Oxidative stress in corneal injuries of different origin: Utilization of 3D human corneal epithelial tissue model.

The purpose of the current work was to utilize a three dimensional (3D) corneal epithelial tissue model to study dry eye disease and oxidative stress-related corneal epithelial injuries for the advancement of ocular therapeutics. Air-liquid interface cultures of normal human corneal epithelial cells were used to produce 3D corneal epithelial tissues appropriate for physiologically relevant exposure to environmental factors. Oxidative stress was generated by exposing the tissues to non-toxic doses of ultraviolet radiation (UV), hydrogen peroxide, vesicating agent nitrogen mustard, or desiccating conditions that stimulated morphological, cellular, and molecular changes relevant to dry eye disease. Corneal specific responses, including barrier function, tissue viability, reactive oxygen species (ROS) accumulation, lipid peroxidation, cytokine release, histology, and gene expression were evaluated. 3D corneal epithelial tissue model structurally and functionally reproduced key features of molecular responses of various types of oxidative stress-induced ocular damage. The most pronounced effects for different treatments were: UV irradiation - intracellular ROS accumulation; hydrogen peroxide exposure - barrier impairment and IL-8 release; nitrogen mustard exposure - lipid peroxidation and IL-8 release; desiccating conditions - tissue thinning, a decline in mucin expression, increased lipid peroxidation and IL-8 release. Utilizing a PCR gene array, we compared the effects of corneal epithelial damage on the expression of 84 oxidative stress-responsive genes and found specific molecular responses for each type of damage. The topical application of lubricant eye drops improved tissue morphology while decreasing lipid peroxidation and IL-8 release from tissues incubated at desiccating conditions. This model is anticipated to be a valuable tool to study molecular mechanisms of corneal epithelial damage and aid in the development of therapies against dry eye disease, oxidative stress- and vesicant-induced ocular injuries.

Human Primary Cell-Based Organotypic Microtissues for Modeling Small Intestinal Drug Absorption.

PURPOSE: The study evaluates the use of new in vitro primary human cell-based organotypic small intestinal (SMI) microtissues for predicting intestinal drug absorption and drug-drug interaction. METHODS: The SMI microtissues were reconstructed using human intestinal fibroblasts and enterocytes cultured on a permeable support. To evaluate the suitability of the intestinal microtissues to model drug absorption, the permeability coefficients across the microtissues were determined for a panel of 11 benchmark drugs with known human absorption and Caco-2 permeability data. Drug-drug interactions were examined using efflux transporter substrates and inhibitors. RESULTS: The 3D-intestinal microtissues recapitulate the structural features and physiological barrier properties of the human small intestine. The microtissues also expressed drug transporters and metabolizing enzymes found on the intestinal wall. Functionally, the SMI microtissues were able to discriminate between low and high permeability drugs and correlated better with human absorption data (r2 = 0.91) compared to Caco-2 cells (r2 = 0.71). Finally, the functionality of efflux transporters was confirmed using efflux substrates and inhibitors which resulted in efflux ratios of >2.0 fold and by a decrease in efflux ratios following the addition of inhibitors. CONCLUSION: The SMI microtissues appear to be a useful pre-clinical tool for predicting drug bioavailability of orally administered drugs.

The EpiOcular Eye Irritation Test (EIT) for hazard identification and labelling of eye irritating chemicals: protocol optimisation for solid materials and the results after extended shipment.

