In situ electrochemical analysis of alkaline phosphatase activity in 3D cell 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 im-plement 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 unrestrict-ed access to the enzyme on the complete cell membrane. Gradual decrease of cell prolif-eration, maturation and spheroid formation in the 3D cell culture resulted in slower in-crease 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 fabricat-ed 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 elec-trode surface area and penetration of the microelectrodes into the cell-laden hydrogel.