Speaker
Description
INTRODUCTION
Despite big efforts and capital invested, drug commercialization is severely hampered by high attrition rates impacting all development phases. Cardiac-side effects are the fourth cause of post-marketing drug withdrawal and stand behind almost 30% during the pre-marketing phase. With the blame on current testing systems, the generation of affordable and more representative cardiotoxicity testing platforms is a must to increase public confidence in drugs simultaneous to solid economical growth. Though the advent of human induced pluripotent stem cells (hiPSC) has promised to raise the prediction capacity of novel systems, the costs associated have highlighted the need to implement advanced technology towards decreasing it, whilst increasing throughput. Our aim is to use the micro-contact printing (μCP)-based “Single Cell Adhesion Dot Arrays” (SCADA) substrates1 with human cardiovascular phenotypes to develop a low-cost, high-throughput and easy-to-use system for human cardiotoxicity tracking.
METHODOLOGY
Micropatterned SCADAS were tuned to match the specific biological requirements of the hiPSC- cardiovascular phenotypes Hence, geometry, size and distribution of protein-patterns in combination with diverse cell density plating conditions are analyzed through optical detection by the use of conventional brightfield microscopy. We use small molecule-based differentiation protocols to obtain cardiomyocytes (hiPSC-CMs), smooth muscle cells (hiPSC-SMCs), endothelial cells (hiPSC-EC) and cardiac fibroblast (hiPSC-CFs). Once the best SCADA layout promoting single cell adhesion is selected, they are exposed to the cardiotoxic drug and cellular death monitored by optically assessing detachment.
RESULTS
A total of 5 different hiPSC lines were employed. The differentiation protocols generated highly-pure hiPSC-CMs, hiPSC-SMCs, hiPSC-ECs and hiPSC-CFs, as defined by FACS, IF and RT-qPCR for specific markers. For hiPSC-CMs, 45x25um Matrigel rectangular dots was the best SCADA design. 70-75% of initial dot array occupancy (DAO) is achieved, providing over a thousand cells (events) per SCADA that can be counted for high sensitivity. Collagen type I rectangular dots of bigger dimensions (90x30um) are employed to contain cells hiPSC-SMCs, hiPSC-ECs and hiPSC-CFs, rendering similar optimized conditions. Exposure to the cardiotoxic drugs doxorubicin or epirubicin at different concentrations elicited a drastic DAO decrease throughout the 96h of exposition, proving the capacity of the devised SCADAs to monitor toxicity; in addition, the different detachment kinetics are correlated to the IC50 values for the different lines, which include hiPSCs derived from patients sensitive to doxorubicin. Moreover, these experiments show a higher sensibility of SMCs to the drug than other cardiovascular phenotypes. Finally, different phenotypes are included in a microfluidic chip in a first step towards building a multidimensional testing platform.
CONCLUSIONS
This work lays the foundations for a promising cardiotoxicity testing system, requiring low cell numbers, inexpensive materials and no specialized equipment. The system can uncover phenotype-specific drug sensitivity, and opens the way to high throughput analysis using microfluidics miniaturization, allowing the decrease of sampling volumes and cellular input.
- Garcia-Hernando, Maite et al., Anal Chem. 92, 9658-9665 (2020)
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