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 (<i>e.g.</i>, temperature, pH) or applying external stimuli (<i>e.g.</i>, 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.