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Page 450 Shami-shah et al. Extracell Vesicles Circ Nucleic Acids 2023;4:447-60 https://dx.doi.org/10.20517/evcna.2023.14

specific EVs a monumental challenge.

While conventional techniques have allowed for EV isolation from various biofluids, additional advances
are needed to achieve the separation of rare EV subtypes. Understanding the benefits and limitations of
these techniques further paves a pathway towards utilizing them for sub-type-specific EV isolation. This
review provides a brief overview of various techniques, describes their challenges and limitations, and adds
a potential pathway towards cell-type specific EV isolation.

EV ISOLATION TECHNIQUES
Ultracentrifugation
Ultracentrifugation has been one of the most widely used methods for EV isolation from complex biological
[31]
samples , which is based on the principle of sedimentation, where EVs are separated from other
biomolecules based on their shape, size, and density . Ultracentrifugation involves first centrifuging a
[20]
sample at a low speed of up to 2,000 xg to remove large debris and dead cells. The resulting supernatant is
then centrifuged at 16,500 xg or less to pellet large apoptotic bodies . After removing these larger species,
[31]
the supernatant is placed in an ultracentrifuge and spun at high speeds, typically around 100,000 xg or
greater, for several h . The high g-force generated from the centrifugation separates different components
[32]
of the biofluid with the EVs pelleting at the bottom of the tube. The speed and duration of centrifugation
depend on the size and density of the EVs being isolated from the samples. The pellet can be washed and
resuspended in buffer for downstream analyses or long-term storage [20,32,33] .

Ultracentrifugation has been a broadly used technique for EV isolation due to several advantages-
particularly its adaptability to large volumes of samples. This attribute helps increase EV yield and makes
the approach well-suited for studies where large sample volumes are available, such as cell culture media or
easily accessible biofluids [20,34] . For example, urine has been used to successfully differentiate patients with
prostate cancer from healthy controls using EVs obtained by ultracentrifugation . Furthermore,
[35]
ultracentrifugation is also relatively inexpensive compared to other methods, such as immunoaffinity
isolation.

While ultracentrifugation is a relatively simple technique that does not require any complex
instrumentation, there are several limitations to this method. Ultracentrifugation may result in partial EV
aggregation and degradation as the high centrifugal force required may lead to artificial fusion of smaller
EVs and fission of larger EVs. This not only limits the ability of ultracentrifugation to isolate EVs of
uniform size, but can also lead to the loss of some of their original biomolecular contents . Additionally,
[33]
co-sedimentation of non-EV biomolecules, such as lipoproteins and protein aggregates with similar
buoyancy to that of EVs, can cause yield and purity problems . These disadvantages are only amplified
[20]
when attempting to isolate EVs from sample matrices with higher viscosities, making
ultracentrifugation a less viable option in some biofluids such as plasma [20,36,37] . Moreover, lengthier spin
times also make this technique less efficient and translatable to a clinical setting. Therefore, while
ultracentrifugation may still be a viable option in the clinic when working with large volumes of dilute
samples, this approach is likely incompatible with the analysis of low-abundance EV subtypes that are
present in complex biofluids.

Density gradient centrifugation
Density gradient centrifugation (DGC) is a method that is commonly used in conjunction with
ultracentrifugation to isolate extracellular vesicles (EVs) from biological fluids [23,38] . DGC involves the use of
a density gradient, which is created by layering solutions with a range of different densities in a tube, such as