A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 barrier^3,4,5,6,7), choroid plexus epithelial cells (HIBCPP/PCPEC, blood-cerebrospinal fluid barrier^{8,9,10,11,12,13,14}), colorectal adenocarcinoma cells (Caco-2, gastrointestinal models¹⁵), or airway/alveolar cell lines (pulmonary models^16,17). 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 used^2,18,19. 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 models^23,24. 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.

Protokół

1. Assembly of a basic volt-amperemeter for TEER measurement

1. Prepare a standard USB charger as the 5 V D.C. power supply, a USB extension cord, a microcontroller that will be used as a programmable square wave generator, two standard multimeters that are able to measure alternating current and voltage as root mean square (True-RMS), four cables with banana plugs, a telephone extension cord with a RJ14 female connector including six pins with the inner four wired (6P4C), two short cables, a luster terminal, a 120 kΩ pre-resistor, wire end ferrules, and soldering lugs. The tools required are an insulation stripper, a crimping tool, and a soldering iron.
2. First, connect the USB extension to the microcontroller board.
3. Strip the end insulation of two short cables. Solder one side per cable either directly to pins 0 and 2 of the microcontroller or to soldering lugs, which in turn are clipped on the respective pins. Crimp the other ends to the wire end ferrules and connect them to a luster terminal as depicted in Figure 1.
4. Link the banana plugs to the multimeters. Strip and crimp the other end of each of the four cables.
5. Cut the telephone extension cord in two pieces and dismantle and crimp the conductors of the side containing the female connector. Check for the continuity of the conductors and pins.
6. The first multimeter will be used to measure current in µA (note that the AC mode has to be set explicitly). Connect it in a series with a 120 kΩ pre-resistor to pins five and six of the RJ14 connector, corresponding to the outer electrode pair of the chopstick electrode.
7. Finally, link the second multimeter, which will be used to measure the transepithelial voltage drop in mV, via the luster terminal to pins three and four of the RJ14 connector, corresponding to the inner electrode pair of the chopstick electrode.
8. If desired, mount the installation in a chassis.

2. Programming the microcontroller

1. Modify the provided source code (supplemental coding file 1) as needed. In the given form, pins 0 and 2 will alternate between ground and +5 V with 40 ms half-time of oscillation. Thus, a square wave signal with an amplitude of 5 V and a frequency of approximately 12.5 Hz will be generated. The real values may differ due to the inaccuracy of the microcontroller's time emitter.
2. Connect the microcontroller to a desktop computer via a USB port and upload the source code with matching software²⁵.

3. Recording of voltage oscillograms (optional)

1. Bypass pins five and six of the RJ14 connector with a 1 kΩ test resistor and connect to an oscilloscope.
2. Check for the frequency, peak voltage, and waveform. Digitize and export the data.
3. If desired, record oscillograms from a reference device (EVOM) and the self-assembled voltammeter for comparison.
   NOTE: In this case, the data was recorded with a Digital Storage Scope HM 208. Being a very basic digital oscilloscope, the image could be internally digitized (frozen) but had to be plotted using an analogue PM 8143 X-Y recorder. The image was subsequently scanned.

4. Cell cultivation and TEER measurement

1. Seed Human Choroid Plexus Papilloma (HIBCPP) cells on cell culture filter inserts with a pore size of 3 µm in DMEM/F12 (see Table of Materials) containing 10% fetal calf serum⁹. Grow the cells at 37 °C in a water saturated atmosphere containing 5% CO₂ as described by Dinner et al.⁹.
2. When the filters reach an impedance of 70 Ω ∙ cm², change to serum-free DMEM/F12 and define the timepoint as Day 0.
3. Connect the electrode to the RJ14 port of the self-assembled voltammeter and plug in the USB power supply. Set the multimeters to AC voltage mode (mV) and AC current mode (µA), respectively.
   1. Alternatively, connect the electrode to a commercially available reference device and turn on according to the manufacturer's instructions.
4. Sterilize the electrode in 80% ethanol for 10 min and equilibrate in the appropriate medium for another 10 min.
5. Put the electrode in both compartments of a cell culture filter insert system (the longer part of the electrode in the lower compartment and the shorter part in the upper compartment) containing a HIBCPP cell layer until the measurement values remain constant.
6. For a reference device, note the impedance directly or calculate the impedance according to Ohm's law (R = U/I) for the self-assembled voltammeter. Be aware that electrode angle affects the measurements.
7. Repeat the TEER measurement (steps 3−6) from Day 0 until Day 4.

Wyniki

To compare the operation of a self-assembled voltammeter with its commercially available counterpart, a voltage oscillogram of both devices was recorded.

As shown in Figure 2A, the reference instrument generated a square wave signal with an amplitude of 80 mV and an oscillation time of 80 ms, which corresponds to a frequency of 12.5 Hz, when operating on-load with a 1 kΩ test resistor.

...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
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