Compare 5 Approaches to Milk EV Isolation: Findings From Morozumi et al 2021

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A comparison of 5 isolation methods for milk extracellular vesicles: a summary of the findings of Morozumi et al. 2021.

Extracellular vesicles (EVs) derived from milk have attracted attention for their potential as therapeutics, drug delivery vectors, and utility in monitoring animal health. We recently discussed in detail the importance of milk EVs alongside an explanation of milk composition and its impacts upon pre-processing and considerations for EV isolation. Here, we summarise a published study which assessed how pre-processing methods interact with different EV isolation methods, to determine the cleanest, most efficient combination.1

Method summary

Pre-processing of milk requires the removal of cells and fat globules, which can be achieved by centrifugation. In this study, raw cow milk was centrifuged using two rounds (1,200 xg at 4oC for 10 minutes) prior to attempting casein micelles removal.

Next, three pre-processing steps for the removal of caesin were investigated: centrifugation, acidification (acetic acid) and EDTA dissociation. Each were then combined with the following EV isolation methods:

  1. Ultracentrifugation (UC)  
  1. Membrane affinity purification with ExoEasy Maxi Kit
  1. Size exclusion chromatography (SEC) with a qEV column or EVSecond L70
  1. Polymer based precipitation with ExoQuick-TC or Total Exosome Isolation Kit
  1. Immuno-affinity purification with MagCapture Exosome Isolation Kit

Centrifugation performed worst all around as a casein removal step when it came to EV purity, likely due to EV depletion. For acidification and EDTA dissociation pre-processing steps, qEV isolation gave the purest EV population. As acidification was the most successful across the board, this method of casein removal was taken forward for further analyses.

In terms of purity in this second round of experiments with acidification, qEV and immuno-affinity purification performed best and isolated EVs of almost the exact same mean size. However, qEV ended on top when EV concentration and EV RNA content recovery were considered, whilst immuno-affinity purification performed poorly in these metrics. Isolation by qEV columns also gave the cleanest transmission electron micrographs.

Figure 1. Comparison of EV isolation methods in milk, assessing (A) EV purity (1011 particles/mg of protein), (B) particle concentration (1011 particles per mL of milk), and (C) RNA concentration (ng/mL of milk). Figure adapted from Morozumi et al. 2021

As isolation using qEV columns performed best in all assays, the authors recommended using qEV columns for the isolation of EVs from milk. The authors also recommended acidification for the removal of casein micelles – though they did not study chymosin, which is another promising method for casein micelle removal.  

Learn more about milk EVs: Milk Extracellular Vesicles: Isolation, Function and Future Uses


  1. Morozumi, M., Izumi, H., Shimizu, T., & Takeda, Y. (2021). Comparison of isolation methods using commercially available kits for obtaining extracellular vesicles from cow milk. Journal of dairy science, 104(6), 6463–6471.

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