Ultracentrifugation is currently the most frequently used and reported method for extracting breast milk exosomes and is considered the “gold standard” for exosomes extraction (36). The ultracentrifugation method is based on the difference in the sedimentation speed of exosomes, proteins, cells, and other substances in the sample. Through different centrifugal forces and time, the supernatant and sediment are separated. The separation efficiency of exosomes depends not only on the centrifugal force but also on the type of rotor, κ-factor, and solution viscosity (37). Compared with the horizontal rotor, the fixed-angle rotor has a shorter sedimentation path length and have a better separation effect. However, the disadvantage of this method is that the obtained breast milk exosomes are deposited on the tube wall instead of the bottom, which makes them quite mobile and compromises the yield. The κ-factor can measure the ball-forming efficiency of the rotor. The specific rotor parameters (t-time, in hours, κ-κ factor, s-sedimentation coefficient) need to be obtained to estimate the time required for the sedimentation of breast milk exosomes. The centrifugal conditions used in different laboratories can be compared and standardized (38). Viscosity is also an important parameter of breast milk exosomes. When using ultracentrifugation to process biological liquids, it is necessary to consider the impact of sample viscosity on the sedimentation rate and the size of breast milk exosomes in advance (39).

The ultracentrifugation method is easy to operate and can be followed by entry-level technicians. It is the most common method for breast milk exosome purification, requiring only one time of ultracentrifugation. Besides, the cost of ultracentrifuge is low and the consumption of solvents is minimal. It has gradually become the most popular choice for researchers. The basic idea of using ultracentrifugation to extract breast milk exosomes is as follows. First, the fat globules in the emulsion are removed by preliminary centrifugation, and then casein and cell debris in the emulsion are removed by second centrifugation. After that, the 0.22 μm filter can be used for pre-treatment of the sample to prevent the clogging of the delicate ultrafilter, and the sample is further filtered using proper ultrafilter with certain cut-off molecular weight (MW) to remove apoptotic bodies and small vesicle aggregates (40–42). Centrifugation is normally implemented to accelerate the separation processes, and to dissolve breast milk exosomes in PBS solution. According to experimental conditions, you can choose to further obtain breast milk exosomes with specific MW by ultracentrifugation. Sow milk exosomes were extracted by 110000 g centrifugation with SW41T rotor for 2 h, and it was found that sow milk exosomes were rich in miRNA promoting intestinal epithelial cell proliferation (43, 44). Izumi et al. (37) carried out ultracentrifugation at 100,000 g, 90 min, and washed with PBS to obtain milk exosomes. Later experiments showed that the miRNA in milk exosomes can be used by human macrophages ingest.

Although ultracentrifugation is a widely used method for extracting milk exosomes, it also has shortcomings. The preliminary treatment can remove some contaminants with relatively large physical size, but there are still other impurities in the sample with similar MW to milk exosomes. To further study the breast milk exosomes, especially in the field of proteomics, co-precipitation of protein aggregates in milk requires additional purification steps for qualitative and quantitative mass spectrometry analysis (7). In addition, ultracentrifugation is relatively time-consuming and may cause damage to the breast milk exosomes under high-speed centrifugation conditions, which will have a certain impact on downstream analysis (45).

Density gradient centrifugation is an improvement as compared to the traditional differential centrifugation. Via the introduce of an inert medium into the centrifugation system, the former transfers the exosomes to the corresponding density gradient range by ultracentrifugation for separation. Density gradient centrifugation is further divided into rate zone centrifugation and isopycnic gradient centrifugation. The former requires a precise density gradient centrifugation system (such as iodixanol) before separation, while the latter does not need to be pre-set, but requires a suitable “self-partitioning” centrifugation media (such as cesium chloride). After ultracentrifugation, exosomes can be dissolved and stored in a stable form in their corresponding density gradient range. Density gradient centrifugation does not require repeated centrifugation and can separate various substances in the sample within one time, and the physicochemical properties of exosomes are not affected due to the protection of the inert medium. Therefore, this method can be used to isolate exosomes with excellent integrity, while multiple types of substances in the sample can be simultaneously isolated for comparative studies. For example, Jeppesen et al. used density gradient centrifugation to simultaneously separate small vesicles (including exosomes) and non-vesicle fractions in cell culture fluids for comparative analysis (46). At present, OptiPrep™ iodixanol is normally used as a centrifugation medium for density gradient separation of exosomes, and the isolated exosomes showed high purity and moderate yield (47). In addition, the process of rate zone centrifugation is dynamic as the density of solutes including breast milk exosomes is greater than the density of the gradient medium. To prevent breast milk exosomes from precipitating, high-density spacers are often added to the bottom of the centrifuge tube (36). However, appropriate centrifugation time is also important to avoid impurities with similar densities.

