New Human Corneal Epithelial Cell Line for in vitro Ocular Toxicity Testing 

Reducing Animal Testing to Assess Toxicity during Drug Development

There is a growing movement in the biomedical industry to phase out routine animal testing in preclinical safety studies, driven by ethical concerns, scientific advances, and evolving regulatory requirements. The goal is to replace animal-based toxicity testing with more effective, human-relevant approaches, such as in vitro assays using human-derived cells and in silico models.

A well-known example is the Draize eye irritation test, which has been used on rabbits since the 1960s to assess the irritation potential of chemicals in cosmetics and hair treatments. Due to animal welfare concerns, this test is now banned in many European countries. As a replacement, rabbit-derived corneal epithelial cells are used in the Short Time Exposure (STE) method for ocular irritancy testing. However, species differences mean these results may not accurately reflect human responses.

Human corneal epithelial cells would be more relevant for in vitro toxicity testing, but their use is limited because they stop proliferating after only a few passages in culture. To overcome this challenge, Fukuda and colleagues applied the K4DT method—co-expressing mutant CDK4 (R24C), cyclin D1, and telomerase reverse transcriptase (TERT)—to create a new immortalized human corneal epithelial cell line to evaluate ocular cytotoxicity.

Establishment and Characterization of New Immortalized Human Corneal Epithelial Cell Line

Human corneal epithelial cells from Lifeline® Cell Technology were cultured in OcuLife™ basal medium supplemented with OcuLife™ LifeFactors, then infected with recombinant lentiviruses. The researchers created two experimental cell lines:

• K4D cells – expressing mutant CDK4 and cyclin D1.
• K4DT + T cells – expressing mutant CDK4, cyclin D1, and TERT.

Morphology and F-actin staining showed both recombinant lines closely resembled primary corneal epithelial cells, indicating no toxic effects from the introduced genes.

When cultured over multiple passages, K4D and K4DT + T cells both proliferated beyond passage 1 compared to primary cell controls and showed accelerated growth rates. However, fibroblast-like cells appeared in K4D cultures after passage 3, which were confirmed by EGFP fluorescence to have lost epithelial characteristics. Consequently, K4D cells without TERT were deemed unsuitable for ocular irritant testing.

Finally, the K4DT + T cells were used for in vitro irritation testing using the STE method, originally developed using rabbit-derived epithelial cells. Primary or K4DT + T human corneal epithelial cells were exposed to 0.5, 5% glycolic acid, a known irritant, as well as a PBS control. Exposure to glycolic acid resulted in a significant, dose-dependent decrease in K4DT + T cell viability.

Benzalkonium chloride, the primary preservative chemical additive for eye drops, also showed strong toxicity in  the immortalized corneal epithelial cell line, showing a significant reduction in cell number at all doses tested (0.05, 0.5, 5%). Based on the results of STE method, the toxicity of Benzalkonium chloride at 5, 0.5, 0.05, 0.005, and 0.0005% with an MTS proliferation assay, a colorimetric method for determining the number of viable cells in proliferation, detectable as the absorbance at 490 nm. Even the lowest concentration (0.0005%) significantly reduced viability was observed, demonstrating the high sensitivity of the immortalized cell line to irritants.

Overall, the newly developed K4DT + T immortalized human corneal epithelial cell line provides a reproducible, human-relevant model for detecting ocular toxicity. Its high sensitivity to chemical irritants supports its use in replacing animal-based tests for experimentation and contributes to improving animal welfare in scientific research.

Lifeline® Products Supporting Ocular Research

Lifeline Cell Technology offers a comprehensive portfolio of primary eye cells and specialized media to help researchers achieve high-quality experimental results including:

• Normal Human Corneal Epithelial Cells
• Human Scleral Fibroblasts
• FibroLife Fibroblast Serum Free Medium Complete Kit
• FibroLife S2 Fibroblast Medium Complete Kit
• OcuLife ™ Epithelial Medium Complete Kit

Join us next month for another installment of the Lifeline® blog to see how our cells and culture media are advancing biomedical research worldwide. If you have used our products in your publication, we’d love to feature your work here!