(C-96) C2C12 Myoblast Culture in Glycosaminoglycan-Based Hollow Fibers: A Muscle Fiber Development Platform for Treatment of Volumetric Muscle Loss

Skeletal muscle has an innate ability to self-regenerate. However, in cases of volumetric muscle loss (VML) caused by trauma or surgical events, more than 30% of a muscle’s mass may be lost. This condition exceeds the regenerative capacity of the remaining muscle mass and surgical interventions are needed to rescue the remaining tissue. The current gold standard of VML treatment involves autologous muscle flap transfers, but results are limited by graft tissue availability and donor site morbidity. Some tissue engineering approaches involving scaffolds composed of decellularized extracellular matrix (dECM) have shown promise, but numerous challenges remain.

We propose the development of myoblast-seeded, glycosaminoglycan (GAG)-based hollow fibers as an alternate tissue engineering strategy. Similar to previously described microcapsules [1], these hollow fibers are composed of a hyaluronan (HA)-chitosan ionic complex and can be loaded with cells and a variety of interior matrices including collagen and/or dECM. We hypothesize that these hollow fibers will allow uniaxial and aligned differentiation of myotube bundles , with incorporated ECM components Providing support for cell growth and myogenic protein expression.

In this study, we sought to generate hollow fibers with diameters under 200 µm, capable of supporting growth and differentiation of a myoblast cell line. We also sought to explore the effects of interior ECM gel composition on cell growth and morphology.

The hollow fibers are an insoluble ionic complex, made from the interaction of polycationic chitosan (CTS) and polyanionic glycosaminoglycans (GAG). Hollow fiber formation and cell encapsulation were accomplished simultaneously using an in-house designed flow-focusing chip (Fig. 1). C2C12 myoblasts (from ATCC) were suspended in the GAG or GAG-ECM solution of interest (8-10 million cells/ml solution) and coaxially extruded with an external sheath of chitosan solution. Ionic interaction between the GAG and the coaxial chitosan flow generated an ionic complex membrane that formed the wall of the hollow fiber. Four GAG formulations were investigated: 1.5% Haluronan (HA), 1.5% HA + 1.0 mg/ml Collagen Type I (HA-collagen), 1.5% HA + 1.0 mg/ml decellularized muscle matrix (HA-dECM), and 1.5% HA + 1.0 mg/ml collagen + 1.0 mg/ml dECM (HA-collagen-dECM). . 10 million cells/mL. After formation, cell-seeded hollow fibers were surface stabilized by washing with 0.1 wt% polygalacturonic acid and normal saline before culturing in L-DMEM containing 10%FBS. Media was changed one hour afterwards to remove excess GAG. Fibers were kept in cultured at 37°C, 95% air/5% CO2, with medium changes every four days.

We are able to generate acellular hollow fibers with diameter ranging between 200 um (Figure 2B) to greater than 1 mm (Figure 2C). We obtained cell-seeded hollow fibers with a diameter as low as 60 um. These results are advantageous as the average diameter of a myofiber is typically 100 um.

In the myoblast-seeded HA-collagen fibers (8 million cells/ml), cell elongation was present at 12 hours post-seeding (Figure 3A), with cells visibly orienting themselves along the axis of the hollow fiber. Cell viability was confirmed at day 4 using Calcein Red Orange, with some myotube formation visible as well (Figure 3C). This confirmed the ability of the myoblasts to differentiate in the 3D environment created within the hollow fibers.

To answer the question of whether addition of dECM would help accelerate myoblast differentiation and produce polynucleated myotubes, we tested four conditions seeded at 10 million cells/ml each: HA, HA-collagen, HA-dECM, and HA-collagen-dECM. Cell elongation was observed in three conditions, namely HA-collagen (Figure 4D), HA-dECM (Figure 4G), and HA-collagen-dECM (Figure 4K). Using Calcein Red Orange, we were able to confirm cell viability in all four conditions at day 6 (Figure 4C,F,I,L). We observed differing cell morphologies across all four conditions. The ability of cells to extend or contract depends on the architecture and stability of the interior gel.

Our experiments thus far have shown that addition of collagen, and dECM to our GAG formulation is able to support cell differentiation, and all conditions support cell viability in vitro. We were also able to confirm cell elongation in all conditions except for HA only, which is expected. We are currently investigating the timing of differentiation in HA-dECM, HA-collagen, and HA-collagen-dECM. Current cultures are ongoing, and results on differentiation in these intrafiber environments will be reported.