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.
Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells
Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expressing cell lines. To clarify the nature of the observed impedimetric effect we performed an equivalent circuit analysis as well as analyzed the role of cell morphology and receptor internalization. Finally, an antagonist based extensive molecular signal pathway analysis revealed small alterations of the actin cytoskeleton as well as the inhibition of at least L-type calcium channels as major reasons for the observed NPY-induced impedance increase. Taken together, our novel impedance spectroscopy based NPY-receptor activation monitoring system offers the opportunity to identify signal pathways as well as for novel versatile agonist/antagonist screening systems for identification of novel therapeutics in the field of obesity and cancer.
Impedimetric real-time monitoring of neural pluripotent stem cell differentiation process on microelectrode arrays
In today’s neurodevelopment and -disease research, human neural stem/progenitor cell-derived networks represent the sole accessible in vitro model possessing a primary phenotype. However, cultivation and moreover, differentiation as well as maturation of human neural stem/progenitor cells are very complex and time-consuming processes. Therefore, techniques for the sensitive non-invasive, real-time monitoring of neuronal differentiation and maturation are highly demanded.
Using impedance spectroscopy, the differentiation of several human neural stem/progenitor cell lines was analyzed in detail. After development of an optimum microelectrode array for reliable and sensitive long-term monitoring, distinct cell-dependent impedimetric parameters that could specifically be associated with the progress and quality of neuronal differentiation were identified. Cellular impedance changes correlated well with the temporal regulation of biomolecular progenitor versus mature neural marker expression as well as cellular structure changes accompanying neuronal differentiation. More strikingly, the capability of the impedimetric differentiation monitoring system for the use as a screening tool was demonstrated by applying compounds that are known to promote neuronal differentiation such as the γ-secretase inhibitor DAPT.
The non-invasive impedance spectroscopy-based measurement system can be used for sensitive and quantitative monitoring of neuronal differentiation processes. Therefore, this technique could be a very useful tool for quality control of neuronal differentiation and moreover, for neurogenic compound identification and industrial high-content screening demands in the field of safety assessment as well as drug development.
A novel microfluidic microelectrode chip for a significantly enhanced monitoring of NPY-receptor activation in live mode
Lab-on-a-chip devices that combine, e.g. chemical synthesis with integrated on-chip analytics and multi-compartment organ-on-a-chip approaches, are a fast and attractive evolving research area. While integration of appropriate cell models in microfluidic setups for monitoring the biological activity of synthesis products or test compounds is already in focus, the integration of label-free bioelectronic analysis techniques is still poorly realized. In this context, we investigated the capabilities of impedance spectroscopy as a non-destructive real-time monitoring technique for adherent cell models in a microfluidic setup. While an initial adaptation of a microelectrode array (MEA) layout from a static setup revealed clear restrictions in the application of impedance spectroscopy in a microfluidic chip, we could demonstrate the advantage of a FEM simulation based rational MEA layout optimization for an optimum electrical field distribution within microfluidic structures. Furthermore, FEM simulation based analysis of shear stress and time-dependent test compound distribution led to identification of an optimal flow rate. Based on the simulation derived optimized microfluidic MEA, comparable impedance spectra characteristics were achieved for HEK293A cells cultured under microfluidic and static conditions. Furthermore, HEK293A cells expressing Y1 receptors were used to successfully demonstrate the capabilities of impedimetric monitoring of cellular alterations in the microfluidic setup. More strikingly, the maximum impedimetric signal for the receptor activation was significantly increased by a factor of 2.8. Detailed investigations of cell morphology and motility led to the conclusion that cultivation under microfluidic conditions could lead to an extended and stabilized cell–electrode interface.