TOXicity assessment on neurons and cardiomyocytes by means of FluoRescence Emitting Electrodes

Project Information

TOX-Free

Grant agreement ID: 964518

DOI

10.3030/964518

Project closed

EC signature date 16 December 2020

Start date 1 June 2021

End date 31 May 2025

Funded under

• EXCELLENT SCIENCE - Future and Emerging Technologies (FET)

Total cost

€ 3 035 166,25

EU contribution

€ 3 035 166,25

Coordinated by

FONDAZIONE ISTITUTO ITALIANO DI TECNOLOGIA
Italy

Periodic Reporting for period 2 - TOX-Free (TOXicity assessment on neurons and cardiomyocytes by means of FluoRescence Emitting Electrodes)

Reporting period: 2022-06-01 to 2025-05-31

Summary of the context and overall objectives of the project

Drugs, pesticides and chemical pollutants can potentially cause neurotoxicity and cardiotoxicity. However, we lack robust assays for accurate and sensitive assessment of functional toxicity in the cardiac and central nervous systems. The EU-funded TOX-Free project develops a non-invasive nanotechnology-based technique capable of recording in vitro electrical signals from human stem-cell derived neuronal and cardiac cells. The TOX-Free biosensing technology is based on the "VIrtual CEll" (VICE) concept that transduces electrophysiological activity into optical signals in a label-free way (A. Barbaglia, Advanced Materials, 2021). The VICE biosensor will allow the assessment and quantification of subtle cellular and functional disturbances by toxicants or drugs beyond existing technologies including microelectrode array (MEA) and live-cell imaging. The biosensor will find direct applications in toxicology and pharmacology as well as in basic biology studies.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Overall the project, the consortium has developed and introduced novel technologies to improve cardiac and neuronal in-vitro toxicology. These technologies stem from the original TOX-Free ideas but expanded into different and novel applications that followed the general needs and trends of the toxicology field for neurons and cardiomyocytes.
Regarding cardiotoxicity, the consortium has developed a new device concept for the measurement of cardiac contraction forces with high spatial resolution, down to single cell, in large monolayer cultures. In addition, these measurements have been combined also with electrical recording of field potentials, to move toward multiparametric analyses. Additionally, the consortium has developed protocols for extreme long-time Action Potential recordings from cardiomyocytes on microelectrode arrays (MEA) for enabling new insights into chronic cardiotoxicity. Finally, we demonstrate a novel polymer based transistor that enables the recording of intracellular-like cardiac Action Potentials by exploiting laser cell optoporation. These polymer based transistors can potentially be fabricated and produced with very low-cost techniques and can be more environmental friendly than semiconductor based biosensors. Furthermore, the consortium has established new protocols for assessing cardiotoxicity on MEA by comparing the effects of several compounds on different cardiac cell models derived from human stem cells, determining which model can better resemble the human cardiac model for cardiotoxicity. Finally, we have also presented a new method for measuring in-vitro fiber-like structures of cardiomyocytes that can reveal quantitative information on signal conduction velocity and gap junctions.

Regarding neurotoxicity, we have developed new all-optical technologies based on Raman Spectroscopy for cell phenotyping and for assessing neuronal toxicity at earlier stages than established technologies. These optical technologies enabled us to discriminate differentiation stages of neuronal spheroids with high temporal resolution and enabled us to detect toxic effects of known compounds on neurons at earlier stages without damaging the spheroids. Potentially, these optical technologies can offer the possibility to follow the development of neuronal toxicity over time in complex three-dimensional spheroids or organoids. Moreover, we have also shown that Raman Spectroscopy combined with MEA electrophysiology can reveal subtle toxicological effects in neuronal cultures by correlating the optical and electrical measurements with advanced machine learning algorithms.
Finally, we have also introduced a laser cell poration technology that offers the recording of sub-threshold activity in neuronal culture on MEA in a non-invasive manner, enabling longitudinal analyses.

The consortium has been active in disseminating and communicating these results to the scientific community and the general audience. Among the main activities, the consortium has actively organized and supported the first three editions of the Biocube Meeting, a new winter-school and workshop focused on biophotonics, bioelectronics, and biomechanics.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The technologies introduced by TOX-Free have the potential to go beyond the state-of-the-art represented by established in-vitro electrophysiological recordings, such as patch-clamp, MEA recording, and optical voltage sensitive recordings.
TOX-Free approaches aim at offering label-free and non invasive measurements, with the final goal of preserving the biological samples in their physiological conditions as long as possible. In turn, this improves the reliability of the measurements, because no external agents such as fluorophores or genetic modifying agents are administered to the cells, and enables the long-time monitoring of the same biological samples for studies of chronic cardiotoxicity and neurotoxicity.

The possibility to assess long-term toxicity effects on neurons and cardiomyocytes will have tremendous impact on society. On the one hand, this is due to the lack of current technologies to properly measure chronic toxicity on the same biological samples. On the other hand, it is becoming increasingly important to understand the effects of long-time exposures to many drugs, which have been shown to lead to cardiotoxic issues as in the case of anti-cancer drugs, like for example Doxorubicin.

Moreover, the development of new devices for high-resolution measurements of cardiac contraction forces follows the general trend of the cardiovascular community to focus not only on cardiac electrophysiology, but also on cardiac contractility. Also, the combination of contractility and electrophysiological measurements on the same device follows the present market trend of developing and commercializing multiparametric approaches that can provide comprehensive information from the same biological samples.

Fluorescence imaging of cardiac cells on a VICE biosensor