TY - JOUR
AU - Anders,, Hans-Joachim
AB - Abstract Electric cell-substrate impedance sensing (ECIS) is a quantitative, label-free, non-invasive analytical method allowing continuous monitoring of the behaviour of adherent cells by online recording of transcellular impedance. ECIS offers a wide range of practical applications to study cell proliferation, migration, differentiation, toxicity and monolayer barrier integrity. All of these applications are relevant for basic kidney research, e.g. on endothelial cells, tubular and glomerular epithelial cells. This review gives an overview on the fundamental principles of the ECIS technology. We name strengths and remaining hurdles for practical applications, present an ECIS array reuse protocol, and review its past, present and potential future contributions to preclinical kidney research. acute kidney injury, cell culture, chronic kidney disease, stem cells, technology INTRODUCTION Electric cell-substrate impedance sensing (ECIS) is a quantitative, label-free and non-invasive analytical method that allows monitoring of the behaviour of adherent cells in culture by online recording of transcellular impedance. Since Giaever and Keese first reported the basic principles of ECIS in 1984, the system has been subsequently optimized to its current form [1]. Meanwhile, an increasing number of practical applications are in use in different research domains. While the physical theory underlying the ECIS technology appears complicated, experimentation, data acquisition and analysis are straightforward. This review provides an introduction to the physicochemical principles underlying the ECIS technology, lists strengths and remaining hurdles of this method, and introduces a range of practical applications in kidney research. In addition, we give an overview of the work being done with ECIS in the kidney domain and propose future perspectives with this evolving technology. PRINCIPLES OF ECIS Simply spoken, ECIS measures changes of voltages across cell monolayers that are acquired between two gold electrodes embedded into the cell culture dish (Figure 1). Different array formats accommodate different experimental strategies (Figure 1A) but the fundamental electrophysical principle remains identical. In a direct current (DC) system, Ohm’s law (V = R × I) describes the relationship between voltage (V), current (I) and resistance (R) within an electric circuit. But because a DC would damage both cells and electrodes in these types of arrays, an alternating current (AC) is applied, where Ohm’s law no longer applies. Under AC conditions, the magnitude of electrical impedance |Z| can be described using the actual resistance (R) and the reactance (X), also called reactive power: |Z|=√(R2+X2). The magnitude of the reactive power is dependent on frequency (f) and capacitance (C), which is described as X = (2 × π × f × C) − 1. The latter refers to the ability of an electrode to store an electrical charge. Hence, |Z| can be described using resistance and capacitance, rather than reactance. Taken from the equation above, impedance, resistance and capacitance change according to frequency. An ECIS device will classically deliver either capacitance or resistance as a readout value, dependent on the frequency chosen (Figure 1D and E). FIGURE 1 Open in new tabDownload slide Schematics of ECIS arrays and impedance measurement. (A–C) Schematics of ECIS arrays drawn to scale. (A) Typical eight-well ECIS-array, where each well is independently connected to the impedance measurement unit. (B) The number of electrodes varies between the different arrays (four examples shown), and hence the overall monitored area per well (given in square millimetre). (C) Independent from the array, each electrode is typically 250 µm in diameter. (D, E) Principles underlying ECIS-derived impedance measurements. An AC is generated between the electrode and the respective counter-electrode in the same well. To close the circuit, the electrical current is forced to travel through the medium. (D) The electrical current at frequencies <2 kHz is mainly travelling in between cells, thereby reflecting the barrier resistance (Rb), whereas at frequencies >40 kHz (E) it is forced through the cell bodies (Cm). FIGURE 1 Open in new tabDownload slide Schematics of ECIS arrays and impedance measurement. (A–C) Schematics of ECIS arrays drawn to scale. (A) Typical eight-well ECIS-array, where each well is independently connected to the impedance measurement unit. (B) The number of electrodes varies between the different arrays (four examples shown), and hence the overall monitored area per well (given in square millimetre). (C) Independent from the array, each electrode is typically 250 µm in diameter. (D, E) Principles underlying ECIS-derived impedance measurements. An AC is generated between the electrode and the respective counter-electrode in the same well. To close the circuit, the electrical current is forced to travel through the medium. (D) The electrical current at frequencies <2 kHz is mainly travelling in between cells, thereby reflecting the barrier resistance (Rb), whereas at frequencies >40 kHz (E) it is forced through the cell bodies (Cm). A cell adhering to an ECIS electrode acts like an electric insulator [2]. The more cells attached to the electrode, the higher the impedance. Vice versa, when (dying) cells detach from the electrode surface, impedance decreases. At the same time, capacitance decreases with cell growth, as cell coverage prevents the storage of an electrical charge inside the electrode. As the capacitance provides an overall measure of electrode coverage, it is a suitable parameter for measuring cell density or changes during cell proliferation, cell migration or cell detachment [2]. Resistance, on the contrary, is more indicative of the barrier function of cells, i.e. the integrity of cell–cell contacts [3, 4]. STRENGTHS OF ECIS Real-time monitoring and recording system ECIS allows quantification of cell–cell contacts and cell coverage in real time without mechanical manipulations (Figure 2A and D). This feature allows defininition of the ideal time point for initiating interventions such as applying an injurious stimulus. Upon that, the cellular response can be closely monitored and the duration of exposure adapted to the cellular response, i.e. changes in impedance (Figure 2B and E). This situation is often encountered when stimulating primary cells with highly cytotoxic, yet pathophysiological relevant agents, e.g. extracellular histones (Figure 2E). Continuous impedance monitoring by ECIS enables one to neatly quantify the level of injury and hence can be reproduced in future experiments. After an injury, the monolayer defect might close due to migration and proliferation of adjacent cells, which again is detectable via impedance. For example, the change of impedance in primary tubular epithelial cells treated with histones to induce cell injury allowed identification of the ideal time point at which to subsequently add recombinant interleukin 22 as a regeneration-promoting agent [5]. Here, ECIS proved to be a superior method allowing close titration and identification of adequate timing of injury and regeneration. Depending on cell type, culture conditions and injury settings, the real-time monitoring and recording of the cell population can be stretched up to several days. FIGURE 2 Open in new tabDownload slide Practical applications in ECIS. (A) Monitoring of cell attachment and proliferation after seeding cells in real time by measuring changes in impedance at multiple frequencies over time. (B) Quantification of effect sizes of chemical or electrical forms of injury and subsequent regeneration. (C) Standardization of cell migration studies by using an electric fence approach. (D) Quantifying differences in the doubling time of various cell types as a hallmark of cell characterization. (E) Precise titration of exposure times to injurious stimuli to achieve standardized cellular responses and to quantify regenerative processes in the same cell population. FIGURE 2 Open in new tabDownload slide Practical applications in ECIS. (A) Monitoring of cell attachment and proliferation after seeding cells in real time by measuring changes in impedance at multiple frequencies over time. (B) Quantification of effect sizes of chemical or electrical forms of injury and subsequent regeneration. (C) Standardization of cell migration studies by using an electric fence approach. (D) Quantifying differences in the doubling time of various cell types as a hallmark of cell characterization. (E) Precise titration of exposure times to injurious stimuli to achieve standardized cellular responses and to quantify regenerative processes in the same cell population. In-depth analysis of cell behaviour Changes of impedance can imply different interpretations depending on the frequency applied [6]. At frequencies up to 2 kHz most of the electric current is passing in between adjacent cells (i.e. barrier resistance, Rb) and below them (i.e. resistance under cells, Ru). Here, changes in impedance are indicative of subtle changes in morphology in the sub-nanometre to micrometre range, e.g. micromotion or cell–cell contacts [7]. At frequencies >40 kHz, the current