Strategies to Enhance Extracellular Vesicle Production

Abstract

Extracellular vesicles (EVs) are sub-micrometer lipid vesicles secreted from parental cells with their information such as DNA, RNA, and proteins. EVs can deliver their cargo to recipient cells and regulate the signaling pathway of the recipient cells to determine their destiny. Depending on the cargo of EVs, the recipient cells can be changed into abnormal state or be relieved from diseases. Therefore, EVs has been spotlighted as emerging therapeutics in biomedical research. However, slow EV secretion rate is the major limitation for the clinical applications of EVs. EV secretion is highly environmental dependent and can be regulated by various stimulants such as chemicals, oxygen levels, pH, radiation, starvation, and culture methods. To overcome the limitation of low productivity of EVs, EV stimulation methods have been widely studied and applied to massive EV productions. Another strategy is the synthesis of artificial EVs from cells by physical methods such as nitrogen cavitation, extrusion via porous membrane, and sonication. These physical methods disrupt cellular membrane and reassemble the membrane to lipid vesicles containing proteins or drugs. In this review, we will focus on how EV generation can be enhanced and recent advances in large scale EV generation strategies.

Introduction

Extracellular vesicles (EVs) are lipid vesicles including exosomes, microvesicles and apoptotic bodies with 50-5000 nm sizes secreted from cells [1, 2]. EVs contain molecular constituents of parental cells such as nucleic acids [3, 4] (DNA, mRNA, small noncoding RNA and DNA fragments), proteins [5] (membrane proteins and cytosolic proteins) and small molecules [6] (metabolite, hormones). Therefore, analysis of EVs can provide the information of their parental cells, which can make EV as a circulating marker [7]. The recent findings also showed that EVs are messengers of their parental cells to communicate with recipient cells by delivering their cargo [8]. The cargo can be transferred to recipient cells and regulate them by perturbing signaling pathways. Depending on the contents of cargo or origin of EVs, EVs can provide different outcomes to the recipient cells. Tumor-derived EVs have been reported to transform the recipient cells to tumor cells [9]. EVs from stem cells can cure diseases, showing their potentials for therapeutics [10]. In addition to natural EVs, small molecules, protein, RNA, and DNA can be loaded to EVs to increase the treatment potency. In contrast to lipid particle, a conventional drug delivery system, EVs exhibit the immune tolerance [11, 12], long half-lives with low toxicity, and blood brain barrier infiltrations when they are injected in vivo. These merits make EVs as an attractive drug delivery system or therapeutics. However, there are several limitations of hiring EVs for therapeutics: slow EV secretion, heterogeneity of EV size and composition, requiring massive cellular culture system [13]. Especially, slow EV secretion rate is the major hurdle for their application toward therapeutics [14]. To overcome this limitation, various EV induction methods have been studied to increase EV yields. This review will focus on various EV stimulation methods and their application for EV production.

Stimulation of EV secretion

EVs can deliver messages of their originating cells to other cells in the body. Environmental stress increases EV release and changes the composition of EVs. Resulting EVs can propagate the stress signals to other cells and alert them to prepare for those stresses. Recent studies have unveiled the factors that control the release rate and the composition of EVs in various cells. This section will discuss about those factors such as protein regulations, thermal and oxidative stress, hypoxia, pH, radiation, starvation, chemicals, and culture environment (Table 1).

Table 1.

Various EV stimulations and their mechanisms

Type of EV stimulation	EV stimulant	Mechanism	References
Protein regulation	Cortactin	MVB trafficking regulation	[15, 16]
	Rab27a, Rab27b	MVB docking regulation	[17]
	PIKfyve	Phosphatidylinositol phosphorylation	[18]
	Eukaryotic translation initiation factor 3 subunit C	Transcription regulation	[19]
	Liver kinase B1	Energy hometostasis regulation	[20]
	ISG15	TSG101 ISGylation	[24]
Thermal and oxidative stress	Heat stress at 40 °C	Accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants	[25, 26]
	Hydrogen peroxide (50–100 µM)	Oxidative stress	[25]
Hypoxia	Low oxygen	Upregulation of Rab27a and downregulation of Rab7, LAMP1/2, NEU-1	[27–29]
pH	Low proton concentration	Not known	[30]
Radiation	γ-irradiation	DNA damage, p53 activation	[33–35]
Starvation	Low glucose level	Metabolic pathway activation	[37, 38]
Chemical treatment	Doxorubicin, foscan	DNA damage	[38]
	Gemcitabine	Upregulation of miRNA-155	[39]
	Melphalan	DNA alkylation	[40]
	Sodium iodoacetate, 2,4-dinitrophenol	Oxidative phosphorylation inhibition, glycolysis inhibition	[41]
	CI-1033, PF-00299804	EGFR inhibition	[42]
	Dithiothreitol, paraformaldehyde	Cell membrane blebbing	[43]
	Palmitic acid	Lipoapoptosis	[44]
3D culture	3D spatial architecture	Environment dependent cell growth	[45]

