We adjusted the component concentrations to match those of human cerebrospinal fluid, providing metabolic substrates necessary for individual cell types growing in the absence of others. Ionic concentrations were chosen to match human cerebrospinal fluid osmolality 45, thus providing extracellular sodium concentrations necessary for proper electrophysiological function. We followed a rationale matched by recent efforts to improve electrophysiological activity in iPS derived neurons46. Iron salts were excluded, instead using iron-loaded transferrin in Neurobasal, in order to avoid dangerous redox cycling through Fenton chemistry. Glutamate was omitted to avoid the excitotoxicity of this amino-acid in dissociated cultures 47. We included additional pyruvate48, and lactic acid 49 as energy-providing and neuroprotective substrates in addition to the more canonical oxidative substrates (glucose, galactose and glutamine/Glutamax). Biotin and lipid-loaded albumin (Albumax) were added to support de novo synthesis lipid bilayers (e.g. axons, myelin sheaths).
