Culturing the Largest Organ in a Dish: Skin Models for Toxicity and Efficacy Testing Speaker: Patrick Hayden March 10, 2018 at 2:00 PM Convention Center—Stars at Night Ballroom Foyer
Posters:
Use of In Vitro Human Intestinal Microtissues as Relevant Models for Drug Permeation and Metabolism in the Gut (Poster #7) Presenting Author: Patrick Hayden March 10, 2018 from 12:00 Noon to 2:00 PM Convention Center—Stars at Night Ballroom Foyer
The Role of Oxidative Stress in Dry Eye Disease: Investigations Using an In Vitro Organotypic Human Corneal Tissue Model (EpiCorneal) (Poster #8) Presenting Author: Patrick Hayden March 10, 2018 from 12:00 Noon to 2:00 PM Convention Center—Stars at Night Ballroom Foyer
Pre-Validation of an Acute Inhalation Toxicity Assay Using the EpiAirway In Vitro Human Airway Model (Poster #9) Presenting Author: Patrick Hayden March 10, 2018 from 12:00 Noon to 2:00 PM Convention Center—Stars at Night Ballroom Foyer
Platform Session: Autoimmunity/Hypersensitivity/Inflammation Wednesday, March 14 • 8:00 AM to 10:45 AM • CC Room 304
In Vitro 3D-Human Small Intestinal Tissue to Study Ligand-Induced Acute and Chronic Inflammation in the Gastrointestinal Tract
M. Klausner1, T. Landry1, Z. Stevens1, J. Markus2, P. Hayden1, and S. Ayehunie1. 1MatTek Corporation, Ashland, MA; and 2MatTek In Vitro Life Science Laboratory, Bratislava, Slovakia. 9:55 AM-10:10 AM • Abstract Number 2649
Intestinal epithelium is known to be involved in innate immune responses by recognizing potential pathogens through cellular pattern recognition receptors (PRRs). Abnormal innate immune responses have been implicated in the pathogenesis of inflammatory bowel diseases (IBD). To investigate PRR responses on the intestinal mucosa, we exposed an in vitro 3D human small intestinal (SMI) microtissue (EpiIntestinal, SMI-100-FT) model to various Toll-like receptor (TLRs) and Node-like receptor (NOD) ligands. The SMI microtissues are cultured using human intestinal fibroblasts and enterocytes and their 3-dimensional polarity and morphology mimics that of native in vivo tissues. Characterization of the microtissues included evaluation of structural features, barrier properties, and expression of drug transporters and drug metabolizing enzymes. Interestingly, ligands to TLR4 (LPS) and NOD2 (Muramyl dipeptide; MDP) were able to induce gene expression of proinflammatory cytokines such as IL-1β, IL-6, and RANTES in a synergistic manner. Prolonged exposure of EpiIntestinal tissue to IL-1β also resulted in reduced tissue membrane integrity, which may be a precursor for IBD-like disease, and led to further induction of pro-inflammatory cytokines and chemokine gene expression (IL-6 and CCL20), which are known to stimulate acquired immune cell responses including release of TNF-α and IFN-Ƴ. To simulate the effect of immune cell responses on the intestinal epithelium, we also exposed the EpiIntestinal tissue to TNF-α and IFN-, which resulted in the reduction of membrane integrity and the release of proinflammatory cytokines. The effect of TNF-α and IFN- on the intestinal epithelium was further exacerbated if antigen-presenting cells such as dendritic cells were incorporated into the 3D intestinal tissue model. In summary, our results suggest that the EpiIntestinal tissue is capable of modeling innate immune responses and can be a useful tool to study the complex interactions of human intestinal epithelium with microbiome in vitro in the induction IBD-like disease.
