Biofabricated in vitro models hold unrealized potential in accelerating drug discovery and reducing new drug costs, but current models are overly simplified and lack physiological relevance. Certain platforms capable of mimicking in vivo conditions face hurdles such as complexity, high costs, and non‐standardized applications. Existing cell sheet engineering methodologies, despite being physiologically relevant, have technical limitations like creating suitable 3D forms with proper anchorage. An innovative platform is introduced here, designed for in vitro modeling, which capitalizes on 2D culture systems and scaffold‐free cell sheets to create self‐assembling 3D tissue constructs. It provides precise control over cellular behavior, especially ECM production and remodeling. Scaffold‐free muscle fibers and lung epithelial cell cultures served as examples. This platform can fuse various scaffold‐free models to construct assembloids mimicking organ structures and functions. Different tissues can be linked via a fluidic system for body‐level simulations. Moreover, ECM‐rich constructs from cell sheets can create organ‐level ECM scaffolds, and the resulting cell sheet‐derived ECM (CSdECM) can be solubilized to facilitate 2D cellular behavior. This versatile platform, compatible with other cell sheet engineering techniques, is set to revolutionize in vitro modeling.

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.

Bioprinting is rapidly developing to be a powerful tool in tissue engineering for both whole tissue printing and development of in vitro models that can be used in drug discovery, toxicology and as in vitro bioreactors. Nevertheless, the ability to create complex 3D culture systems with different types of cells, extracellular matrices integrated with perfusable channels has been a challenge. Here we develop an approach that combines xurography of a scaffold material (cellulose) with extrusion printing of bioinks on it, followed by assembly in a layer by layer fashion to create complex 3D culture systems that could be used as in vitro models of biological processes. This new method, termed as ExCeL can recapitulate the complexities of natural tissues with proper 3D distribution of cells, ECMs, and different molecules while providing the whole structure with mechanical stability, direct and easy access to the cells, even the ones that are positioned deep in to the bulk of the structure, without the need to fix or section the samples. Briefly, bioprinting of predefined patterns with feature size of ~ 1mm has been made possible by treating paper with the hydrogel's crosslinker and printing cell embedded hydrogel that will solidify immediately upon contact with the paper. These impregnated layers can be used as single layers or in a layer-by-layer approach by stacking them (here up to 4 layers) for applications such as cell migration and proliferation in 3D structures composed of collagen or alginate as well as their response to exposure to FBS was assessed. By cutting the paper in certain patterns, printing hydrogel on the rest of it, and stacking them, features like embedded channels could be formed that will provide cells will better mass transfer in thick structures. This technique provides biologists with a unique tool to perform sophisticated in-vitro assays.