2.3. In Vitro Cytotoxicity Testing

All handling, housing, and surgical procedures of the mice were approved by the University of Texas Institutional Animal Care and Use Committee. Cytotoxicity assays were carried out as previously described [42] and in accordance with the ISO protocol “10993-5: Biological evaluation of medical devices” using both NTC 929 fibroblasts (ATCC, Manassas, VA, USA) and embryonic day 15 (E15) mouse-derived cortical neurons. Briefly, 50 and 100% concentration LCE extract was evaluated against Tygon-F-4040-lubricant tubing extract (positive control) and cell medium (negative control) [42]. In accordance with the ISO protocol, materials were said to ‘pass’ if normalized cell viability percentages exceeded 70% following 24-h incubation with material extracts.

Cortices were surgically dissected from E18 mouse embryos, dissociated, and cultured as previously described [4,5]. Prior to seeding, 24 well polystyrene plates (Greiner Bio-One, Kremsminster, Austria) were treated with 50 μg/mL poly-d-lysine (PDL) (Sigma-Aldrich, Saint Louis, MO, USA) and 20 μg/mL laminin to facilitate cell adhesion. Cells were seeded at a density of 100,000 cells/well and incubated at 37 °C, 10% CO₂, and 95% humidity in proliferation medium (Dulbecco’s Modified Eagle Medium, GlutaMAX, B-27, ascorbic acid, and 10% horse serum). Serum was reduced to 0% over the span of 5 days to avoid over-proliferation and ganglionation of supporting cells. Fibroblasts were sub-cultured prior to seeding in a 24 well polystyrene plate. Cells were incubated at 37 °C, 10% CO₂, and 95% humidity in complete medium (Dulbecco’s Modified Eagle Medium and 10% horse serum). Material extracts were made by soaking strips of LCE (3 cm²/mL) in normal cell medium (Dulbecco’s Modified Eagle Medium) at 37 °C, 10% CO₂, and 95% humidity for 24 h. When cells formed a semi-confluent layer, cell medium was exchanged for 50% or 100% material extract concentrations. Cells were incubated in extract for 24 h prior to using a LIVE/DEAD cytotoxicity kit for mammalian cells according to manufacturer’s protocol (Thermo Fisher, L3324, Waltham, MA, USA). Briefly, cells were stained with 2 μM Calcein-AM and 4 μM Ethidium homodimer for live and dead cells, respectively. Images were collected using a 10× objective on an inverted microscope (Nikon Ti eclipse, Nikon, Tokyo, Japan). Cell counts were carried out using a boutique ImageJ (NIH, Bethesda, MD, USA) macro, which applied a 2.0 Gaussian blur before locating local intensity maxima. Cells that were stained with both dyes were marked as “dual-stained” and were regarded as part of the dead cell count based on a MATLAB code.