Exosome Isolation: Purification and Characterisation Tools

Exosome Isolation: Purification and Characterisation Tools

WHAT ARE EXOSOMES? 

Exosomes are a type of extracellular vesicle (EV) typically 30-150nm in diameter secreted by most human cells and enable cell-to-cell communication. Exosomes are found in abundance in body fluids including blood, urine, saliva, milk, semen, bile juice, ascites, cystic, bronchoalveolar and gastrointestinal lavage fluid and play a significant role in the transfer of biomolecules like proteins, lipids, RNA, DNA.

Exosomes have been found to be involved in multiple biological roles including immune responses, pregnancy, cardiovascular diseases, central nervous system-related diseases, and cancer progression. The molecular cargo they carry can reflect their cell origin so they are considered to be promising biomarkers for the diagnosis of cancer and other various diseases. 

WHY ARE EXOSOMES IMPORTANT?

The study of exosomes is an active area of research which are being explored as a tool for disease diagnosis and treatment. Exosomes can be engineered to deliver diverse therapeutic payloads, including short interfering RNAs, antisense oligonucleotides, chemotherapeutic agents, and immune modulators, with the ability to direct their delivery to a desired target. Research into the characterisation of lipid nanoparticles highlights the importance of understanding and refining these delivery mechanisms for therapeutic applications. Ongoing research is enhancing our ability to harness their therapeutic and diagnostic potential. The need for more standardised purification and analytical procedures to study exosomes will likely reveal their functional heterogeneity.

Exosomes are being explored for their potential use as drug delivery vehicles of therapeutic RNA into specific parts of the body such as the brain in the treatment of neurological disorders including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. Research into the role of exosomes in therapeutic applications has highlighted their potential for safe and efficient delivery systems. While there are many benefits in exploring the use of exosomes for the safe and efficient delivery of their cargo, major challenges related to their isolation are highlighting development opportunities.

Therapeutic Applications of Exosomes and Associated Challenges

Therapeutic ApplicationTarget DiseasesKey Challenges
Drug Delivery VehiclesNeurological Disorders: Alzheimer’s, Parkinson’s, Huntington’sEfficient Targeting and Delivery
RNA TherapeuticsCancer TreatmentPurity and Yield of Isolated Exosomes
Immune ModulationAutoimmune DiseasesHeterogeneity in Size and Composition
Biomarker DiscoveryCancer, Cardiovascular DiseasesLack of Standardised Isolation Techniques

WHAT ARE THE CHALLENGES IN ISOLATION AND CHARACTERISATION? 

Reliable isolation and characterisation of extracellular vesicles (EVs) are critical for advancing drug discovery and play a key role in disease detection and treatment. The ability to quickly identify elevated exosome concentrations, as detailed in the comparative analysis of exosome isolation methods, can signal the onset or progression of disease, offering valuable diagnostic insights. Additionally, leveraging the unique properties of EVs as drug-delivery vehicles presents an exciting and innovative approach to combating various diseases.

However, EVs extracted from bodily fluids in response to specific conditions are highly heterogeneous, varying in size, composition, and function. This complexity makes the characterisation of these vesicles an especially challenging yet essential task for researchers.

The need for precise and accurate characterisation of exosomes will continue to grow as our knowledge of the heterogeneity of EVs, their cargo, and their functions evolves. Exosome identification and isolation have the potential to substantially improve our understanding of the basic biology of exosomes and their use in applied science and technology. Such knowledge will inform the therapeutic potential of exosomes for various diseases, including cancer and neurodegenerative diseases.

With increasing potential for their clinical utilisation, it has become imperative to optimise exosome isolation methods for maximum yield, purity and assay reproducibility. Besides the traditional ultracentrifugation method, there are currently several commercial exosome isolation kits developed based on different principles such as charge neutralisation-based precipitation, gel-filtration, affinity purification using magnetic beads, etc. However, these methods can generate significant challenges in exosome isolation, particularly with yield, purity and quality. 

Challenges of Current Exosome Isolation Methods:

  • Different exosome isolation methods can yield different amounts of exosomes
  • The size distribution of exosomes can significantly vary among preparations from different isolation methods.
  • Exosomes isolated from different isolation methods can show differences in their zeta potential
  • The quality of exosomes can differ from different isolation methods affecting functional applications
  • High cost of disposable modules/consumables, high shear stress involved and inflexible sample volumes

There is therefore a need for a new technology to improve exosome purification in terms of efficiency, purity, yield, speed and robustness. A standardised approach to exosome isolation and characterisation will enable research dependability and translational success. For more information, read this comprehensive analysis of exosome isolation methods.

Clog-Free Ultra Fast Exosome Purification With Exodus

Traditional methods for isolating exosomes, while effective for size-based separation, often suffer from clogged membrane pores, which disrupt continuous processing. These limitations are addressed in recent advances in exosome isolation technologies. To solve this issue, the EXODUS system uses a unique design that combines advanced oscillation technology with a dual-membrane filter.

Comparison of Traditional Exosome Isolation Methods vs. EXODUS System

FeatureTraditional MethodsEXODUS System
PurityModerate; impurities often remainHigh; impurities effectively filtered
YieldVariable and inconsistentHigh and reproducible
Processing SpeedTime-consuming (hours to days)Ultra-fast (minutes to hours)
Risk of CloggingHigh due to membrane pore blockagesLow; clog-free design
ScalabilityLimited to small volumesScalable for high-throughput workflows
Cost of OperationHigh; frequent consumable replacementsCost-efficient; durable components
Ease of UseManual steps prone to errorsAutomated and user-friendly
CompatibilityLimited sample typesBroad compatibility across samples

HERE’S HOW IT WORKS

  • Smart Filtration: The system uses periodic negative and air pressure switching to create oscillations on a nanoporous membrane. This allows small impurities, like proteins and nucleic acids, to pass through, while exosomes are retained in a central chamber.
  • Anti-Clogging Technology: Double harmonic oscillators generate waves and fluid movement that keep particles suspended in liquid. This prevents clogging and particle aggregation, ensuring smooth and efficient operation.

The result is a fast, reliable, and clog-free way to purify exosomes, ideal for research and clinical applications. Recent advancements in automatic, label-free exosome isolation have demonstrated significant improvements in efficiency and yield, offering a breakthrough for researchers.

The EXODUS Exosome Isolation System is an automated platform designed to streamline the purification and enrichment of exosomes. This system leverages advanced nanofiltration and oscillation technologies to provide a fast, efficient, and highly reliable solution for exosome research.

Key Features of the Exodus System:

High Purity and Yield:

  • Ensures consistent recovery of high-quality exosomes suitable for downstream applications, such as biomarker discovery, therapeutic development, and basic research.

Dual-Membrane Nanofiltration:

  • Incorporates a nanoporous membrane for precise, label-free separation.
  • Effectively captures exosomes while filtering out impurities like free nucleic acids and proteins.

Clog-Free Operation:

  • Designed to overcome common issues with membrane blockages found in traditional methods.
  • Supports continuous and ultra-fast isolation, ideal for high-throughput workflows.

Automated and User-Friendly:

  • Intuitive interfaces and automated protocols make it accessible for researchers at any level of expertise.

WHY CHOOSE THE EXODUS SYSTEM?

The EXODUS system addresses key challenges in exosome isolation by providing a reliable, scalable, and reproducible solution. It is ideal for a wide range of applications, including disease biomarker identification, Therapeutic exosome production and Fundamental studies on exosome biology.

With its advanced, compact design, the EXODUS system empowers researchers to focus on advancing their exosome studies, reducing manual intervention, and improving data consistency.

Once exosomes have been isolated and purified, they need to be characterised.

Exosome Characterisation Techniques

WHAT IS NTA? 

Nanoparticle Tracking Analysis (NTA) is the method of choice and a standard tool that has assisted exosome researchers for over a decade, providing detailed insights into NTA for exosome characterisation. NTA visualises and measures the light scattering from individual EVs moving under Brownian motion offering high-resolution size and concentration characterisation of EVs in their natural environment. 

How is NTA Useful?

  • Understand the role exosomes play in disease, and how they can be utilised in diagnostic or therapeutic applications, with size and concentration data. 
  • Optimise isolation and purification methods with detailed insight into exosome heterogeneity. Changes in size distribution can often indicate disease stage, which is important in diagnosis.
  • Easily assess batches to confirm production and sample consistency of EV samples
  • Detect subpopulations of intact vesicles, common and specific biomarkers, and cargo using fluorescence capabilities. 

