A comparison of human skeletal muscle cell maturation in 2D versus 3D culture: A quantitative proteomic study

Area of Science:

• Biomedical Engineering
• Cell Biology
• Muscle Physiology

Background:

• Three-dimensional (3D) culture systems more accurately represent native muscle tissue compared to traditional 2D monolayers.
• Understanding the impact of culture duration and conditions on muscle cell maturity in 3D is vital for advancing research.

Purpose of the Study:

• To compare the differentiation, maturity, and functional characteristics of human skeletal muscle cells cultured in 2D versus 3D environments.
• To identify optimal culture durations for 3D muscle constructs used in functional studies.

Main Methods:

• Human skeletal muscle cells were cultured in 2D and 3D hydrogel models for up to 21 days.
• Quantitative proteomic analysis and functional measurements (passive tension, contractile force) were performed.
• Myoblast differentiation and myotube development were assessed.

Main Results:

• Myoblasts differentiated into myotubes by day 8 in both 2D and 3D cultures.
• 3D constructs showed a slow-twitch phenotype at day 8, shifting to a fast-twitch phenotype by day 21.
• By day 21, 3D cultures exhibited enhanced mitochondrial maturity and extracellular matrix remodeling, but reduced contractile forces compared to earlier time points.

Conclusions:

• 3D culture promotes skeletal muscle myotube maturity, with distinct phenotypic and functional changes over 21 days.
• Culture duration significantly impacts the characteristics of 3D muscle constructs.
• 8 days of differentiation in 3D constructs appears optimal for peak contractile force, offering valuable models for interventions targeting muscle function preservation.