Our work is supported by the Swiss National Science Foundation (SNF), the Swiss Initiative in Systems Biology (SystemsX.ch), the Muscular Dystrophy Association USA (MDA), the Association Fran&ccedil;aise contre les Myopathies (AFM), the United Mitochondrial Disease Foundation (UMDF), the Gebert-R&uuml;f Foundation Rare Diseases Programme (GRF), the Swiss Society for Research of the Muscle Diseases (SSEM/FSRMM), the Swiss Life "Jubil&auml;umsstiftung f&uuml;r Volksgesundheit und medizinische Forschung", the Roche Research Foundation and the University of Basel.

1. Preparation of the Primary Human Muscle Cell Culture
<ol>
 <li>Establish the human muscle cell cultures according to the explantation-re-explantation technique4. First, remove non-muscle tissue from the biopsies. Then, embed 1 mm3 muscle explants in a coagulum of plasma, which allows fibroblasts to emerge from the explants.</li>
 <li>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 ...

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We have nothing to disclose.

A physiological in vitro tool to study muscle cell function in normal and pathological context is of highest interest for the myologists, because muscle cell cultures usually do not recapitulate the importance of multiple cells and cell-type connections. The addition of purified motor neurons to muscle cells is not enough to achieve a functional motor unit since the presence of Schwann cells is required for the innervation to be efficient5 and it is not topologically (3D + special distribution) releva...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalog Number</td>
 </tr>
 
 <tr>
 <td>HBSS</td>
 <td>Gibco/Invitrogen</td>
 <td>14170</td>
 </tr>

 <tr>
 <td>MEM</td>
 <td>Gibco/Invitrogen</td>
 <td>31095</td>
 </tr>

 <tr>
 <td>Medium 199 </td>
 <td>Gibco/Invitrogen</td>
 <td>31153</td>
 </tr>

 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Fetal Clone Perbio</td>
 <td>SH30066.03</td>
 </tr>

 <tr>
 <td>Insuline</td>
 <td>Sigma</td>
 <td>I9278</td>
 </tr>

 <tr>
 <td>Human EGF</td>
 <td>Sigma</td>
 <td>E9644</td>
 </tr>

 <tr>
 <td>Human FGF</td>
 <td>Sigma</td>
 <td>F0291</td>
 </tr>

 <tr>
 <td>Penicillin/streptomycin solution</td>
 <td>Gibco</td>
 <td>15140</td>
 </tr>
 </tbody>
</table>

a functional motor unit in the culture dish: co-culture of spinal cord explants and muscle cells

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 missing1. 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 Askanas2. 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 diseases3, 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.

Watch this Scientific Journal Video about A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells at JoVE.com

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

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

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Neuroscience

15.2K visualizações.  University of Basel. Células musculares cultivadas são um modelo inadequado para recapitular músculo inervado In vivo. Uma unidade de motor funcional pode ser reproduzida In vitro Por inervação diferenciados humanos células musculares de ratos embrionários primários usando explantes da medula espinhal. Este artigo descreve como co-culturas de explantes da espinal medula e células musculares são estabelecidos.