Real-time Impedance Research Articles

Label-free monitoring of embolotherapy via catheter electrochemical impedance spectroscopy

Catheter-based embolization has become a widely adopted minimally-invasive treatment for a broad range of applications. However, assessment of embolization endpoints requires x-ray fluoroscopic monitoring, exposing patients and physicians performing embolization procedures to harmful ionizing radiation. Moreover, x-ray fluoroscopy assessment of embolization endpoints is low sensitivity, subjective, and may not reflect the actual physiology of blood flow reduction, thus providing little oversight of the embolization procedure. Inspired by the observation that the dielectric properties of blood differ from those of fluids injected during the embolization procedure, a customized angiographic catheter was created with embedded electrodes for catheter-based electrochemical impedance spectroscopy as a way to monitor embolization. Real-time electrochemical impedance spectroscopy was performed in a phantom and compared to visual and videographic monitoring. Electrochemical impedance spectroscopy was able to sense endpoints of embolization, including stasis, reflux, and persistent flow. This new technique offers a label-free method of sensing embolization progress with potentially higher sensitivity and reproducibility compared to x-ray, as well as offer substantial reduction in x-ray exposure to patients and physicians.

219: Feasibility Study on Real-Time Bioelectric Impedance Monitoring for Evaluating Body Water Status in Murine Sepsis Models during Extracorporeal Membrane Oxygenation

219: Feasibility Study on Real-Time Bioelectric Impedance Monitoring for Evaluating Body Water Status in Murine Sepsis Models during Extracorporeal Membrane Oxygenation

Lymphotoxin beta-activated LTBR/NIK/RELB axis drives proliferation in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the intrahepatic (iCCA) or extrahepatic (eCCA) bile ducts with poor prognosis and limited treatment options. Prior evidence highlighted a significant contribution of the non-canonical NF-κB signalling pathway in initiation and aggressiveness of different tumour types. Lymphotoxin-β (LTβ) stimulates the NF-κB-inducing kinase (NIK), resulting in the activation of the transcription factor RelB. However, the functional contribution of the non-canonical NF-κB signalling pathway via the LTβ/NIK/RelB axis in CCA carcinogenesis and progression has not been established. Human CCA-derived cell lines and organoids were examined to determine the expression of NF-κB pathway components upon activation or inhibition. Proliferation and cell death were analysed using real-time impedance measurement and flow cytometry. Immunoblot, qRT-PCR, RNA sequencing and insitu hybridization were employed to analyse gene and protein expression. Four invivo models of iCCA were used to probe the activation and regulation of the non-canonical NF-κB pathway. Exposure to LTα1/β2 activates the LTβ/NIK/RelB axis and promotes proliferation in CCA. Inhibition of NIK with the small molecule inhibitor B022 efficiently suppresses RelB expression in patient-derived CCA organoids and nuclear co-translocation of RelB and p52 stimulated by LTα1/β2 in CCA cell lines. In murine CCA, RelB expression is significantly increased and LTβ is the predominant ligand of the non-canonical NF-κB signalling pathway. Our study confirms that the non-canonical NF-κB axis LTβ/NIK/RelB drives cholangiocarcinogenesis and represents a candidate therapeutic target.

Esophageal Per-Oral Endoscopic Myotomy (E-POEM): Future Directions and Perspectives.

POEM is a mature procedure endorsed by societal guidelines as a first line therapy for achalasia and spastic esophageal disorders. Nonetheless, several questions remain, including expanding indications for POEM, periprocedural evaluation and management, and the optimal POEM technique to enhance clinical success while mitigating risk for reflux. There is uncertainty regarding several technical aspects of the POEM myotomy; though aggregating evidence supports the use of real-time impedance planimetry to guide the myotomy. While post-POEM reflux remains a concerning long term sequela, there is an increasing focus on the potential role of endoscopic anti-reflux interventions. Lastly, with the widespread adoption of POEM, we continue to witness ongoing efforts to standardize post-procedural care and training in this procedure. POEM is no longer a novel but rather established procedure. Yet, this technique has continued to evolve, with the aim of optimizing treatment success while reducing adverse events and risk for post-procedural reflux.