The 7th Amendment to the EU Cosmetics Directive and the EU REACH Regulation have reinforced the need for in vitro ocular test methods. Validated in vitro ocular toxicity tests that can predict the human response to chemicals, cosmetics and other consumer products are required for the safety assessment of materials that intentionally, or inadvertently, come into contact with the eye. The EpiOcular Eye Irritation Test (EIT), which uses the normal human cell-based EpiOcular™ tissue model, was developed to address this need. The EpiOcular-EIT is able to discriminate, with high sensitivity and accuracy, between ocular irritant/corrosive materials and those that require no labelling. Although the original EpiOcular-EIT protocol was successfully pre-validated in an international, multicentre study sponsored by COLIPA (the predecessor to Cosmetics Europe), data from two larger studies (the EURL ECVAM-COLIPA validation study and an independent in-house validation at BASF SE) resulted in a sensitivity for the protocol for solids that was below the acceptance criteria set by the Validation Management Group (VMG) for eye irritation, and indicated the need for improvement of the assay's sensitivity for solids. By increasing the exposure time for solid materials from 90 minutes to 6 hours, the optimised EpiOcular-EIT protocol achieved 100% sensitivity, 68.4% specificity and 84.6% accuracy, thereby meeting all the acceptance criteria set by the VMG. In addition, to satisfy the needs of Japan and the Pacific region, the EpiOcular-EIT method was evaluated for its performance after extended shipment and storage of the tissues (4-5 days), and it was confirmed that the assay performs with similar levels of sensitivity, specificity and reproducibility in these circumstances.

Development of the EpiOcular(TM) eye irritation test for hazard identification and labelling of eye irritating chemicals in response to the requirements of the EU cosmetics directive and REACH legislation.

The recently implemented 7th Amendment to the EU Cosmetics Directive and the EU REACH legislation have heightened the need for in vitro ocular test methods. To address this need, the EpiOcular(TM) eye irritation test (EpiOcular-EIT), which utilises the normal (non-transformed) human cell-based EpiOcular tissue model, has been developed. The EpiOcular-EIT prediction model is based on an initial training set of 39 liquid and 21 solid test substances and uses a single exposure period and a single cut-off in tissue viability, as determined by the MTT assay. A chemical is classified as an irritant (GHS Category 1 or 2), if the tissue viability is ≤ 60%, and as a non-irritant (GHS unclassified), if the viability is > 60%. EpiOcular-EIT results for the training set, along with results for an additional 52 substances, which included a range of alcohols, hydrocarbons, amines, esters, and ketones, discriminated between ocular irritants and non-irritants with 98.1% sensitivity, 72.9% specificity, and 84.8% accuracy. To ensure the long-term commercial viability of the assay, EpiOcular tissues produced by using three alternative cell culture inserts were evaluated in the EpiOcular-EIT with 94 chemicals. The assay results obtained with the initial insert and the three alternative inserts were very similar, as judged by correlation coefficients (r²) that ranged from 0.82 to 0.96. The EpiOcular-EIT was pre-validated in 2007/2008, and is currently involved in a formal, multi-laboratory validation study sponsored by the European Cosmetics Association (COLIPA) under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The EpiOcular-EIT, together with EpiOcular's long history of reproducibility and proven utility for ultra-mildness testing, make EpiOcular a useful model for addressing current legislation related to animal use in the testing of potential ocular irritants.

In vitro reconstructed 3D corneal tissue models for ocular toxicology and ophthalmic drug development.

Testing of all manufactured products and their ingredients for eye irritation is a regulatory requirement. In the last two decades, the development of alternatives to the in vivo Draize eye irritation test method has substantially advanced due to the improvements in primary cell isolation, cell culture techniques, and media, which have led to improved in vitro corneal tissue models and test methods. Most in vitro models for ocular toxicology attempt to reproduce the corneal epithelial tissue which consists of 4-5 layers of non-keratinized corneal epithelial cells that form tight junctions, thereby limiting the penetration of chemicals, xenobiotics, and pharmaceuticals. Also, significant efforts have been directed toward the development of more complex three-dimensional (3D) equivalents to study wound healing, drug permeation, and bioavailability. This review focuses on in vitro reconstructed 3D corneal tissue models and their utilization in ocular toxicology as well as their application to pharmacology and ophthalmic research. Current human 3D corneal epithelial cell culture models have replaced in vivo animal eye irritation tests for many applications, and substantial validation efforts are in progress to verify and approve alternative eye irritation tests for widespread use. The validation of drug absorption models and further development of models and test methods for many ophthalmic and ocular disease applications is required.

In vitro three-dimensional organotypic culture models of the oral mucosa.