As a special form of ultracentrifugation, density gradient centrifugation can remove protein aggregates with different densities and obtain high-purity breast milk exosomes. The disadvantage is that the operation is complicated and time-consuming. The principle underlying the density gradient method is similar to that of ultracentrifugation, except that the crude exosomes obtained from density gradient centrifugation need to be placed in a density gradient medium and centrifuged for further purification. The density of the HBM exosomes layer is 1.13–1.19 g/mL, which is their average density within the density gradient (48). This layer is thus carefully aspirated and redissolved in PBS. Currently, proteomics studies of breast milk exosomes tend to use the sucrose density gradient method (24, 49–52). The more classic method is the extraction method of Reinhardt when he studied the proteomics of bovine milk exosomes in 2012 (51). In this method, the purified milk exosomes were obtained in 43%/35% layer after centrifugation under four sucrose density gradients of 80, 43, 35, and 5%. In this way, the separation of milk exosomes from non-vesicle particles was successfully achieved. Therefore, this method separates vesicles from particles of different densities and can extract HBM exosomes existing in low content. This method has high purity, and the buffer effect of the separation medium can also reduce the damage to HBM exosomes by centrifugal force, which is suitable for functional research, marker detection, and content analysis of exosomes. Exosomes have been successfully isolated from cell culture supernatant and HBM using this method. However, this method is more complicated to operate, which limits its separation efficiency and its application in clinical and diagnostic research.

The polymer precipitation method, also known as the chemical precipitation method, is originally used in virus research. The mechanism of this method is to combine water molecules by introducing drainage polymers, such as polyethylene glycol (PEG) (53), to change the dispersion status of the sample system so that components with low solubility can precipitate and thus be separated (54). By adding proper precipitant to the supernatant, breast milk exosomes precipitation can be obtained by centrifugation. Zhou et al. (55) extracted HBM exosomes with ExoQuick Precipitation Kit and showed that it was rich in immune-related miRNA. Lukasik et al. (56) extracted HBM exosomes and sow milk exosomes using precipitation kits and analyzed their miRNA, it is indicating that mammalian milk exosomes are rich in miRNA. Rani et al. (57) used the same precipitation kits to extract exosomes from HBM. By establishing a new in vitro digestion model and doing cell experiment, it was shown that HBM exosomes protect miRNA from severe digestion processes. Thus, HBM exosomes can cross the intestinal barrier into blood circulation and obtain cell function.

Both chemical precipitation and ultracentrifugation are non-specific methods for separating breast milk exosomes. That is why its pretreatment and post purification process is important. Subcellular particles such as lipoprotein can be removed in the pre-separation step, and the polymer can be removed by the Sephadex G-25 column in the post-separation step. Aside from the other components which are hard to remove from the sample, the precipitation reagent itself also brings in interferences, such as PEG, which can cause severe noise during proteomic analysis. PEG is an ion inhibitor and can lead to + 44 ion series in the mass spectrum (58). As a non-volatile solute, PEG can prevent matrix crystallization, but will pollute the liquid chromatographic column of matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) (59). However, such a dilemma can be solved by choosing proper sample pre-treatment methods such as gel digestion or proteomics research (60, 61). Other unsolved issues in the precipitation method using PEG include low purity and recovery, miscellaneous protein impurities (false positive), uneven particle size, polymer contaminants, and spoilage of HBM exosomes caused by chemical additives such as Tween-20, etc. These unsolved issues will affect the biological activity of HBM exosomes to a certain extent.

