Regulating Fibrinolysis to Engineer Skeletal Muscle from the C2C12 Cell Line

Abstract

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.

Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro

“…GM was replenished daily for 3 days, at which point the cells were confluent and the media was switched to differentiation media (DM) consisting of high glucose DMEM containing 2% Horse serum (Sigma–Aldrich), 1% Penicillin‐streptomycin and 0.5 mg/ml 6‐aminocaproic acid. Following 48 hr in DM, media was switched to maintenance media (MM) in accordance with previous reports (Khodabukus & Baar, 2009) consisting of high glucose DMEM, 7% FBS, 1% penicillin‐ streptomycin, and 0.5 mg/ml 6‐aminocaproic acid. MM was changed daily for the duration of the experiment (14 days) and was supplemented with 1, 5, or 20 mM of L‐leucine (Sigma–Aldrich) and/or rapamycin (100 nM; Millipore, Hertfordshire, UK) from day 9 onward for functional, morphological, and mRNA analyses.…”

Generation and Use of Cultured Human Primary Myotubes

“…Furthermore, the current methods of generating muscle constructs result in myooids which exhibit significant deficits in force production compared to that of native muscle (Dennis et al, 2001). The absence of fibroblasts in constructs generated from C 2 C 12 cells also resulted in a force deficit manifesting in a 35% reduction in peak twitch force after tetanic stimulation (Khodabukus & Baar, 2009). The authors noted that addition of 3T3 fibroblasts to C 2 C 12 myotubes (ratio 1:5) prior to seeding attenuated this force deficit but lead to a 50% decrease in specific force.…”

“…Consistent with this, Hinds et al, (2011) found that the use of a hydrogel matrix consisting of Matrigel and fibrin enhanced myotube formation and density within the construct which was positively correlated with force production. Whilst no single extracellular matrix has been identified conclusively as superior, the use of fibrin gels with aprotinin, a plasminogen inhibitor and the naturally occurring cross-linking agent, genipin, have been shown to enhance force production and time-to-failure of constructs by way of controlled degradation of this matrix (Khodabukus & Baar, 2009). Moreover, fibrin gels provide a more flexible scaffold medium into which cells migrate and proliferate both on top of and within the fibrin gel to increased specific force production (Dennis et al, 2001;Huang et al, 2005).…”

“…Methods utilising C 2 C 12 myotubes have since been optimised to support an extended period for which the constructs can be maintained in culture (Khodabukus & Baar, 2009). The protocol reported below is based on the optimised procedure.…”

“…Electrical stimulation of constructs has been successfully demonstrated using clonal C 2 C 12 myotubes in a number of studies (Khodabukus & Baar, 2009;Langelaan et al, 2010;Park et al, 2008). However, in our experience the process of electrically stimulating constructs of human primary myotubes has proven difficult.…”