Isolation of subepithelial zone cells from patients with Salzmann’s degeneration and in normal state

Relevance Salzmann nodular degeneration (SND) is accompanied by the appearance of protruding opaque pathological foci in the subepithelial layer of the cornea. As the process progresses, it spreads to deeper layers of the cornea and can lead to the formation of persistent stromal opacities and severe visual impairment [1, 2]. SND occurs mainly against the background of chronic inflammation of the anterior surface of the eye, but idiopathic cases also occur [3]. Symptomatic conservative therapy is considered among the treatment methods at the early stages of SND, while surgical treatment is indicated for lesions of the optical zone of the cornea. In this case, the recurrence rate can reach 31%, which indicates the need to search for more effective therapeutic approaches. Cell cultures are a convenient model system for studying pathological processes in vitro and assessing the effectiveness of therapeutic drugs [4]. For the study of the cell model system, it is important to compare cell cultures from pathological and healthy tissue, which will allow us to identify specific changes characteristic of pathology, on the basis of which more effective treatment methods can be developed. Objective Obtaining and characterizing cell cultures obtained from the pathological zone of the cornea of ​​patients with UDZ and from healthy tissue of the same localization. Material and methods Samples of the subepithelial zone of the cornea obtained as a result of keratectomy for UDZ or excised with a keratome from a cadaver eye were dissociated in a solution of 2 mg / ml collagenase type I for 4-6 hours, after which they were cultured in a complete RPMI-1640 nutrient medium with the addition of 10% FBS under standard CO2 incubator conditions. Upon reaching a confluent monolayer, the cells were passaged for further cultivation and analysis. Morphological characteristics of cells were assessed using light microscopy, the population doubling rate was calculated based on the increase in the area of ​​the cell layer in the first passages, the expression of surface and intracellular markers was studied using immunocytochemical staining and flow cytometry. Results Primary cell cultures were obtained from healthy and pathological corneal tissue. Both types of cultures contained cells of both epithelial and stromal morphotypes. The populations differed in the doubling rate and the level of expression of epithelial differentiation markers and keratocyte activation markers. Conclusion Cell cultures isolated from the pathological zone of the cornea of ​​patients with UDZ, as well as from healthy corneal tissue of the cadaver eye, were obtained and characterized. Comparative analysis of these cultures revealed differences in their morphology, population doubling rate, and expression of epithelial differentiation markers and keratocyte activation. Key words: cell model; Salzmann's nodular degeneration; corneal cell culture