Transcytotic Intercellular Gelation Enables Cell Spheroids

Cell spheroids bridge the discontinuity between <i>in vitro</i> systems and<i> in vivo</i> animal models, but inducing cell spheroids by nanomaterials remains an inefficient and poorly understood process. We used cryo-EM to determine the atomic structure of helical nanofibers self-assembled from D-peptides, and used fluorescent imaging to show that endo/exocytosis of enzyme-responsive D-peptide assemblies results in intercellular gels that enable cell spheroids. Specifically, D-phosphopeptides self-assemble to form nanoparticles to undergo endocytosis. The nanoparticles, being resistant to proteolysis and partially dephosphorylated, go through exocytosis to the cell surface and turn into helical nanofibers, which act as the artificial matrices of intercellular gels to induce cell spheroids from either suspended or adherent cells. No cell spheroids form without the endo- or exocytosis, the phosphate triggers, or the shape-switching of the peptide assemblies. This study, coupling transcytosis and morphological transformation of peptide assemblies to form intercellular gels in situ, mimics biogenesis to form intercellular matrices, and demonstrates a biomimetic approach for regenerative medicine and tissue engineering.