Improving morphological and functional properties of enteric neuronal networks in vitro using a novel upside-down culture approach.

The enteric nervous system (ENS) comprises millions of neurons and glia embedded in the wall of the gastrointestinal tract. It not only controls important functions of the gut but also interacts with the immune system, gut microbiota, and the gut-brain axis, thereby playing a key role in the health and disease of the whole organism. Any disturbance of this intricate system is mirrored in an alteration of electrical functionality, making electrophysiological methods important tools for investigating ENS-related disorders. Microelectrode arrays (MEAs) provide an appropriate noninvasive approach to recording signals from multiple neurons or whole networks simultaneously. However, studying isolated cells of the ENS can be challenging, considering the limited time that these cells can be kept vital in vitro. Therefore, we developed an alternative approach cultivating cells on glass samples with spacers (fabricated by photolithography methods). The spacers allow the cells to grow upside down in a spatially confined environment while enabling acute consecutive recordings of multiple ENS cultures on the same MEA. Upside-down culture also shows beneficial effects on the growth and behavior of enteric neural cultures. The number of dead cells was significantly decreased, and neural networks showed a higher resemblance to the myenteric plexus ex vivo while producing more stable signals than cultures grown in the conventional way. Overall, our results indicate that the upside-down approach not only allows to investigate the impact of neurological diseases in vitro but could also offer insights into the growth and development of the ENS under conditions much closer to the in vivo environment.NEW & NOTEWORTHY In this study, we devised a novel approach for culturing and electrophysiological recording of the enteric nervous system using custom-made glass substrates with spacers. This allows to turn cultures of isolated myenteric plexus upside down, enhancing the use of the microelectrode array technique by allowing recording of multiple cultures consecutively using only one chip. In addition, upside-down culture led to significant improvements in the culture conditions, resulting in a more in vivo-like growth.

Nanoimprint lithography-based replication techniques for fabrication of metal and polymer biomimetic nanostructures for biosensor surface functionalization.

Nanostructuring is a promising and successful approach to tailor functional layers and to improve the characteristics of biosensors such as signal transmission and tighter cell-surface coupling. One of the major objectives in biosensing and tissue engineering is the development of interfaces that mimic the natural environment of biosystems composed of extracellular matrix biomolecules. Nevertheless, effective techniques to reconstruct the random distribution of these biomolecules are still not well established. For this reason, the presented work demonstrates different methods based on nanoimprint lithography to replicate randomly distributed natural nanostructures with complex geometries into different polymers and metals. The fidelity of the replicated nanostructures has been evaluated by atomic force microscopy and the attributes of the fabrication processes have been discussed. Finally, different replication techniques have been combined for the biomimetic nanostructuring of the dielectric passivation layer as well the metal electrode surface to develop novel whole-surface-nanostructured microelectrode arrays.