Enhancing\nCell Aggregation and Migration via Double-Click\nCross-Linking with Azide-Modified Hyaluronic Acid

We present a novel approach to the formation of cell\naggregates\nby employing click chemistry with water-soluble zwitterionic dibenzo\ncyclooctadiyne (WS-CODY) and azide-modified hyaluronic acid (HA-N<sub>3</sub>) as a linker to facilitate rapid and stable cell aggregation.\nBy optimizing the concentrations of HA-N<sub>3</sub> and WS-CODY,\nwe achieved efficient cross-linking between azide-modified cell surfaces\nand HA-N<sub>3</sub>, generating cell aggregates within 10 min, and\nthe resulting aggregates remained stable for up to 5 days, with cell\nviability maintained at approximately 80%. Systematic experiments\nrevealed that a stoichiometric balance between HA-N<sub>3</sub> and\nWS-CODY is important for effective cross-linking, highlighting the\nroles of both cell–surface azide modification and HA in the\naggregate formation. We also investigated the genetic basis of altered\ncell behavior within these aggregates. Transcriptome analysis (RNA-seq)\nof aggregates postcultivation revealed a marked fluctuation of genes\nassociated with ‘cell migration’ and ‘cell adhesion’,\nincluding notable changes in the expression of <i>HYAL1</i>, <i>ICAM-1</i>, <i>CEACAM5</i> and <i>RHOB</i>. These findings suggest that HA-N<sub>3</sub>-mediated cell aggregation\ncan induce intrinsic cellular responses that not only facilitate cell\naggregate formation but also modulate cell–matrix interactions.\nWe term this phenomenon ‘chemo-resilience’, The simplicity\nand efficacy of this click chemistry-based approach suggest it may\nhave broad applicability for forming cell aggregates and modulating\ncell–matrix interactions in tissue engineering and regenerative\nmedicine.