A high-throughput 3D conjunctival spheroid model for standardized in vitro testing.

New Approach Methodologies (NAMs) based on human cells are increasingly needed to improve the physiological relevance, reproducibility, and ethical acceptability of preclinical ocular surface research. We developed a reproducible and scalable three-dimensional in vitro conjunctival spheroid model using primary human conjunctival epithelial cells and conjunctival fibroblasts as a human-relevant, scaffold-free test system. Agarose-based microwell arrays were fabricated via a combination of custom-made high-resolution 3D printing and polydimethylsiloxane (PDMS) replica molding, enabling the formation of uniform microwells suitable for spheroid culture and parallel production of size-controlled microtissues. Primary human conjunctival epithelial cells and fibroblasts were isolated from donor tissue obtained during routine ophthalmic surgeries and expanded under defined culture conditions. Fibroblast spheroids were first generated within agarose microwells at controlled seeding densities, resulting in stable and size-controlled aggregates. Subsequently, conjunctival epithelial cells were seeded onto pre-formed fibroblast spheroids to establish bilayered conjunctival spheroids that mimic native tissue organization. Spheroid development, morphology, and viability were systematically characterized using optical microscopy, live/dead assays, histological staining, immunofluorescence, and advanced imaging techniques including scanning electron microscopy (SEM), cryo-SEM, and transmission electron microscopy (TEM). Quantitative image analysis demonstrated consistent spheroid size and shape over time, while viability assays confirmed high cell survival. Histological and ultrastructural analyses revealed organized cellular architecture and extracellular matrix deposition, indicative of functional tissue-like constructs. By combining primary human cells, scaffold-free assembly, and microwell-based scalability, this platform contributes to the implementation of the 3Rs and provides a fit-for-purpose NAM for dry eye disease-related research, conjunctival inflammation studies, and early-stage preclinical compound testing.