{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T08:49:34Z","timestamp":1792831774462,"version":"4.5.0"},"reference-count":133,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,6,11]],"date-time":"2025-06-11T00:00:00Z","timestamp":1749600000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Boehringer Ingelheim Pharmaceuticals"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>In recent years, the field of skeletal muscle tissue engineering has experienced significant advancements, evolving from traditional two-dimensional (2D) cell cultures to increasingly sophisticated three-dimensional (3D) engineered constructs. While 2D models have provided foundational insights into muscle cell biology, emerging 3D platforms aim to better recapitulate the complex native muscle environment, including mature muscle fibers, supportive vasculature, and native-like extracellular matrix (ECM) composition. Here, we provide a comprehensive review of current in vitro skeletal muscle models, detailing their design principles, structure, and functionalities as well as the advantages and limitations inherent to each approach. We put a special emphasis on 3D engineered muscle tissues (EMTs) developed through advanced bioengineering strategies and note that design criteria such as scaffold selection, perfusion system incorporation, and co-culture with supporting cell types have significantly enhanced tissue maturity and complexity. Lastly, we explore the application of these engineered models to disease studies, highlighting models of both mendelian muscle disorders and common polygenic diseases and the potential of these platforms for drug discovery and regenerative therapies. Although an ideal in vitro model that fully recapitulates native muscular architecture, vascularization, and ECM complexity is yet to be realized, we identify current challenges and propose future directions for advancing these bioengineered systems. By integrating fundamental design criteria with emerging technologies, this review provides a roadmap for next-generation skeletal muscle models poised to deepen our understanding of muscle biology and accelerate therapeutic innovation.<\/jats:p>","DOI":"10.3390\/cells14120882","type":"journal-article","created":{"date-parts":[[2025,6,11]],"date-time":"2025-06-11T08:18:06Z","timestamp":1749629886000},"page":"882","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":29,"title":["From 2D Myotube Cultures to 3D Engineered Skeletal Muscle Constructs: A Comprehensive Review of In Vitro Skeletal Muscle Models and Disease Modeling Applications"],"prefix":"10.3390","volume":"14","author":[{"given":"Tianxin","family":"Cao","sequence":"first","affiliation":[{"name":"Cardiovascular-Renal-Metabolic Diseases Research Department, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-7019-758X","authenticated-orcid":false,"given":"Curtis R.","family":"Warren","sequence":"additional","affiliation":[{"name":"Cardiovascular-Renal-Metabolic Diseases Research Department, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1007\/164_2016_53","article-title":"Assembly and Maintenance of Myofibrils in Striated Muscle","volume":"235","author":"Sanger","year":"2017","journal-title":"Handb. 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