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Development of a human corneal epithelium model utilizing a collagen v…

작성자 관리자 작성일 2023-02-24
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Development of a human corneal epithelium model utilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing eye irritant chemicals


Abstract

The brief TEER (trans-epithelial electrical resistance) assay after exposing chemicals to corneal epithelium in vivo is known as a suitable method for evaluating corneal irritancy and permeability quantitatively and continuously. A collagen vitrigel membrane we previously developed is a thin (about 20 μm thick) and transparent membrane composed of high density collagen fibrils equivalent to connective tissues in vivo, e.g. corneal Bowman’s membrane. To develop such a TEER assay system in vitro utilizing a human corneal epithelial model, HCE-T cells (a human corneal epithelial cell line) were cultured on the collagen vitrigel membrane substratum prepared in a Millicell chamber suitable for TEER measurement. Human corneal epithelium model possessing 5–6 cell layers sufficient for TEER assay was successfully reconstructed on the substratum in the Millicell chamber by culturing the cells in monolayer for 2 days and subsequently in air–liquid interface for 7 days. The exposure of chemicals to the model induced the time-dependent relative changes of TEER in response to the characteristic of each chemical within a few minutes. These results suggest that the TEER assay using the human corneal epithelial model is very useful for an ocular irritancy evaluation as an alternative to the Draize eye irritation test.

Highlights

► A human corneal epithelium model was reconstructed on a collagen vitrigel membrane. ► Thin collagen vitrigel membrane was used as a substitute for Bowman’s membrane. ► The model appropriate for TEER assay was composed of 5–6 HCE-T cell layers. ► The changes of epithelial barrier function after exposing chemicals were measured. ► The brief TEER assay using the model was useful for an ocular irritancy evaluation.


Abbreviations

BCOP
bovine corneal opacity and permeability
ICE
isolated chicken eye
IRE
isolated rabbit eye
SIRC
Statens Serum Institut rabbit corneal
STE
short time exposure
TEER
trans-epithelial electrical resistance
DMEM
Dulbecco’s modified Eagle Medium
F-12
nutrient mixture F-12
FBS
fetal bovine serum
EGF
epidermal growth factor
DMSO
dimethyl sulfoxide
PET
polyethylene terephthalate
ERS
electrical resistance system
GHS
globally harmonized system
NI
non-irritant
EDTA
ethylenediaminetetraacetic acid
PBS
phosphate buffered saline
ADMET
absorption, distribution, metabolism, excretion and toxicity

Keywords

Corneal epithelium
Eye irritation tests
Collagen vitrigel membrane
TEER
HCE-T cells

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