Cellular Electrical Impedance Research Articles

Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

Despite the vaccination campaigns for COVID-19, we still cannot control the spread of SARS-CoV-2, as evidenced by the ongoing circulation of the Omicron variants of concern. This highlights the need for broad-spectrum antivirals to further combat COVID-19 and to be prepared for a new pandemic with a (re-)emerging coronavirus. An interesting target for antiviral drug development is the fusion of the viral envelope with host cell membranes, a crucial early step in the replication cycle of coronaviruses. In this study, we explored the use of cellular electrical impedance (CEI) to quantitatively monitor morphological changes in real time, resulting from cell-cell fusion elicited by SARS-CoV-2 spike. The impedance signal in CEI-quantified cell-cell fusion correlated with the expression level of SARS-CoV-2 spike in transfected HEK293T cells. For antiviral assessment, we validated the CEI assay with the fusion inhibitor EK1 and measured a concentration-dependent inhibition of SARS-CoV-2 spike mediated cell-cell fusion (IC50 value of 0.13 μM). In addition, CEI was used to confirm the fusion inhibitory activity of the carbohydrate-binding plant lectin UDA against SARS-CoV-2 (IC50 value of 0.55 μM), which complements prior in-house profiling activities. Finally, we explored the utility of CEI in quantifying the fusogenic potential of mutant spike proteins and in comparing the fusion efficiency of SARS-CoV-2 variants of concern. In summary, we demonstrate that CEI is a powerful and sensitive technology that can be applied to studying the fusion process of SARS-CoV-2 and to screening and characterizing fusion inhibitors in a label-free and non-invasive manner.

Cellular Electrical Impedance as a Method to Decipher CCR7 Signalling and Biased Agonism.

The human C-C chemokine receptor type 7 (CCR7) has two endogenous ligands, C-C chemokine ligand 19 (CCL19) and CCL21, displaying biased agonism reflected by a pronounced difference in the level of β-arrestin recruitment. Detecting this preferential activation generally requires the use of separate, pathway-specific label-based assays. In this study, we evaluated an alternative methodology to study CCR7 signalling. Cellular electrical impedance (CEI) is a label-free technology which yields a readout that reflects an integrated cellular response to ligand stimulation. CCR7-expressing HEK293 cells were stimulated with CCL19 or CCL21, which induced distinct impedance profiles with an apparent bias during the desensitisation phase of the response. This discrepancy was mainly modulated by differential β-arrestin recruitment, which shaped the impedance profile but did not seem to contribute to it directly. Pathway deconvolution revealed that Gαi-mediated signalling contributed most to the impedance profile, but Gαq- and Gα12/13-mediated pathways were also involved. To corroborate these results, label-based pathway-specific assays were performed. While CCL19 more potently induced β-arrestin2 recruitment and receptor internalisation than CCL21, both chemokines showed a similar level of Gαi protein activation. Altogether, these findings indicate that CEI is a powerful method to analyse receptor signalling and biased agonism.

Electrical impedance-based contractile stress measurement of human iPSC-Cardiomyocytes

Heart failure fundamentally results from loss of cardio myocyte contractility. Developing new methods that quantify the contractile stress of the human cardiomyocyte would facilitate the study of the molecular mechanism of heart failure and advance therapy development, to improve the current five year survival for these patients. The measurement of cellular electrical impedance measurement was recently applied to monitor cardiomyocyte beating rate and rhythm, for the study at cellular maturation, and for drug screening. However, due to the lack of a quantified relationship between the impedance signal and contractile stress, change of cardiomyocyte contractile stress cannot genuinely be quantified from impedance measurements. Here, we report the first quantitative relationship between contractile stress and impedance, which enables the accurate prediction of cardiomyocyte contractility using impedance signals. Through simultaneous measurement of beating human iPSC-cardiomyocytes using impedance spectroscopy and atomic force microscopy, a power-law relationship between impedance and contractile stress was established with a confidence level of 95%. The quantitative relationship was validated using pharmacology known to alter cardiomyocyte contractility and beating (verapamil, using clinically relevant concentrations of 0.05 μM, 0.10 μM, and 0.15 μM). The contractile stress values as measured by AFM were 9.04 ± 0.14 kPa (0.05 μM), 7.72 ± 0.11 kPa (0.10 μM) and 6.23 ± 0.17 kPa (0.15 μM), and as predicted by impedance using the derived power-law relationship were 9.39 kPa, 7.76 kPa, and 6.05 kPa with a relative error of 3.73%. Our power-law relationship is the first to describe a quantitative correlation between contractile stress and impedance, broadening the application of electrical impedance measurement for characterizing complex cardiac functions (beating rate, beating rhythm and contractile stress).

Optimization of in vitro trophoblast assay for real-time impedimetric sensing of trophoblast-erythrocyte interactions in Plasmodium falciparum malaria.

