In situ electrochemical analysis of alkaline phosphatase activity in 3D cell cultures

•2D and 3D pyrolytic carbon microelectrodes were fabricated.•Gelatin hydrogels with Saos-2 cells were deposited on the electrodes.•Alkaline phosphatase activity in the 3D cell cultures was measured electrochemically.•Initial activity was higher in 3D cell cultures but decreased due to spheroid formation.•With 3D carbon microelectrodes 2-fold higher signals were recorded in the 3D cultures. In bioelectrochemistry, cells are typically cultured in monolayers on 2D electrodes and methods such as electrochemical impedance spectroscopy (EIS) or voltammetry are used to detect changes in the cell population or responses to external stimuli. Here, we implement an electrochemical assay for the in situ monitoring of alkaline phosphatase (ALP) activity of Saos-2 cells cultured in a 3D gelatin hydrogel matrix with a thickness of 300 µm deposited on pyrolytic carbon electrodes. After cell seeding, ALP activity for cells in the hydrogel was higher compared to 2D cell cultures probably due to unrestricted access to the enzyme on the complete cell membrane. Gradual decrease of cell proliferation, maturation and spheroid formation in the 3D cell culture resulted in slower increase and eventually a decrease in ALP activity over time. Finally, 3D microstructured electrodes and 3D carbon pillar microelectrodes with a height of 225 µm were fabricated and applied for ALP detection directly in the 3D cell cultures. This resulted in higher electrochemical signals compared to 2D electrode configurations due to increased electrode surface area and penetration of the microelectrodes into the cell-laden hydrogel. [Display omitted]