Use of a serum-free reconstituted epidermis as a skin pharmacological model

Doucet, Olivier; Robert, Cécile; Zastrow, L.

doi:10.1016/0887-2333(96)00011-2

Cited by 28 publications

(11 citation statements)

References 25 publications

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“…38 and adapted to reconstituted tissues 13 including the 3D-HCE by Doucet et al 11 The assay measures the activity of the mitochondrial enzyme succinyl dehydrogenase, which is expressed in living cells. The signal generated is proportional to the number of metabolically active cells.…”

Section: Mtt Testmentioning

confidence: 99%

“…10 It was proposed as a useful alternative model to the classic Draize test 11,12 for the assessment of eye irritation potential of chemicals and cosmetic products. The toxicokinetic approach described by Doucet et al 12,13 was based on the MTT test procedure previously published on 3D-reconstituted epidermal and corneal models. The MTT test has already been shown to constitute a rapid and cost-effective method of screening for surface toxicity of topical agents using monolayer cell cultures 14 and the procedure used on 3D-models provided a good correlation between in vitro results and MMAS values that correspond to the MAS obtained after 24 hours or more in vivo.…”

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confidence: 99%

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Multiple Endpoint Analysis of the 3D-Reconstituted Corneal Epithelium after Treatment with Benzalkonium Chloride: Early Detection of Toxic Damage

Pauly

Méloni²,

Brignole-Baudouin

et al. 2009

Invest. Ophthalmol. Vis. Sci.

113

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Fluorescence techniques conjugated with confocal microscopy on 3D-reconstructed corneal epithelia were well suited for the investigation of toxicological markers such as cell junction alteration, apoptosis, cell activation, and proliferation and gave relevant results compared with the known human data. They complement the new sensitive MTT test and improve the operating potential of this new, valuable 3D model in ophthalmic toxicology.

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Section: Mtt Testmentioning

confidence: 99%

mentioning

confidence: 99%

Multiple Endpoint Analysis of the 3D-Reconstituted Corneal Epithelium after Treatment with Benzalkonium Chloride: Early Detection of Toxic Damage

Pauly

Méloni²,

Brignole-Baudouin

et al. 2009

Invest. Ophthalmol. Vis. Sci.

113

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“…Several investigators have utilized artificial reconstructed skin or mucosa substitutes for clinical or basic science studies to evaluate the functional aspects of mucosa and the cytotoxic effects to radiation7, 14, 15, 17. These systems utilized an irradiated xenogeneic feeder layer of cells and/or medium that contained animal serum and pituitary extract.…”

mentioning

confidence: 99%

Development of an in vitro model for radiation-induced effects on oral keratinocytes

Tobita

Izumi

Feinberg

2010

International Journal of Oral and Maxillofacial Surgery

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Changes in epithelial cell activity and the production of pro-inflammatory cytokines were examined utilizing an organotypic culture system as an in vitro model to study the effects of radiation on oral keratinocytes to simulate what is thought to occur in radiation-induced oral mucositis. Monolayer cultures of oral keratinocyte were irradiated by varying the dose. Cell injury was assessed using a colony forming efficiency (CFE) assay. Third passage oral keratinocytes were seeded onto AlloDerm ® to form a 3D construct of an ex vivo produced oral mucosa equivalent (EVPOME) which was irradiated with 0, 1, 3 and 8 Gy. Formalin-fixed sections of the EVPOME were used for histology and immunohistochemistry to examine proliferative capacity. Epithelial cell viability of EVPOME was measured by MTT assay. Spent culture medium was used to determine post-radiation pro-inflammatory cytokine production. Basal cells became more swollen and pyknotic as radiation increased, implying loss of cell viability also determined by MTT assay. The number of Ki-67 immunopositive cells and CFE showed negative correlation with radiation, indicating loss of cell proliferative capacity. The production of pro-inflammatory cytokines, IL-1α and IL-8, tended to increase in a radiation dose dependent manner. The EVPOME lacking submocosal cellular components was a useful model. Keywordsradiation-induced oral mucositis; in vitro assay system; organotypic culture; pro-inflammatory cytokine Oral mucositis is a severe side effect for patients undergoing radiation and/or chemotherapy for head and neck tumors, clinically characterized by erythema, ulceration and pseudomembrane formation of the oral mucosa resulting in ulcers producing severe pain and Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 7,14,15,17 . These systems utilized an irradiated xenogeneic feeder layer of cells and/or medium that contained animal serum and pituitary extract. These cells and/or supplements add an unknown element to the culture conditions through cross-contamination with prions and/or slow viruses, and the culture medium for the cells is not chemically well defined. NIH Public AccessIn response to these limitations the authors developed a tissue-engineered three-dimensional (3D) human stratified oral mucosa, an ex vivo produced oral mucosa equivalent (EVPOME) in a culture system devoid of animal serum products, pituitary extracts and a xenogeneic irradiated cell feeder layer 10 . EVPOME can be utilized as a more accurate representation of cellular behavior in situ to assess the effects of irradiation on cell behavior, cell...

