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 various sources have been selected to assess the damaging effects of chemicals and UV radiation on HCECs^2,3. pHCEC lines are available from companies that provide these cells from donor tissues of adults. Primary cells can be treated with dispase and gently scraped off the cornea for culture⁵. The cells are then tested for viruses and contamination and then shipped cryopreserved in 10% dimethyl sulfoxide.

The advantage of primary cell lines is that the cells are genetically identical to the cells of the donor. This is ideal, as an in vitro model should mimic the in vivo tissue as much as possible. The disadvantage of primary cell lines is that they have a limited number of cell divisions or passages⁶. The limited number of cells available restricts the number of experiments that can be conducted with a single primary culture, increasing the cost of the experiments.

Immortalized cell lines have also been used in cell culture toxicity models. However, unlike the primary cell line obtained from in vivo tissue, the immortalized cell line has been genetically altered. Immortalized cells are created by incorporating the genes of a virus into the DNA of primary cells^6,7,8. Cells with successful viral gene incorporation are selected for the immortalized cell line. The advantage of immortalization is that it allows for indefinite rapid proliferation, providing an unlimited number of cells to perform multiple experiments using the same cell line. This allows for consistency between experiments and reduces the cost.

In addition to changes in the genes that limited cell proliferation, changes in the expression of genes of critical functionality could also occur⁹. Therefore, the disadvantage of using immortalized cells is that they may no longer represent the original in vivo cells in terms of their response to various external stimuli¹⁰. Comparisons have involved observing the toxic effects of chemicals on primary and immortalized human corneal keratocytes¹¹, as well as immortalized HCECs and rabbit corneal epithelial primary cells¹². The comparison between the effects of toxins on primary human keratocytes and immortalized keratocytes showed no significant differences¹¹. Using the methods detailed in this article, the effectiveness of these assays to assess the toxicity of UV radiation and ocular toxins on pHCECs and iHCECs will be determined.

Three ocular toxins commonly used in in vitro assays were selected: BAK, H₂O₂, and SDS. BAK is a cationic preservative commonly used in ophthalmic solutions^13,14, H₂O₂ is commonly used to disinfect contact lenses¹⁵, and SDS is an anionic surfactant found in detergents and shampoos¹⁴. Similar to ocular toxins, UV radiation can also cause significant damage to HCECs³. In addition, overexposure to UV can cause an ocular condition known as photokeratitis characterized by symptoms of tearing, light sensitivity, and a feeling of grittiness¹⁶.

There is an unlimited number of primary cell cultures that can be used and various immortalized cell lines that have been developed. Therefore, an investigation was undertaken to compare primary HCECs to an immortalized HCEC line to determine similarities and differences between models that incorporate these types of cells.

This investigation used microscopy to assess possible differences between pHCECs and iHCECs on cell physiological response to UV and toxins. The effects of UV radiation and chemicals on cell metabolic activity and inflammatory cytokine release for the two cell lines were also evaluated. The importance of determining the differences among the two cell lines is to understand the optimal use of these cell lines for evaluating: 1) the effect of UV radiation on cells, 2) the effects of toxins on cells, and 3) the resulting changes to metabolism, cell viability, and cell cytokine release for future studies.

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 incubate the flask at 37 °C until the cells are fully detached. Neutralize the dissociation solution with DMEM/F12 with serum and then centrifuge the cells at 500 × g. Remove the supernatant medium and resuspend the cells in HOEM medium.

2. Determination of cell size using confocal microscopy

1. Seed both pHCECs and iHCECs onto collagen-coated Petri dishes with glass-bottom coverslips at a concentration of 1 × 10⁵ with 1 mL of HOEM. Grow pHCECs and iHCECs, one set of cultures for 24 hours and another set of cultures for 48 h, in a 37 °C incubator with 5% CO₂.
2. After the incubation period, stain the cells with 500 µL of annexin staining buffer solution containing calcein (4 µM), ethidium homodimer-1 (8 µM), and annexin V (5 µL in 500 µL buffer-Alexa Fluor 647 conjugate) for 20 min at 37 °C. After staining the cells, adjust the microscope for capturing the excitation/emission wavelengths of 488/515 nm for calcein-AM, 543/600 nm for ethidium homodimer-1, and 633/665 nm for annexin V using a confocal laser scanning microscope.
3. Obtain the images and take Z-stacks to assess cell size in three dimensions.
   1. To acquire the two-dimensional (2D) and three-dimensional (3D) images, find the area to image with an apochromat 40x/1.2 water objective. With the Argon (488) (first scan), HeNe1 (543) (second scan, and HeNe2 (633) (third scan) lasers, scan in three separate channels by using the beam splitters, HFT 488/543/633, NFT 635 Vis, and NFT 545 LP560, LP505.
   2. Use multi-track sequential scanning to reduce crosstalk.
      NOTE: LP505 is used for channel 2 with the 488 laser to capture the emission of the calcein dye. LP560 is used in channel 3 with the 543 laser to capture the emission of the ethidium homodimer 1. NFT 635 is used with the 633 laser to capture the emission of the annexin V. The purpose of the HFT is to separate the excitation and emission light. NFT splits light by reflecting light < specified wavelength to a separate channel while letting light > a specified wavelength through to a second channel. LP filters block shorter wavelengths than the specified wavelength and let the longer wavelengths through.
   3. To image in 3D, set the confocal to Z-stack and scan at least 20 frames.

