Neuronal differentiation of NT2 cells in a fully controlled bioreactor. Neuronal differentiation was induced by addition of retinoic acid (RA) from day 3 onwards (RA treatments). Phase contrast photomicrographs of neurospheres harvested at day 9 (A), day 16 (B) and day 23 (C) of the bioreactor culture. By day 23, neurosphere composition was analyzed by cryosection immunofluorescence microscopy - double labeling of nestin (red) and βIII-Tub (green) (C1). Harvested neurospheres were further cultured in mitotic inhibitory (MI) conditions, on poly-D-lysine and Matrigel-coated surfaces. Cultures were visualized by phase contrast microscopy 1 day after plating (D,E,F) and characterized by immunofluorescence microscopy 3 days after plating (G,H,I). Double labeling of nestin (red) and βIII-Tub (green). Phase contrast and immunofluorescence images of cultures derived from neurospheres harvested at day 9 (D,G), day 16 (E,H) and day 23 (F,I). Scale bars: 100 μm.