EXOSOME RESEARCH SERVICES

IMMUNOSTEP through its extensive experience in this field knows how to help you with your Exosome Research.

Introduction to Exosomes

Exosomes are small (~50‐150 nm) extracellular vesicles (EVs) released from all cell types and found in body fluids and cell culture supernatants. Exosomes are generated by fusion of a specialized endosome, the multivesicular body (MVB), with the plasma membrane (Fig.1). Exosomes have been proposed to provide means for intercellular exchange of macromolecules, allowing the transfer of proteins, lipids, mRNA, miRNA and DNA, contributing to intercellular communication in relevant biological processes, including apoptosis, antigen presentation, angiogenesis, inflammation, and coagulation; playing therefore an important role in the development of several diseases, and specifically, modulating cancer microenvironment and the immune response. In addition, exosomes have recently emerged as a new source of potential non‐invasive biomarkers for various diseases, since they can be easily obtained from body fluids such as urine, blood, saliva or breastmilk and their composition may be directly dependent on the physiological and/or pathological state of the patient. Even the number of secreted exosomes can change with the onset of different pathologies, so the detection of quantity variations could be of great relevance for diagnosis, especially in patients with cancer. Isolation and characterization of exosomes from body fluids can provide very valuable information for early detection, disease monitoring and development of effective treatments against cancer and autoimmune diseases, among others. Moreover, a deeper knowledge of the exosome biology can also accelerate the use of these extracellular vesicles in fields such as regenerative medicine, vaccines and monoclonal antibodies, where they could play an important role as delivery systems, helping to increase the effectiveness of the treatments.

Fig.1 Exosome biogenesis (J Proteomics. 2019 Apr 30;198:87-97). 




Immunostep offers its clients a wide variety of comprehensive exosome services, contributing significantly to facilitate the achievement of objectives in the scientific, diagnostic and therapeutic development areas. 

Exosome Isolation/Purification

With respect to starting material for isolation Evs the most common body fluids employed are plasma, serum, urine and milk and regarding the starting volume by sample type, majority of researchers that report a starting volume >100 ml used cell culture media, whilst those researchers that report a starting volume <1 ml, studied plasma.  About isolation methods, ultracentrifugation remains by far the most widely used primary method, followed by a combination of isolation techniques. However when the starting volume is little or sample correspond to a body fluids researchers employ more frequently methods like precipitation, size exclusion chromatography and magnetic beads (Fig.2). 

Fig.2 Techniques used for the isolation of extracellular vesicles (J Extracell Vesicles. 2016 Oct 31;5:32945). 

Immunostep, has developed a series of protocols for the isolation and purification of -quality exosomes derived from multiple sources including cell culture samples and all human proximal fluids, including plasma, urine, cerebrospinal fluid, amniotic fluid, bronchoalveolar lavage fluid, synovial fluid, sperm , saliva, malignant and pleural effusions of ascites and breast milk.

Protocols for Exosome Purification 

  • Ultracentrifugation: Based on density and size under centrifugal force is generally considered being the gold standard method for extracellular vesicles isolation, although it may produce some aggregation of EVs.

  • Precipitation: based on EVs solubility, is a quick method, with a high recovery although it usually precipitates non-EV material.

  • Size Exclusion Column (SEC): probably the most suitable method, alone or in combination, to isolate exosomes from biological samples and in particular from plasma / serum, since it allows the separation of exosomes from important contaminants such as proteins and HDL. Additionally, Immunostep has columns that allow separation between EVs-exosomes and small EVs-exosomes.

Fig.3 Representative elution profile for SEC exosome purification. In red, the vesicle elution profile whilst in blue the proteins eluted in later fractions than vesicles.  

  • Immunoaffinity capture: Based on the use of capture beads, it allows the specific isolation of exosomes based on the membrane display of a specific marker. Example: CD63, EpCAM, PD-L1, etc..


