Novel method for fabrication of skeletal muscle construct from the C2C12 myoblast cell line using serum‐free medium AIM‐V

Abstract

We have fabricated muscle tissue from murine myoblast cell line C2C12 by modifying the previously reported method. Fabrication of skeletal muscle tissue has been performed in many ways including the use of a biodegradable scaffold, a collagen gel-embedded culture, or cell sheet tissue engineering, but the extent of tension generation remains low. Recently, a new skeletal muscle tissue engineering technique involving self-dissociation of a cell sheet from a laminin-coated polydimethylsiloxane surface was reported which mostly involved a primary cell culture or co-culture of C2C12 and 10T1/2 cells. In this study, we succeeded in fabricating muscle tissue using C2C12 cells alone by enhancing cell-cell attachment by the use of serum-free medium AIM-V. C2C12 cells were seeded on to a laminin-coated PDMS surface in a 35 mm culture dish with two silk sutures of 5 mm in length each pinned at two places 18 mm apart. Then, cells were allowed to differentiate in AIM-V, and the cells started to dissociate in a sheet-like manner after 5-8 days of differentiation. The cells remained attached to the silk sutures, and tissue having a cylindrical morphology was fabricated. After the cylindrical morphology had been obtained, the medium was changed to DMEM supplemented with 2% horse serum, followed by culture for an additional 5-8 days for maturation. Tissue fabricated using this method was excitable with electric pulse stimulation and the generated active tension was approximately 1.4x greater than that reported previously for a co-culture of C2C12 and 10T1/2 cells. Immuno-fluorescence study revealed the presence of a sarcomere structure within the fabricated tissue, and Western blotting confirmed the expression of muscle specific-proteins. The increased active tension generation compared to that with the previously reported method is probably attributable to the increased proportion of myogenic cells in the tissue. Myooid fabricated from mono-culture of C2C12 will be useful in the muscle study, especially in the area where gene modification is needed.

Designing of a Si-MEMS device with an integrated skeletal muscle cell-based bio-actuator

“…To visualize the myotubes, cytochemical experiments were performed as previously described (Fujita et al 2009b). Briefly, C2C12 cells were fixed in PBS containing 4% paraformaldehyde for 10 min, permeabilized with PBS containing 2% of Triton X-100, washed with PBS three times, and then incubated with the primary antibody for 1 h. A monoclonal anti-α actinin (sarcomeric) antibody (EA-53; Sigma, Saint Louis, MO) was used at the dilution of 1:1000.…”

In vitro drug testing based on contractile activity of C2C12 cells in an epigenetic drug model

“…Thus, artificial skeletal muscle constructs should mimic the architecture of native muscle and exhibit contractile force generation. Using primary myoblasts and/or a C2C12 myoblast cell line, several researchers have developed three-dimensional (3D) culture systems that are available for contractile force measurement910, and tissue-engineered skeletal muscle constructs have been applied in drug testing1112, atrophy modeling713 and muscular disease modeling6. In a previous study, we developed a method for fabricating functional skeletal muscle tissue constructs using a magnetic force-based tissue engineering (Mag-TE) technique1415, in which C2C12 cells were labeled with magnetite cationic liposomes (MCLs)16 as a functional magnetic nanoparticle, and assembled by applying a magnetic field to form a cell-dense and aligned fascicle-like structure15.…”

Visualizing the appearance and disappearance of the attractor of differentiation using Raman spectral imaging

“…It is of interest whether Raman spectra from myotubes differ from those of myoblasts; however, long-term differentiation of C2C12 on the silica substrate failed as they detached 4–5 days after induction of differentiation. This is possibly due to the tension generated by differentiating cells reported previously16. Furthermore, myotubes tend to position above undifferentiated myoblasts, increasing the difficulty of Raman observation.…”