How Are Lipids Extracted and Exosomes Isolated in Lipidomics Research?

In lipidomics research, the extraction of lipids and the isolation of exosomes represent two essential procedures. The following summarizes widely adopted techniques for both processes:

 

Lipid Extraction

Two classical protocols are commonly employed: the Folch and Bligh-Dyer methods.

 

1. Folch Method

Samples are mixed with a chloroform–methanol solution at a 2:1 (v/v) ratio, followed by the addition of saline to induce phase separation. This results in a biphasic system: the upper aqueous/methanol phase and the lower organic (chloroform–lipid) phase. The lower phase is collected and evaporated to yield purified lipids.

 

2. Bligh-Dyer Method

Samples are treated with chloroform, methanol, and water in a ratio of 1:2:0.8. After thorough mixing and centrifugation, the organic phase containing lipids is separated. Compared to the Folch method, the Bligh-Dyer method uses reduced solvent volumes and is better suited for small sample quantities.

 

Exosome Isolation

Several methodologies are available for the isolation of exosomes, including:

 

1. Differential Centrifugation

This multistep process involves sequential centrifugation at increasing speeds: low-speed centrifugation removes cells and large debris; medium-speed centrifugation eliminates apoptotic bodies and larger vesicles; ultracentrifugation subsequently pellets exosomes.

 

2. Density Gradient Centrifugation

Often applied after ultracentrifugation, this method uses density gradients (e.g., sucrose or iodixanol) to further purify exosomes based on their buoyant densities.

 

3. Size-Exclusion Chromatography (SEC)

SEC separates particles based on hydrodynamic size using a porous matrix. This approach minimizes protein contamination and preserves exosomal bioactivity.

 

4. Immunoaffinity-Based Isolation

Exosomes can be selectively captured using antibodies targeting specific surface markers, enabling high-purity isolation based on exosome-associated proteins.

 

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