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1Biozentrum, University of Basel
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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.
Arnold, A. S., Christe, M., Handschin, C. A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells. J. Vis. Exp. (62), e3616, doi:10.3791/3616 (2012).
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.
1. Preparation of the Primary Human Muscle Cell Culture
2. Isolation of the Rat Embryonic Spinal Cord Explants
The whole procedure to isolate the spinal cord explants is performed under a binocular microscope, in Hank's Balanced Salt Solution (HBSS, Gibco/Invitrogen ref 14170) containing 10% Fetal Bovine Serum (FBS).
3. Innervation of the Human Muscle cells with Embryonic Rat Spinal Cord Explants
4. Maintenance of the Heterologous Nerve-muscle Co-cultures
5. Representative Results
As soon as two days after the innervation, it is possible to see the neurites emerging from each explant and establishing contacts with the muscle cells, represented by the button-like structures (Fig 1).
As early as 4-6 days after the innervation, a few individual fibers will start to contract.
The contractions persist for many months, maintained simply by changing the culture medium twice a week.
After about 2 weeks, the spinal cord explant itself degenerates, but the innervation is conserved. This is normal. A fine layer of nerve component stays at the surface of the muscle cell layer. This nervous network is sufficient to maintain the functionality of the motor unit.
If the explants float in the medium the day after innervation, they will never adhere. This happens when the muscle cell layer is not sufficiently confluent, or when the medium is added too roughly. Sometimes, the explant adheres but no neurites will emerge. This is likely due to the lack of DRG attached to it. In that case as well, the innervation will not be established. Manipulate the dishes very carefully the first week, as the explants can still detach easily. To exchange the fusion media, add the liquid drop-wise with a minimal speed to avoid cell detachment.
Figure 1. Spinal cord explants -muscle cell co-cultures from day 1 to day 15.
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) relevant The protocol described here has been used successfully to establish innervated muscle cells in vitro and results in a complete NMJ maturation that can be easily assessed.
Murine models are now available for many neuromuscular diseases. Unfortunately, many of these models are limited in terms of translation to the disease in human patients, for example the mouse models for spinal muscular atrophy, Duchenne muscular dystrophy or amyotrophic lateral sclerosis. Spinal muscular atrophy is caused by a mutation in the SMN1 gene, which in part can be mitigated by the presence of a second SMN gene in the human genome6. In contrast, the mice genome contains only one gene for SMN, and its deletion is embryonically lethal7. Thus, in order to develop mouse spinal muscular atrophy models, a human SMN2 copy was artificially introduced into the Smn knockout mice8,9. These animals still represent the closest genetic model to the human pathology, but the phenotype observed in those mice is due to the expression of an SMN2 gene that mice normally do not express10. In the case of Duchenne muscular dystrophy, the mdx mice have a mutated dystrophin gene similar to human patients, but the outcome is much less severe compared to the symptoms observed in patients11. The most widely used mouse model for amyotrophic lateral sclerosis, the SOD1 mice, so far disappointed in revealing insights into disease mechanisms and therapeutic approaches 12,13. Therefore, spinal cord explants-muscle cell co-cultures to study the function, genomic and proteomic characteristics of the muscle coming from patients, in a completely physiological setting, represent an invaluable tool to study such complex diseases. Furthermore this hetero-species model helps to discriminate between neurogenic and myogenic pathological mechanisms. 3,14,15
The co-culture presented here also has some limitations. For example, immunohistochemistry experiments on the muscle cells are difficult to perform on this structure, because the nerve terminal embodied by the explant covers the postsynaptic NMJ apparatus. This system can also be used to assess the neurotrophic or myotonic effects of drugs in an integrated and relatively mature culture system, but it does not allow high-throughput screening, as the volume would be far too big. Curiously, the homologous nerve-muscle co-culture with both the nerve and muscle components coming only from mouse or rat is not functional and fails to result in muscle contractions. Recently however, homologous innervation between mouse satellite muscle cells and mouse spinal cord explants has been successfully performed by modification of the date of embryo harvesting (Callizot, Steinschneider and colleagues, unpublished results). Further progress in establishing routine protocols for homologous co-cultures might help to overcome the species limitation that exists for this technique today.
Despite these limitations, we describe here a method that enables the study of many aspects of muscle and motor neuron physiology; a technique which is not difficult to perform, does not require any special material and that results in stable spinal cord explants- muscle co-cultures that can easily be maintained and studied for many months.
Certain aspects of the protocol are of particular importance and need special care to ensure a proper innervation. First, the rat embryos have to be between 13 and 14 ED of age. Second, 500,000 cells per 35-mm dish ensures the correct level of cell confluence to facilitate adherence of the explants that are added to the muscle cells immediately when cell fusion starts. Finally, as emphasized in the protocol, the addition of the medium after the deposit of the explants on the muscle cells is the most difficult part of this procedure and should be performed with extreme care.
We have nothing to disclose.
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 Française contre les Myopathies (AFM), the United Mitochondrial Disease Foundation (UMDF), the Gebert-Rüf Foundation Rare Diseases Programme (GRF), the Swiss Society for Research of the Muscle Diseases (SSEM/FSRMM), the Swiss Life "Jubiläumsstiftung für Volksgesundheit und medizinische Forschung", the Roche Research Foundation and the University of Basel.
|HBSS||GIBCO, by Life Technologies||14170|
|MEM||GIBCO, by Life Technologies||31095|
|Medium 199||GIBCO, by Life Technologies||31153|
|Fetal Bovine Serum||Fetal Clone Perbio||SH30066.03|
|Penicillin/streptomycin solution||GIBCO, by Life Technologies||15140|
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