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

Abstract

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.

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

“…While there have been many attempts to reconstruct the NMJ in lab-on-chip devices (see [51,52] for recent reviews), our study involves an approach that combines the use of an axotomy-purposed microfluidic device, which in turn allows compartmentalization of the neuronal and muscular components while allowing axonal manipulation, with optogenetic control of both muscular and neuronal activity. Cultured C2C12-ChR2 cells in our devices embedded in hydrogels displayed contractile properties similar to those seen in similar cultures of C2C12 cells [46,47] (i.e., fatigue after electrical/optical stimulation or contraction properties).…”

Development of a simple and versatile in vitro method for production, stimulation, and analysis of bioengineered muscle

“…These culture models are useful for studying muscle function in health and disease, or as screening platforms for clinical research. In order to mimic in vivo muscle physiology, they must recapitulate relevant features of skeletal muscle architecture and function, such as the formation of aligned and compacted muscle fibers, and contractile ability in response to electrical or chemical stimulation [ 3 ].…”

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

“…The spinal neuromuscular circuit and neuromuscular diseases (NMD) have been thoroughly studied for years paying special attention to the α-motoneuron and its connection with skeletal muscle (SkM) cells 1 . In addition the role of other cells as Schwann cells 2 – 6 , astrocytes 7 , microglia 8 , 9 , oligodendrocytes 10 , 11 , interneurons 12 , and γ-MN 13 , has been considered.…”

“…There has only recently been published a protocol to differentiate intrafusal and extrafusal muscle fibres 47 . Furthermore, there is a small literature on experiments performing in vitro coculture of sensory neurons with skeletal muscle cells 43 , 48 – 50 , in contrast with the vast collection of studies on the neuromuscular motor pathway 1 . Also, while most of these publications focusing on the neuromuscular sensory afferents are performed with rodent tissue, so do not accurately mimic human conditions, they have helped elucidate the role of several molecules on proprioception and mechanotransduction.…”