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Klingeborn et al. Page 3

publications returned 4,584 articles (Dec. 14, 2016), 1,205 of which were published in 2016
and 36 slated to be published in 2017, at the time this review was written (Fig. 1). The
cancer field in particular has taken the lead investigating exosomes and other EVs for novel
approaches to therapy, new mechanistic understanding of tumorigenesis, tumor signaling,
novel biomarkers, and modulation of metastasis (Dhondt et al., 2016; Lopatina et al., 2016;
Srivastava et al., 2016). For example, modulation of the extracellular matrix (ECM) by
matrix metalloproteinases (MMPs), annexins and proteoglycans on exosomes have been
shown to increase metastasis (Hakulinen et al., 2008; Sakwe et al., 2011; Stepp et al., 2015;
You et al., 2015). This ECM-modulating exosomal activity may also play an important role
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in eye diseases where pathological ECM remodeling is an integral part of the disease
mechanism, such as in glaucoma and AMD (Bowes Rickman et al., 2013; Roy Chowdhury
et al., 2015).

2. Methods for exosome isolation – pros and cons

A number of methods can be used for isolating exosomes and small EVs. However, the EV
preparations resulting from the different methods span a wide range of purities and
properties. Thus, it is very important to choose the isolation method that is appropriate for
the downstream analysis methods that will be used or the experiments that will be done with
the isolated EVs. In particular, complex biological fluids such as plasma, serum and urine
pose difficulties for EV isolation.
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Following is a brief discussion of the most common exosome isolation methods with regard
to their pros and cons:

2.1. Differential ultracentrifugation
Differential ultracentrifugation is considered to be the gold standard for exosome and small
EV isolation (Thery et al., 2006). This technique is based on a scheme of sequential
centrifugations of the exosome-containing fluid and the resulting supernatants: 200 g, 2,000
g, 10,000 g, and 100,000 g. The resulting 100,000 g pellet, which contains exosomes, other
small to medium-sized EVs, lipoprotein particles and large protein aggregates, is
resuspended in PBS and centrifuged again at 100,000 g to control for artifactual trapping of
materials. The final pellet is resuspended or lysed depending on the downstream analysis
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method. It is a relatively simple methodology; however, the resulting pellet, although
enriched for, does not purely consist of exosomes. Other drawbacks include the need for
access to an ultracentrifuge and a relatively low yield compared to PEG precipitation as
discussed below.

2.2. Polyethylene glycol (PEG) precipitation
Precipitation of viruses by PEG solutions is a method that has been used for over 40 years
(Adams, 1973; Lewis and Metcalf, 1988; Yamamoto et al., 1970). Recent adaptations of
PEG precipitation for isolation of EVs have shown that the most efficient protocols for
exosome precipitation utilize PEG polymers with average molecular weights of 6,000 or
8,000 Da (PEG-6000 and PEG-8000) (Antes et al., 2013; Vlassov et al., 2013). PEG
precipitation-based kits such as ExoQuick (System Biosciences) and Total Exosome
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Prog Retin Eye Res. Author manuscript; available in PMC 2018 July 01.