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Isolation of extracellular vesicles: Determining the correct approach (Review).

Szatanek R, Baran J, Siedlar M, Baj-Krzyworzeka M - Int. J. Mol. Med. (2015)

Bottom Line: Although vast knowledge on the subject of EVs has accumulated over the years, there are still fundamental issues associated with the correct approach for their isolation.The aim of this reveiw was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches.The realization of this 'uniqueness' is the first step in the right direction for the complete assessment of EVs.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Immunology and Transplantology, Jagiellonian University Medical College, 30-663 Krakow, Poland.

ABSTRACT
The discovery of extracellular vesicles (EVs) has revised the interpretation of intercellular communication. It is now well established that EVs play a significant role in coagulation, inflammation, cancer and stem cell renewal and expansion. Their release presents an intriguing, transporting/trafficking network of biologically active molecules, which are able to reach and modulate the function/behavior of the target cells in a variety of ways. Moreover, the presence of EVs in various body fluids points to their potential for use as biomarkers and prognostic indicators in the surveillance/monitoring of a variety of diseases. Although vast knowledge on the subject of EVs has accumulated over the years, there are still fundamental issues associated with the correct approach for their isolation. This review comprises the knowledge on EV isolation techniques that are currently available. The aim of this reveiw was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches. The realization of this 'uniqueness' is the first step in the right direction for the complete assessment of EVs.

No MeSH data available.


Related in: MedlinePlus

Size distribution of particles obtained by nanoparticle tracking analysis (NTA) from cell culture medium sample supplemented with fetal bovine serum (FBS) and from freshly opened cell culture medium.
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f1-ijmm-36-01-0011: Size distribution of particles obtained by nanoparticle tracking analysis (NTA) from cell culture medium sample supplemented with fetal bovine serum (FBS) and from freshly opened cell culture medium.

Mentions: When isolating EVs from conditioned cell culture media one has to consider the presence of an additional, ‘artificial’ EV source, namely fetal bovine serum (FBS) or any other supplement for that matter, which is routinely added to cell cultures (19,20). Our results obtained by nanoparticle tracking analysis (NTA) showed freshly prepared cell culture medium supplemented with FBS to have a substantial particle population (mostly <100 nm in size) already present before the actual use (Fig. 1). This observation alone clearly demonstrates that the downstream EV isolation from conditioned cell culture media carries along the risk of obtaining, apart from proper EVs, also those from FBS, which obviously, obscures the final results. This observation is in accordance with observations of other groups, which have also shown FBS to contain vesicles that may be later isolated along the actual EV (19–21). To counter that problem, many groups apply filters to remove EVs from the media/FBS or use a long ultracentrifugation step. In an elegant study, Lötvall et al showed that the removal of FBS-originated EVs is critical for further downstream experiments, since these vesicles are capable of inducing effects similar to those of EVs isolated from the actual cell line culture media (21). Thus, it has been proposed that a 16 h-ultracentrifugation step at 100,000 × g or greater for FBS is absolutely obligatory for a complete FBS vesicle depletion, since shorter centrifugation steps are insufficient (19,21). An alternative may be the use of an exosome-free FBS, which is already commercially available, but is still rather expensive. Some also postulate the use of bovine serum albumin (BSA) instead of the standard FBS (20).


Isolation of extracellular vesicles: Determining the correct approach (Review).

Szatanek R, Baran J, Siedlar M, Baj-Krzyworzeka M - Int. J. Mol. Med. (2015)

Size distribution of particles obtained by nanoparticle tracking analysis (NTA) from cell culture medium sample supplemented with fetal bovine serum (FBS) and from freshly opened cell culture medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494580&req=5

f1-ijmm-36-01-0011: Size distribution of particles obtained by nanoparticle tracking analysis (NTA) from cell culture medium sample supplemented with fetal bovine serum (FBS) and from freshly opened cell culture medium.
Mentions: When isolating EVs from conditioned cell culture media one has to consider the presence of an additional, ‘artificial’ EV source, namely fetal bovine serum (FBS) or any other supplement for that matter, which is routinely added to cell cultures (19,20). Our results obtained by nanoparticle tracking analysis (NTA) showed freshly prepared cell culture medium supplemented with FBS to have a substantial particle population (mostly <100 nm in size) already present before the actual use (Fig. 1). This observation alone clearly demonstrates that the downstream EV isolation from conditioned cell culture media carries along the risk of obtaining, apart from proper EVs, also those from FBS, which obviously, obscures the final results. This observation is in accordance with observations of other groups, which have also shown FBS to contain vesicles that may be later isolated along the actual EV (19–21). To counter that problem, many groups apply filters to remove EVs from the media/FBS or use a long ultracentrifugation step. In an elegant study, Lötvall et al showed that the removal of FBS-originated EVs is critical for further downstream experiments, since these vesicles are capable of inducing effects similar to those of EVs isolated from the actual cell line culture media (21). Thus, it has been proposed that a 16 h-ultracentrifugation step at 100,000 × g or greater for FBS is absolutely obligatory for a complete FBS vesicle depletion, since shorter centrifugation steps are insufficient (19,21). An alternative may be the use of an exosome-free FBS, which is already commercially available, but is still rather expensive. Some also postulate the use of bovine serum albumin (BSA) instead of the standard FBS (20).

Bottom Line: Although vast knowledge on the subject of EVs has accumulated over the years, there are still fundamental issues associated with the correct approach for their isolation.The aim of this reveiw was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches.The realization of this 'uniqueness' is the first step in the right direction for the complete assessment of EVs.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Immunology and Transplantology, Jagiellonian University Medical College, 30-663 Krakow, Poland.

ABSTRACT
The discovery of extracellular vesicles (EVs) has revised the interpretation of intercellular communication. It is now well established that EVs play a significant role in coagulation, inflammation, cancer and stem cell renewal and expansion. Their release presents an intriguing, transporting/trafficking network of biologically active molecules, which are able to reach and modulate the function/behavior of the target cells in a variety of ways. Moreover, the presence of EVs in various body fluids points to their potential for use as biomarkers and prognostic indicators in the surveillance/monitoring of a variety of diseases. Although vast knowledge on the subject of EVs has accumulated over the years, there are still fundamental issues associated with the correct approach for their isolation. This review comprises the knowledge on EV isolation techniques that are currently available. The aim of this reveiw was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches. The realization of this 'uniqueness' is the first step in the right direction for the complete assessment of EVs.

No MeSH data available.


Related in: MedlinePlus