A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Abstract

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing 1 . These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas 2 . To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context. Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre-and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases 3 , and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context. Video LinkThe video component of this article can be found at http://www.jove.com/video/3616/ Protocol Establish the human muscle cell cultures according to the explantation-re-explantation technique 4 . First, remove non-muscle tissue from the biopsies. Then, embed 1 mm 3 muscle explants in a coagulum of plasma, which allows fibroblasts to emerge from the explants. 2. The embedded explants are transferred onto a plasma-gelatin coated dish and let them grow in normal growth medium (MEM supplemented with 25% medium 199, 10% fetal bovine serum [FBS], 1% penicillin/streptomycin, 10 μg/ml insulin, 10 ng/ml human epidermal growth factor [EGF], 12.5 ng/ml human f...

Induced Formation and Maturation of Acetylcholine Receptor Clusters in a Defined 3D Bio-Artificial Muscle

“…Previous studies on in vitro NMJ development and innervation are mostly based on 2D culture systems [11–13]. Here, we propose that a tissue-engineered BAM can be used as a 3D in vitro system to study NMJ formation and muscle innnervation.…”

A system for studying mechanisms of neuromuscular junction development and maintenance

“…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.…”

In Vitro Innervation as an Experimental Model to Study the Expression and Functions of Acetylcholinesterase and Agrin in Human Skeletal Muscle

“…Many different homologous and heterologous co-culture models have been described to study innervation of animal skeletal muscle cells. However, as regards in vitro innervation of human skeletal muscle cells, heterologous co-culture with embryonic rat spinal cord has been the most widely used approach [ 9 , 23 , 24 , 25 , 27 , 35 , 42 , 46 , 82 , 103 , 104 , 105 , 106 , 107 , 108 , 109 ]. Alternative approaches to innervate human skeletal muscle cells include co-culture with embryonic mouse spinal cord [ 110 ], ventral part of embryonic rat spinal cord [ 111 ] or dissociated embryonic rat spinal cords [ 22 ].…”

“…In this model, rat embryos are dissected to obtain spinal cord explants, which are needed as a source of motor neurons, while human skeletal muscle cells are obtained from biopsies or discarded surgical material. The preparation of co-cultures and time course of its development are schematically depicted in Figure 1 A (technical aspects are reviewed in [ 109 ]). The typical co-culture with spinal cord explant and contraction units is shown in Figure 1 B.…”