Figure 1.

Overview of the differentiation steps and experimental design. (A) Timeline of the development of the neurospheroids from the differentiation process to the transfer and growth onto the MEAs. (B) Main steps performed at DAY 3/DIV 0: Collection of cells and hanging-drop technique. Early-stage neurons and astrocytes are harvested the same day in two separate tubes. Consequently, cells are counted and mixed in a 1:1 ratio in a 1 mL vial at a final concentration of 5,330,000 cell/mL. Therefore, 15 µL of the cell suspension were plated on 15 µL medium drops in the lid of a petri dish. Finally, the lid was gently placed back on the petri dish, half-filled with DPBS, and then stored in the incubator. (C) Experimental protocol for chemical neuromodulation and electrical stimulation.