Monday, August 2, 2010

Trans Epithelial Electric Resistance (TEER) Measurements


World Precision Instruments Inc

Category: TEER Measurement | 26/04/2006 - 10:53:49

The confluence of the cellular monolayer is quickly determined by a sharp increase in TEER. Recently there has been a significant surge of interest in introducing a combined electrode for resistance measurements in the Millipore 96-well PAMPA (parallel artificial membrane permeability assay) plate.

TEER measurement technology, which was first introduced by WPI in the mid-1980's, has since been perfected and expanded to include a range of TEER related manual and automatic instrumentation.

EVOM

EVOM - Epithelial Voltohmmeter

  • Manual TEER measurement of epithelial cells in 24- and 96-well plates
  • Battery powered electrically isolated meter
  • AC current avoids adverse effects on tissue
  • Compatible with Endohm chambers
  • STX2 manual electrode enclosed with meter
  • STX2 can be sterilized with EtO, alcohol or a bactericide

The EVOM was the first instrument designed specifically to perform routine TEER (Trans Epithelial Electric Resistance) measurement in tissue culture research.

The battery-powered EVOM produces an AC current that avoids adverse effects on tissues, which can otherwise be caused, by a DC current -- including electrode metal deposits.

The EVOM has an easy-to-read LC display and is also available with a BNC recorder output (EVOMX). Input impedance of the EVOM is greater than 1010 ohm, and resistance ranges from 0 to 1999 ohm; and 0 to 20,000 ohm can be monitored using the switchable range setting located on the instrument front panel.

One unique feature of the EVOM is that resistance readings are unaffected by membrane capacitance and membrane voltage. The membrane voltage reading range is ±200 mV. EVOM comes complete with the popular STX2 "Chopstick" electrode set. The STX2 consists of a fixed pair of double electrodes, 4 mm wide and 1 mm thick.

Each stick of the electrode pair contains a silver/silver-chloride pellet for measuring voltage and a silver electrode for passing current. The small size of each electrode is designed to facilitate placement of the electrodes into cell culture wells.

STX2 can be used with all tissue culture inserts currently on the market. For more accurate quantitative measurements and/or for lower resistance measurements (e.g., endothelial tissue cultures) the EVOM can be used in conjunction with the optional Endohm chambers.

EVOMX-STX100M

EVOMX

  • BNC output for Data Acquisition System
  • Toggle switch for continuous measurement

EVOMX is a modified version of the EVOM and is recommended for use with STX100 series electrodes. Its measurement switch and circuits have been modified to remain ON continuously.

This enables the user to have one hand free to move the electrode while using the other to note the readings. A BNC recorder output connector is provided on the EVOMX to permit direct recording of TEER measurements to a chart recorder or computer-based data-acquisition system.

STX100 Electrodes

  • Designed for 24-well HTS plate (Corning Costar and BD Falcon) and with 96-well plates (Millipore and BD Falcon)
  • Improved accuracy down to 5 Ohm
  • Sterilized with EtO, alcohol or bactericide

With the development of a High Throughput Screening (HTS) protocol for faster drug discovery, a new line of cell culture filter plates have been introduced by several major cell culture insert manufacturers.

These HTS plates normally have either 24 or 96 individual cell culture inserts "bonded" together as one plate so that it can be handled by a robot apparatus. In response to these developments, WPI has developed an automatic REMS system and a manual electrode, STX100, for TEER measurements using HTS plates.

STX100's design is based on the same reliable design principle as the universally used STX2 electrode, with several important modifications. The size of the electrode tip has been reduced to 1.5 mm to facilitate positioning through the narrower slit of the HTS plate. The STX100 electrode itself is constructed using a stronger material for higher durability and maximum usage applications. The bottom section of the electrode is shaped to fit neatly into the "keyhole" shaped filter well.

This enables the STX100 electrode to produce increased accuracy and reproducibility of TEER readings (±5Ω) compared to the standard STX2. Several versions of STX100 are available, designed to fit the Corning Costar 24-well HTS plate, the Falcon 24 well HTS plate, the Millipore Multiscreen CaCo 96-well plate, and BD Falcon HTS 96-multiwell plate. Measurement can be directly performed when the HTS plate is in either a common or divided tray, reducing the possibility of contamination as well as mechanical damage to the cultured cells.

EVOMX-Multi96

Multi-96

  • Resistance measurement with 96-well PAMPA (parallel artificial membrane permeability assay) plates

Non-cell based 96-well PAMPA assays have been designed for predictive drug candidate testing. To ensure that the donor/acceptor fluxes are not due to porous or unstable hexadecane layers, the stability of the hexadecane membrane can be tested at the end of the incubation period by electrical resistance measurements.

To conduct the measurement a PAMPA plate is transferred to MULTI-96. Like the other Endohm models (designed for individual cell culture cups), the base of MULTI-96 is a fluid- filled receptacle for the PAMPA plates, also serving as the bottom half of the electrode pair.

Inserting the top electrode into each well allows the user to obtain the resistance of the synthetic membrane layer of that well. MULTI-96 provides a stable and reproducible reading of tissue culture resistance. EVOMX is recommended for use with MULTI-96.

ENDOHMS98-rev

ENDOHM

  • TEER measurement of endothelial cell cultures in individual cups
  • Compatible with EVOM and EVOMX
  • Improved accuracy of 1-2 Ohm
  • Accommodates 6mm, 12mm, 24mm cups and Costar Snapwell cup
  • Sterilized with EtO, alcohol or a bactericide

Using WPI's EVOM resistance meter, Endohm chambers provide reproducible resistance measurements of endothelial tissue in culture cups. Transfer cups from their culture wells to the Endohm chamber for measurement rather than using hand-held electrodes. The chamber and the cap each contain a pair of concentric electrodes: a voltage-sensing silver/silver chloride pellet in the center plus an annular current electrode.

