A Method for the Isolation of Exosomes from Human and Bovine Milk

Abstract

Scope: Milk provides a natural means of nutrient supply to infants. Exosomes are an important component of milk that are not only being studied for their promise in translational medicine but also in infant nutrition. They also play important roles in intercellular communication and immune function in mammary glands and are able to transfer their materials to the recipient. Therefore, the isolation of high-quality exosomes is an important aspect of exosome research.

Methods and results: This study is a technical study, which provides a detailed methodology for the isolation and enrichment of exosomes from milk. In this study, we evaluate the suitability of using the exosome enrichment method that we have recently published for bovine milk, on human milk. We initially isolated extracellular vesicles from human and bovine milk on a fresh set of samples, using ultracentrifugation, and then exosomes were subsequently enriched via size exclusion chromatography (SEC). Following isolation and enrichment, exosomes from both species were characterized by particle concentration (nanoparticle tracking analysis, NTA), morphology (transmission electron microscopy, TEM), and the presence of exosomal markers (immunoblotting and mass spectrometry using information dependant acquisition (IDA)). The key exosomal characteristics of spherical/donut-shaped morphology, the presence of exosomal markers, e.g., FLOT-1 and the tetraspanins, CD9 and CD81), and particle concentration were confirmed in both human and bovine milk exosomes.

Conclusion: We conclude that our robust exosome enrichment method, previously published for bovine milk, is suitable for use on human milk.

Figure 1

Flowchart showing methods for the isolation of extracellular vesicles (EVs) from milk and subsequent exosome enrichment. EVs were isolated from human and bovine milk by differential ultracentrifugation. Unpasteurised milk was centrifuged at 3000 rcf followed by 0.25M EDTA (1 : 1; v/v) treatment to remove excess casein and supernatants (S/N) subsequently centrifuged at 12,000, 30,000, 70,000, and 100,000 rcf, respectively. The pellet obtained after the sequential centrifugation process contains EVs. After reconstitution in PBS, the EV suspension was used for exosome enrichment. 600 μl of EV suspension was introduced on top of a SEC column (qEV column) and processed via SEC to obtain 16 fractions as described in the flowchart.

Figure 2

Nanoparticle tracking analysis (NTA) to determine the particle concentration (particles/ml), yield (particles), and particle concentration per volume of milk of the exosomes obtained after enrichment, for pooled fractions 7–10 (n = 3 experimental replicates). (a) The table indicates concentration (particles/ml) in PBS for both human and bovine milk exosomes (n = 3 per group). (b) The table depicts the total yield (particles) obtained from both bovine and human milk in a 2 ml exosome solution in PBS (n = 3 per group). (c) The figure displays concentration of exosome particles extrapolated to the initial volume of bovine and human milk (10 ml and 9 ml, respectively). The concentration is expressed as particles per ml of milk. The Mann–Whitney U (unpaired) test revealed no significant differences between the two groups (error bars ±SEM).

Figure 3

Immunoblotting for the presence of exosomal marker FLOT-1 (49 kDa). (a, b) The presence of FLOT-1 in exosomes in the pooled samples (fraction 7–10) for both human and milk exosome samples. The blots also indicate presence of FLOT-1 individual exosome fractions 7, 8, 9, and 10 for both species (highlighted in yellow box). The SEC void volumes (i.e., fractions 1–6) were pooled into two groups, F1–3 and F4–6, for ease of running SDS-PAGE on a single gel. (a) Bovine. (b) Human.

Figure 4

Transmission electron microscopy displaying exosomal morphology and approximate size. Representative electron micrographs of pooled exosome isolations by size exclusion chromatography. (a, b) The presence of intact spherical/donut-shaped particles, for both bovine and human exosomes, respectively. Scale bar 200 nm. (a) Bovine. (b) Human.