Scalable XTEN-Based Coiled-Coil Hydrogels for Injectable Ocular Cell Delivery

Abstract

Limbal stem cell deficiency (LSCD) disrupts corneal epithelial renewal and can lead to progressive vision loss. Although existing cell-based therapies can restore corneal epithelial cells, they do not directly reconstitute the paracrine stromal support provided by the healthy limbal niche. Limbal fibroblasts (LFs) maintain this niche through secretion of keratinocyte growth factor (KGF) and other stromal mediators. This work evaluates a recombinant protein-based coiled-coil hydrogel, built from an intrinsically disordered XTEN polypeptide backbone flanked by self-assembling pentameric coiled-coil domains, as a scalable and storage-compatible injectable platform for LF delivery. The hydrogel was produced at multi-gram scale and characterized across four translationally relevant endpoints: formulation concentration, long-term protein storage, preformed gel freeze-thaw stability, and cell compatibility. Compared with a 10% w/v formulation, a 5% w/v formulation retained yielding and recovery behavior while providing a softer matrix better suited for fine-gauge injection. Protein stock stored in solution at -80°C for approximately two years retained its gel-forming capacity, and preformed gels tolerated frozen storage at -20°C or -80°C for at least four weeks without a statistically detectable change in viscoelastic behavior. Primary LFs from three donors maintained greater than 98% viability after encapsulation and 27G needle injection. Finally, gel-encapsulated LFs retained paracrine KGF secretory function and responded to inflammatory stimulation consistent with physiological wound-healing signaling, while cocultured human limbal epithelial cells maintained corneal-specific marker expression. These findings support the continued evaluation of this recombinant coiled-coil hydrogel as a mechanically versatile and storage-compatible injectable carrier for limbal fibroblast delivery and motivate further studies of post-injection cell function.