The newest instrument, NanoSight Pro has evolved to include intelligent NS XPLORER software and enhanced sensitivity. Powered by machine learning, the NanoSight Pro provides guided workflows, automated processing, and automated particle identification to provide easy, quick, and accurate analysis. High sensitivity detection enables very detailed information about samples in both the light scatter and fluorescence modes. For further details, explore the benefits of using nanoparticle tracking analysis, a key tool for exosome characterisation. This allows NanoSight Pro to detect exosomes as small as 30nm in just minutes. Furthermore, samples can be recovered in their native form after the measurement. NTA can detect the presence of antigens on EVs by applying fluorescently labelled antibodies. By enabling specific detection of these biomarkers and cargo, users can better understand the behaviour of extracellular vesicles and decode their messages.

WHAT IS DLS? 

Dynamic Light Scattering (DLS), also known as Photon correlation spectroscopy, is a non-invasive, well-established technique for measuring the size and size distribution of molecules and particles typically in the submicron region, and with the latest Malvern Panalytical  Zetasizer technology, lower than 1nm. DLS is a complementary technique to NTA and can be used for measuring the size of exosomes. The method employs a monochromatic laser that passes through a liquid suspension of particles. Time-dependent fluctuations in the intensity of scattered light caused by Brownian motion of particles are observed and their velocity or translational diffusion coefficient is measured which can be converted into a hydrodynamic diameter. 

While both DLS and NTA follow the Brownian motion of dispersed light from the target particles, they operate in different ways. NTA measures individual particle diffusion. DLS measures changes in the intensity of scattered light on a bulk sample. Both methods offer several different benefits and therefore by combining the two techniques users can take advantage of the complementary information they provide.

  • NTA can often provide higher resolution size measurements, but DLS can offer a faster assessment of the mean size and polydispersity. For perfectly monodisperse samples both DLS and NTA should give the same result.
  • DLS is most suitable for particle sizes larger than ~1 micron, for quality control of nanoparticle production and for early detection of aggregates.
  • NTA is most suited for polydisperse distributions where users require a higher resolution of peaks and want to measure the concentration of nanoparticles.
  • For NTA, users can selectively look at only a fluorescently tagged part of the distribution, while in DLS fluorescence can affect the measurements and require a filter (e.g. quantum dots).
  • NTA can detect samples 10-1000 times more dilute than DLS.
  • DLS can handle a wider concentration range without dilution.

The Zetasizer Advance range of instruments are the most widely used dynamic light scattering (DLS) instruments measuring particle and molecular size, particle charge and particle concentration from less than a nanometer to several microns. Combining novel measurement capabilities together with an artificial intelligence (AI) led approach to data quality assessment, the new Zetasizer systems help gain more insight and further confidence to reliably characterise the size and surface charge of colloids, biomolecular nanoparticles; screen protein formulations for colloidal stability and the presence of aggregates; and, assess the shelf-life and stability of complex formulations. 

Building on the legacy of the industry-leading Zetasizer Nano series, the three core models, Zetasizer Lab, Zetasizer Pro and Zetasizer Ultra, can be tailored and quickly upgraded to suit specific application needs.

Each benefit from the latest advances includes:

  • Adaptive Correlation, a statistically driven approach to produce the best correlation data, without the need for sample filtering to deliver reliable, faster size measurements and added confidence in results.
  • Multi-Angle Dynamic Light Scattering (MADLS) for calibration-free measurement of particle concentration.
  • Novel constant current zeta mode – allows for high ionic strength measurements previously not achievable. Improved zeta sensitivity requires much lower sample concentrations for a zeta measurement, saving precious material.
  • Size Quality Guidance – AI-guided data quality advice allows even a novice without any prior light-scattering knowledge to make sense of sizing data.
  • The fluorescence filter wheel allows for extended applications with fluorescent samples, like quantum dots. Polarisation filters, both vertical and horizontal polarisation components can be detected, potentially gaining insights into particle rotational diffusion.
  • Novel 3 μL low volume size cell, lowers sample volume and extends concentration range.
  • Works with OmniTrust: Malvern Panalytical’s compliance solution for the regulated environment

In addition to the above, more than 100k publications are using the Zetasizer. 

Would you like to learn more about Exosome isolation characterisation toolsContact our team for expert consultation.

Contact us for a personal demonstration within your lab using the Malvern Zetasizer Ultra, Malvern NanoSight and EXODUS auto exosome isolation system today! Contact us.