Exploring Milk EVs: Isolation and Characterization Technology

Introduction

Compared with EVs from blood or cell culture media, milk-derived EVs feature abundant sources, high yield and excellent in vivo tolerance, supporting both basic mechanism research and translational delivery applications. Nevertheless, commercial milk processing and inconsistent purification protocols can alter vesicle integrity, cargo-loading capacity and biological activity, leading to poor experimental reproducibility. Standardized material screening and multi-step purification are therefore essential for reliable milk EV studies.

Raw Milk Selection: Effects of Processing on EV Activity

Post-harvest thermal processing significantly affects the functional performance of milk EVs. Recent studies have confirmed that pasteurization impairs vesicle membrane structure and degrades surface functional proteins, thereby weakening the drug encapsulation and delivery capability of milk EVs. In curcumin delivery assays, EVs isolated from pasteurized milk exhibited substantially lower loading efficiency than those from untreated raw milk, indicating that industrial heat treatment compromises EV bioactivity.

Raw bovine milk without heating, skimming or sterilization better preserves native vesicle morphology, surface biomarkers and intrinsic biological functions. It minimizes batch variations introduced by commercial manufacturing procedures and ensures consistent quality across comparative experiments. For drug delivery and functional verification studies, raw milk is currently the preferred starting material for milk EV preparation (Table 1).

Table 1. Source and Drug-Loading Performance of Milk EVs

Common Isolation and Purification Strategies for Milk EVs

No universal standard protocol has been established for milk EV isolation. Similar to other biological fluids, mainstream approaches include differential centrifugation, density gradient centrifugation, size-exclusion chromatography (SEC), immunoaffinity capture, membrane filtration and polymer precipitation. However, milk contains high concentrations of casein and whey proteins, which severely interfere with vesicle purification and reduce sample purity.

Acidification pretreatment has been validated as an effective impurity removal strategy. Mild acid treatment induces selective precipitation of casein contaminants, effectively reducing background protein interference. Combining acidification with centrifugation or chromatographic purification markedly improves EV yield, purity and structural integrity, representing a practical and cost-effective composite workflow for milk EV preparation. Nevertheless, traditional composite protocols relying on repeated centrifugation and manual column operation suffer from low repeatability, severe vesicle loss and residual protein contamination, which greatly limits functional and quantitative research of milk EVs (Table 2-3).

Table 2. Abundance of Purified EVs Derived from Different Sources

Table 3. Common Isolation Technologies for Milk EVs

Standard Characterization Workflow for Milk EVs

Biomarker Verification by Western Blotting

Western blotting is routinely used for qualitative identification. Typical positive markers for milk EVs include tetraspanins (CD9, CD63, CD81), vesicle biogenesis proteins (Alix, Tsg101) and characteristic functional proteins (Flotillin 1, HSP90). Detection of these canonical markers confirms the identity of isolated vesicles and excludes non-vesicle contaminants.

Particle Size and Concentration Quantification

Nanoparticle Tracking Analysis (NTA) serves as the primary quantitative method to determine EV particle concentration and size distribution. Dynamic Light Scattering (DLS) complements NTA by evaluating hydrodynamic diameter and sample polydispersity, reflecting the dispersion stability of purified vesicles.

Morphological Observation

Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) are applied to visualize the typical cup-shaped morphology of EVs and assess structural integrity. Microscopic imaging e

ffectively excludes broken vesicles and aggregated impurities, providing intuitive morphological evidence of qualified samples.

Functional and Traceability Characterization

For delivery-oriented research, flow cytometry and microarray platforms enable high-throughput antigen-based profiling. Fluorescent labeling and in vitro/in vivo tracing assays are widely used to evaluate cargo loading efficiency, vesicle distribution and sustained-release performance, supporting functional validation of milk EVs as delivery vectors.

Figure 1. Characterization and Identification Technology System of Milk EVs

Application Prospects of Milk EV Delivery Systems

Unlike blood-derived EVs mainly explored as disease biomarkers, milk EVs are highly suitable for oral delivery and regenerative medicine research. Their scalable yield, good biocompatibility and high stability make them ideal natural nanocarriers for small-molecule drugs, proteins and nucleic acids. Accurate control of raw material sources and standardized purification and characterization pipelines are critical to promoting their further translational applications in disease therapy and tissue repair.

To overcome low purity, low recovery and inconsistent batches in traditional milk EV extraction, UPPER Biotech’s automated ExoFaster-500 EV isolation platform enables one-click label-free purification with three specialized SEC columns tailored to different experimental priorities.

Protocol used:

1. Prepare 10% citric acid solution (10.94 g monohydrate citric acid in 100 mL water); acetic acid can be used as an alternative.

2. Centrifuge raw milk at 10,000 × g, 4 °C for 30 min; remove the top fat layer and collect the middle aqueous phase.

3. Adjust sample pH to 4.6 with citric acid or acetic acid, incubate 15 min, then centrifuge again (10,000 × g, 4 °C, 30 min) to precipitate casein. Neutralize supernatant to pH 7 with 1 N NaOH and filter through a 0.45 μm membrane.

4. Concentrate filtrate 10–20× via ultrafiltration (5,000 × g, room temperature, 30 min).

5. Load concentrated sample onto ExoFaster-500 instrument and run the serum/plasma separation program paired with your preferred column for EV purification.

   
   

Column selection guide:

• Standard Column: Balanced yield and purity for general EV research

• High-Purity Column: Minimal protein contamination for proteomics & sensitive functional assays

• High-Concentration Column: Optimized for maximum EV particle recovery

Performance:

Column Type	Total EV Concentration	Total Protein	EV Purity (particles/μg protein)
Standard Column	3.45×10¹¹ EV/mL	0.10 mg/mL	3.34×10⁹
High-Purity Column	1.90×10¹¹ EV/mL	0.02 mg/mL	1.04×10¹⁰
High-Concentration Column	Not tested	Not tested	Not tested

Both characterized columns produce sharp NTA particle peaks with mean sizes ~118–120 nm, and >94% vesicles fall within the canonical 30–200 nm EV size window. Compared with differential ultracentrifugation, ExoFaster-500 reduces protein and lipid contaminants, delivers consistent particle size, minimizes manual errors and vesicle structural damage to preserve milk EV native bioactivity and drug-loading capacity. NTA and WB validation confirm intact EVs with strong expression of canonical markers CD9, CD63, TSG101 and Alix, nearly undetectable impurity markers, and excellent run-to-run repeatability, fully supporting functional testing, drug delivery development and multi-omics analysis.

References

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