View our posters:
Poster Session: Safety Assessment: Drug Discovery Monday, March 12 • 9:15 AM to 4:30 PM • CC Exhibit Hall
Permeability Screening of Drugs Using a Reproducible In Vitro 3D-Human Small Intestinal Tissue Model
S. Ayehunie1, T. Landry1, Z. Stevens1, J. Markus2, M. Klausner1, and P. Hayden1. 1MatTek Corporation, Ashland, MA; and 2MatTek In Vitro Life Science Laboratory, Bratislava, Slovakia. Poster Board Number: P784 • Abstract Number: 1558 Displayed: 9:15 AM–4:30 PM • Author Attended: 1:30 PM–3:00 PM (Block C)
Availability of human primary cell-based 3D small intestinal (SMI) microtissues that recapitulate structural and functional mimicry with the in vivo counterpart is critical to predict the safety and bioavailability of compounds intended for oral administration. Here we describe reproducibility and drug permeability of a newly developed in vitro 3D-human small intestinal (SMI) microtissue model. The SMI microtissues are cultured using primary human intestinal fibroblasts and enterocytes and their 3-dimensional polarity and morphology mimics that of native in vivo tissues. Characterization of the microtissues included evaluation of structural features, barrier properties, and expression of drug transporters and drug metabolizing enzymes. The quality and reproducibility of tissue production was compared in two independent production facilities (MatTek, Ashland, MA, USA and IVLSL, Bratislava, Slovakia by measuring TEER and Lucifer Yellow (LY) leakage. To evaluate the suitability of the microtissues for drug absorption, the apparent permeability coefficient (Papp) values for a panel of 18 benchmark drugs with known human absorption values were measured. Drug-drug interactions were examined using drugs known to be substrates or inhibitors of efflux transporters. Results showed that tissues are highly reproducible with physiological TEER values averaging 146.4±20.8 W*cm2 (% CV=14.2%) and 142.1±13.8 W*cm2 (CV=9.7%) in the USA (N=128 lots) and Slovakia (N=9 lots) facilities. The microtissues also expressed drug transporters and metabolizing enzymes known to be present in vivo. Drug permeation results showed that the intestinal microtissues could discriminate between low and high permeability drugs with 94% accuracy. The in vitro Papp values also correlated well with human absorption data (r2 = 0.85). SMI microtissues exposed to drug efflux transporter substrates resulted in drug efflux ratios of > 2.0, indicating active efflux transport involvement. Simultaneous treatment of microtissues with efflux transporter substrates and inhibitors reduced the drug efflux ratio while increasing the bioavailability of the test drug, providing further evidence of drug efflux transporter activity. In conclusion, the SMI microtissues appear to be a useful pre-clinical tool for predicting drug safety and bioavailability of orally administered drugs.
Poster Session: Epigenetics Tuesday, March 13 • 9:15 AM to 4:30 PM • CC Exhibit Hall
Characterization of Whole Smoke and Smokeless Tobacco Exposure to a 3D Human Oral Tissue Model
B. Keyser1, M. Bachelor2, A. Maione2, and W. Fields1. 1RAI Services Company, Winston-Salem, NC; and 2MatTek Inc., Ashland, MA. Poster Board Number: P260 • Abstract Number: 1927 Displayed: 9:15 AM–4:30 PM • Author Attended: 3:00 PM–4:30 PM (Block D)
Oral disease is frequently associated with viral and environmental exposures as well as oral hygiene. The use of tobacco is an additional risk factor in the development of oral disease. The goals of this study were to evaluate cytotoxicity, inflammatory response and oxidative stress in response to extracts of CORESTA smokeless tobacco reference products (CRPs) exposed to a 3D human oral buccal model, EpiOral™, as well as to determine the potential cytotoxicity of whole smoke in this model.
CRPs for snus (CRP1), moist snuff (CRP2) and dry snuff (CPR3) were each extracted in complete artificial saliva (CAS) with a ratio of 300 mg of CRP to 1 mL of CAS. CRP extracts were sterile-filtered and stored at –80°C until the time of exposure. CRP extracts (15 – 300 mg/ml) were applied to the apical side of EpiOral™ tissues for 24 or 48 hours continuously. Cytotoxicity (3-(4,5-d-2-yl)-2,5-diphenyltetrazolium bromide; MTT), oxidative stress (8-isoprostane) and inflammatory response (cytokine release; IP10, IL-1α, IL-8) were measured after each time point. Whole smoke exposures were conducted by exposing the EpiOral™ tissues to either 3R4F whole smoke (Health Canada Intense smoking regimen, 0.5 L/min dilution airflow, 20 mL/min vacuum) generated by a VITROCELL® VC1® smoking robot or concurrent clean air control up to 2.5 hours and cytotoxicity was measured 24 hours post exposure.
Exposure of whole smoke to EpiOral tissues induced a dose-dependent response in cytotoxicity (IC50 of 9 cigarettes/54 minutes of exposure), in contrast with < 15% cytotoxicity observed in tissues exposed to CRPs for significantly longer time (24 or 48 hrs). However, while the CRP extracts elicited minimal cytotoxicity, time- and dose-dependent effects on oxidative stress and inflammatory response were observed. The release of 8-isoprostane and each cytokine was greater at 48 hours than 24 hours. In addition, a differential response between each of the CRPs was observed for 8-isoprostane at 48 hours of exposure.
