MatTek scientists will be attending and presenting posters at the Society of Toxicology Annual Meeting in Nashville, Tennessee. Read more to see what they’ve been working on and request copies of their posters. We can’t wait to see you at SOT 2023!
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Bioengineering of Novel Organotypic 3D Human Liver/Hepatocyte Tissue Model for Drug-Induced Liver Injury/Toxicity Studies (P136)
S. Ayehunie, C. Holm, D. Sazer, M. Frare, A. Armento, and Y. Kaluzhny. MatTek Life Sciences, Ashland, MA.
Session: Wednesday, March 22 | 2:30PM – 4:15PM
Development of a human primary cell-based 3D organotypic hepatocyte/liver tissue model that can be cultured for weeks with polarized hepatic morphology and maintains high-level expression of major liver-associated drug metabolizing enzymes is an attractive alternative platform to study acute and chronic liver toxicity and drug-induced Liver injury. In this study, we developed a human 3D hepatocyte tissue model using adult primary hepatocytes. To reconstruct the model, hepatocytes were seeded onto cell culture inserts and fed with a specialized medium to form polarized and well-differentiated hepatocyte tissue structures with defined apical and basolateral surfaces. Tissue morphology was characterized by histology, albumin expression, and release were evaluated by immunohistochemistry and ELISA. To monitor changes in gene expression levels for drug-metabolizing enzymes associated with first-pass metabolism during the culture period, qPCR was performed on days 0, 10, 16, and 23. Utility of the tissue model for drug toxicity studies was demonstrated by dosing the reconstructed liver tissue with 100 uM of 5 model drugs (SN38, Bosentan, Diclofenac, Fialuridine, and Tolcapone) that are known to have adverse effects on the liver in humans. Outcome measurements for liver toxicity include increased levels of alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) release, two of the biomarkers with clinical relevance in liver function tests. Characterization of the tissue model showed that 3D columnar hepatocyte tissue formation (histology), hexagonal cellular structure (topical view imaging), albumin production (immunohistochemistry), and albumin release to the basolateral and apical sides (ELISA). qPCR results demonstrated high-level expression of enzymes involved in drug metabolism such as CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, and CYP4A11. Interestingly repeated application of Fialuridine, a drug intended for hepatitis B treatment that was abruptly terminated due to induction of liver failure or causing of severe liver toxicity during clinical trials, showed an increase in ALT and AST levels in a time-dependent manner at days 5 and 7 indicative of drug-induced liver injury (DILI). The positive control SN38, a metabolite of the cancer drug Irinotecan, also showed an increase in ALT and AST levels. The development of this novel 3D human liver tissue model using primary adult hepatocytes creates an opportunity to study liver physiology in an in vitro tissue platform. The developed liver model can also play a key role in screening drug candidates that are in the development pipeline for their liver toxicity potential. Such models will have an impact on the development of new approach methodologies (NAMs) to identify adverse effects of therapeutic candidates and to reduce animal use for experimentation.
Organotypic 3D Primary Human Nasal Tissue for Infection with SARS-CoV-2 Variants and Drug Safety and Efficacy Studies (Talk)
Seyoum Ayehunie1, Samantha Durand1, Julia Oh2, Kie-Hoon3, Brett Hurst3, Mitch Klausner1, Alex Armento1. MatTek Life Sciences, 200 Homer Avenue, Ashland, MA. 2 The Jackson Laboratory, 10 Discovery Drive, Farmington, CT 860-837-2014. 3Utah State University, 5600 Old Main Hill, Logan, UT 84322-5600.
Session: Thursday, March 23 | 8:30AM – 11:00AM
Abstract: The nasal mucosa serves as the first line of defense against inhaled chemicals, drugs, and respiratory infections in the nasal cavity. Here we developed a novel in vitro primary human cell-based 3D human nasal epithelial tissue (NET) cultured on microporous membrane cell culture inserts at an air-liquid interface (ALI). The NET was characterized by histology, barrier function (TEER), viability (MTT), infection with viruses, and response to toxic chemicals. Histological and immunohistochemical evaluation of the in vitro NET model showed a polarized, ciliated, and multilayered tissue that expresses epithelial cell markers including the SARS-CoV-2 entry-related /proteins/genes (ACE-2 and TMPRSS2). To monitor reproducibility, TEER results from N=21 lots were analyzed and the mean TEER value for the lots was 423±67 Ω*cm2. Single-cell sequencing of the differentiated NET cultures also confirms the presence of differentiated cell types (goblet, club cells, basal, multiciliated cells, and not previously identified cells (NPIC)). The NET tissue model was infectable with SARS CoV-2 variants (Beta, Delta, and Omicron) and the maximum infection was noted around Day 4, which is slightly faster than the 5 days observed for tracheal-bronchial tissues. A dose-dependent response was noted when the infected NET tissues were treated with drugs such as Remdesivir and EIDD1931. Tissue toxicity was monitored by Cell Counting Kit-8 (CCK-8). To evaluate the utility of the nasal tissue model for toxicological studies of respiratory irritants, we tested the effect of a known mucous membrane irritant, butylamine, following 4 hr topical exposure to 0.5 and 2 mg/mL of the test article. The data showed a reduction in barrier by 12.3 ± 5.8 and 33.2% ± 0.0, respectively, compared to the vehicle control (corn oil), which is indicative of toxicity caused by the test article. In short, this novel NET model can be added to the toolbox of 3D respiratory tissue models to predict viral infection, drug safety, and toxicity of chemical inhalants at an important respiratory entry site.
Nanotoxicity of Engineered Nanoparticles following Acute and Chronic Exposures of the Small Intestinal Tissue Model (P632)
S. Ayehunie, K. Causey, A. Armento, and Y. Kaluzhny. MatTek Life Sciences, Ashland, MA.
Session: Wednesday, March 22 | 9:00AM – 10:45AM
Abstract: Despite the expanding number of applications for engineered nanoparticles (ENPs), human health concerns associated with ingested nanoparticles are poorly understood. In this study, we utilized a 3D human small intestinal (SMI) tissue model to develop a physiologically relevant test system to assess the nanotoxicity of ingestible nanomaterials. For dose-response experiments, we tested ENPs of various sizes: 1) Copper oxide (CuO), 2) Zinc oxide (ZnO), 3) titanium oxide (TiO2), 4) silver (Ag), 5) Aluminum (Al), 6) iron oxide (FeO), and 7) single wall carbon nanotubes (SWCNT) at 4 different concentrations. Nanotoxicity was monitored using the following endpoints: 1) tissue viability (MTT), 2) barrier integrity (TEER), 3) structural damage (histology), 4) cytokine release (BioPlex ELISA) and 5) DNA damage (Comet assay) to monitor genetic perturbations in the gut epithelium. In all the experiments, tissues were exposed to 40 ul of different doses of sonicated nanoparticles under rocking conditions for 4 hr. After 4 hrs, dosed tissues were further cultured overnight under static conditions. For chronic exposure, tissues were exposed every other day and cultured for a total of 7- 18 days (4-8 repeat dose applications). Using IC15 (a concentration that reduces barrier function or tissue viability by 15%) as a cutoff, we observed a dose-response reduction of barrier integrity and tissue viability for CuO and ZnO following acute exposure. However, acute exposure to Titanium oxide did not induce toxicity for the concentrations tested. Furthermore, culture supernatants collected at 24 hr of the culture period were also analyzed for inflammatory cytokines and the result showed a dose-dependent release of IL-8 for CuO and ZnO. Tissues exposed to lower doses of nanoparticles that were not found to be toxic in acute exposure studies showed toxicity following repeat applications. For instance, repeated exposure of tissues to CuO (50 nm; 125 ug/ml) showed toxicity on days 7, 14, and 18. FeO (5 nm, 900 ug/ml, Gold (2 um, 125 ug/ml), and TiO2 (15 nm, 900 ug/ml) showed toxicity on days 14 and 18. Analysis of the comet assay of the chronic exposure showed an increase in DNA tail length in tissues treated with SWCNT (125 and 900 µg/mL) and CuO (125 and 900 µg/mL). Overall, the TEER measurement was a sensitive endpoint compared to the MTT tissue viability assay. In summary, the use SMI tissue model to examine the toxicity profile of ingested nanotoxicity will also enhance our understanding of nanoparticle cellular deposition and absorption/permeation through the intestinal tissues. The acute and chronic exposure results will play a key role in dose/design studies to generate a physiologically relevant data set for the food and pharmaceutical industries and to provide greater insight into in vivo responses.
EpiOcular Time-to-Toxicity Test Method for Eye Hazard Subcategorization (P251)
Silvia Letasiova1, Lenka Hudecova1, Jan Markus1, Yulia Kaluzhny2, Els Adriaens3, Mitch Klausner2 1MatTek Europe, Mlynske Nivy 73, 82105 Bratislava, Slovakia, 2MatTek Life Sciences, 200 Homer Ave, Ashland, MA, USA, 3Adriaens Consulting BVBA, Bellemdorpweg 95, 9981 Aalter, Belgium
Monday, March 20 | 10:45AM – 12:00PM
For a long time, assessment of serious eye damage/eye irritation relied on the use of laboratory animals – in vivo Draize eye irritation test, 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 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. However, this method could not distinguish between chemicals causing serious eye damage and less-severe eye irritation. Recently, OECD TG 492B has been accepted which describes an in vitro procedure allowing the identification of chemicals that: a) do not require labeling for serious eye damage or eye irritancy (No Category or No Cat), b) can cause serious eye damage (Category 1 or Cat 1), and c) are eye irritants (Category 2 or Cat 2) according to the UN GHS ocular hazard categories. The EpiOcular™ time-to-toxicity test method was developed for eye hazard identification of liquid and solid chemicals according to the three UN GHS categories. The method is based on the results of several specialized projects. The CON4EI project resulted in the prediction models for liquids and solids based on a set of 80 chemicals (38 liquids and 42 solids). Additional chemicals were tested within the ALT4EI project, and another set of chemicals was added in 2022. This resulted in a robust final set of 144 reference chemicals – 78 liquids and 66 solids, to confirm the proposed testing strategy. The performance criteria established by the OECD expert group on eye/skin irritation/corrosion and phototoxicity for all 144 chemicals were met. Using the proposed testing strategy for liquids, we were able to correctly identify 78.7% of Cat 1 (N=27), 63.5% of Cat 2 (N=26), and 82.0% of No Cat (N=25). Using the proposed testing strategy for solids, we correctly predicted 75.0% of Cat 1 (N=28), 59.4% of Cat 2 (N=16), and 80.3% of No Cat (N=22). Overall, 76.8% of Cat 1 (N=55), 61.9% of Cat 2 (N=42), and 81.2% of No Cat (N=47) were correctly predicted. The EpiOcular™ time-to-toxicity test method is a novel approach for subcategorizing both liquid and solid compounds. The developed prediction models have proven to be capable of successfully distinguishing chemicals (substances and mixtures) into 3 UN GHS ocular hazard categories: No Cat, Cat 2, and Cat 1.
Multi-species 3D Airway Tissue Models for Translational Inhalation Toxicity Studies (P516)
S. Ayehunie, R. Jackson, S. Durand, K. Coen, T. Landry, M. Klausner, Y. Kaluzhny, and A. Armento. MatTek Life Sciences, Ashland, MA, USA
Wednesday, March 22 | 2:30PM – 4:30PM
Abstract: Scalable and reproducible in vitro 3-dimensional organotypic models from different species are needed for translational studies in order to develop reliable alternatives to traditional in vivo animal inhalation toxicity tests. The aim of this study is to compare toxicity responses of 3D airway tissue models constructed using primary tracheobronchial epithelial cells harvested from rat, primate, and human tissues. Primary cells from the different species were expanded in monolayer culture and seeded onto microporous membrane inserts to reconstruct 3D organotypic tissue models. Tissues were characterized for the polarity of epithelial cells (histology), epithelial cell markers (IHC), barrier integrity (transepithelial electrical resistance, TEER measurement), and functionality in inhalation toxicological studies by testing 3 well-characterized chemical toxicants (CT). Polyethylene glycol was used as the positive control and either water or corn oil was used as a negative control. CT (100 µL) was applied to the apical surface, and tissue inserts were sealed with insert caps (MILICEL-MTK-CAP, MatTek Life Sciences) for 4 hr to mimic in vivo rat exposure experiments. After 4 hr, dosed tissues were washed and allowed to recover for 20 hr at 37oC. Analysis of the 3D tissues from the different species revealed: well-polarized epithelium with a physiological TEER value of >300 Ώ*cm2, cilia formation on the apical surfaces, and mucin production mimicking the airway microenvironment. Acute exposure to CT for 4 hr showed varying levels of tissue viability and membrane integrity by MTT and TEER assays, respectively. While the effective dose concentration that reduces tissue viability by 50% (ED-50) for vinyl acetate and chloroacetaldehyde are comparable (<2 mg/tissue) for all species, the ED-50 value for toluene showed differences: human >20 mg, primate 16.2±1.7 mg, and rat 13.8±0.1mg. Based on the MTT viability and TEER values the test chemicals were rank-ordered from high to minimal toxicity: chloroacetaldehyde > vinyl acetate > toluene >propylene glycol and vehicle control. While the human and primate airway models showed comparable MTT values, the rat airway tissue was more sensitive to the higher concentration of toluene. Although more chemicals need to be tested, the multispecies 3D airway tissue models will be vital translational tools to predict airway inhalation toxicity and to bridge the in vitro in vivo knowledge gap to reliably predict human responses, while providing an alternative approach to animal experimentation.
High-Throughput Screening of Chemicals for Respiratory Toxicity Using a 3-Dimensional (3D) Tissue Model (P496)
Mitchell Klausner, Yulia Kaluzhny, George R. Jackson, Alex Armento, MatTek Life Sciences, Ashland, MA, USA
Wednesday, March 22 | 2:30PM – 4:15 PM
A 3D, highly differentiated, tracheal bronchial tissue (TBT) has been previously used to screen chemicals for respiratory toxicity (Jackson, et al, Prevalidation of an acute inhalation toxicity test using the EpiAirway in vitro human airway model, Appl In Vitro Tox, 2018). In the current study, the TBT was cultured using a new, 96-well high throughput (HTP) cell culture insert plate with a microporous membrane bottom. H&E-stained histological cross-sections of the tissue cultured on the HTP plate showed a pseudo-stratified morphology with ciliated and mucous-producing cells, similar to the standard model produced in a 24-well, individual insert format. Likewise, transepithelial electrical resistance (TEER) values for the HTP tissues were not significantly different (p=0.19) than measurements made on the TBT grown on the 24-well individual inserts: 861 ± 175 (n=16 lots) vs. 998 ± 339 (n=41 lots) Ω*cm2, respectively. The utility of the HTP tissues for toxicological testing was investigated using 8 respiratory irritants (RI), which ranged from GHS Category 1 (most toxic) to GHS Category 5 (least toxic). 4 doses of each RI were applied to the apical side of the tissues for 3 hours, and tissue viability was determined using the MTT assay, without any recovery period. The dose of each RI which reduced tissue viability by 25% (ED-25) was mathematically interpolated and compared with previously reported results obtained using the 24-well format tissues. A strong correlation (r2=0.967) was observed between ED-25 values for the HTP tissues and the ED-25 values previously reported for the individual inserts. Thus, we anticipate that the HTP tissues will be useful for toxicological screening of the large numbers of formulations typically investigated during the drug development/ formulation process.
In Vitro Evaluation of Local Tolerance of Vaginal Formulations and Medical Devices with a 3D Human Epithelial Model (P688)
S. Ayehunie1, C. Pellevoisin2, T. Landry1, A. Armento1, Y. Kaluzhny1, and M. Klausner1. 1MatTek Life Sciences, Ashland, MA; and 2MatTek Europe, Bratislava,, Slovakia.
Session: Tuesday, March 21 | 9:00AM-10:45AM
Abstract: The goal of the study is to validate the utility of the 3D human in vitro vaginal tissue model as an alternative for the rabbit vaginal irritation (RVI) test requested for the regulatory evaluation of drugs and devices in contact with the vaginal mucosa. A double-blinded study was conducted in vitro and in vivo with N=14 coded test articles (TAs) including materials intended to be in contact with vaginal tissues in the form of preservatives, contraceptives, solvents, viscosity enhancers, antiseptics, and cleansing agents (surfactants). The TAs were topically applied in vitro and in vivo at 2% dose with 5 repeat exposures over 6 days. Dose volumes were proportionally adjusted based on the surface area and N=5 rabbits and N=3 EpiVaginal tissues were used per TA. While the RVI score was used to monitor in vivo irritation; MTT, TEER, and histological analysis were used as endpoints for the in vitro assays. The results showed that four TAs including two known irritants, benzalkonium chloride (BZK) and nonoxynol 9 (N9) were predicted as irritants by MTT viability and TEER (<50% reduction). While BZK was identified as a mild/severe irritant in the RVI assay, the effect of N9 in rabbits was highly variable. Based on the rabbit data, we further examined the long-term reproducibility and predictivity of the in vitro assay by determining the effect using tissue reconstructed from cells harvested from multiple donors (n=4). The variability of the reconstructed 3D vaginal tissues (EpiVaginal™) from the cells of the 4 donors was monitored using 55 test articles commonly used in feminine care products. Triplicate EpiVaginal tissues were topically exposed to TAs at a single concentration (2%) and incubated for 24 hr. After the 24 hr exposure, tissues were rinsed with PBS and examined for tissue viability (MTT assay) and for barrier integrity (TEER) measurements. The results showed that 5 of the 55 test articles were found to be irritant in both the MTT and TEER assays in all four donors. These TAs were Gynol containing 2% N9, benzalkonium chloride, sodium dodecyl sulfate, acetic acid, and cremophor. These test articles resulted in <50% tissue viability and <50% of the TEER values versus the saline controls. Only 1 test article, copper sulfate, showed low MTT viability (9.5%) but the TEER values for 3 of 4 donors were non-irritating (>50%). Miconazole nitrate resulted in MTT values > 50% in 2 out of 4 donors (avg viability for 4 donors = 57.5%) but had TEER ≥ 50% for all donors. Of the remaining test articles, 45 were found to be non-irritants in both MTT and TEER assays in all four donors. In conclusion, the MTT and TEER assays from the four donors were highly reproducible and a decrease in MTT and TEER appears to be useful endpoints for preclinical toxicity screening of chemicals and feminine care products. The use of this in vitro system to assess the safety of topically applied vaginal care products, drugs, and vaginally inserted medical devices will be cost-effective and could replace the use of animals for experimentation.