Sequestration of Plasmodium falciparum-infected erythrocytes (IEs) is responsible for the pathophysiology of placental malaria, leading to serious complications such as intrauterine growth restriction and low birth weight. However, it is an experimental challenge to study the biology of human placenta. Conventional cell culture-based in vitro placental models rely on immunostaining techniques and high-magnification microscopy is limited in providing real-time quantitative analysis. Impedimetric sensing in combination with cell culture may offer a useful tool. In this paper, we report that real-time label-free measurement of cellular electrical impedance using xCELLigence technology can be used to quantify the proliferation, syncytial fusion, and long-term response of BeWo cells to IEs cytoadhesion. Specifically, we optimized key experimental parameters of cell seeding density and concentration of forskolin, a compound used to promote cell syncitiation, based on electrical signals and immunostaining results. Prolonged time of infection with IEs that led to cell-cell junction vanishment in BeWo cells and release of inflammatory cytokines were monitored in real time by continuous change in electrical impedance. The results suggest that the impedimetric technique is sensitive and can offer new opportunities for the study of cellular responses of trophoblast cells to IEs. The developed system can provide potentially a high-throughput screening tool of anti-adhesion or anti-inflammatory drugs for placental malaria infections.

A Review of Electrical Impedance Characterization of Cells for Label-Free and Real-Time Assays

The aim of this review is to discuss label-free and real-time assays characterizing the electrical impedance of cells for biomedical and pharmacological applications. Electrical impedance measurements can be used to non-destructively monitor cellular behavior by analyzing the electrical cell-substrate impedance, which is determined by cellular morphology and distribution on the sensing electrode. Cellular electrical impedance measurements can be used to analyze the effects of various therapeutic agents, nanoparticles, naturally derived bioactive compounds, chemotaxins, or other stimulants on cellular cytotoxicity, motility, migration, invasion, and proliferation. This method is becoming increasingly widespread, with applications ranging from basic studies of the cell cycle, cell signaling, chronic disease, and pathophysiological mechanisms to smart cell monitoring and high-throughput screening, which are required by the pharmacological industry.

A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance.

Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus.

Gap junction blockage promotes cadmium-induced apoptosis in BRL 3A derived from Buffalo rat liver cells.

Gap junctions mediate direct communication between cells; however, toxicological cascade triggered by nonessential metals can abrogate cellular signaling mediated by gap junctions. Although cadmium (Cd) is known to induce apoptosis in organs and tissues, the mechanisms that underlie gap junction activity in Cd-induced apoptosis in BRL 3A rat liver cells has yet to be established. In this study, we showed that Cd treatment decreased the cell index (a measure of cellular electrical impedance) in BRL 3A cells. Mechanistically, we found that Cd exposure decreased expression of connexin 43 (Cx43), increased expression of p-Cx43 and elevated intracellular free Ca2+ concentration, corresponding to a decrease in gap junctional intercellular communication. Gap junction blockage pretreatment with 18β-glycyrrhizic acid (GA) promoted Cd-induced apoptosis, involving changes in expression of Bax, Bcl-2, caspase-3 and the mitochondrial transmembrane electrical potential (Δψm). Additionally, GA was found to enhance ERK and p38 activation during Cd-induced activation of mitogen-activated protein kinases, but had no significant effect on JNK activation. Our results indicated the apoptosis-related proteins and the ERK and p38 signaling pathways may participate in gap junction blockage promoting Cd-induced apoptosis in BRL 3A cells.

Assessment of molecular events during in vitro re-epithelialization under honey-alginate matrix ambience

Re-epithelialization is one of the most important stages of cutaneous regeneration and its success requires supportive micro-ambience which may be provided with suitable bio-matrix. Biocompatibility and efficacy of such bio-matrix in re-epithelialization could be explored by multimodal analysis of structural and functional attributes of in vitro wound healing model including evaluation of prime molecular expressions of the epithelial cells during repair. Present study examines the influence of honey-alginate and alginate matrices on re-epithelialization in keratinocyte (HaCaT) population in a 2-D wound model. Cellular viability, proliferation and cell–cell adhesion status were assessed during wound closure using live/dead cell assay and by evaluating expressions of Ki67, p63 and E-cadherin along-with % change in cellular electrical impedance. Efficacy of honey-alginate matrix in comparison to only alginate one was demonstrated by a quicker reduction in wound gap, improved cellular viability, enhanced expressions of Ki67, p63 and its isoforms (TAp63, ΔNp63) as well as E-cadherin. Faster restoration of electrical attribute (% of impedance change) after wounding also indicated better impact of honey-alginate matrix in re-epithelialization.

