Extracellular vesicles (EVs) are revolutionising our understanding of cell-to-cell communication. These ubiquitous messengers encased in a lipid bilayer membrane, are nano-sized packages actively released by virtually all cell types. Ranging from a mere 30 nanometers to a surprising 5 microns in diameter, EVs bridge the gap between cells, acting as a sophisticated communication network within the body. They shuttle a diverse cargo of biomolecules, including proteins, lipids, and nucleic acids (mRNA, miRNA), that can significantly influence the recipient cell’s phenotype and behaviour. This intricate communication system plays a critical role in numerous physiological processes, from orchestrating immune responses to facilitating tissue repair and development.
Despite their shared role as cellular messengers, EVs exhibit remarkable heterogeneity. They differ significantly in their biogenesis, size, and cellular origin. This diversity necessitates their classification into distinct categories. The three main classes of EVs are:
The success of therapeutic EVs hinges on a critical choice: the cell source. Different cell types produce EVs with unique cargo – a cocktail of proteins, lipids, and nucleic acids – that shape the therapeutic potential of these nano-messengers. EVs can be harnessed from two main sources: body fluids and in vitro cell cultures.
Our bodies harbour a wealth of EVs in various fluids, including plasma, urine, and even milk. Plasma-derived EVs, with their anti-inflammatory, antioxidant, and pro-angiogenic (blood vessel promoting) properties, are being explored for treating diverse conditions, such as cardiac damage, osteoarthritis, wounds, ageing, and muscle injuries. Platelet-enriched plasma (PRP) emerges as another exciting source, offering EVs with regenerative potential. PRP-derived EVs can promote cell proliferation and blood vessel formation, aiding tissue repair after injuries.
Breast milk extends far beyond its role as a source of essential nutrients for newborns. Recent research highlights the presence of a unique population of EVs within breast milk. These human milk-derived EVs possess a remarkable repertoire of therapeutic effects, including promoting gut maturation, mitigating intestinal damage, modulating the immune system, and even attenuating viral infections. This underscores the remarkable versatility of EVs and their potential to significantly impact infant health.
By harnessing the potential of EVs isolated from bodily fluids, researchers are unlocking a novel class of therapeutic tools. These naturally occurring messengers hold immense promise for revolutionising how we approach the treatment and management of various diseases and conditions.
EVs can also be isolated from cell cultures for therapeutical purposes. Mesenchymal stem cells (MSCs) are the rockstars of this approach, particularly valued for their immunomodulatory properties. MSC-derived EVs have a knack for suppressing excessive immune responses, making them prime candidates for treating inflammatory diseases. The therapeutic efficacy of MSC-derived EVs has been investigated in many diseases, including osteoarthritis, wound healing, and liver injury.
Beyond MSCs, the world of neural cells holds promise. Studies reveal that EVs isolated from neural cells like microglia and astrocytes, or even neural stem cells, can exert neuroprotective effects. This translates to potential improvements in motor and cognitive function after traumatic brain injury.
Almost all immune cell types, including T cells, B cells, dendritic cells, macrophages, and neutrophils, have been shown to produce EVs. These immune cell-derived EVs are pivotal players in orchestrating and modulation of immune responses. These EVs typically contain the same markers as their parental cells. For instance, NK-derived EVs contain typical NK cell markers and can deliver a cargo of cytotoxic proteins, including perforin, and granzymes that destroy target cells.
By strategically selecting the cell source, researchers can harness the unique therapeutic properties of EVs. From immunomodulation to tissue regeneration, these tiny powerhouses hold immense potential for revolutionising medicine.
Figure 2: Therapeutic EVs sources and their possible clinical applications.
Credit: Sanz-Ros J, Mas-Bargues, C et. al., doi: 10.3390/ijms24032344
Reproduced under the Creative Commons license
The burgeoning field of EV research hinges on the development of robust isolation techniques. To fully elucidate the biological roles of EVs and translate their potential into clinical applications, researchers require methods that preserve their inherent heterogeneity. These methods leverage the distinct biophysical and biochemical properties of EVs, including size, density, morphology, and surface marker expression. Selecting the optimal isolation technique is paramount, as it directly influences the purity, yield, and ultimately, the functional integrity of the isolated EV population.
