The authors received no funding for this work.

1. Culture of pHCECs and iHCECs
<ol>
	<li>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 &#181;M epinephrine, 0.4% cell media supplement P (Table of Materials), 5 &#181;g/mL rh insulin, 5 &#181;g/mL apo-transferrin, and 100 ng/mL hydrocortisone hemisuccinate in collagen-1 coated culture flasks (18 mL in a 75 cm2 culture flask).</li>
	<li>Change the ...

<ol>
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J., Sivak, J. G., Jones, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+ultraviolet-induced+damage+in+human+corneal,+lens,+and+retinal+pigment+epithelial+cells.">In vitro ultraviolet-induced damage in human corneal, lens, and retinal pigment epithelial cells.</a> Molecular Vision. 17, 237-246 (2011).</li><li>Hakkarainen, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+cytotoxic+effects+of+marketed+ophthalmic+formulations+on+human+corneal+epithelial+cells.">Acute cytotoxic effects of marketed ophthalmic formulations on human corneal epithelial cells.</a> International Journal of Pharmaceutics. 511 (1), 73-78 (2016).</li><li>Spurr, S. J., Gipson, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+corneal+epithelium+with+Dispase+II+or+EDTA.+Effects+on+the+basement+membrane+zone.">Isolation of corneal epithelium with Dispase II or EDTA. Effects on the basement membrane zone.</a> Investigative Ophthalmology &amp; Visual Science. 26 (6), 818-827 (1985).</li><li>Kahn, C. R., Young, E., Lee, I. H., Rhim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+corneal+epithelial+primary+cultures+and+cell+lines+with+extended+life+span:+in+vitro+model+for+ocular+studies.">Human corneal epithelial primary cultures and cell lines with extended life span: in vitro model for ocular studies.</a> Investigative Ophthalmology &amp; Visual Science. 34 (12), 3429-3441 (1993).</li><li>Araki-Sasaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+SV40-immortalized+human+corneal+epithelial+cell+line+and+its+characterization.">An SV40-immortalized human corneal epithelial cell line and its characterization.</a> Investigative Ophthalmology &amp; Visual Science. 36 (3), 614-621 (1995).</li><li>Griffith, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+human+corneal+equivalents+constructed+from+cell+lines.">Functional human corneal equivalents constructed from cell lines.</a> Science. 286 (5447), 2169-2172 (1999).</li><li>Toouli, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+mammary+epithelial+cells+immortalized+by+simian+virus+40+T-Antigen+or+by+the+telomerase+catalytic+subunit.">Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit.</a> Oncogene. 21 (1), 128-139 (2002).</li><li>Kaur, G., Dufour, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines:+Valuable+tools+or+useless+artifacts.">Cell lines: Valuable tools or useless artifacts.</a> Spermatogenesis. 2 (1), 1-5 (2012).</li><li>Zorn-Kruppa, M., Tykhonova, S., Belge, G., Diehl, H. A., Engelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+corneal+cell+cultures+in+cytotoxicity+testing.">Comparison of human corneal cell cultures in cytotoxicity testing.</a> Altex. 21 (3), 129-134 (2004).</li><li>Huhtala, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+an+immortalized+human+corneal+epithelial+cell+line+and+rabbit+corneal+epithelial+cell+culture+in+cytotoxicity+testing.">Comparison of an immortalized human corneal epithelial cell line and rabbit corneal epithelial cell culture in cytotoxicity testing.</a> Journal of Ocular Pharmacology and Therapeutics. 18 (2), 163-175 (2002).</li><li>Pisella, P. J., Fillacier, K., Elena, P. P., Debbasch, C., Baudouin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+effects+of+preserved+and+unpreserved+formulations+of+timolol+on+the+ocular+surface+of+albino+rabbits.">Comparison of the effects of preserved and unpreserved formulations of timolol on the ocular surface of albino rabbits.</a> Ophthalmic Research. 32 (1), 3-8 (2000).</li><li>Youn, H. Y., Moran, K. L., Oriowo, O. M., Bols, N. C., Sivak, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+and+UV-B-induced+damage+of+the+cultured+bovine+lens.">Surfactant and UV-B-induced damage of the cultured bovine lens.</a> Toxicology in vitro. 18 (6), 841-852 (2004).</li><li>Hughes, R., Kilvington, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+hydrogen+peroxide+contact+lens+disinfection+systems+and+solutions+against+Acanthamoeba+polyphaga.">Comparison of hydrogen peroxide contact lens disinfection systems and solutions against Acanthamoeba polyphaga.</a> Antimicrobial Agents and Chemotherapy. 45 (7), 2038-2043 (2001).</li><li>Delic, N. C., Lyons, J. G., Di Girolamo, N., Halliday, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damaging+effects+of+ultraviolet+radiation+on+the+cornea.">Damaging effects of ultraviolet radiation on the cornea.