High Content Drug Testing Platform Using Matured Human Engineered Cardiac Tissue

The current drug development process is both very slow (15 year average) and costly ($1.5B/drug average). Despite this hefty investment, drugs are routinely withdrawn from the market due to serious toxicities and adverse cardiovascular effects, indicating a failure of the current drug screening methods to fully detect and characterize drug effectiveness and toxicity on human cardiomyocytes (CMs). Safety screen assessments performed on cell cultures and animal models do have predictive value but the results obtained may not correlate with clinical risk. Specifically, this entire framework predicts only 75-90% of the effects of a drug in a human patient with the result that many promising drugs are abandoned early in development and some cardiotoxic drugs go to market. A potential explanation is that much of the safety and efficacy testing has relied upon animal studies and little information has been gathered from human preparations. The applicability of animal studies is limited by the fundamental cardiovascular differences amid species. Similarly, cardiac ion channel in vitro screens frequently involve non-cardiac cells (Chinese hamster ovary, CHO or human embryonic kidney, HEK) that have been engineered to overexpress an individual target ion channel, most commonly the human ether-a-go-go (hERG) channel (I_Kr current). However, interference with other currents or currents in combination could also contribute to cardiac complications necessitating an assessment protocol that evaluates the effect of a drug on the collective behaviour of ion channels in a cardiac tissue.

To address these limitations we have developed the Biowire II platform, a human cardiac tissue array using CMs derived from human induced pluripotent stem cells (hiPSCs) that: 1) captures the physiological hallmarks of the adult human myocardium, 2) is situated in an inert plastic platform compatible with current drug screening equipment and practices in the pharmaceutical industry and 3) enables on-line non-destructive readouts of contractile force and Ca²⁺ transients, as well as the collective behaviour of ion channels.

In the Biowire II platform, immature human CMs and support cells in a hydrogel matrix were seeded into inert polystyrene microwells fitted with a pair of flexible polymer wires for tissue alignment. Upon compaction, the human cardiac tissues were electrically field stimulated over the course of weeks and were demonstrated to have highly organized myofibril ultrastructure, improved electrical properties (ET, MCR), a positive force-frequency relationship (1-4Hz), post-rest potentiation, a recorded conduction velocity of 35.7 cm/s and an action potential profile with human adult-like characteristics (I_to1 notch, plateau, a rapid upstroke velocity and resting membrane potential of -70mV). The Biowire II platform was also demonstrated to be amenable to various cell sources (CDI iCell CMs and CMs²; human embryonic stem cell derived Hes2 and Hes3 CMs; hiPSC BJ1D and C2A CMs), and the engineered human cardiac tissues obtained using the Biowire II platform were both physically and functionally reproducible. Using non-destructive force readouts, the chronotropic and inotropic effects of isoproterenol, nifidipine and thapsigargin were captured. Whole cell patch clamp recordings from the intact Biowire II tissues demonstrated the effects of dofetilide, flecainide and 4-Aminopyridine on action potential. Taken together, the inert plastic Biowire II platform can generate high fidelity human engineered cardiac tissues that possess structural and functional maturation hallmarks of human adult myocardium and can be used as an in vitro drug testing platform to provide non-destructive contractile and calcium handling readouts as well as to interrogate the effect of drugs on the collective ion channel behaviour of human cardiac tissue.