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-dropintegrated 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 hangingdrop 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±0.1μM),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.