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HCE-T cell-based corneal epithelial model: Scale- down to 96-well insert plates

HCE-T cell line is the most extensively characterized human-derived cell line of corneal epithelium. 3D HCE-T models are commonly cultured on 12-well insert plates and are widely used for permeability and biocompatibility testing [1, 2]. The aim of this research is transfer of a 3D HCE-T model from 12 to 96-well insert plate, in order to develop a high-throughput screening (HTS) model of human corneal epithelium. The cells were stained using 4′, 6-diamidino-2-phenylindole (DAPI, Invitrogen, USA). Fluoroshield™ mounting medium with DAPI was purchased from Sigma-Aldrich (Germany) and 3-[4, 5-dimethylthiazol-2-yl]-2, 5- diphenyl tetrazolium bromide (MTT) was purchased from Carbosynth (UK). For nanoemulsion (NE) preparation the following substances were used: ibuprofen (Hubei Biocause Pharmaceutical, China), Miglyol® 812 (Kemig, Croatia), lecithin S45 (Lipoid, Germany), LMW chitosan (Sigma-Aldrich), Kolliphor® EL (BASF, Germany), glycerol (T.T.T., Croatia) and purified water (SG, Germany). HCE-T cells (RIKEN Cell Bank, Japan) were cultivated in DMEM/F12 medium (Sigma-Aldrich) supplemented with FBS (5%, Capricorn, Germany), insulin (5 μg/ml, Sigma-Aldrich), dimethyl sulfoxide (0.5%, Applichem, Germany), epidermal growth factor (10 ng/ml, Sigma-Aldrich) and antibiotic-antimycotic solution (Sigma-Aldrich) at 37 °C in a humidified atmosphere with 5% CO2. The cells were seeded (1×104 cells per well) on polycarbonate membranes of a 96-well insert plate (PSHT004S5, Merck, Germany) pre-coated with rat tail type I collagen (Sigma-Aldrich) and human fibronectin (Sigma- Aldrich). The cells were cultivated submerged in the medium (75 µL apical and 250 µL basolateral volume) during 7 days and were subsequently exposed to the air-liquid interface (ALI) for 3 days. The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) at different time points after seeding and the cell nuclei were stained with DAPI. The membranes with cells were mounted on slides using Fluoroshield™ mounting medium with DAPI and the cover slips were sealed with nail polish. The cell nuclei were imaged using confocal fluorescence microscope (ImageXpress® Micro Confocal, Molecular Devices, USA) at 60× magnification. Ophthalmic nanoemulsions (NEs) were produced using microfluidizer (Microfluidics LM20, USA) at 1000 bar and 5 cycles and their composition is shown in table 1. The NEs were diluted 10× in HBSS buffer (pH 6) and the HTS model was exposed to the diluted formulations 30 min at 37°C. The viability of the model was determined using MTT assay and the results are compared with the results obtained previously on a 12-well plate HCE-T model. The HCE-T model was successfully cultivated on a 96- well insert plate. After a 7-day cultivation period in submerged conditions a confluent monolayer was formed and 3 days after ALI exposure multiple layers are observed (Figure 1a). However, HTS HCE-T model was characterized with fewer layers (3-5) than the 12-well plate model [1]. Furthermore, the uppermost layer of the HTS model was not confluent after 3 days of ALI exposure. The viability of the HTS HCE-T model after treatment with ophthalmic NEs was high (about 80%). However, it was lower than the viability of a 12-well plate model treated with the same formulations, under the same conditions (Figure 1b) [2]. A HTS 3D HCE-T model of human corneal epithelium can be cultivated using 96-well insert plates. However, slower proliferation rate and greater sensitivity to the ophthalmic NE formulation is observed in comparison to the standard 12-well plate HCE-T model. In conclusion, further optimisation of cultivation conditions is necessary to obtain higher similarity with the standard 12-well plate HCE-T model. References: 1. M. Juretić et al., HCE-T cell-based permeability model: A well-maintained or a highly variable barrier phenotype?, Eur. J. Pharm. Sci., 2017. vol. 104, pp. 23–30 2. B. Jurišić Dukovski et al., Functional ibuprofen-loaded cationic nanoemulsion: Development and optimization for dry eye disease treatment, Int. J. Pharm., 2020. vol. 576, 118979

HCE-T, ophthalmic nanoemulsion, multilayer, MTT

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