A Novel 3D Label-Free Monitoring System of hES-Derived Cardiomyocyte Clusters: A Step Forward to In Vitro Cardiotoxicity Testing
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.
A novel 96-well multielectrode array based impedimetric monitoring platform for comparative drug efficacy analysis on 2D and 3D brain tumor cultures
Aggressive cancer entities like neuroblastoma and glioblastoma multiforme are still difficult to treat and have discouraging prognosis in malignant stage. Since each tumor has its own characteristics concerning the sensitivity towards different chemotherapeutics and moreover, can obtain resistance, the development of novel chemotherapeutics with a broad activity spectrum, high efficacy and minimum side effects is a continuous process. Sophisticated in vitro assays for comprehensive prediction of in vivo drug efficacy and side effects represent an actual bottleneck in the drug development process. In this context, we developed a novel in vitro 2D and 3D multiwell–multielectrode device for drug efficacy monitoring based on direct real-time impedance spectroscopy measurement in combination with our unique 96-well multielectrode arrays and microcavity arrays. For demonstration, we used three neuro- and glioblastoma cell lines that were cultured as monolayer and multicellular tumor spheroids for recapitulating in vivo conditions. Using our novel 96-well multielectrode array based system it was possible to detect time and concentration dependent responses concerning treatment with doxorubicin, etoposide and vincristine. While all tested chemotherapeutics revealed high potency for apoptosis induction in neuroblastoma cells, etoposide was ineffective for glioblastoma cell lines. Determination of IC50 values allowed us to compare drug efficacy in 2D and 3D culture models and moreover, revealed chemotherapeutic and tumor cell line specific activity patterns. These pharmacokinetic patterns are of great interest in the context of preclinical drug development. Thus, impedance spectroscopy based monitoring systems could be used for the fast in vitro based in vivo prediction of novel anti-tumor drugs.
A tubular electrical impedance tomograph (EIT) with micrometric dimensions was fabricated by using rolledup nanotechnology. This approach gives access to EIT devices with tunable sizes in the sub-100 µm range. EIT images of silicon dioxide microparticles were obtained as proof of principle. These devices could enable the impedimetric analysis of biological micro-scale objects, such as single cells or small cell clusters.
In recent years, there has been a growing interest in the analysis of single cells. Single-cell studies give valuable insights in the variability of biological cells in one and the same cell population, and can give new information about their fundamental properties. Impedance measurements are especially well-suited for these analyses, as they are labelfree and non-destructive. Tomographic measurements in particular are of interest since they can additionally provide spatial information in real time. EIT studies of single cells call for appropriate measurement chambers, whose sizes should be similar to that of the object to be studied. This can easily be achieved using rolled-up nanotechnology.