MatTek scientists will be attending and presenting posters at the 2022 European Congress on Alternatives to Animal Testing, in Linz, Austria September 26-28. Read more to see what we’ve been working on and request copies of our posters.
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High throughput intestinal tissues and on newly fabricated 96-well culture plates
(Abstract #177/Poster #65)
Zachary Stevens, Paul Kearney, Mitch Klausner, Alex Armento, Marek Puskar, Seyoum Ayehunie.
Viewing: Wednesday, September 28
High throughput 96-well tissue culture plates that allows culture of three-dimensional physiological tissue systems of small intestinal (SMI) organotypic tissue on a newly fabricated membrane support was 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 as measured by transepithelial electrical resistance (TEER). Histological specimens were collected on weekly basis. To monitor well-to-well tissue reproducibility, TEER measurement was performed every week on all 96-well tissues. Histological and immunological stains showed that tissues fixed at all time points (> 3 months) were structurally similar to the standard EpiIntestinal tissue structure and morphology. The 3D tissues are well polarized and stratified with villi like structural formation. Immunohistochemical staining also shows epithelial marker (CK19), tight junction formation (ZO-1), and brush border marker 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 a viable EpiIntestinal tissue model. Availability of intestinal organotypic tissue that can be cultured for extended period of time (3 months) can be used for chronic exposure experiments. This result extends our previously published tissue utility time point from 42 days to 90 days. In conclusion, the newly fabricated plates support reconstruction of EpiIntestinal tissues for extended time period 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.
Evaluating ocular side effects of systemic medications utilizing the in vitro reconstructed human corneal epithelial tissue model
(Abstract #176/ Poster #75)
Yulia Kaluzhny, Viktor Karetsky, Miriam Kinuthia, Mitchel Klausner, Alex Armento
Viewing: Wednesday, September 28
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.
Evaluation of phototoxic potential and ability to form ROS of selected compounds by using EpiDerm Phototoxicity test.
(Abstract #172/ Poster #77)
Martina Nalezinkova, Lenka Hudecova, Jan Markus, Silvia Letasiova
Viewing: Wednesday, September 28
Evaluation of phototoxic potential of compounds and mixtures isa crucial step in the safety assessment of cosmetic, pesticide and pharmaceutical products absorbing UV and visible light. The validated and regulatory accepted in vitro assay, the 3T3 NRU PT(OECD TG 432), provides high level of sensitivity. However, it has been reported that this monolayer model also generates high rate of false positive results due to the lack of barrier properties naturally appearing in the human skin or other targeted complex tissues. In vitro reconstituted human skin models are increasingly being investigated for their usability in hazard identification and safety testing, because of their organotypic structure with a functional stratum corneum that allows for assessment of bioavailability of topically applied compounds and mixtures.
An in vitro phototoxicity test using the human reconstructed epidermis model EpiDerm™ (EpiDerm™ H3D-PT) has been developed and pre-validated almost 20 years ago and can be used either as standalone method for the phototoxicity testing of topically applied materials, or in combination with the 3T3 NRU PT, to minimize the potentially false positive results from this assay. In June 2021, OECD Test Guideline (TG) 498: In vitro Phototoxicity: Reconstructed Human Epidermis Phototoxicity test method was adopted. OECD TG 498 is a stand-alone method for evaluating the phototoxic potential of a test chemical after topical application in reconstructed human epidermis (RhE) in the presence and absence of simulated sunlight. EpiDerm™ H3D-PT is currently the only tissue model accepted under this test guideline.
The aforementioned method is based on evaluating the viability of exposed and irradiated tissues. However, another key determinant of photoreactive changes in cells is presence of reactive oxygen species (ROS). Therefore, we have selected a set of 13 compounds with known phototoxic potential and analyzed them using EpiDerm™ phototoxicity test. Moreover, we have studied the ability to form ROS using dichlorofluorescin diacetate assay (DCFH-DA) in combination with the UVA irradiation and the exposure to the test compound. Using this approach all 13 compounds were correctly identified as either having or not having phototoxic potential.
These preliminary results confirm that EpiDerm™ H3D-PT is a reliable test for the detection of phototoxicity and prediction of the phototoxic potential of selected substances and suggest that it can be also used to identify the formation of ROS after UVA irradiation. To confirm these results and to further examine potential of such combined method we will apply this approach to an expanded set of phototoxic and non-phototoxic compounds.