Extracellular vesicles as “pro-coagulant dust” were identified by Wolf from blood platelets in Wolf (1967), and then in Pan and Johnstone (1983) were among the first scientists who described EVs (Pan and Johnstone, 1983). EVs are a heterogeneous group of vesicles containing different cargos (Abels and Breakefield, 2016). They can transfer different biomolecules such as lipids, proteins, and nucleic acids between different cells, distinguishing their significant role in cell-cell communications (Raposo and Stoorvogel, 2013). EVs are generated and released by almost all types of cells and are classified as exosomes, macrovesicles, and apoptotic bodies (Gurunathan et al., 2021; Pournaghi et al., 2021). Also, ectosomes, shedding vesicles, and microparticles are other types of EVs involved in inter/intra cellular communications (Gurunathan et al., 2019). EVs have attracted tremendous attention from both basic and clinical fields of study during the last decade due to their putative and significant role in several physiological and pathological processes (Jadli et al., 2020). EVs have been isolated from different body fluids such as blood, urine, tears, saliva, etc. (Akers et al., 2013). Some disorders such as inflammatory diseases can modify the EVs and change their numbers, content, composition, and function (Knijff-Dutmer et al., 2002). It has been shown that microbial infections can change the production and release process of EVs in infected cells (Rodrigues et al., 2018). Moreover, some EVs derived from immune cells can play a key role in induction of inflammation (Spencer and Yeruva, 2021).

Macrovesicles (MVs), also known as microparticles, are small membranous vesicles released from almost all cell types including mesenchymal stem cells, endothelial cells, some immune cells, etc. (Cocucci et al., 2009). Their size is ranging from 100 to 1000 nm, and they are formed by direct outward blebbing and pinching of the cell membrane. The production of EVs is regulated by physiological and/or pathological processes (Lynch and Ludlam, 2007; Sedgwick and D’Souza-Schorey, 2018). Initially, MVs are considered as cell debris, however, they recently were recognized as mediators of inter/intra cellular communication tools (Camussi et al., 2010). MVs can carry various bioactive molecules such as cytokines and chemokines, which highlight their antimicrobial potential and also their role in host defense against pathogenic microorganisms (Timár et al., 2013).

Apoptotic cell-derived EVs (ApoEVs) are another class of EVs released from apoptotic cells and contain cell organelles and nuclear materials (Gurunathan et al., 2019). ApoEVs are divided into two subtypes, including large apoptotic bodies (ApoBDs) with a diameter range of 1000–5000 nm and small apoptotic microvesicles (ApoMVs) with <1000 nm (Caruso and Poon, 2018). ApoEVs are important because they accelerate apoptotic cell clearance and also have a role in intercellular communication and immune modulation (Zitvogel et al., 2010; Poon et al., 2014). ApoEVs act as a key regulator of antigen presentation process, antimicrobial immunity against pathogens, and modulator of the dendritic cells’ response against viral infections (Winau et al., 2006; Schiller et al., 2008; Caruso and Poon, 2018).

Exosomes (30-150 nm in diameter) are the third group of EVs isolated from a variety of body fluids and released by the fusion of multivesicular bodies (MVBs) with the plasma membrane (Simons and Raposo, 2009; Babaei and Rezaie, 2021; Mobarak et al., 2021). Some exosomes are generated and released from various cells in response to different stimuli, but others are continuously produced and released (Mathivanan et al., 2010). They contain various types of cargo molecules which are engaged in the biogenesis and transportation ability of exosomes (Zhang et al., 2019). Exosomes have been implicated in a variety of biological functions, including elimination of old and disused biomolecules (Harding et al., 2013), involvement in tumor progression especially in angiogenesis and metastases (Rak, 2010; Hood et al., 2011), antigen presentation (Bobrie et al., 2011), differentiation of some immune cells to modulate immune responses (Zhang and Grizzle, 2011), and facilitating the spread of some pathogenic microbes or elimination of microbes through interaction with recipient cells (Furuyama and Sircili, 2021; White et al., 2021).

