MatTek scientists will be attending and presenting posters at the XVIth International Congress of Toxicology, in Maastricht, Netherlands September 18-21. Read more to see what we’ve been working on and request copies of our posters.
Evaluation and Sub-categorization of Ocular Irritants Using the EpiOcular Tissue Model – Prediction Models for Liquids and Solids (Abstract ID # 798 / Poster #P12-43) Silvia Letasiova1, Lenka Hudecova1, Jan Markus1, Yulia Kaluzhny2, Mitch Klausner2 1 MatTek Europe, Bratislava, Slovakia. 2 MatTek Life Sciences, Ashland, MA, USA
Viewing: Tuesday, September 20
Abstract Assessment of serious eye damage/eye irritation originally involved the use of laboratory animals (OECD TG 405). In 2015, a new test guideline (OECD TG 492) was accepted which enables the use of an in vitro procedure based on the reconstructed human cornea-like epithelium (RhCE) to distinguish between chemicals (substances and mixtures) not requiring classification and those that must be labeled for eye irritation or serious eye damage. Chemicals identified as requiring classification for eye irritation/serious eye damage must be further tested to distinguish between eye irritants and those causing serious eye damage. There have been several projects focused on the development of tiered testing strategies for eye irritation assessment which takes into account all drivers of classification. The goal of these projects has been to develop a testing strategy to subcategorize chemicals that: a) do not require labeling for serious eye damage or eye irritancy (No Category), b) can cause serious eye damage (Category 1 or Cat 1), and c) are eye irritants (Category 2 or Cat 2) [1,2]. In the current project, a set of 13 chemicals (7 liquids and 6 solids) that are listed as proficiency chemicals in draft OECD TG 492B were tested using the RhCE model, EpiOcular. We used a testing strategy developed in the CON4EI project and confirmed in the ALT4EI project, which combines the most predictive time-points of EpiOcular time-to-toxicity neat and dilution protocols. Liquids and solids were tested separately with different methodologies and prediction models. The set of chemicals consisted of 4 Cat 1 chemicals, 5 Cat 2 chemicals, and 4 No Cat chemicals. Using the proposed testing strategy, we were able to correctly identify 100% of Cat 1 chemicals (4/4), 100% of Cat 2 chemicals (5/5), and 100% of No Cat chemicals (4/4). The testing strategy proposed in CON4EI and verified in ALT4EI projects to achieve optimal prediction for all three categories – prediction models for liquids and solids seems to be a very promising tool in an integrated testing strategy (ITS) that can discriminate chemicals to No Cat, Cat 2 and Cat 1.
Evaluating ocular side effects of systemic medications utilizing the in vitro reconstructed human corneal epithelial tissue model (Abstract ID # 928 / Poster #P12-52) Yulia Kaluzhny, Viktor Karetsky, Miriam Kinuthia, Mitchel Klausner, Alex Armento
Viewing: Tuesday, September 20
Abstract
Chronic use of systemic medications can cause light sensitivity, pain, corneal edema/inflammation, and/or cytotoxicity. Animal tests are often poor predictors of human responses. There is a worldwide need for physiologically relevant, human primary cell-based tissue models to address ocular safety for the evaluation of new drug formulations. We have utilized an in vitro reconstructed EpiCorneal™ tissue model to analyze the effect of frequently used drugs with known adverse ocular side effects. EpiCorneal tissues are cultured using normal human corneal epithelial cells, express site-specific mucins and tight junctions, and attain morphology, barrier properties (Transepithelial electrical resistance or TEER > 1000±200 Ω*cm2), and gene expression comparable to the in vivo human cornea. Tissue performance, evaluated by TEER and tissue viability (MTT assay), were comparable after 24h and 96h under simulated shipping conditions. The effects of Chlorpromazine hydrochloride (CPZ), a common psychotropic agent; Hydroxychloroquine sulfate (HCQ), an anti-inflammatory / anti-malaria drug; Alfuzosin hydrochloride (ALF), an antihypertensive drug; and Fosamax (Alendronate Sodium, FOS), a common anti-osteoporosis agent, were investigated. Endpoints included MTT, TEER, histology, and LDH and cytokine release. Tissues were incubated in the medium containing physiologically relevant concentrations of the drugs for up to 72h. EpiCorneal tissue model is valuable for evaluating formulations with negligible irritation potential. It is suitable for rapid drug screening, will model systemic and topical drug exposure, improve the predictivity of human responses, and be more cost-effective and reproducible than animal methods. It will facilitate drug discovery worldwide by allowing screening and optimization of pharmaceuticals prior to clinical studies.