mainly passes through the cell bodies (i.e. membrane capacitance, Cm), providing reliable information about cell coverage [8] (Figure 1D and E). Label-free and non-invasive assessment of barrier integrity Radio- or fluorescence-labelled markers are frequently used to investigate the barrier function of cell monolayers in vitro. While the sensitivity of radioisotopes is high, they require complex safety measures in handling and storing. Also, short half-lives of specific isotopes contradict long-term storage. Fluorophore-based probes, on the contrary, are often less sensitive for subtle changes in monolayer permeability and might suffer from instability and poor specificity [9]. In addition, tracer compounds per se can affect barrier integrity [9]. In contrast, ECIS is a label-free, non-invasive method with high sensitivity, minimal effort and low complexity in setup and handling [3–5]. Visual control of ECIS arrays During an ECIS, experiment cells can be observed microscopically, as the ECIS arrays are sufficiently thin to allow for bright field and phase contrast microscopy. For instance, Rother et al. were able to combine ECIS with high-quality fluorescence microscopy, assuring data efficacy and continuity [10]. REMAINING HURDLES Some technical hurdles remain. Commercially available ECIS arrays have a relatively small representative area (Figure 1B and C). Depending on the array size (e.g. 8- or 96-well plates) and the number of electrodes per well, the monitored area varies between only 0.06% and 4.9% in 8-well plates and 0.8% and 12.4% in 96-well plates, respectively. Thus, reliable measurements can only be obtained when the cells are homogenously distributed or have formed a continuous monolayer, which is notoriously difficult with primary cell isolates, especially isolates consisting of numerous cell types. ECIS arrays acquiring impedance readings from <1% of the covered surface area easily produce non-representative results. Increasing the number of replicates can address this problem, but another hurdle, the significant costs for each array, limit this evasion strategy. For example, Applied Biophysics sells a set of equipment at a price of 54 000–67 000 euros, and costs for a single experiment range from 88 to 645 euros, depending on the type of array. However, reusing arrays multiple times is feasible. Upon concluding an experiment, rinsing the wells with water, applying trypsin digestion for several hours, incubating for 2 h with 0.01% sodium hypochlorite, rinsing with water and sprinkling the arrays with 70% ethanol before air drying allows the reuse of ECIS arrays at least five times without relevant impact on data quality. It has to be kept in mind that ECIS measurements only provide indirect evidence on cell behaviour, without any information about processes at the molecular level. For example, distinguishing between hyperplasia and hypertrophy is impossible. When investigating drug effects on regeneration of a cell barrier after injury, additional techniques, such as cell cycle analyses and cell counting, are needed to draw valid conclusions and to avoid inadequate interpretations. Finally, not all cell types grow well on the gold-coated array plates. The use of different coatings can support cell growth and potentially helps to avoid artificial cellular responses, which was shown, for example, for ECIS cultures of podocytes and glomerular endothelial cells [11, 12]. Finding the appropriate conditions is usually a matter of trial and error that can be tedious and expensive. We summarize reported coating conditions for primary renal cells and cell lines in ECIS experiments in Table 1. Table 1 ECIS plate coatings used for renal cell lines or primary cells Cell type Species Coating References Cell lines  Podocytes transfected with large T antigen/SV40 Human Uncoated, cystein + fibronectin, cystein + collagen [13] [14] [15]  Glomerular endothelial cells Human Uncoated, cystein + collagen [14] [16]  Mesangial cells transfected with large T antigen/SV40 + human telomerase Human Uncoated [17]  LLC-pig kidney 1 cells Porcine Uncoated [18]  Human kidney 2 cells Human Uncoated [19]  Wistar rat kidney proximal tubule 0293 cells Rat Uncoated [20]  Tubular cells transfected with large T antigen/SV40 Mouse Uncoated [21]  Madin-Darby Canine Kidney (MDCK) cells Human Uncoated [22]  Norvegicus rattus kidney 52 cells Human Collagen IV, laminin [23]  Vero cells Monkey Uncoated [24] Primary cells  Podocytes Human Uncoated [4]  Podocytes Mouse Uncoated, collagen IV, fibronectin, BSA [4],[25] [11]  Glomerular endothelial cells Human Cystein, laminin, fibronectin, collagen IV [26] [12]  Glomerular endothelial cells Mouse Uncoated [27]  Tubular epithelial cells Mouse Uncoated [27]  Mixed kidney cells Rat Uncoated [28]  Renal progenitor cells Human Uncoated Unpublished own data Cell type Species Coating References Cell lines  Podocytes transfected with large T antigen/SV40 Human Uncoated, cystein + fibronectin, cystein + collagen [13] [14] [15]  Glomerular endothelial cells Human Uncoated, cystein + collagen [14] [16]  Mesangial cells transfected with large T antigen/SV40 + human telomerase Human Uncoated [17]  LLC-pig kidney 1 cells Porcine Uncoated [18]  Human kidney 2 cells Human Uncoated [19]  Wistar rat kidney proximal tubule 0293 cells Rat Uncoated [20]  Tubular cells transfected with large T antigen/SV40 Mouse Uncoated [21]  Madin-Darby Canine Kidney (MDCK) cells Human Uncoated [22]  Norvegicus rattus kidney 52 cells Human Collagen IV, laminin [23]  Vero cells Monkey Uncoated [24] Primary cells  Podocytes Human Uncoated [4]  Podocytes Mouse Uncoated, collagen IV, fibronectin, BSA [4],[25] [11]  Glomerular endothelial cells Human Cystein, laminin, fibronectin, collagen IV [26] [12]  Glomerular endothelial cells Mouse Uncoated [27]  Tubular epithelial cells Mouse Uncoated [27]  Mixed kidney cells Rat Uncoated [28]  Renal progenitor cells Human Uncoated Unpublished own data Open in new tab Table 1 ECIS plate coatings used for renal cell lines or primary cells Cell type Species Coating References Cell lines  Podocytes transfected with large T antigen/SV40 Human Uncoated, cystein + fibronectin, cystein + collagen [13] [14] [15]  Glomerular endothelial cells Human Uncoated, cystein + collagen [14] [16]  Mesangial cells transfected with large T antigen/SV40 + human telomerase Human Uncoated [17]  LLC-pig kidney 1 cells Porcine Uncoated [18]  Human kidney 2 cells Human Uncoated [19]  Wistar rat kidney proximal tubule 0293 cells Rat Uncoated [20]  Tubular cells transfected with large T antigen/SV40 Mouse Uncoated [21]  Madin-Darby Canine Kidney (MDCK) cells Human Uncoated [22]  Norvegicus rattus kidney 52 cells Human Collagen IV, laminin [23]  Vero cells Monkey Uncoated [24] Primary cells  Podocytes Human Uncoated [4]  Podocytes Mouse Uncoated, collagen IV, fibronectin, BSA [4],[25] [11]  Glomerular endothelial cells Human Cystein, laminin, fibronectin, collagen IV [26] [12]  Glomerular endothelial cells Mouse Uncoated [27]  Tubular epithelial cells Mouse Uncoated [27]  Mixed kidney cells Rat Uncoated [28]  Renal progenitor cells Human Uncoated Unpublished own data Cell type Species Coating References Cell lines  Podocytes transfected with large T antigen/SV40 Human Uncoated, cystein + fibronectin, cystein + collagen [13] [14] [15]  Glomerular endothelial cells Human Uncoated, cystein + collagen [14] [16]  Mesangial cells transfected with large T antigen/SV40 + human telomerase Human Uncoated [17]  LLC-pig kidney 1 cells Porcine Uncoated [18]  Human kidney 2 cells Human Uncoated [19]  Wistar rat kidney proximal tubule 0293 cells Rat Uncoated [20]  Tubular cells transfected with large T antigen/SV40 Mouse Uncoated [21]  Madin-Darby Canine Kidney (MDCK) cells Human Uncoated [22]  Norvegicus rattus kidney 52 cells Human Collagen IV, laminin [23]  Vero cells Monkey Uncoated [24] Primary cells  Podocytes Human Uncoated [4]  Podocytes Mouse Uncoated, collagen IV, fibronectin, BSA [4],[25] [11]  Glomerular endothelial cells Human Cystein, laminin, fibronectin, collagen IV [26] [12]  Glomerular endothelial cells Mouse Uncoated [27]  Tubular epithelial cells Mouse Uncoated [27]  Mixed kidney cells Rat Uncoated [28]  Renal progenitor cells Human Uncoated Unpublished own data Open in new tab PRACTICAL APPLICATIONS ECIS is increasingly used in many biomedical fields to address questions regarding wound healing, drug risk assessment, barrier integrity and cell differentiation (Figure 3A and B). FIGURE 3 Open in new tabDownload slide Application of ECIS in biomedical research. (A) Around 27 years after ECIS technology was established, the preclinical research community began to recognize the potential contribution of the method to the field, which is shown by an increased number of publications using ECIS. On the contrary, kidney-associated research did not implement the technology with a similar trend. (B) Tissues studied using ECIS in relation to each other. FIGURE 3 Open in new tabDownload slide Application of ECIS in biomedical research. (A) Around 27 years after ECIS technology was established, the preclinical research community began to recognize the potential contribution of the method to the field, which is shown by an increased number of publications using