Several isolation methods are currently developed for the isolation and purification of EVs in bulk (Witwer et al., 2013). Sequential centrifugation and ultracentrifugation are the conventional methods to isolate EVs in cell culture media or body fluids (Théry et al., 2006). Gradient ultracentrifugation based on sucrose density is also used to minimize protein contamination (Tauro et al., 2012). Chromatography is another tool that can be employed to purify exosomes based on their size and dimensions or surface markers such as CD9, CD63, CD81, and EpCAM (Böing et al., 2014; Oksvold et al., 2015). Once isolated, the purified EVs are characterized. Currently, several methods are developed to analyze the EVs and their content for both research and clinical purposes. These methods include transmission and scanning electron microscopy (TEM and SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), resistive pulse sensing (RPS), flow cytometry, fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), microfluidics, and electrochemical biosensor-based devices (Théry et al., 2006; Woo et al., 2016).

The content, or cargo, of EVs varies and extremely depends on the parental cells and recently new databases including ExoCarta, Vesiclepedia, exoRBase, EVmiRNA, and EVpedia were developed to classify them (Liu et al., 2019). These databases provide information about the content of EVs such as lipids, proteins, miRNAs, and other components. Also they provide useful information about the isolation and characterization of EVs (Jadli et al., 2020). The proteome of the EVs is affected by their biogenesis. For example, ESCRT proteins (Alix, TSG101, HSC70, and HSP90β) regulate the biogenesis and transportation of some EVs and thus these proteins are expected to be found in EVs regardless of the type of the originating cells (Théry et al., 2001; Van Niel et al., 2006). Therefore these proteins can be used as marker for the detection and characterization of EVs (Doyle and Wang, 2019). Some tetraspanin families of proteins such as CD63, CD9, and CD81 are commonly found in EVs and also used as marker proteins both for the detection and purification of EVs (Witwer et al., 2013). Along with the exosome and some EVs surface markers, EVs carry certain biomolecules such as mRNA, miRNA (Zhou J. et al., 2013), cytokines, and antigen presentation molecules (MHC-I, MHC-II) (Gutiérrez-Vázquez et al., 2013) which contribute to the physiological and pathological function of exosomes (Dini et al., 2020). Microbial EVs contain different cargoes based on their origin. The EVs of Gram-negative bacteria contain cytoplasmic proteins, nucleic acids, virulence factors (e.g., toxins), peptidoglycan, and inner membrane. The EVs of Gram-positive bacteria contain membrane-associated virulence proteins, fatty acids, lipoteichoic acid, phospholipids, and some components similar to the Gram-negative EVs (Yu et al., 2018; Bose et al., 2020).

As mentioned above, EVs are mainly responsible for inter/intracellular communications. It was shown that EVs could interact with target cells and therefore they make an impact on cell physiology, phenotype, and function (Simons and Raposo, 2009; Mardpour et al., 2018). Also they can mediate the horizontal transfer of genetic materials (Natasha et al., 2014). Due to the widespread and cell-specific availability of some types of EVs, particularly exosomes, in almost all body fluids, they can be considered as biomarkers (Zhang et al., 2019). Moreover, EVs can be used as delivery vehicles for the efficient transfer of biological therapeutic agents across different biological barriers to desired cells (Haney et al., 2015). In addition, EVs can be applied in regenerative medicine, tissue engineering and cell homeostasis (Gurunathan et al., 2021). They play critical roles in immunoregulation, including antigen presentation, immune activation, immune suppression, and also immune tolerance via exosome-mediated inter/intracellular communications. They also play pivotal role in the host defense against viral and microbial infections (Gurunathan et al., 2021). Documented evidence has shown that host cells-derived EVs and even EVs derived from bacteria can mediate the crosstalk between pathogen and innate immune cells, and thus modulate the innate immune responses of the host (Munich et al., 2012).

