Single-cell patterning represents a key approach to decouple and better understand the role and mechanisms of individual cells of a given population. In particular, the bottom-up approach of engineering neuronal circuits with a controlled topology holds immense promises to perceive the relationships between connectivity and function. In order to accommodate these efforts, highly flexible SU-8 cantilevers with integrated microchannels have been fabricated for both additive and subtractive patterning. By directly squeezing out single cells onto adhesive surfaces, controlled deposition with a spatial accuracy of 5 μm could be achieved, while subtractive patterning has been realized by selective removal of targeted single cells. Complex cell patterns were created on substrates pre-patterned with cell-adhesive and repulsive areas, preserving the original pattern geometry for long-term studies. For example, a circular loop with a diameter of 530 μm has been realized using primary hippocampal neurons, which were fully connected to their respective neighbors along the loop. Using the same cantilevers, the versatility of the technique has also been demonstrated via in situ modification of already mature neuronal cultures by both detaching individual cells of the population and adding fresh ones, incorporating them into the culture.

Spinal cord injury (SCI) patients suffer from diverse gait deficits depending on the severity of their injury. Gait assessments can objectively track the progress during rehabilitation and support clinical decision making, but a comprehensive gait analysis requires far more complex setups and time-consuming protocols that are not feasible in the daily clinical routine. As using inertial sensors for mobile gait analysis has started to gain ground, this work aimed to develop a sensor-based gait analysis for the specific population of SCI patients that measures the spatio-temporal parameters of typical gait laboratories for day-to-day clinical applications. The proposed algorithm uses shank-mounted inertial sensors and personalized thresholds to detect steps and gait events according to the individual gait profiles. The method was validated in nine SCI patients and 17 healthy controls walking on an instrumented treadmill while wearing reflective markers for motion capture used as a gold standard. The sensor-based algorithm (i) performed similarly well for the two cohorts and (ii) is robust enough to cover the diverse gait deficits of SCI patients, from slow (0.3 m/s) to preferred walking speeds.

Cells in vitro behave differently if cultured in a 2D or 3D environment. In spite of the continuous progress over the recent years, methods available for realizing 3D cultures of primary neurons are still fairly complex, limited in throughput and especially limited in compatibility with other techniques like multielectrode arrays (MEAs) for recording and stimulating the network activity with high temporal precision. In this manuscript, a paper-based approach is presented using cellulose filter paper as a mobile substrate for 3D cultures of primary rat hippocampal and cortical neurons. Acting as 3D scaffolds for network development, filter membranes with different surface treatments were prepared to control network homogeneity and laser cut to change the network topology through spatial confinement. The viability of the prepared cultures was comparable to that of reference 2D cultures for over 4 weeks, and the mechanical stability of the paper substrates made it possible to transfer the cultures to MEA chips in an on-demand manner. Once the cultures were successfully transduced with a gene-encoded calcium indicator and transferred to a MEA chip, the optical and electrical signals of neuronal activity were simultaneously recorded and combined to study the different activity patterns with high spatiotemporal resolution. The high-throughput nature of the presented approach makes it a valuable tool for investigating the intimate relationship between topology and function, by studying the intrinsic parameters influencing network synchronization and signal propagation through the different activity patterns of 3D neural cultures with arbitrary topology. The developed platform provides a robust and simple alternative to existing 3D culturing technologies for neurons.