ECIS. On the contrary, kidney-associated research did not implement the technology with a similar trend. (B) Tissues studied using ECIS in relation to each other. Cell attachment, migration and proliferation The ECIS technology is often used for monitoring cell physiology such as attachment, migration and proliferation [29]. The simplest method to quantify hyperplasia or hypertrophy with ECIS is shown in Figure 2A and D. Adherent cells are spread in the well, and impedance is monitored until it becomes stable, which indicates the establishment of an intact monolayer. The optimal cell number for seeding depends largely on cell size and proliferation speed and hence has to be titrated upfront. In our experience, 40 000–50 000 cells/0.8 cm2 are a suitable starting point to study the proliferation of primary murine renal tubular cells. Wegener et al. proposed to use the time it takes to reduce the baseline capacitance of the medium alone by 50% (t1/2) as a parameter of proliferation (Figure 1E) [8]. As another option, one can use the area under curve to quantify differences, especially whenever the healing process does occur in a non-linear fashion [30]. ‘Wound healing’ is frequently monitored using scratch assays, where a pipette tip is used to injure cells monolayers. However, such scratches often create voids with jagged edges and occasionally cause cells to pile-up densely alongside. In contrast, ECIS offers the use of an electric fence (Figure 2C). Cells are seeded in ECIS arrays with a continuous electric current running through the electrodes, thereby inhibiting cell attachment in a central area. Upon assuring cell confluency around the electrodes microscopically, the electric fence is switched off and the cells are allowed to ‘close the gap’. Alternatively after reaching confluency, the cells growing within the diameter of the electrodes can be injured by applying repeated pulses of high voltages (Figure 1B). The subsequent ‘wound closure’ can then be monitored with the voltage range used for impedance measurements. Although fencing leads to highly reproducible wound sizes, it is important to keep in mind that depending on the ECIS array used, such fence wounds are relatively small, which may be a downside for certain questions. Toxicity testing The ECIS system is frequently being used in drug development and risk assessment in different fields such as cancer research, virology, pulmonology and nephrology. The system is capable of detecting subtle damages in intact cell monolayers, by indicating early loss of barrier integrity (loss of cell–cell contacts) or cell dysfunction (i.e. loss of micromotion) [7], which are either low-dose effects or precede actual cell death. In this regard, the sensitivity of ECIS is superior to other assays widely used to assess cytotoxicity. When working with natural extracts, colorimetric assays tend to be also rather unspecific due to compound-related interferences. Fallarero et al. identified complex kinetic profiles for a variety of Betula pendula extracts that were not detectable using classical assays of cytotoxicity [31]. Apart from testing cytotoxicity, ECIS can be employed for the discovery of compounds that attenuate cytotoxicity [13, 32]. In general, such studies can prove to be difficult to conduct, as the damage inflicted on the cells needs to be substantial enough for the drug to be able to exert its effect, but also low enough to leave a sufficient number of surviving and non-senescent cells, which are able to respond to the drug. These titration efforts can be lengthy and costly. If the injury is inflicted in an ECIS setting, the cellular response to the injurious agent can be monitored in real time and stopped at any given moment to establish highly reproducible lethal dose rates (e.g. LD50) [5, 30]. Barrier function and cell invasion ECIS is especially useful for cell types that form barriers, as changes in these barriers can be quantified easily by measuring impedance at different frequencies. In that, ECIS is similar to transepithelial/endothelial electrical resistance (TEER) assays, which rely on porous filter inlays, only utilizing the standard solid substrate ECIS arrays. The electrical resistance measured in TEER is composed of two components, the cell–cell and the cell–substrate resistance, where only the former quantifies the barrier integrity. Compared with TEER, ECIS systems can distinguish the two components, thereby examining the actual barrier integrity [33]. An additional advantage of ECIS over conventional TEER assays is the possibility for further investigations using immunostaining and microscopy to study