4.8. Neurospheres Migration Assay

Neural stem cells (NSCs) composing the neurospheres were cultured using adapted protocols [64,65]. Cell viability was assessed using the MTT assay from adapted protocols of Bresciani et al. [66]. NSCs were first proliferated in standard DMEM medium supplemented with epidermal growth factors E9644 (EGF, Sigma, Saint-Louis, MO, USA) until confluence was reached (about 5–6 days). Cells were detached using Trypsin-EDTA 25200-056 (Gibco, Waltham, MA, USA) and seeded in 96-well Corning Ultra low attachment plates 7007 (Corning Inc., Corning, NY, USA) at 1.25 × 10⁶ cells. The medium was changed every two days by removing half of the medium, and the plates were shaken at 150 rpm for 10 min before adding fresh DMEM medium enriched with EGF. Once neurospheres were well-formed, the medium was replaced with DMEM/F12 21331-020 (Gibco, Waltham, MA, USA) containing 1% P/S supplemented with 1% N2 17502-048 (Gibco, Waltham, MA, USA) to allow differentiation into neuroglial cells. Neurospheres were then transferred into a 24-well plate with a glass coverslip coated with Poly-L-Lysine P7405 (PLL, Sigma, Saint-Louis, MO, USA) and matrigel^® 356237 (BD Biosciences, Heidelberg, Germany) to allow migration.

Attached neurospheres were non-exposed (vehicle condition) or treated with 50 µg/mL of a purified extract of marennine from Haslea ostrearia during 48 h. These conditions correspond to the highest values of concentration and exposure duration used on 2D cultures. Phase-contrast photographs of each well were taken by a microscope using a 10× objective (Nikon ECLIPSE TS2, Nikon Inc., Melville, NY, USA). Measurements were performed according to the protocol previously described [67]. Briefly, to assess the size of each neurosphere, the area was measured. To determine the migration of cells, we measured the area covered by migrated cells using the Image J^® software (NIH Image, Bethesda, MD, USA [68]).