Diffusion-based culture and real-time impedance monitoring of tumor spheroids in hydrogel microwells of a suspended membrane under microfluidic conditions

Tumor spheroids are widely studied for in vitro modeling of tumor growth and responses to anticancer drugs. However, current methods are mostly limited to static and perfusion-based cultures, which can be improved by more accurately mimicking pathological conditions. Here, we developed a diffusion-based dynamic culture system for tumor spheroids studies using a thin membrane of hydrogel microwells and a microfluidic device. This allows for effective exchange of nutrients and metabolites between the tumors and the culture medium flowing underneath, resulting in uniform tumor spheroids. To monitor the growth and drug response of the spheroids in real-time, we performed spectroscopic analyses of the system's impedance, demonstrating a close correlation between the tumor size and the resistance and capacitance of the system. Our results also indicate an enhanced drug effect on the tumor spheroids in the presence of a low AC electric field, suggesting a weakening mechanism of the spheroids induced by external perturbation.

A Blockchain-Based Secure Sharing Scheme for Electrical Impedance Tomography Data

Real-time electrical impedance tomography (EIT) data sharing is becoming increasingly necessary, due to the extensive use of EIT technology in various sectors, including material analysis, biomedicine, and industrial process monitoring. The prevalence of portable EIT equipment and remote imaging technology has led to a predominance of centralized storage, Internet protocol transmission, and certificates from certificate authorities (CA) in telemedicine data. This has resulted in compromised data security, network communication delays, high CA maintenance costs, increased risks of medical data privacy breaches, and low security. Therefore, this paper offers a consortia blockchain-based method for exchanging EIT data that addresses security and integrity concerns during data storage and exchange, while maintaining transparency and traceability. Proprietary re-encryption techniques are employed to guarantee traceability when exchanging anonymous data, enabling precise control over data access. This scheme serves to protect both data and identity privacy, as well as to trace the actual identity of potential malicious users, while also thwarting any coordinated efforts between partially trusted parties and data requesters seeking unauthorized access to confidential information. Additionally, a combination of blockchain and InterPlanetary File System (IPFS) distributed storage technology is utilized to ease the burden of EIT data storage. The feasibility and effectiveness of the proposed solution were validated through a series of experiments, demonstrating its ability to effectively prevent data tampering and misuse, reduce data management costs, and enhance the efficiency and quality of data sharing.

Assessing pulmonary function in ALS using electrical impedance tomography

Objective We sought to determine whether thoracic electrical impedance tomography (EIT) could characterize pulmonary function in amyotrophic lateral sclerosis (ALS) patients, including those with facial weakness. Thoracic EIT is a noninvasive, technology in which a multi-electrode belt is placed across the chest, producing real-time impedance imaging of the chest during breathing. Methods We enrolled 32 ALS patients and 32 age- and sex-matched healthy controls (HCs) without underlying lung disease. All participants had EIT measurements performed simultaneously with standard pulmonary function tests (PFTs), including slow and forced vital capacity (SVC and FVC) in upright and supine positions and maximal inspiratory and expiratory pressures (MIPs and MEPs, respectively). Intraclass correlation coefficients (ICCs) were calculated to assess the immediate reproducibility of EIT measurements and Pearson’s correlations were used to explore the relationships between EIT and PFT values. Results Data from 30 ALS patients and 27 HCs were analyzed. Immediate upright SVC reproducibility was very high (ICC 0.98). Correlations were generally strongest between EIT and spirometry measures, with R values ranging from 0.64 to 0.82 (p < 0.001) in the ALS cohort. There were less robust correlations between EIT values and both MIPs and MEPs in the ALS patients, with R values ranging from 0.33 to 0.44. There was no significant difference for patients with and without facial weakness. There were no reported adverse events. Conclusion EIT-based pulmonary measures hold the promise of providing an alternative approach for lung function assessment in ALS patients. Based on these early results, further development and study of this technology are warranted.

Abstract 3248: Metabolic rewiring shifts engineered TCR T cell bioenergetics to enhance cytotoxicity

