Human-Derived Corneal Epithelial Cells Expressing Cell Cycle Regulators as a New Resource for in vitro Ocular Toxicity Testing

Abstract

The Draize test has been used on rabbits since the 1960s to evaluate the irritation caused by commercial chemicals in products such as cosmetics or hairdressing products. However, since 2003, such tests, including the Draize test for cosmetics, have been prohibited in European countries because they are considered problematic to animal welfare. For this reason, replacement of in vivo methods with the alternative in vitro methods has become an important goal. In this study, we established a corneal epithelial cell line co-expressing a mutant cyclin-dependent kinase 4 (CDK4), Cyclin D1, and telomerase reverse transcriptase (TERT). The established cell line maintained its original morphology and had an enhanced proliferation rate. Furthermore, the cells showed a significant, dose-dependent decrease in viability in an irritation test using glycolic acid and Benzalkonium chloride. These cells can now be shared with toxicology scientists and should contribute to increasing the reproducibility of chemical testing in vitro.

Keywords: cell cycle regulators; corneal epithelial cells; cyclin D1; cyclin-dependent kinase 4; immortalization; telomerase reverse transcriptase.

Figure 1

Cell cycle analysis of primary, K4D, and K4DT cells. (A) Cell cycle analysis of primary cells. (B) Cell cycle analysis of K4D cells. (C) Cell cycle analysis of K4DT + T cells.

Figure 2

Assessment of glycolic acid (0.5 and 5%) as an irritant in primary and K4DT + T cells using the STE method. (A) The number of primary and K4DT cells. The data are expressed as the mean, with the error bars representing the standard error. Two stars indicate a statistical significance of more than 1%. (B) Cell morphology of PBS-treated (control) primary cells (left panel) and 5% glycolic acid-treated primary cells (right panel). (C) Cell morphology of PBS-treated K4DT + T cells (left panel) and 5% glycolic acid-treated K4DT + T cells (right panel).

Figure 3

Detection of toxicity of Benzalkonium chloride using the STE method. (A) The number of K4DT cells. The data are expressed as the mean, with the error bars representing the standard error. Two stars indicate a statistical significance of more than 1%. (B) Cell morphology of PBS-treated (control) K4DT cells (left panel) and 0.05% Benzalkonium chloride-treated K4DT cells (right panel).

Figure 4

Detection of toxicity of Benzalkonium chloride using MTS assay. (A) The absorbance of K4DT cells. The data are expressed as the mean, with the error bars representing the standard error (n = 16). Two stars indicate a statistical significance of more than 1%. (B) The appearance of 96 microplates after 1-h incubation at 37°C after the addition of MTS reagent.

Figure 5

Schematic representation of the different expression vectors used in this study and confirmation of genomic integration and protein expression. (A) Structure of the expression vectors used to establish the K4D and K4DT + T cell lines. LTR, long terminal repeat; CMV, cytomegalovirus promoter; Hygro, Hygromycin-resistant gene. (B) Confirmation of the genomic integration of the TERT expression cassette by PCR: (1) primary cells; (2) K4D cells; (3) K4DT + T cells. The upper panel shows the specific 500-bp PCR product confirming the presence of the TERT expression cassette, whereas the lower panel shows the 400-bp PCR product derived from the human Tuberous Sclerosis Type II gene.

Figure 6

Cell morphology of primary, K4D, and K4DT + T cells. (A) Cell morphology using phase contrast microscopy at passage 0. (B) Morphological observation of the cytoskeleton (F-actin) and the nucleus, following staining with rhodamine-labeled phalloidin and DAPI, respectively. Left panel: EGFP fluorescence. Middle panel: phalloidin staining. Right panel: merged images of the phalloidin, DAPI, and EGFP stains.

Figure 7

Detection of introduced proteins with western blotting. (A) Primary, K4D, and K4DT+T cells were applied into the western blotting using anti-CDK4, anti-Cyclin D1, anti-tubulin antibodies. The triplicated technical replicates were examined. (B) Quantitation results of the signal intensity with Image J software. The signal intensity of primary, K4D, and K4DT cells were shown in the graphs.

Figure 8

Cell growth status and morphological observation of primary, K4D, and K4DT + T cells. (A) Cell growth and sequential passaging of primary, K4D, and K4DT + T cell. Cell growth is represented by the cumulative population doubling value. (B) Morphological observation at passage 1 in the population doubling assay. (C) Cell morphology of K4D and K4DT + T cells at passage 3. Lower magnification of K4D (left panels) and K4DT + T cells (right panels). (D) Higher magnification of K4D cells (left panels) and K4DT + T cell (right panels).

Figure 9

Cell morphology and EGFP expression of K4D cells at passage 3. (A) K4D cells at passage 3. Cell morphology by differential interference contrast microscopy (DIC, left panel), EGFP fluorescence (EGFP, middle panel), and merged images (merge, right panel). (B) High-magnification view of K4D cells at passage 3 by DIC (left panel), EGFP fluorescence (EGFP, middle panel), and merged images (right panel).

Figure 10

Immunostaining of K4D cells expressing EGFP. (Upper left) EGFP expression in K4D cells at passage 3. (Upper right) Immunostaining of cytokeratin 3/2p in K4D cells detected with Alexa 568. (Middle left) Nuclear counterstaining with DAPI. (Middle right) The merged picture among EGFP, cytokeratin 3/2p, and DAPI channels. (Lower left) Cell morphology of difference in contrast (DIC). (Lower right) The merged picture among EGFP, cytokeratin 3/2p, DAPI, and DIC channels. Note that fibroblast-like cells that are indicated by arrows were neither negative for EGFP and cytokeratin 3/2p.