Impedance-based cell monitoring: barrier properties and beyond
© Benson et al.; licensee BioMed Central Ltd. 2013
Received: 1 October 2012
Accepted: 19 December 2012
Published: 10 January 2013
In multicellular organisms epithelial and endothelial cells form selective permeable interfaces between tissue compartments of different chemical compositions. Tight junctions which connect adjacent cells, control the passage of molecules across the barrier and, in addition, facilitate active transport processes. The cellular barriers are not static but can be deliberately modulated by exposure to specific external stimuli. In vitro models representing the essential absorption barriers of the body are nowadays available, thus allowing investigation of the parameters that control permeability as well as transport processes across those barriers. Independent of the origin of the barrier forming cells, techniques are needed to quantify their barrier integrity. One simple assay is to measure the permeability for given hydrophilic substrates possessing different molecular weights like sucrose or dextrans. However, this technique is time-consuming and labor-intensive. Moreover, radioactive or fluorescently-labeled substrates are needed to allow easy analytical detection. Finally, if transport processes are investigated, the standard permeant may interfere with the transport process under investigation or might even alter the barrier integrity by itself. Thus, independent, non-invasive techniques are needed to quantify the barrier integrity continuously during the experiment. Such techniques are available and are mainly based on the measurement of the transendothelial or transepithelial electrical resistance (TEER) of barrier forming cells grown on porous membranes. Simple devices using two sets of electrodes (so-called Voltohmeters) are widely used. In addition, an easy-to-use physical technique called impedance spectroscopy allows the continuous analysis of both the TEER and the electrical capacitance giving additional information about the barrier properties of cells grown on permeable membranes. This technique is useful as a quality control for barrier forming cells. Another impedance-based approach requires cells to be grown directly on solid, micro-structured electrodes. Here, we will discuss the physical background of the different techniques; advantages, disadvantages, and applications will be scrutinized. The aim is to give the reader a comprehensive understanding concerning the range and limits of the application, mainly focusing on endothelial cells.
KeywordsBarrier forming cells Endothelium Epithelium Blood–brain barrier Electrical resistance TEER Impedance spectroscopy
A characteristic feature of epithelial as well as endothelial cell layers is the formation of intercellular junctions resulting in a tight cellular barrier separating the apical (luminal) from the basolateral (abluminal) side. These cell layers form selectively permeable interfaces between compartments of different chemical composition, thus controlling diffusion along the paracellular way as well as transport processes through intracellular pathways. This is guaranteed by the so-called tight junctions (intercellular connections) that seal the intercellular cleft . An intact barrier is crucial for the physiological activities of the corresponding tissue. However, the barrier is not static but can be modulated by specific stimuli to open and close selectively, thus allowing controlled passage from the blood to the brain or vice versa[2–4]. Developing methods to overcome the barrier is an important issue and highly relevant for medical treatment of diseases within the barriers. Drug delivery systems or strategies to open the barrier temporarily will help to allow medication to cross the blood–brain and the blood-CSF (cerebrospinal fluid) barrier as well as other barriers such as in the intestine, the kidney, the testis and the placenta. Adequate in vitro models are nowadays available, based on cell cultures grown on permeable supports . These are of major importance since the setup offers full access to both the apical and the basolateral compartments. In vitro models can, in principle, be based on primary cells [6, 7] or cell lines [8, 9]. However, in case of blood–brain barrier (BBB) models most cell lines do not express barrier properties similar to in vivo conditions . Thus care has to be taken if such cells are used for transport experiments.
Since the presence of an intact barrier is crucial for reliable in vitro experiments, techniques had to be developed to quantify the barrier integrity. One possibility is to measure the permeability for small hydrophilic substances like radioactively labeled sucrose or low molecular weight fluorescent dyes . A straightforward method is the measurement of the TEER. A scrutiny of different TEER measurements will be given here. The major focus will be on techniques that allow automated long-term monitoring of barrier-relevant parameters including the dynamic processes within the tight junction network.
TEER measurement according to Ohm`s law
Automated impedance-based cell monitoring under physiological conditions using the cellZscope® device
Basics of the technique
Based on the equivalent circuit, the corresponding modeling computer software can be employed to determine automatically best fit parameters and to extract the TEER and the capacitance Ccl as readout parameters. Further possible contributions to the cell layer`s total impedance (for example the cell membrane resistance change) can be neglected in this first order approximation. For a deeper understanding of the mathematical difference between resistance and impedance, imaginary numbers and vectors have to be considered .
Applications of impedance measurements using filter systems
The effect of glucocorticoids on the properties of the blood-brain barrier
Impedance measurement as quality control
Techniques that quantify barrier properties have to achieve a good correlation with the permeability measurements of small, polar substances that are not substrates of BBB transporters. For impedance measurements, a constantly low sucrose permeability (value of p = 10-7 cm/s), which is characteristic for the BBB in vivo, was found in cells with TEER values > 600-800 Ω · cm2, while cells exhibiting a lower TEER showed a more variable permeability . When performing transport experiments, it is of great importance to have an online control of barrier properties since a disruption of barrier integrity might result in false interpretation of data.
