High Throughput Drug Testing Platform for Long Term Cardiotoxicity Monitoring Using High Fidelity Engineered Cardiac Tissue

Introduction

An average of $1.5 billion is spent to bring a new drug to the market, while a large portion of the drugs is withdrawn at the later stage of the development. Up to 20% of recent drug withdrawals were due to cardiac toxicity and proarrhythmic effects. Thus, tissue-specific toxicity screening with a human cardiac model is necessary to weed out the unqualified drug candidates.

Materials and Methods

To fabricate the platform, hot embossing technique with polystyrene and poly(methyl methacrylate) will be used to generate micro-wells with dimension of 5mm by 1mm by 300m, which will fit in a single well of a96 well-plate shown in figure1. Every micro-well will have two auto-fluorescent and flexible wires made of poly (octamethylene maleate (anhydride) citrate) positioned at the edges of the well. After cell seeding, cardiac tissue will compact around wires and suspend in the well. Adjacent to both short ends of the well, a pair of electrodes will be printed and connected with electrical stimulators (Grass88X). The shape changes of wires will be recorded by optical and fluorescence high speed camera during tissue contraction in the device. By tracking the deflection of the wire with Image J, beam deflection equation will be used to measure tissue generated contractive forces. In addition, beating frequency, amplitude, contraction and relaxation times will be easily acquired. The accuracy of the device will be validated by comparing the calculated forces and the ones measured using a force transducer. This device can eliminate the common drawback of post deflection design, in which tissue often slips off the posts. The design will also prevent inconveniently measuring positions of tissue on posts, which is a crucial parameter in force calculation. In the next stage, drugs with known effects (e.g.,verapamil, nifedipine, norepinephrine, isoproterenol, lidocaine, caffeine, E-4301, etc.) will be tested to confirm ECT responds similarly to human adult cardiac tissue. Then, a group of drugs approved but withdrawn from the market (cisparide, terfenadine, rosiglitazone) will be tested to show the sensitivity of the device. Negative control will be ketaconazole with no cardiotoxicity. In the final stage, a library of 367 kinase inhibitors obtained from GlaxoSmithKline will be used to test the influence of different signaling pathways on ECT function and survival, to further explore possible drug targets.

Results

Significant improvement of electrical properties with excitation threshold and maximum capture rate on day 21 as end point measurements has been shown. Fluorescence images on cellular protein expression on day 21 after seeding (end point assessment): Gap junction protein, connexin 43 (Cx43) (Red); sarcomere protein Cardiac troponin T (cTnT) (Green) and F-actin (Green); a-actinin (infrared red). Staining for f-actin and -actinin showed co-localized unique striation structures 2. Video was taken at different time point to quantify contraction forces of the spontaneous beating. Contractile forces increased rapidly in the first week and approached a plateau in the second and third week. Force per cross-section was calculated from deflection at day 21 (end point assessment) with pacing frequency from 0.5Hz to 3Hz. After normalizing to forces generated at 0.5Hz, positive FFR has been shown. For preliminary drug testing, representative tissue beating pattern at a given concentration of drugs in comparison with the control. Norepinephrine addition at 10mM resulted in the increase in contraction frequency. E4301 addition at 100nM resulted in an occasional after beat or prolonged relaxation time. Isoproterenol had slight positive inotropic action (increase in contractility) at 100 nM and 1mM, and a slight negative inotropic effect (decrease in contractility) at 10 mM.

Discussion and Conclusion

This project has successfully designed a cardiotoxicity specific drug screening platform in a 96 well-plate format with terminally differentiated ECT. The design eliminates the use of PDMS. The device will realize the miniaturization of ECT and be compatible with three different stages of tissue cultivation and drug testing, which are miniaturized cardiac tissue formation, electrical stimulationtoenhance tissue maturationand automated high throughput drug screening with contractile force readout.

References

1. Nunes, S.S., et al., Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes. Nat Methods, 2013. 10(8): p. 781-7.

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