Overcoming technical challenges in extracellular vesicle research

Extracellular vesicles (EVs) released by cells are highly heterogeneous in size, cargo, membrane composition, biogenesis, and—of critical importance for biomarker discovery and molecular diagnostics—biological function. EVs, thought to aid in local and long-distance cell communication, are released by most cells and are present in all biofluids, suggesting their crucial role in both normal and disease states. However, many vital aspects of EV function, including cargo selection and loading, the release of EVs into extracellular space, recognition and acceptance by other cells, and high efficiency in delivering bioactive molecules to the recipient cells, remain a mystery.

EV researchers are consistently turning to morphological, proteomic, genomic, and transcriptomic analyses to unravel the mysteries of EVs and better understand their biology as well as their potential use in diagnostic and therapeutic applications. Companies like Alpha Nano Tech are helping these researchers by providing them with expertise and complete EV characterization services. We recently had the opportunity to talk to Dr. Daria Filonov, chief scientific officer (CSO) at Alpha Nano Tech, about their work on extracellular vesicles.

What is your role at Alpha Nano Tech, and what does the company do?

I'm a CSO at Alpha Nano Tech, a specialty contract research organization focused on the characterization of nanoparticles and extracellular vesicles. With the recent blast of publications dedicated to exosomes and extracellular vesicles, a careful characterization of exosomes has become critically important.

Alpha Nano Tech helps design and execute research projects of any complexity, starting with EV isolation from virtually any biofluid or tissue to comprehensive characterization. The characterization methods include assessing sample purity, particle size, concentration, dispersity, charge, biomarker expression, and protein and RNA profiles. Additionally, we perform structural analysis and size cross-validation with electron microscopy.

What are extracellular vesicles, and why are they important? What first made you interested in exosomes?

Extracellular vesicles are produced by all cell types; have a dual-layer membrane; and carry proteins, RNA, and DNA fragments specific to their origin and condition. They can be classified into a few different types depending on their size and mechanism of origin. Despite all the recent attention, we are just beginning to understand how many different roles EVs can play and their potential applications. EVs are actively studied for discoveries in tissue-specific diagnostics of various diseases; as an anti-inflammatory, regenerative, and tissue-healing agent; as a component of drug resistance, angiogenesis, and fertilization mechanisms; and as a drug carrier and transmitter of antiviral activities. These are just a few examples of how exosomes can be used, and the possibilities are endless.

Emerging exosome science caught my attention years ago as it combines the excitement of working on cutting-edge biology and medicine with the joy of applying the newest nanoscale research solutions from physics.

Chief scientific officer Daria Filonov, PhD, at Alpha Nano Tech

What is Alpha Nano Tech's mission with respect to exosome research? Tell us about the work that you do at Alpha Nano Tech with extracellular vesicles, specifically exosomes.

Our company's mission is to provide scientific and technical expertise in nanomaterials and exosomes. Being in constant connection with leading analytical instrument producers and key industry leaders, we keep pace with the most recent developments and solutions available on the market so we can serve our clients with the most advanced tools. We also serve as a quality control and R&D site to producers and developers of instruments, commercial kits, and assays. Most of the projects we work on are innovative by nature and require extensive research and development to assist pioneers in this field.

Isolation of exosomes is an important step in their characterization. What are the challenges with exosome isolation, and how are you overcoming them?

Indeed, exosome isolation is the first and the most critical step. We have had an opportunity to try most of the kits and techniques available on the market to understand how each method works and the results they produce for different input materials. Moreover, as a service provider, we have a chance to tease out sources of interlaboratory variation when different labs use the same approach on similar source material.

The key parameters for any approach, in my opinion, are availability, time requirements, efficiency, specificity, and integrity of the resulting EVs. For example, popular and easy-to-use polyethylene glycol (PEG) precipitation kits provide a good yield yet suffer from binding of the polymers to the EV surface, often leading to a change in EV size and surface properties. This approach is good regarding cost, setup, and time requirements, but it lacks specificity and may impact the physical characteristics of purified EVs.

