A major consideration when identifying lead compounds from drug screens is to measure their cardiotoxicity. Numerous lead compounds and drugs developed for different therapies, ranging from cancer to psychotic disorders, have been found to interfere with normal cardiac physiology, and have thus been retracted from early- and late-stage drug trials as well as from the drug market.1, 2
Of the various forms of cardiotoxicity, one that has received considerable attention is toxicity associated with the hERG (human ether-a-go-go related gene
ion channel. Drug-induced block of hERG channels, or disruption of their normal surface membrane expression, can lead to prolongation of the electrocardiographic QT interval, resulting in long QT syndrome and an increased risk of cardiac arrhythmia in the form of torsades de pointes
The importance of hERG-related toxicity is best illustrated by the promiscuity of hERG channels to bind myriad compounds, including anti-cancer (e.g., arsenic trioxide),4
antimalarial (e.g., chloroquine),5
antipsychotic (e.g., chlorpromazine),6
antihistamine (e.g., astemizole),7
and antiarrhythmic (e.g., quinidine, dofetilide)8, 9
drugs, all of which acutely block or disrupt the membrane trafficking of hERG channels. As a consequence of the broad hERG-related toxicity from various therapeutics, evaluation of the effect of new drugs on hERG function is now required by the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) regulators, and is a mandatory step in the development of novel pharmaceuticals.10
Often, compound libraries undergo multiple screenings, i.e., high-throughput chemical library screening is employed early in development with the final lead compound(s) later undergoing rigorous evaluation using GLP (Good Laboratory Practice) compliant methods.
Currently, most hERG screening assays employ traditional well-plate formats and ultimately a GLP-compliant gold standard patch clamp electrophysiology approach to test for potential drug toxicity on model cell lines that stably express hERG ion channels.11–13
Typical procedures involve selecting single cells for patch clamp analysis with drug applied acutely to test for a reduction in hERG channel current,12
or mixing the drug of interest into cell culture plates for a desired time and then selecting single cells for patch clamp analysis or lysates of cells for protein chemistry. Recently, live cell Western analysis using infrared scanning technology was also employed to assess hERG surface membrane expression in a well-plate format.14
Although a variety of new screening assays (e.g., automated patch clamp, radioligand displacement, flux assays, and HERG-Lite®15
) have demonstrated increased throughput capabilities, there remains a need to further improve efficiency and lower cost of these assays. It has been estimated that the costs of drug development – from discovery to market – is between ~US$800 million and $2 billion per new drug, with a sizable portion of the costs consumed by synthesizing, storing, and testing numerous compounds that inevitably fail to reach market.16–19
These costs can be lowered by reducing the volume of compounds necessary to perform large screening assays during the discovery stage of the development process. Microfluidics offers a low-volume alternative platform for testing drug toxicity of cultured cells. Microfluidic systems consist of arrays of microchannel geometries that can be designed for cell culture and tailored to model controlled microenvironments. Beyond the basic benefits of reduced reagent consumption and the potential for increased throughput, microfluidic systems allow precise spatial and temporal control of cell-cell and cell-matrix interactions. Because of the benefits of microfluidic cell culture, a number of studies in the past several years have investigated and characterized cell growth, maintenance, function, and response of various cell types in microfluidic devices.20–29
However, to our knowledge, the properties of hERG-expressing cell lines in microfluidic cell culture have yet to be characterized. To prove that microfluidic channels are suitable for cell culture and analysis of hERG-expressing cells, and to demonstrate the various advantages of microfluidic methods, it is necessary to properly characterize and validate the specific functional responses of these cells in microchannels.
In addition to characterizing the biological responses of hERG-expressing cells within microchannel environments, there is also a need to continue developing microfluidics technology that is more amenable to biological experimentation in the laboratory. An often-cited drawback of microfluidic systems, according to biologists, is their inherent complexity and need for external equipment, on-chip pumps and valves, and connecting tubes that are required for fluid handling.30
While some of these accessories can provide additional functionality and control within microchannels, they often lead to loss of efficiency and usability, and to increased probability of technical failure (e.g., leaks). One technique that is both effective for handling flows in microchannels and amenable to biological experimentation is the use of surface tension-driven passive pumping. Passive pumping relies on dispensing precise volumes of droplets at the microchannel inlets and outlets, and allowing differences in surface tension at droplet interfaces to produce a pressure gradient within the microchannel.31, 32
This method is convenient for biological assays because it only requires simple pipetting operations, and can be integrated with automated liquid handling systems for high-throughput applications.25
Furthermore, biological assays performed using microchannels can be analyzed using laser scanners and plate readers, as recently demonstrated,23
and this further establishes microfluidics as a viable alternative for integration in the biology laboratory. Continued progress in this area is essential to the ongoing development of useful technology that can serve the biology research community.
Our objective in this study was to investigate the growth and culturing properties of HEK (human embryonic kidney) cells stably transfected with hERG channel protein towards the development of a high-throughput, reliable, and less costly platform for screening the cardiac toxicity of drug compounds. We cultured non-transfected and stably transfected HEK cells in polystyrene flasks, as well as in microchannels made from two different plastics (polystyrene (PS) and cyclo-olefin polymer (COP)) and a silicone elastomer (polydimethylsiloxane (PDMS)).We performed cell proliferation assays, immunofluorescent staining procedures, traditional Western blot analyses, and live-cell Western analyses coupled with infrared detection to establish microchannels as a viable culturing environment for hERG-expressing cells. In addition to the abovementioned assays, electrophysiological clamp patch experiments were performed on microchannel-cultured cells to verify the functional properties of the hERG ion channels after culturing in microchannels. After performing the necessary validation tests, hERG-expressing cells were further treated with various drugs to determine the suitability of microchannels for drug toxicity testing. Results demonstrated the potential for microfluidic screening and suggest that PS and COP (i.e., thermoplastic) microchannels may be more appropriate than PDMS devices because it does not undesirably absorb hydrophobic drug molecules. The use of passive pumping for fluid handling and infrared laser scanning for biological readout in our platform obviates the need for external pumps and tubing that are commonly used in other microfluidic systems, and allows the use of existing biology laboratory infrastructure and equipment for analysis.