In Vivo‐Like Scaffold‐Free 3D In Vitro Models of Muscular Dystrophies: The Case for Anchored Cell Sheet Engineering in Personalized Medicine

Abstract Progress in understanding the underlying mechanisms of muscular dystrophies is hindered by the lack of pathophysiologically relevant in vitro models. Here, an entirely scaffold‐free anchored cell sheet engineering platform is used to create patient‐specific three‐dimensional (3D) skeletal muscle in vitro models. This approach effectively replicates mature muscle phenotypes and tissue‐ and disease‐specific extracellular matric (ECM). Models were developed using primary cells from healthy individuals and patients with Duchenne Muscular Dystrophy and Myotonic Dystrophy Type 1. Through a combination of quantified histological staining (Hematoxylin & Eosin, Movat's Pentachrome, Masson's Trichrome) and immunostaining (desmin, myosin heavy chain, laminin, and dystrophin), it was demonstrated that the models formed mature constructs closely resembling their respective in vivo conditions. Proteomics analysis revealed that the models exhibited appropriate upregulation and downregulation of disease‐relevant pathways. Models of diseased tissues accurately reflected key phenotypic features of the diseases, including alterations in muscle fiber integrity and ECM composition. Upon treatment with therapeutically beneficial drugs, significant changes in their proteomic profiles were documented, highlighting the models’ potential for drug screening. This novel in vitro modeling approach, unlike other 3D techniques that rely on exogenous biomaterials that interfere with natural cellular behaviors, provides a promising platform for studying muscular dystrophies.