Collectively, the data suggest that the EpiOral™ three-dimensional human cell culture model may be useful in differentiating smokeless tobacco products and evaluating between tobacco product categories.
Poster Session: Emerging Technologies Tuesday, March 13 • 9:15 AM to 4:30 PM • CC Exhibit Hall
Use of a 3D Full-Thickness Skin Equivalent and Mass Spectrometry Imaging for Evaluating Disposition of Topically Applied Drugs
M. Klausner1, M. Bachelor1, A. Armento1, C. Walsh2, E. Seeley2, and H. Goodman2. 1MatTek Corporation, Ashland, MA; and 2Protea Biosciences, Morgantown, WV. Sponsor: P. Hayden. Poster Board Number: P482 • Abstract Number: 2145 Displayed: 9:15 AM–4:30 PM • Author Attended: 3:00 PM–4:30 PM (Block D)
Human skin equivalents are a useful tool for the evaluation of percutaneous permeation and absorption of topical actives. Standard evaluation typically involves determining quantities of active that (1) traverse the skin equivalent into a receiver solution below the tissue over a given period of time (2) remain on the apical surface and (3) are retained within the tissue. Incorporating the use of mass spectrometry imaging (MSI) into this type of study allows for localization of endogenous and exogenous compounds along with their relative concentrations in thin tissue sections. Utilization of MSI technology has the added benefit of identifying metabolites of the parent drug compound, some of which may cause localized or systemic toxicity. In this study, an over the counter (OTC) retinol complex was applied topically to EpiDermFT, a metabolically active, full thickness skin tissue. Retinol, formulation components, and metabolites of the parent compound were localized in the epidermal or dermal layers of the skin equivalent following a single 24 hour treatment. The use of EpiDermFT in combination with MSI technology for the study of percutaneous permeation and absorption studies can be utilized in the development of cosmetic and pharmaceutical-based actives to better understand drug disposition and the associated metabolites that may result following topical application.
Poster Session: Alternatives to Mammalian Models I Tuesday, March 13 • 9:15 AM to 4:30 PM • CC Exhibit Hall
Detecting GI Toxicity Earlier Than in Dog: Developing an In Vitro Assay to Predict Clinical Diarrhea
M. Peters1, H. Barthlow1, C. Scott1, A. Armento2, and S. Ayehunie2. 1AstraZeneca, Waltham, MA; and 2MatTek, Ashland, MA. Poster Board Number: P711 • Abstract Number: 2357 Displayed: 9:15 AM–4:30 PM • Author Attended: 1:30 PM–3:00 PM (Block C)
Comparison of In Vitro Rat and Human Mucociliary Airway Models for Inhalation Toxicity Testing
P.J. Hayden1, G.R. Jackson1, A. Maione1, C. Roper2, and M. Klausner1. 1MatTek Corporation, Ashland, MA; and 2Charles River Laboratories, Edinburgh, United Kingdom. Poster Board Number: P720 • Abstract Number: 2366 Displayed: 9:15 AM–4:30 PM • Author Attended: 1:30 PM–3:00 PM (Block C)
Accurate assessment of inhalation toxicity potential is important for developing new inhalable therapeutics, evaluating environmental air pollutants, and for establishing safe handling, labeling, and emergency response procedures for chemicals. Traditionally, animal models have been used for assessing inhalation toxicity. However, there is growing awareness that in vivo animal models may not always accurately predict human outcomes. Therefore, development of in vitro organotypic airway tissue models constructed from primary human cells has become a high priority in the scientific community. Yet, validation of these systems for predicting in vivo human response is problematic because the available in vivo inhalation toxicity data has been primarily produced using rodents.
To address this issue, we have developed a scalable in vitro organotypic model of rat mucociliary airway epithelium. This model will allow for the direct comparison of chemical toxicity responses with previously developed in vitro human airway models. Conducting airways epithelial cells were isolated from 8-week old, male, CD rats (Charles River) and expanded in culture. The cells were seeded onto microporous membrane inserts and cultured at the air-liquid interface for up to 27 days. H&E stained histological evaluation demonstrated a high level of organotypic mucociliary differentiation including a pseudostratified epithelial structure with copious development of functional cilia. Robust barrier function was demonstrated by development of transepithelial electrical resistance of up to 800 Ω x cm2. A comparison of the toxicity responses observed in the rat vs. human airway models was conducted using 14 chemicals with in vivo GHS inhalation toxicity classifications ranging from categories 1-5. The airway tissue models were exposed to 4 doses of each test chemicals for 3 hrs, followed by measurement of tissue viability. IC75 concentrations were determined from the dose-response data. Results show that rat and human tissue responses were strikingly similar for each chemical. Future work will expand the range of chemicals tested to further define the comparative responses. These results suggest that in vitro rat airway models will be a useful tool to facilitate rodent to human translation of in vitro inhalation toxicology data, and ultimately a more complete transition to human-based in vitro models of inhalation toxicity.