</a> Photochemistry and Photobiology. 93 (4), 920-929 (2017).</li><li>Ahuja, D., Saenz-Robles, M. T., Pipas, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SV40+large+T+antigen+targets+multiple+cellular+pathways+to+elicit+cellular+transformation.">SV40 large T antigen targets multiple cellular pathways to elicit cellular transformation.</a> Oncogene. 24 (52), 7729-7745 (2005).</li><li>Tong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pin1+inhibits+PP2A-mediated+Rb+dephosphorylation+in+regulation+of+cell+cycle+and+S-phase+DNA+damage.">Pin1 inhibits PP2A-mediated Rb dephosphorylation in regulation of cell cycle and S-phase DNA damage.</a> Cell Death &amp; Disease. 6, 1640 (2015).</li><li>Muller, P. A., Vousden, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+mutations+in+cancer.">p53 mutations in cancer.</a> Nature Cell Biology. 15 (1), 2-8 (2013).</li><li>Seshacharyulu, P., Pandey, P., Datta, K., Batra, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatase:+PP2A+structural+importance,+regulation+and+its+aberrant+expression+in+cancer.">Phosphatase: PP2A structural importance, regulation and its aberrant expression in cancer.</a> Cancer Letters. 335 (1), 9-18 (2013).</li><li>Yang, D., Okamura, H., Morimoto, H., Teramachi, J., Haneji, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+phosphatase+2A+Calpha+regulates+proliferation,+migration,+and+metastasis+of+osteosarcoma+cells.">Protein phosphatase 2A Calpha regulates proliferation, migration, and metastasis of osteosarcoma cells.</a> Laboratory nvestigation; A Journal of Technical Methods and Pathology. 96 (10), 1050-1062 (2016).</li><li>Xie, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+and+inactivation+of+the+PP2A+complex+promotes+the+proliferation+and+angiogenesis+of+hemangioma+endothelial+cells+through+activating+AKT+and+ERK.">Disruption and inactivation of the PP2A complex promotes the proliferation and angiogenesis of hemangioma endothelial cells through activating AKT and ERK.</a> Oncotarget. 6 (28), 25660-25676 (2015).</li><li>Mallet, J. D., Rochette, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wavelength-dependent+ultraviolet+induction+of+cyclobutane+pyrimidine+dimers+in+the+human+cornea.">Wavelength-dependent ultraviolet induction of cyclobutane pyrimidine dimers in the human cornea.</a> Photochemical &amp; Photobiological Sciences. 12 (8), 1310-1318 (2013).</li><li>Cejkova, J., Cejka, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+oxidative+stress+in+corneal+diseases+and+injuries.">The role of oxidative stress in corneal diseases and injuries.</a> Histology and Histopathology. 30 (8), 893-900 (2015).</li><li>Wang, S. Q., Balagula, Y., Osterwalder, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoprotection:+a+review+of+the+current+and+future+technologies.">Photoprotection: a review of the current and future technologies.</a> Dermatologic Therapy. 23 (1), 31-47 (2010).</li><li>Svobodova, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+after+acute+exposure+of+mice+skin+to+physiological+doses+of+UVB+and+UVA+light.">DNA damage after acute exposure of mice skin to physiological doses of UVB and UVA light.</a> Archives of Dermatological Research. 304 (5), 407-412 (2012).</li><li>Kennedy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraviolet+irradiation+induces+the+production+of+multiple+cytokines+by+human+corneal+cells.">Ultraviolet irradiation induces the production of multiple cytokines by human corneal cells.</a> Investigative Ophthalmology &amp; Visual Science. 38 (12), 2483-2491 (1997).</li><li>Kode, A., Yang, S. R., Rahman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+cigarette+smoke+on+oxidative+stress+and+proinflammatory+cytokine+release+in+primary+human+airway+epithelial+cells+and+in+a+variety+of+transformed+alveolar+epithelial+cells.">Differential effects of cigarette smoke on oxidative stress and proinflammatory cytokine release in primary human airway epithelial cells and in a variety of transformed alveolar epithelial cells.</a> Respiratory Research. 7, 132 (2006).</li><li>Epstein, S. P., Chen, D., Asbell, P. 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The author, Lyndon W. Jones, over the past 3 years, through CORE, has received research support or lectureship honoraria from the following companies: Alcon, Allergan, Allied Innovations, Aurinia Pharma, BHVI, CooperVision, GL Chemtec,i-Med Pharma, J&amp;J Vision, Lubris, Menicon, Nature's Way, Novartis, Ophtecs, Ote Pharma, PS Therapy, Santen, Shire, SightGlass SightSage, and Visioneering. Lyndon Jones is also a consultant and/or serves on an advisory board for Alcon, CooperVision, J&amp;J Vision, Novartis, and Ophtecs. The other authors have nothing to disclose.

