In vitro modelling of human proprioceptive sensory neurons in the neuromuscular system.

Proprioceptive sensory neurons (pSN) are an essential and undervalued part of the neuromuscular circuit. A protocol to differentiate healthy and amyotrophic lateral sclerosis (ALS) human neural stem cells (hNSC) into pSN, and their comparison with the motor neuron (MN) differentiation process from the same hNSC sources, facilitated the development of in vitro co-culture platforms. The obtained pSN spheroids cultured interact with human skeletal myocytes showing the formation of annulospiral wrapping-like structures between TrkC + neurons and a multinucleated muscle fibre, presenting synaptic bouton-like structures in the contact point. The comparative analysis of the genetic profile performed in healthy and sporadic ALS hNSC differentiated to pSN suggested that basal levels of ETV1, critical for motor feedback from pSN, were much lower for ALS samples and that the differences between healthy and ALS samples, suggest the involvement of pSN in ALS pathology development and progression.

Rapid Manufacturing of Multilayered Microfluidic Devices for Organ on a Chip Applications.

Microfabrication and Polydimethylsiloxane (PDMS) soft-lithography techniques became popular for microfluidic prototyping at the lab, but even after protocol optimization, fabrication is yet a long, laborious process and partly user-dependent. Furthermore, the time and money required for the master fabrication process, necessary at any design upgrade, is still elevated. Digital Manufacturing (DM) and Rapid-Prototyping (RP) for microfluidics applications arise as a solution to this and other limitations of photo and soft-lithography fabrication techniques. Particularly for this paper, we will focus on the use of subtractive DM techniques for Organ-on-a-Chip (OoC) applications. Main available thermoplastics for microfluidics are suggested as material choices for device fabrication. The aim of this review is to explore DM and RP technologies for fabrication of an OoC with an embedded membrane after the evaluation of the main limitations of PDMS soft-lithography strategy. Different material options are also reviewed, as well as various bonding strategies. Finally, a new functional OoC device is showed, defining protocols for its fabrication in Cyclic Olefin Polymer (COP) using two different RP technologies. Different cells are seeded in both sides of the membrane as a proof of concept to test the optical and fluidic properties of the device.

Neuromuscular Activity Induces Paracrine Signaling and Triggers Axonal Regrowth after Injury in Microfluidic Lab-On-Chip Devices.

Peripheral nerve injuries, including motor neuron axonal injury, often lead to functional impairments. Current therapies are mostly limited to surgical intervention after lesion, yet these interventions have limited success in restoring functionality. Current activity-based therapies after axonal injuries are based on trial-error approaches in which the details of the underlying cellular and molecular processes are largely unknown. Here we show the effects of the modulation of both neuronal and muscular activity with optogenetic approaches to assess the regenerative capacity of cultured motor neuron (MN) after lesion in a compartmentalized microfluidic-assisted axotomy device. With increased neuronal activity, we observed an increase in the ratio of regrowing axons after injury in our peripheral-injury model. Moreover, increasing muscular activity induces the liberation of leukemia inhibitory factor and glial cell line-derived neurotrophic factor in a paracrine fashion that in turn triggers axonal regrowth of lesioned MN in our 3D hydrogel cultures. The relevance of our findings as well as the novel approaches used in this study could be useful not only after axotomy events but also in diseases affecting MN survival.

Challenges and Future Prospects on 3D in-vitro Modeling of the Neuromuscular Circuit.

Movement of skeletal-muscle fibers is generated by the coordinated action of several cells taking part within the locomotion circuit (motoneurons, sensory-neurons, Schwann cells, astrocytes, microglia, and muscle-cells). Failures in any part of this circuit could impede or hinder coordinated muscle movement and cause a neuromuscular disease (NMD) or determine its severity. Studying fragments of the circuit cannot provide a comprehensive and complete view of the pathological process. We trace the historic developments of studies focused on in-vitro modeling of the spinal-locomotion circuit and how bioengineered innovative technologies show advantages for an accurate mimicking of physiological conditions of spinal-locomotion circuit. New developments on compartmentalized microfluidic culture systems (cµFCS), the use of human induced pluripotent stem cells (hiPSCs) and 3D cell-cultures are analyzed. We finally address limitations of current study models and three main challenges on neuromuscular studies: (i) mimic the whole spinal-locomotion circuit including all cell-types involved and the evaluation of independent and interdependent roles of each one; (ii) mimic the neurodegenerative response of mature neurons in-vitro as it occurs in-vivo; and (iii) develop, tune, implement, and combine cµFCS, hiPSC, and 3D-culture technologies to ultimately create patient-specific complete, translational, and reliable NMD in-vitro model. Overcoming these challenges would significantly facilitate understanding the events taking place in NMDs and accelerate the process of finding new therapies.