Real-Time Bioimpedance Sensing of Antifibrotic Drug Action in Primary Human Cells

Abstract

Fibrotic diseases are among the most serious health issues with severe burdens due to their chronic nature and a large number of patients suffering from the debilitating effects and long-term sequelae. Collagenase treatment is a nonsurgical option but has limited results. To date, there is no potent noninvasive solution for fibrosis. Part of the reason for this is the lack of appropriate in vitro live cell screening tools to assess the efficacy of new therapeutical agents. Here, we demonstrate the utility of a cell-based electrochemical impedance biosensor platform to screen the efficacy of potential antifibrotic compounds. The platform employs a label-free and noninvasive strategy to detect the progression of fibrosis and the potency of the antifibrotic molecules in real-time. The fundamental principle that governs this novel system is that dynamic changes in cell shape and adhesion during fibrosis can be measured accurately by monitoring the changes in the impedance. This is achieved by growing the cells on a transparent interdigitated indium tin oxide (ITO) electrodes. It was demonstrated by monitoring the efficacy of a model antifibrotic compound, PXS64, on cells collected from patients with Dupuytren's contracture. We confirmed the validity of the developed biochemical impedance biosensor as an tool for in vitro screening of antifibrotic compounds and provided quantitative information on subcellular influences of the examined chemical molecules using correlative microscopy analyses that monitor the average cell area, cell morphology, and the amount and directionality of the deposited extracellular matrix protein collagen and measurement of cytosolic Ca changes.

TGF‐β‐induced fibrotic stress increases G‐quadruplex formation in human fibroblasts

“…By monitoring Fura-2 AM fluorescence, we observed a significant~3.9-fold increase in intracellular Ca 2+ levels in stimulated scar-forming Dupuytren fibroblasts compared to unstimulated controls, confirming an increased flux in intracellular Ca 2+ during scar formation ( Fig. 2A) [27]. Significant increase in intracellular Ca 2+ levels was also observed in primary fibroblasts obtained from healthy individuals following TGF-b stimulation (Fig.…”

“…A–C). This reduction in collagen 1 deposition was also associated with a significant ~ 72.6% decrease in intracellular Ca 2+ levels and ~ 73% reduction in BG4 foci (Fig. ), further confirming the association between scar formation and G‐quadruplexes.…”

“…The amount of intracellular calcium in cells was measured using Fura‐2 AM fluorescence as previously described . Fluorescence at λ ex 340 and 380 nm and λ em 510 nm emission was measured on an inverted Nikon TE2000‐U microscope at 1‐min intervals with an exposure of 50 ms using a Hamamatsu Orca ER digital camera (Hamamatsu Photonics, Hamamatsu city, Japan) at 37 °C.…”

Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy

“…[8,9] Herein, fluorescence microscopy was coupled with aconventional three electrode electrochemical cell to investigate the potential dependent redox switch of organic fluorophores, thus demonstrating ap owerful tool to characterize the emissive properties of fluorescent and redox active molecules. This typically means ITO, [20][21][22][23][24][25] although studies of less common transparent materials are of interest. [15] This often means the fluorescent species are immobilized.…”

“…[18,19] Secondly,s urfaces that are both compatible with high-resolution light microscopy and are electroactive are required. This typically means ITO, [20][21][22][23][24][25] although studies of less common transparent materials are of interest. [9,26] Of the molecules that have been shown to undergo electrochemical switching of their fluorescence, improvements are needed in photostability [27] and fluorescence quantum yields.…”