Enhancing Stem Cell‐based Toxicity Assays by Engineering the Culture Niche in a High‐throughput, Assay‐agnostic Manner

Abstract

Human induced pluripotent stem cell (hiPSC)‐based assays hold great promise for ameliorating human disease and improving the translation of new therapies, but they have shortcomings that hamper their ability to accurately model human physiology. For example, hiPSC‐Cardiomyocytes (CMs) mis‐classify several compounds of known cardiotoxicity. One likely source of this is the maturity of the cells; most culture techniques result in hiPSC‐CMs that express fetal phenotypes, leading to inaccurate or difficult‐to‐translate results. While engineering the extracellular matrix (ECM) has demonstrated improvements to both structural and functional phenotypes, it is often hampered by challenges with integration into industry‐standard assays or instrumentation. Further, the ability to engineer increasingly intricate and multi‐faceted artificial culture environments often drives decisions that trade off biological complexity with experimental throughput. Here, we developed bioengineering techniques to create biomimetic patterns that mimic the shape and structure of the ECM on a transparent polymer‐coated glass layer. The resultant structure directs focal adhesion assembly mechanisms in individual cells to drive higher‐ordered structuring of 2D tissues. This forms 2D anisotropic syncytia from iPSC‐CMs in industry‐standard microplate formats that are compatible with transmitted light microscopy. The method is extended to micro‐electrode arrays (MEA) in a manner that maintains both the quality of MEA recordings and baseline electrophysiological properties. iPSC‐CMs grown this way exhibit several structural and biochemical phenotypes including adult protein isoform expression, myofibril alignment, and sarcomere spacing, length, and width. We show that biomimetic cues enhance the electrophysiological response of cardiomyocytes to various drugs of known effect. Since ECM structuring recapitulates the polarized nature of cells and their concomitant gap junctions, we used the gap junction blocker carbenoxolone for validation studies. Polarized tissues enable differential conduction block measurements between longitudinal and transverse directions. Our MEA data show that patterned tissues exhibit increased sensitivity to carbenoxolone over unpatterned constructs (IC50’s of 14.71 nM versus 398.1 nM, respectively) comparing favorably to clinically relevant unbound concentrations reported in the low nM range. Our biomimetic engineering approach is instrument‐ and assay agnostic, effective on a variety of cell types and lines (including neuronal cells; examples to be presented), and compatible with high‐throughput approaches.Support or Funding InformationNational Institutes of Health Small Business Innovation Research Grant.(A) decellularized ex‐vivo cardiac tissue slices demonstrate a polarized meshwork of extracellular matrix (ECM) fibers; (B) our biomimetic patterning technique recapitulates the nanoscale structure and orientation of the ECM; (C) the technique can be expanded to the surface of high‐throughput MEAs without degradation to electrical signal detection while maintaining the nanoscale pattern (inset); (D) FCDI iCell2 Cardiomyocytes cultured on traditional plastic surfaces show unstructured morphology; (E) iCell2 Cardiomyocytes cultured on our biomimetic surfaces recapitulate the anisotropic structure of adult heart tissue.Figure 1(A) sarcomere lengths (B) and widths of cells on biomimetic nanosurfaces (green) compared to conventional surfaces (gray) are increased; (C) adult isoforms of structural proteins are enhanced with biomimetic nanopatterning; (D) patterned cells exhibit higher oxygen consumption rate; (E) patch clamp data demonstrate increased action potential duration on patterned tissues; (F) asymmetric conduction on patterned tissues enhances the sensitivity of MEA assays to conduction blockers;Figure 2