Real-time characterization of uptake kinetics of glioblastomavs.astrocytes in 2D cell culture using microelectrode array

Abstract

Extracellular measurement of uptake/release kinetics and associated concentration dependencies provides mechanistic insight into the underlying biochemical processes. Due to the recognized importance of preserving the natural diffusion processes within the local microenvironment, measurement approaches which provide uptake rate and local surface concentration of adherent cells in static media are needed. This paper reports a microelectrode array device and a methodology to measure uptake kinetics as a function of cell surface concentration in adherent 2D cell cultures in static fluids. The microelectrode array simultaneously measures local concentrations at five positions near the cell surface in order to map the time-dependent concentration profile which in turn enables determination of surface concentrations and uptake rates, via extrapolation to the cell plane. Hydrogen peroxide uptake by human astrocytes (normal) and glioblastoma multiforme (GBM43, cancer) was quantified for initial concentrations of 20 to 500 μM over time intervals of 4000 s. For both cell types, the overall uptake rate versus surface concentration relationships exhibited non-linear kinetics, well-described by a combination of linear and Michaelis-Menten mechanisms and in agreement with the literature. The GBM43 cells showed a higher uptake rate over the full range of concentrations, primarily due to a larger linear component. Diffusion-reaction models using the non-linear parameters and standard first-order relationships are compared. In comparison to results from typical volumetric measurements, the ability to extract both uptake rate and surface concentration in static media provides kinetic parameters that are better suited for developing reaction-diffusion models to adequately describe behavior in more complex culture/tissue geometries. The results also highlight the need for characterization of the uptake rate over a wider range of cell surface concentrations in order to evaluate the potential therapeutic role of hydrogen peroxide in cancerous cells.

Fabrication and ex vivo evaluation of activated carbon–Pt microparticle based glutamate biosensor

“…We may be able to further elucidate on the characteristics of astrocyte-mediated glutamate concentration gradient by printing a linear array of glutamate biosensors and measuring at specific distances simultaneously. The measurement from biosensor array may be useful for quantifying the relationship between the glutamate concentration at the cell surface and the glutamate uptake rate [14,38,39].…”

Ex vivo electrochemical measurement of glutamate release during spinal cord injury

“…Connect working, reference and counter electrodes to the potentiostat with test hook clips.Apply +0.5 V to the working electrode versus the pseudo-Ag/AgCl reference electrode and record current. Note: The choice of holding potential depends on the electrochemical sensor used. Applying +0.5 V vs. Ag/AgCl is an adequate holding potential oxidase/Pt-based electrochemical biosensors [1,8,9]. Another holding potential typically used for this class of biosensors is +0.7 V vs. Ag/AgCl.Wait at least 20 min (1200 s) after applying the 0.5 V potential for non-Faradaic current to decrease.At 20 min (1200 s), simulate spinal cord injury by compressing about 70 N with forceps for 5 s at the part of the spinal cord segment immediately in front of where the glutamate biosensor is inserted (Fig.…”

“…Note: The choice of holding potential depends on the electrochemical sensor used. Applying +0.5 V vs. Ag/AgCl is an adequate holding potential oxidase/Pt-based electrochemical biosensors [1,8,9]. Another holding potential typically used for this class of biosensors is +0.7 V vs. Ag/AgCl.…”