Protein regulations

Protein signaling pathways help cellular systems to sustain homeostasis and to defend themselves from environmental changes. Generation and composition of EVs can be also controlled by the regulation of protein signaling pathway. Therefore, a number of proteins have been reported as regulators of EV generation. Sinha et al. reported the high expression level of cortactin in EVs from head and neck squamous carcinoma cells [15]. Another research showed that knockdown or overexpression of cortactin regulated the secretion of EV, but not that of microvesicle, or ectosome [16]. Furthermore, proteomic analysis showed no difference between control cell-derived EVs and cortactin knockdown cell-derived EVs. Mechanistic study showed that cortactin did not directly regulate the EV generation in these cells. It rather regulated multivesicular bodies (MVB) trafficking, docking, and the release of EVs from invadopodia of the cells. Another MVB related EV stimulating proteins are Rab GTPases. Among those GTPases, Rab27a and Rab27b were associated with MVB docking at the plasma membrane resulting the stimulation of EV secretion [17]. When Rab27a and Rab27b were silenced, EV secretion was reduced without modifying the composition of EVs. PIKfyve, a phosphatidylinositol phosphorylating kinase has been reported as another regulator of EV generation [18]. PIKfyve inhibition by apilimod or knockdown of PIKfyve by siRNA increased the EV secretion and secretory autophagy, demonstrating that these pathways are closely related. The inhibition of PIKfyve led to blocking of lysosomal fusion with both MVBs and autophagosomes. Eukaryotic translation initiation factor 3 subunit C (EIF3C) is highly expressed during human hepatocellular carcinoma (HCC) tumor progression. EIF3C upregulation in HCC increased EV secretion for potentiating tumor angiogenesis [19]. Liver kinase B1 (LKB1) plays a critical role in EV generation of lung cancer cells. The elevated EVs by LKB1 triggered the cancer cell migration by down-regulation of migration-suppressing miRNAs including miR-125a, miR-126 and let 7b [20]. In addition to EV regulation by protein itself, it has been reported that posttranslational modification of protein is closely related to the cellular protein sorting process into EVs and the regulation of EV generation [21–24]. ISG15 is analogous to ubiquitin and conjugated to target proteins by E1, E2 and E3 ligases. This process, called ISGylation, was revealed as an EV generation controller [24]. When MVB protein TSG101 was modified with ISG15, MVB was co-localized with lysosomes. This process accelerated the aggregation and degradation of MVB resulting the reduction of EV secretion.

Thermal and oxidative stress

Thermal and oxidative stresses were reported as another way to stimulate cells to secrete more EVs. When Jurkat cells (T cell lymphocyte cell line) and Raji cells (B cell lymphocyte cell line) were subjected to heat stress at 40 °C, EVs from the cells were increased by threefold and 22-fold in comparison to the those from cells at 37 °C. For oxidative stress, the high concentration of hydrogen peroxide (50–100 µM) stimulated Jurkat and Raji cells to release 15-fold and 32-fold EVs compared to the cells in normal culture condition. These stresses generated tumor cell-derived EVs with soluble NKG2D ligand that suppressed the immune response toward tumor cells [25]. This observation might explain the NK-cell dysfuction in leukemia/lymphoma patients. Therefore, adverse effect of hyperthermal anti-cancer therapies should be considered. The mechanism of EV stimulation by thermal and oxidative stresses has been reported due to accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants [26].

Hypoxia

Hypoxic condition increased the EV secretion of ovarian cancer cells by promoting a secretory lysosome phenotype [27]. Upregulating Rab27a and downregulating Rab7, LAMP1/2, NEU-1 of the cancer cells were the critical factors of the increment of EV generation in hypoxia. The secreted EVs had high levels of STAT3 and FAS proteins which could trigger tumor progression and metastasis of recipient cells. Breast cancer cells in hypoxia increased the secretion of EVs containing hypoxia-related miRNA-210 in hypoxia-inducible factor 1α (HIF-1α) dependent manner [28]. This report showed the critical role of HIF-1α in EV generation. Moreover, glioblastoma-derived EVs in hypoxic condition induced angiogenesis and tumor growth by transferring hypoxia-related RNAs and proteins [29]. Collectively, hypoxia can stimulate the secretion of EVs in tumor cells for their survivals to overcome oxygen deficient microenvironment.

pH

The low pH (< 6.0) is another EV stimulating factor [30]. Acidic pH could generate EVs with high level of cholesterol in their membrane and make more EVs to fuse the recipient cells. Moreover, low pH led to generate EVs with more caveolin-1, which can transfer an aggressive phenotype to recipient cells. Ban et al. monitored EV generations in pH 4, 7, and 11. pH 4 increased not only the concentration of EV itself, but also the concentration of protein and RNA inside the EVs. However, high pH reduced EV secretion as well as the amounts of proteins and RNAs inside the EVs [31]. In addition to EV generation, acidic pH also stimulated the fusion between EV and recipient cells [30, 32]. These studies suggested importance of pH in EV generation and their trafficking.

Radiation

γ-irradiation increased the cellular EV secretion from melanoma cancer cells. The EVs stimulated dendritic cells to be matured by regulation of various immune related proteins for their defense from cancer cells, which can be crucial for designing EV based cancer vaccine [33]. A proteomic approach identified that TSAP6 proteins control γ-irradiation mediated EV generations in lung cancer cell lines. Mechanistic study revealed that γ-irradiation induced DNA damage of cells and activated p53 proteins. Subsequently, p53 controlled the downstream protein TSAP6, which resulted increment of the EV generation [34]. In addition to lung cancer cell lines, prostate cancer cells under irradiation also secreted more EVs in a p53-dependent manner [35].

Starvation

Serum starvation stimulated monocyte cells (THP-1) to release more EVs containing high expression level of TSG101, leaflet phospholipids, and monocyte markers (CD18, CD14) [36]. Monocyte-derived EVs resulted cell surface thrombogenicity and endothelial hemostatic balance disruption. In addition, tube formation was observed by the monocyte-derived EV treatment. Cardiomyocytes under glucose starvation also released more EVs [37]. Glucose starvation generated EVs containing more proteins and miRNAs related to metabolic pathway and energy acquisition. Cardiomyocytes-derived EVs under glucose starvation could increase not only proliferation of endothelial cells, but also their angiogenesis. This result suggested that EVs from starvation condition could be a trigger of neovascularization to compensate acute situation such as cardiac injury. Another group also reported starvation stimulated EV secretion in prostate cancer cell, PC3 [38].

