(PDF) Progress, opportunity, and perspective on exosome isolation - Efforts for efficient exosome-based theranostics Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3684 Theranostics 2020; 10(8): 3684-3707. doi: 10.7150/thno.41580 Review Progress, opportunity, and perspective on exosome isolation – efforts for efficient exosome-based theranostics Dongbin Yang1#, Weihong Zhang2#, Huanyun Zhang1, Fengqiu Zhang3, Lanmei Chen4, Lixia Ma5, Leon M. Larcher6, Suxiang Chen6, Nan Liu7, Qingxia Zhao8, Phuong H.L. Tran9, Changying Chen10, Rakesh N Veedu6,11, Tao Wang2,6,11 1. Department of Neurosurgery of Hebi People s Hospital; Hebi Neuroanatomical Laboratory, Hebi, 458030, China. 2. School of Nursing, Zhengzhou University, Zhengzhou, 450001, China. 3. Henan Key Laboratory of Ion-beam Bioengineering, Zhengzhou University, Zhengzhou, China, 450000. 4. Guangdong Key Laboratory for Research and Development of Nature Drugs, School of Pharmacy, Guangdong Medical University, Zhanjiang 524023, China. 5. School of Statistics, Henan University of Economics and Law, Zhengzhou 450046, China. 6. Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth 6150, Australia. 7. General Practice Centre, Nanhai Hospital, Southern Medical University, 528244, Foshan, China. 8. School of Medicine, Wake Forest University, Winston Salem, NC 27101, USA. 9. School of Medicine, and Centre for Molecular and Medical Research, Deakin University, 3216, Australia. 10. The First Affiliated Hospital of Zheng Zhou University, Zhengzhou 450001, China. 11. Perron Institute for Neurological and Translational Science, Perth 6009, Australia #These authors contribute equally to this work.  Corresponding author: Tao Wang, wangtaomary@zzu.edu.cn; Rakesh N Veedu, R.Veedu@murdoch.edu.au; Changying Chen, changying@zzu.edu.cn. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2019.10.29; Accepted: 2020.02.08; Published: 2020.02.19 Abstract Exosomes are small extracellular vesicles with diameters of 30–150 nm. In both physiological and pathological conditions, nearly all types of cells can release exosomes, which play important roles in cell communication and epigenetic regulation by transporting crucial protein and genetic materials such as miRNA, mRNA, and DNA. Consequently, exosome-based disease diagnosis and therapeutic methods have been intensively investigated. However, as in any natural science field, the in-depth investigation of exosomes relies heavily on technological advances. Historically, the two main technical hindrances that have restricted the basic and applied researches of exosomes include, first, how to simplify the extraction and improve the yield of exosomes and, second, how to effectively distinguish exosomes from other extracellular vesicles, especially functional microvesicles. Over the past few decades, although a standardized exosome isolation method has still not become available, a number of techniques have been established through exploration of the biochemical and physicochemical features of exosomes. In this work, by comprehensively analyzing the progresses in exosome separation strategies, we provide a panoramic view of current exosome isolation techniques, providing perspectives toward the development of novel approaches for high-efficient exosome isolation from various types of biological matrices. In addition, from the perspective of exosome-based diagnosis and therapeutics, we emphasize the issue of quantitative exosome and microvesicle separation. Key words: Exosome, microvesicle, extracellular vesicle, microfluidic, diagnosis, separation Ivyspring International Publisher Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3685 1. Exosomes and two obstacles for exosome-based basic and applied investigations The physiological function of the human body relies on effective and precise cell communication [1]. Apart from contact-dependent and soluble molecule- mediated signal transduction, we have expanded our knowledge of cell communication in recent years to include the role of exosomes as a new form of signaling system [2]. Exosomes are small extracellular vesicles with diameters between 30–150 nm that feature a double-layer lipid membrane structure. Exosomes can be produced by almost all kinds of cells under both physiological and pathological conditions, and are widely distributed through easily accessible body fluids such as blood, saliva, breast milk or urine. The discovery of exosomes was first reported in 1987 [3]. For many years, exosomes were assumed to be \"junk” produced during the maturation process of cells [3]. However, with the recent isolation of various proteins, lipids and genetic materials (e.g., miRNA, mRNA, DNA molecules as well as long-noncoding RNAs) from different types of exosomes [4], their crucial roles in cell communication and epigenetic regulation have been recognized [2]. Importantly, whether under pathological or physiological condi-tions, exosome contents are finely regulated by their parental cells to pass information from the parental cells to other cells for specific functions [5]. In turn, the functional states of the parental cells can be estimated by analyzing their exosome contents [5], which lays the foundation for exosome-based diagnosis, especially non-invasive liquid biopsy. Apart from in disease diagnosis, exosome application also features in various biomedical