A comparative analysis of cultivation techniques of epithelial stem cells of cornea and creation of biomedical cell product (biocomposite) on the basis biocompatible matrix and stem cells

Abstract

In recent years the autologous stem cells of front cornea epithelium (CSC) are increasingly used in the treatment of degenerative diseases of this transparent part of the outer shell of the eye. Limb is considered as a specialized storage for these cells in eye. A number of techniques for isolation of stem cells from limb epithelium and their cultivation in vitro were developed. Goal of the work: To analyze the possibilities of two methods of cultivation of epithelial stem cells front epithelium of cornea (CSC), explants and suspension ones by criteria of efficiency of proliferation, time consumption and constancy of morphology and function parameters of cell cultures. To study of biocompatibility of CSC with commercial gel preparations for its application as an ingredient of biocomposite to use in clinical practice. The primary multilayer CSC cultures were obtained by two methods (explant and suspension) from biopsies of intact parts of cornea limb of 10 patients at the age from 39 to 73 years suffering different types of ceratopathies. Under comparative analysis of CSC cultures their viability, proliferation potential and immunophenotype were investigated. It was shown that explant method is more effective as compared with suspension one as it allows to gain the significant amount of cells with epithelium like morphology. The cell population was presented primarily by epithelium CSC: 95% of cells synthesized K19+ protein. CSC proliferation index of cells isolated by explant method was by 3.5-4 times higher, the time of doubling of population at cultivation in DMEM/F12 medium with 10 ng/ml of EGF was 48 h. Using explant method from one limb biopsy (1 х2 mm) a biomass of CSC in amount of 4 ± 1,2 mln during 3-4 weeks (1-2 passages) was obtained. It was show under investigation for biocompatibility of cells with a number of commercial gel preparations that preparation Provisk is the best on this indicator and this allows to recommend it as a carrier for cells when biomedical cell product are produced.