Isolation, expansion, and characterization of mesenchymal stem cells from adult rat bone marrow

Introduction: Mesenchymal stem cells (MSCs) have been studied and applied extensively because of their ability to self-renew and differentiate into various cell types. Aim of the study :To isolate, increase the expansion rate and define rat bone marrow MSCs by immunohistochemical and electron microscopic studies. Materials and methods:Twenty adult male albino rats were used and divided equally into two groups. The bone marrow was harvested and flushed out from the long bones of each group. In group 1, the cells were cultured using complete medium containing Dulbecco’s modified Eagle’s medium, 1% antibiotics, and 10% fetal bovine serum. In group 2, the cells were cultured in complete medium supplemented with 8% fetal bovine serum and 2% autologous rat serum.When the primary culture became nearly confluent, the adherent cells were subcultured. After 12 days from the primary culture, aliquots o f the cells were prepared for Giemsa stain, transmission electron microscopy, and immunohistochemical staining for CD44, CD105, and CD34. Results:The adherent cells in both groups were heterogeneous in appearance, and most of them were spindle or star-shaped with vesicular nuclei. Some cells were binucleated. The MSCs in group 2 reached confluency more rapidly than those in group 1. After passaging of group 2, the adherent cells became more homogeneously fibroblast-like in appearance. Immunostaining of MSCs revealed a positive reaction for CD44 and CD105 and a negative reaction for CD34. Ultrastructural examination revealed that the native MSCs appeared with many pseudopodia, the nucleus was eccentric, and the inner zone of the cytoplasm was rich in free ribosomes, many mitochondria, few rough endoplasmic reticulum, with obvious Golgi complex, and some lysosomes. Some MSCs showed no pseudopodia with central nucleus. Others appeared with two euchromatic elliptical nuclei and a nucleolus in one of them. Conclusion:MSCs can be easily purified, cultured, and expanded more rapidly using autologous rat serum.