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Updated: Jul 16, 2025
Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media
Published on: May 13, 2022
A protocol for isolating and imaging large extracellular vesicles or midbody remnants from mammalian cell culture
Sungjin Park1, Smit A Patel1, Elizabeth E Torr1
1Department of Genetics, University of Wisconsin, Madison, WI 53706, USA.
Abstract:
Traditionally, midbody remnants (MBRs) are isolated from cell culture medium using ultracentrifugation, which is expensive and time consuming. Here, we present a protocol for isolating MBRs or large extracellular vesicles (EVs) from mammalian cell culture using either 1.5% polyethylene glycol 6000 (PEG6000) or PEG5000-coated gold nanoparticles. We describe steps for growing cells, collecting media, and precipitating MBRs and EVs from cell culture medium. We then detail characterization of MBRs through immunofluorescent antibody staining and immunofluorescent imaging.
Insights
This study introduces a cost-effective method for isolating midbody remnants (MBRs) and extracellular vesicles (EVs) from cell cultures using polyethylene glycol (PEG) or PEG-coated gold nanoparticles, replacing ultracentrifugation.
Area of Science:
- Cell Biology
- Biotechnology
- Nanotechnology
Background:
- Midbody remnants (MBRs) and extracellular vesicles (EVs) are crucial in cellular processes.
- Traditional isolation methods like ultracentrifugation are costly and time-consuming.
Purpose of the Study:
- To develop an efficient and affordable protocol for isolating MBRs and large EVs from mammalian cell culture media.
- To present an alternative to ultracentrifugation for MBR and EV isolation.
Main Methods:
- Utilized 1.5% polyethylene glycol 6000 (PEG6000) for precipitation.
- Employed PEG5000-coated gold nanoparticles for precipitation.
- Described cell culture, media collection, and precipitation steps.
- Detailed MBR characterization using immunofluorescent antibody staining and imaging.
Main Results:
- Successfully isolated MBRs and large EVs using PEG and PEG-coated gold nanoparticles.
- Demonstrated a viable alternative to ultracentrifugation for MBR/EV isolation.
- Characterized isolated MBRs via immunofluorescence.
Conclusions:
- The developed protocol offers a more accessible and economical approach for MBR and EV isolation.
- This method facilitates further research into the biological roles of MBRs and EVs.