In pursuit of the ideal isolation strategy, researchers are continuously refining existing techniques and exploring novel approaches. The ultimate goal lies in achieving high-purity EV isolates with preserved structural and functional characteristics, allowing for a comprehensive understanding of their biological roles and therapeutic potential. Here, we will explore the most commonly utilised EV isolation methods:
Table 1: Different techniques for isolating extracellular vesicles.
| Isolation Techniques | Mechanism | Advantages | Disadvantages |
| Differential Centrifugation | Uses high centrifugal force to separate EVs based on size and density. | – Considered the “gold standard” – Yields intact EVs | – Time-consuming – Prone to operator error – May damage EVs – Low purity (requires additional steps) |
| Density Gradient Centrifugation | Similar to differential centrifugation, but separates EVs based on their buoyant density in a gradient medium. | – Higher purity than differential centrifugation – Separates EVs by subpopulations | – More complex and expensive – Requires specialised equipment |
| Ultrafiltration | Uses membranes with different pore sizes to separate EVs based on size. | – Relatively simple and fast – Can be automated | – May clog membranes – Lower purity – May damage EVs |
| Polymer-based Precipitation | Uses polymers to precipitate EVs out of solution. | – Fast and inexpensive – Can be used with small sample volumes | – Lower purity – May not be suitable for all EV types |
| Size Exclusion Chromatography (SEC) | Separates EVs based on their size as they flow through a column packed with beads. | – Gentle on EVs – Good for separating EVs from protein contaminants | – Lower yield compared to centrifugation methods – May not separate all EV subpopulations |
| Immunoaffinity Isolation | Uses antibodies specific to EV surface markers to capture EVs. | – High purity – Isolates specific subpopulations of EVs | – Expensive – Requires knowledge of specific EV surface markers |
| Microfluidic Devices | Utilises microchannels to manipulate and isolate EVs based on size, density, or electrical properties. | – Precise control over EV isolation – Potential for automation – High potential for future development | – Relatively new technology – Limited availability of commercial devices – May require specialised expertise |
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Physical analysis of exosomes is crucial in the field of exosome research, particularly when exploring their therapeutic potential. Physical analysis helps determine the size, density, surface molecules, and purity which is essential for understanding how exosomes behave in the body and their potential for targeted delivery. This also provides a standardized way to characterise exosomes, allowing researchers to compare results across different studies and laboratories, which is vital for ensuring the reproducibility and reliability of therapeutic approaches.
Nanoparticle Tracking Analysis (NTA): This technique allows for the determination of exosome size and concentration. It combines the properties of both laser light scattering microscopy and Brownian motion to obtain size distributions of particles in liquid suspension. The machine is normally equipped with one or more lasers and an optical microscope connected to a digital camera. Particles are visualised by the light they scatter upon laser illumination, and the Brownian motion of individual exosomes is monitored. The software analyses the video frame-by-frame, tracking the movement of each exosome. By applying the Stokes-Einstein equation, which relates particle size to its Brownian motion, the software calculates the hydrodynamic diameter of each exosome. The software generates a size distribution profile, revealing the number of exosomes at various size ranges. Additionally, by analysing the total number of tracked particles within a defined volume, NTA can estimate the exosome concentration in the sample.
Dynamic Light Scattering (DLS): Like NTA, dynamic light scattering, or DLS, uses the scattered light due to the Brownian motion of particles to estimate particle size and concentration. However, DLS measures the fluctuations in the intensity of scattered light caused by the Brownian motion of particles in a solution. By analysing the pattern of fluctuations in the scattered light intensity, DLS can determine the size distribution of the particles in the solution, including the size range of exosomes present. The data obtained from DLS analysis are typically presented as a size distribution plot, which shows the relative abundance of exosomes at different size ranges within the sample. While DLS is a powerful technique for analysing the size distribution of exosomes, it only provides estimation to the average hydrodynamic diameter of the entire particle population in the sample. Unlike NTA, DLS doesn’t provide information on individual exosome sizes.