tight junction protein expression within the same plate. Also, to study transient barrier dysfunctions, ECIS—due to its continuous readout—is more suitable for obtaining high-resolution data than TEER [34]. Using ECIS, it has been demonstrated that some drugs, cytokines, immune cells and viruses disrupt the barrier function of epithelial or endothelial cells. Moreover, ECIS has also been used to study transmigration mechanisms in co-cultures. For example, Goc et al. quantified the micro-invasive behaviour of prostate cancer cells into endothelial cell monolayers using ECIS-derived resistance readings [35]. Differentiation and stem cell biology ECIS can also monitor stem cell differentiation. Bagnaninchi and Drummond used adipose tissue-derived stem cells and demonstrated distinguishable impedance kinetics in differentiated adipocytes, differentiated osteocytes and undifferentiated stem cells [36]. Park et al. reported differences in impedance changes in human mesenchymal stem cells cultured in neural differentiation media versus standard media [37]. Corradetti et al. dissected the different growth kinetics of murine embryonic stem cells in response to paracrine stimuli by investigating ECIS-measured resistance [38]. Given the increasing interest in stem cell biology, the potential use of ECIS in this context awaits further exploration. USING ECIS IN STUDIES ON KIDNEY PHYSIOLOGY AND PATHOPHYSIOLOGY Glomerulus ECIS has mainly been applied for investigating the barrier function in monolayers of glomerular epithelial or endothelial cells. Nevertheless, Saleem et al. found that angiotensin II impairs podocyte barrier integrity, while angiotensin receptor blocker abolished the same effect [32]. This way the authors could conclude that angiotensin receptor blockers not only reduce glomerular filtration pressure but also directly sustain podocyte barrier integrity. Henao et al. used ECIS to demonstrate that sFlt-1, which is found in high levels in patients with preeclampsia, impairs podocyte barrier function [39], offering a new explanation for damage to the glomerular filtration barrier in preeclampsia. Furthermore, interferon-β was shown to decrease barrier integrity of podocytes [4], while having opposite effects on endothelial cells [40]. These in vitro ECIS studies offer a mechanistic explanation for contradictory observations reported in different glomerular injury models [4, 40]. In vitro microfluidic systems are used to recapitulate in vivo microvessels as well as possible. Bevan et al. managed to combine the benefits of ECIS and microfluidic chambers to investigate the role of laminar shear stress on endothelial barrier integrity [41]. Thereby, they found that shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase, which in turn can affect podocyte barrier integrity. Proximal tubules Among the different kidney compartments, proximal tubular cells are particularly vulnerable to nephrotoxic agents. Hence, toxicity screenings for drugs or environmental substances are frequently performed using ECIS on these cells [20, 21]. The impact of cytokines such as tumour necrosis factor alpha (TNFα) can also be studied. For example, Amoozadeh et al. used ECIS to demonstrate that TNFα has a biphasic effect on proximal tubular epithelial cells, where it negatively affects epithelial barrier function during injury but it accelerates the subsequent epithelial repair [42]. Distal tubules and collecting duct Kakiashvili et al. employed ECIS on MDCK cells to study the epithelial growth factor receptor-dependent role of TNFα on cell proliferation during wound healing and fibrogenesis [43]. As another example, Donaldson et al. found a mutation of the Src homology 3 domain in the Nephrocystin-1 (NPHP1) gene, which encodes the protein nephrocystin, to affect epithelial cell–cell junctions and barrier function as a possible explanation for the causative role of this gene in juvenile nephronophthisis [44]. They revealed that MDCK cells stably expressing a nephrocystin mutant with the respective deletion have a reduced ability to establish tight junctions, leading ultimately to a disruption of tubular epithelial cell polarity and cyst formation [44]. COMBINING ECIS WITH OTHER TECHNOLOGIES Several efforts have been made to integrate impedance sensing into more sophisticated devices. For example, ECIS was maximized to generate a large 2D matrix to closely study cell motility, an aspect of great interest, e.g. in the characterization of cancer cell metastases [45]. Zhang et al. developed a stretchable ECIS biosensor simulating the dynamic environment of organisms, which are exposed to either