Development of tissue-engineered skeletal muscle manufacturing variables

Area of Science:

• Tissue engineering
• Biomaterials science
• Skeletal muscle physiology

Background:

• Three-dimensional (3D) tissue-engineered models offer superior physiological relevance compared to traditional 2D cell cultures for drug and material testing.
• Understanding manufacturing variables is crucial for optimizing 3D skeletal muscle models and ensuring consistent, reproducible results.
• Reducing resource use in model development is essential for cost-effective preclinical research.

Purpose of the Study:

• To investigate how key manufacturing variables influence the myotube characteristics of a C2C12 skeletal muscle model.
• To assess the impact of manufacturing parameters on resource utilization and model consistency.
• To establish a benchmark for consistent production across scaled tissue-engineered constructs.

Main Methods:

• C2C12 murine myoblasts were cultured within tethered collagen scaffolds under controlled tension and medium conditions.
• Collagen polymerizing area length-to-width ratios and cell seeding densities were systematically varied.
• A factorial design of experiments was employed to identify optimal manufacturing parameters.
• Scaled constructs were compared to assess consistency across different model sizes.

Main Results:

• Collagen scaffold length-to-width ratios greater than one significantly reduced cell-matrix attachment and remodeling forces (p < 0.05), impacting cell fusion.
• Optimal conditions (4 million C2C12s/ml, polymerizing area width 150% of anchor point) yielded favorable myotube characteristics and reduced model rupture.
• Scaled constructs demonstrated no significant differences in characteristics compared to larger models.
• Consistent formation of approximately 20 myotubes with <25° alignment variation was achieved in the central region.

Conclusions:

• Initial manufacturing variables critically influence the formation and characteristics of engineered skeletal muscle tissue.
• The study established a benchmark model for consistent, reproducible skeletal muscle tissue engineering.
• This optimized model serves as a potential cost-effective preclinical testbed for drug and material evaluation.