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Updated: Aug 20, 2026
Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells
Published on: July 22, 2021
Assessment of ocular irritation by image processed quantification of cell injury in human corneal cell cultures and
Maria Engelke1, Jens Patzke, Svitlana Tykhonova
1Institute for Biophysics, University of Bremen, Bremen, Germany. mengelke@uni-bremen.de
Abstract:
Currently, there are no accepted alternative tests for the replacement of animals in ocular irritation testing. This study focused on the quantification of cellular viability as a measure of toxic events in immortalised human corneal cell cultures and a three-dimensional corneal construct. Simultaneous vital dye staining by calcein AM and ethidium homodimer-1 was used to provide "live" and "dead" probes, respectively. For further quantification, we have developed image processing tools to evaluate digital images obtained from confocal fluorescence scanning microscopy measurements. Based on the finding that ocular irritation can be related to the extent of cell injury at the various cell layers of the cornea, we extended our studies from corneal cell cultures to an in vitro human corneal equivalent system comprising epithelial, stromal keratocyte and endothelial layers. Our results showed that the microscopic measurement of cellular injury by using either cell cultures or in vitro corneal constructs, combined with image processed quantification, can provide insight into the extent of the toxic effects.
Insights
This study quantifies cellular viability in human corneal models to assess ocular irritation. Microscopic analysis and image processing offer a promising alternative to animal testing for toxic effects.
Area of Science:
- Toxicology
- In vitro models
- Ocular irritation testing
Background:
- Animal testing for ocular irritation lacks accepted alternatives.
- Developing in vitro methods is crucial for ethical and efficient toxicity assessment.
- Cellular viability assays are key indicators of toxic events.
Purpose of the Study:
- To quantify cellular viability as a measure of toxic events in human corneal cell cultures and a 3D corneal construct.
- To establish an alternative method for ocular irritation testing.
- To correlate cell injury with ocular toxicity.
Main Methods:
- Utilized immortalised human corneal cell cultures and a 3D corneal construct.
- Employed simultaneous vital dye staining (calcein AM for live, ethidium homodimer-1 for dead cells).
- Developed image processing tools for confocal fluorescence scanning microscopy analysis.
Main Results:
- Quantified cellular viability and injury in corneal cell cultures and 3D constructs.
- Demonstrated that microscopic measurement of cell injury correlates with toxic effects.
- Image processing enhanced the quantification of cellular damage.
Conclusions:
- Microscopic measurement of cellular injury in vitro, combined with image processing, provides insight into ocular toxic effects.
- This approach shows potential as an alternative to animal testing in ocular irritation assessment.
- Further validation of these in vitro methods is warranted for regulatory acceptance.
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