Control protocol for robust in vitro glial scar formation around microwires: Essential roles of bFGF and serum in gliosis

Previously, we reported an in vitro cell culture model that recreates many of the hallmarks of glial scarring around electrodes used for recording in the brain; however, the model lacked the reproducibility necessary to establish a useful characterization tool. This methods paper describes a protocol, modeled on protocols typically used to culture neural stem/precursor cells, that generates a predictable positive control of an intense scarring reaction. Six independent cell culture variables (growth media, seeding density, bFGF addition day, serum concentration in treatment media, treatment day, and duration of culture) were varied systematically and the resulting scars were quantified. The following conditions were found to give the highest level of scarring: Neurobasal medium supplemented with B27, 10% fetal bovine serum at treatment, 10 ng/ml b-FGF addition at seeding and at treatment, treatment at least 6 days after seeding and scar growth of at least 5 days. Seeding density did not affect scarring as long as at least 500,000 cells were seeded per well, but appropriate media, bFGF, and serum were essential for significant scar formation-insights that help validate the in vitro-based approach to understanding glial scarring. With the control protocol developed in this study producing a strong, reproducible glial scarring positive control with every dissection, this culture model is suitable for the in vitro study of the mechanisms behind glial scarring and neuroelectrode failure.