Impedance Imaging of Cells and Tissues:Design and Applications

Due to their label-free and noninvasive nature,impedance measurements have attracted increasing interest in biological research.Advances in microfabrication and integrated-circuit technology have opened a route to using large-scale microelectrode arrays for real-time,high-spatiotemporal-resolution impedance measurements of biological samples.In this review,we discuss different methods and applications of measuring impedance for cell and tissue analysis with a focus on impedance imaging with microelectrode arrays in in vitro applications.We first introduce how electrode configurations and the frequency range of the impedance analysis determine the information that can be extracted.We then delve into relevant circuit topologies that can be used to implement impedance measurements and their characteristic features,such as resolution and data-acquisition time.Afterwards,we detail design considerations for the implementation of new impedance-imaging devices.We conclude by discussing future fields of application of impedance imaging in biomedical research,in particular applications where optical imaging is not possible,such as monitoring of ex vivo tissue slices or microelectrode-based brain implants.

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.

Integrating impedance-based growth-rate monitoring into a microfluidic cell culture platform for live-cell microscopy

Growth rate is a widely studied parameter for various cell-based biological studies.Growth rates of cell populations can be monitored in chemostats and micro-chemostats,where nutrients are continuously replenished.Here,we present an integrated microfluidic platform that enables long-term culturing of non-adherent cells as well as parallel and mutually independent continuous monitoring of(i)growth rates of cells by means of impedance measurements and of(ii)specific other cellular events by means of high-resolution optical or fluorescence microscopy.Yeast colonies were grown in a monolayer under culturing pads,which enabled high-resolution microscopy,as all cells were in the same focal plane.Upon cell growth and division,cells leaving the culturing area passed over a pair of electrodes and were counted through impedance measurements.The impedance data could then be used to directly determine the growth rates of the cells in the culturing area.The integration of multiple culturing chambers with sensing electrodes enabled multiplexed long-term monitoring of growth rates of different yeast strains in parallel.As a demonstration,we modulated the growth rates of engineered yeast strains using calcium.The results indicated that impedance measurements provide a label-free readout method to continuously monitor the changes in the growth rates of the cells without compromising high-resolution optical imaging of single cells.