ChanTest ChansPorter Assay

 


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Issue 4

ChanTest® ChansPorter™ Assay
Ussing Assay for Higher Throughput Measurement of Electrogenic Transporter Currents


The Ussing Chamber Assay (UCA) is an electrophysiological assay that uses epithelial voltage clamp technology to measure net electrolyte, nutrient and drug transport across epithelial tissues. Ex vivo transport activity by essentially all epithelia in the body has been evaluated with this assay. Cultured epithelial cells (primary and cell lines) capable of forming polarized epithelia are used extensively for in vitro UCAs in drug discovery, physiology and toxicology evaluations.

ChanTest developed the capability to regularly perform high-quality UCAs at an unprecedented scale. ChanTest UCAs include three independent, 24-chamber systems dedicated to cystic fibrosis, R&D, and contract research testing with the capacity to simultaneously test 72 epithelia grown on SnapWell™ filter inserts. A fourth 24-chamber system is used for cGMP compliant release assays. The epithelia are derived from cultures of patient primary cells and human and animal origin cell lines. ChanTest generated culture and process infrastructure to repeatedly record from 96 Cystic Fibrosis human Bronchial Epithelia (CFhBE) derived epithelia per day, four days per week, using two UCA systems. Incorporating this level of throughput, using human bronchial epithelia, into discovery screening and profiling processes can increase productivity several fold, eliminating false positives and negatives arising from animal cell lines. Time to results are also shortened significantly, saving time and cost.

The ChanTest TECC-24 system is another embodiment of the UCA in development at ChanTest to improve productivity. TECC-24 is a semi-automated UCA system that utilizes a cartesian robot, a 24-channel epithelial voltage clamp (TECC-24), and 24-well microplates containing CFhBE epithelia grown on permeable support. A manual TECC-4 4channel system was used to validate 24-well microplate CFhBE cultures and optimize culture conditions, experimental methods and analysis software. Validation of corrector assays on the TECC-4 system was completed in 2012.

Figure 1 – A standard Modular Ussing Chamber (one of 24 in each system) used for primary Ussing assays is shown above. For clarity, chamber is shown without electrodes, solution or epithelium on the Snapwell insert. Each chamber is comprised of two half-chambers that sandwich the insert at the interface between them so that the epithelium on the insert separates the solutions in the half chambers.

Using these systems, a large group of cell-based assays has been thoroughly tested and validated by ChanTest, and is now suitable for immediate use in high-throughput measurement applications. The following information illustrates the completion of assay testing and validation of performance under optimized conditions.

Primary Ussing Assays
ChanTest validated primary UCAs for human cystic fibrosis transmembrane conductance regulator (CFTR) chloride ion channels and for human epithelial sodium channels (ENaC). The human CFTR UCA utilizes CFhBE primary cells derived from patients homozygous for the ΔF508 mutation. The human ENaC UCA utilizes normal human bronchial epithelia (NhBE) derived from normal patient lung tissue.  Both assays are validated and the experimental results are shown below. Corrector test articles suppress the trafficking defect caused by the ΔF508-CFTR mutation. Data from a CFhBE cell plating validation study for ΔF508-CFTR corrector assays are shown in Figure 2. Success was determined statistically using Dunnett’s test and Student’s t-test and Z factors were calculated. Statistical comparisons of ΔF508-CFTR short circuit current (ISC) peak and steady state responses to channel modulators were significantly different between uncorrected vehicle treated and positive control Test Article (TA) treated epithelia.

Figure 2 - Cell plating validation experiment for corrector assays: bronchial epithelia from a cystic fibrosis (CF) patient homozygous for ΔF508-CFTR treated with correctors C4 and CF-106951 and vehicle (control) for 48 hours at 37ºC in the incubator.  Currents were aligned to 0 µA at time indicated by the yellow star.  The large negative short circuit current appearing after the epithelia are voltage clamped at the beginning of the record is produced by sodium ions moving through apical ENaC channels (sodium absorption) and is blocked by apical 30µM amiloride. The negative forskolin, IBMX and genistein activated and CFTRinh-172 inhibited short circuit current is chloride ions moving through apical ΔF508-CFTR channels (chloride secretion).

Figure 3 - Cell plating validation experiment for ENaC assays in NHBE: ENaC amiloride cumulative concentration-response is shown for each epithelium.  Each epithelium acts as its own control and the data are normalized by the 10-µM-amiloride response.  Measurement is made at 35ºC.

Secondary Ussing Assays
The secondary Ussing assays currently in development are designed to study intestinal CFTR chloride channel function and to measure electrogenic transporters (PEPT1 and Na-K ATPase) and non-electrogenic transporters (Caco-2).

If you have questions about the ChanTest® ChansPorter™ Assay, or how ChanTest can help your organization with drug discovery and development needs, helping to increase productivity, and shorter time to results, please contact us directly.