In this study, we found a significant reduction of Aβ40 and Aβ42 in the conditioned media of 3D neuro-spheroids exposed to BACE1 or γ-secretase inhibitor. This finding is consistent with findings in 2D cultures, indicating that the direction of the drug effect is identical under 2D and 3D conditions, as expected. However, the magnitude of reduction in 3D cultures was less than that observed in 2D neurons exposed to the same concentration of inhibitors. We quantified drug exposure of 3D neuronal spheroids after two-day of treatment and found it to be less than the dosing concentrations (Fig 9). We reasoned that neurons enclosed inside of spheroids had less overall exposure to drugs. During the early stages of treatment, before the concentration of compounds inside and outside of spheroids reach equilibrium, significant amounts of Aβ may have been generated inside the spheroids and eventually released to the media. The alternative interpretation of these data, that 3D configuration of neurons alters their response to drugs, is contradicted by our result showing the same direction of drug effect and is not supported by