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%), ...

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 effects1,2,3,4. These models typically evaluate physiological effects such as changes to the cell&#39;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 HCECs2,3. 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 cells6,7,8. 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 solutions13,14, 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>75 cm2 Vented Flask</td>
			<td>Corning</td>
			<td>354485</td>
			<td>This is the BioCoat brand, collagen-coated</td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>costar</td>
			<td>3370</td>
			<td></td>
		</tr>
		<tr>
			<td>alamarBlue</td>
			<td>Fisher Scientific</td>
			<td>dal 1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin Staining buffer solution</td>
			<td>Invitrogen, Burlington, ON, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin V</td>
			<td>Invitrogen, Burlington, ON, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 100 microscope with a Zeiss confocal laser scanning microscope 510 system</td>
			<td>Carl Zeiss Inc., Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 48 Well plates</td>
			<td>Corning</td>
			<td>354505</td>
			<td>This is the BioCoat brand, collagen-coated</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>BioTek</td>
			<td>CYT5MPV</td>
			<td>Can read fluorescence from 280 - 700 nm (for assay 540/590)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hycone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>glass bottom coverslips</td>
			<td>MatTek Corporation, Ashland, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human Corneal Epithelial Cells</td>
			<td>University of Ottawa</td>
			<td>N/A</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Human Ocular Epithelia Media (HOEM) with the following supplements:</td>
			<td>Millipore, Billerica, MA, USA</td>
			<td>SCMC001</td>
			<td>Epigro base media supplemented with 6 mM L-Glutamine, 0.002% EpiFactor O (cell media supplement O in article) , 1.0 &#956;M Epinephrine, 0.4% EpiFactor P (cell media supplement P in article), 5 &#956;g/mL rh Insulin, 5 &#956;g/mL Apo- Transferrin, and 100 ng/mL Hydrocortisone Hemisuccinate in Collagen-1 coated culture flasks (BioCoat, Corning, Tewksbury, MA, USA).</td>
		</tr>
		<tr>
			<td>Live Dead calcien and ethidium homodimer</td>
			<td>Invitrogen, Burlington, ON, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MesoScale Discovery (MSD) QuickPlex SQ 120 instrument</td>
			<td>Rockville, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MSD Human Proinflammatory Panel II (4-Plex) V-Plex assay</td>
			<td>Rockville, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100x concentration so add 1 mL to each 99 mL of media</td>
		</tr>
		<tr>
			<td>Primary Corneal Epithelial Cells</td>
			<td>Millipore, Billerica, MA, USA</td>
			<td>SCCE016</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax fluorescence multi-well plate reader</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express (cell disassociation solution)</td>
			<td>Fisher Scientific</td>
			<td>12605036</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining the toxicity of uv radiation and chemicals on primary and immortalized human corneal epithelial cells

