HIGH-THROUGHPUT DRUG-SCREENING USING HUMAN CARDIOMYOCYTES IN MECHANICALLY CONTROLLED ENVIRONMENTS

KIM, GYU-TAE 1, 2; SHAFIEYAN, YOUSEF 1; SHEN, TRONG 1, 3; PLAKHOTNIK, JULIA 4; MAYNES, JASON 4; HINZ, BORIS 1, 3

1. Faculty of Dentistry, University of Toronto; 
2. Cardiovascular Sciences Collaborative Specialization, University of Toronto; 
3. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 
4. Departments of Anesthesia and Biochemistry, University of Toronto

Background: Drug side-effects are a major risk for patients and one of the most common reasons for drug recall from the market. One of the most common reasons for failing drug trials is related to cardio toxic side-effects. Better preclinical test using human cardiomyocytes (CM) as sentinel cells in high throughput screening (HTS) are needed to detect drug side-effects earlier. However, no currently available HTS assay can directly assess CM contraction amplitude. We developed a HTS platform to quantify deformations (wrinkles), created by cell contractions on silicone polymer surfaces. The wrinkling silicone polymer substrates can be produced in a stiffness ranging from soft healthy to stiff diseased heart. Proof-of-principle tests with one substrate stiffness already demonstrated the suitability of the wrinkling assay to quantify contractions of induced pluripotent stem cell (iPSC)-derived CM.

Hypothesis: Tuning culture surface stiffness to the mechanical conditions of the heart will enhance the validity of in vitro drug tests with human iPSC-derived CM.

Objective: To evaluate HTS with human iPSC-derived CM in wrinkling assays of different pathophysiological stiffness and in different drug treatment scenarios.

Methods: Wrinkling silicone polymer substrates with stiffness corresponding to healthy and diseased heart were cast onto the bottom of 96-well plates, covalently functionalized with matrix protein, and seeded with human iPSC-derived CM. CM beating frequency, amplitude, and regularity of contraction was quantified as a function of substrate stiffness and in response to a panel of established drugs. Wrinkling device performance was benchmarked against an industry gold-standard cardiotoxic test device (xCELLigence). In addition to modulating CM beating with drugs, we performed electrical stimulation (pacing) with different input frequencies and voltages. Further, CM contraction was simultaneously analyzed with calcium transients to assess cardiac excitation-contraction in proof-of-principle tests.

Results: Tests performed with 9 established drugs in 5 concentrations validated the wrinkling device for its capacity to detect dose-dependent drug effects on CM beating. Compared to xCELLigence, the wrinkling assay showed higher sensitivity and the unique ability to measure contraction amplitude changes. The wrinkling assay succeeded where xCELLigence failed to reveal expected drug effects, e.g., showing dose-dependent decrease in response to the cardiac myosin activator omecamtiv mecarbil. Culture on either soft or stiff wrinkling substrates modulated maturity of CM sarcomere structure and, consequently, responsiveness to drug treatment. The wrinkling contraction assay also reliably detected changes in rate and amplitude of CM contraction modulated by acute electrical pacing, and was compatible with simultaneous calcium analysis.

Conclusion: We validated the wrinkling HTS assay for its ability and high sensitivity to measure changes in rate and amplitude of CM contractions. The stiffness-tuneable culture material of the device allows to assess CM contraction under different mechanical pathophysiological conditions.

Impact: The wrinkling assay fills the gap of HTS test to measure CM contraction amplitude and provides additional features, such as pathophysiological mechanical conditions to improve in vitro drug tests.