In this review article, we first discussed the biogenesis and the fate of EVs. Then, EV-based therapy was described and recent developments in understanding the potential application of stem cell-derived EVs in infectious diseases were recapitulated. In addition, the mechanisms by which EVs were exploited to fight against infectious diseases were highlighted. Finally, the deriver challenges that exists in the translation of stem cell-derived EVs into the clinical arena were explored.

The biogenesis of the exosomes is well-defined as compared to the other types of EVs. The biogenesis of exosomes is a multistep biological process regulated through different signaling pathways (Abels and Breakefield, 2016). Biogenesis of exosomes initiates with the formation of early endosomes followed by second inward budding of the endosomal membrane which leads to the formation of the late endosomes. Late endosomes or intraluminal vesicles (ILVs) follow the endocytic pathway for the generation of exosomes (Sluijter et al., 2014). In the final stage, the generated ILVs are released as exosomes into the extracellular space via exocytosis (Jadli et al., 2020). Some endogenous molecules such as small GTPase Ral (Hyenne et al., 2018) and adiponectin/T-cadherin (Obata et al., 2018) and also some microbes including viral infections and Gram-positive and Gram-negative bacterial infections as extrinsic factors can influence the biogenesis of exosomes (Crenshaw et al., 2018). For the biogenesis of exosomes, different protein sorting mechanisms have been identified, among them endosomal sorting complex transport (ESCRT)-dependent pathway (Frankel and Audhya, 2018) and ESCRT-independent pathway (Babst, 2011) are two widely explained mechanisms (Figure 1).

To release exosomes, multiple cellular steps are required to be completed including formation of MVBs, transportation of MVBs to the plasma membrane, and fusion of MVBs with the plasma membrane. Several molecules have been engaged in these processes (Rabinovich et al., 2000). MVB can either fuse with a lysosome to degrade their cargo or fuse with the plasma membrane, leading to exosome release. ISGylation, a post-translational ubiquitin-like modification, is one of the signals that regulates the MVBs’ fate. ISGylation of MVB proteins promotes fusion of MVBs with lysosomes, which promoting the exosome release (Chen et al., 2003).

Following the release and secretion of EVs to the extracellular environment, the EVs’ ability to interact with recipient cells and capacity to deliver their contents such as proteins, lipids, and nucleic acids into the recipient cells can determine the role of EVs in physiological and pathological processes (Jadli et al., 2020). Several mechanisms have been introduced for the uptake of exosomes, including phagocytosis, macropinocytosis, clathrin-mediated endocytosis (CME), caveolin-dependent endocytosis (CDE), and plasma membrane fusion (Tamura et al., 2016; Sun et al., 2018). Phagocytosis is in charge of the internalization of bacteria, EVs, and others components. Toll-like receptors (TLRs), scavenger, and complement receptors, as specific targets on the cell surface, participate in invaginations around the material intended for internalization (Wei et al., 2021). Macropinocytosis is another mechanism characterized by plasma membrane ruffling and is induced by growth factors or other signal stimulations. Membrane ruffles form a cup-like structure that seals at its distal tips to construct a relatively large endosome (Canton, 2018). The resulting vesicles contain extracellular fluid and small particles (Wei et al., 2021). CME is a receptor-mediated endocytic process used to transport a wide range of cargo molecules from the cell surface to the interior. Clathrin and adaptor protein 2 (AP2) complexes are necessary for the formation of clathrin-coated vesicles and then the internalization of cargo molecules (Palmirotta et al., 2018). CDE requires the caveolin, a dimeric protein, in the plasma membrane to facilitate the raft-mediated endocytosis (Melo et al., 2015). CDE is another mechanism for the uptake of EVs, but the precise mechanism of internalization may differ depending on the type of EVs and recipient cells (Nanbo et al., 2013). Fusion is the last mechanism of EVs internalization, which enables EVs membrane to directly merge with the cell plasma membrane and transfer cargo molecules to recipient cells (Jadli et al., 2020).

