MatTek scientists exhibited and presented posters at the 2024 Eurotox Congress in Copenhagen, Denmark.
Read on to see what we’ve been working on, including our new kidney model and multispecies airway tissues, and download copies of our posters.
Poster Presentations:
3D Primary Human Kidney Tissue Model for Nephrotoxicity (P02-35)
Viktor Karetsky, Joseph Finelli, Yulia Kaluzhny, Michell Klausner, Alex Armento, Seyoum Ayehunie | MatTek Life Sciences, Ashland, MA
Session: PV01, Poster topic: P02 New approach methodologies: 3D models, stem-cells, organ-on-chip
Abstract
Background: Currently renal toxicity accounts for only 2% of drug failure during the preclinical phase but nearly 20% of drug withdrawal is associated with kidney damage during human clinical trials. This discrepancy emphasizes the need for a more physiological and functional alternative assay system that can predict and recapitulate drug induced kidney injury/toxicity in humans. The proximal tubular (PT) region is the most common site for compound and nutrient reabsorption and is highly susceptible to drug and toxin damage. 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 goal of this study is to develop a novel physiologically relevant and functional primary human kidney-cell-based three-dimensional (3D) organotypic tissue model that can accurately predict drug-induced human nephrotoxicity. Methods: 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 9-23. Receptor mediated FITC-albumin uptake and transpeptidase hydrolytic activity of glutamyl transpeptidase (GGT1) and leucine aminopeptidase (LAP) were assayed on days 9-16. Results: The PTEC organotypic tissues organize into characteristic tubular structures, develop a barrier with mean TEER values of 169±33.4 W×cm2 between days 9 to 23 of the culture period. Histological features show epithelial layer with tubular structure. Immunohistochemical analysis of the 3D kidney model stained positive for tight junction proteins ZO-1, and clauidn-1. At a gene level, the polarized organotypic tissues expressing brush border proteins megalin, cubilin, and villin, together with water channel AQP1 and GGT1 on the apical side and sodium-potassium ATPase pump on the basolateral side. Transmission electron microscopy confirms brush border formation. Real-time qPCR also confirmed that tissues express a panel of PTEC-specific markers that are necessary for renal clearance, secretion, and reabsorption: aminopeptidase CD13, p-glycoprotein (PgP; MDR1), multidrug resistance proteins MRP 1, 3, 4, and 5), 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 and time dependent receptor mediated uptake of FITC-albumin by the PTEC tissues was observed by fluorescent microscopy. Kidney uptake of albumin was inhibited by addition of BSA (competitive binding utilizing a common receptor for albumin) or the drug chlorpromazine, an inhibitor of the calthrin-dependent endocytosis. Hydrolytic activity was monitored by the conversion of γ-Glutamyl-p-nitroanilide (GPNA) and L-leucine-p-nitroanilide (LLNA), substrates for GGT1 and LAP using spectrophotometric assays of p-nitroaniline (PNA) following 30 min incubation. Specific transpeptidase hydrolytic activity was inhibited in the presence of an irreversible inhibitor acivicin (1.2mM) by 88.8% (GPNA) and 35.0% (LLNA). Glucose uptake was also enhanced by the addition of sodium chloride. Furthermore, treatment of the 3D kidney model with Cisplatin, a known nephrotoxin that causes acute and chronic kidney injury, shows compromise in PTEC barrier integrity and reduced viability of tissues in a time and concentration dependent manner. Conclusions: The reconstructed in vitro 3D PTEC organotypic tissue is physiological in terms of structure, barrier properties, gene expression, and tissue functionality mimicking the in vivo human PT region. This model is anticipated to be a useful tool to evaluate human nephrotoxicity and to perform mechanistic studies that can improve the predictivity of human responses to pharmacological compounds. This model will help establish confidence in modeling drug induced kidney damage/injury and reduce animal use for experimentation.
