The use of soft materials as substrate for neural probes aims at achieving better compliance with the surrounding neurons while maintaining minimal rejection. Many strategies have emerged to enable such probes to penetrate the cortex, among which the use of resorbable polymers. We performed several tests involving two resorbable polymers considered most promising: polyethylene glycol (PEG) and silk fibroin (SF) from Bombyx Mori silkworms. Our coating method provides a repeatable, uniform structure optimized for a stress-reduced insertion of a parylene-C neural probe. Standard compression tests as well as in vitro and in vivo insertion assessments show that both SF and PEG-coated probes are stiff enough to avoid the buckling effect during insertion in the cortex. However, with a buckling force of 300 mN and a mechanical holding in vitro of tens of minutes, we assess silk fibroin to be more reliable for practical handling. In vivo first try-outs in mouse brain showed neither buckling issues of the probe nor undesired alteration of the signal recording. Moreover, we evidenced two distinct time scales in the bioresorption of our polymer coatings: silk fibroin degrades itself in a matter of weeks and PEG dissolves itself within seconds in the presence of water. We then present a hybrid PEG and SF coating that could be used as a drug delivery system with different time scales to reduce both the acute and the chronic body reaction.

The realization of 3D architectures for the study of cell growth, proliferation, and differentiation is a task of fundamental importance for both technological and biological communities involved in the development of biomimetic cell culture environments. Here we report the fabrication of 3D freestanding scaffolds, realized by multiphoton direct laser writing and seeded with neuroblastoma cells, and their multitechnique characterization using advanced 3D fluorescence imaging approaches. The high accuracy of the fabrication process (≈200 nm) allows a much finer control of the micro- and nanoscale features compared to other 3D printing technologies based on fused deposition modeling, inkjet printing, selective laser sintering, or polyjet technology. Scanning electron microscopy (SEM) provides detailed insights about the morphology of both cells and cellular interconnections around the 3D architecture. On the other hand, the nature of the seeding in the inner core of the 3D scaffold, inaccessible by conventional SEM imaging, is unveiled by light sheet fluorescence microscopy and multiphoton confocal imaging highlighting an optimal cell colonization both around and within the 3D scaffold as well as the formation of long neuritic extensions. The results open appealing scenarios for the use of the developed 3D fabrication/3D imaging protocols in several neuroscientific contexts.

3D fabrication techniques are rapidly expanding in the field of scaffold development for cell culture and tissue engineering. Herein we report the realization of free-standing PEGDA hydrogel architectures by using two-photon lithography. The morphological and immunofluorescence characterization of neuro2A cells revealed a tridimensional colonization featuring multiple neuritic extensions per cell as well as the expression of β-tubulin neuronal marker and actin microfilaments. The results open new perspectives in the continuous quest for structured biomaterials able to provide a favorable environment to cells and at the same time not interfering with imaging protocols necessary for a clear scenario of the cell seeding. RECEIVED

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The continuous miniaturization of electronic components and the emergence of nanobiotechnology has opened new perspectives to monitor electrical activities at the single cell level. Here, we describe the creation of very high surface-to-volume ratio passive devices (vertical nanowire probes) using large-scale fabrication process, allowing to follow the electrical activity of mammalian neurons. Based on conventional silicon processing, the silicon nanowires were silicided in platinum in order to improve their electrochemical performances and to guarantee their biocompatibility. Very high signal to noise ratio was achieved (up to 2000) when measuring spontaneous action potentials. Moreover, this bio-platform was used to record the impact of various bio-chemical and electrical stimulations on neuronal activity. To conclude, this study proposes a thorough comparison of the characteristics and performances of these new nanowire-based nanoprobes with the main alternative systems published up to now.

We report on the plasma etching of thick (~23µm) Parylene C structures. Parylene C is a transparent polymer that benefits from high biocompatibility, flexibility and chemical inertness, and has gained increased attention over the years in the biomedical field. In the manufacturing process, highly defined structuration steps of Parylene C are essential, but techniques based on laser, scalpel and wet etching have shown to be unsuitable for properly cut structures. Plasma etching remains nowadays the most widespread option, though fast etching rate, lack of residues and high aspect ratios are still hard to achieve. To overcome these issues, the selection of both mask material and plasma conditions is crucial. Here, three masks-metal, positive and negative photoresists-are tested as stencils, and several plasma parameters are briefly studied in order to obtain the highest etching rate while maintaining good coverage. We showed that increasing the RF power up to a considerable 2800W while maintaining a moderate physical contribution (bias power, pressure, temperature), is optimal in the achievement of fast PaC etching without inducing thermal stress. Besides, the addition of a short fluorinated plasma in the midst of the process is shown to alleviate residues. For the first time, negative photoresist Intervia Bump Plating (BPN) coating followed by ICP 1-RIE 2 are used in order to pattern Parylene C-based structures, with a clean cut, vertical profile and fast etching rate (~0.87±0.06 µm/min) and a selectivity of 0.5. This solution was carried out to release unitary Parylene-based neural probes from a silicon wafer. Finally, cytotoxicity assays on these neural implants were performed to make sure that no trace of mask or stripper residues would jeopardize device biocompatibility.