Chemicals

A phototherapeutic or doxorubicin (Dox) treatment increased EV release [38]. Photosensitizer, foscan induced more than 400-fold EV enhancement compared to control within 1 h. Cytotoxic Dox induced 30 times higher EV shedding in compared to control in 24 h. Anticancer drug, gemcitabine induced miRNA-155 upregulation in pancreatic cancer cells, which led to EV secretion enhancement. miRNA-155 containing EV transformed recipient cancer cells to be resistant toward gemcitabine [39]. Melphalan, DNA alkylating agent induced secretion of EVs in multiple myeloma cells. The secreted EVs stimulated interferon-γ production in natural killer cells by activating TLR2/HSP 70 dependent NF-kB pathway [40]. EVs from cancer cell lines were increased by the treatment of sodium iodoacetate (IAA; glycolysis inhibitor) plus 2,4-dinitrophenol (DNP; oxidative phosphorylation inhibitor) [41]. Ex vivo study was performed to monitor EV secretion in the presence or absence of IAA/DNP with minced or intact kidneys from C57BL/6 mice. IAA/DNP increased the EV release from tissue explants into culture medium. In vitro and ex vivo results were further validated in vivo by injecting IAA/DNP into mice. IAA/DNP stimulated the levels of EVs in the blood compared to control mice. EGFR kinase inhibitors such as CI-1033 and PF-00299804 increased emission of EVs containing EGFR, phospho-EGFR, and genomic DNA. The phosphor-EGFR, ERK, and AKT levels of EVs were varied according to the type of EGFR inhibitors. This suggested targeted therapy could change EV protein compositions [42]. Sulfhydryl-blocking using DTT (dithiothreitol) and PFA (paraformaldehyde) has been reported as EV stimulating agent [43]. Sulfhydryl-blocking agent caused the cell membrane blebbing to generate plasma membrane-derived vesicles and increased more than 10 times higher EVs compared to natural secretion. In addition to the small molecules, saturated fatty acid has been reported as a stimulant of EVs from renal tubular epithelial cells [44]. Palmitic acid, a common saturated fatty acid caused lipoapoptosis and EV release simultaneously. Confocal microscopy demonstrated the autocrine uptake of the palmitic acid induced EVs by recipient cells. Thus, diverse chemical mediated cellular stress induces EV release and propagates the stress signal to surrounding cells via EV for the cellular survival.

3D culture system

Although various EV stimulation methods have been suggested, most of them give stresses to cells and perturb the contents of EVs from the cells. To overcome this limitation, 3D cell culture system has been established. Rocha et al. developed 3D cell culture system using agarose microwell array and compared 2D versus 3D culture system for EV production [45]. Gastric cancer cell-derived EVs in 2D and 3D culture system were collected and their contents such as RNA and protein was analyzed. Overall upregulation of miRNA and downregulation of proteins in 3D culture was observed. They also found that the 3D cell culture system generated more EVs than the 2D culture system. The size distribution of EVs from 3D cell culture showed distinct patterns compared to that of EVs from 2D cell culture system. This report suggested the importance of cellular culture system for producing EVs. Since 3D culture is closer to physiological environment than 2D culture, this finding demonstrated the importance of spatial architecture on EV generation and their contents.

Artificial EVs

Although EV generation can be increased by above mentioned cellular stimulation methods, large scale EV generation is still hard to be achieved. To tackle this issue, artificial EV generation technologies have been developed for reproducibility and high yield of EV production. These methods use the physical forces or chemicals to break cells and release the cellular components. With reconstitution of the released lipids, proteins and nucleic acids, artificial EVs can be generated in large quantity. In this section, the physical and chemical methods such as nitrogen cavitation, extrusion through porous membrane, sonication, dissolving in high pH will be discussed (Table 2).

Table 2.

A list of large-scale EV generation methods

Type of generated EV	Generation method	Generation mechanism	References
Artificial EV generation	Nitrogen cavitation	Nitrogen gas-mediated bubble formation within the cells	[46, 47]
	Extrusion via porous membrane	Pushing cell through porous membrane using extruder	[48]
	Sonication	Cell rupture with hypotonic lysis, mechanical disruption and formation of artificial EV with sonication	[50]
	High pH solution and sonication	Alkaline solution to dissolve cellular membrane. Following pH neuralization and sonication	[51]
	Artificial EV synthesis	Plasma membrane protein extraction and assembly with cholesterol and synthetic choline-based phospholipids	[52, 53]
Large scale natural EV generation	Dithiothreitol, paraformaldehyde	Sulfhydryl-blocking	[43]
	Stimulation of red blood cells	Treatment of calcium ionophore A23187	[54]
	Using bioreactor	Maximizing cell culture efficiency on large surface area	[56–58]
	Using 3D scaffold in bioreactors	Complex architecture combined with flow or mechanical stimulation	[60, 61]
	Cellular nanoporation	A transient electrical stimulation	[62]

Nitrogen cavitation

Cavitation is changing a pressure rapidly in a liquid to form many small vapor-filled cavities. Collapse of cavities can give an intense forcing for breaking materials. Nitrogen cavitation is one of the cavitation methods using nitrogen gas to generate the forces. Wang et al. harnessed nitrogen cavitation to generate EVs from white blood cells for the first time (Fig. 1A) [46]. In a cavitation container, nitrogen gas was injected to cell suspension, which allowed nitrogen dispersion into the cell under high pressure of 350 psi. When the pressure was dropped rapidly, nitrogen inside the cells formed bubbles within the cells. Expanding bubbles broke the cell and released cellular component to the solution. Broken cellular membrane spontaneously formed vesicles with broad range of particle sizes. Through a membrane with a pore size of 200 nm, extrusion of the vesicle gave a uniformed size of artificial EVs. The yield of artificial EV was 16 times higher than natural EV generations [47].