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“…[35] based upon the reduction of tetrazolium salts as originally discovered by Altman [36], [37]. The assay was then redeveloped for multilayers by Doucet in 1996 [38], adapted again by Van Goethem in 2006 to improve accuracy [15], and finally reworked by Pauly to further improve apical sensitivity in 2009 [39]. To allow the MTT assay to be used for our larger and curved stratified model, Pauly’s protocol was further modified as follows.…”

Section: Methodsmentioning

confidence: 99%

Development of a Curved, Stratified, In Vitro Model to Assess Ocular Biocompatibility

et al. 2014

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PurposeTo further improve in vitro models of the cornea, this study focused on the creation of a three-dimensional, stratified, curved epithelium; and the subsequent characterization and evaluation of its suitability as a model for biocompatibility testing.MethodsImmortalized human corneal epithelial cells were grown to confluency on curved cellulose filters for seven days, and were then differentiated and stratified using an air-liquid interface for seven days before testing. Varying concentrations of a commercial ophthalmic solution containing benzalkonium chloride (BAK), a known cytotoxic agent, and two relevant ocular surfactants were tested on the model. A whole balafilcon A lens soaked in phosphate buffered saline (BA PBS) was also used to assess biocompatibility and verify the validity of the model. Viability assays as well as flow cytometry were performed on the cells to investigate changes in cell death and integrin expression.ResultsThe reconstructed curved corneal epithelium was composed of 3–5 layers of cells. Increasing concentrations of BAK showed dose-dependent decreased cell viability and increased integrin expression and cell death. No significant change in viability was observed in the presence of the surfactants. As expected, the BA PBS combination appeared to be very biocompatible with no adverse change in cell viability or integrin expression.ConclusionsThe stratified, curved, epithelial model proved to be sensitive to distinct changes in cytotoxicity and is suitable for continued assessment for biocompatibility testing of contact lenses. Our results showed that flow cytometry can provide a quantitative measure of the cell response to biomaterials or cytotoxic compounds for both the supernatant and adherent cell populations. As a specifically designed in vitro model of the corneal epithelium, this quantitative model for biocompatibility at the ocular surface may help improve our understanding of cell-material interactions and reduce the use of animal testing.

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Use of a serum-free reconstituted epidermis as a skin pharmacological model

Cited by 28 publications

References 25 publications

Multiple Endpoint Analysis of the 3D-Reconstituted Corneal Epithelium after Treatment with Benzalkonium Chloride: Early Detection of Toxic Damage

Multiple Endpoint Analysis of the 3D-Reconstituted Corneal Epithelium after Treatment with Benzalkonium Chloride: Early Detection of Toxic Damage

Development of an in vitro model for radiation-induced effects on oral keratinocytes

Development of a Curved, Stratified, In Vitro Model to Assess Ocular Biocompatibility

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