Enhancing Cell Aggregation and Migration via Double-Click Cross-Linking with Azide-Modified Hyaluronic Acid

We present a novel approach to the formation of cell aggregates by employing click chemistry with water-soluble zwitterionic dibenzo cyclooctadiyne (WS-CODY) and azide-modified hyaluronic acid (HA-N₃) as a linker to facilitate rapid and stable cell aggregation. By optimizing the concentrations of HA-N₃ and WS-CODY, we achieved efficient cross-linking between azide-modified cell surfaces and HA-N₃, generating cell aggregates within 10 min, and the resulting aggregates remained stable for up to 5 days, with cell viability maintained at approximately 80%. Systematic experiments revealed that a stoichiometric balance between HA-N₃ and WS-CODY is important for effective cross-linking, highlighting the roles of both cell–surface azide modification and HA in the aggregate formation. We also investigated the genetic basis of altered cell behavior within these aggregates. Transcriptome analysis (RNA-seq) of aggregates postcultivation revealed a marked fluctuation of genes associated with ‘cell migration’ and ‘cell adhesion’, including notable changes in the expression of HYAL1, ICAM-1, CEACAM5 and RHOB. These findings suggest that HA-N₃-mediated cell aggregation can induce intrinsic cellular responses that not only facilitate cell aggregate formation but also modulate cell–matrix interactions. We term this phenomenon ‘chemo-resilience’, The simplicity and efficacy of this click chemistry-based approach suggest it may have broad applicability for forming cell aggregates and modulating cell–matrix interactions in tissue engineering and regenerative medicine.