Fig. 1.

Artificial EV generation. A Scheme of EV generation by nitrogen cavitation and their purification. Reprinted with permission from ref [46]. B Cells were pushed through porous membrane using extruder to generate EVs

Extrusion via porous membrane

Extrusion is a process of pushing cell through porous membrane using extruder (Fig. 1B). Jang et al. reported monocyte-derived artificial EV generation using extruder with a serial porous membrane (10 μm, 5 μm and 1 μm filters) [48]. Resulting EVs were loaded with Dox for anti-cancer therapy in mouse model. The properties of artificial EVs were similar to naturally secreted EVs. However, the yield of EV mimetic nanovesicles was more than 100-fold higher than that of naturally secreted EVs. During the extrusion process, chemotherapeutics and fluorescent imaging molecules could be loaded inside the EVs efficiently.

Sonication

Sonication has been widely used for conventional liposome preparation [49]. Thamphiwatana et al. harnessed sonication for artificial macrophage like EV preparations [50]. The mouse macrophages were ruptured and the resulting membranes were purified through multiple processes of hypotonic lysis, mechanical disruption and differential centrifugation. They used sonication to form membrane vesicles and subsequently fused them with poly(lactic-co-glycolic acid) (PLGA) for artificial EVs. The macrophage like EVs bound to proinflammatory cytokines and inhibited the inflammation cascades in sepsis mouse model.

High pH solution and sonication

EV mimetic ghost nanovesicles loaded with anti-inflammatory drug, dexamethasone was prepared using alkaline solution and sonication [51]. Alkaline solution dissolved cellular membrane in the solution, which could be the outer shell of ghost nanovesicles. In the presence or absence of drugs, following pH neuralization and sonication made the membrane compartment self-assembled for ghost nanovesicles (Fig. 2A). The nanovesicles had similar properties of naturally released EVs. Using this method, more than 200-fold EVs were produced compared to the normal cell culture method. The nanovesicles could be loaded only with the desired drug for the maximum efficacy without any intracellular components, such as RNA, proteins and nucleic acids. The nanovesicles showed effective anti-inflammatory activity to mitigate outer membrane vesicle‐induced sepsis.

Fig. 2.

Artificial EV generation. A High pH solution and sonication. Cells were lysed with high pH solution and sonication. Membrane was purified with ultracentrifuge. Purified membrane pellet was mixed with drugs or proteins to generate artificial EVs. B Biomimetic artificial EV generation. (left panel) Extraction of proteolipid from cells. (middle panel) Assembly of plasma membrane proteins with cholesterol and synthetic choline-based phospholipids. (right panel) Vesicular formulation of biomimetic artificial EVs. DPPC, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine; DSPC, 1, 2-distearoyl-sn-glycero-3-phosphocholine; DOPC, 1, 2-dioleoyl-sn-glycero-3-phosphocholine; CHOL, cholesterol. Reprinted with permission from ref [53]

Biomimetic artificial EVs

Top-down synthetic strategy to make artificial EVs was reported (Fig. 2B) [52, 53]. Plasma membrane proteins were extracted from leukocyte and assembled with cholesterol and synthetic choline-based phospholipids. Controlling ratios of membrane proteins-to-synthetic phospholipids generated various types of artificial EVs, called leukosomes. 1:300 ratio of protein to lipids provided promising stability, protein integrity and membrane fluidity. During the vesicle synthesis, drug or cytosolic protein could be injected to the vesicles for therapeutic purposes. Leukosomes preferentially targeted inflamed vasculature, which enabled the selective drug delivery to inflamed tissues. This report offered an option to synthesize customized artificial EVs for clinical purposes.