pulsation, bending or stretching to enable meaningful investigations on cell behaviour under more physiological relevant settings [46]. Liu et al. coupled ECIS with a biosensor for resonant frequencies enabling simultaneous assessments of cell adhesion and viability in parallel to classical ECIS readouts [47]. ECIS can also be combined with a light-addressable potentiometric sensor to register extracellular acidification as a measure for the cellular metabolism alongside impedance, which could be useful for the increasing interest in metabolic profiling of cells [48]. Performing ECIS on cell lines expressing a stress-inducible reporter protein is an interesting development that may offer numerous attractive applications also in kidney research [49]. Technically feasible but up until now not widely used, is an ECIS-assisted single-cell measurement. By combining the ECIS-technology with a Boyden chamber design, Nguyen et al. provided kinetic information about cell migration and invasion processes in 3D extracellular matrixes using a cancer cell line [50]. With a similar intention, Susloparova et al. addressed the limited spatial resolution of ECIS by combining it with ion-sensitive field-effect transistors [51], thereby creating an impedance spectroscopy device. This enabled them to study cell adhesion and detachment processes on a single-cell level. A potential new and wide area of use for ECIS comes with the rapid developments in more sophisticated in vitro models of whole organs, i.e. organ-on-a-chip. With the ECIS providing continuous monitoring of cell growth and well-being in a non-invasive manner, this might be a supreme technique to follow the growth of different barrier-forming cell types in more complex 3D structures. Within this field of research, microscopy is so far the method of choice when it comes to assessing barrier integrity and differentiation of cells. Here, ECIS would serve as a valuable additional read-out that enables quantification of epithelialization and differentiation, thereby increasing the reproducibility of these experiments. SUMMARY ECIS is widely used across all biomedical research domains but has not yet been extensively explored in kidney research. Being a label-free, non-invasive technique that enables the quantification of cell–cell contacts and cell coverage in real time and in an easy-to-use fashion, ECIS comes with many advantages over other established methods addressing questions of cell viability, barrier integrity and proliferation. The possibility of combining quantitative ECIS readouts with microscopy and numerous other analytical methods offers a wide range of analytical opportunities from which kidney researchers could easily benefit. Incorporating ECIS into other analytical platforms or methods is affirmative for the growing impact of this technology on in vitro research, and yields profound insight into the cell physiology and pathophysiology. A number of limitations, as with any analytical method, remain, the significant costs being one of them. However, reuse protocols can help to minimize this aspect. We conclude that ECIS enriches our spectrum of available analytic tools in in vitro kidney research. FUNDING T.I. was supported by the Uehara Memorial Foundation and the Japanese Society of Clinical Pharmacology and Therapeutics. Z.B.Z. was supported by a Chinese Scholarship Council. M.K.Ś. was supported by a fellowship provided by the European Renal Association – European Dialysis and Transplant Association (ERA-EDTA). H.-J.A. was supported by the Deutsche Forschungsgemeinschaft (AN372/14-3, 23-1, 24-1, 27-1) and the BMBF (REPLACE-AKI 031L0071). CONFLICT OF INTEREST STATEMENT None declared. REFERENCES 1 Giaever I , Keese CR. Monitoring fibroblast behavior in tissue culture with an applied electric field . Proc Natl Acad Sci USA 1984 ; 81 : 3761 – 3764 Google Scholar Crossref Search ADS PubMed WorldCat 2 Keese CR , Wegener J , Walker SR et al. Electrical wound-healing assay for cells in vitro . Proc Natl Acad Sci USA 2004 ; 101 : 1554 – 1559 Google Scholar Crossref Search ADS PubMed WorldCat 3 Andersen K , Kesper MS , Marschner JA et al. Intestinal dysbiosis, barrier dysfunction, and bacterial translocation account for CKD-related systemic inflammation . 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TI - Electric cell-substrate impedance sensing in kidney research
JO - Nephrology Dialysis Transplantation
DO - 10.1093/ndt/gfz191
DA - 0016-04-10
UR - https://www.deepdyve.com/lp/oxford-university-press/electric-cell-substrate-impedance-sensing-in-kidney-research-o0krSfUotl
SP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -