Сell model for experimental research in keratoconus pathogenesis

Abstract

INTRODUCTION: Certain mineral elements dismetabolism is known to play an important part in the pathogenesis of the corneal degenerative and dystrophic diseases, as metalloenzymes participate in connective tissues metabolism affecting their properties. Thus, in keratoconus corneal tissue is depleted in iron, copper and zinc, what could be the underlying cause of cornea biomechanical properties impairment. Keratoconus modeling is complicated and practically not reproducible in animals, therefore cell models elaboration is very much in demand. Definite mineral elements content must be reduced in order to simulate pathological changes specific for keratoconic corneas.

PURPOSE: The aim of this study was an elaboration of a cell model suitable for keratoconus pathogenesis research. This goal achieving involved solving the following tasks: 1) to develop a tissue-engineered system that mimic healthy corneal stroma; 2) to develop a technique for selective depletion of the nutrient medium by mineral elements involved in keratoconus pathogenesis; 3) to assess the possibility of the designed tissue-engineered system growth in the depleted nutrient medium.

MATERIAL AND METHODS: The study was carried out with the primary culture of human keratocytes, which were used to build tissue-engineered systems of three types: on silicone, on a membrane, without a carrier. Cell cultures morphology was evaluated by light and electron microscopy. The nutrient media were zinc depleted via decationization of fetal bovine serum using two types of ion-exchange resins; mineral elements concentrations were evaluated by means of inductively coupled plasma mass spectrometry.

RESULTS: The tissue system engineered without a carrier in form of cell sheet was chosen as the most convenient model. The decationization of the serum by means of Chelex 100 resin was shown to be a successful method for tenfold zinc concentration reduction in the nutrient medium. Keratocytes cultivation in the form of cell sheet on a zinc-depleted medium was successfully approved.

CONCLUSION: Elaborated tissue-engineered system could be considered as a model of the corneal stroma under specific for keratoconus conditions of zinc depletion.

Keywords

cell sheets / tissue model / keratoconus / zinc / mineral elements depletion / ICP-MS

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Subbot A.M., Novikov I.A., Patejuk L.S., Kobzeva A.V., Avetisov S.J.