A defined long-term in vitro tissue engineered model of neuromuscular junctions

Abstract

Neuromuscular junction (NMJ) formation, occurring between motoneurons and skeletal muscle, is a complex multistep process involving a variety of signaling molecules and pathways. In vitro motoneuron-muscle co-cultures are powerful tools to study the role of different growth factors, hormones and cellular structures involved in NMJ formation. In this study, a serum-free culture system utilizing defined temporal growth factor application and a non-biological substrate resulted in the formation of robust NMJs. The system resulted in long-term survival of the co-culture and selective expression of neonatal myosin heavy chain, a marker of myotube maturation. NMJ formation was verified by colocalization of dense clusters of acetylcholine receptors visualized using alpha-bungarotoxin and synaptophysin containing vesicles present in motoneuron axonal terminals. This model will find applications in basic NMJ research and tissue engineering applications such as bio-hybrid device development for limb prosthesis and regenerative medicine as well as for high-throughput drug and toxin screening applications.

Protocol and cell responses in three-dimensional conductive collagen gel scaffolds with conductive polymer nanofibres for tissue regeneration

“…The results for the first time suggested that the conductive collagen gels were biocompatible with both cell types, which could be found in a neuromuscular junction tissue. A co-culturing study of skeletal muscle and motor neuron cells, which was used to model neuromuscular function [53,54], could also be done on these types of scaffold, because they have biomimetic structure inherited from collagen and attract cell adhesion via a 2 b 1 integrin cellular receptor, which contributes to collagen polymerization, for type I collagen fibrils [55]. They also possess comparable biocompatibility to collagen gel (control) when using low concentration (0.5 wt%) of PANI or PEDOT and facilitate electricity to pass through the conductive collagen gel.…”

A system for studying mechanisms of neuromuscular junction development and maintenance

“…5). Other studies had used lengthy protocols by which formation of NMJs was achieved after 20-25 days or even longer time periods, if we take into account the time needed for either prior motor neuron or myofiber differentiation (Das et al, 2010;Southam et al, 2013). In addition, in these studies, the NMJs and myofibers did not reach the same stage of differentiation as that described in this manuscript.…”

“…Much attention has been given to the development and improvement of the neuronal compartment with the emergence of embryonic stem cells (Chipman et al, 2014;Das et al, 2007;Soundararajan et al, 2007) and induced pluripotent stem cells (Bohl et al, 2015;Burkhardt et al, 2013;Egawa et al, 2012); however, the muscle counterpart has been underexploited and its potential underestimated. Previous studies failed to produce convincing data on muscle differentiation either because muscle cell lines, such as C2C12, incapable of producing highly differentiated myofibers, were used or because the muscle compartment was overlooked (Arnold et al, 2012;Chipman et al, 2014;Das et al, 2009Das et al, , 2010Umbach et al, 2012). The use of such cell lines might also preclude synaptogenesis and/or synaptic differentiation.…”

“…To overcome these problems, different in vitro coculture systems have been set up in which motor neuron and skeletal muscle are grown together in order to recapitulate the formation and eventual disruption of the NMJ. To date, co-culture methods established from various species have been described, including mouse (Morimoto et al, 2013;Zahavi et al, 2015), rat (Das et al, 2010;Southam et al, 2013), Xenopus (Lu et al, 1996;Peng et al, 2003) and chick (Frank and Fischbach, 1979), and also heterologous co-cultures built from motor neuron and muscle cells obtained from different species, such as rat-human , mouse-human (Son et al, 2011) and mouse-chick (Soundararajan et al, 2007). However, these co-culture methods resulted in the formation of immature myofibers (thin muscle fiber, with centrally localized nuclei and no transversal triads) with immature sarcomeric structures (Das et al, 2007(Das et al, , 2009Southam et al, 2013).…”

Mechanistic investigation of adult myotube response to exercise and drug treatment in vitro using a multiplexed functional assay system

“…These cantilevers, as well as the coverslip controls used in this study, were coated with an amine-terminated alkylsilane, (3-trimethoxysilyl propyl) diethylenetriamine (DETA) (United Chemical Technologies, Bristol, PA) using methods previously described (6,13,14,39). DETA is an analog of spermidine, which is known to promote long-term survival of cells in vitro, and has been previously validated in the culture of various cell types (9,23).…”