Bio-protocol · 10 authors, 5 centres
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This paper describes a novel in vitro assay using patient-derived tears to study their effects on human corneal epithelial cells. Tears from patients with Persistent Epithelial Defect (PED) induced morphological changes, increased MMP9 activity, and altered gene expression of inflammatory and corneal homeostasis markers compared to healthy controls. This method provides a non-invasive, disease-specific model for studying ocular pathology and testing potential therapeutics.
**Background:** Tears are a complex biological fluid that maintain ocular homeostasis and protect against pathogens. Their composition changes in disease, making them a valuable source of biomarkers. However, the functional impact of tears on corneal epithelial cells is poorly understood. This protocol presents an in vitro assay using patient-derived tears to study their effects on human corneal limbal epithelial (HCLE) cells, providing a more physiologically relevant model than single-factor treatments.
**Methods:** Tears were collected from healthy controls and Persistent Epithelial Defect (PED) patients using Schirmer strips. Proteins were extracted by grinding strips in PBS, vortexing, and centrifuging at 16,000× g for 5 min at 4°C. Protein concentration was measured by NanoDrop at 280 nm. HCLE cells were cultured in keratinocyte-serum free medium (KSFM) with growth factors, then seeded at 50,000 cells/well in 12-well plates. After 20 h (60-70% confluency), cells were washed and treated with 60-100 μg/mL of tear proteins in KSFM without growth factors for 48 h. Morphological changes were observed using an EVOS imaging system. Culture supernatants were analyzed for MMP9 activity by gelatin zymography: samples were run on 8% acrylamide gels containing 1 mg/mL gelatin, incubated in 1% Triton X-100, then developing buffer overnight at 37°C, stained with Coomassie blue, and destained. RNA was extracted using Qiagen Mini-RNA Isolation kit, and 800 ng of RNA was converted to cDNA using Verso cDNA Synthesis kit. Real-time PCR was performed in duplicate using SYBR Green on an Azure Biosystems qPCR system with cycling: 95°C for 3 min, then 40 cycles of 95°C for 15 s and 60°C for 20 s. Gene expression was normalized to geometric mean of housekeeping genes RPL10 and beta-actin, using the ΔΔCt method. Statistical significance was assessed by unpaired t-test (p < 0.05).
**Key Results:** HCLE cells treated with PED tear proteins showed morphological changes: cells became more rounded, with decreased cell number and increased vacuoles, disrupting normal epithelial stratification compared to control cells (cuboidal, stratified). Gelatin zymography revealed increased MMP9 activity in culture supernatants from PED tear-treated cells compared to controls. Gene expression analysis (N=3) showed significant upregulation of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, TGFβ1, TGF-β2) and altered expression of corneal homeostasis markers (E-cadherin, beta-catenin, desmoglein) and transcription factors (KLF4, GPX4, NCO4) in PED tear-treated cells versus controls (p < 0.05).
**Clinical Implications:** This assay provides a non-invasive, patient-specific in vitro model to study ocular diseases using tears. It can be used to investigate disease mechanisms, identify biomarkers, and test drug efficacy in a physiologically relevant context. The method is cost-effective, reproducible, and can track disease progression over time. Limitations include the need for a minimum tear migration of 5 mm for protein isolation and the requirement for careful handling to avoid protein degradation. Future applications may extend to other ocular conditions such as dry eye, glaucoma, and diabetic retinopathy.