Development of a Cell-Based Approach to Assess the Pulmonary Fibrosis of Nanomaterials
G.M. Hilton1, B. RothenRutishauser2, H. Barosova2, S. Chortarea2, F. Zerimariam2, M.J. Clift3, V. Stone4, P. Hayden5, A. Maione5, M. Sharma1, and A.J. Clippinger1. 1PETA International Science Consortium Ltd, London, United Kingdom; 2Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland; 3In Vitro Toxicology Group, Swansea University Medical School, Swansea, United Kingdom; 4HeriotWatt University, Edinburgh, United Kingdom; and 5MatTek Corporation, Ashland, MA. Poster Board Number: P723 • Abstract Number: 2369 Displayed: 9:15 AM–4:30 PM • Author Attended: 1:30 PM–3:00 PM (Block C)
Inhalation exposure to multi-walled carbon nanotubes (MWCNTs) has been linked to adverse health effects in vivo, such as pulmonary fibrosis. The monetary, ethical, and scientific concerns associated with the 90-day rodent inhalation test, required to assess the substances of concern under many jurisdictions, warrants development and use of non-animal approaches, such as predictive human cell cultures. Additionally, thorough characterization of the test material is critical throughout the use of in vitro assays to understand concentration- or form-dependent effects. Presented here are results from including alveolar epithelial cells (A549), fibroblasts (MRC-5), and macrophages (THP-1). Cells were ALI; The MWCNTs were characterized using a combination of qualitative and quantitative methods ultraviolet-visible-near infrared spectroscopy, bright field and enhanced dark field microscopy, and quartz crystal microbalance. Cytotoxicity was not observed in mono-cultures, but tumor necrosis factor-alpha, and 1 beta) a epithelial cells, pulmonary endothelial cells, fibroblasts, and THP-1 cell lineMatTek Corp)The goal of this work is to develop an in vitro testing strategy using human-relevant methods to predict pulmonary toxicity and to enable effective risk assessment of substances including MWCNTs.
Detection of Reactive Chemicals and Oxidants Using an Organotypic Human Airway Model with Nrf2 Reporter Activity: Application to Evaluation of Tobacco Products
A. Maione, G.R.Jackson, O. O’Connell, J. Foisy, M. Klausner, and P.J.Hayden. MatTek Corporation, Ashland, MA. Poster Board Number: P726 • Abstract Number: 2372 Displayed: 9:15 AM–4:30 PM • Author Attended: 1:30 PM–3:00 PM (Block C)
The Nrf2 transcription factor controls expression of enzymes involved in defense against electrophilic and oxidative damage. In the current work, an organotypic model of human airway epithelium containing a luciferase reporter for Nrf2 activation was developed. The reporter model was characterized with 12 reference chemicals, whole tobacco smoke (WTS) and electronic cigarette vapor (ecig). Primary human tracheobronchial epithelial cells (NHBE) from 2 donors were tranduced with a lentiviral vector Nrf2 luciferase reporter. Stably transduced cells were expanded and cyropreserved to produce large pools of reporter-expressing cells which were then utilized to produce differentiated organotypic airway epithelial models. Organotypic structure and barrier properties of reporter models were found to be similar to untransduced models, as determined by histological evaluation and barrier assessment. The airway reporter models were exposed to test chemicals by apical application of solutions containing up to 0.6% DMSO. A smoking machine exposed the models to WTS or ecig vapor. Luciferase activity was evaluated using a commercial kit and a microplate luminometer. Toxicity was evaluated by LDH release. Dose response experiments were performed to determine a range that spanned non-toxic to moderately toxic concentrations. Test chemicals included: isothiocyanates (sulforaphane), oxidants (H2O2, menadione) and electrophilic chemicals (acrolein, iodoacetamide, nitrobenzylbromide, cinnamaldehyde, dinitrochlorobenze, t-butylhydroquinone), parthenolide, quercetin, and cyclophosphamide (precursor of acrolein metabolite). Only slight Nrf2 activity was obtained following treatment with sulforaphane, a reversible thiol binding chemical. H2O2 and menadione produced only weak activation over the entire span of doses. However, strongly electrophilic chemicals (acrolein, iodoacetamide, nitrobenzylbromide, cinnamaldehyde, dinitrochlorobenze, t-butylhydroquinone) elicited strong induction of Nrf2. WTS also induced strong Nrf2 activation. Ecig vapor produced only weak activation. The results demonstrate that the Nrf2 airway reporter model is a highly sensitive detector of reactive electrophilic chemicals or mixtures including WTS. The model may prove useful for safety evaluation of new generation nicotine delivery products.