Fig.4:  Immunecapture beads. Schematic representation, not to scale, of the caputure bead assay. Sci Rep. 2019 Feb 14;9(1):2042. 



In summary, Immunostep we have developed a wide range of protocols for exosome isolation/purification  from all kind of samples, providing a customized solution for each project, with high purity and optimal recovery results and compatible with any downstream processing or analysis.

Exosome Characterization. 

Immunostep has implemented and developed several characterization and validation methods, for both research and clinical purposes, that allow analyze purity and composition (Fig. 3) and to quantify exosomal cargo, which contains critical information that has proven tremendously promising for diagnostic, prognostic and even therapeutic use in a variety of disease areas.

Fig. 3: Exosome Composition. Exosomes are unique in their molecular composition, at the level of proteins, lipids and nucleic acids, and as we mention before this molecular content is a reflection of the state of precursor cell at the time exosome is generated.  J Proteomics. 2019 Apr 30;198:87-97. 

These methods include: 

  • Transmission electron microscopy (TEM)

  • Scanning electron microscopy (SEM)

  • Nanoparticle tracking analysis (NTA)

  • Western blot (WB)

  • Enzyme-linked immunosorbent assay (ELISA)

  • Flow cytometry

  • Fluorescence-activated cell sorting (FACS).

  • Multiplexing bead arrays.

  • Gene expression analysis (mRNA and microRNA).

Fig. 3: Electronic microscopy. Exosomes isolated by a capture bead.

Sci Rep. 2019 Feb 14;9(1):2042. 

Fig. 4: Nanoparticle tracking analysis (NTA). Size and particle concentration analysis.  Sci Rep. 2019 Feb 14;9(1):2042. 




Fig. 5: Bead-based Array Principle. Exosome bead array allow detection up to 10 exosomal surface markers plus one isotype controls. J Proteomics. 2019 Apr 30;198:87-97. 

Exosome characterization and analysis presents a tremendous clinical opportunity and specifically in the area of liquid biopsy. However, realizing the potential requires detecting and characterizing exosomes with high accuracy and reproducibility, which can be challenging for any project or research. Immunostep makes available to its clients all their experience and technical capabilities in the field of exosomes, to make the clinical use of these EVs a reality.

Exosome Production

Exosomes are useful as vectors for drugs and as active substance for cell therapy. Thus exosomes are composed of cell membranes, rather than synthetic polymers, and as such better tolerated by the host, at the same time that reduces exposure of the content to host proteases and antibodies. Additionally has the ability to cross the blood-brain barrier allowing the cargo delivery in nervous central systems.  Finally MSC-derived exosomes has a great potential for regenerative medicine and cell therapy. However, technical challenges in obtaining sufficient amounts of exosome have limited further progress in studies and clinical application.

In this sense, Immunostep offers Large-Scale Exosome Production, thanks to the use of Hollow fiber membrane systems.

How can we help you?

The Immunostep service team has a long experience in the production, isolation and characterization of exosomes and make available all this experience at your disposal. Below are some examples of finished projects:. 

Service

Description

1

Isolation, characterization and subsequent analysis of miRNAs expression in plasma samples

Exosome Isolation combining SEC and capture beads for the analysis of miRNAs (mir-16-5p,

mir-126-3p, mir-30c-5p and mir-19a-3p)

2

MSC-Derived Exosomes production and characterization

Isolation and expansion of adipose-derived mesenchymal stromal cells for MSC-Derived Exosomes production for regenerative medicine purposes.

3

Exosome samples comparative by bead custom make array platform.

Semi-quantitative analysis of markers of tumor exosomes in a battery of CSF samples by custom make multiplexing bead array

4

Isolation and characterization of exosomes from melanoma. 

Isolation and characterization of protein markers (WB, FCM) of exosomes from Melanoma.

5

Isolation and characterization of specific PD-L1 exosomes

Isolation and characterization of tumor exosomes exosome thanks to the use of PD-L1 capture beads in A375-derived exosomes and plasma.