Nanoparticles at the blood–brain barrier7
Using impedance measurements, it is also possible to ascertain the toxicity of nanomaterials. Wagner et al. have combined different oximes for treatment of organophosphate poisoning with human serum albumin nanoparticles to enable transport across the BBB. Using impedance spectroscopy, they were able to verify the presence of an intact barrier during their transport studies. Additionally, impedance measurements were used to assess the toxicity of the used nanoparticles underlining the results obtained from cytotoxicity assays.
Inflammatory cells cross the blood–brain barrier without opening the tight junctions
Pericytes and astrocytes regulate the blood–brain barrier integrity
In vivo, the BBB properties are not solely due to the properties of capillary endothelial cells but are also induced by surrounding pericytes and astrocytes. The in vitro induction of BBB properties by astrocytes was characterized by determining the resistance in a co-culture-model of endothelial cells and astrocytes [24, 25]. For all conditions tested, it was found that astrocytes increase BBB integrity. Also, the influence of pericytes on the integrity of the BBB is still under discussion. Employing TEER measurements, it was found that pericytes can either decrease or increase the integrity of the barrier, depending on their state of differentiation . With the aid of resistance measurements, Nakagawa et al. were able to ascertain the best culture conditions for the establishment of a triple co-culture model of endothelial cells with astrocytes and pericytes . The authors were able to show that the presence of astrocytes and pericytes resulted in an increase of TEER compared to the mono-culture.
Electrical cell substrate impedance sensing
Electric cell-substrate impedance sensing (ECIS, Applied BioPhysics inc, Troy NY, USA) is an impedance-based method, which can be used as a tool for real-time monitoring of the cell behaviour such as adherence, mobility and growth on solid substrates . This technique allows investigation of the invasive nature of cancer cells, the barrier function of endothelial cells, cell-cell and cell-matrix interactions, signal transduction for modern drug discovery and wound-healing processes.
In ECIS, cell layers are grown to confluence not on porous membranes but directly on integrated gold-film electrodes. The close proximity of the cell monolayer to the thin gold electrodes results in high sensitivity measurements. However, it is important to realize that there is no basolateral fluid compartment present due to the adherence of the cells to the electrode. This excludes the employment of the ECIS setup in transport or transfer experiments. There are also fundamental differences in the measured impedance data that need to be considered when comparing results obtained with membrane-based experimental setups such as the cellZscope . This will be enlightened by some basic calculations in the following paragraph.
Cell attachment and cell growth
Extracellular matrix coating of ECIS electrodes
Two-path impedance spectroscopy
So far we have only considered changes in the paracellular resistance and the sub-epithelial resistance caused by the cell attachment. However, solutes may also be transported via a transcellular way crossing the apical and the basolateral membrane. Thus, it might become desirable to determine the para- and the transcellular resistance separately. This is especially important if ion fluxes via ion channels are involved. Krug et al. have developed a technique based on impedance spectroscopy which enables them to differentiate between the transcellular and paracellular pathways. In combination with flux measurements using e.g. fluorescein as a paracellular marker they are able to determine the transcellular resistance as well .
Electrical resistance measurements are valuable tools to quantify barrier properties. Impedance spectroscopy is a new non-invasive technique to monitor not only barrier function but also processes, like cell growth and cell differentiation. The main advantage of impedance spectroscopy is the automated monitoring process. Two main systems have to be distinguished. One is the cellZscope device, using standard cell culture inserts with semi-permeable membranes as substrates for cell growth, thus allowing simultaneous transport investigations. The second is the ECIS system, where the cells are directly grown on the electrode thus losing their basolateral compartment. However, in addition the value α which characterises the cell-matrix interaction, can be determined. Moreover, since higher currents might be applied locally, cells can be locally destroyed to allow measurements of wound healing. Thus, both experimental methods are important, their use depending on the scientific question under investigation.
Impedence Of Cell-Matrix Contacts
Capacitance Of The Cell Membrane
Capacitance Of The Electrodes
Capacitance Of The Membrane In ECIS
Confocal Laser Scanning Microscopy
Electrical Cell Substrate Impedance Sensing
Normal Rat Kidney
Poly(Butyl)Cyanoacrylate - Nanoparticles
Porcine Brain Capillary Endothelial Cells
Ohmic Resistance Of Cell-Cell Contacts
Ohmic Resistance Of The Membrane
Transendothelial Electrical Resistance
Tumor Necrosis Factor - α
We like to thank Dr. Boris Anzcykowski from Nanoanalytics GmbH for helpful discussions and continuous support. The input of Prof Dr. Joachim Wegener, Institute for Analytical chemistry, Chemo- and Biosensors at the University of Regensburg in the development of the impedance spectroscopy is especially acknowledged.
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