Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Madison T. Jones; Samantha W. Manioci; Ashley E. Russell

doi:10.3791/63614

Chromatografia wykluczająca wielkość do oddzielania pęcherzyków zewnątrzkomórkowych od kondycjono...

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Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Chromatografia wykluczająca wielkość do oddzielania pęcherzyków zewnątrzkomórkowych od kondycjonowanych pożywek do hodowli komórkowych

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10:46 min

May 13, 2022

DOI: 10.3791/63614-v

Madison T. Jones¹, Samantha W. Manioci¹, Ashley E. Russell^1,2

¹Department of Biology, School of Science,Penn State Erie, The Behrend College, ²Magee Womens Research Institute—Allied Member

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Overview

This protocol demonstrates the efficient separation of extracellular vesicles (EVs) from conditioned cell culture media using size exclusion chromatography. The methodology allows researchers to analyze vesticular cargo and examine intracellular communication mechanisms.

Key Study Components

Area of Science

Cell Biology
Extracellular Vesicles
Chromatography Techniques

Background

Extracellular vesicles play a critical role in intercellular communication.
Characterizing EVs can provide insights into their biological functions.
Using size exclusion chromatography enables effective separation from proteins.
This method is user-friendly and straightforward for researchers.

Purpose of Study

To provide a reliable protocol for EV extraction.
To facilitate the characterization of EV content.
To enhance understanding of EV-mediated communication in biological systems.

Methods Used

The study employs size exclusion chromatography to separate EVs from conditioned media.
It utilizes various centrifugation steps to process the media effectively.
Sample concentrations are adjusted using ultra filtration units.
Critical steps include monitoring sample retention and washing the column.
A representative western blotting is used to verify EV marker expression.

Main Results

Strong expression of CD markers (CD9, CD63, CD81) was observed in specific EV fractions.
No expression of certain proteins (GM130, calnexin) in EV fractions indicated successful separation.
These findings support the method's effectiveness for concentrating EVs and characterizing their content.

Conclusions

The study demonstrates a reliable method for isolating EVs, enhancing research capabilities.
This method lays a foundation for further studies into EV functions and signaling pathways.
Understanding EVs can aid in developing therapeutic strategies targeting intracellular communication.

Frequently Asked Questions

What are the advantages of using size exclusion chromatography for EV isolation?

Size exclusion chromatography allows for gentle separation of extracellular vesicles from serum or conditioned media without significant protein denaturation, making it ideal for downstream analysis.

How are the extracellular vesicles prepared before chromatography?

The conditioned media is first centrifuged at various speeds to remove cellular debris and large particles, ensuring that only the desired vesicular fractions proceed to chromatography.

What types of data can be obtained from the isolated EVs?

Isolated EVs can yield molecular readouts such as protein content analysis, post-translational modifications, and potential signaling pathways involved in cellular communication.

Can this method be adapted for different biological samples?

Yes, the procedure can be modified to accommodate various fluids such as plasma, urine, or other cell culture media, allowing broad applicability in EV research.

Are there any limitations to this method?

While effective, the method may not fully separate all extracellular proteins from EVs, which could lead to some co-isolation and potential interference in downstream analyses.

How is the effectiveness of EV separation validated?

Validation is typically performed using western blot analysis to detect specific EV markers, confirming successful isolation of target vesicles.

Protokół tutaj pokazuje, że pęcherzyki zewnątrzkomórkowe mogą być odpowiednio oddzielone od kondycjonowanych pożywek do hodowli komórkowych za pomocą chromatografii wykluczającej rozmiar.

Oddzielenie pęcherzyków zewnątrzkomórkowych od płynów, takich jak pożywki do hodowli komórkowych, może pozwolić naukowcom scharakteryzować ładunek pęcherzykowy i zbadać ich mechanizmy komunikacji wewnątrzkomórkowej. Technika ta pozwala na wystarczające oddzielenie EV i białek zewnątrzkomórkowych od kondycjonowanych pożywek do hodowli komórkowych, co jest proste i przyjazne dla użytkownika. Na początek umieść PBS w łaźni wodnej o temperaturze 37 stopni Celsjusza.

Obserwować kolby do hodowli komórkowych pod mikroskopem złożonym z soczewką obiektywową 10 x i 100 x obserwować kolby kontrolne i kolby do hodowli komórkowej poddane działaniu tunikamycyny. Zanotuj wszelkie różnice między kolbami. Usunąć pożywkę z kolby i umieścić ją w probówce o pojemności 50 mililitrów.

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