Abstract Background: Adoptive T cell-based transfer has shown tremendous success in hematological cancers. However, an important obstacle to using cell and gene therapy for solid tumors is the aggressive tumor microenvironment (TME). To significantly improve prognosis of subjects undergoing immunotherapy, it is essential to incorporate unique methods to drive T cell energy production and metabolism towards empowering T cells to dismantle the suppressive TME. This can promote anti-tumor effects and persistence of the transfused engineered T cells. In the present study we present a simple metabolic preconditioning process that improves T cell performance and bioenergetics. Methods: Using the Agilent xCELLigence RTCA eSight, and Seahorse XF Analyzer we assessed the killing efficiency, and bioenergetics of engineered T-cells after pretreating with Arginine. Briefly, engineered TCR T-cells against MART-1 were generated by transducing CD3+ T-cells (Hemacare, Seattle, WA) with retrovirus SAMEN-DMF5 with a CD34 marker gene, against MART-1. The TCR T cells were pre-conditioned in 6mM Arginine for 7 days, followed by a killing assay using MART-1 expressing melanoma cell line as target cells (624.38) at the E:T ratio of 5:1. Target melanoma cells, 624.38 cells, were engineered to express a red-fluorescent nuclear protein. The comparison was made with transduced T-cells grown in RPMI (no added amino acid supplementation, and non-transduced T cells. The cytotoxicity assay was assessed using real-time impedance and live cell imaging data collected on xCELLigence RTCA eSight. Concurrently, the metabolic profiling of the engineered T-cells was examined by the XF96 Analyzer. Results: Supplementing the growth medium with 6 mM Arginine significantly increased the potency of MART-1 engineered T cells (up to 6-fold) and bioenergetic studies revealed increased spare respiratory capacity, basal respiration, and ATP production via mitochondrial respiration. Conclusions: Arginine pre-conditioning improved TCR T cell potency through metabolic rewiring favoring mitochondrial respiration. For Research Use Only. Not for use in diagnostic procedures RA45236.3483333333 Citation Format: Rashmi Pillai, Xiaoyu Zhang, Yama Abassi. Metabolic rewiring shifts engineered TCR T cell bioenergetics to enhance cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3248.

Abstract 4699: Evaluating extracellular matrix invasion and cytotoxicity of natural killer cells in a novel co-culture assay

Abstract Background: The complex tumor microenvironment (TME) in solid tumors presents unique challenges for lymphocytes used in cancer immunotherapy. The extracellular matrix (ECM) in TME can limit the recruitment of lymphocytes and restrict access to cancer cells. This study utilized Matrigel to study the kinetics of natural killer cell invasion and cytotoxicity. We hypothesized that the kinetics of tumor cell killing by the NKs would depend on the distance for invasion and rely on matrix-metalloproteinases (MMPs) for remodeling the ECM. Methods: Increasing volumes (50-100 µL/well) of Matrigel (6 mg/mL) representing increasing distance for invasion was layered over MCF7-red target tumor cells expressing nuclear-localized mKate2 (red fluorescent protein). NK92 (Creative Bioarray) or eGFP-NK92 (BPS Bioscience) were seeded over the solidified Matrigel layer at E:T of 3:1. The broad spectrum MMP inhibitor, Ilomostat (GM6001, 2 µM and 10 µM), was added to the wells to investigate to role of MMPs. Label-free impedance and live cell imaging data was collected in real time on xCELLigence RTCA eSight to evaluate NK cell invasion and cytotoxicity. Percent cytolysis was calculated on the RTCA software from normalized cell impedance readings and imaging data using the formula [(1-(treated/untreated)*100]. Results: Impedance reading increases as the MCF7-red cells settle, attach, and proliferate. The readings plateau as the cells reach confluence. The addition of Matrigel modulates the impedance signature of MCF7 cells. Importantly, Matrigel delays cytolysis by NK92 cells added at 24h. The delay increases with increasing volume of Matrigel representing increasing invasion distance. The time to achieve 60% killing with respect to controls (KT60) was 67, 76 and 89 hours for 50, 75 and 100 µL/well, respectively. Consistent with the role of MMPs in invasion, percent cytolysis calculated independently from normalized impedance readings and live cell imaging data confirmed both delayed and reduced target cell killing by GFP-NK92 cells in the presence of Ilomostat. Interestingly, only a few GFP-NK92 were detected by live cell imaging when there was increased target cell death. These included highly active NK cells that made multiple contacts with different MCF7 red targets in clusters suggesting serial killing activity. Conclusions: The observations that only a few NK92 cells are detected during the killing of susceptible target cells and that cytolysis is not abrogated by the inhibition of MMPs suggest a potential role for distal effectors like cytokines, independent of invasion. This is a novel 2.5D assay that demonstrates the potential of RTCA eSight platform for studying the invasion and cytotoxicity function of various immune cells through both real-time impedance recording and live-cell time-lapse imaging simultaneously. (For Research Use Only. Not for use in diagnostic procedures. RA45236.3472453704) Citation Format: Jyothi Thyagabhavan Mony, Xiaoyu Zhang. Evaluating extracellular matrix invasion and cytotoxicity of natural killer cells in a novel co-culture assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4699.

