Interfacing Stretchable Electronics and Engineered Neuronal Cultures for In Vitro Mechano-Neurobiology

Engineered neuronal networks have become a valuable tool to study neural cultures <i>in vitro</i>. The ability to control the growth and connectivity of neuronal networks in a regulated environment allows to study basic processes such as memory formation and learning, or to test the effect of drugs on the nervous system. However, most engineered neuronal networks are cultured on stiff substrates, not reflecting the soft and stretchable nature of the majority of biological tissues.<br/>To address this issue, we present a modular device interfacing stretchable electronics and polydimethylsiloxane (PDMS) microstructures to study the mechanobiology of engineered neuronal networks <i>in vitro</i>. The device fabrication is based on two previously published technologies developed in our group: firstly, a stretchable multielectrode array (MEA) is produced by the patterning of porous polyvinylidene difluoride (PVDF) membranes with photolithography, followed by filtration of nanowires and nanoparticles through the patterned areas to create the tracks and electrodes. These conductive patterns are then transferred on semi-cured PDMS in a layered fashion to form the stretchable MEA¹. The electrodes are characterized using cyclic voltammetry and impedance spectroscopy. Then, a PDMS axon-guiding microstructure² is aligned on the multielectrode array and glued onto it using a thin film of diluted PDMS. The axon-guiding microstructure is produced using a standard soft lithography process: two-layer SU-8 masters are fabricated by spin coating and patterning. PDMS is spin-coated on the masters, resulting in an approximately 250 µm thick patterned microstructure. The first, top layer of the structure has circular landing spots open on top to enable the placement of neurons and neural spheroids into the desired nodes of a network. The second, bottom thin layer is patterned so that axons from neurons seeded in different nodes gradually merge with each other unidirectionally to form an artificial nerve, while cell bodies cannot penetrate into this six µm thick closed compartment of the microstructure. The mechanical properties of the device are investigated by performing a peeling test to characterize the interface bonding strength between the two layers. In addition, a cyclic stretching test is carried out to characterize the device electromechanically.<br/>Neurons can then be cultured in the microstructure and their axons grow in a controlled and confined manner in microchannels, forming engineered neuronal cultures. The biocompatibility of the device is tested by live/dead staining. We envision using the stretchable MEA to stimulate the neurons electrically under various strain conditions for mechano-neurobiology studies.<br/>References:<br/>1. Renz, A., Lee, J., Tybrandt, K., Brzezinski, M., Lorenzo, D., & Cerra Cheraka, M. et al. (2020). Opto-E-Dura: A Soft, Stretchable ECoG Array for Multimodal, Multiscale Neuroscience. <i>Advanced Healthcare Materials</i>, 9(17), 2000814<br/>2. Forró, C., Thompson-Steckel, G., Weaver, S., Weydert, S., Ihle, S., & Dermutz, H. et al. (2018). Modular microstructure design to build neuronal networks of defined functional connectivity. <i>Biosensors And Bioelectronics</i>, 122, 75-87