Microfluidic cell culture and its application in high-throughput drug screening: cardiotoxicity assay for hERG channels

Abstract

Evaluation of drug cardiotoxicity is essential to the safe development of novel pharmaceuticals. Assessing a compound's risk for prolongation of the surface electrocardiographic QT interval and hence risk for life-threatening arrhythmias is mandated before approval of nearly all new pharmaceuticals. QT prolongation has most commonly been associated with loss of current through hERG (human ether-a-go-go related gene) potassium ion channels due to direct block of the ion channel by drugs or occasionally by inhibition of the plasma membrane expression of the channel protein. To develop an efficient, reliable, and cost-effective hERG screening assay for detecting drug-mediated disruption of hERG membrane trafficking, the authors demonstrate the use of microfluidic-based systems to improve throughput and lower cost of current methods. They validate their microfluidics array platform in polystyrene (PS), cyclo-olefin polymer (COP), and polydimethylsiloxane (PDMS) microchannels for drug-induced disruption of hERG trafficking by culturing stably transfected HEK cells that overexpressed hERG (WT-hERG) and studying their morphology, proliferation rates, hERG protein expression, and response to drug treatment. Results show that WT-hERG cells readily proliferate in PS, COP, and PDMS microfluidic channels. The authors demonstrated that conventional Western blot analysis was possible using cell lysate extracted from a single microchannel. The Western blot analysis also provided important evidence that WT-hERG cells cultured in microchannels maintained regular (well plate-based) expression of hERG. The authors further show that experimental procedures can be streamlined by using direct in-channel immunofluorescence staining in conjunction with detection using an infrared scanner. Finally, treatment of WT-hERG cells with 5 different drugs suggests that PS (and COP) microchannels were more suitable than PDMS microchannels for drug screening applications, particularly for tests involving hydrophobic drug molecules.

Figure 1

Culture of HEK and WT-hERG cells in polystyrene (PS) straight microchannels (A) and PDMS elliptical microchannels (B). Both HEK and WT-hERG cell lines displayed similar morphologies whether they were cultured in PS flasks, commercially available PS (and COP) microchannels, or in-house fabricated PDMS microchannels (C). Scalebar = 200 µm.

Figure 2

HEK and WT-hERG cell proliferation was determined by BrdU staining (A) and TO-PRO-3 nuclear staining (B and C) directly in microchannels. Both cell lines readily incorporated BrdU after 24 h incubation in microchannels. Staining of nuclei using TO-PRO-3 showed increases in cell counts from 24 to 48 h for both cell lines using PDMS (B) or PS (C) microchannels. In PS microchannels, cell seeding densities of 300 (WT 300 and HEK 300) and 450 (WT 450 and HEK 450) cells/mm² both resulted in increased TO-PRO-3 intensities. (WT 450 and HEK 450 staining images not shown). Scalebar = 200 µm.

Figure 3

Characterization of hERG expression and K⁺ channel function after microchannel culture. (A) Presence of hERG was verified by immunofluorescent staining of WT-hERG cells. Scalebar = 25 µm.(B) hERG expression was also verified after culturing in PDMS microchannels by conventional Western blot analysis. (C) hERG channel function after microchannel culture was verified by standard patch clamp tests to demonstrate expected electrophysiological properties of WT-hERG cells.

Figure 4

Fluoxetine treatment of WT-hERG cells in PS microchannels. (A) Phase contrast images of cultured HEK cells (no treatment only) and WT-hERG cells in PS microchannels after treatment with 0, 30, and 60 µM doses of fluoxetine. Scalebar = 200 µm. (B) Live-cell western analysis using infrared detection to quantitatively measure hERG expression on cell membranes. Red stain is Syto 60 (nuclei) and green stain is hERG protein. (C) Average normalized fluorescence intensity values for hERG staining. ANOVA analysis showed significant differences (P < 0.01) between all conditions (brackets in graph). “WT con” = WT-hERG cells with no fluoxetine treatment; “WT 30” = WT-hERG cells with 30 µM fluoxetine treatment; “WT 60” = WT-hERG cells with 60 µM fluoxetine treatment. hERG fluorescence intensity was normalized to Syto 60.

Figure 5

Conventional Western blot analysis of hERG protein expression after fluoxetine treatment of HEK and WT-hERG cells cultured in polystyrene microchannels.

Figure 6

Live-cell Western analyses for various drug treatments of WT-hERG cells in COP microchannels. (A) Stained images of aspirin (ASA) and ivermectin (Iver) treatments. (B) Average normalized fluorescence intensity values for hERG staining of ASA and ivermectin treatments. (C) Stained images of acetaminophen (ACE) and arsenic trioxide (As₂O₃) treatments. (D) Average normalized fluorescence intensity values for hERG staining of ACE and As₂O₃ treatments. hERG fluorescence intensity was normalized to Syto 60. Significant statistical difference (P < 0.001) indicated by brackets.