Three-dimensional, organotypic models of the oral mucosa have been developed to study a wide variety of phenomena occurring in the oral cavity. Although a number of models have been developed in academic research labs, only a few models have been commercialized. Models from academic groups offer a broader range of phenotypes while the commercial models are more focused on the oral and gingival mucosa. The commercialized models are manufactured under highly controlled conditions and meet the requirements of quality standards, which leads to high levels of reproducibility. These in vitro models have been used to evaluate the irritancy of oral care products such as toothpastes, mouthwashes, and mucoadhesives. The effects of cigarette smoke on oral cavity tissues have been studied and compared to those of e-cigarettes. Oral tissue models have facilitated investigation of the mechanisms of oral mucositis and oral candidiasis and have been used to examine transbuccal drug delivery rates and the absorption of nanoparticles. Infection studies have investigated the effects of HIV-1 along with the effects of commensal and pathogenic bacteria. More recently, a differentiated oral tissue model has been shown to express the ACE2 receptor, which is known to be important for the receptor-mediated entry of the SARS-CoV-2 coronavirus into human cells and tissues. Hence, oral mucosal models may find application in determining whether viral infection of the oral mucosa is possible and whether such infection has implications vis-a-vis the current COVID-19 pandemic. As is apparent, these models are used in a broad variety of applications and often offer advantages versus animal models in terms of reproducibility, avoiding species extrapolation, and the ethical concerns related to human and animal experimentation. The goals of this paper are to review commercially available models of the human buccal and gingival mucosa and highlight their use to gain a better understanding of a broad range of phenomena affecting tissues in the oral cavity.

Human small intestinal organotypic culture model for drug permeation, inflammation, and toxicity assays.

The gastrointestinal tract (GIT), in particular, the small intestine, plays a significant role in food digestion, fluid and electrolyte transport, drug absorption and metabolism, and nutrient uptake. As the longest portion of the GIT, the small intestine also plays a vital role in protecting the host against pathogenic or opportunistic microbial invasion. However, establishing polarized intestinal tissue models in vitro that reflect the architecture and physiology of the gut has been a challenge for decades and the lack of translational models that predict human responses has impeded research in the drug absorption, metabolism, and drug-induced gastrointestinal toxicity space. Often, animals fail to recapitulate human physiology and do not predict human outcomes. Also, certain human pathogens are species specific and do not infect other hosts. Concerns such as variability of results, a low throughput format, and ethical considerations further complicate the use of animals for predicting the safety and efficacy xenobiotics in humans. These limitations necessitate the development of in vitro 3D human intestinal tissue models that recapitulate in vivo-like microenvironment and provide more physiologically relevant cellular responses so that they can better predict the safety and efficacy of pharmaceuticals and toxicants. Over the past decade, much progress has been made in the development of in vitro intestinal models (organoids and 3D-organotypic tissues) using either inducible pluripotent or adult stem cells. Among the models, the MatTek's intestinal tissue model (EpiIntestinal™ Ashland, MA) has been used extensively by the pharmaceutical industry to study drug permeation, metabolism, drug-induced GI toxicity, pathogen infections, inflammation, wound healing, and as a predictive model for a clinical adverse outcome (diarrhea) to pharmaceutical drugs. In this paper, our review will focus on the potential of in vitro small intestinal tissues as preclinical research tool and as alternative to the use of animals.

Intralaboratory and interlaboratory evaluation of the EpiDerm 3D human reconstructed skin micronucleus (RSMN) assay.

A novel in vitro human reconstructed skin micronucleus (RSMN) assay has been developed using the EpiDerm 3D human skin model [R. D. Curren, G. C. Mun, D. P. Gibson, and M. J. Aardema, Development of a method for assessing micronucleus induction in a 3D human skin model EpiDerm, Mutat. Res. 607 (2006) 192-204]. The RSMN assay has potential use in genotoxicity assessments as a replacement for in vivo genotoxicity assays that will be banned starting in 2009 according to the EU 7th Amendment to the Cosmetics Directive. Utilizing EpiDerm tissues reconstructed with cells from four different donors, intralaboratory and interlaboratory reproducibility of the RSMN assay were examined. Seven chemicals were evaluated in three laboratories using a standard protocol. Each chemical was evaluated in at least two laboratories and in EpiDerm tissues from at least two different donors. Three model genotoxins, mitomycin C (MMC), vinblastine sulfate (VB) and methyl methanesulfonate (MMS) induced significant, dose-related increases in cytotoxicity and MN induction in EpiDerm tissues. Conversely, four dermal non-carcinogens, 4-nitrophenol (4-NP), trichloroethylene (TCE), 2-ethyl-1,3-hexanediol (EHD), and 1,2-epoxydodecane (EDD) were negative in the RSMN assay. Results between tissues reconstructed from different donors were comparable. These results indicate the RSMN assay using the EpiDerm 3D human skin model is a promising new in vitro genotoxicity assay that allows evaluation of chromosome damage following "in vivo-like" dermal exposures.

