Rat Adipose-Derived Stem Cell Isolation and Characterization

MSCs were obtained from AT of 6-8 weeks old male rats (180 ± 20 g) as previously described by De Luna-Saldivar et al. [4 (link)]. The isolated ASCs were cultured in Dulbecco’s modified eagle’s medium (DMEM, Sigma-Aldrich, St Louis, MO, USA) containing 10% fetal bovine serum (FBS, Biosolutions International, Melbourne, Australia) and 1% penicillin–streptomycin mixture (Nacalai Tesque, Kyoto, Japan) at 37 °C in a 5% CO₂ humidified incubator. The medium was changed twice a week. Cells at 80%–85% confluence were trypsinized and passaged until the fourth passage. Then the cells were washed and suspended in PBS for use.
Characterization of ASCs for the expression of the cell surface markers, MSC markers (CD90, CD105 and CD73) and hematopoietic markers (CD34 and CD45), by FCM was performed [25 (link)]. Furthermore, characteristics of ASCs for their expression to CD73, CD105, CD90, CD34 and CD45 genes were confirmed by qRT-PCR as described later.