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Reconstruction of Novel 3D Models of the Three Parts of the Human Small Intestine for Drug Toxicity (P24-07)
Zachary Stevens1, Jonathan Cheong2, Mitchell Klausner1, Alex Armento1, Seyoum Ayehunie1
1MatTek Life Sciences, Ashland, MA and 2Genentech, San Francisco, CA
Session: PV03, Poster topic: Gut microbiota and toxicity
Abstract
Background: Gastrointestinal (GI) toxicity is a common adverse event that limits pharmaceutical development across diverse therapy areas. The mechanisms of drug induced gastrointestinal (GI) toxicity are often poorly understood, in part due to the lack of physiologically relevant in vitro models that recapitulate the role of the three parts/segments of the small intestine. Traditional in vitro cell cultures approach utilizes immortalized human colorectal Caco-2 cells cultured for about 21 days for assessing ADME properties but have limitations in that it is cell based (colon derived), lacks major drug transporters and drug metabolizing enzymes, not fully polarized structural features, and not predictive of GI toxicity even though it has been in use for more than 5 decades. To mimic the physiology and functionality of the human gut, we previously developed the small intestinal model utilizing cells derived from the ileum. However, there is a need by the pharma industry for in vitro 3D models representing the other parts of the small intestine (duodenum, and jejunum). Having models of the different parts of the small intestine will allow identification of the primary site of absorption for each drug candidate to predict efficacy or identify compounds with lower risk and/or aid to develop dosing schedules that mitigate the risks. Methods: State-of-the-art tissue culture and tissue engineering methods were used to reconstruct 3D GI models representing the duodenum, jejunum, and ileum of the human small intestine. The 3D tissue models use primary cells and were characterized morphologically by histology and functionally by performing drug permeation studies. Gene expression levels of drug transporters and drug metabolizing enzymes were quantified by qPCR & protein quantification analyses were performed by LC/MS. The activities of the transporters were evaluated using Vinblastine as substrate for P-gP and MRP. Results: The 3D tissues developed structural features resembling villi, with barrier function. The mRNA data from the 3D tissue models agreed well with published data showing high expressions of SLCO2B1, SLC16A1 and SLC15A1 by the three segments of the small intestine. Some of the genes transcribed to proteins include: 1) drug transporters such as P-glycoprotein (PgP, MDR1), Multi-drug resistant protein (MRP-3), breast cancer resistant protein (BCRP, ABCG2). Further analysis of the mRNA data reveals P-gP, MRP-2, and ABCG2 expressions in the ileum model were lower compared to the duodenum and Jejunum models. On the other hand, SLC15A1 Peptide transporter 1 (PepT-1) expression was greater in the ileum model compared to the others (Ileum > jejunum > duodenum). Expression of drug metabolizing enzymes such as CYP3A4, CYP2C9, UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1), and Carboxylesterase 1 (CES-1) was maintained in each segment at a comparative level. However, CYP2C9 expression was more pronounced in the jejunum and the rank order was jejunum > duodenum > ileum. The efflux ratio for Vinblastine decreased when the MK571 (MRP inhibitor) and Elacridar (P-gP inhibitor) were added with Vinblastine. Treatment of the tissues with Raloxifene (an anti-osteoporotic drug, CYP3A4 substrate) showed formation of raloxifene-6-glucuronide demonstrating the presence of the phase II glucuronidase enzymes in the tissue models. When Zafirlukast, an inhibitor of the glucuronidase enzymes, was added with Raloxifene, raloxifene-6-glucuronide formation was reduced. Conclusion: These results suggest that the reconstructed tissues from the three segments of the small intestine will serve as useful tools to predict both investigational and traditional GI drug safety and absorption in the GI tract. In addition, use of these models will reduce animal use and improve the pre-clinical drug development process.
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Toxicological Aspects of in vitro Oral Mucosa Wound Healing Models (P02-41)
Jennifer Molignano1, Marek Puskar2, George Robert Jackson1, Silvia Letasiova2, Alex Armento1, Seyoum Ayehunie1, Mitch Klausner1 | 1MatTek Life Sciences, Ashland, MA and 2MatTek Europe, Bratislava, Slovakia
Session: PV01, Poster topic: P02 New approach methodologies: 3D models, stem-cells, organ-on-chip
Abstract
Background: Wounds in the oral cavity tissues can result from surgical procedures, accidents, or canker sores. Oral wounds present a site for potential infection and increased sensitivity to oral irritants. Untreated wounds can lead to pathogen invasion, chronic pain, poor cosmetic outcomes, and in some cases, extended hospitalization. Thus, damaged oral mucosal tissue needs to be treated and healed as soon as possible. The objective of this project is to develop new in vitro oral wound healing models and determine their toxicological profile using common dentifrice materials. Methods: Two tissue models were used in this study (both produced by MatTek Corporation and MatTek Europe): a) EpiOral (ORL-200), which consists of normal, human-derived oral epithelial cells cultured to form a highly differentiated model of human (non-cornified) buccal tissue, and b) EpiGingival (GIN-100), a cornified model of the gingival mucosa. On Day 0, tissues were wounded using a 3 mm biopsy punch (representing 11.6% of total tissue area) and wound closure was monitored using brightfield microscopy, histological cross sections, and transepithelial electrical resistance (TEER) measurements. The toxicological profile of the tissues was probed using the common dentifrice additive, sodium dodecyl sulfate (SDS), at 1%. The exposure time which decreased the tissue viability to 50% (ET-50) of wounded tissues (WT) and non-wounded tissues (NWT) was determined. Results: Histological cross sections and brightfield microscopy of the WT showed that the wounds extend down to the underlying inert microporous culture membrane (Figure 1 & 2). Within 2 days post-wounding (PW) for the ORL-200 tissue and 4 days PW for the GIN-100 tissue basal cells migrate into the wound to reestablish a continuous monolayer (Figure 1). Immediately following wounding, TEER for WT decreases to <20% of the NWT controls, but TEER increases as wound healing proceeds (Figure 3). Wounding also changes the toxicological profile of the tissues. For the ORL-200 model, the ET-50s for the WT and NWT were 27.8 and 49.1 mins, respectively, which represents a 76% increase in ET-50 for the NWT vs WT. By contrast, in the cornified GIN-100 model, the ET-50s were 54.5 and 124.5 mins, respectively, which is 128% increase in ET-50 for NWT vs WT (Table 1). Conclusions: These results demonstrate successful development of wound models for tissues of the oral cavity. Wound healing can be monitored with brightfield microscopy, TEER, histology, and sensitivity to a common dentifrice additive. These models will be useful for testing new therapeutic compounds designed to hasten wound closure in the oral cavity and for determining toxicity profiles in barrier-compromised oral cavity tissues. Additionally, wounding has been shown to decrease the ET-50 of ORL-200 and GIN-100 tissues indicating that it could be a useful tool when a more sensitive tissue model is required, such as for testing many in-use tobacco and oral care products that typically give low responses in NWT.
Note: Additional results were obtained after submission of the abstract and are included in this poster.
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Use of 3D Airway Tissue Models Cultured from Rat, Primate, or Human Cells for Translational Inhalation Studies (P01-73)
Ayehunie1, G. R. Jackson1, K. Coen1, T. Landry1, J. Markus2, S. Letasiova2, M. Klausner1, and A. Armento1 | 1MatTek Corporation, Ashland, MA; and 2MatTek Europe, Bratislava, Slovakia.