Further development of the EpiDerm 3D reconstructed human skin micronucleus (RSMN) assay.

The upcoming ban on testing of cosmetics in animals by the European Union's 7th Amendment to the Cosmetics Directive will require genotoxicity safety assessments of cosmetics ingredients and final formulations to be based primarily on in vitro genotoxicity tests. The current in vitro test battery produces an unacceptably high rate of false positives, and used by itself would effectively prevent the use and development of many ingredients that are actually safe for human use. To address the need for an in vitro test that is more predictive of genotoxicity in vivo, we have developed an in vitro micronucleus assay using a three-dimensional human reconstructed skin model (EpiDerm) that more closely mimics the normal dermal exposure route of chemicals. We have refined this model and assessed its ability to predict genotoxicity of a battery of chemicals that have been previously classified as genotoxins or non-genotoxins based on in vivo rodent skin tests. Our reconstructed skin micronucleus assay correctly identified 7 genotoxins and 5 non-genotoxins, demonstrating its potential to have a higher predictive value than currently available in vitro genotoxicity tests, and its utility as part of a comprehensive in vitro genotoxicity testing strategy.

Prevalidation of an Acute Inhalation Toxicity Test Using the EpiAirway In Vitro Human Airway Model.

Introduction: Knowledge of acute inhalation toxicity potential is important for establishing safe use of chemicals and consumer products. Inhalation toxicity testing and classification procedures currently accepted within worldwide government regulatory systems rely primarily on tests conducted in animals. The goal of the current work was to develop and prevalidate a nonanimal (in vitro) test for determining acute inhalation toxicity using the EpiAirway™ in vitro human airway model as a potential alternative for currently accepted animal tests. Materials and Methods: The in vitro test method exposes EpiAirway tissues to test chemicals for 3 hours, followed by measurement of tissue viability as the test endpoint. Fifty-nine chemicals covering a broad range of toxicity classes, chemical structures, and physical properties were evaluated. The in vitro toxicity data were utilized to establish a prediction model to classify the chemicals into categories corresponding to the currently accepted Globally Harmonized System (GHS) and the Environmental Protection Agency (EPA) system. Results: The EpiAirway prediction model identified in vivo rat-based GHS Acute Inhalation Toxicity Category 1-2 and EPA Acute Inhalation Toxicity Category I-II chemicals with 100% sensitivity and specificity of 43.1% and 50.0%, for GHS and EPA acute inhalation toxicity systems, respectively. The sensitivity and specificity of the EpiAirway prediction model for identifying GHS specific target organ toxicity-single exposure (STOT-SE) Category 1 human toxicants were 75.0% and 56.5%, respectively. Corrosivity and electrophilic and oxidative reactivity appear to be the predominant mechanisms of toxicity for the most highly toxic chemicals. Conclusions: These results indicate that the EpiAirway test is a promising alternative to the currently accepted animal tests for acute inhalation toxicity.

New Human Organotypic Corneal Tissue Model for Ophthalmic Drug Delivery Studies.