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)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) 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, H2O2, 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.

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...

Watch this Scientific Journal Video about Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells at JoVE.com

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

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

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

This method can be used to evaluate in vitro the toxicity of new ophthalmic product formulations. By refining the concentrations of chemicals and product formulations using ocular cells in vitro, the use of animals for assessing new product safety can be minimized. In vitro assays evaluate toxicity endpoints that cannot be assessed in vivo, such as determining the effect of a chemical on mitochondrial function, cell membrane integrity, and enzyme activity.Evaluate cell toxicity in vitro is key to understanding potential mechanisms of toxicity. This is essential for establishing a maximum tolerable dose of therapeutic chemicals in the eye. Demonstrating the procedure will be Nijani Nagaarudkumaran, a researcher from the Jones Laboratory.Begin by seating both pHCECs and iHCECs onto collagen coated Petri dishes with glass bottom cover slips at a concentration of 1 x 10 to the 5th with 1 milliliter of human ocular epithelium medium, or HOEM. Grow pHCECs and iHCECs 1 set of cultures for 24 hours and another set for 48 hours in a 37-degree-Celsius incubator with 5%carbon dioxide. After incubation, stain the cells with 500 microliters of annexin staining buffer solution containing 4 micromolar calcein, 8 micromolar ethidium homodimer 1, and annexin 5 for 20 minutes at 37 degrees Celsius.After staining the cells, adjust the confocal laser scanning microscope to capture the excitation and emission wavelengths, as described in the text manuscript. Obtain the two-dimensional and three-dimensional images following the instructions in the manuscript. Seed the cells at 5 x 10 to the 4th per milliliter of HOEM in each well of a 24-well collagen 1 coated culture plate and incubate at 37 degrees Celsius with 5%carbon dioxide for 3 hours.After incubation, reduce the volume of the medium in the wells to 300 microliters. Next, expose the cells to UVA radiation at 6.48 watts per square meter, and UVB radiation at 1.82 watts per square meter at 37 degrees Celsius for 5 and 20 minutes in separate experiments. After UV radiation, add 200 microliters of fresh HOEM to each well and incubate for 20 hours at 37 degrees Celsius with 5%carbon dioxide.The next day, collect the cell supernatant in each well and transfer it into sterile 2-milliliter polypropylene tubes. Freeze the supernatant at minus 80 degrees Celsius. 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 interleukin 6, interleukin 8, interleukin-1 beta, and TNF-alpha.Prepare 10%metabolic assay reagent in DMEM and F12. Replace the culture medium in each well with 1 milliliter of the 10%metabolic assay solution and incubate at 37 degrees Celsius with 5%carbon dioxide for 4 hours. Measure the fluorescence of each solution using a fluorescent plate reader at 530 nanometers excitation and 590 nanometers emission wavelengths.Seed cells at 5 x 10 to the fourth per milliliter of HOEM in each well of a 24-well collagen 1 coated culture plate. Incubate at 37 degrees Celsius with 5%carbon dioxide for 3 hours. After incubation, remove the medium and expose the cells to 1 milliliter of chemical toxins, which include 0.001%benzalkonium chloride, or BAK, 0.01%hydrogen peroxide, and 0.0025%sodium dodecyl sulfate in PBS for 5 and 15 minutes.After exposure to the chemical toxins, remove the toxins from