*4.3. Cell Culture Conditions and Differentiation*

The differentiation process of PC12 cells was performed according to the protocol established by Greene and Tischler with some modifications [58]. PC12 cells were grown in 25 or 75-cm2 culture flasks in a CO2-incubator (37 ◦C, 5% CO2 and 95% humidity) in RPMI-1690 medium supplemented with 10% FBS and 100 μg/mL penicillin-streptomycin. The cells were used at logarithmic growth between passage 4 and 20. The PC12 cells were dissociated with TrypLE (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), seeded on type I collagen-coated 96-well plates in a concentration of 5 × 103 cells per well and incubated for 24 h prior to differentiation in order to let the cells adhere to the plates' surface. For differentiation, cells were treated with 100 ng/mL of NGF-*β* freshly dissolved in RPMI 1640 media supplemented with 2% FBS and 100 μg/mL penicillin-streptavidin. For the bioassays, cells were treated with NGF-*β* for 5 days. Medium and NGF were replenished every 48 h. The differentiation process was analyzed with a holo-tomographic microscope (3D Cell Explorer, Ecublens, Switzerland). The morphological changes of NGFtreated PC12 cells versus untreated PC12 cells are shown in Figure 8. Further, the described experiments were performed after 5 days of human NGF-*β*-induced differentiation.

**Figure 8.** The representative microphotograph of a non-differentiated PC12 cells (**A**); the representative microphotographs of PC12 cells differentiated into neuron-like cells: after 2 days (**B**), after 4 days (**C**), and after 5 days (**D**) of incubation with human NGF-*β*. The morphological changes during the differentiation process were analyzed with the 3D Cell Explorer free-label microscope.
