Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

Summary

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

Abstract

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H₂O₂), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α) assay, and cells were evaluated for viability using fluorescent dyes.

The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H₂O_2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

Introduction

In vitro toxicology studies are performed to predict the toxic effects of chemicals and other agents that can cause damage to cells. In the assessment of toxicity to the cornea, human corneal epithelial cells (HCECs) have been used in models for evaluating these effects^1,2,3,4. These models typically evaluate physiological effects such as changes to the cell's metabolic activity, cell proliferation, and other cell functions such as the production and release of inflammatory cytokines. For these toxicology studies, cells from var....

Protocol

1. Culture of pHCECs and iHCECs

1. Grow the pHCECs and iHCECs in human ocular epithelial medium (HOEM) with the following supplements: 6 mM L-glutamine, 0.002% cell media supplement O (Table of Materials), 1.0 µM epinephrine, 0.4% cell media supplement P (Table of Materials), 5 µg/mL rh insulin, 5 µg/mL apo-transferrin, and 100 ng/mL hydrocortisone hemisuccinate in collagen-1 coated culture flasks (18 mL in a 75 cm² culture flask).
2. Change the medium in the flasks every 2-3 days by removing the medium after the cells grow to ~80% confluence. Add dissociation solution without phenol red and i....

Results

Cell size
The primary and immortalized HCECs were visualized with three fluorescent dyes, which reflect three different stages of cell viability. Live cells are green (calcein-AM), dead cells are red (ethidium homodimer-1), and apoptotic cells are yellow (annexin V-computer-adjusted color for better visualization of the fluorescence signal). Live cells contain esterases in the cell cytoplasm and convert calcein-AM to calcein. Dead cells have cell membranes that are permeable to ethidium homodimer-1. .......

Discussion

Potential differences in the use of two types of HCECs were assessed. Cells were placed in the same medium (HOEM) at identical concentrations of cells and then exposed to short and long periods of UV radiation and three ocular toxins. Doses of UV radiation and chemicals were selected based on their physiological effects, which were damaging enough to the cells to produce intermediate responses that could be compared. Exposure times of 5 and 20 min for UV radiation and 5 and 15 min for the selected doses of BAK (0.001%), .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
75 cm2 Vented Flask	Corning	354485	This is the BioCoat brand, collagen-coated
96 well plate	costar	3370
alamarBlue	Fisher Scientific	dal 1025
Annexin Staining buffer solution	Invitrogen, Burlington, ON, Canada
Annexin V	Invitrogen, Burlington, ON, Canada
Axiovert 100 microscope with a Zeiss confocal laser scanning microscope 510 system	Carl Zeiss Inc., Germany
Corning 48 Well plates	Corning	354505	This is the BioCoat brand, collagen-coated
Cytation 5	BioTek	CYT5MPV	Can read fluorescence from 280 - 700 nm (for assay 540/590)
Fetal Bovine Serum	Hycone	SH30396.03
glass bottom coverslips	MatTek Corporation, Ashland, MA, USA
Human Corneal Epithelial Cells	University of Ottawa	N/A	SV40-immortalized human corneal epithelial cells from Dr. M. Griffith (Ottawa Eye Research Institute, Ottawa, Canada) and have been characterized Griffith M, Osborne R, Munger R, Xiong X, Doillon CJ, Laycock NL, Hakim M, Song Y, Watsky MA. Functional human corneal equivalents constructed from cell lines. Science. 1999;286:2169-72.
Human Ocular Epithelia Media (HOEM) with the following supplements:	Millipore, Billerica, MA, USA	SCMC001	Epigro base media supplemented with 6 mM L-Glutamine, 0.002% EpiFactor O (cell media supplement O in article) , 1.0 μM Epinephrine, 0.4% EpiFactor P (cell media supplement P in article), 5 μg/mL rh Insulin, 5 μg/mL Apo- Transferrin, and 100 ng/mL Hydrocortisone Hemisuccinate in Collagen-1 coated culture flasks (BioCoat, Corning, Tewksbury, MA, USA).
Live Dead calcien and ethidium homodimer	Invitrogen, Burlington, ON, Canada
MesoScale Discovery (MSD) QuickPlex SQ 120 instrument	Rockville, MD, USA
MSD Human Proinflammatory Panel II (4-Plex) V-Plex assay	Rockville, MD, USA
Penicillin Streptomycin	Gibco	15140-122	100x concentration so add 1 mL to each 99 mL of media
Primary Corneal Epithelial Cells	Millipore, Billerica, MA, USA	SCCE016
SpectraMax fluorescence multi-well plate reader	Molecular Devices, Sunnyvale, CA, USA
TrypLE Express (cell disassociation solution)	Fisher Scientific	12605036