Document resource
Purpose (i) To develop a Reconstructed human Cornea-like Epithelium (RhCE) model for assessing ocular irritation in vitro. (ii) To demonstrate technical proficiency of the model as an in vitro irritation test method according to OECD Test Guideline 492. (iii) To employ the RhCE model for evaluating potential irritancy of medical devices intended for corneal storage.Methods Immortalized Human Primary Corneal Epithelium Cells were cultured on PET microporous inserts in a chemically defined medium. At confluence, cells were exposed to Air-Liquid Interface (ALI) to induce the formation of an epithelium-like barrier. Functional parameters of the model were established according to OECD 492 Guideline, investigating a) Morphology, b) Viability, c) Barrier Function, and d) Reproducibility. Morphological evaluation was conducted using standard histological techniques. Tissue viability was quantified with colorimetric MTT assay. To investigate epithelial barrier integrity and function, transepithelial electrical resistance (TEER) was recorded and IC50 value (i.e. the SDS concentration that reduced by 50% the viability of the model) was estimated with MTT assay. Reproducibility of the RhCE model as a test method was assessed by calculating inter-batch standard deviation of tissue models treated with DPBS (negative control) and neat ethanol (positive control). Technical proficiency of the model was verified using a set of reference chemical compounds covering the whole range of irritancy categories. Negative and positive controls were simultaneously included in each test to establish% viability (with respect to the negative control) and sensitivity (with respect to the positive control) of the developed RhCE model. The medical devices Tissue-C, Carry-C, Eusol-C and Kerasave, intended for human corneal storage, were also assessed for irritancy by employing the model.Results Immortalized human corneal epithelium cells reached confluence within 3–4 days in culture in the PET inserts and developed an epithelium-like structure when cultured under ALI for additional 4–7 days. Sections of the RhCE model exhibited stratified cellular organization, containing viable cellular layers and a non-keratinized surface. The viability of the RhCE model was maintained up to 15 days of the total culture duration. An increase in TEER recording was observed since ALI was introduced and increased in an ALI time-dependent manner, as an indicator of the growing barrier. Higher TEER values were obtained when ALI was introduced immediately after confluence rather than postponing it. Estimated IC50 values were found to fall within the range of the IC50 values of the validated reference RhCE test methods. Prolonged ALI duration resulted in increased IC50 in an ALI time-dependent manner. RhCE proficiency studies demonstrated the model’s ability to differentiate between irritants and non-irritants. Tested medical devices for corneal storage were deemed as non-irritant by the developed in vitro RhCE irritation test method.Conclusions Viability, barrier function, morphology and reproducibility of the novel RhCE model are consistent with OECD 492 Guidelines. The developed RhCE model test method shows promise for advancing to pre-validation and validation studies.