Poster Session: Respiratory Toxicology: Tobacco Products Wednesday, March 14 • 9:15 AM to 4:30 PM • CC Exhibit Hall
The Use of Human 3D Reconstructed Bronchial Tissue to Study the Effects of Cigarette Smoke and E-Cigarette Aerosol on a Wide Range of Cellular Endpoints
L. Czekala1, M. Stevenson1, L. Simms1, N. Tschierske1, A. Maione2, and T. Walele1. 1Imperial Tobacco Ltd, Bristol, United Kingdom; and 2MatTek Corp., Ashland, MA. Sponsor: F. Ayala-Fierro Poster Board Number: P325 • Abstract Number: 2810 Displayed: 9:15 AM–4:30 PM • Author Attended: 10:45 AM–12:15 PM (Block B)
In 2015, Public Health England concluded e-cigarettes are around 95% less harmful than smoking tobacco cigarettes and in 2016 the Royal College of Physicians stated e-cigarettes should be promoted widely as a substitute for smoking. However, some recent data has indicated that e-cigarette aerosol can potentially produce reactive oxygen species which may give rise to inflammation, DNA damage and reduced cell viability. In this study, biological endpoints were investigated in the EpiAirwayTM 3D in vitro model (MatTek Corp), a highly differentiated human airway culture derived from primary tracheal/bronchial epithelial cells. EpiAirwayTM tissue was exposed at the air-liquid interface to whole smoke from a conventional tobacco cigarette and aerosol generated from an e-cigarette device using two different e-liquids (commercial and experimental e-liquid both with 2.4% nicotine). Smoke or aerosol was generated using a VITROCELL® VC 1 smoking machine following Health Canada Intense and CRM°81 regime, respectively. The integrity of the epithelial barrier of tissues, was measured before and 24 hours after exposure using trans-epithelial electrical resistance. Tissue viability was assessed using the MTT assay. Conditioned media were collected 24 hours after the exposure, to determine tissue secretion of the pro-inflammatory cytokines IL-6 and IL-8. The 8-Isoprostane assay evaluated oxidative stress response. Tissue morphology was assessed by H&E staining. The TUNEL assay was used to detect apoptotic cells; γH2AX assay to detect DNA double-strand breaks and Ki67 staining was used as a marker of cell proliferation. Under the experimental conditions, cigarettes impaired barrier function and reduced cell viability to approximately 30% after exposure to 45 puffs and induced secretion of IL-6 and IL-8. By contrast, the e-cigarette aerosol, up to 400 puffs, did not alter barrier function, cellular viability or cytokine secretion compared to air matched controls. Unlike cigarettes, our initial results suggest a lack of cytotoxicity and pro-inflammatory responses to e-cigarette aerosols. In conclusion, the 3D lung model appears to show the clear and substantial differences between these product types on in vitro biological endpoints.
Poster Session: Alternatives to Mammalian Models III: Liver, Ocular, and Skin Alternatives Wednesday, March 14 • 9:15 AM to 4:30 PM • CC Exhibit Hall
Characterization of Drug Metabolizing Enzymes, Transporters, and Permeation in a Human Organotypic Corneal Tissue Model.
Y. Kaluzhny, M. Kinuthia, T. Truong, A. Lapointe, P. Hayden, and M. Klausner. MatTek Corporation, Ashland, MA. Poster Board Number: P794 • Abstract Number: 3161 Displayed: 9:15 AM–4:30 PM • Author Attended: 1:30 PM–3:00 PM (Block C)
The corneal barrier is vitally important for eye protection, but also presents a significant challenge for delivery of ophthalmic drugs. Most current studies utilize excised animal corneas that are not suitable for rapid drug screening and also have poor species extrapolation and standardization.
