Experimental outline for NT2 cell sampling and characterization during expansion (A) and differentiation (B) in fully controlled bioreactors. (A) In expansion runs, cells were harvested from days 0 (inoculum), 3 and 6 and immediately characterized by flow cytometry. Harvested cells were plated on glass coverslips and processed for immunofluorescence microscopy analysis after 2 days or plated in tissue culture flasks for induction of neuronal differentiation. For this, cultures were treated with retinoic acid (RA) for 5 weeks, splitted and further cultured in mitosis inhibitory (MI) conditions. After 12 days in MI, the neurons were harvested, identified by immunofluorescence microscopy using neuronal markers and neuronal differentiation efficiencies were calculated. (B) In differentiation runs, the addition of RA was initiated at day 3 of bioreactor culture and prolonged for 3 weeks. Neurospheres were harvested at day 9, 16 and 23. The latest were analyzed by cryosection immunofluorescence microscopy. All neurosphere harvested were plated in static culture flasks and cultured in MI conditions. After 3 days, cultures were characterized by immunofluorescence microscopy and after 7 days and neuronal differentiation efficiencies were calculated.