Session: PV01, Poster topic: In vitro methodologies & screening
Abstract
Background and Purpose: In vitro models of the respiratory tract are highly differentiated and have been commercially available since 2000. These models have been widely used for toxicological, respiratory infection, tobacco safety, and inhaled drug delivery studies. Availability of tissue models representing the different segments of the respiratory tract were instrumental in gaining insight into the underlying mechanisms of SARS-CoV-2 infection and for screening of anti-viral compounds. Despite these useful applications, there are large databases of animal toxicity data which are not directly translatable to data obtained from the human in vitro airway tissue models due to species differences. Methods: To close this translational gap, cells harvested from both rat and non-human primate (rhesus monkey) tissues were utilized to develop models similar to EpiAirway, the tracheobronchial tissue model offered by MatTek that is cultured with normal human cells. The tissues were characterized for structure (histology), epithelial cell markers (IHC), barrier integrity (transepithelial electrical resistance, TEER measurement), and functionality (inhalation toxicological studies). To verify the reliability of these models worldwide, quality control (QC) data for tissue lots produced in the US and Europe were compared. Results: The animal cell-derived 3D tissues exhibited similar characteristics to human tissues including: well polarized epithelia with physiological TEER values of >300 Ώ*cm2, cilia formation on the apical surfaces, and mucin production mimicking the airway microenvironment. Acute exposure to 4 chemical toxicants (CT) showed species-specific changes in tissue viability and membrane integrity as measured by MTT and TEER assays, respectively. The effective dose concentration that reduces tissue viability by 50% (ED-50) for vinyl acetate (VA) and chloroacetaldehyde (CA) were both <2 mg/tissue and the ED-50 for propylene glycol (PG) was > 20 mg/tissue for all species. However, the ED-50 values for toluene (T) showed differences between the species: 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: CA > VA > T > PG and the vehicle controls (water and corn oil). TEER values from standardized QC tests averaged 1094 ± 325 (n=141 lots) in the US vs. 913 ± 238 (n=64 lots) in Europe. TEER values were not statistically different (p < 0.001). Conclusions: 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 a worldwide alternative approach to animal experimentation.
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Prevalidation of the EpiDerm RhE Model in the EpiSensA Assay for in vitro Skin Sensitization (P06-22)
C. Pellevoisin1, K. Guntur1, C Romero1, J. Markus2, B. De Servi3, M. Meloni3, T. Landry1, S. Letasiova2, M. Klausner1
1 MatTek Corporation, Ashland, Massachusetts, USA, 2 MatTek Europe, Bratislava, Slovakia, 3 VitroScreen, Milano, Italy
Abstract
The Epi2SensA is a similar method to the Epidermal Sensitization Assay (EpiSensA) in the 2024 version of the “OECD TG 442D: In Vitro Skin Sensitisation” addressing the Adverse Outcome Pathway Key Event on Keratinocyte activation. Epi2SensA uses the EpiDerm reconstructed human epidermis (RhE) model from MatTek as the experimental system instead of the LabCyte RhE model in the validated reference method (VRM), EpiSensA. Thanks to the air liquid interface and metabolic activity of RhE models, the method showed better performances for pre/pro-haptens as well as for lipophilic substances compared to already 2D validated methods. The EpiDerm model is already validated in several OECD test guidelines (TG431, TG439, TG498) and is available in the majority of OECD member countries which is an important parameter for the implementation of this OECD test guideline. The Epi2SensA, like the EpiSensA, is based on gene expression quantification of four biomarkers related to the induction of skin sensitization: activating transcription factor 3 (ATF3) and interleukin 8 (IL‐8) which reflect the inflammatory response of keratinocytes; glutamate-cysteine ligase, modifier subunit (GCLM) and DnaJ (Hsp40) homolog, subfamily B, member 4 (DNAJB4) which reflect the induction of cytoprotective gene pathways. The prediction model of the assays is based on the modulation of the expression of the four target genes quantified by quantitative real-time PCR analysis after topical exposure of test chemicals. The chemical is classified as skin sensitizer if the fold induction of the expression of at least one of the genes exceeded the respective cut‐off value: 15‐fold for ATF3, 2‐fold for GCLM or DNAJB4, and 4-fold for IL‐8. The VRM protocol was optimized for the EpiDermTM model with a set of chemicals including positive controls (Clotrimazole, 4-NBB), skin sensitizers (eugenol, 2-aminophenol, imidazolidinyl urea, methyl methacrylate) and non skin sensitizer chemicals (benzyl butyl phthalate, salicylic acid, and sodium dodecyl sulphate). The predictivity of the Epi2SensA has been further verified with the 20 chemicals of the performance standard. The me-too validation of the Epi2SensA is underway with 3 naïve laboratories, Burleson Research Technologies (BRT) in the US, Eurofins BioPharma Product Testing in Germany and the Food and Drug Safety Center Hatano Research Institute (FDSC) in Japan. Project 4.172 of the OECD work plan for the Me-Too validation of the EpiDerm model for the EpiSensA method was submitted by France and Japan.
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