Large scale EV generation

Along with artificial EVs, natural EV generation methods have been developed using chemical, electrical and 3D-environmental simulation. These strategies can be used for large scale EV generations from cancer cells, normal cells, and even stem cells for therapeutic purposes. The merit of these approaches is that natural EV contents having therapeutic effects can be preserved. Moreover, natural EVs have lower chance to trigger immune response in vivo when they are used as therapeutics [10]. This section will discuss about the natural EV generation methods using chemicals, red blood cells, cellular nanoporation, bioreactors and 3D scaffolds (Table 2).

Chemicals

Massive number of EVs with therapeutics were prepared with sulfhydryl-blocking using DTT (dithiothreitol) and PFA (paraformaldehyde) as above mentioned [43]. Sulfhydryl-blocking generated EVs with desirable size for Dox delivery in mouse model. Sulfhydryl-blocking increased more than tenfold of EVs than naturally released EVs within 2 h. EVs produced by this strategy showed improved cellular uptake and intracellular release of Dox compared to liposomes. In tumor mouse model, they showed that the EV inhibited tumor growth and improved survival rate. This fast and scalable EV generation method showed their potential toward clinical application.

Red blood cells-derived EVs

Usman et al. devised a new strategy for production of large-scale EVs from red blood cells (RBCs) [54]. By the treatment of calcium ionophore A23187, 5–10 × 10¹³ EVs from 200 ml of RBC were obtained. Anti-sense oligonucleotide, Cas9 RNA, and guide RNA were loaded to EVs from RBCs for therapeutic purposes. The RNA delivery with RBC-derived EVs was successful in human cells and xenograft mouse models. RBC-derived EVs were also used for carriers of small molecule drug, camptothecin [55]. The camptothecin loaded EVs showed higher retention time and good cytotoxicity toward lung carcinoma cells. Since RBCs have no nuclear and mitochondrial DNA, EVs from RBCs can be used safely without the risk of transferring oncogenic DNA and retrotransposon elements. Abundance of RBCs is another advantage for using them as EV sources. Considering the blood transfusions in clinic, RBC-derived EVs can be considered as safe drug delivery systems. For storage issue, aggregation or morphological change of the EVs were not observed after 1–3 freeze–thaw cycles. This high durability of EVs from RBCs makes them as a promising therapeutic.

Bioreactor

Bioreactors were initially designed for the large-scale cell culture by maximizing surface compared to conventional 2D culture system. Recently, those methods also have been utilized for increasing EV yields by maximizing cell culture efficiency without any stress stimulation on cells. Bioreactor systems could enhance EV yields up to 40-fold than 2D cell culture systems [56]. Although more media and frequent culture was required, these methods allowed an efficient cell expansion without perturbing cellular functions. Representative 3D culture bioreactor systems are microcarriers [57] and hollow-fibre bioreactors [58]. The microcarriers are small beads with various types of material, pore size, and surface charge. A certain type of microcarriers can be choose for the optimal culture condition for specific cell types. The cells attached to the microcarriers were cultured in spinner flask with a rotating plastic paddle for mixing [59]. The sizes of flask were ranging from 100 mL to 20 L, which enabled large scale cell culture for massive EV generations with small volume of culture media (Fig. 3A). This method yielded large amount of MSCs and their EVs without perturbing their pluripotency. A drawback of this method was that MSCs became more metabolically active with more nutrient consumption. Hollow-fibre bioreactor is another type of bioreactor for maximizing surface (Fig. 3B). The hollow-fibre has a reservoir bottle with hollow and semi-permeable fibres [58]. The diameter of the tube-shape fibres are 200 µm. The glass reservoir bottle with fibres in it has inlet and outlet for media injection, gas flow and harvesting cellular products such as EVs. The fresh media could be injected to the reservoir automatically via pumping systems through closed circuit, which could reduce the risk of contaminations. The exchange of media and CO₂ was achieved with the porous barriers of the fibres.

Fig. 3.

The scheme of bioreactors. A Microcarriers containing the cells were incubated in spinner flask. B Hollow-fibre bioreactor and its cross section with cells inside.

