A Novel 3D Label-Free Monitoring System of hES-Derived Cardiomyocyte Clusters: A Step Forward to In Vitro Cardiotoxicity Testing

Abstract

Unexpected adverse effects on the cardiovascular system remain a major challenge in the development of novel active pharmaceutical ingredients (API). To overcome the current limitations of animal-based in vitro and in vivo test systems, stem cell derived human cardiomyocyte clusters (hCMC) offer the opportunity for highly predictable pre-clinical testing. The three-dimensional structure of hCMC appears more representative of tissue milieu than traditional monolayer cell culture. However, there is a lack of long-term, real time monitoring systems for tissue-like cardiac material. To address this issue, we have developed a microcavity array (MCA)-based label-free monitoring system that eliminates the need for critical hCMC adhesion and outgrowth steps. In contrast, feasible field potential derived action potential recording is possible immediately after positioning within the microcavity. Moreover, this approach allows extended observation of adverse effects on hCMC. For the first time, we describe herein the monitoring of hCMC over 35 days while preserving the hCMC structure and electrophysiological characteristics. Furthermore, we demonstrated the sensitive detection and quantification of adverse API effects using E4031, doxorubicin, and noradrenaline directly on unaltered 3D cultures. The MCA system provides multi-parameter analysis capabilities incorporating field potential recording, impedance spectroscopy, and optical read-outs on individual clusters giving a comprehensive insight into induced cellular alterations within a complex cardiac culture over days or even weeks.

Impedance-based cellular assays for regenerative medicine

“…Arrays of microcavities have been designed by the Robitzki group to host 3D cardiomyocyte clusters derived from embryonic stem cells and assess them in real time with EIS and field potential measurements [83]. Both chronotropic and action potential duration prolongation effects have been detected with this system.…”

Impedimetric real-time monitoring of neural pluripotent stem cell differentiation process on microelectrode arrays

“…EIS is an innovative technique that is broadly used for label-free and real-time monitoring in biosensors (Katz and Willner 2003;Ong et al 2001). Complex cellular processes such as cell death/degeneration Jahnke et al 2013b;Poenick et al 2014;Seidel et al 2012), motility (Haas et al 2010) and signaling in single cells and multidimensional structures in vitro and in vivo (Jahnke et al 2013a) can be analyzed.…”

“…Furthermore, distinct effective temporal intervals for the DAPT effect could be determined that are in explicit accordance with the literature(Borghese et al 2010).Taken together, we developed an EIS platform specifically designed for quantitative precursor-specific neuronal network differentiation monitoring that provides the capability for an easy scale-up to industrial standardized 96-well plates. In this setting, EIS can serve as a quality control instrument for the design and optimization of neural stem/progenitor cell differentiation protocols and therewith neuronal cultures before performing assays, especially when combined to hybrid field potential-based neuron electrophysiology recording(Jahnke et al 2013b). Subsequently, the samemeasurement system can be applied for functional pharmacological investigations e.g.…”

Heart-on-a-chip based on stem cell biology

“…Therefore, in vitro models, which mimic native microenvironment of heart, are developed to understand stem cell conductivity. MEA and impedance spectroscopy (EIS) are used for measurements of extracellular recording of field potential from beating cardiomyocytes , Meissner et al, 2012, Fendyur and Spira, 2012, Jahnke et al, 2013, Myers, et al, 2013, Takeuchi et al, 2013. The microdevices, integrated with electrodes for measurements of electrical conductivity of cells, enable non-invasive and multisite monitoring of cells functions under exposure of various factors.…”