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In JoVE (1)
Other Publications (15)
- The Journal of Investigative Dermatology
- Endocrine Research
- Toxicology in Vitro : an International Journal Published in Association with BIBRA
- Regulatory Toxicology and Pharmacology : RTP
- Archives of Oral Biology
- Toxicology in Vitro : an International Journal Published in Association with BIBRA
- Journal of Virology
- Mutation Research
- Mutation Research
- Toxicology in Vitro : an International Journal Published in Association with BIBRA
- Toxicology
- AIDS Research and Human Retroviruses
- Toxicology in Vitro : an International Journal Published in Association with BIBRA
- Toxicology
- Alternatives to Laboratory Animals : ATLA
Articles by Mitchell Klausner in JoVE
JoVE General
An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model
In this video, we demonstrate the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients.
Other articles by Mitchell Klausner on PubMed
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.
Intranasal Delivery of Recombinant Human Parathyroid Hormone [hPTH (1-34)], Teriparatide in Rats
The aim of this study was to explore the nasal route as an alternative to daily subcutaneous injections of hPTH (1-34). Anesthetized rats were surgically prepared and nasally dosed with aqueous solutions of hPTH (1-34). Plasma samples were assayed by radioimmunoassay and data generated fit to two-(intravenous) and one-(intranasal) compartment pharmacokinetic models using WinNonlin. The toxicity of hPTH (1-34) solution administered to the rats was assessed by screening its effect on transepithelial electrical resistance, potential difference, paracellular marker permeation, tissue viability, and protein leakage using the EpiAirway tissue model. The intranasal absorption of hPTH (1-34) was rapid; the absorption rate constants (alpha) were 33.2+/-24 h(-1) [without bovine serum albumin (BSA)] and 9.8+/-5.1 h(-1) (with 1% BSA). The maximum plasma concentrations (Cmax): 151+/-24 pg/mL (without BSA) and 176+/-37 (with 1% BSA) were attained within approximately 15 min. The intranasal bioavailabilities (Fabs) were 12.1+/-3.4% (without BSA) and 17.6+/-1.5% (with 1% BSA). The hPTH (1-34) formulation administered to the rats had no detrimental effect on the EpiAirway tissue epithelial electrical parameters and functional integrity. Based on the results of this study, the nasal route appears to be a prospective alternative to subcutaneous injections of hPTH (1-34).
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.
Ensuring Quality of in Vitro Alternative Test Methods: Current Practice
In Vitro toxicology methods are being validated and adopted by regulatory agencies for use as alternatives to animal testing. Such methods may use ex vivo tissues or bioconstructs, some of which may be proprietary. Users of the data from these methods need to be reassured that the assays or assay components used in their studies provide consistent, good quality data over time, matching the standards achieved during the validation process. This paper presents an overview of approaches currently used by representatives of a manufacturer and a contract testing laboratory to ensure that the results from in vitro alternative methods are reproducible and of high quality over time. These approaches include full characterization of cells or tissues, sampling of each lot of manufactured bioconstructs for performance, and regular use of controls and benchmark chemicals to provide assurance of consistency of assay performance.
Antimicrobial Barrier of an in Vitro Oral Epithelial Model
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.
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.
Activated CD34-derived Langerhans Cells Mediate Transinfection with Human Immunodeficiency Virus
Langerhans cells (LCs) are a subset of dendritic cells (DCs) that reside within epidermal and mucosal tissue. Because of their location, LCs are potentially the first cells to encounter human immunodeficiency virus (HIV) during sexual transmission. We report that LCs purified from CD34(+)-derived DCs can facilitate the transinfection of target cells but only after activation. Virions were observed in an intracellular compartment that contains several tetraspanins, in addition to the unique LC markers langerin and CD1a. This reveals that the trafficking of HIV within LCs is reminiscent of that which occurs in mature monocyte-derived DCs and that it varies with the activation state of the cell. The observation that activated LCs can mediate transinfection suggests a potential role for these cells in the known increase in HIV transmission associated with sexually transmitted infections that would cause inflammation of the genital lining.
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.
Microvesicating Effects of Sulfur Mustard on an in Vitro Human Skin Model
Bis-(beta-chloroethyl) sulfide (SM) is a potent skin vesicant previously used for chemical warfare. Progress in determination of the mechanistic basis of SM pathology, and development of prophylactic and/or therapeutic countermeasures to SM exposure has been hampered by lack of physiologically relevant models of human skin. The current work evaluated a newly developed tissue engineered full-thickness human skin model in a completely in vitro approach to investigation of SM-induced dermal pathology. The model was first characterized with regard to overall morphology, lipid composition, basement membrane (BM) composition and ultrastructural features that are important targets of SM pathologic activity. Well-developed BM ultrastructural features were observed at the dermal-epidermal junction (DEJ), thus demonstrating successful resolution of a primary deficiency of models previously evaluated for SM studies. Studies were then conducted to evaluate histopathological effects of SM on the model. Good replication of in vivo effects was observed, including apoptosis of basal keratinocytes (KC) and microblister formation at the DEJ. Tissue engineered skin models with well-developed basement membrane structures thus appear to be useful tools for in vitro mechanistic studies of SM vesicant activity and development of preventive/therapeutic approaches for SM pathology.