Tunable Resistive Pulse Sensing (TRPS): This emerging technique offers a high-resolution analysis of exosome size, concentration and zeta potential as they pass through a size-tunable nanopore. TRPS measures the change in electrical current as individual exosomes pass through the nanopore. As individual exosomes pass through the pore, they briefly block some of the currents. TRPS measures these current blockades. The magnitude and duration of the current blockade are related to the size of the particle that caused it. By analysing these blockades, TRPS can provide information on individual exosome sizes. The resolution capabilities of TRPS are much higher than NTA as NTA struggles to properly resolve subpopulations of multimodal samples and has been shown to overestimate particle concentration.
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Table 2: Comparison of different techniques for physical analysis of EVs.
| Tunable Resistive Pulse Sensing (TRPS) | NTA (Nanoparticle Tracking Analysis) | Dynamic Light Scattering (DLS) |
| Single-particle technique | Single-particle technique | Ensemble technique |
| Particle size is directly proportional to blockade magnitude; each particle is measured individually | Brownian motion is tracked for individual particles and the resulting diffusion coefficient is used to calculate the hydrodynamic diameter | Analysis of the fluctuations in scattered light intensity caused by Brownian motion of particles in solution |
| Using appropriately sized nanopores, particles can be measured across a wide size range (40 nm–11 µm) | Smaller size range (approximately 10 – 2000 nm) | Wider size range (1 nm –10 µm) – but strong limitations for multimodal samples |
| Automated data processing with user-friendly data visualisation interface | Advanced data analysis required | Simple protocols |
| Can resolve populations in multimodal samples | Resolution of multimodal samples is relatively limited | Cannot resolve multimodal samples |
| Not dependent on optical properties of particles and dispersant | Accurate knowledge of the optical properties of particles and dispersants is required | Accurate knowledge of the optical properties of particles and dispersants is required |
Each technique has its own strengths and limitations in exosome analysis. NTA is known for its high resolution and sensitivity, DLS offers rapid analysis suitable for high-throughput applications, and TRPS provides absolute size measurements with high resolution. The choice of technique depends on the specific requirements of the study and the characteristics of the exosome sample being analysed.
Given their rich content of biomolecules like lipids, proteins, and nucleic acids (including microRNA, messenger RNA, and DNA), along with small molecules inherited from parent cells, EVs play pivotal roles in physiological and pathological processes such as cell proliferation, immune modulation, angiogenesis, and tissue repair. Consequently, they are increasingly recognised as promising nanotherapeutics in regenerative medicine.
Indeed, EVs offer substantial advantages over conventional stem cell therapies in regenerative medicine, including multiple therapeutic effects, efficient cargo delivery, enhanced biocompatibility and safety, as well as improved reproducibility and stability. The therapeutic mechanisms of native EVs primarily involve cell-surface interactions with target cells and subsequent cargo transfer, activating signalling pathways that mitigate disease effects and promote regenerative functions.
Beyond native EVs, the field is actively exploring the potential of engineered EVs. These customised EVs offer a new frontier in targeted therapy. By incorporating specific therapeutic agents into their cargo, engineered EVs can act as targeted delivery vehicles. This approach unlocks the potential for a more potent and progressive regenerative effect compared to native EVs alone.
The future of regenerative medicine holds immense promise with EVs as the cornerstone. Research is actively investigating how EVs can be harnessed to promote healing and restore function in a wide range of organs, including the brain, heart, skin, bones and cartilage, liver, and kidneys. As research progresses, EVs have the potential to revolutionise the way we approach tissue regeneration and restore health.
Figure 3: Schematic illustration of the EVs therapeutic mechanisms and regeneration applications in various organs including brain, heart, bone, liver, kidney, and skin.
Credit: Li M., Fang F., et. al., doi: 10.7150/thno.72812
Reproduced under the Creative Commons license
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