Immunophenotypic characteristics and karyotype analysis of bone marrow-derived mesenchymal stem cells of rabbits during in vitro cultivation.

Abstract

The aim of this study was to establish the immunophenotypic profile and karyotypic stability of bone marrow mesenchymal stem cells (MSCs) of rabbits at the early passages in vitro following the application of different methods of dissociation of cellular material. MSCs were obtained from the femur bone marrow of three clinically healthy rabbits under general anaesthesia. Bone marrow aspirate was seeded in Petri dishes and cultured in a CO2 incubator with 5% CO2 at 37.0oC using a standard procedure. Immunohistochemical detection of nuclear proteins, cytoskeletal proteins and cell adhesion were performed by immunohistochemical analysis and karyotype analysis of MSCs following the enzyme and chelating methods of dissociation of the cell monolayer. The results of the immunophenotypic analysis of rabbit bone marrow MSCs showed that at the first, seventh, twelfth, and eighteenth passages these cells express markers of mesenchymal, muscle, epithelial and nerve cells. The choice of the enzyme or chelating method of dissociation of a culture of rabbit mesenchymal stem cells affects their cytogenetic variability. Dissociation of the MSCs monolayer with ethylenediaminetetraacetic acid produces a cell culture with fewer quantitative and qualitative changes in the chromosome apparatus as compared to the enzyme method. Rabbit MSCs express markers of mesenchymal (vimentin, actin), muscle, epithelial and nerve (E-cadherin, N-cadherin) cells that are essential for differentiation of these cells. The chelating method of dissociation of a culture of rabbit mesenchymal stem cells, using ethylenediaminetetraacetic acid during cultivation, is more advantageous than the enzyme method of dissociation because it leads to less cytogenetic variability.