A Plasmacytoid Dendritic Cell (CD123+/CD11c-) Based Assay System to Predict Contact Allergenicity of Chemicals
A predictive allergenicity test system for assessing the contact allergenicity of chemicals is needed by the cosmetic and pharmaceutical industry to monitor product safety in the marketplace. Development of such non-animal alternative assay systems for skin sensitization and hazard identification has been pursued by policy makers and regulatory agencies. We investigated whether phenotypic and functional changes to a subset of dendritic cells (DC), plasmacytoid DC (pDC), could be used to identify contact allergens. To achieve this goal, normal human DC were generated from CD34+ progenitor cells and cryopreserved. Frozen DC were thawed and the pDC fraction (CD123+/CD11c-) was harvested using FACS sorting. The pDC were cultured, expanded, and exposed to chemical allergens (N=26) or non-allergens (N=22). Concentrations of each chemical that resulted in >50% viability was determined using FACS analysis of propidium iodide stained cells using pDC from 2 to 5 donors. Expression of the surface marker, CD86, which has been implicated in dendritic cell maturation, was used as a marker of allergenicity. CD86 expression increased (> or =1.5-fold) for 25 of 26 allergens (sensitivity=96%) but did not increase for 19 of 22 non-allergens (specificity=86%). In a direct comparison to historical data for the regulatory approved, mouse local lymph node assay (LLNA) for 23 allergens and 22 non-allergens, the pDC method had sensitivity and specificity of 96% and 86%, respectively, while the sensitivity and specificity of the LLNA assay was 83% and 82%, respectively. In conclusion, CD86 expression in pDC appears to be a sensitive and specific indicator to identify contact allergenicity. Such an assay method utilizing normal human cells will be useful for high throughput screening of chemicals for allergenicity.
HIV Type 1 Fails to Trigger Innate Immune Factor Synthesis in Differentiated Oral Epithelium
The oral mucosa is relatively resistant to human immunodeficiency virus type 1 (HIV-1) transmission. The mechanisms contributing to this resistance remain incompletely understood, but may include HIV-induced synthesis of innate immune factors. We used fully differentiated oral epithelium as a surrogate for the oral mucosa in vivo, exposed it to X4- and R5-tropic HIV-1 in culture, and quantified mRNA expression of six innate immune factors. Neither virus increased expression of human beta defensin 2 (hBD-2) mRNA over supernatants from uninfected lymphoblast controls. HIV-1 also failed to induce mRNA of four additional innate immunity-related genes. Similar results were obtained with oral monolayer epithelial cells. Interestingly, the X4-tropic virus inhibited mRNA expression of hBD-2, and of three of the other factors, at higher dosages in the differentiated oral epithelium but not the monolayers. The failure of HIV-1 to induce innate immune factors in the differentiated epithelium was not due to a lack of tissue penetration, as we detected fluorescence-tagged virions up to 30 mum deep from the apical surface. HIV-1 does not trigger de novo innate immune factor synthesis in oral epithelium, pointing to the role of a constitutive innate immunity for protection against HIV-1 in the oral cavity.
Evaluation of EpiDerm Full Thickness-300 (EFT-300) As an in Vitro Model for Skin Irritation: Studies on Aliphatic Hydrocarbons
The aim of this study was to understand the skin irritation effects of saturated aliphatic hydrocarbons (HCs), C9-C16, found jet fuels using in vitro 3-dimensional EpiDerm full thickness-300 (EFT-300) skin cultures. The EFT-300 cultures were treated with 2.5microl of HCs and the culture medium and skin samples were collected at 24 and 48h to measure the release of various inflammatory biomarkers (IL-1alpha, IL-6 and IL-8). To validate the in vitro results, in vivo skin irritation studies were carried out in hairless rats by measuring trans epidermal water loss (TEWL) and erythema following un-occlusive dermal exposure of HCs for 72h. The MTT tissue viability assay results with the EFT-300 tissue show that 2.5microl/tissue ( approximately 4.1microl/cm(2)) of the HCs did not induce any significant changes in the tissue viability for exposure times up to 48h of exposure. Microscopic observation of the EFT-300 cross-sections indicated that there were no obvious changes in the tissue morphology of the samples at 24h, but after 48h of exposure, tridecane, tetradecane and hexadecane produced a slight thickening and disruption of stratum corneum. Dermal exposures of C12-C16 HCs for 24h significantly increased the expression of IL-1alpha in the skin as well as in the culture medium. Similarly, dermal exposure of all HCs for 24h significantly increased the expression of interleukin-6 (IL-6) and IL-8 in the skin as well as in the culture medium in proportion to the HC chain length. As the exposure time increased to 48h, IL-6 concentrations increased 2-fold compared to the IL-6 values at 24h. The in vivo skin irritation data also showed that both TEWL and erythema scores increased with increased HCs chain length (C9-C16). In conclusion, the EFT-300 showed that the skin irritation profile of HCs was in the order of C9C10C11C12
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-1β, 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-1β 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-1β release appear to be useful endpoints for preclinical screening of topically applied chemicals and formulations for their vaginal irritation potential.
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.
