Spheroid Sheets: A Scalable Platform for Producing Tissue Membrane Constructs

Bottom-up tissue engineering using cell spheroids offers many advantages in recapitulating native cell-cell and cell-matrix interactions. Many tissues, such as cartilage, bone, cardiac muscle, intestine, and neural tissues, have been tissue-engineered using cell spheroids. However, previous methods for spheroid assembling, such as mold casting, hydrogel-based bioprinting, or needle array, either lack control over final tissue geometry or face challenges in scalability and throughput. In this protocol, we describe a robust and scalable tissue engineering method for assembling cell spheroids into a thin, planar spheroid sheet. The spheroids are sandwiched between two flexible meshes held by a frame, facilitating uniform spheroid fusion while ensuring nutrient exchange and ease of handling. We demonstrate this method by producing thin cartilage tissue from human mesenchymal stem cells undergoing chondrogenic differentiation. This approach offers a practical platform for producing thin membrane-like tissue constructs for many research and therapeutic applications. Key features • Spheroid-based tissue engineering: Utilizing cell spheroids to build various membrane-like tissues. • Controlled tissue thickness: Frame and mesh constrain thickness and guide lateral fusion of spheroids, enabling uniform and thin tissue for efficient nutrient diffusion. • Scaffold-free construct: After the thin tissue membrane is formed, the frame and mesh can be removed. • Mechanical support: Meshes enable easy handling and can aid in transplantation of the constructs, for example, by allowing them to be wrapped or sutured.