the wells, rinse with 1 milliliter of PBS, and add 1 milliliter of HOEM to each well. After 20 hours of incubation, perform a metabolic assay by replacing the medium with 1 milliliter of a 10%metabolic assay solution and incubating at 37 degrees Celsius with 5%carbon dioxide for 4 hours. Measure the fluorescence of each solution using a fluorescent plate reader at 530 nanometers excitation, and 590 nanometers emission wavelengths.Transfer the cell supernatants from the wells following the 20-hour incubation into separate sterile 2-milliliter polypropylene tubes and freeze them at minus 80 degrees Celsius. Use the same multiplex platform used to assess the cytokines from the UV-treated cells following the kit's instructions to quantify interleukin 6, interleukin 8, interleukin-1 beta and TNF-alpha. Seed cells at 1 x 10 to the 5th per milliliter of HOEM in each well of a 24-well collagen 1 coated culture plate and incubate at 37 degrees Celsius with 5%carbon dioxide for 24 hours.After incubation, remove the medium and expose the cells to 1 milliliter of chemical toxins, including 0.001%BAK, 0.005%BAK, and 0.01%BAK in PBS for 5 minutes. After exposure, remove the chemical toxins, rinse the wells with 1 milliliter of PBS and add 1 milliliter of HOEM to each well. After 20 hours of incubation, stain the cells with 500 microliters of annexin staining buffer solution containing calcein, ethidium homodimer, and annexin 5 for 20 minutes at 37 degrees Celsius.Adjust the microscope to measure intensities at excitation and emission wavelengths of 630 and 675 nanometers for annexin 5, 495 and 515 nanometers for calcein AM, and 528 and 617 nanometers for ethidium 1 staining. Then acquire images of the cells using the fluorescence microscope. The iHCECs were found to be small cells that range from 10 to 20 micrometers and the pHCECs were in the range of 20 to 50 micrometers after 24 hours of growth.Similar differences in size range were observed for iHCECs and pHCECs after 48 hours of growth. Compared to the non-UV exposed cells, the metabolic activity of irradiated pHCECs was significantly reduced at 20 minutes of exposure. For iHCECs, the metabolic activity decreased for cells irradiated at both 5 and 20 minutes.Therefore, it took a longer exposure time to reduce the metabolic activity of the pHCECs than the iHCECs. The maximum cytokine release by the iHCECs was at 5 minutes of UV exposure. The maximum cytokine release for pHCECs occurred at 20 minutes of UV exposure.In terms of total amounts of inflammatory cytokines released the pHCECs released substantially more interleukin-1 beta, interleukin 8, and TNF-alpha than the iHCECs, whereas the iHCECs released more interleukin 6. Both pHCECs and iHCECs showed a change in the release of interleukin 6 after exposure to all three chemicals. BAK caused a decrease in the release of interleukin 6 compared to the control for both primary and immortalized HCECs.Hydrogen peroxide caused an increase in the release of interleukin 6 from iHCECs, and a decrease in the release from pHCECs. Both pHCECs and iHCECs were significantly damaged at 0.005%and 0.1%of BAK. After performing in vitro assessments of ocular cell toxicity, further animal models of ocular toxicity could be undertaken to determine the effect of an ocular product on corneal nerves or the ocular immune system.These techniques are essential for determining the mechanisms of chemical and product formulation toxicity on the eye and will help minimize animal use for product development testing.

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Research

JoVE Journal

Biology

1.9K ビュー.  University of Waterloo. この方法は、新しい眼科用製品製剤の毒性をin vitroで評価するために使用できます。in vitroで眼細胞を使用して化学物質や製品製剤の濃度を精製することにより、新製品の安全性を評価するための動物の使用を最小限に抑えることができます。インビトロアッセイは、ミトコンドリア機能、細胞膜の完全性、酵素活性に対する化学物質の影響を決定するなど、in vivoでは評...