Real-Time Monitoring the Effect of Cytopathic Hypoxia on Retinal Pigment Epithelial Barrier Functionality Using Electric Cell-Substrate Impedance Sensing (ECIS) Biosensor Technology

Abstract

Disruption of retinal pigment epithelial (RPE barrier integrity is a hallmark feature of various retinal blinding diseases, including diabetic macular edema and age-related macular degeneration, but the underlying causes and pathophysiology are not completely well-defined. One of the most conserved phenomena in biology is the progressive decline in mitochondrial function with aging leading to cytopathic hypoxia, where cells are unable to use oxygen for energy production. Therefore, this study aimed to thoroughly investigate the role of cytopathic hypoxia in compromising the barrier functionality of RPE cells. We used Electric Cell-Substrate Impedance Sensing (ECIS) system to monitor precisely in real time the barrier integrity of RPE cell line (ARPE-19) after treatment with various concentrations of cytopathic hypoxia-inducing agent, Cobalt(II) chloride (CoCl2). We further investigated how the resistance across ARPE-19 cells changes across three separate parameters: Rb (the electrical resistance between ARPE-19 cells), α (the resistance between the ARPE-19 and its substrate), and Cm (the capacitance of the ARPE-19 cell membrane). The viability of the ARPE-19 cells and mitochondrial bioenergetics were quantified with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and seahorse technology, respectively. ECIS measurement showed that CoCl2 reduced the total impedance of ARPE-19 cells in a dose dependent manner across all tested frequencies. Specifically, the ECIS program’s modelling demonstrated that CoCl2 affected Rb as it begins to drastically decrease earlier than α or Cm, although ARPE-19 cells’ viability was not compromised. Using seahorse technology, all three concentrations of CoCl2 significantly impaired basal, maximal, and ATP-linked respirations of ARPE-19 cells but did not affect proton leak and non-mitochondrial bioenergetic. Concordantly, the expression of a major paracellular tight junction protein (ZO-1) was reduced significantly with CoCl2-treatment in a dose-dependent manner. Our data demonstrate that the ARPE-19 cells have distinct dielectric properties in response to cytopathic hypoxia in which disruption of barrier integrity between ARPE-19 cells precedes any changes in cells’ viability, cell-substrate contacts, and cell membrane permeability. Such differences can be used in screening of selective agents that improve the assembly of RPE tight junction without compromising other RPE barrier parameters.

Relative Contribution of Different Mitochondrial Oxidative Phosphorylation Components to the Retinal Pigment Epithelium Barrier Function: Implications for RPE-Related Retinal Diseases

“…First, to determine the effect of specific mitochondrial inhibitors on the spreading of ARPE-19 cells over the electrode, the capacitance across the ARPE-19 cells was calculated as a surrogate measurement ( Figure 2 , Figure 3 and Figure 4 ). To do so, the frequency at which the maximum ARPE-19 cell’s spreading over the electrode occurs was selected to be 64,000 Hz based on our previously published data [ 31 ]. Second, given that the capacitance across the cells decreases in an inverse relationship to the extent of spreading, determining the effects of the mitochondrial inhibitors on the electrical properties of the cells were best performed once the cells had finished spreading across the substrate, and thus had reached a state of electrical quiescence.…”

“…Next, we sought to determine the effect of specific mitochondrial inhibitors on the overall barrier functionality of ARPE-19 cells by measuring total electrical resistance across the cells. To achieve this goal, the frequency where the total resistance is at a maximum was determined to be 4000 Hz, as in our previously reported study [ 31 ]. Thereafter, the ARPE-19 cell groups’ total resistances were measured for 100 h after treatment with different mitochondrial inhibitors ( Figure 5 , Figure 6 and Figure 7 ).…”

“…When faced with mitochondrial inhibition and dysregulated energy production, the cell responds in a multiplicity of ways, with the cell behavior and expression response changing over time. The Electric Cell-substrate Impedance Sensing (ECIS) biosensing technology is uniquely positioned to investigate how the cells dynamically respond to mitochondrial insults, as it monitors the impedance of cells in real-time using a constant 1µA alternating current (AC) [ 30 , 31 ]. The ECIS also is capable of monitoring the spreading activity of cells via capacitance measurements, as well as monitor a cell monolayer’s total electrical resistance.…”

“…The ECIS also is capable of monitoring the spreading activity of cells via capacitance measurements, as well as monitor a cell monolayer’s total electrical resistance. The use of the ECIS then also extends to the investigation of multiple components of the RPE barrier, as the technology uses total resistance measurements to calculate electrical resistance at both the paracellular and basolateral cell surfaces as well as cell membrane capacitance [ 30 , 31 ].…”

Functional optimization of electric cell-substrate impedance sensing (ECIS) using human corneal epithelial cells

“…We had previously published on ECIS assessment of the human retinal pigment epithelial cell line, ARPE-19, which is widely used as an alternative for primary retinal pigment epithelial cells. This cell line is grown in DMEM/F12 medium as a standard 12,13 . Further, the literature revealed studies of different types of epithelial cells (retinal epithelial cells 12 and kidney epithelial cells 14 ) also utilized DMEM/F12 media supplemented with 10% FBS.…”