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Page 95 Rutter et al. Extracell Vesicles Circ Nucleic Acids 2023;4:90-106 https://dx.doi.org/10.20517/evcna.2023.04

[59]
barrier to fungal EV isolation . If grown for too long, mycelial pellets may contribute to cellular
contamination in EV preparations. The inner core of these pellets is dense and allows for poor oxygen
diffusion. Eventually, cells in the core undergo apoptosis, which could taint EV preparations with
[65]
cytoplasmic contamination .

Depending on the organism, fungal phytopathogens have been grown from 16 h to 4 weeks, although 72 h
is the most common period [Figure 1]. Fungal tissues in the culture are pelleted at 4,000 to 8,000 rpm and
the supernatant is processed for EVs. One low-speed spin (~10,000 to 15,000 x g lasting 30 minutes) is used
to pellet any large particles or potential cellular debris [Figure 1]. The supernatant can then be concentrated
using an ultrafiltration system and the EVs pelleted at 100,000 x g for one hour [Figure 1]. Some studies also
include filtration through a 0.22-0.45 µm filter before or after the low-speed centrifugation step, as well as
multiple washes of the EV pellet [Figure 1].

Yields for DUC are typically low, which requires some laboratories to pool multiple samples to obtain
[52]
enough material for analyses . Using size exclusion chromatography (SEC) instead of DUC, Garcia-Ceron
et al. was able to double or even triple the amount of EVs isolated from Fov cultures compared to
experiments using DUC . SEC allows large volumes of supernatant to be processed for EVs while better
[62]
preserving their integrity by avoiding centrifugal forces that could damage vesicles or cause aggregates.
Another method for improving yield is to generate protoplasts. For example, little to no EVs were found in
the culture supernatant of C. higginsianum. Only when the cell walls were digested could EVs be isolated .
[59]
This technique has also been used to effectively isolate EVs from the filamentous, opportunistic human
pathogen Aspergillus fumigatus . Although it improves yield, the more fragile nature of protoplasts means
[66]
a higher risk of cytoplasmic contamination. Removing the cell wall could also influence the protein cargo of
[66]
EVs as the fungus responds to stress and attempts to generate new cell walls .

EV pellets are heterogeneous in nature, containing both different populations of EVs and non-vesicular
components. Purification is encouraged when attempting to define a biomarker for EVs or determine
functionality . SEC can be used in tandem with DUC as a way to purify EV pellets away from non-vesicular
[4]
[57]
components . This method is fairly successful at removing non-vesicular components but cannot
completely eliminate some soluble proteins and lipoproteins [67,68] . Density gradient ultracentrifugation using
sucrose or iodixanol, an iso-osmotic compound with low toxicity, is another common technique for
purifying EV samples [24,52,59] . This method is more successful at isolating EVs away from non-vesicle
components but can reduce yield . Both SEC and density gradient ultracentrifugation have the added
[68]
benefit of being able to separate different populations of vesicles [24,59,67] .

EV CHARACTERISTICS
After isolation, EV pellets are characterized to confirm the presence of membrane vesicles. Common
analyses include negative staining transmission electron microscopy (TEM), dynamic light scattering (DLS)
or nano-particle tracking (NTA) and, when possible, immunoblots for protein EV markers.

Fungal EVs can range from 20 to 1,000 nm in diameter . When negatively stained for TEM, EVs from
[69]
fungal phytopathogens appear as cup-shaped objects ranging from 50 to 500 nm in diameter [55,56] [Table 1].
NTA of EVs from fungal phytopathogens has recorded diameters ranging from ~90 to 300 nm [24,52,55,56,59,62] ,
while DLS of EVs from Alternaria infectoria recorded two populations of EVs, one with a diameter of 50 nm
and the other with a diameter of 100 nm [Table 1]. These slightly different measurements no doubt
[60]
depend upon the species of fungi and the growth conditions, but they also have much to do with the
method of measurement. TEM allows researchers to observe a wide range of particles, but measurements