Amphetamine increases vascular permeability by modulating endothelial actin cytoskeleton and NO synthase via PAR-1 and VEGF-R

Abuse of amphetamine-type stimulants is linked to cardiovascular adverse effects like arrhythmias, accelerated atherosclerosis, acute coronary syndromes and sudden cardiac death. Excessive catecholamine release following amphetamine use causes vasoconstriction and vasospasms, over time leading to hypertension, endothelial dysfunction or even cardiotoxicity. However, immediate vascular pathomechanisms related to amphetamine exposure, especially endothelial function, remain incompletely understood and were analyzed in this study. Pharmaco-pathological effects of acute d-amphetamine-sulfate (DAM) were investigated ex vivo using contraction–force measurements of rat carotid artery rings and in vitro using label-free, real-time electrochemical impedance spectroscopy (EIS) on endothelial and smooth muscle cells. Specific receptor and target blocking was used to identify molecular targets and to characterize intracellular signaling. DAM induced vasodilation represented by 29.3±2.5% decrease in vascular tone (p<0.001) involving vascular endothelial growth factor receptor (VEGF-R) and protease activated receptor 1 (PAR-1). EIS revealed that DAM induces endothelial barrier disruption (−75.9±1.1% of initial cellular impedance, p<0.001) also involving VEGF-R and PAR-1. Further, in response to DAM, Rho-associated protein kinase (ROCK) mediated reversible contraction of actin cytoskeleton resulting in endothelial barrier disruption. Dephosphorylation of Serine1177 (−50.8±3.7%, p<0.001) and Threonine495 (−44.8±6.5%, p=0.0103) of the endothelial NO synthase (eNOS) were also observed. Blocking of VEGF-R and PAR-1 restored baseline eNOS Threonine495 phosphorylation. DAM induced vasodilation, enhanced vascular permeability and actin cytoskeleton contraction and induced eNOS hypophosphorylation involving VEGF-R, PAR-1 and ROCK. These results may contribute to a better understanding of severe adverse cardiovascular effects in amphetamine abuse.

Development of a real-time impedance matching system for ion cyclotron resonance heating in experimental advanced superconducting tokamak

To achieve stable operation of an ion cyclotron resonance heating (ICRH) system in the Experimental Advanced Superconducting Tokamak (EAST), a real-time impedance matching system needs to be established to respond to antenna load variation during long pulse discharges. A new impedance matching method based on capacitors was proposed in this study. By considering the reflected voltage of the transmission line as the feedback parameter, the real-time impedance-matching system can quickly control the motors based on a programmable logic controller to determine the minimum reflection voltage. A real-time impedance matching system was successfully used on the test platform in the laboratory and on the ICRH system in EAST. A significant result is that we can match the variable impedance within 1 s by suitably adjusting the motor controller to ensure high-power and long-pulse operation of the ICRH system to satisfy the requirements of the EAST experiment.

Towards inferring positioning of straight cochlear-implant electrode arrays during insertion using real-time impedance sensing.

Cochlear-implant electrode arrays (EAs) are currently inserted with limited feedback, and impedance sensing has recently shown promise for EA localisation. We investigate the use of impedance sensing to infer the progression of an EA during insertion. We show that the access resistance component of bipolar impedance sensing can detect when a straight EA reaches key anatomical locations in a plastic cochlea and when each electrode contact enters/exits the cochlea. We also demonstrate that dual-sided electrode contacts can provide useful proximity information and show the real-time relationship between impedance and wall proximity in a cadaveric cochlea for the first time. The access resistance component of bipolar impedance sensing has high potential for estimating positioning information of EAs relative to anatomy during insertion. Main limitations of this work include using saline as a surrogate for human perilymph in ex vivo models and using only one type of EA.

Online monitoring of epithelial barrier kinetics and cell detachment during cisplatin-induced toxicity of renal proximal tubule cells.