Purpose: The purpose of the current work was to develop a physiologically relevant, in vitro human three-dimensional (3D) corneal epithelial tissue model for use in ophthalmic drug development. Methods: Normal human corneal epithelial cells were cultured at the air-liquid interface to produce the 3D corneal tissue model. Corneal barrier was determined by measuring transepithelial electrical resistance (TEER). Quantitative PCR arrays were utilized to investigate expression of 84 phase I/II metabolizing enzymes and 84 drug transporter genes. Permeability was evaluated using model compounds with a wide range of hydrophobicity, molecular weight, and excipients. Finally, different formulations of latanoprost and bimatoprost were administered and drug absorption and tissue viability and integrity were investigated. Results: Histologic assessment and TEER of the corneal tissue model revealed tissue structure, thickness, and barrier formation (1000 ± 146 Ω·cm2) comparable to native human corneal epithelium. The 3D corneal tissue expressed tight junctions, mucins, and key corneal epithelial detoxification enzymes. Drug-metabolizing enzyme and transporter gene expression in 3D corneal tissue and excised human corneal epithelium were highly correlated (r2 = 0.87). Coefficients of permeation for model drugs in the tissue model and excised rabbit corneas also showed a high correlation (r2 = 0.94). As expected, latanoprost and bimatoprost free acids had much lower permeability (Papp = 1.2 × 10-6 and 1.9 × 10-6) than the corresponding prodrugs (Papp = 2.5 × 10-5 and 5.6 × 10-5), respectively. The presence of 0.02% benzalkonium chloride in ophthalmic formulations significantly affected tissue barrier and viability. Conclusions: The newly developed 3D corneal tissue model appears to be very useful for evaluation of corneal drug permeability and safety during ophthalmic drug development.

Organotypic human oral tissue models for toxicological studies.

Three-dimensional models of the human oral epithelia have been developed to test the irritation of oral-care products and to provide systems to study the pathology of the oral cavity. The in vitro tissue models, cultured using normal oral epithelial cells and serum free medium, adopt a buccal or gingival phenotype. The buccal tissue (designated ORL-200) is 8-12 cell layers thick and non-cornified; the gingival tissue (designated GIN-100) is 9-13 layers thick and cornified at the apical surface. The tissues express cytokeratins 13 and 14 similar to their corresponding native oral tissues. The MTT viability assay was used to assess inter-lot and intra-lot reproducibility. The MTT average intra-lot coefficient of variation (CV) was less than 10% for both tissues and the time required to reduce tissue viability by 50% (ET-50) following application of 1% Triton-X 100 averaged 1.02+/-0.33 h (n=26) and 7.97+/-0.80 h (n=14) for the buccal and gingival tissues, respectively. The utility of the buccal tissue for irritation studies was examined by testing prototype dentifrice formulations and commercially available products including mouthwashes, toothpastes, and oral cleansers. Use of the MTT ET-50 assay and cytokine release clearly differentiated between the formulations and the oral care products. In conclusion, the oral tissue models represent highly reproducible, non-animal means to screen the irritation potential of newly developed oral care products and should be useful to study the innate immunity, biology, and pathology of the oral mucosa.

Organotypic human vaginal-ectocervical tissue model for irritation studies of spermicides, microbicides, and feminine-care products.

A three-dimensional organotypic vaginal-ectocervical (VEC) tissue model has been developed to test the irritation of topically applied spermicides, microbicides, and vaginal-care products. The in vitro tissue model was reconstructed using normal VEC epithelial cells and is well stratified, containing differentiated basal, suprabasal, intermediate, and superficial cell layers similar to in vivo tissue. The intermediate and superficial cell layers contain glycogen, and the expression of cytokeratins 13 and 14 in the tissue also parallels that of native tissue. The MTT viability assay and histological assessment were used to test inter-lot and intra-lot reproducibility. The MTT average intra-lot coefficient of variation (CV) was less than 10% and the time required to reduce tissue viability by 50% (ET-50) following application of 1% Triton X-100 averaged 1.25+/-0.24h (n=23) upon completion of the 11-day culture period and 1.30 h+/- 0.19 for the same tissues stored overnight at 4 degrees C on agarose gels. The utility of the VEC model for irritation studies was examined by testing commercially available products using the MTT assay and histological assessment. The average ET-50 values ranged between 1.8 and 2.7h for feminine washes, 3.9-6.7 h for spermicides, 6.8-18 h for anti-itch creams, and >18 h for douches, lubricants, and anti-fungal creams. Studies of cytokines released from VEC cultures following product application showed that elevated concentrations of IL-1alpha and IL-1beta were associated with toxicity of test materials. In conclusion, the VEC tissue model is a highly reproducible, non-animal means to assess the irritation of contraceptives, microbicides, and vaginal-care products.

