The conjunctiva is a crucial ocular tissue that contributes to tear film stability and immune defense. Its epithelium contains specialized goblet cells that secrete mucins, particularly MUC5AC. In vitro models of the conjunctiva are needed to study diseases like dry eye, allergies, and autoimmune conditions. However, recreating goblet cells in vitro has been challenging. Previous models lacked full differentiation, including cup-like mucus-filled structures and proper mucin expression. This study aimed to develop a full-thickness 3D conjunctiva model with functional goblet cells using primary human cells.
Primary conjunctival epithelial cells and fibroblasts were isolated from human biopsies obtained from 9 adult donors undergoing glaucoma or retinal detachment surgery (no other ocular pathology). Epithelial cells were characterized in 2D for morphology and doubling time using three media (Pro1, E1, A1). 3D epithelial models (rhConE) were generated by seeding epithelial cells on inserts and culturing at the air-liquid interface with Pro3, E3, or A3 media. Full-thickness models (FTConM) were created by combining a compressed collagen I hydrogel containing 4.5 × 10^4 fibroblasts (6.43 × 10^4 cells/mL) with 5.0 × 10^5 epithelial cells seeded on top. FTConM were cultured in Pro10 medium. Models were analyzed at days 10, 15, and 20 using histology (H&E, Alcianblue-PAS), immunofluorescence for cytokeratins (CK1, CK13, CK14, CK19), E-cadherin, vimentin, and MUC5AC, and RT-qPCR for MUC5AC and MUC16 expression relative to GAPDH. Barrier function was assessed by impedance spectroscopy (TEER at 1000 Hz). MTT assays measured viability.
- Epithelial cells in 2D retained cobblestone morphology up to passage 4 and expressed CK13 and CK19. Doubling time varied by media and passage.
- rhConE formed stratified epithelia (4-6 layers) but showed pathologic keratinization and no goblet cell differentiation. TEER values increased over time, peaking at day 18 in A3 medium (2992 ± 1259 Ω·cm²) compared to Pro3 (1233 ± 874 Ω·cm²) and E3 (1344 ± 442 Ω·cm²). At day 20, all media showed comparable TEER.
- FTConM formed non-keratinized stratified epithelia of 7-9 cell layers. Alcianblue and PAS staining revealed mucus-filled goblet cells with secretion. Goblet cells were visible from day 10 and fully differentiated by day 15.
- Immunofluorescence showed CK1 and CK14 predominantly in basal cells by day 20; CK13 and CK19 were expressed throughout all epithelial layers. In FTConM, CK19 expression was strong across all layers, whereas in rhConE it was only in single cells. E-cadherin confirmed cell junctions, and vimentin stained fibroblasts.
- Confocal microscopy of whole mounts showed MUC5AC-positive vesicles around nuclei of goblet cells.
- RT-qPCR: MUC5AC was expressed at all time points but significantly lower than native tissue (p < 0.05). MUC16 expression at day 15 was significantly lower than native (p < 0.05), but by day 20, expression was not significantly different from native (p = 0.491).
- Media comparison (Pro3 vs Pro10) on rhConE: Pro10 increased basal cell layers and PAS staining but did not induce goblet cell differentiation. TEER values showed no significant differences between Pro3 and Pro10.
**Clinical Implications**
This full-thickness 3D conjunctiva model represents a significant advancement in ocular tissue engineering, achieving goblet cell differentiation and mucus production that mimics native conjunctiva. It provides a reproducible, human-derived platform for studying goblet cell biology, conjunctival diseases (e.g., dry eye, allergies, autoimmune conditions), and drug testing. The model's use of primary cells and defined components reduces reliance on animal experiments and enables personalized medicine approaches using patient-derived cells. Future applications include investigating goblet cell differentiation pathways, disease mechanisms, and therapeutic screening.