Organotypic Small Intestinal Model for Long-Term High-Throughput Screening Toxicity Studies (Abstract ID # 686 / Poster #P18-06) Puskar1, J. Markus1, Z. Stevens2, P. Kearney2, M. Klausner2, A. Armento2, S. Ayehunie2 1 MatTek Europe, Bratislava, Slovakia 2 MatTek Life Sciences, Ashland, MA, USA
Viewing: Tuesday, September 20
Abstract
High throughput 96-well tissue culture plates that allow the culture of three-dimensional physiological tissue systems of small intestinal (SMI) organotypic tissue on a newly fabricated membrane support have been developed. The plates were used to reconstruct human primary intestinal tissues and cultures were followed for over 3 months by monitoring tissue histology and barrier integrity (measured by transepithelial electrical resistance, TEER). Histological specimens were collected on a weekly basis. To monitor well-to-well tissue reproducibility, TEER measurements were performed every week on all 96-well tissues. Histological and immunological stains showed that tissues fixed at all time points (>3 months) were similar to the standard EpiIntestinal tissue structure and morphology. The 3D tissues are well polarized and stratified with villi-like structural formation. Immunohistochemical staining shows markers for epithelial cells (CK19), tight junction (ZO-1), and brush borders (villin) similar to the standard tissue model. The barrier integrity measurement (TEER) demonstrated high well-to-well reproducibility (weekly average %CV <20%) for up to 90 days of the culture period. The weekly measurement of TEER for all the wells/tissues were within a physiological range of 160-300 ohms*cm2. This value was within our QC criteria for the EpiIntestinal tissue model. The availability of intestinal organotypic tissue that can be cultured for extended periods of time (>3 months) can be used for chronic exposure experiments. These results extend our previously published tissue utility period from 42 days to 90 days. In conclusion, the newly fabricated plates support the reconstruction of EpiIntestinal tissues for extended time periods that can be used for hazard identification of chemicals and nanoparticles in a high throughput format and to study drug safety and efficacy following chronic exposures or multiple applications of compounds.
Development and evaluation of in vitro inhalation model to predict acute respiratory toxicity of mists and volatile liquids (Abstract ID # 831 / Poster #13-24) Kaluzhny2, L. Hudecova1 , J. Markus1, G. R. Jackson2, O. Gabriel2, S. Letasiova1, M. Klausner2, P. Kearney2, A. Armento2 1 MatTek Europe, Bratislava, Slovakia 2 MatTek Life Sciences, Ashland, MA, USA
Viewing: Tuesday, September 20
Abstract
Acute respiratory toxicity (ART) testing is required to assess the health effects of inhaled substances. OECD accepted methods utilize GHS categorization that is based on animal death. There is no validated in vitro ART assay, even though animal tests have limitations as predictors of human responses and are discredited based on ethical grounds. The goals of this work were to develop physiologically relevant ART test(s) using the EpiAirway™ tissue model, demonstrate interlaboratory transferability, and correlate the results to the established categorization systems relevant to human respiratory irritation. Test articles (TA, n=53) were applied to EpiAirway tissues (0.6 cm2) at MatTek (USA) and MatTek IVLSL (Slovakia) with ART protocols developed for exposure to mists/sprays (Direct Application Protocol, DAP) and vapors/volatile liquids (Vapor Cap Protocol, VCP). In both protocols, tissues were exposed for 4 hours to 4 fixed doses of the TA (0.5, 2, 10, 20 mg/tissue, diluted in corn oil or water). In the DAP, TAs were applied to the apical tissue surface, and in the VCP, to an absorbent material