Characterization of the ISO Database of Reference Chemicals for Interlaboratory Studies to Demonstrate the Applicability of OECD Methods to Assess Skin Sensitization of Medical Devices (P297)
Christian Pellevoisin1, Ahn Nguyen 2, Ron Brown2, Rose-Marie Jenvert3, Hervé Groux4, Peter Cornelis5, Michelle H. Lee5, Austin Zdawczyk6, Joseph Carraway6, Emma Pedersen7, Joel Cohen8, David Waeckerlin9, Audrey P. Turley5, Sebastian Hoffmann10 and Kelly Coleman11 1MatTek Life Sciences, USA, 2Risk Science Consortium, USA, 3SenzaGen, Sweden, 4ImmunoSearch, France, 5Nelson Lab, USA, 6NAMSA, USA, 7Key2Compliance, Sweden, 8Gradient Corp, USA, 9Sonova AG, Switzerland, 10seh-cs, Germany, 11Medtronic, USA
Session: Wednesday, March 22 | 10:45AM – 12:00PM
The skin sensitization potential of medical devices is typically assessed using animal-based test methods such as the Guinea Pig Maximization Test (GPMT) or the Local Lymph Node Assay (LLNA). There is increased interest in using the non-animal methods from the OECD test guideline 442 series, however, these methods have been validated with neat chemicals and need to be evaluated and qualified before they can be accepted for evaluation of medical devices. The ISO Technical Specification 11796 under development in Working Group 8 (WG8) of ISO Technical Committee 194 (ISO/TC 194) provides the framework to demonstrate the applicability of these methods to assess the skin sensitization potential of medical devices. Due to the lack of positive and negative reference materials to conduct prevalidation and interlaboratory studies, test samples will be prepared from polar and non-polar extracts of a negative silicone polymer spiked with skin-sensitizing or non-sensitizing chemicals. A database of 29 reference chemicals, 22 skin sensitizers, and 7 non-sensitizers, representative of raw materials and/or leachables found in some medical devices has been established. The structural, physicochemical, and mechanistic domains of the candidate compounds have been characterized and compared to the OECD reference database used to validate the defined approaches in the OECD guideline 497. The skin sensitizer chemicals of the ISO database cover a range of potency from weak to extreme. All the mechanistic reaction domains known to be associated with sensitization are represented although, as in the OECD database, no reaction domain alerts were identified for nearly half of the chemicals. Statistical analysis of the distribution of values for the physicochemical properties considered relevant to skin sensitization in the OECD Guideline 497 (e.g., octanol-water partition coefficient) showed a similarity between the curated database and the OECD database. Particular attention was paid to the quality of the historical animal and human toxicological data and searches were performed in the scientific literature and in several reference databases. The EC3 value derivate from the dose-response curve in the LLNA was used to define the spiking concentration for 19 of the skin sensitizers. For 2 chemicals, more conservative values were used from human and animal data and for 1 chemical the concentration was based on a weight of evidence approach from vivo and vitro data. The exercise broadly illustrates how reference compounds can be selected for the validation and qualification of New Approach Methods (NAMs) for medical devices. To date, there are 9 in vitro and in chemico methods in the OECD TG 442 guidelines, and two methods based on the use of a reconstituted human epidermis model are accepted in the OECD Test Guideline Program. The framework of the ISO TS 11796 will ensure that the same process and evaluation criteria will be applied to all these methods when conducting prevalidation and interlaboratory studies to demonstrate their applicability to assess skin sensitization of medical devices
Validation of Physiologically Relevant In Vitro Human Inhalation Toxicity Tests for Volatile Liquids, Mists, and Sprays (P115)
Y. Kaluzhny1, G. R. Jackson1, J. Marcus2, P. Kearney1, S. Letasiova2, M. Klausner1, and A. Armento1. 1MatTek Life Sciences, Ashland, MA; and 2MatTek Europe, Bratislava, Slovakia.
