Reversible Cell Layering for Heterogeneous Cell Assembly Mediated by Ionic Cross-Linking of Chitosan and a Functionalized Cell Surface Membrane

Current heterogeneous cell assembly techniques in coculture systems rely on irreversible cell layering or a cell separation membrane. However, the techniques possess major drawbacks of inefficiency in direct interactions of the assembled cell layers and cell separation following coculture, which hamper characterization and therapeutic applications of the cells following coculture. Here, we develop a reversible cell layering platform for assembly of heterogeneous cells that allows both active direct cell–cell interactions and facile cell separation. Anionic maleimide-chondroitin-sulfate is grafted onto the surface membrane of myogenic C2C12 cells and human mesenchymal stem cells (hMSCs) to modify the surface charge of the cells without cytotoxicity. A highly porous chitosan thin film is formed in situ interspacing between the heterogeneous cell layers via ionic cross-linking of cationic chitosan and anionic functionalized cells, forming compactly assembled double-layered cell constructs. The chitosan film ...