Rapid scaffold-free cell sheet formation and their patterning as building blocks of complex 3D tissue constructs.

Three-dimensional (3D) cell cultures offer superior potential in replicating native tissue microenvironments by better supporting cell-cell and cell-extracellular matrix (ECM) interactions that are critical for guiding cellular behavior and functionality in engineered tissues. Among 3D approaches, scaffold-free techniques have gained attention for their ability to produce high-cellular density, and well-organized tissue-like constructs. In particular, cell sheets are uniquely suited for regenerative applications due to their contiguous architecture, large-area coverage, and integration potential with host tissues. However, current biofabrication methods for cell sheet production often require altering culture conditions (e.g., temperature, pH) or applying external stimuli (e.g., magnetic or electrical fields), which can damage cells, compromise sheet integrity, or demand costly, non-adaptable equipment. Here, we present a rapid, self-assembly-based technique using unmodified polydimethylsiloxane (PDMS) molds as culture vessels. When seeded at a critical cell density, adherent cells spontaneously self-assemble into planar 3D cell sheets within 6 hours, without substrate modification or specialized equipment. Our qRT-PCR analysis revealed significant upregulation of E-cadherin in cell sheets, confirming that cell-cell adhesion, rather than cell-substrate anchorage, drives sheet formation. We showed that our technique is versatile, supporting the creation of large-area and patterned sheets, stacked multi-layer constructs, and co-culture configurations. Notably, fibroblast cell sheets, demonstrated progressive ECM production, with histological analysis confirming collagen deposition over time. Overall, our approach preserves cell viability and function while offering a simple, rapid, and cost-effective alternative to conventional methods for fabricating cell sheets. This platform holds broad potential for applications in tissue engineering, regenerative medicine, disease modeling, and cultivated meat production.