The height of the top electrode can be adjusted to fit cell culture cups of different manufacture. Endohm's symmetrically apposing circular disc electrodes, situated above and beneath the membrane, allow a more uniform current density to flow across the membrane than with STX2 electrodes. The background resistance of a blank insert is reduced from 150 Ω (when using WPI's hand-held STX2 electrodes) to less than 5 Ω.

With Endohm's fixed electrode geometry, variation of readings on a given sample is reduced from 10-30 Ω with STX2 electrodes (depending on the experience of the user) to 1-2. Compared with other resistance measurement methods, Endohm with EVOM offers a much more con-venient and economic solution to "leaky tissue" measurement. Because of the uni-form density of the AC square wave current from EVOM, errors owing to electrode polarization or membrane capacitance are largely eliminated.

Endohm together with EVOM offers the most accurate and economical endothelial ohmmeter now avail-able. To date, cups from Costar, Millipore, ICN Biomedicals, and Falcon have been tested. Endohm chambers may be sterilized with EtO, alcohol or a bactericide (also see: Cidex, Microsurgery section); not autoclavable.

REMS2002

Automated TEER Measuring System

The REMS AutoSampler automates measurements of electrical resistance of transepithelial, transendothelial or Caco-2 cell membranes being grown to confluence on microporous filters of high throughput screening (HTS) 24- and 96-well microplates. It is a PC-controlled, tissue resistance measurement system that offers reproducibility, accuracy, flexibility and ease-of-operation for this kind of measurement.

Automated measurement of tissue resistance in cell culture microplates provides the important advantages of speed, precision, decreased opportunity for contamination and the instant availability of measured resistance data on a computer. These measurements are useful in applications such as drug bioavailability studies and studies on the mechanisms of drug transport.

The main components of the REMS AutoSampler include: the robotic sampler that moves the electrode over each well of the microplate, the electrode which is located on the robotic arm, a base plate for the 24- and 96-well tray, a Windows-based data acquisition card, the REMS interface unit and the REMS software to operate the system on a Windows-based computer.

The REMS AutoSampler automates TEER measurements previously made with WPI's EVOM Epithelial Voltohmmeter. Automated tissue resistance measurements up to 20 kΩ can be performed on 24- or 96-well HTS microplates. Microplates presently supported include the Corning Costar HTS Transwell-24, Falcon HTS Multiwell insert systems, and Millipore Multiscreen™ CaCo 96-well plate.

The REMS AutoSampler is designed to facilitate integration with other robotic systems. Special locating bars are installed on the REMS base platform that allow other system robots to place an HTS tray into a precise location on the REMS base.

The REMS AutoSampler will automatically measure and record tissue resistance from a user-specified matrix of culture wells on the microplate. According to the specified sequence, the robotic arm moves over the identified wells taking TEER measurements. By means of a x-y-z locating system, the electrode-containing arm is positioned precisely and reproducibly over each well.

The ability of the REMS AutoSampler to reproducibly and precisely locate the electrode results in highly reproducible TEER measurements. TEER measurements are stored in the computer as the electrode moves from one well to the next. The Windows-based software provides user-friendly features to acquire, display and store the tissue resistance measurements.

The REMS electrode is very compact and robust in design. Each of two rod-shaped probes, 1.5 mm in diameter, consists of a pair of electrodes: one electrode for injecting current and the other for measuring the voltage. The use of two pairs of electrodes eliminates the error caused by the electrode-liquid interface.

To take a measurement, the robot inserts one probe into the center of the filter well and the other into the opening slot of the 24- or 96-well plate. The use of AC current to measure resistance provides several advantages over DC current, including:

  • Absence of offset voltages on measurements;
  • There is a zero net current being passed through the membrane and therefore it is not adversely affected by a current charge;
  • No electrochemical deposition of electrode metal.

The REMS AutoSampler also features a rinse and calibration check station. If occasional rinsing of the REMS electrode is required it may be sent to a rinse station by pressing the rinse station button on the menu bar. The rinse station can also function as a calibration check station when fitted with a calibration cell containing a synthetic membrane (WPI's optional CALICELL-HTSF).

The use of this calibration cell, which mimics a confluent epithelial membrane's resistance in fluid, provides a quick and effective test to determine when the REMS measurement system is fully initialized following start-up and to check its functionality during operation.

REMS KitREMS Kit

  • Give your HTS system the ability to perform REMS TEER measurements

WPI's REMS TEER measurement system is also available in a fully customizable package that does not include the robot. The REMS-KIT is designed to enable manufacturers and users of robotic and HTS systems to incorporate TEER measurement capability into their own automated protocols. Essentially the REMS-KIT provides the same TEER measuring system as the REMS but without the robot positioner.

Control over TEER measurement is accomplished using the DDE protocol. Virtually any Windows-compatible programming language that uses the DDE protocol (including LabView and Visual Basic) can be used.

The REMS-KIT is designed for use with Corning Costar HTS Transwell-24, Falcon HTS Multiwell Insert System and Millipore MultiscreenTM CaCo 96-well plates. The system includes the following components:

  • REMS TEER electrode with 5-ft cable
  • Dummy TEER electrode for training robot
  • REMS TEER measurement electrode interface unit
  • Windows PCI A/D data acquisition card
  • Interface software using the DDE protocol
  • Instruction Manual

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