The size exclusion is a method of extracting breast milk exosomes according to the size of exosomes. At present, the extraction of milk exosomes by the size exclusion method is mostly combined with ultracentrifugation. Milk fat globules, cell fragments, and other substances are removed in the early stage. Exosomes are then obtained by ultra-centrifugation, which is then eluted and purified by a chromatographic column (61, 62). Blans et al. (27) successfully isolated extracellular vesicles from human and bovine milk by the size exclusion method, and found that the expression of marker proteins such as CD9, CD63, and CD81 monoclonal antibodies were higher by the size exclusion method without ultracentrifugation. Vaswani et al. extracted breast milk exosomes by ultracentrifugation combined with the size exclusion method and iodixanol density gradient method (63, 64). The results showed that ultracentrifugation combined with the size exclusion method had higher HBM exosomes yield.

The main advantages of size exclusion method are simple operation, high enrichment efficiency, good reproducibility, and high sample purity. In addition, the size exclusion method relies on gravity flow and low pressure to separate breast milk exosomes, so it can obtain unruptured breast milk exosomes with regular shape. However, the chromatographic column limits the sample loading amount, generally no more than 0.5 mL, which reduces the extraction efficiency of breast milk exosomes. That is why the method is time-consuming and is not suitable for batch processing. This method is only suitable for functional study, marker detection and content analysis of breast milk exosomes. In addition, multiple chromatographic columns are normally used due to the existence of protein and lipid interference in these exosomes, which limits the application of this method (65).

Proteomics research of milk exosomes revealed that aside from common proteins derived from different cells, breast milk exosomes also contain cell-specific proteins, which can be used as markers for their immune separation. The surface of HBM exosomes contains many targeting markers, such as CD9, CD63, and CD81 (27, 54), or annexin from the four-transmembrane protein family (66), which can be selectively combined with specific antibodies for their extraction. At present, immunocapture methods are available in the form of various kits. The most commonly used immunocapture kits are Enzyme-linked immunosorbent assay (ELISA), and immunomagnetic beads are becoming popular in recent years (28). ELISA uses purified antibodies, such as CD9 antibody, to coat a microplate, and sample incubation is carried out using the microplate to extract breast milk exosomes. The immunomagnetic bead method uses CD9, CD63, and other antibodies to modify the surface of the magnetic beads, and sample incubation with the addition of these magnetic beads is conducted to adsorb HBM exosomes with the corresponding surface ligand via antibody-antigen interaction. Compared with the ELISA microplate, the magnetic beads have a larger contact surface area and is easy to disperse in large size sample, and the obtained exosomes tend to stay in their intact structure due to the rapid magnetic separation (67).

The most representative extraction method in immunocapture is magnetic bead extraction, and the main advantage is that magnetic beads provide a large surface area for exosome capture to improve the extraction efficiency. Because magnetic bead extraction can separate exosomes from specific cells, and this method is highly specific, the purity of the product is high and is suitable for marker detection and clinical diagnosis of exosomes (68, 69). However, its application is limited to samples with a certain surface target for immune adsorption and the cost is high. In addition, the ELISA method and the immunomagnetic bead method require multiple times of washing during the extraction process, which contributes to the loss of exosomes (70). At present, there are few reports on the extraction of breast milk exosomes by the immunocapture method. Considering the complexity of the components of HBM, higher requirements are put forward for the pretreatment and purification of HBM. At the same time, the immunocapture method is not suitable for the processing of a large number of samples. Therefore, it is necessary to consider pre-processing of HBM and then concentrating it to achieve better results. In addition, this method has some problems, such as harsh use and storage conditions.