Another widely used method is ultracentrifugation. This approach provides high yields of particles but requires specialized equipment not available in every lab. It is time-consuming and does not yield pure EVs. It works well for relatively clean biofluids, like cell-culture media, which doesn't contain serum or other proteins. For more complex matrices like plasma, ultracentrifugation results in co-sedimentation of protein particles and lipoproteins that are about the same size as exosomes.

Many methods have a common problem of producing EVs with high background protein content. These proteins can strongly interfere with subsequent analysis. For example, if we are doing proteomics analysis of plasma-derived exosomes, but the major proteins in the sample are albumin and IgGs, they will hinder the signal from minor exosome-specific proteins, and we won't detect them.

Another problem we frequently face is poor yield from low-input biofluids, such as cerebrospinal fluid. How do we solve these problems? Well, first of all, choosing the right isolation method is critical. There are several factors to consider: the nature of biofluids, the mechanism of isolation, and the throughput of the method. Depending on the goals of a specific project, we often optimize the protocol, doing additional size exclusion, ultrafiltration, or affinity purification steps. It is rare for a method to work perfectly straight out of the box for every application.

We have had a great experience with Capturem columns from Takara Bio for several sample types, including plasma, urine, saliva, and cell-conditioned media. The basic protocol involves a four-step process: preclearing the sample, loading the supernatant onto a membrane that binds exosomes, washing, and eluting. The elution step can be repeated to improve yield. Also, the elution volume can be adjusted to get a more concentrated sample if necessary. That is a very handy feature for optimizing concentration to the required volumes for optimal fluorescent nanoparticle tracking analysis (fNTA), western blot, or transmission electron microscopy (TEM) analysis. The protocol takes 30 minutes at the most, and there is no fraction collection involved or any specialized equipment needed. The first step, preclearing, is essential for producing samples with minimal contamination from non-EV particles. In the next step, the sample is loaded onto the isolation column, and exosomes are bound to the membrane. This helps remove not only nonmembrane particles but also the majority of soluble proteins. One parameter to consider when designing the protocol is the capacity of the membrane: overloading the column with a large sample volume or concentration will lead to a loss in yield.

Why do you think your customers choose your services? What are the challenges of working with exosomes, and what makes Alpha Nano Tech the right partner for their project?

The quality and reliability of the data produced by Alpha Nano Tech, combined with our knowledge and experience, make it the company of choice. With so many obstacles to overcome during startup, an R&D team with limited experience in exosome research can spend a few years to achieve what we can deliver in weeks. Our quality control system is a vital and fundamental part of the company and supported by professionals with over twenty years of experience in GLP and GMP facilities. Second, the cost of having our assistance is much lower than acquiring instruments, training and supporting dedicated scientists, and keeping an annual budget for consumables and maintenance.

Lastly, we aim for the fastest possible turnaround time. We have no extra fees for "rush" analysis, as we try to complete everything as soon as possible. Of course, we recognize the importance of supporting basic research and offer a 50% discount on academic projects, accepting orders even for a single sample, and treating our clients equally.

We don't simply want the job done; we want our clients to succeed, and we want to help them advance their research. We are scientists and take pride in our research.

What are your thoughts on the future of extracellular vesicle research?

The deeper our understanding of the genesis and role of EVs, the faster we can expect the breakthroughs in technology and therapeutics. This is a great field to be in right now. We are looking forward to seeing how exosomes revolutionize the field of regenerative and personalized medicine.

And, we are here to help in overcoming any challenges.

Thank you, Daria, for sharing your insights and explaining how Alpha Nano Tech is helping advance EV research. Daria used our Capturem Extracellular Vesicle Isolation Kit to quickly isolate highly pure EVs from various biofluids, including plasma, urine, saliva, and cell-conditioned media, for downstream analyses.