To fulfill the need for a physiologically relevant, human-based in vitro system to study ophthalmic drug delivery, we developed an organotypic 3D corneal tissue model. The model contains normal human corneal epithelial cells that grow at the air-liquid interface and develop a tight barrier (1000 ±250 Ω*cm2)that is comparable to the in vivo human cornea, express tight junctions, mucins, and key corneal detoxification enzymes.
Utilizing a PCR gene array we investigated the expression of 84 Phase I/II metabolizing enzymes and 84 drug transport related genes in the organotypic 3D tissue model and in the isolated human corneal epithelium. High correlation coefficients of r2=0.87 were obtained between gene expression of EpiCorneal tissues and normal human corneal epithelium.
Corneal permeability was evaluated using model compounds with a wide range of hydrophobicity, molecular weight, and excipients. The correlation of permeation coefficients to excised animal corneas for model drugs (r2) was 0.84. Various ophthalmic formulations, including Latanaprost and Bimatoprost eye drops, were administered and their effect on drug absorption, tissue viability and integrity was investigated. As expected, Latanoprost free acid had much lower permeability (Papp=8.0×10-6) than its prodrug isopropyl ester form (Alcon, Papp=2.5×10-4). The presence of 0.02% BAC in ophthalmic solutions significantly affected tissue barrier and viability (analyzed by MTT and LY leakage assays), while BAC-free formulation didn’t have an effect on tissue integrity and viability. Permeability of Bimatoprsot in BAC-free Krebs-Ringer buffer (KRB) was 6.3×10-5 and 3.7×10-4 in the vehicle containing 0.02% BAC (Lumigan, Allergan).
In summary, the model demonstrates in vivo like structure, barrier and drug metabolizing/transporter expression. Tissue permeability, as well as effects on viability and barrier of various known ophthalmic formulations/excipients is similar to excised corneas. EpiCorneal tissue model may be useful in the evaluation of corneal drug permeability and safety during the development of new ophthalmics.
The Role of Oxidative Stress in Dry Eye Disease: Utilization of Human Organotypic Corneal Tissue Model.
Y. Kaluzhny, M.W. Kinuthia, T. Truong, A. Lapointe, P. Hayden, and M. Klausner. MatTek Corporation, Ashland, MA. Poster Board Number: P795 • Abstract Number: 3162 Displayed: 9:15 AM–4:30 PM • Author Attended: 1:30 PM–3:00 PM (Block C)
Oxidative damage plays an important role in many ocular diseases, including dry eye disease (DED). Current methods used to investigate the mechanisms of corneal injuries utilize monolayer cell cultures or animals that result in poor data extrapolation, or low throughput and high cost. As a result, there is a need for more physiologically relevant, human-based in vitro models for ocular research.
This study evaluated the utility of an in vitro reconstructed 3D tissue model to study oxidative stress (OS) and DED. The constructs are comprised of normal human corneal epithelial cells that are cultured at the air-liquid interface to produce tissues similar to in vivo corneal structure and express tight junctions, mucins, and key corneal detoxification enzymes: dehydrogenases, reductases, glucoronosyltransferases, and CYP P450.
OS was generated by UV or desiccating stress conditions (DSC) to stimulate morphological, cellular, and molecular changes relevant to DED. Reactive oxygen species (ROS), lipid oxidation, cytokine release, barrier function, tissue viability, histology, and gene expression were evaluated. UV and DSC caused increased ROS, oxidation of lipids, release of IL8 and upregulation of proinflammatory genes. Application of topical lubricants improved tissue morphology, barrier function, and normalized IL8 release.
Utilizing a PCR gene array we investigated the effect of UV irradiation (60 mJ of UVB, 2h post-incubation) and DSC (60% RH, 40°C, and 5% CO2, 24h) on the expression of 84 genes related to OS. 6 genes were >2-fold upregulated in UV-treated cultures compared to untreated control, including genes involved in ROS metabolism, peroxidases, and serine peptidase inhibitor SPINK1. 15 genes were >2-fold upregulated in DED tissues compared to control, including antioxidants and PTGS2 (COX-2) peroxidases, SPINK1, OS responsive gene HMOX1, and other genes involved in superoxide metabolism – ALOX12 and NOS2. In both UV-irradiated and DED tissues OS pathway signature genes, SPINK1 and HMOX1 were upregulated.
The in vitro reconstructed normal human corneal tissue model structurally and functionally reproduces OS and DED markers. Gene expression changes in OS-exposed EpiCorneal tissues closely parallel in vivo changes associated with inflammatory response. This model is anticipated to be a useful tool to study molecular mechanisms of ocular surface damage, DED, and to evaluate new corneal drug formulations.
