Regulating fibrinolysis to engineer skeletal muscle from the C2C12 cell line.
Muscles engineered from transformed cells would be a powerful model for the study of muscle physiology by allowing long-term in vitro studies of muscle adaptation. However, previously described methods either take >5 weeks to produce a tissue or use collagen as a scaffold, which decreases the specific force of the muscle, making it hard to measure the function of the constructs. The aim of this study was to rapidly engineer muscle using the C2C12 cell line in fibrin, which has a stiffness similar to muscle tissue, allowing accurate functional testing. Both the protease inhibitor aprotinin and the natural cross-linker genipin increased the length of time that muscle could be cultured, with genipin increasing the time in culture to 10 weeks. The function of the tissues was significantly affected by the batch of serum (64-78%) or thrombin (41%), the differentiation medium (78%), and the seeding protocol (38%), but was unaffected by initial cell number. Strikingly, different C2C12 clones produced up to a 3.6-fold variation in force production. Under optimal conditions, the tissues form in 10.4+/-0.3 days and remain fully functional for 5 weeks over which time they continue to mature. The optimized model described here provides rapid, reliable, and functional tissues that will be useful in the study of skeletal muscle physiology.
Khodabukus
Alastair
A
Functional Molecular Biology Lab, Division of Molecular Physiology, University of Dundee, Dundee DD1 5EH, United Kingdom.
Baar
Keith
K
D000818:Animals; D002460:Cell Line; D005337:Fibrin / Q000737:chemistry / Q000494:pharmacology*; D005342:Fibrinolysis / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D018482:Muscle, Skeletal / Q000737:chemistry / Q000187:drug effects / Q000254:growth & development*; D032446:Myoblasts / Q000166:cytology* / Q000502:physiology*; D009924:Organ Culture Techniques / Q000379:methods*; D023822:Tissue Engineering / Q000379:methods*
D005337:Fibrin