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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - CellStretch (A Cardiomyocyte Workout:Using Dielectric Elastomer Actuators for Mechanical Stimulation of in-vitro Cells)

Teaser

Summary

Most of what is known in the realm of cell biology is the result of painstaking experiments of in vitro cell cultures. Removing tissue from a living organism and placing it into the controlled environment of a petri dish allows for complete control of the experiment. No longer obscured by the complexity of an entire living body, in vitro cell cultures enabled physiologists to use microscopy, biochemistry, and electrophysiology to reveal the basic biological functions of both tissues and single cells. This led to great advances in our understanding of cell metabolism, toxicology, drug screening, fertility, and genetics. Although temperature, chemical composition, and electrical signaling can be precisely controlled, glassware is not only hard but also static. There is no bending or stretching, no movement. And what is life without motion? Cells â€˜feelâ€™ their surroundings, push back against external perturbations and self-organize to assert themselves in a dynamic world. But a Petri dish is dead: a hard, unyielding boundary condition, a sterile well where cells are cultured and experimented on from hours to days, until they die.

We developed in this project a mechanically active device for new kinds of in vitro cell physiology experiments. Through the use of Dielectric Elastomer Actuators (DEA), a stretchable membrane replaces the petri dish. Cells can be grown on such â€˜artificial musclesâ€™ and exposed to well defined strains and strain rates; this represents a much closer approximation to their native environment. DEAs expand the experimental parameter space of in vitro studies to include the all-important mechanical degree of freedom; this addresses the mechanosensitive response of living cells.

In this context, we were able to develop not only a mechanically dynamic cell culture well, but also an integrated a micro electrode array (MEA) enabling electrical sensing and stimulation in addition to the mechanical dynamics. This allowed us to monitor the electromechancial feedback of a strand of cardiomyocytes by tracking the impulse propagation. We were able to determine not only the sensitivity to strain amplitude but also the response to changing strain rates.

The underlying mechanisms of arrhythmias causing cardiac failure, currently the number one cause of death in the EU, can now be studied with the inclusion of mechano-electrical feedback (MEF). Adverse mechano-electrical feedback in the heart is dramatically illustrated by accidents where people die after the thorax is struck powerfully, e.g. by a football. Much subtler, but quantitatively much more relevant, ad-verse mechano-electrical feedback is thought to occur in diseased hearts in patients with high blood pressure, valvular heart diseases, and infarction. The CellStretch project developed a fundamentally new device that can both generate the strain rates associated with traumatic interactions and simultaneously track the electrophysiology of the cell strand.

Work performed

The primary focus of CellStretch was to integrate the DEA technology, combined with compliant electrodes, to enable new kinds of experiments to study mechano-electrical feedback mechanisms in vitro. To do this, a modular system was developed:
I. Single culture wells that include the mechanically active substrate upon which the cells are grown. Here the cells can be stretched and also make contact with extra-cellular electrodes that sense the field potential (essentially the derivative of the action potential as observed by a group of cells firing synchronously);
II. The single wells interface with a motherboard. This can be in the form of a single well holder or in an array architecture. The motherboard interfaces with a DAQ, and a high voltage power supply is used to drive the DEA. Options include on-board thermometry as well as signal buffering or amplification; and
III. The readout electronics, consisting of signal conditioning and amplification, filtering, and the DAQ, in addition to the input triggers for electrical stimulation.

It was shown that strain amplitudes exceeding 15% are possible, which is the upper limit typically observed in vivo. Furthermore, there is complete control of the strain rate, making it possible to apply period strain and physiological strain patterns as well as mechanical trauma. The results were submitted for publication and presented at scientific conferences (EuroEAP 2016; Biointerfaces International, 2016; NanoBioTech Montreux 2016).

We were able to track the impulse propagation, in particular the velocity, while periodically imposing a tensile strain on the strand. We could show that the net result was a velocity increase with regards to strain. The simplest case model, based on the Cable Theory, is used to illustrate that the velocity change can be explained without activating strain-sensitive ion channels (although the model cannot exclude this possibility). The results are exciting in that they demonstrate the stability of the heart muscle cells to endure cyclic strain, as one would expect of the driving mechanism of an organ that beats without rest over a lifetime. Given the minimal feedback of the myocytes themselves, these results define the baseline for the search for regulatory feedback mechanisms, failure of which can underlie cardiac arrhythmias.

Final results

The platform developed will facilitate a new class of biology experiments targeting the mechanical degree of freedom. With such a high degree of control, with strains exceeding 20% possible and rates of up to 1000 /s, practically any physiological or even traumatic strain event can be reproduced in a controlled and repeatable manner. While the focus of CellStretch was on the mechano-feedback mechanisms of cardiomyocytes, the method is applicable to many cell types. There is strong interest in applying this technique to nerve cells: It is possible to measure the impulse propagation under mechanical loads, in particular rates, currently inconceivable with any other technology. This means that events such as blunt force impact to the spinal cord or brain can now me observed in vitro.

The modular architecture, which can be built into an array, makes it possible to conduct higher throughput experiments needed for screening experiments. This would include the testing of drugs for pharmaceutical purposes, toxicology screening, or even genetic sequencing studies. For example, it is known that high strain rates affect the cell membrane permeability. This tool provides the ideal method for systematically mapping this sensitivity.

The modular architecture implemented allows us to envision further developments, some of which have already been initiated during the CellStretch project. Additional add-on technologies can be integrated to leverage capability. These could include superfusion to create a miniature incubator, or optical elements for lighting and photon stimulation. The addition of lensless optics could create a self-contained system that does not require bulky incubators or microscopes.

This tool will certainly continue to shed light on mechano-electrical feedback mechanisms and deepen our understanding of the underlying causes of arrhythmias, the primary cause of heart failure. As significant is the potential to reveal the mechanisms that lead to tragic injuries of the nervous system due to blunt force trauma.