Home › Cell Biology › Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model
Cell Biology JoVE (Open Access) Citable · DOI

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

DOI: 10.3791/52979-v
What you'll learn
  • ✓ Prepare and culture reconstructed human cornea-like epithelial tissue models
  • ✓ Execute standardized test article exposure and rinsing protocols
  • ✓ Measure cell viability using MTT assay to classify irritant potential
  • ✓ Interpret results to discriminate GHS ocular hazard categories
Protocol

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Difficulty
advanced
Total time
~2–3 days (Day 0 tissue preparation, Day 1 exposure/rinsing/viability assay, Day 2 optional for solid test articles)
Model organism
Reconstructed human cornea-like epithelial (RhCE) tissue model
Biosafety
BSL-1

Steps

1
Prepare RhCE tissue and equilibrate culture conditions

Set up reconstructed human cornea-like epithelial tissue models on appropriate culture supports and equilibrate to assay-ready conditions prior to test article exposure.

▶ 01:43
2
Expose tissue to test article

Apply test article (liquid or solid chemical) directly to RhCE tissue surface for the specified contact duration to assess irritant response.

▶ 03:08
3
Rinse tissue to remove test article residue

Thoroughly rinse RhCE tissue with saline or appropriate buffer to remove excess test material and halt chemical exposure.

▶ 04:42
4
Quantify cell viability via MTT assay

Incubate treated RhCE tissue with MTT reagent and measure absorbance to determine mitochondrial metabolic activity as a proxy for tissue viability.

▶ 06:13
5
Classify ocular hazard based on viability data

Interpret MTT absorbance values against reference thresholds to assign test article to GHS irritant category (Category 1/2 or No Category).

▶ 08:19
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