Total internal reflection fluorescence microscopy (TIRFM) as a powerful tool to follow dynamic events at the cell membrane

Abstract

The aim of this study was to isolate and characterize rat Adipose Derived Mesenchymal Stem Cells (AD-MSCs) in order to evaluate their proliferative potential and their ability to differentiate in different cell types. AD-MSCs and Derived Mesen- chymal Stem Cells (BM-MSCs) have the same characteristics in terms of plasticity. The advantage of adipose tissue is that it is an easier accessible source and it offers a large amount of MSCs by less invasive surgical tecniques. MSCs were obtained from subcutaneous adipose tissue of Wistar rats. First of all microbiological controls were made to exclude the presence of bacteria or fungi in the tissue. Adipose tissue was mechanically and enzimatically fragmented and stomal cell fraction was seeded in adherent culture flasks in DMEM 20% FBS. After 48h the medium was replaced. Cells were characterized by evaluating: 1)their ability to adhere to the plastic; 2) the clonogenic potential by Colony Forming Unit (CFU) assay; 3) their ability to differentiate in 3 mesodermal lineages (adipocytes, osteocytes and chondrocytes). AD-MSCs are able to differenti- ate in adipocytes, osteocytes and chondrocytes as confirmed by Oil Red'O staining, von Kossa staining and histological analysis respectively. This first characterization is essential for the second part of our study in which we are planning to use AD-MSCs in vivo to restore renal function after an induced ischemic damage in experimental animals.