NeuPES was a multidisciplinary project that had two main wings: Engineering and Neurobiology.
In the engineering part we focused on the development of an in vitro electrical stimulation system to apply well-defined electrical field to the cells. We have developed and characterized nanostructured electrodes (nanocolumn arrays) made of titanium, gold, and platinum. The fabrication was done using the Glancing Angle Deposition (GLAD) configuration with magnetron sputtering deposition, which is a scalable method to produce nanostructured electrodes. We characterized the surface morphology using scanning electron microscope (SEM) and calculated the roughness using atomic force microscope (AFM), fig. A and B.
Based on our central hypothesis, the increase in surface area would improve the electrical properties of the electrodes. We measured the impedance of the system using Electrochemical Impedance Spectroscopy and calculated the charge injection capacity of those electrodes from cyclic voltammetry tests: the impedance of the system decreases significantly when the nanocolumn arrays are used. Moreover, the non-faradic (capacitive) mechanism has superior role in charge cargo than the faradic one in case of nanostructured electrodes, which is beneficial since faradic charges are responsible for electrode erosion, water splitting and generation of toxic by-products,fig. C and D.
We have also built an accelerated ageing system to predict lifetime of electrodes after implantation that simulates the body condition in vitro: tests will be performed in the near future.
We have generated a computation model using COMSOL Multiphysics to define the actual delivered current and electrical field of our system, demonstrating a significant improvement in the uniformity of the delivered electrical field to the cells when nanocolumns are used, fig. E.
Based on these results, we have designed an imaging-compatible, miniaturized device for in vitro electrical stimulation of the cells, which benefits research in neurobiology where one deals with limited resources (cells and growth factors) and needs multiple replications for the experiments as well as in situ imaging. We secured the intellectual property of this invention (fig. F) under the Spanish Patent Application (P202030626 in June 2020. This innovation has been analysed by the European Commission's Innovation Radar and consequently published in their platform. Currently, we are actively looking for industrial partners.
In the neurobiology part, we have focused on defining the best electrical stimulation protocol that induces pro-regenerative and neuroprotective responses after ischemic condition with and without electrical stimulation and we have assessed the results in protein expression level. First, we stablished a stroke model in vitro (oxygen glucose deprived condition, OGD) using neurons, astrocyte and mix cultures from rat cerebral cortex. We found different conditioning periods for different cultures (e.g. neurons are more sensitive to OGD compared to astrocytes) and validated the efficiency when more than 50% of cell death was obtained. Then, we applied electrical stimulation and analysed the effect, althoughmore studies are needed since the number of assessed cells was too small.
These preliminary results motivated the next step:to define the optimum electrical stimulation protocol. We have designed an in-situ calcium imaging experiment to observe the effect of different parameters in live cells when stimulated with electrical signals. We run a brief experiment right before the end of this project and observed that the oscillation of calcium changed when electrical stimulation was applied
We have presented the results of this work in the conference proceedings eCells and Materials (eCM periodicals) at the Tissue Engineering and Regenerative Medicine International Society conference (TERMIS). We have organized a symposium at the conference of the European Society for Biomaterials, ESB-2019) and presented a contribution there. We have disseminated this idea to the general public using a leading webpage (Sparrho) and currently we are preparing scientific manuscripts to be published in 2021.
