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    Size exclusion chromatography for exosome isolation


    The isolation and characterization of exosomes from biological fluids can provide valuable information for early detection, disease monitoring and treatment development. As we have addressed in previous articles exosome isolation will be an essential process in order to use exosome in research and diagnosis, and as a consequence of that we can say there have been an important progress in the last years in exosome isolation techniques development worldwide. In this sense, size exclusion chromatography (Exosome purification columns) is one of the more efficient techniques for exosome isolation, the efficiency of exosome isolation columns in contaminant separation is higher than in other exosome isolation techniques, which means that the isolation process will be more successful. In this article, we will be describing everything you need to know about this exosome isolation techniques, and how to use exosome isolation columns (discover more about exosomes isolation and characterization methods and specific markers).

    How to isolate exosomes?

    Extracellular vesicles of all sizes and specifically exosomes (EVs) have been isolated using a wide range of different enrichment techniques such as differential ultracentrifugation, size-exclusion chromatography, ultrafiltration, polyethylene glycol-based precipitation, immunoaffinity capture (exosome capture beads) or by using microfluidics, and different commercial exosome isolation kits based on flow cytometry.

    Each of these previously mentioned methods of isolation have presented advantages and disadvantages, because each of them work differently in order to separate the exosomes and the different types of extracellular vesicles to use them in a variety of applications such as regenerative medicine, or as an active ingredient in cosmetics.

    To the question how to purify exosome from cell culture, or how to perform the isolation of exosome from blood plasma here, we will be describing one of the commonly used methods of exosome isolation, with a focus on size-exclusion chromatography.

    The chromatography process implies the use of a stationary phase packed  in a column, which enables the passage of a liquid mobile phase. This contains the analyte in an aqueous buffer solution, which passes through of the column at a rate proportional to its size. With complex mixtures composed of particles of different sizes, the larger molecules are separated from the gel and are recovered quickly, while the smaller particles are impeded and elute much later.

    What is size exclusion chromatography?

    As we have been describing so far, size exclusion chromatography is a technique for the isolation of macromolecules in which a porous gel allows the separation of different elements (solutes) according to their molecular sizes.

    The gel material used is composed of small silica particles or polymers, which have pores where molecules are trapped, so that larger molecules are retained and elute quickly, while smaller molecules can penetrate and are retained for a longer time.

    The main advantage of this method for extracellular vesicles purification and thanks to Immunostep exosome isolation columns kit, is that exosomes are isolated in a single step, the protocol takes little time and allows efficient removal of contaminating proteins as well as separation of exosome subtypes. As a result of this process, extracellular vesicles isolated by this method are obtained with high purity.

    Some of the disadvantages we can analyze about size exclusion chromatography are that samples are collected in several fractions, and these must be characterized to ensure the detection of extracellular vesicles and proteins.

    Furthermore, aseptic working conditions are necessary to avoid microbial contamination and a large sample loading volume (>1 ml) can lead to inefficient separation, so small sample volumes (~0.5 ml) per run are necessary to obtain optimal results.

    Exosome isolation columns with the highest precision

    Size exclusion chromatography (SEC) has been described as the most efficient method to isolate extracellular vesicles in a single step, with good recovery and almost complete removal of possible contaminants in exosome samples such as proteins and lipoproteins.

    In this sense, Immunostep has developed a very efficient assay based on Size exclusion chromatography (SEC), becoming this kit one of the most reliable methods for contaminant removal from exosomes, because it is able to remove contaminants of minimal size from exosomes.

    Specifically, this kit is an essential tool if we are looking to perform nucleic acid analysis or in vitro assays from a plasma and/or urine sample volumes of less than 1 mL up to a maximum volume of 20 mL.

    Furthermore, if we start from a sample volume greater than 20 mL, we could also combine Exosome Isolation columns with other techniques. For instance, we could perform a differential ultracentrifugation and then the method that best suits us depending on the purpose of its application.