Microfluidic technology is a recently developed technique for high demanding separation tasks. The microfluidic chips are designed for small-volume liquid samples and can act as lab-on-a-chip to achieve separation and reaction in their dedicated designed channels within a short time, which can be applied to exosome separation and downstream analysis. Microfluidic technology is a promising direction of liquid biopsy for its high efficiency and easy operability. Exosomes are targeted by the binding with specific antibodies immobilized on the inner capture surface of microfluidics. By making surface modifications on the inner walls of these tunnels, the separation of exosomes using microfluidics can be achieved based on different chromatographic principles. At present, microfluidic technology is mainly divided into three categories, which are separation based on size, separation based on immunoaffinity, and dynamic separation (29, 71).

Size-based milk exosomes separation devices usually include nano filters, nanoporous membranes, or nanoarrays, which are usually placed in cross-sections or fabricated on the substrate of a microfluidic channel. When the sample fluid flows through the channel, these exosomes will be trapped in the structures to achieve the separation effect. The principle of the extraction method of milk exosomes based on immunoaffinity is similar to the separation by immunocapture. By modifying the surface of the microchannel with antibodies or affinity particles or immuno-magnetic beads, the microfluidic device with immunoaffinity is used to collect these milk exosomes. According to specific biomarkers on the surface of milk exosomes, these exosomes containing the target protein are specifically separated from other extracellular vesicles (50). Considering the limitations and shortcomings of extraction methods based on size and immunoaffinity, some studies have combined or integrated the use of external forces such as electric field, acoustic force, and hydrodynamic force into the microfluidic design for faster and easier extraction of milk exosomes. These methods can be classified as dynamic separation methods. Zhao et al. (72) used a dialysis membrane of 30 nm pore size to capture exosomes on the membrane surface, while allowing proteins to flow through the membrane. Specifically, exosomes are manipulated by applying an electric field to a porous dialysis membrane (72). In this device, non-target nanoparticles were driven away, and exosomes were captured on the membrane concurrently by electrical forces. This method showed 65% exosomes recovery and 83.6% protein removal in a relatively short time (≈30 min). However, this method requires optimized electrical conditions for different liquid samples. Furthermore, the device has a complicated structure that need to be further simplified. Meanwhile, Lee et al. proposed a continuous and contactless “acoustic nanofilter” system for extraction of exosomes based on ultrasonic standing waves, in which these exosomes are separated by size and density through applying different acoustic forces (73). In this device, an acoustic field is applied across the sample flow field, forming pressure nodes at either side of the channel. Larger particles experience a much stronger acoustic force that moves more rapidly across the channel to the pressure nodes, and are removed by “sheath flows” within these regions. Because the filtration operates in a continuous-flow mode, the risk of channel clogging is minimized. This method has achieved a 80% recovery for exosomes and a 90% recovery for larger microvesicles from the cell culture medium. One remarkable advantage of this device is that particles of different sizes can be separated by tuning the magnitude and frequency of the acoustic field, as well as the flow rate. Complicated nanolithography is avoided in the fabrication of this device, although electrical and acoustic transducers are essential components. Furthermore, relevant technical acoustics knowledge is required for the proper operation of the device. Using another strategy, Yang et al. (30) applied flow field-flow fractionation (FlFFF) technology for the separation of subcellular species, including exosomes. In this case, the external force employed is a hydrodynamic force, and separation by FlFFF is achieved within a rectangular microchannel. Sample components of cell homogenates are carried by migration flow along the channel axis to the detector, with a separate flow moving across the channel, which results in extraction between sample components along one wall of the channel. Smaller particles diffuse faster and move further than larger particles against the channel wall in a direction opposite to the crossflow and smaller particles ultimately migrate faster down the channel, since the flow velocity of the parabolic flow profile increases as the distance from the channel wall increases.

