Optimized method for extraction of exosomes from human primary muscle cells

Area of Science:

• Cell biology
• Biochemistry
• Endocrinology

Background:

• Skeletal muscle functions as an endocrine organ, releasing myokines and extracellular vesicles (EVs) like exosomes and microvesicles.
• These EVs influence physiological processes, impacting regeneration, aging, and myopathies.
• Studying the muscle secretome requires reliable methods for EV isolation from primary human myoblasts.

Purpose of the Study:

• To optimize in vitro cultivation of human myoblasts for EV research.
• To compare exosome extraction efficiency between ultracentrifugation and a modified polymer-based precipitation method.
• To establish a scalable method for isolating muscle-derived exosomes, overcoming limitations of cell number and senescence.

Main Methods:

• Human myoblasts were cultured under optimized conditions.
• Exosomes were isolated using standard ultracentrifugation and a modified polymer-based precipitation with extra washing steps.
• Vesicle characterization involved CD marker analysis, density gradient centrifugation, electron microscopy, and NanoSight tracking.

Main Results:

• Both methods yielded exosomes positive for CD63, CD82, and CD81, with similar density and morphology.
• The modified polymer-based precipitation method was significantly more efficient, requiring over 30 times fewer myoblasts.
• Isolated exosomes were successfully internalized by recipient cells, including human myotubes and iPSC-derived motor neurons.

Conclusions:

• Modified polymer-based precipitation offers an optimized, high-yield strategy for exosome extraction from human myoblasts.
• This method overcomes limitations of cell number and senescence, enabling broader research into muscle-derived EVs.
• The optimized protocol facilitates the study of exosome content and subpopulations for therapeutic and diagnostic applications.