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

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 CD۹ as a marker. Purified exosomes were labeled with a PKH۲۶ red fluorescent labeling kit. The labeled exosomes were incubated with SKOV۳ ovarian tumor cells for ۱۲ h at ۳۷°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 ۳۰-۱۰۰ nm. Exosome size measurement by dynamic light scattering analysis also showed a single bell-shaped size distribution with a peak of ~۸۰ nm. Western blot analysis also demonstrated the presence of CD۹ (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 CD۹. Florescent microscopy showed that PKH۲۶-labeled exosomes could be taken up by SKOV۳ 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.

کلیدواژه ها:

Mesenchymal stem cells ، Exosomes ، Differential centrifugation ، cellular uptake ، Tumor cells ، اگزوزوم ، جذب سلولی ، سلول های بنیادی مزانشیمی ، سانتریفوژ افتراقی ، سلول های توموری