Protocol for differential centrifugation-based separation and characterization of apoptotic vesicles derived from human mesenchymal stem cells
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Updated: Aug 28, 2025
Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells
Published on: September 23, 2022
Da Chen1, Zixuan Zhao1, Kai Zhang2
1State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, China; Department of Orthodontics, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
Abstract:
Apoptotic vesicles (apoVs) are specific extracellular vesicles generated during apoptosis and play important roles in multiple physiological and pathophysiological settings. Here, we present a protocol using differential centrifugation to separate apoVs from human mesenchymal stem cells (MSCs) after induction of apoptosis. We describe how to characterize apoV size and morphology by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), and determination of specific biomarker expression by immunoblotting. Our protocol will be useful for preparing apoVs for further functional analysis. For complete details on the use and execution of this protocol, please refer to Zheng et al. (2021).
Insights
This study details a protocol for isolating apoptotic vesicles (apoVs) from human mesenchymal stem cells. The method uses differential centrifugation and characterization techniques like NTA and TEM for further functional analysis.
Area of Science:
- Extracellular Vesicle Biology
- Cellular Biology
- Apoptosis Research
Background:
- Apoptotic vesicles (apoVs) are crucial extracellular vesicles released during programmed cell death.
- These vesicles have significant roles in both normal physiological processes and various disease states.
- Understanding apoVs requires reliable methods for their isolation and characterization.
Purpose of the Study:
- To present a detailed protocol for the isolation of apoptotic vesicles (apoVs).
- To enable the separation of apoVs from human mesenchymal stem cells (MSCs) following apoptosis induction.
- To facilitate further functional studies of apoVs.
Main Methods:
- Differential centrifugation was employed to isolate apoVs from MSCs.
- Nanoparticle tracking analysis (NTA) was used to determine apoV size and concentration.
- Transmission electron microscopy (TEM) was utilized for morphological assessment.
- Immunoblotting was performed to confirm the expression of specific biomarkers.
Main Results:
- A reproducible protocol for isolating apoVs from apoptotic MSCs was established.
- Characterization confirmed the size and morphology of the isolated apoVs.
- Biomarker analysis validated the identity of the isolated vesicles as apoVs.
Conclusions:
- The presented protocol provides a robust method for preparing apoVs.
- This methodology supports further investigation into the functional roles of apoVs.
- The protocol is valuable for researchers in the fields of cell biology and extracellular vesicle research.