Antimicrobial barrier of an in vitro oral epithelial model.

OBJECTIVE: Oral epithelia function as a microbial barrier and are actively involved in recognizing and responding to bacteria. Our goal was to examine a tissue engineered model of buccal epithelium for its response to oral bacteria and proinflammatory cytokines and compare the tissue responses with those of a submerged monolayer cell culture. DESIGN: The tissue model was characterized for keratin and beta-defensin expression. Altered expression of beta-defensins was evaluated by RT-PCR after exposure of the apical surface to oral bacteria and after exposure to TNF-alpha in the medium. These were compared to the response in traditional submerged oral epithelial cell culture. RESULTS: The buccal model showed expression of differentiation specific keratin 13, hBD1 and hBD3 in the upper half of the tissue; hBD2 was not detected. hBD1 mRNA was constitutively expressed, while hBD2 mRNA increased 2-fold after exposure of the apical surface to three oral bacteria tested and hBD3 mRNA increased in response to the non-pathogenic bacteria tested. In contrast, hBD2 mRNA increased 3-600-fold in response to bacteria in submerged cell culture. HBD2 mRNA increased over 100-fold in response to TNF-alpha in the tissue model and 50-fold in submerged cell culture. Thus, the tissue model is capable of upregulating hBD2, however, the minimal response to bacteria suggests that the tissue has an effective antimicrobial barrier due to its morphology, differentiation, and defensin expression. CONCLUSIONS: The oral mucosal model is differentiated, expresses hBD1 and hBD3, and has an intact surface with a functional antimicrobial barrier.

Characterization of a Hormone-Responsive Organotypic Human Vaginal Tissue Model: Morphologic and Immunologic Effects.

Estrogen and progesterone regulate proliferation and differentiation of epithelial cells in the female genital tract. We investigated the effects of these hormones on reconstructed human organotypic vaginal epithelial tissue models (EpiVaginal). We ascertained that epithelial cells in the tissue models express estrogen and progesterone receptors. Treatment with estradiol-17ß (E(2)) significantly increased epithelium thickness and transepithelial electrical resistance (TEER), whereas progesterone (P) treatment resulted in thinning of the epithelium and decreased TEER when compared with untreated controls. Exposure to E(2) increased (1) the expression of the progesterone receptor B (PR-B), (2) accumulation of glycogen in suprabasal cells, (3) epithelial differentiation, and (4) the expression of a number of gene pathways associated with innate immunity, epithelial differentiation, wound healing, and antiviral responses. These findings indicate that EpiVaginal tissues are hormone responsive and can be used to study the role of female reproductive hormones in innate immune responses, microbial infection, and drug delivery in the vaginal mucosa.

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model.

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&A) for liquids, 100.0/68.4/ and 84.6% SS&A for solids, and 97.6/68.3/ and 83.1% for overall SS&A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Gene expression profile of tissue engineered skin subjected to acute barrier disruption.

The main function of the skin is to protect the body from infection, dehydration, and other environmental insults by creating an impermeable barrier of cornified cell layers, the stratum corneum. In contrast to cells in culture, tissue-engineered skin equivalents contain well-developed basal, spinous, granular, and cornified cell layers providing an excellent model to study the tissue response to barrier disruption. After 7 d of culture at the air-liquid interface the barrier of the tissues was disrupted by short exposure to acetone and the global gene expression profile of the tissues was evaluated using DNA microarrays. We found that tissue-engineered skin responds to barrier disruption by a two-wave dynamic response. Early on, the cells upregulate signal transducing, stress, proliferation, and inflammation genes to protect the tissue and possibly to communicate the damage to the immune system and neighboring tissues. At later times, pro-inflammatory cytokines and some growth-related genes are significantly reduced but enzymes that participate in lipid synthesis increase, suggesting that the epidermal cells attempt to restore the lost barrier. Quantitative immunostaining for the proliferation antigen Ki67 revealed that barrier disruption by acetone increased proliferation by 4-fold in agreement with the microarray data and previous in vivo studies. Our work suggests that functional genomics may be used in tissue engineering to understand tissue development, wound regeneration, and response to environmental stimuli. A better understanding of engineered tissues at the molecular level may facilitate their application in the clinic and as biosensors for toxicologic testing.

