Enhanced cell sheet engineering through combination of single cells and spheroids on liquid interface using perfluorocarbon.

Cell sheet engineering provides a scaffold-free strategy for fabricating cohesive tissue constructs, but challenges remain in maintaining structural integrity and mimicking complex tissue architectures. This study demonstrated perfluorodecalin-based liquid-liquid interfaces, known for their inertness and stability, as a simple, and efficient platform for fabricating cell sheets. Using single cells, spheroids, and their combination, we evaluated methods to enhance sheet formation. Single cells formed cohesive sheets at high densities (4 × 10⁶ cells/well) but exhibited limited long-term stability due to nutrient constraints. Spheroids formed robust sheets at lower densities (2 × 10⁶ cells/well), whereas higher densities impaired fusion. The mixed approach combined the advantages of both, improving uniformity, mechanical stability, and spheroid fusion, while mimicking muscle-like structures with vascular networks. Additionally, the cell sheets retained adipogenic and chondrogenic differentiation potential, highlighting their functional viability. These findings establish liquid interfaces as a practical and versatile platform for tissue engineering, regenerative medicine, and in vitro modeling.