Eukaryotic cells generate a heterogeneous group of EV subtypes, recognized by biogenesis mechanism and their size (Sedgwick and D’Souza-Schorey, 2018; Théry et al., 2018). EVs have been observed in all body fluids of humans (Raposo et al., 1996; Lässer et al., 2011; Street et al., 2012; Roberts and Kurre, 2013; Hiemstra et al., 2014) and are generated by various cell types (Raposo et al., 1996; Barry et al., 1998; Van Niel et al., 2001; Lai and Breakefield, 2012; Peinado et al., 2012). EVs are responsible for developing the functional range of the bioactive molecules secreted by cells, improving their stability, and boosting their ability to achieve better localized concentrations (Liu et al., 2019; Guo et al., 2021). EVs have an important role in delivery and orchestration of antimicrobial responses from host immune system. In infectious diseases, the cells such as epithelial cells and macrophages have the first contact with the EVs of pathogens that containing bioactive molecules and induce host pro-inflammatory responses (Hui Winnie et al., 2018; Li et al., 2018; White et al., 2021). The stimulated immune cells during infections may produce EVs that convey antimicrobial agents (Kesimer et al., 2009; Hu et al., 2013; Timár et al., 2013) or play decoy roles to protect host cells by binding and coating toxins secreted from bacterial pathogens (da Cruz et al., 2020) (Table 1). Production of EVs by a variety of host cells may be enhanced during infectious diseases (da Cruz et al., 2020). Different types of microorganisms and viruses have all been reported to directly stimulate host EVs secretion (Kim et al., 2012; Antwi-Baffour et al., 2020; Mehanny et al., 2020). The enhancement of host EVs secretion has been shown following an extracellular challenge of alveolar epithelial cells with heat-sacrificed bacteria, providing that the activator of this response was bacterial CpG DNA that bond to endosomal receptors of TLR9 (Keller et al., 2020). Host endothelial cells and macrophages infected by bacterial agents are similarly promoted to release EVs (Bhatnagar et al., 2007; Hui Winnie et al., 2018; Li et al., 2018). Besides increasing the production of EVs, infections can alter the contents of EVs produced by host cells (Bhatnagar et al., 2007). MSCs-derived EVs promote healing process in diabetic foot by loading some bioactive molecules including growth factors, nucleic acids, and proteins. Also, as a vehicle for non-bioactive substances like antibiotics can inhibit the bacterial growth and accelerate improvement in the diabetic wound repair in bacteria-associated diabetic foot ulcers (Raghav et al., 2021).

Host EVs produced by immune cells have been demonstrated to elicit strong antimicrobial effects in in vitro and in vivo conditions (Timár et al., 2013; Wang C. et al., 2019). These antimicrobial potencies are contributed to various bacteriostatic and bactericidal compounds present in the EVs cargo (Table 1) (Timár et al., 2013; Hiemstra et al., 2014). For instance, EVs obtained from human urinary tract are usually enriched in proteins with immune functions, such as bacteriostatic proteins like mucin-1, fibronectin, CD14, and also bactericidal proteins such as calprotectin, dermcidin and lysozyme C (Hiemstra et al., 2014). Moreover, such EVs hindered the growth of probiotic and uropathogenic strains of E. coli as well as its laboratory-adopted strains, and mediated the bactericidal functions through a lytic process (Hiemstra et al., 2014). Also, EVs are assumed to stabilize bioactive components like RNAs, and affect the non-lethal pathways of controlling microbial behaviors (Liu et al., 2016). EVs released from the host cells upon exposure to pathogenic microbes may also be able to safeguard cells from microbial assaults via efficient imitating the targets of their toxins and functioning as decoys (Keller et al., 2020).

