Abstract
Purpose :
Diabetic Keratopathy (DK) is a diabetic complication affecting the ocular surface structure and function. Current research on in vitro disease models for DK is insufficient, particularly those lacking multi-cell integration. In this study, we built a biomimetic in vitro DK model incorporating corneal epithelium, nerves, stroma, and endothelium.
Methods :
The model was constructed in Transwell inserts. A corneal stromal cells (CSCs)-laden collagen membrane was affixed to the upper surface. After 2 days of culture, corneal endothelial cells (CEnCs) were seeded on the undersurface. Then, dorsal root ganglion organoids (DRGO) were introduced 2 days later. After 7 days, corneal epithelial cells (CEpCs) were added. Following 5 days of air-liquid interface culture, cells were maintained with (HG) or without (NC) the addition of high glucose (55mM) for an additional 10 days (Fig. 1A). The structure was observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Cell integration was assessed using confocal microscopy. Trans-epithelial electrical resistance (TEER) was measured to evaluate barrier function, and a cytokine microarray was employed to analyze the expression of relevant factors in the culture medium.
Results :
The model exhibited a multi-layered structure, encompassing corneal epithelial layers, a stromal layer, and a monolayer of endothelium (Fig. 1B). Nerves were observed within the epithelial and anterior stromal layers. The endothelium formed tight junction structures (Fig. 1C). SEM results revealed increased CEpCs death, disrupted nerve bundle structures, and CEnCs extracellular matrix deposition in the HG compared to the NC. Fluorescent images demonstrated nerve bundle disintegration in the HG, while the NC appeared relatively normal (Fig. 1D). TEER results indicated elevated barrier function in NC and HG, but it was weakened under HG (Fig. 1E). The heatmap illustrated differential factor expression levels in the culture medium. Inflammatory factors (such as IL-6, IL-8, TNF-α) were upregulated in the HG, while the neurotrophic factor NT-4 was downregulated. These findings closely resembled the clinical features of DK.
Conclusions :
We constructed a biomimetic multi-cellular DK in vitro model, which simulates the disease characteristics and corneal cell alterations seen in DK.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.