Adapted from ref [14]

3D scaffold in bioreactors

Although bioreactors can enhance the yield of EV generations, it still does not meet the demands on large scale EVs for clinical applications. Recently, 3D-printed scaffold combined with bioreactors was used for establishing complex cellular architecture. Patel et al. designed a scaffold with large culture surface area for culture medium perfusion and material transport using 3D printing (Fig. 4A) [60]. The scaffold structure allowed media to flow through the scaffold and gave the shear stresses (1.5 × 10^–2 dyn/cm²) near the reported physiological pressure values for endothelial cells. Human dermal microvascular endothelial cells (HDMEC) in this system could increase more than 100-fold EV production compared to 2D culture system. However, the ratio of CD63-positive EVs in whole EV population produced by this system was reduced than that of 2D culture system. These data suggested substantial increment of non-exosomal EV population from this system. The data also indicated that EV generation was increased as a cost of protein per each EV. Compared to 2D system, the amount of protein per EV was reduced in bioreactor system. They suggested this observation could be explained by flow stress-induced cell membrane shedding. Guo et al. also developed cell type dependent 3D scaffolds for bioreactors (Fig. 4B) [61]. Human dental pulp stem cells (DPSCs) and mesenchymal stem cells (MSCs) were seeded on 3D Fibra-Cel scaffolds in the bioreactors for flow stimulation. In case of skeletal muscle cells (SkMCs), elastic stretchable scaffold was used to mimic muscle tissues. Both systems dramatically increased the EV production compared to 2D culture systems. Mechanistic study revealed that this phenomenon was mediated by yes-associated protein (YAP). YAP protein is associated with mechanosensing and activation of canonical Wnt signaling pathway. Finally, DPSC-derived EVs was treated to neuron cells to monitor their therapeutic efficacy. DPSC-derived EVs treatment increased axonal sprouting, showing a potential as damaged spinal code repairing therapeutics.

Fig. 4.

Large scale EV generations A Scheme of perfusion bioreactor. (left panel) 3D printed scaffold is connected to a peristaltic pump and media reservoir. The media is circulating at 4 mL/min flow rate. (right panel) The structure 3D printed scaffold. Reprinted with permission from ref [60]. B Bioreactors with flow stimulation or cyclic stretching. Mechanically stimulated EVs from stem cells are actively secreted from the 3D scaffolds. The blue arrowheads indicate the directions of flow within the bioreactors. Reprinted with permission from ref [61]. C Scheme of cellular nanoporation. The cellular nanoporation system consists of a nanochannel (small black rectangles). Plasmid DNA in the culture media is transfected to cells through nanochannels under transient electrical pulses. Attached cells secrete large amount of plasmid containing EVs [62]

Cellular nanoporation

Yang et al. reported a transient electrical stimulation technology for large-scale EV generation. They developed a cellular nanoporation chip to stimulate cells to generate EVs with the tranfection of therapeutic agent such as mRNA, miRNA, and shRNA simultaneously (Fig. 4C) [62]. The nanochannels with 500 nm nanopore enabled transient electrical pulse to transfer DNA plasmid from culture media to attached mouse embryonic fibroblast (MEF). Unlike other stress inducing strategy, cellular nanoporation enabled EV production without cellular damages. They also suggested the mechanism of EV stimulation by external stress. They found that focal cellular damage and local heating from nanoporation upregulated heat shock proteins and elevated intracellular calcium concentrations, which led to massive EV productions. This research would be a clue for the precise molecular mechanism in stimulation of EV productions. With the help of simple mRNA packaging process, the EVs produced by nanoporation could inhibit tumor growth and prolong animal survival.

Conclusion

EV recently has drawn the attention as next generation therapeutics. EVs’ immunotolerance, long half-lives with low toxicity, excellent delivery capacity support their potentials as therapeutics. However, slow EV secretion rate is the major hurdle for their clinical applications. During past decades, large scale EV productions has been explored based on mechanistic studies of EV generations. Chemical, physical and environmental stimulation methods have been tried for the EV production stimulation. However, none of them stands out as a solution for clinical applications. Each method has their own pros and cons, and many researchers are still pursuing new methods for practical EV preparation. Along with EV generation methods, isolating homogenous EVs and engineering EV technology should be considered for hiring EV toward therapeutics.

Author contributions

Juhee Hahm, B.S., Jonghoon Kim, Ph.D. and Jongmin Park, Ph.D. wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Ethical statement

There are no animal experiments carried out for this article.