Quality assurance for in vitro alternative test methods: quality control issues in test kit production.

In vitro toxicology methods are being adopted by regulatory agencies worldwide. Many of these methods have been validated by using proprietary materials, often in the form of test kits. Guidelines for the use of Good Laboratory Practice methods for in vitro methods have been proposed. However, users of the data from these methods also need to be reassured that the proprietary materials and the test kits will provide consistent, good quality data over time, not just during the validation process. This paper presents an overview of the methods currently used by representatives of kit manufacturers and contract testing laboratories to ensure that the results from methods that utilise test kits are reproducible over time and across different types of test materials. This information will be valuable as a basis for future discussion on the need for formalised oversight of the quality of these materials.

Full Thickness EpiDerm: a dermal-epidermal skin model to study epithelial-mesenchymal interactions.

Unlike previous dermal-epidermal models, Full Thickness EpiDerm is cultured in an easily manipulated cell culture insert, and the tissue extends from wall to wall. In terms of ease of use, these characteristics greatly facilitate the testing of potential allergens or irritants in that direct topical application is possible. Topical exposure to the common surfactant, 1% Triton X-100, resulted in MTT tissue viability dose-response curves that fell within the normal range of the keratinocyte-only tissue, EpiDerm. Currently, in order to produce a standardised, reproducible organotypic tissue, all lots of EpiDerm are compared to a reference database of effective time-50 (ET-50) values, i.e. the time of exposure after which viability is reduced to 50% following exposure to 100 microl of Triton X-100. The database average (184 tissue lots) is 6.74 +/- 0.99 hours (+/- 1 SD); initial lots of the full thickness tissue, tested in an identical manner, averaged 7.79 +/- 1.24 hours (n = 11). Histological cross-sections of the full thickness tissue showed an epidermal layer that is very similar to EpiDerm and native epidermis on a fibroblast-containing collagen matrix dermis-like layer. Irradiation of the tissue with ultraviolet light induced an increase in collagenase (MMP-1) release. Based on these initial results, investigation of the tissue response to stimuli specifically affecting the dermis or epidermal/dermal "cross-talk" will probably prove informative.

Development of an in vitro alternative assay method for vaginal irritation.

The vaginal mucosa is commonly exposed to chemicals and therapeutic agents that may result in irritation and/or inflammation. In addition to acute effects, vaginal irritation and inflammation can make women more susceptible to infections such as HIV-1 and herpes simplex virus 2 (HSV-2). Hence, the vaginal irritation potential of feminine care formulations and vaginally administered therapeutic agents is a significant public health concern. Traditionally, testing of such materials has been performed using the rabbit vaginal irritation (RVI) assay. In the current study, we investigated whether the organotypic, highly differentiated EpiVaginal™ tissue could be used as a non-animal alternative to the RVI test. The EpiVaginal tissue was exposed to a single application of ingredients commonly found in feminine hygiene products and the effects on tissue viability (MTT assay), barrier disruption (measured by transepithelial electrical resistance, TEER and sodium fluorescein (NaFl) leakage), and inflammatory cytokine release (interleukin (IL)-1α, IL-1ß, IL-6, and IL-8) patterns were examined. When compared to untreated controls, two irritating ingredients, nonoxynol 9 and benzalkonium chloride, reduced tissue viability to <40% and TEER to <60% while increasing NaFl leakage by 11-24% and IL-1α and IL-1ß release by >100%. Four other non-irritating materials had minimal effects on these parameters. Assay reproducibility was confirmed by testing the chemicals using three different tissue production lots and by using tissues reconstructed from cells obtained from three different donors. Coefficients of variation between tissue lots reconstructed with cells obtained from the same donor or lots reconstructed with cells obtained from different donors were less than 10% and 12%, respectively. In conclusion, decreases in tissue viability and barrier function and increases in IL-1α and IL-1ß release appear to be useful endpoints for preclinical screening of topically applied chemicals and formulations for their vaginal irritation potential.