Isolation, culture and detection of mesenchymal stem cells in tissues from critical limb ischemia model in rat

INTRODUCTION: Rat mesenchymal stem cells (MSCs) represent a small portion of the cells in the stromal compartment of bone marrow and have the potential to differentiate into new blood vessel and other tissues. MSCs transplantation in tissues from critical limb ischemia model in rat may represent a therapeutic applications of vascular regeneration. AIM: The aim of this study was to isolate with a simple method the rat bone marrow stromal cells. Then the adherent cells were labeled with 5-bromo-2-deoxyuridine (BrdU, Sigma) and injected in the gastrocnemius and adductor muscle of ischemic hind limbs in order to demonstrate their presence in the critical limb ischemia model in rat. MATERIAL AND METHODS: MSCs were isolated from Wistar rats, 8 weeks of age. The MSCs were labeled in vitro for later identification by adding 10 μg/mL 5-bromo-2 –deoxyuridine (BrdU, Sigma). RESULTS: Small colonies of fibroblast-like cells were seen after several days of primary culture. These colonies increased in size and were subcultured after 15–18 days. CONCLUSION: The MSCs obtained in this study presented a stable undifferentiated phenotype under normal culture conditions. MSCs are easy to isolate, culture, and detect in in vivo culture. These cells are characterized by high plasticity and could have an important role in angiogenesis.