title: Microvesicles preparation from mesenchymal stem cells.
abstract: BACKGROUND
  Extracellular vesicles are particles ranged from 30 nm to 5μm and subcategorized into three groups; exosomes, microvesicles and apoptotic bodies, each of which have different biological impact. Lack of a standard method for the detection and isolation of MVs has led to a challenging issue that is a worth considering. In this study, we isolated MVs from the conditioned medium of UC-MSCs by four different schemes of ultracentrifugation.
  
  
  METHODS
  We examined the efficacy of differential centrifugation ranging from 10,000×g to 60,000×g on UCMSCs- derived microvesicles yield and purity. The fractions were evaluated by Dynamic Light Scattering (DLS) method, total protein quantification and flow cytometry.
  
  
  RESULTS
  UC-MSCs were spindle cells that adhered to plastic culture flasks. These cells expressed MSC markers such as CD44 and CD73, whereas were negative for hematopoietic markers CD45 and CD34. UC-MSCparticles were successfully isolated. Particles were heterogeneous vesicles of approximately 50 to 1250 nm in diameter that bear the surface-expressed molecules UC-MSCs such as; CD90, CD106, CD166 and CD44, and negative for CD34, CD63, and CD9. According to the results of DLS method, centrifugation at 10,000, 20,000, 40,000 and 60,000 ×g, all gave MVs of less than 1000 nm. It is of notion that only at the centrifugation rates of 40,000 and 60,000×g, particles of less than 100 nm in diameter were also obtained.
  
  
  CONCLUSIONS
  The choice of exact speed greatly influences the purity of MVs and their yield. Our findings indicate that centrifugation at 20,000×g is appropriate for the purification of UC-MSC-MVs.
authors: PhD student of Hematology, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran. Cellular and Molecular Research Center, Yasuje University of Medical Sciences, Yasuje, Iran. fariba.rad89@gmail.com.
authors: Fariba
authors: F
authors: Rad
authors: Professor of Immunology, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran, & Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. pourfa@yahoo.com , pourfa@IBTO.com.
authors: Ali Akbar
authors: AA
authors: Pourfathollah
authors: Associate Professor of Immunology, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran. f.yari@ibto.ir.
authors: Fatemeh
authors: F
authors: Yari
authors: PhD of Hematology, Hematology, Oncology and Stem Cell Transplantation Research Center, Tehran University, Medical Sciences, Tehran, Iran. saeedm_58@yahoo.com.
authors: Saeed
authors: S
authors: Mohammadi
authors: Associate Professor of Immunology, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran. m.kheirandish@ibto.ir.
authors: Maryam
authors: M
authors: Kheirandish
keywords: DLS method differential centrifugation; exosomes; extracellular vesicles; microvesicles