Real-time and non-invasive assessment of tissue health is crucial for maximizing the potential of microphysiological systems (MPS) for drug-induced nephrotoxicity screening. Although impedance has been widely considered as a measure of the barrier function, it has not been incorporated to detect cell detachment in MPS with top and bottom microfluidic channels separated by a porous membrane. During cell delamination from the porous membrane, the resistance between both channels decreases, while capacitance increases, allowing the detection of such detachment. Previously reported concepts have solely attributed the decrease in the resistance to the distortion of the barrier function, ignoring the resistance and capacitance changes due to cell detachment. Here, we report a two-channel MPS with integrated indium tin oxide (ITO) electrodes capable of measuring impedance in real time. The trans-epithelial electrical resistance (TEER) and tissue reactance (capacitance) were extracted from the impedance profiles. We attributed the anomalous initial increase observed in TEER, upon cisplatin administration, to the distortion of tight junctions. Cell detachment was captured by sudden jumps in capacitance. TEER profiles illuminated the effects of cisplatin and cimetidine treatments in a dose-dependent and polarity-dependent manner. The correspondence between TEER and barrier function was validated for a continuous tissue using the capacitance profiles. These results demonstrate that capacitance can be used as a real-time and non-invasive indicator of confluence and will support the accuracy of the drug-induced cytotoxicity assessed by TEER profiles in the two-channel MPS for the barrier function of a cell monolayer.

Validation of Electrical Impedance Technique in Real-Time Using the xCELLigence as an Interference-Free Method for Assessing Cytotoxicity of Silver and Hydroxyapatite Nanoparticles

This study demonstrated the utility of real-time electrical impedance, utilizing the xCELLigence system to assess the cytotoxic effects of silver nanoparticles (AgNPs) and hydroxyapatite nanoparticles (HANPs) on A549 cells. Method validation confirmed the accuracy and reliability of cell index (CI) measurements. The NPs were thoroughly characterized to confirm their elemental composition, size, and morphology through energy dispersive spectroscopy, transmission electron microscopy, and dynamic light scattering. Cytotoxicity assays revealed a concentration-dependent response. Exposure to AgNPs resulted in a significant change in CI, with a pronounced increase followed by a sharp decrease in the first few hours after treatment at the highest concentration of 50 μg mL–1. This pattern suggested an initial cell response to AgNPs exposure, possibly indicating mechanisms such as apoptosis or necrosis, followed by a decrease in cell adhesion. Conversely, HANPs demonstrated a tendency to restore CI at higher concentrations (1-100 μg mL–1) before 48 h, suggesting a potential recovery in cell proliferation capacity. This underscores the diverse cell responses to NPs, emphasizing the sensitivity of the method in detecting subtle cytotoxic effects. These findings highlight the efficacy of real-time electrical impedance in dynamic nanoparticle cytotoxicity assessments, surpassing traditional assays. Adapting to high-throughput screening enhances nanomaterial safety evaluations.

Endogenous bystander killing mechanisms enhance the activity of novel FAP-specific CAR-Tcells against glioblastoma.

CAR-T cells are being investigated as a novel immunotherapy for glioblastoma, but clinical success has been limited. We recently described fibroblast activation protein (FAP) as an ideal target antigen for glioblastoma immunotherapy, with expression on both tumor cells and tumor blood vessels. However, CAR-T cells targeting FAP have never been investigated as a therapy for glioblastoma. We generated a novel FAP targeting CAR with CD3ζ and CD28 signalling domains and tested the resulting CAR-T cells for their lytic activity and cytokine secretion function invitro (using real-time impedance, flow cytometry, imaging and bead-based cytokine assays), and in vivo (using a xenograft mimicking the natural heterogeneity of human glioblastoma). FAP-CAR-T cells exhibited target specificity against model cell lines and potent cytotoxicity against patient-derived glioma neural stem cells, even when only a subpopulation expressed FAP, indicating a bystander killing mechanism. Using co-culture assays, we confirmed FAP-CAR-T cells mediate bystander killing of antigen-negative tumor cells, but only after activation by FAP-positive target cells. This bystander killing was at least partially mediated by soluble factors and amplified by IL-2 which activated the non-transduced fraction of the CAR-T product. Finally, a low dose of intravenously administered FAP-CAR-T cells controlled, without overt toxicity, the growth of subcutaneous tumors created using a mixture of antigen-negative and antigen-positive glioblastoma cells. Our findings advance FAP as a leading candidate for clinical CAR-T therapy of glioblastoma and highlight under-recognised antigen nonspecific mechanisms that may contribute meaningfully to the antitumor activity of CAR-T cells.

Label-free high-throughput impedance-activated cell sorting.

