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.

Poly(4-vinylaniline)/Polyaniline Bilayer-Functionalized Bacterial Cellulose for Flexible Electrochemical Biosensors.

A bacterial cellulose (BC) nanofibril network is modified with an electrically conductive polyvinylaniline/polyaniline (PVAN/PANI) bilayer for construction of potential electrochemical biosensors. This is accomplished through surface-initiated atom transfer radical polymerization of 4-vinylaniline, followed by in situ chemical oxidative polymerization of aniline. A uniform coverage of the BC nanofiber with 1D supramolecular PANI nanostructures is confirmed by Fourier transform infrared, X-ray diffractogram, and CHN elemental analysis. Cyclic voltammograms evince the switching in the electrochemical behavior of BC/PVAN/PANI nanocomposites from the redox peaks at 0.74 V, in the positive scan and at -0.70 V, in the reverse scan, (at 100 mV·s-1 scan rate). From these redox peaks, PANI is the emeraldine form with the maximal electrical performance recorded, showing charge-transfer resistance as low as 21 Ω and capacitance as high as 39 µF. The voltage-sensible nanocomposites can interact with neural stem cells isolated from the subventricular zone (SVZ) of the brain, through stimulation and characterization of differentiated SVZ cells into specialized and mature neurons with long neurites measuring up to 115 ± 24 µm length after 7 days of culture without visible signs of cytotoxic effects. The findings pave the path to the new effective nanobiosensor technologies for nerve regenerative medicine, which demands both electroactivity and biocompatibility.

Silk sericin-enhanced microstructured bacterial cellulose as tissue engineering scaffold towards prospective gut repair.

As a first step towards the production of functional cell sheets applicable for the regeneration of gut muscle layer, microstructured bacterial cellulose (mBC) was assessed for its ability to support the growth of enteric nervous system (ENS) and gut smooth muscle cells (SMCs). To improve the cellular response, mBC was modified with silk sericin (SS) which has renowned abilities in supporting tissue regeneration. While SS did not impair the line structures imparted to BC by PDMS templates, similarly to the patterns, it affected its physical properties, ultimately leading to variations in the behavior of cells cultured onto these substrates. Enabled by the stripes on mBC, both SMCs and ENS cells were aligned in vitro, presenting the in vivo-like morphology essential for peristalsis and gut function. Interestingly, cell growth and differentiation remarkably enhanced upon SS addition to the samples, indicating the promise of the mBC-SS constructs as biomaterial not only for gut engineering, but also for tissues where cellular alignment is required for function, namely the heart, blood vessels, and similars.

Poly(4-vinylaniline)/polyaniline bilayer functionalized bacterial cellulose membranes as bioelectronics interfaces.

Bacterial cellulose (BC) fibers are chemically functionalized with poly(4-vinylaniline) (PVAN) interlayer for further enhancement of electrical conductivity and cell viability of polyaniline (PANI) coated BC nanocomposites. PVAN is found to have promoted the formation of a uniform PANI layer with nanofiber- and nanorod-like supramolecular structures, as an overall augmentation of PANI yield. Compositional and microstructural analysis indicates a PVAN/PANI bilayer of approximately 2 µm formed on BC. The solid-state electrical conductivity of such synthesized BC nanocomposites can be as high as (4.5 ± 1.7) × 10-2 S cm-1 subject to the amounts of PVAN chemically embraced. BC/PVAN/PANI nanocomposites are confirmed to be thermally stable up to 225 °C, and no signs of cytotoxicity for SVZ neural stem cells are detected, with cell viability up to 90% on BC/PVAN/PANI membranes. We envisage these new electrically conductive BC/PVAN/PANI nanocomposites can potentially enable various biomedical applications, such as for the fabrication of bioelectronic interfaces and biosensors.

Carbon Nanotube-Reinforced Poly(4-vinylaniline)/Polyaniline Bilayer-Grafted Bacterial Cellulose for Bioelectronic Applications.

Microbial cellulose paper treated with polyaniline and carbon nanotubes (PANI/CNTs) can be attractive as potential flexible capacitors in terms of further improvements to the conductivity and thermal resistance. The interactions between PANI and CNTs exhibit new electrochemical features with increased electrical conductivity and enhanced capacity. In this study, PANI/CNTs was incorporated into a flexible poly(4-vinylaniline)-grafted bacterial cellulose (BC/PVAN) nanocomposite substrate for further functionalization and processability. PANI/CNTs coatings with a nanorod-like structure can promote an efficient ion diffusion and charge transfer, with a significant enhancement of the electrical conductivity after CNTs reinforcement of 1 order of magnitude up to (1.0 ± 0.3) × 10-1 S·cm-1. An escalating improvement of the double charge capacity (∼54 mF) of the grafted BC nanocomposites was also detected through electrochemical analysis. The multilayered electrical coatings also reinforce the thermal resistance, preventing anticipated thermal degradation of the BC substrate. The cell viability and differentiation assays using neural stem cells (SVZ cells) testified to the cytocompatibility of the grafted BC nanocomposites, while inducing neuronal differentiation over 7 days of culture with a neurite that was 77 ± 24.7 µm long. This is promising for meeting the requirements in the construction of high-performance bioelectronic devices that can actively interface biologically, providing a friendly environment for cells while tuning the device performance.

Microstructured Multilevel Bacterial Cellulose Allows the Guided Growth of Neural Stem Cells.

Repeated photolithographic and etching processes allow the production of multileveled polymer microstructures that can be used as templates to produce bacterial cellulose with defined surfaces on demand. By applying this approach, the bacterial cellulose surface obtains new properties and its use for culturing neural stem cells cellulose substrate topography influences the cell growth in a defined manner.