CHARACTERISTICS OF CULTURES OF CELLS OBTAINED FROM HUMAN LIMB
Mariya Alexandrovna SUROVTSEVA1, Igor Alekseevich ISKAKOV2, Olga Vladimirovna POVESHCHENKO1, Alexandr Petrovich LYKOV1, Irina Innokentievna KIM1, Evgeniya Viktorovna YANKAYTE1, Vladimir Iosifovich KONENKOV1, Valeriy Vyacheslavovich CHERNYKH2
1Research Institute for Clinical and Experimental Lymphology - a Branch of Federal Research Center of the Institute of Cytology and Genetics of SB RAS
2S. Fyodorov Eye Microsurgery Federal State Institution of Minzdrav of Russia, Novosibirsk Branch
Keywords: лимб, эксплант, амниотическая мембрана, лимбальные стволовые клетки, мультипотентные мезенхимальные стволовые клетки, limb, explant, amniotic membrane, limbal stem cells, multipotent mesenchymal stem cells
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.
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