Increasing the amount of FGF2 passages of neural precursor cells has been shown to increase the yield of the neural induction38. Hence we explored the possibility of using higher passage numbers of NPCs by a limited amount of FGF2 passages (6 or 12 passages, passaging twice a week) before cryopreservation and final plating. Notably, live cell calcium imaging showed that even a limited amount of FGF2 passages (for 6 or 12 times) decreased neuronal network functionality (Fig. 3d), lowering the percentage of active neurons (p < 0.0001), bursting frequency (p < 0.0001) and synchronicity (p < 0.0001). Therefore, FGF2 passaging is not recommended and we did not use passaged NPCs for other experiments in this study.