Tuesday, March 21 | 2:30PM – 4:15PM
In vivo animal models are currently accepted by regulatory authorities for acute respiratory toxicity (ART) testing. However, animal tests have been discredited as predictors of human responses on physiological and ethical grounds. The goal of this work was to develop physiologically relevant ART tests utilizing the EpiAirway™ tissue model, to demonstrate correlation to OECD-accepted GHS categorization, and investigate interlaboratory reproducibility. Test articles (TA, n=53) were applied to EpiAirway tissues produced at MatTek (USA) and MatTek Europe (Slovakia) with two ART protocols, the Direct Application Protocol (DAP) for exposure to mists/sprays, and the Vapor Cap Protocol (VCP) for exposure to vapors/volatile liquids. In both protocols, tissues were exposed for 4h to 4 fixed doses (diluted in corn oil or water) to mimic in vivo rat exposure; followed by 20h post incubation. The effects on tissue viability (MTT assay) and barrier properties (Transepithelial Electrical Resistance, TEER) were determined. The effective doses which reduced tissue viability by 25% (ED-25) or by 50%(ED-50) were mathematically interpolated for the DAP and VCP methods, respectively, and correlated to the GHS categories. In the DAP, TAs were applied to the apical surface. Using the MTT assay, the DAP discriminated between GHS Cat.1&2/3&4/5&NC with a Sensitivity/Specificity/Accuracy (S/S/A) of63.5/76.1/69.8% (MatTek) and 63.8/76.1/70.0% (MatTek Europe). Utilizing the changes in TEER, the DAP discriminated between GHS categories with a S/S/A of 65.9/76.7/71.3% (MatTek) and 64.1/76.6/70.3% (MatTek Europe). The correlation coefficient between the two laboratories was R2 =0.91 for MTT and 0.76 for TEER. In the VCP, TAs were applied to an absorbent material in a special cap that forms a tight seal above the tissue allowing exposure to TA vapor. Using the MTT assay, the VCP discriminated between GHS categories with S/S/A of 70.8/83.2/77.0 (MatTek) and 71.9/83.2/77.5% (MatTek Europe). Utilizing the changes in TEER, the VCP discriminated between GHS categories with a S/S/A of 64.4/78.5/71.5 (MatTek) and 67.1/80.1/73.6 (MatTek Europe). The correlation coefficient between the laboratories was R2 =0.93 for MTT and 0.84 for TEER. Using the MTT assay, both the VCP and DAP demonstrated good predictivity of GHS categories and high interlaboratory reproducibility. Both protocols provide robust and efficient, physiologically relevant, organ-specific in vitro tests that can improve the predictivity of human responses, reduce the number of animals being used to assess respiratory toxicity, and help establish confidence for regulatory applications.
Development of In Vitro 3D Human Kidney Proximal Tubular Epithelial Tissue Model (P114)
Y. Kaluzhny, J. Finelli, Z. Stevens, A. Armento, and S. Ayehunie. MatTek Life Sciences, Ashland, MA.
Session: Tuesday, March 21 | 2:30PM – 4:15PM
Abstract: The proximal tubular (PT) region is the most common site for a compound-specific kidney injury. PT region is responsible for essential kidney functions, including reabsorption of low molecular weight proteins, solutes, and glucose; secretion of acids; and clearance of administered medications. The ultimate goal of this project is to develop a novel physiologically relevant primary human kidney-cell-based 3-dimensional (3D) organotypic tissue model for the prediction of human nephrotoxicity. Human primary proximal tubular epithelial cells (PTEC) were isolated and expanded in a monolayer culture prior to seeding onto microporous membrane inserts to reconstruct a 3D organotypic tissue model. 3D tissues were analyzed by histology, barrier integrity (transepithelial electrical resistance, TEER), immunostaining, and qPCR on days 5 to 30. Receptor-mediated FITC-albumin uptake and transpeptidase hydrolytic activity of glutamyl transpeptidase (GGT1) and leucine aminopeptidase (LAP) were assayed on days 10 to 16. The PTEC organotypic tissues organize into characteristic tubular structures, develop a barrier with TEER 110.2+/-33.3 Ω·cm2 on day 9 and stain positive for tight junction proteins ZO-1, claudin-1, and occludin. The organotypic tissues differentiate into polarized epithelium expressing brush border proteins megalin and cubilin together