Application of the Reconstructed Rabbit Corneal Epithelium Model to Assess the <i>in Vitro</i> Eye Irritancy Test of Chemicals

Abstract

The rabbit corneal epithelium model (RCE model) was developed as a three-dimensional in vitro model to replace animal testing for the assessment of eye irritation. In the model, a stratiˆed culture of rabbit corneal epithelial cells is grown at the air-liquid interface on collagen gel that acts as a parabasal membrane. Histological cross-sections show that the structure of the RCE model closely parallels that of the rabbit corneal epithelium. The eye irritation potency of test samples is estimated from the measurement of viability using the MTT assay in conjunction with the RCE model. A set of 30 chemicals belonging to diŠerent families with known in vivo Draize score was investigated with the in vitro eye irritation test using the RCE model in order to internally validate the protocol. Use of the RCE model at concentrations of 0.05％, 0.50％, and 1.00％ and the calculation of the IC 50 and percentage of viability allowed the irritants to be divided into four classes. The performance of the in vitro eye irritation test at a concentration of 0.50％ using the RCE model was characterized by good sensitivity (92.3％), good speciˆcity (100％), and good accuracy (93.3％) compared with the irritation classiˆcation predicted by in vivo Draize score at concentrations of 10％ and 100％. These results indicate that the RCE model may provide a useful and sensitive in vitro eye irritation test as an alternative method to the Draize test.

An overview of current techniques for ocular toxicity testing

“…In vitro cultured epithelium is capable of retaining the in vivo repair mechanisms found in the native cornea (Davila et al, 1998), although these mechanisms are not always given the level of attention they deserve (Dholakiya and Barile, 2013). Epithelial models can be constructed from animal cells (commonly SIRC cells (Ubels and Clousing, 2005)) such as in the STE test, human epidermal cells, or human corneal cells, which are usually cultured in defined medium on cell culture membranes using air-lifting techniques (Alépée et al, 2013;Cotovio et al, 2007;Kaluzhny et al, 2011;Matsuda et al, 2009) to create a 3D stratified epithelium. Cytotoxicity following topical exposure is generally used as an endpoint (Curren and Harbell, 2002), and epithelial models have the potential to identify non-classified/nonirritating substances from mild irritants (Scott et al, 2010).…”

“…Thus, rabbit corneal cells or mouse fibroblasts are often utilized as an alternative source. Matsuda et al (2009) cultured rabbit corneal epithelium (RCE) cells onto collagen hydrogels, which acts as a perabasal membrane. To validate the model 30 chemicals with known degrees of eye irritation (from Draize testing), ranging from non-irritating to severely irritating were tested.…”

“…To validate the model 30 chemicals with known degrees of eye irritation (from Draize testing), ranging from non-irritating to severely irritating were tested. Inconsistencies occurred when testing acids and alcohols, which was thought to be due to a pH dilution, the volatility of the alcohol, or a reaction with the buffer solution prior to testing (Matsuda et al, 2009).…”

“…Test substances are directly applied to the models, and cytotoxicity is measured using MTT. Substances that cause the most rapid injury to cells generally have higher irritation potentials (Matsuda et al, 2009). The original protocol has since been developed into a single time-point protocol known as the EpiOcular TM eye irritation test (EIT) (Pfannenbecker et al, 2012).…”

3',8"-biisokaempferide, a cytotoxic biflavonoid and other chemical constituents of Nanuza plicata (Velloziaceae)

“…Models using cultured cells to evaluate the cytotoxicity of chemicals have the advantage of being simple and quick with a low evaluation cost. 22 The investigation of new molecules for glioblastoma treatment is relevant because despite current advances in therapy, including surgical resection followed by radiation and chemotherapy, the prognosis for patients remains poor. 23 In this paper, the cytotoxicity of 1 to human glioblastoma GL-15 cells was evaluated in vitro.…”