Transforming growth factor-β transiently induces vimentin expression and invasive capacity in a canine mammary gland tumor cell line

The epithelial–mesenchymal transition (EMT) is a crucial event that occurs during cancer metastasis and can be induced by transforming growth factor-β (TGF-β) in various tumor cells in vitro. However, little is known about the effects of TGF-β in canine mammary gland tumors (CMGTs). Here, we investigated the role of TGF-β in CMGT. We observed that treatment of the CMGT cell line CHMp13a with TGF-β1 leads to transient induction of the mesenchymal marker vimentin. Real-time measurements of cellular electrical impedance also showed that CMGT invasiveness is transiently increased by TGF-β1 treatment, but is reversed after prolonged stimulation. This phenomenon is similar to the mesenchymal–epithelial transition (MET, the reverse phenomenon of EMT), and a process that is implicated in the establishment of secondary metastatic lesions.

Use of cellular electrical impedance sensing to assess in vitro cytotoxicity of anticancer drugs in a human kidney cell nephrotoxicity model

Nephrotoxicity is one of the major concerns for anticancer drug safety because most drugs are metabolized and excreted by the kidneys. Convenient tools able to perform rapid in vitro cytotoxicity analysis and identify drug side effects in kidney cells during early phases of drug discovery could be beneficial to drug development programs. Here we developed an electrical cell-substrate impedance sensing system (ECIS) capable of continuously measuring the dosage and time response of human proximal tubular epithelial (HK2) cells exposed to four drugs throughout the experimental period. These drugs induced HK2 cell apoptosis/death in a dose-dependent manner, although with very different dose-response effects. DDP (50 μM) was the most cytotoxic and induced obvious HK2 cell apoptosis rapidly after exposure. The other three drugs had much lower cytotoxicity, even at concentrations approaching 1 mM. The results obtained from our ECIS system correlated well with conventional in vitro assays such as flow cytometry and cell viability assays. Notably, the continuous and automatic measurements provided by ECIS system allow for better resolution for drugs with different temporal toxicity profiles. Furthermore, we investigated the effect of DDP's antidotes, glutathione and sodium subsulfite, on DDP-induced cytotoxicity, both of which decreased nephrotoxicity of DDP in a dose-dependent manner. Overall this study illustrates the convenience of ECIS for direct, continuous assessment of the cytotoxicity of anticancer drugs in vitro. ECIS has the potential to become a useful, non-invasive analytical method for early evaluation of drugs and antidotes of toxins.

Electrical Impedance as a Novel Biomarker of Myotube Atrophy and Hypertrophy

Measuring myotube thickness is a physiological and unbiased approach for screening therapeutic compounds that prevent skeletal muscle atrophy or induce hypertrophy. However, an accurate cell thickness estimate is often quite challenging because of the extreme heterogeneity of the myotube cellular population and therefore the lack of a regular distribution of perturbed myotubes. Here the authors present a novel method to evaluate changes in myotube thickness via measuring cellular electrical impedance. They demonstrate that both qualitative and quantitative changes in electrical impedance as a function of cellular adhesion in real time correlate well with variation in myotube thickness caused by atrophy or hypertrophy agents. Conversely, pharmacologically blocking myotube hypertrophy prevents changes in electrical impedance. Thus, impedance can be used as a reliable and sensitive biomarker for myotube atrophy or hypertrophy. Application of this technique to drug screening might be beneficial in finding novel treatments preventing muscle atrophy and other diseases associated with any morphological change in cell shape.

Cellular electrical impedance spectroscopy: an emerging technology of microscale biosensors

Electrical impedance spectroscopy, owing to its label-free, noninvasive and easy miniaturization nature, has shown great potential in cellular biochemical sensing and cell-based diagnostics. In this article, cutting-edge technologies of electrical impedance spectroscopy are reviewed according to different sensing mechanisms, including monitoring of ion release from live cells, utilization of nonconductive cellular membranes, and detection of the spatial distribution of cells. The most successful applications where the electrical impedance sensors have been proven effective are introduced, including investigation of anticancer drug therapies, wound healing, cytotoxicity evaluation and measurement of blood rheological behavior. Furthermore, the research advances that yield enhanced sensitivities and functionalities are introduced. Finally, the commercialization challenges and the critical barriers that hinder the sustaining development of cellular electrical impedance spectroscopy are discussed.

Distinguishing between activated and nonactivated eosinophils by AC impedance measurements.

A cellular electrical impedance device which can detect the activated state of eosinophils has been developed and tested. This impedance device consists of a small gold electrode (50 microns x 50 microns) and a large gold electrode (1.5 cm x 0.5 cm) on a glass substrate, and it was fabricated by standard photolithographic techniques. Eosinophils, which belong to the granulocytic class of white blood cells, exhibit different physical properties when they change from the nonactivated state to the activated state. Hypothetically, these changes should correspond to a change in the measured electrical impedance. In this paper, data from the measured electrical impedance of eosinophils is presented. The measurements show that the average impedance of the activated eosinophils is 26% lower than the average impedance of the nonactivated eosinophils. Statistical analysis of the measured data shows that there is a significant difference between the measured impedances of activated and nonactivated eosinophils.