Multi-analyte biosensor interface for real-time monitoring of 3D microtissue spheroids in hanging-drop networks

Microfluidics is becoming a technology of growing interest for building microphysiological systems with integrated read-out functionalities.Here we present the integration of enzyme-based multi-analyte biosensors into a multi-tissue culture platform for‘body-on-a-chip’applications.The microfluidic platform is based on the technology of hanging-drop networks,which is designed for the formation,cultivation,and analysis of fluidically interconnected organotypic spherical three-dimensional(3D)microtissues of multiple cell types.The sensor modules were designed as small glass plug-ins featuring four platinum working electrodes,a platinum counter electrode,and an Ag/AgCl reference electrode.They were placed directly into the ceiling substrate from which the hanging drops that host the spheroid cultures are suspended.The electrodes were functionalized with oxidase enzymes to enable continuous monitoring of lactate and glucose through amperometry.The biosensors featured high sensitivities of 322±41 nA mM^(−1) mm^(−2) for glucose and 443±37 nA mM^(−1) mm^(−2) for lactate;the corresponding limits of detection were below 10μM.The proposed technology enabled tissue-size-dependent,real-time detection of lactate secretion from single human colon cancer microtissues cultured in the hanging drops.Furthermore,glucose consumption and lactate secretion were monitored in parallel,and the impact of different culture conditions on the metabolism of cancer microtissues was recorded in real-time.

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.