3. Exposure of cells to UV radiation

1. Seed the cells at 5 × 10⁴/mL of HOEM in each well of a 24-well collagen-1 coated culture plate and incubate at 37 °C with 5% CO₂ for 3 h. After the incubation period, reduce the volume of the medium in the wells to 300 µL. Next, expose the cells to UV radiation (both UVA at 6.48 W/m² and UVB at 1.82 W/m² as both UVA and UVB tubes are turned on in the incubator at the same time) in a 37 °C incubator for 5 and 20 min in separate experiments.
2. After the UV radiation, add 200 µL of fresh HOEM to each well and incubate for 20 h at 37 °C with 5% CO₂.
3. After incubation, collect the cell supernatant in each well and transfer it into sterile 2 mL polypropylene tubes. Freeze at -80 °C. To determine the cytokine levels released by the pHCECs and iHCECs after UV exposure, use a multiplex cytokine assay and follow the kit's instructions to quantify the following four cytokines: IL-6, IL-8, IL-1β, and TNF-α.
4. Add metabolic assay reagent to the cells in the wells.
5. Prepare 10% metabolic assay reagent in DMEM/F12. Replace the culture medium in each well with 1 mL of the 10% metabolic assay solution and incubate at 37 °C with 5% CO₂ for 4 h.
6. Measure the fluorescence of each solution using a fluorescent plate reader at 530/590 nm excitation/emission wavelengths.

4. Exposure of cells to chemical toxins

1. Seed cells at 5 × 10⁴/mL of HOEM in each well of a 24-well collagen-1 coated culture plate and incubate at 37 °C with 5% CO₂ for 3 h. After the incubation period, remove the medium and expose the cells to 1 mL of chemical toxins (BAK 0.001%, H₂O₂ 0.01%, and SDS 0.0025% in phosphate-buffered saline (PBS)) for 5 and 15 min.
2. After exposure to the chemical toxins, remove the toxins from the wells, rinse with 1 mL of PBS, and add 1 mL of HOEM to each well. After 20 h of incubation, perform a metabolic assay by replacing the medium with 1 mL of a 10% metabolic assay solution and incubating at 37 °C with 5% CO₂ for 4 h. Measure the fluorescence using a fluorescence multi-well plate reader at 530/590 nm excitation/emission wavelengths.
3. Transfer the cell supernatants from the wells following the 20 h incubation into separate sterile 2 mL polypropylene tubes and freeze at -80 °C. Quantify cytokines released by the pHCECs and iHCECs after exposure to the chemicals. Use the same multiplex platform used to assess the cytokines from the UV-treated cells. Use a multiplex cytokine assay following the kit's instructions to quantify the following four cytokines: IL-6, IL-8, IL-1β, and TNF-α.

5. Examination of cells exposed to various concentrations of BAK

1. Seed cells at 1 × 10⁵/mL of HOEM in each well of a 24-well collagen-1 coated culture plate and incubate at 37 °C with 5% CO₂ for 24 h. After the incubation period, remove the medium and expose the cells to 1 mL of chemical toxins (BAK 0.001%, BAK 0.005%, and BAK 0.01% in PBS) for 5 min.
2. After exposure, remove the chemical toxins, rinse the wells with 1 mL of PBS, and add 1 mL of HOEM to each well. After 20 h of incubation, stain the cells with 500 µL of annexin staining buffer solution containing calcein (4 µM), ethidium homodimer-1 (8 µM), and annexin V (5 µL in 500 µL buffer-Alexa Fluor 647 conjugate) for 20 min at 37 °C. Adjust the microscope to measure intensities of annexin V, calcein AM, and ethidium-1 staining at excitation/emission wavelengths of 630/675 nm, 495/515 nm, and 528/617 nm, respectively. Image the cells using the fluorescence microscope

6. Data analysis

1. Carry out a normality test and a test for equal variances before performing an analysis of variance (ANOVA), Welch ANOVA, or Kruskal-Wallis test. Use the appropriate post-hoc test for comparing each group tested. Set p < 0.05.

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

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