adherent plates (Corning, Costar, UK) 24 h prior to transfection (n=3 replicates). 1 h prior to transfection, the media was removed and replaced with OptiMEM (Gibco, UK). In the meantime, polyplex particles were formulated by mixing a specified amount of branched 25kDa PEI (Sigma-Aldrich, Ireland) and pDNA (fixed at a dose of 2 μg) to give an N/P ratio of 10. The PEI-pDNA polyplexes were then suspended in OptiMEM and added to the monolayer and incubated at 37°C for 15 min. After 15 min, an additional 1 ml of OptiMEM was added and the cells were incubated for approximately 4 h. After this transfection period, the OptiMEM was aspirated out and the cell monolayer was rinsed with PBS before being supplemented with complete media and incubated at 37 0 C to allow expression of the transgene.

MSCs were seeded at a density of 5x10 4 cells per well in 6 well adherent plates (Corning, Costar, Ireland) 24 h prior to transfection. Media was removed from cells 1 h prior to transfection and cells were washed in PBS (Sigma-Aldrich, Ireland) and 1 mL of OptiMEM (Gibco, Ireland) was added.
Nanoparticles were made as described in Section 2.1.2 and, following complexation, OptiMEM was added in a 1:1 ratio to the nanoparticle mixture to produce the transfection medium of which 500 µL was added to each well. After 4 h, transfection media was removed, cells were washed twice in PBS and growth media was replenished.