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

Area of Science:

• Ocular surface research
• Tissue engineering
• In vitro models

Background:

• Need for physiologically relevant, reproducible, and ethical preclinical ocular research.
• Limitations of current methods in ocular surface research.
• Growing demand for New Approach Methodologies (NAMs) using human cells.

Purpose of the Study:

• To develop a reproducible and scalable three-dimensional (3D) in vitro conjunctival spheroid model.
• To create a scaffold-free test system using primary human cells for ocular research.
• To establish a New Approach Methodology (NAM) for preclinical ocular surface studies.

Main Methods:

• Fabrication of agarose microwell arrays using 3D printing and PDMS replica molding.
• Isolation and expansion of primary human conjunctival epithelial cells and fibroblasts.
• Generation of bilayered conjunctival spheroids by seeding epithelial cells onto fibroblast spheroids.

Main Results:

• Consistent spheroid size and shape over time confirmed by quantitative image analysis.
• High cell survival demonstrated through viability assays.
• Organized cellular architecture and extracellular matrix deposition observed in histological and ultrastructural analyses.

Conclusions:

• The developed 3D in vitro conjunctival spheroid model is reproducible, scalable, and uses primary human cells.
• This scaffold-free platform supports the 3Rs (Replacement, Reduction, Refinement) in animal testing.
• The model is suitable for dry eye disease research, conjunctival inflammation studies, and preclinical compound testing.