A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency
Summary
Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.
Abstract
Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.
Introduction
Epithelial and endothelial cells function as cellular boundaries, separating the apical and basolateral sides of the body. If they are connected through tight junctions, passive substance diffusion through the paracellular spaces is restricted1, resulting in the formation of a selectively permeable barrier. Several artificial barrier systems have been developed2 using microvascular endothelial cells (HBMEC, blood-brain barrier3,4,5,6,7), choroid plexus epithelial cells (HIBCPP/PCPEC, blood-cerebrospinal fluid barrier8,9,10,11,12,13,14), colorectal adenocarcinoma cells (Caco-2, gastrointestinal models15), or airway/alveolar cell lines (pulmonary models16,17). These systems typically consist of cells grown in a monolayer on permeable membranes (i.e., filter insert systems) to allow access to the apical and basolateral sides. It is important that the integrity of the model system matches the in vivo conditions. Hence, several techniques have been developed to analyze barrier function by measuring paracellular diffusion of tracer compounds across the cell layer. These substances include radiolabeled sucrose, dye-labeled albumin, FITC-labeled inulin, or dye-labeled dextrans2. However, chemical dyes can make cells unusable for further experiments. To monitor barrier systems noninvasively, measurement of transepithelial/transendothelial electrical resistance (TEER) across a cellular monolayer can be used2,18,19. Because bipolar electrode systems are influenced by the electrode polarization impedance at the electrode-electrolyte interface, tetrapolar measurements are generally used to overcome this limitation20. The underlaying technique is a four-terminal sensing (4T) that was first described in 1861 by William Thomson (Lord Kelvin)21. In brief, the current is injected by a pair of current-carrying electrodes while a second pair of voltage-sensing electrodes is used to measure the voltage drop20. Nowadays, so-called chopstick electrodes consist of a pair of double electrodes, each containing a silver/silver-chloride pellet for measuring voltage and a silver electrode for passing current2. The electrical impedance is measured between the apical and the basolateral compartment with the cell layer in between (Figure 1). A square wave signal at a frequency of typically 12.5 Hz is applied at the outer electrodes and the resulting alternating current (AC) measured. Additionally, the potential drop across the cell layer is measured by the second (inner) electrode pair. Electrical impedance is then calculated according to Ohm's law. TEER values are normalized by multiplying impedance and cell layer surface area and are typically expressed as Ω ∙ cm2.
There are systems in which cells and electrodes are arranged in a more sophisticated way, but are also based on the 4T measuring principle and can be used with the same measurement devices. EndOhm systems, for example, in which the filter is inserted, contain a chamber and cap with a pair of concentric electrodes with the same structure as the chopstick electrode. The shape of the electrodes allows for a more uniform current density flow across the membrane, thereby reducing variation between readings. Even more complex (but also more accurate) is an Ussing chamber, where a cell layer separates two chambers filled with Ringer's solution22. The chamber itself can be gassed with oxygen, CO2, or N2, and stirred or supplemented with experimental substances. As ion transport across the cell layer occurs, a potential difference can be measured by two voltage-sensing electrodes near the tissue. This voltage is cancelled out by two current-carrying electrodes placed next to the cell layer. The measured current will then give the net ion transport and the transepithelial resistance, which reflects barrier integrity, can be determined22. TEER measurement can also be applied on body-on-a-chip systems that represent barrier-tissue models23,24. These systems mimic in vivo conditions of the cells and often consist of several types of cells, stacked on top of each other in layers.
The following protocol explains how to set up a cost-effective and reliable voltammeter with programmable output frequency that produces no statistically significant differences in TEER compared to commercially available measurement systems.
Protocol
Representative Results
Discussion
Before a self-made voltammeter can be used in a daily routine, it is essential to check the device for proper function. In our case, a half-time of oscillation of 40 ms (12.5 Hz) was programmed, but the effective oscillation time turned out to be 60 ms (16.7 Hz). This inaccuracy of the microcontroller's time emitter had no detectable impact on TEER measurements. It might be best to determine the actual frequency using the frequency setting of one of the multimeters. If any deviation is found, the source code can be a…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Herman Liggesmeyer and Marvin Bende for their expert advice in electrotechnics and informatics.
Materials
| 120 kOhm resistor | General (generic) equipment | ||
| Banana plug cables | General (generic) equipment | ||
| Cables | General (generic) equipment | ||
| Chopstick electrode | Merck Millicell | MERSSTX01 | |
| Chopstick electrode (alternative) | WPI World Precision Instruments | STX2 | |
| Crimping tool | General tool | ||
| Digispark / ATtiny85 | AZ-Delivery Vertriebs GmbH | Digispark Rev.3 Kickstarter | |
| DMEM:F12 | Gibco (Thermo Fisher) | 31330038 | |
| Fetal calf serum (FCS)/Fetal Bovine Serum (FBS) | Life Technologies | 10270106 | |
| Filter inserts 3µm translucent | Greiner Bioone | 662631 | |
| HIBCPP | Hiroshi Ishikawa / Horst Schroten | ||
| Insulation stripper | General tool | ||
| Luster terminal | General (generic) equipment | ||
| Oscilloscope | HAMEG | Digital Storage Scope HM 208 | |
| Plotter | PHILIPS | PM 8143 X-Y recorder | |
| Software Arduino | https://www.arduino.cc | Arduino 1.8.9 | |
| Soldering iron | General tool | ||
| Soldering lugs | General (generic) equipment | ||
| Telephone cable with RJ14 (6P4C) connector | General (generic) equipment | ||
| Test resistor | Merck Millicell | MERSSTX04 | |
| True-RMS multimeters | VOLTCRAFT | VC185 | |
| USB charger | General (generic) equipment | ||
| USB extension cord | General (generic) equipment | ||
| Voltohmmeter for TEER measurement | WPI World Precision Instruments | EVOM | |
| Voltohmmeter for TEER measurement (alternative) | Merck Millicell | ERS | |
| Wire end ferrules | General (generic) equipment |
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