Due to their nanometer to micrometer scale, detection of EVs by means of currently available lab equipment is challenging. Also, the tissue sources of the MSCs have an impact on the EVs characteristics. It was shown that the origin of the MSCs has an impact on the amounts and sizes of EVs. Exosome samples isolated by ultracentrifugation (UC) and tangential flow filtration (TFF) showed abundant expression of CD81 and CD9 markers and depletion of calnexin as compared to parental cells, but CD63 marker was only expressed on UC-isolated EVs (Haraszti et al., 2018). Therefore, different isolation methods produce different populations of EVs, and thus emphasize the need for standardized good manufacturing practices (GMPs). Because of different components of EVs, some databases such as ExoCarta, Vesiclepedia, and EVpedia have been established in recent years (Keerthikumar et al., 2016; Pathan et al., 2019). These databases have valuable resources for the identification of different EVs, but some specific and universal markers for each EV have not yet been provided. Therefore, further studies are required for the precise characterization of EVs and determining their identity.

Since EVs are derived from different cells, including MSCs, they may contain some impurities. For example, fetal bovine serum (FBS) is often added to the MSCs culture media. It was shown that FBS fractions contain RNA molecules and deep sequencing of these fractions showed that 13.6% and 21.7% of the RNA in cell pellet and supernatant mapped to the human genome, respectively (Wei et al., 2016). Some laboratories employed the UC for the removal of the majority of EVs and miRNAs found in the FBS (Shelke et al., 2014; Wei et al., 2016). Because of xenobiotic contents of FBS, some manufacturers used human platelet lysate instead of FBS. Human platelet lysate is xenobiotic-free and fibrinogen-depleted and can be used for MSCs culture in GMP studies. It was shown that UC could be able to eliminate the serum-derived RNAs in the lysates (Pachler et al., 2017b). It should be noted that EVs isolated by UC and anion exchange chromatography have similar markers and size distribution, but EVs enriched by TFF are not similar (Heath et al., 2018). Other methods were developed to improve the EVs’ purity include quantification of protein to particle ratio (Webber and Clayton, 2013) and protein to lipid ratio (Osteikoetxea et al., 2015); however, these methods still have some limitations.

Many factors influence the content and amount of EVs released by cells and there are currently no standard protocols for the isolation and storage of EVs (Lener et al., 2015). Also, there is MSC donor-to-donor and batch-to-batch variation (Russell et al., 2018). It was shown that EVs are highly sensitive to cell stress, and their content may be changed in response to stress. For example, treatment of human placental cells with tunicamycin induces ER stress in these cells and leads to the release of EVs containing HSP70 and HMGB1 (Collett et al., 2018). The content of the cell culture media, the composition of the serum added to the media, and the drug interactions affect the EVs’ integrity (Wiest and Zubair, 2020). Isolation methods also change the composition of EVs. Haraszti et al. showed that when MSCs were grown in 3D culture conditions and isolated by TFF, displayed a different protein content compared to other methods of culturing and isolation (Haraszti et al., 2018). Most studies focus on UC as a gold standard for the isolation of EVs. Busatto et al. (2018) reported that when they used the US for isolation, the size distribution and albumin purity of samples changed significantly from batch-to-batch, but using TFF, in contrast, showed less batch-to-batch differences. These studies highlighted the factors affecting the reproducibility of EVs and emphasized the need for the development and validation of isolation methods.

Sterility tests are assays performed by manufacturers to determine the microbial contaminations. Strict donor eligibility criteria and screening methods for diseases are the first steps for the determination of sterility and safety of the products. The companies generating EVs must comply with FDA Title 21, Part 610 (Wiest and Zubair, 2020). Microbial contamination is important not only for the safety of the recipients, but also some microbes produce and secret EVs which might interfere with the EVs in the therapeutic product (Quah and O’neill, 2007; Giri et al., 2010). The size of some viral particles is similar to EVs which raises some challenges for EV isolation by size-dependent techniques such as TFF and other chromatography-based methods (Matthews, 1975). TFF is better than UC for reducing the risk of contamination because TFF method can be conducted in a closed system, but UC technique requires multiple steps and requires multiple transfer of the fractions to the new containers (Wiest and Zubair, 2020).