in a specially designed cap that forms a tight seal above the tissue allowing exposure to TA vapor. The effects on tissue viability (MTT assay) and barrier properties (Transepithelial Electrical Resistance, TEER) were determined. The effective doses (ED) which reduced tissue viability by 25% (ED-25) or by 75% (ED-75) were mathematically interpolated for the DAP and VCP methods, respectively. The ED-25 and ED-75 were correlated to the acute irritation Health Effects (HE) Codes (HE14/15/16) listed by OSHA and to standard GHS criteria. Using the MTT assay, the DAP discriminated between HE14/15/16&NH with a Sensitivity/Specificity/Accuracy (S/S/A) of 74.3/86.7/80.5% (MatTek) and 72.3/85.2/78.7% (MatTek IVLSL); correlation to GHS Cat.1&2/3&4/5&NC gave results of 58.8/74.6/66.7% (MatTek) and 67.9/78.2/73.0% (MatTek IVLSL) S/S/A. The VCP discriminated between HE codes with S/S/A of 80.9/90.5/85.7% (MatTek) and 77.6/90.0/83.8% (MatTek IVLSL); correlation to GHS was 70.1/82.9/76.5 S/S/A (MatTek) and 71.1/82.2/76.7% (MatTek IVLSL). Both protocols demonstrated high predictivity of human HE Codes that were published by the US Occupational Safety and Health Administration (OSHA). Good inter-laboratory reproducibility was observed for the VCP and DAP methods. The VCP and DAP provide physiologically relevant, organ-specific in vitro tests that can improve the predictivity of human responses and significantly reduce the number of animals being used.
Organotypic 3D primary human nasal tissue model for toxicological studies (Abstract ID #1166 / e-poster) S. Ayehunie1, M. Debatis1, Z. Stevens1, J. Oh2, Y. Kaluzhny1, C. Pellevoisin1, A. Armento1 1 MatTek Life Sciences, Ashland, MA, USA 2 The Jackson Laboratory, Farmington, CT
Abstract
The nasal mucosa serves as the first line of defense against inhaled chemicals, drugs, and respiratory infections. Here we developed a novel in vitro primary human cell-based 3D human nasal epithelial tissue (NET) cultured on a cell culture insert with a porous (0.4μm) membrane bottom at an air-liquid interface (ALI). The NET model was characterized by histology, barrier function (TEER), viability (MTT), toxicity, and infection with viruses. Histological and immunohistochemical evaluation of the in vitro NET showed a polarized, multilayered tissue that expresses markers of epithelial cells (CK19), tight junctions (ZO-1andClaudin-1), mucin (MUC5B and MUC5AC), SARS-CoV-2 entry-related/proteins/genes (ACE-2 and TMPRSS2), and cilia (alpha-tubulin). Single-cell sequencing of the differentiated NET also confirmed the presence of differentiated cell types (goblet, club cells, basal, multi-ciliated cells, and not previously identified cells (NPIC). The NPIC were enriched in response to allergic rhinitis/respiratory syncytial virus infection (increase in IL1 RL 1, MMP9, MMP10) and some laminins and integrins. Infection of the NET with viruses also showed high expression of IDO1, which is associated with innate antiviral immune functions. To monitor reproducibility, the MTT and TEER results from N=9 lots were analyzed: MTTOD values were>1.0 (mean OD=1.7±0.2) and TEER values were 300.9±42.4Ω*cm2 (n=9 lots).To evaluate the utility of the nasal tissue model for toxicological studies, we tested the effect of a known mucous membrane irritant, butylamine, following 4hr topical exposure to 0.5mg/mL and 2mg/mL of the test article. The irritant reduced the barrier integrity to 33.2%±0.0 and 12.3%±5.8, respectively, compared to the vehicle control (corn oil), which is indicative of toxicity of the test article. In short, this novel NET model can be added to the toxicologist’s toolbox of 3D respiratory tissue models to predict the safety of chemical inhalants, therapeutic candidates, viral and bacterial infections at the inhalation entry site.
