Co‐assembled Protein Nanosheets for the Expansion of Mesenchymal Stem Cells with Enhanced Multipotency and Functionality on Printable Liquid Microcarriers

Human mesenchymal stem cells (hMSCs) hold great translational potential for regenerative medicine and tissue engineering. However, their clinical application is hindered by the rapid loss of pluripotency during large-scale expansion on conventional substrates. Here we present co-assembled protein nanosheets-stabilized liquid microcarriers that enable scalable hMSC expansion while preserving pluripotency and functionality. Uniformly sized liquid microcarriers are produced using a custom dynamic droplet printing device. When aged appropriately, these liquid microcarriers support robust hMSC adhesion, rapid proliferation, and maintenance of pluripotency and therapeutic function. Enhanced multipotency retention is mediated by intermediate cytoskeletal tension and nucleus localization of Yes-associated protein, regulated by the nanoscale mechanical properties of the protein nanosheets. Moreover, non-enzymatic cell harvesting through gentle centrifugation preserves high cell viability and functionality. This study establishes engineered protein nanosheets-stabilized liquid microcarriers as a promising platform for scalable, non-disruptive stem cell expansion with significant therapeutic translation potential.