    Size exclusion chromatography for exosome isolation: Exosome Isolation columns by Immunostep

    The characterization of exosomes requires in most cases the isolation of intact exosomes, and, in this sense, a large number of methods have been developed for the enrichment of exosomes from biological fluids.

    As we have described before, Immunostep offers some of the most common techniques for exosomes isolation with all the guarantees. Below we describe some of the main advantages of Immunostep Exosome isolation columns kit:

    ►  Great performance separating exosomes from important contaminants: Immunostep reusable Size Exclusion Columns allows the rapid isolation of extracellular vesicles (EVs) from cell culture supernatants and complex biological fluids. Each column efficiently removes background proteins, lipids, solutes, and other contaminants to improve the sensitivity and accuracy of downstream analysis while maintaining the biological properties of the EVs.

    ►  Fast and efficient exosome isolation kit: The vesicles can be isolated in 15 minutes thanks to these columns, and this allows EV samples to be collected and analyzed very fast. These Columns are available ready to use, and quality assured so your isolations are reproducible from column to column and batch to batch.

    ►  Reusable exosome isolation columns and efficient packaging: Immunostep columns have been developed to facilitate their use, and for this reason it comes with a “smart box” which easily transform to a practical column rack. 

    ►  Improved exosome isolation and recovery: A comparison was made using columns competitor, obtaining Immunostep columns a better performance in terms of recovery. Discover more about exosome isolation columns performance.

    How to use exosome isolation columns?

    Immunostep has developed SEC columns for EVs isolation from complex biological fluids such as: plasma, serum, urine, and cerebrospinal fluid. The protocol is very easy and efficient, and we describe it bellow:

    Column equilibration

    First of all, rinse the column with 21 mL of running buffer. Make sure enough time is given for the column to be in the operational temperature range. It is important not to allow the column to run dry, and to have into consideration that the top filter must stay wet. After that, use only fresh filtered (0.2 μL) buffer to avoid particulate contamination.

    Sample fraction collection

    When the column is used according to the protocol, for 500μl fractions, the first four fractions (2.0 mL), is the void volume which does not contain vesicles. These vesicles elute predominantly in fractions 5, 6 and 7 with removal of protein contamination. Fractions beyond 8 usually contain higher protein and lower vesicle levels.

    Sample fraction collection

    After the collection of the vesicle fractions, rinse the column with 21 mL of running buffer to rinse out all the protein and small molecules before the next sample application. After that, rinse the column with 10 mL of a bacteriostatic agent and store as indicated in the storage section.

    Isolation Column cleaning

    Cleaning is performed in order to remove any precipitated proteins or other contaminants that can build up on the column. Cleaning is performed using 0.5 ml of sodium hydroxide (500 mM NaOH). Note that the column should never be stored in sodium hydroxide. Then, equilibrate the columns immediately after the cleaning with 10 ml of water followed by 21 ml of running buffer.

    Fraction collection concentration

    If the volume of the starting sample is small and / or the concentration and EVs is low in the sample, it is possible that the EVs will be much diluted in the collected fractions. In many cases, for downstream processing it is necessary to concentrate the fractions, for that, we recommended to use an Amicon filter (100kDa).

    If you are interested in this or any other exosome isolation kit or method we can help you, contact us and let us know more about your needs.



    1. Sidhom K, Obi PO, Saleem A. A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option? Int J Mol Sci. 2020;21(18):6466. Published 2020 Sep 4. doi:10.3390/ijms21186466
    2. Liu DSK, Upton FM, Rees E, et al. Size-Exclusion Chromatography as a Technique for the Investigation of Novel Extracellular Vesicles in Cancer. Cancers (Basel). 2020;12(11):3156. Published 2020 Oct 27. doi:10.3390/cancers12113156
    3. Immunostep, Technical Data Sheet, 2022. Available online: http://exosomes.imaisd.es/wp-content/uploads/2021/10/TDS-Exosome-Isolation-Columns-SEC.pdf