Isolation, Characterization and Cellular Uptake of Exosomes Derived from Bone Marrow Mesenchymal Stem Cells

Authors
K. Pakravan 1
S. Babashah 1
M. Javan 2
M. Sadeghizadeh 1
S. J. Mowla 1
M. Sadeghizadeh 1
1 Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
2 Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Abstract
Objective: Cell-derived microvesicles are described as a new mechanism for cell-to-cell communication. Stem cell-derived exosomes have been described as a new mechanism for the paracrine effects of mesenchymal stem cells (MSCs). In this regard, exosomes may play a relevant role in the intercellular communication between MSCs and tumor cells.
Methods: Exosomes were purified from the conditioned medium of MSCs by differential centrifugation. Exosome size and morphology were examined by scanning electron microscope and sized with dynamic light scattering (DLS). Western blot analysis confirmed the exosomes by using CD9 as a marker. Purified exosomes were labeled with a PKH26 red fluorescent labeling kit. The labeled exosomes were incubated with SKOV3 ovarian tumor cells for 12 h at 37°C, and we used an inverted fluorescence microscope to monitor cellular uptake.
Results: Scanning electron microscopy revealed that the purified MSCs-derived exosomes had a spherical shape with a diameter of approximately 30-100 nm. Exosome size measurement by dynamic light scattering analysis also showed a single bell-shaped size distribution with a peak of ~80 nm. Western blot analysis also demonstrated the presence of CD9 (a representative marker of exosomes) in the purified exosomes. These data confirmed that the vesicles isolated from MSCs-conditioned media were the exosomes based on their size and presence of the protein marker CD9. Florescent microscopy showed that PKH26-labeled exosomes could be taken up by SKOV3 tumor cells with high efficiency.
Conclusion: Our approach for isolation, characterization and cellular uptake of exosomes derived from MSCs is valuable and a prerequisite for future studies that intend to discover exosome function in tumor cells. The ability to study the biology of exosome uptake in cancer cells could provide opportunities for functional studies of these natural nanovesicles and their contents in cancer therapy.

Keywords

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Pathobiology Reserach
Volume 19, Issue 3
November 2016
Pages 17-31