The major limitation of microfluidics is the sample size and the recovery rate. Although size-based microfluidics can produce homogeneous exosomes that are not contaminated by non-exosome proteins and other extracellular vesicles, the recovery rate is low. The microfluidic separation method based on immunoaffinity achieves high exosome purity but can only isolate milk exosomes rich in target proteins. The dynamic extraction method can achieve the uniformity of milk exosomes size after extraction. However, it requires expensive equipment, such as microfluidic devices, and professional technicians (71). Besides, microfluidic technology is difficult to be applied on a large scale due to the lack of standardization, large-scale clinical sample testing, and relevant method validation (72). Ultimately, the extraction of breast milk exosomes based on microfluidic technology has great potential for the development of clinical devices that can be used in hospitals for diagnostics, as well as portable personal pre-diagnostic devices used in various samples, such as HBM and bovine breast milk (74).

HBM provides essential nutrients for newborn growth and development and contains a variety of bioactive ingredients that can affect gastrointestinal tract development in breastfed infants. HBM also contains mRNA, microRNA, and lncRNA, most of which are encapsulated in human breast milk-derived exosomes and have an impact on important development-related biological functions (75). Exosomes found in preterm colostrum (PC) and term colostrum (TC) promoted VEGF protein expression, induced the proliferation and migration of small intestinal epithelial cells (FHCs) and induced angiogenic responses in endothelial cells (76). Exosomal circRNAs found in human colostrum (HC) may regulate VEGF signaling, and intestinal development (77, 78). HBM exosomes can influence gastrointestinal protection, and are important in the prevention and treatment of gastrointestinal diseases, which can regulate the metabolism of biological substances and promote the growth and development of infants (55, 79–83). HBM exosomes exert beneficial effects in preventing necrotizing enterocolitis (NEC) by reducing inflammation and injury to the intestinal epithelium as well as by restoring intestinal tight-junction proteins. HBM exosomes can promote intestinal maturation and microbiome establishment, which act as mediators for probiotics to function. HBM exosomes may cooperate with probiotics to regulate the intestinal microbiota of infants (18, 84–92), so as to improve the resistance to intestinal pathogens (Table 2).

HBM exosomes also have immune function (93), and these exosomes are known to regulate B cell, T cell, monocyte development, and to control the innate immune response and cytokine production. HBM exosomes contribute to physiological and therapeutic functions. Several studies have reported that HBM is rich in immune-regulating miRNA, such as let-7a, miR-21, miR-141, miR-146a, and miR-148a. Further research shows that exosomal miRNA may transfer genetic material to the infant, which can be protected from digestion in the baby’s gastrointestinal tract and circulate into the blood (Table 3). The Gabrielson group demonstrated that human breast milk-derived exosomes inhibited CD3-induced production of IL-2, IFN-γ, and TNF-α in peripheral blood mononuclear cells (PBMCs) while increasing the number of FoxP3 + CD4 + CD25 + regulatory T cells in PBMCs. They also reported that MUC-1 expressed on human breast milk-derived exosomes binded to DC-SIGN on monocyte-derived dendritic cells (MDDCs) and blocked viral transfer from MDDCs to CD4 + T cells via clinical research (94). In addition, HBM exosomes possess different phenotypes, depending upon maternal sensitization and lifestyle, and different phenotypes influence allergy development in children differentially (95). For example, exosomes derived from mothers with an anthroposophic lifestyle are associated with a lower prevalence of allergic sensitization than those from non-anthroposophic mothers. These data suggest that HBM exosomes potentially influence the immune system. The results revealed that exosomes from colostrum are highly enriched in proteins implicated in the innate immune response, inflammatory response, acute-phase response, platelet activation, cell growth, and complement activation, whereas proteins implicated in transport and apoptosis are enriched in exosomes from mature milk (96). Proteomic studies support the immunomodulatory function of HBM exosomes. Antigen peptide MHC complexes are presented to T cells through direct presentation or cross presentation to mediate their immune response to antigens, which indicate the importance of colostrum in immune defense and immune system development in infancy.

