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Updated: Mar 15, 2026
Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells
Published on: September 23, 2022
Microvesicles preparation from mesenchymal stem cells
Fariba Rad1, Ali Akbar Pourfathollah2, Fatemeh Yari3
1PhD 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.
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.
Conclusion:
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.
Insights
Optimizing microvesicle (MV) isolation from UC-MSCs is crucial. Centrifugation at 20,000×g effectively purifies these MVs, enhancing yield and purity for research applications.
Area of Science:
- Cell Biology
- Biotechnology
- Nanotechnology
Background:
- Extracellular vesicles (EVs), including exosomes, microvesicles (MVs), and apoptotic bodies, vary in size and biological function.
- Standardized methods for isolating MVs are lacking, posing a significant challenge in research.
- This study focuses on isolating MVs from umbilical cord-derived mesenchymal stem cells (UC-MSCs).
Purpose of the Study:
- To evaluate different ultracentrifugation schemes for isolating MVs from UC-MSCs.
- To determine the optimal centrifugation speed for maximizing MV yield and purity.
- To characterize the isolated UC-MSC-derived MVs.
Main Methods:
- UC-MSCs were cultured, and MVs were isolated using four different ultracentrifugation speeds (10,000×g to 60,000×g).
- MV fractions were analyzed using Dynamic Light Scattering (DLS) for size, total protein quantification, and flow cytometry for marker expression.
- UC-MSCs were characterized for stem cell markers (CD44, CD73) and hematopoietic markers (CD45, CD34).
Main Results:
- UC-MSCs expressed MSC markers (CD44, CD73) and lacked hematopoietic markers (CD45, CD34).
- Isolated particles ranged from 50 to 1250 nm and expressed UC-MSC surface markers (CD90, CD106, CD166, CD44), while lacking CD34, CD63, and CD9.
- All tested centrifugation speeds yielded MVs <1000 nm; speeds of 40,000×g and 60,000×g additionally produced particles <100 nm.
Conclusions:
- Centrifugation speed significantly impacts the purity and yield of isolated MVs.
- A centrifugation speed of 20,000×g is identified as optimal for purifying UC-MSC-derived MVs.
- This optimized method facilitates reliable MV isolation for further studies.
