One possibility for this discrepancy is the bioavailability of the inhibitors. In the 2D environment, all neurons are exposed to the same concentration of inhibitors evenly; in 3D environment, surface neurons within each spheroid may be exposed to higher drug concentrations than internal cells. To determine whether these two inhibitors were permeable to neuro-spheroids, we collected the same number of neuronal spheroids exposed to BACE1 or γ-secretase inhibitor and extracted drugs for LC-MS/MS quantification (Fig 9C). We found that these compounds accumulated and remained inside of neuro-spheroids at ~30% (γ-secretase inhibitor, Fig 9C, red) to ~40% (BACE1 inhibitor, Fig 9, blue) of dosing concentration, suggesting that the reduction of drug efficacy was related to decreased exposure to drugs.