Due to cell-free nature, EVs are hypothesized to be safer than other products, but there are limited data about the safety profile of EV-containing compounds (Wiest and Zubair, 2020). A short-term safety study was conducted by Montaner-Tarbes et al. (2018), where they treated healthy pigs with EVs derived from pigs with the porcine syndrome. They found that healthy pigs treated with EVs have no signs of the disease (Montaner-Tarbes et al., 2018). Until recently, adverse events (AEs) or toxicity related to EVs-based treatments are rarely reported (Nassar et al., 2016; Saleh et al., 2019).

Extracellular vesicles are very sensitive to temperature and pH of the storage buffer (Wiest and Zubair, 2020). The ideal temperature for the long-time storage of EVs is –80°C (Otrokocsi et al., 2014). It has been shown that the quantity of EVs decreases in a time-dependent manner when they are stored at room temperature or 4°C following isolation. Also, the results of the light-scattering analysis demonstrated a notable time-dependent increase in the structural changes of EVs when they stored for a long time at –20°C (Otrokocsi et al., 2014). Changes in the pH of the storage buffer can induce EVs’ aggregation and loss of their functionality (Lener et al., 2015). The storage buffer is also an important factor for maintaining the EVs’ functionality. The phosphate-buffered saline was exploited in the majority of published studies as the EVs storage buffer (Li et al., 2019; Marcoux et al., 2019), but others also have used sucrose buffer (Busatto et al., 2018), lactated Ringer’s solution (Pachler et al., 2017b), and PBS supplemented with trehalose (Bosch et al., 2016) for the storage of EVs. The type of EVs and their application may require different storage buffers and maintaining conditions.

Despite the difficulties in identifying active components in the EVs, potency determination becomes more popular in the last few years in preclinical studies (Wiest and Zubair, 2020). Due to the pleiotropic effects of proteins and RNAs contained within EVs, identifying the active ingredients in exosome therapy is challenging. EVs’ potency assays are promising methods to overcome the challenges of identifying an active ingredient. The basics of many potency assays is the release of pro-inflammatory cytokines by M1-phenotype macrophages. To do this, EVs must be added to the culture of M1-phenotype macrophages and the desired inflammatory marker is measured based on the dose of EVs (Pachler et al., 2017a; Willis et al., 2018). These methods are used frequently for the potency evaluation but further research is needed to validate their applications.

Large-scale production of EVs for clinical applications needs scale-up culture of MSCs, but long-term passaging may result in losing clonal and differentiation capacity of cells (Raghav et al., 2021). Therefore, it is necessary to develop new methods for reliable expansion of MSCs to mass-produce EVs for clinical use. Also, large-scale culture of MSCs in bioreactor requires the addition of some ingredients such as fibronectin for cell adherence purposes. Fibronectin as an ingredient has some complications because this protein makes clogging in filter pores and interferes with the size-based selection of EVs using the TFF method. Therefore the conditioned media need an extra centrifugation step, which make the risk of contamination more (Wiest and Zubair, 2020).

For the production of EVs in large quantities, the companies demand a standardized manufacturing process which must comply with GMP regulations (Mobarak et al., 2021). The GMP-grade production of EVs is a process that depends on the cell type, culture media, cultivation, and purification methods. In the case of cell type, five cell types including bone marrow and adipose tissue-derived MSCs, monocyte-derived dendritic cells (DCs), human cardiac progenitor cells, and HEK293 cells have been used in GMP-grade for production of EVs. Cultivation methods employ both static and dynamic systems. Flask based systems are static but bioreactor systems are dynamic (Rezaie et al., 2021). In the GMP-grade production of EVs, bioreactor systems are preferred due to the dynamic monitoring system (Akbari and Rezaie, 2020). In the case of purification of GMP-grade products, a number of steps including filtration for the removal of cell debris, centrifugation for enrichment of the conditioned media, and isolation of EVs from the media should be performed. Differential centrifugation, despite its complications, is the preferred method for concentrating the conditioned media (Salimi et al., 2020).