Neonatal immune system development is not mature yet and is vulnerable to pathogenic bacteria, neonatal necrotizing enterocolitis, neonatal sepsis, and other infectious diseases. HBM contains a large number of antibacterial components (89), immune cells, and immune regulatory factors, which can promote the maturation of the immune system and regulate immune tolerance and inflammatory response. The discovery of miRNA in HBM with immunological protection provides a new possibility for the prevention and treatment of neonatal infectious diseases. miRNA-146a in breast milk can regulate the expression of Toll-like receptor 4 (TLR4) through a negative feedback mechanism (17). Yang et al. have found that the miRNA-146a content in neonatal monocytes is significantly higher in 24 h after stimulation with lipopolysaccharide, and has protective effects on infected newborn. It can reduce the incidence of neonatal septic shock and chronic infection. The contents of miRNA-146a and miRNA-223 in the blood of patients with sepsis decreased significantly, which can be used as biomarkers for the diagnosis of sepsis. The decline of miRNA-150 and miRNA-574-5p is related to the severity of sepsis and can be used to evaluate the prognosis of sepsis. The contents of miRNA-146a, miRNA-223, miRNA-150, and miRNA-574-5p in HBM are high. Breastfeeding may play an anti-infective role by increasing these miRNAs level in the blood of infants (97).

HBM exosomes can resist enteroviruses, and exosomes in colostrum can neutralize at least one or several enteroviruses, including poliovirus types 1, 2, and 3, Coxsackievirus A9, B3 and Be, ECHO virus types 6 and 9, and rotavirus. HBM exosomes also can resist respiratory viruses and can resist the infection of influenza virus, parainfluenza virus, adenovirus, and respiratory syncytial virus. In addition to the above viruses, it has been reported that HBM exosomes contain resistance factors to herpes simplex virus and show activity against human immunodeficiency virus, cytomegalovirus, HIV-1, and hepatitis C virus, which is related to their ability to prevent virus-cell adhesion or replication (98, 99). In short, the antiviral characteristics of HBM exosomes can be summarized as follows. firstly, there are specific and non-specific immune components against a variety of viruses in HBM exosomes. Secondly, SIgA plays a major role in these antiviral substances. Thirdly, the titer of antiviral substances in colostrum is high, but it decreases significantly in mature milk.

HBM exosome surface expression of MHC molecules, costimulatory molecules can activate in vivo antigen-specific T cells to play their immune role. In vitro experiments showed that T cells could not be activated directly by exosomes but by the aid of dendritic cells (DC). That is, exosomes secreted by a DC carrying antigen-MHC molecular complex are phagocytosed by another DC, and the DC is stimulated by some signal to mature. The mature DC can be used as a medium to activate specific T cell immune response. In this process, the main role of HBM exosomes is to present the antigen-MHC molecular complex to the DC that is not exposed to the antigen. In this way, the number of DC-carrying antigens increases, and more T cells are activated to cause a strong immune response. In antitumor immunity, HBM exosomes carrying tumor antigen are phagocytosed by DC and use DC as a medium to activate tumor antigen-specific T cells, thus playing an antitumor role (1, 100). With the significant advancements in the research of nucleic acids, proteins, and microbiota, HBM is now known to contain distinct bioactive molecules along with renowned nutritional components, and their therapeutic roles are becoming acknowledged. In particular, HBM exosomes have gained much attention owing to their inherent antitumor activities. These exosomes modulate immune function and suppress the proliferation of various cancer cells in vitro and in vivo, and their chemotherapeutic function mainly comes from the miRNA with immune-regulatory and tumor-suppressive activities (15).

The fusion of exosomes with liposomes can increase the loading capacity and also retain the targeting capability. Therefore, HBM exosomes have come into focus as “natural carriers” for certain drugs (101). Drug carriers include liposomes, dendrimers, and polymers because they are easy to digest, non-toxic, and stable (102, 103). Aside from all these attributes, HBM exosomes are considered as a promising material to act as drug carriers due to their natural low cytotoxicity, high bioavailability, low immunogenicity, and autophagy in exosomes provides a theoretical approach for the treatment of lysosomal related diseases (104). According to the study of Kim et al., HBM exosomes can serve as oral delivery vehicles for chemotherapeutic agents and siRNAs, and further modification of these exosomes with a tumor-targeting ligand enables the targeted delivery of drugs to the tumor sites (93, 105).