with water channel AQP1 and GGT1 on the apical side and sodium-potassium ATPase pump on the basolateral side. Real-time qPCR analysis confirmed that tissues express a panel of PTEC-specific markers that are necessary for renal clearance, secretion, and reabsorption: aminopeptidase CD13, multi-drug resistance proteins MRP2/4, CYP450 enzymes, glucose transporters SGLT1/2, multidrug and toxin extrusion transporter MATE1, organic cation and anion transporters OCT1/2, OCTN1/2, and OATP4C1, urate transporter URAT1, and sodium phosphate co-transporter NP2. Specific concentration-dependent and time-dependent receptor-mediated uptake of FITC-albumin by PTEC organotypic tissues were observed by fluorescent microscopy. Significant conversion ofγ-Glutamyl-p-nitroanilide (GPNA, 2.5 mM) and L-leucine-p-nitroanilide (LLNA, 3 mM), substrates for GGT1 and LAP respectively, were detected via spectrophotometric monitoring of p-nitroaniline (PNA) following 30 min incubation. Specific transpeptidase hydrolytic activity was inhibited in the presence of an irreversible inhibitor acivicin (1.2 mM) by 88.8% (GGT1) and 35.0% (LAP). The reconstructed in vitro 3D PTEC organotypic tissue morphology, barrier properties, gene expression, and tissue performance resemble the in vivo human PT region. This model is anticipated to be a valuable tool to evaluate human nephrotoxicity and its mechanisms, improve the predictivity of human responses to pharmacological substances, and help establish confidence in drug development and testing.
Development of an In Vitro Test Method for Irritation of Medical Devices Used in the Oral Cavity (P291)
Marek Puskar1, Jennifer Molignano2, Christian Pellevoisin2 Silvia Letasiova1 and Mitch Klausner2 1 MatTek Europe, Bratislava, Slovakia, 2 MatTek Life Sciences, Ashland, MA, USA
Session: Wednesday, March 22 | 10:45AM – 12:00PM
The irritation of any medical device (MD) contacting oral tissues (gingival, buccal, lingual, etc) needs to be evaluated. The objective of this project is to develop and validate an in vitro assay to assess the oral irritation of medical devices. This assay will replace the historical in vivo assay performed on Syrian hamsters. The ISO 10993-23 standard requires that MDs be evaluated using an in vitro irritation test based on reconstructed human epidermis (RhE) before animal or human patch testing is performed. However, for testing of medical devices with intended contact with oral tissues, the RhE models are not appropriate, and the ISO standard recommends the use of other in vitro models with relevant cells or tissues. The EpiOral model consists of normal, human-derived oral epithelial cells cultured to form a multilayered, highly differentiated model of the human buccal tissue. Produced commercially for more than 15 years, several methods have been developed to study oral penetration, drug delivery, and irritancy of oral care products such as toothpaste, mouthwashes, and orthodontic devices. To assess the feasibility of an in vitro method, initial experiments tested solutions of irritant chemicals contained in MDs used in the oral cavity. Solutions with increasing concentrations of ethanol, lactic acid, methyl methacrylate, sodium dodecyl sulfate (SDS), phosphoric acid, sodium hypochlorite, hydrogen peroxide, and chlorhexidine digluconate in sodium chloride or sesame oil were applied to the EpiOral model. The time required to reduce tissue viability by 50% (ET-50), was determined. The results showed a clear relationship between tissue viability and exposure time and between ET-50 and the concentration of the irritant chemical. Compared to historical in vivo data, the in vitro method classified the samples containing an irritant at the expected concentration. In addition, the ET50s allowed differentiation between strong and mild irritants. The data demonstrate that this in vitro assay has equivalent or superior performance to in vivo method. The next step of the project is to assess the irritation potential of several marketed medical devices, some of which are known to induce irritant responses in vivo. We welcome other stakeholders (producers of medical devices, regulators, and other interested parties) to join us as we further develop the assay method and move it into the validation process.