Speaker
Description
Introduction: To date, no conjunctival spheroids have been reported, despite the conjunctiva’s vital role in maintaining ocular surface homeostasis and contributing to various ocular surface diseases. The conjunctiva is a dynamic, multi-cellular mucosal tissue that functions in barrier homeostasis, and tear film stabilization. However, physiologically relevant 3D in vitro models that replicate native structure and function remain lacking. Such models are crucial for advancing mechanistic studies and drug development, while also aligning with the 3Rs principles (Replacement, Reduction, and Refinement). Here, we present a novel approach to generate functional 3D conjunctival spheroids using honeycomb-inspired agarose microwells, enabling scalable and reproducible production for ocular research and therapeutic screening.
Methods: To fabricate honeycomb-inspired microwells, a 2% agarose solution was cast onto 3D-printed micropillars and subsequently sterilized using UV treatment for 4 hr. In this study, we employ primary human conjunctival fibroblast and epithelial cells. Initially, the primary conjunctival fibroblasts were seeded into the agarose molds at densities ranging from 1,000 to 5,000 cells per microwell to generate spheroids of different sizes, followed by centrifugation to promote aggregation. After spheroid formation, conjunctival epithelial cells were seeded on top to facilitate epithelium formation. A series of analyses were conducted, including spheroid size measurement, cell count, viability testing (live/dead staining), and histological staining to evaluate spheroid morphology and epithelial layer formation.
Results and discussion: Honeycomb-inspired agarose microwells facilitated the scalable and robust formation of spheroids. The model demonstrated the ability to mass-produce spheroids of uniform size, which could be easily tuned by adjusting the cell number. The size of the spheroid was tunable with the varying cell number. Live/dead staining indicated excellent cell viability within the spheroids, particularly those formed by 3000 cells. Histological staining confirmed the stromal-like structure and multilayered conjunctival epithelium. Additionally, immunostaining with specific conjunctival markers such as CK13, MUC5AC, and CK19 validated the epithelial identity and functional relevance of the spheroids.
Conclusion: This 3D conjunctival spheroid model enables scalable, reproducible, and cost-effective production of physiologically relevant tissue structures. Using primary human cells, it offers potential for personalized medicine and serves as a valuable in vitro platform for ocular surface research and drug screening.
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