Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC169
Establishment of a motoneuron-myotube co-culture in a 3D fibrin gel model of skeletal muscle
S. L. Passey1, A. S. Smith2,1, V. Mudera3, L. Greensmith2, K. Baar4, M. P. Lewis1,5
1. Muscle Cellular and Molecular Physiology Research Group, ISPAR Bedford, University of Bedfordshire, Bedford, United Kingdom. 2. The Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, London, United Kingdom. 3. UCL Institute of Orthopaedics and Musculoskeletal Science, London, United Kingdom. 4. Functional Molecular Biology Lab, UC Davis, Davis, California, United States. 5. UCL School of Life and Medical Sciences, London, United Kingdom.
Introduction. There is currently no truly biomimetic in vitro model of skeletal muscle incorporating a functional neuronal input analogous to that seen at the in vivo neuromuscular junction. Establishment of such a model would allow detailed study of neuromuscular biology and skeletal muscle in health and disease whilst greatly reducing reliance on the use of in vivo models. Fibrin cast skeletal muscle shows similar physiological and contractile characteristics to in vivo muscle including the ability to generate force in response to electrical stimulation (1). We have now further developed the fibrin muscle model by the co-culture of primary rat muscle-derived cells (MDCs) and primary rat motoneurons (MNs), with the aim of generating an engineered muscle construct with functional neuronal input. Methods. Fibrin cast skeletal muscles were engineered as described previously (1). Rat MDCs were isolated from the hind limbs of P1 Sprague-Dawley rat pups by collagenase digestion and cultured on fibrin gels. When the MDCs became confluent MNs isolated from the ventral horn of spinal cords from Sprague-Dawley rat E14 embryos were plated at 50,000 cells per gel. Gels were fixed 7 days post MN-seeding and fluorescence immunocytochemistry was used to label the myotubes (desmin), the motoneurons (MAP2, SV2, 2H3), and the Acetylcholine receptors (AChR: TexasRed-Bungarotoxin (BTX)). Results. Microscopic observations reveal formation of a tight bundle of aligned multinucleate myotubes analogous to that seen in in vivo skeletal muscle. Functional maturation of the myotubes was evident, as myotubes developed the striated appearance characteristic of skeletal muscle and spontaneous contractions were observed (n=4). MNs survive within the muscle fibrin cultures for at least 7 days (n=2), and MN survival was shown to be greater in gels seeded with 200,000 MDCs compared to 400,000 MDCS (11.5 vs. 3.65 MN cells/field of view, n=2). BTX clustering and colocalisation with MN markers (n=3) was evident. Discussion. Using an established model of skeletal muscle, we have added extra complexity through the addition of a neuronal input. Fibrin cast skeletal muscles can be rapidly engineered together with MN, producing contractile neuromuscular constructs. No only do MNs survive in the co-culture system, they also induce AChR clustering at sites of contact indicating physiological interactions between the two cell types. These data are the first step towards the formation of organised NMJs. Establishment of a 3D muscle-MN culture system will be of great benefit to the study of NMJ formation, maturation, and function and will reduce reliance on animal models for such studies.
Where applicable, experiments conform with Society ethical requirements