Microdroplet-Engineered Skeletal Muscle Organoids from Primary Tissue Recapitulate Parental Physiology with High Reproducibility.

Achieving high maturity and functionality in in vitro skeletal muscle models is essential for advancing our understanding of muscle biology, disease mechanisms, and drug discovery. However, current models struggle to fully recapitulate key features such as sarcomere structure, muscle fiber composition, and contractile function while also ensuring consistency and rapid production. Adult stem cells residing in muscle tissue are known for their powerful regenerative potential, yet tissue-derived skeletal muscle organoids have not been established. In this study, we introduce droplet-engineered skeletal muscle organoids derived from primary tissue using cascade-tubing microfluidics. These droplet-engineered organoids (DEOs) exhibit high maturity, including well-developed striated sarcomeres, spontaneous and stimulated contractions, and recapitulation of parental muscle fiber types. Notably, DEOs are produced in just 8 d without the need for primary cell culture-substantially accelerating the 50- to 60-d process required by classical organoid models. Additionally, the cascade-tubing microfluidics platform enables high-throughput production of hundreds of uniform DEO replicates from a small tissue sample, providing a scalable and reproducible solution for skeletal muscle research and drug screening.