Toshiaki Takezawa

Animal Immune and Cell Biology Research Unit

［Abstract］

We fabricated a culture model reflecting an architecture of human corneal epithelium using a new biomaterial “collagen vitrigelTM membrane” and developed a sensitive method for testing the safety of chemicals for eyes. The method provides an excellent judgment of safety without animal experiments and would be useful for developing safe cosmetics with less irritancy.

［Keywords］

collagen, vitrigelTM, cornea, eye irritancy test, culture model, three-dimensional culture

［Background］

Safety assessment of the chemical ingredients of cosmetics and medicines for human is mainly performed in experimental animals. However, the results based on experimental animals do not always guarantee chemical safety for humans. Also, the use of animals for the safety assessment of cosmetic ingredients is already prohibited in the EU from the viewpoint of animal protection. Although these reasons have globally promoted the development of test methods without using animals, an excellent eye irritancy test (EIT) method that can entirely replace the animal testing has not been established yet. In this study, we fabricated a culture model of corneal epithelium by three-dimensionally culturing human corneal epithelium-derived cells in a collagen vitrigel membrane chamber in order to develop a testing method that can be used to replace the EIT method using rabbit eyes.

［Results and Discussion］

1. Collagen vitrigel membrane (CVM) is composed of about 100 times high-density collagen fibrils in comparison to the traditional collagen gel. We developed a chamber by fixing a CVM on the edge of a plastic cylinder and succeeded in commercializing a new cell culture ware “CVM chamber” in collaboration with a private company (Fig. 1). The CVM chamber useful for three-dimensional culture of various cells facilitates the fabrication of culture models not only for tissue sheet-types but also for organoid plate-types.
   
2. A human corneal epithelium (HCE) model resembling HCE histologically was successfully fabricated by culturing HCE-derived cells three-dimensionally in a CVM chamber (Fig. 2).
   
3. We developed a novel eye irritancy test (EIT) based on vitrigel to evaluate the presence or absence of ocular irritancy of chemicals using the HCE model. The Vitrigel-EIT method is a rapid and brief test method that can be used to analyze the changes in the patterns of transepithelial electrical resistance (TEER) values within 3 minutes after exposing the chemicals to the HCE model and utilizing the criteria for three indexes to estimate each pattern (Fig. 3).
   
4. The ocular irritancy of 30 test chemicals was analyzed using the Vitrigel-EIT method and an excellent correlation with GHS classification based on animal experiments was confirmed (sensitivity: 100%, specificity: 75%, accuracy: 90%). In addition, immunohistological analyses of chemicals tested as false-positive by the Vitrigel-EIT method were demonstrated to possess mild irritancy.

1. The Vitrigel-EIT method would contribute to the development of safe cosmetics and shampoos with less irritancy.
   
2. We will perform validation of the Vitrigel-EIT method to facilitate registration based on the OECD Test Guideline for manufacturers around the world.
3. We will fabricate culture models of digestive organs and blood vessels in the CVM chamber to develop safe methods for testing medicines and foods.

Fig.1. (A) A nylon membrane ring-embedded CVM. (B) High-density collagen fibrils of a CVM. (C) A CVM chamber.

Fig.2. HCE-model fabricated in a CVM chamber.

Fig.3. Schema of the Vitrigel-EIT method for testing eye irritancy without using experimental animals.

 

[Collaborator]
Hiroyuki Yamaguchi (Kanto Chemical Co., Inc.), Hajime Kojima (National Institute of Health Sciences)

[Reference]

1. Takezawa T, Nishikawa K, Wang P-C (2011) Development of a human cornealepithelium model utilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing eye irritant chemicals Toxicology in Vitro 25(6):1237-1241
   
2. Yamaguchi H, Kojima H, Takezawa T. (2013) Vitrigel-eye irritancy test method using HCE-T cells Toxicological Sciences 135(2):347-355
   
3. Patent application #JP-2012-115262 (Japan)