CHARACTERISTICS OF CULTURES OF CELLS OBTAINED FROM HUMAN LIMB

Author: Surovtseva M.A.¹, Iskakov I.A.², Poveshchenko O.V.³, Lykov A.P.⁴, Kim I.I.⁵, Yankayte E.V.⁶, Konenkov V.I.⁷, Chernykh V.V.⁸

Author Affiliations

Abstract

Limbal epithelial stem cells are localized in the limb, which determine the regeneration of the corneal epithelium, both physiological and reparative. The aim of the study is to characterize the culture of cells isolated by different methods. Materials and methods. Limbal stem cells were isolated from the limbus region of enucleated human eyes. Cell isolation was produced by the enzymatic and explantation methods. Cell cultivation was performed on the amniotic membrane and without it, in DMEM/F12 growth media (Dullbecco’s Modified Eagle Medium/Nutrient F12 Ham) and CECBM (Corneal Epithelial Cell Basal Medium). The morphological assessment and immunophenotyping of cell cultures were performed. Results and its discussion. The obtained culture was distinguished by cell heterogeneity at the enzymatic method of isolating cells from the limbus. The presence of fibroblast-like cells was revealed in the suspension of non-adherent cells, which resembled stromal cells in appearance and growth pattern. Immunophenotyping revealed that most cells carry markers of multipotent mesenchymal stromal cells (CD90⁺/CD73⁺/CD105⁺/CD34^–/CD45^–) and a small number – markers of epithelial cells (p63α⁺, ABCG₂⁺, CK19⁺). Explant culture was more uniform. The cells had a rounded, epithelial-like shape, adhered well to the surface of the amniotic membrane and grew on it. The results of phenotyping indicated that cells are represented mainly by epithelial cells (p63α⁺, ABCG₂⁺, CK19⁺). Conclusion. The culture is represented mainly by MMSC at the enzymatic method of isolating cells from the limbal graft and cultivating them without feeder in the growth medium DMEM/F12. Isolation of limb cells by the explantation method and their cultivation in the selective medium CECBM allow us to obtain mainly the culture of epithelial stem cells.

Key words

limb, explant, amniotic membrane, limbal stem cells, multipotent mesenchymal stem cells

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About Authors (Correspondence):

Surovtseva M.A. – candidate of medical sciences, senior researcher of laboratory of cell technologies, e-mail: mfelde@ngs.ru

Full Text

snmzh_3-2019_surovceva_15-20.pdf

Received: 13/06/2019

Accepted: 13/06/2019