Modeling and measuring glucose diffusion and consumption by colorectal cancer spheroids in hanging drops using integrated biosensors

As 3D in vitro tissue models become more pervasive, their built-in nutrient, metabolite, compound, and waste gradients increase biological relevance at the cost of analysis simplicity. Investigating these gradients and the resulting metabolic heterogeneity requires invasive and time-consuming methods. An alternative is using electrochemical biosensors and measuring concentrations around the tissue model to obtain size-dependent metabolism data. With our hanging-drop-integrated enzymatic glucose biosensors, we conducted current measurements within hanging-drop compartments hosting spheroids formed from the human colorectal carcinoma cell line HCT116. We developed a physics-based mathematical model of analyte consumption and transport, considering (1) diffusion and enzymatic conversion of glucose to form hydrogen peroxide (H 2 O 2 ) by the glucose-oxidase-based hydrogel functionalization of our biosensors at the microscale; (2) H 2 O 2 oxidation at the electrode surface, leading to amperometric H 2 O 2 readout; (3) glucose diffusion and glucose consumption by cancer cells in a spherical tissue model at the microscale; (4) glucose and H 2 O 2 transport in our hanging-drop compartments at the macroscale; and (5) solvent evaporation, leading to glucose and H 2 O 2 upconcentration. Our model relates the measured currents to the glucose concentrations generating the currents. The low limit of detection of our biosensors (0.4 ± 0.1 μM), combined with our current-fitting method, enabled us to reveal glucose dynamics within our system. By measuring glucose dynamics in hanging-drop compartments populated by cancer spheroids of various sizes, we could infer glucose distributions within the spheroid, which will help translate in vitro 3D tissue model results to in vivo.