Cell sorting holds broad applications in fields such as early cancer diagnosis, cell differentiation studies, drug screening, and single-cell sequencing. However, achieving high-throughput and high-purity in label-free single-cell sorting is challenging. To overcome this issue, we propose a label-free, high-throughput, and high-accuracy impedance-activated cell sorting system based on impedance detection and dual membrane pumps. Leveraging the low-latency characteristics of FPGA, the system facilitates real-time dual-frequency single-cell impedance detection with high-throughput (5 × 104 cells per s) for HeLa, MDA-MB-231, and Jurkat cells. Furthermore, the system accomplishes low-latency (less than 0.3 ms), label-free, high-throughput (1000 particles per s) and high-accuracy (almost 99%) single-particle sorting using FPGA-based high-precision sort-timing prediction. In experiments with Jurkat and MDA-MB-231 cells, the system achieved a throughput of up to 1000 cells per s, maintaining a pre-sorting purity of 28.57% and increasing post-sorting purity to 97.09%. These findings indicate that our system holds significant potential for applications in label-free, high-throughput cell sorting.

Digital Sensing Systems for Electromyography.

Surface electromyogram (EMG) signals find diverse applications in movement rehabilitation and human-computer interfacing. For instance, future advanced prostheses, which use artificial intelligence, will require EMG signals recorded from several sites on the forearm. This requirement will entail complex wiring and data handling. We present the design and evaluation of a bespoke EMG sensing system that addresses the above challenges, enables distributed signal processing, and balances local versus global power consumption. Additionally, the proposed EMG system enables the recording and simultaneous analysis of skin-sensor impedance, needed to ensure signal fidelity. We evaluated the proposed sensing system in three experiments, namely, monitoring muscle fatigue, real-time skin-sensor impedance measurement, and control of a myoelectric computer interface. The proposed system offers comparable signal acquisition characteristics to that achieved by a clinically-approved product. It will serve and integrate future myoelectric technology better via enabling distributed machine learning and improving the signal transmission efficiency.

Label-Free and Real-Time Electrical Impedance Monitoring of Macrophage Polarization of THP-1 Monocytes on Indium Tin Oxide Electrode

Label-Free and Real-Time Electrical Impedance Monitoring of Macrophage Polarization of THP-1 Monocytes on Indium Tin Oxide Electrode

Modeling and Real-Time Cartesian Impedance Control of 3-DOF Robotic Arm in Contact with the Surface

Modeling and Real-Time Cartesian Impedance Control of 3-DOF Robotic Arm in Contact with the Surface

Early Failure Detection for CO2 Reduction Electrolyzers Via in-Line Electrochemical Impedance Spectroscopy

The electrochemical CO2 reduction reaction (CO2RR) presents the opportunity to produce chemical feedstocks and fuels directly from CO2. Recent achievements in energy efficiency and current density are approaching the targets required for commercial viability; however, stability remains more than two orders of magnitude below the required 80,000 h. In CO2RR there is a challenge to distinguish the cause of failure among many possibilities. Failure can result from initial setup (e.g. over or under compression of the membrane and electrodes), gradual degradation of components (e.g. cathode and anode catalyst restructuring and dissolution), accumulation of products or byproducts (e.g. alcohols or salt accumulation), or immediate failures (e.g. a hole in the membrane or a short circuit). These failure modes must be addressed to reach the targeted stability needed for the wide-scale adoption of this technology. Early identification and mitigation of these failures would increase the electrolyzer lifetime and the commercial viability of CO2RR electrolyzers.Current techniques to characterize failures of the electrolyzer rely on post-mortem analysis of cell components. The analysis of individual components after operation may not accurately reflect their state in operando. There are techniques that are capable of analyzing the cell in operation (e.g. XAS, Raman, and TEM), but these typically require modified cell architectures and are operated at lower reaction rates. To effectively diagnose failure, the analysis technique should be applied to electrolyzers operating under realistic conditions.Here we implement an in-line real-time electrochemical impedance spectroscopy (EIS) technique to monitor CO2RR electrolyzers during operation. EIS is a non-destructive technique that can provide continuous information on the performance of specific components within the electrolyzer. We characterize common failure modes, such as poor compression, salt formation, catalyst degradation, and short circuits, and their identifying changes to the EIS response. We extract key electrochemical parameters from the EIS response and use them to develop a framework to identify and prevent the most common failure modes. Among other applications, this framework allowed for the detection of anode catalyst degradation 11 h before other indicators, such as cell voltage or product selectivity. This technique and framework can be applied to monitor CO2 electrolyzers as they are scaled and stacked.