fields including drug delivery [6], cell-free vaccine development [7], and regenera-tive medicine [8]. Recently, the application of exo-somes as a potent substitute for maternal cells in immunotherapy and regenerative medicine has been demonstrated with in vivo animal work, serving as the basis for several ongoing clinical studies [9]. Indeed, exosomes hold high potential in the treatment of various diseases; by 2018 exosome-related investiga-tions attracted $250 million (USD) in investments and are expected to exceed $1 billion (USD) by 2021 [10]. Accordingly, there are currently 127 exosome-related clinical trials being registered at Clinicaltrials.gov (versus 26 trails for the year of 2017) involving treat-ment and diagnosis of multiple types of diseases. Considering that the key discovery of genetic material in exosomes was not published until 2007 [2], the speed of clinical translation of exosome-based thera-nostics has far exceeded the original expectations [9]. However, the general atmosphere around exosome-based clinical application is still pessimistic. As addressed by a recent position paper of the International Society for Extracellular Vesicle (ISEV) [9], the explosive attention and substantial capital investment in clinical translation of exosomes is mainly due to open intellectual property space, which provides incentive for early movers. Whether these efforts are successful depends on the solution of several key technical issues, as historically, there have been two main technical hindrances that restrict the basic and applied researches of exosomes [11]. The first is how to simplify the exosome extraction procedure and improve the yield of exosomes; the second is how to effectively distinguish exosomes from other extracellular vesicles, especially from functional microvesicles. In this work, by comprehensively analyzing existing exosome isolation techniques, we provide suggestions and insights for future exosome separation methods and related applications. In addition, from the perspective of exosome-based diagnosis and therapeutics, we also emphasize the issue of quantitative exosome and microvesicle separation. 2. Six major separation strategies exploring different physiochemical properties of exosomes Exosomes are nano-sized extracellular vesicles distributed through vastly complex body fluids, which makes high-yield exosome isolation challeng-ing [12]. For instance, although ultracentrifugation has been the \"gold standard” for exosome separation due to its high processing capacity, high levels of protein aggregate and lipoprotein contamination in exosome samples prepared through this method greatly compromises their quantification and funct-ional analysis [13]. Because a single method fitting a variety of sample sources is not practicable, efforts have been made to exploit different physiochemical and biochemical properties of exosomes. Until now, six classes of exosome separation strategies have been reported, including ultra-speed centrifugation, ultra-filtration, immunoaffinity capture, charge neutraliza-tion-based polymer precipitation, size-exclusion chro-matograph, and microfluidic techniques, with unique sets of advantages and disadvantages for each tech-nique (Table 1). In this section, by analyzing principles, procedures, and advantages and disad-vantages of individual techniques, we provide a panoramic view of current exosome isolation strat-egies. This overview not only facilitates the optimiza-tion of exosome isolation strategies in different appli-cations, but also provides new outlooks for the Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3686 development of novel devices and approaches for efficient exosome isolation. Importantly, as we will discuss in Section 3, although vesicles prepared by current approaches are commonly denoted as exosomes, it should be noted that the term \"exosome” is often used improperly in published articles or clinical trials. Apart from exosomes, the \"exosome samples” prepared via current techniques also include a great number of non-exosome vesicles such as microvesicles, apoptotic bodies and ectosomes [11, 14]. This is because of their vast overlap in physicochemical properties and the currently limited knowledge about the molecular mechanisms of exosome biogenesis and release. Such non-exosome particles, especially functional micro-vesicles, compromise the accuracy and reliability of exosome-based theranostics. For this reason, the 2018 ISEV guideline position paper has suggested that due to the lack of pure exosome separation with current techniques, the commonly used term of exosome should be replaced with the more collective term of extracellular vesicle [15]. As a result, unless specifi-cally stated, the term \"exosome” used in this article denotes a mixture of small extracellular vesicles such as exosomes, apoptotic bodies, microparticles, microvesicles, ectosomes, as well as oncosomes. 2. 1 Ultracentrifugation-the gold standard exosome isolation approach With the capacity to generate centrifugal forces as high as 1,000,000 ×g (100,000–150,000 ×g is commonly used for exosome separation), ultracentri-fugation is an optimal process for separating small particles including bacteria, viruses, and cellular organelles. As such, ultracentrifugation readily trans-lates to exosome isolation and has contributed to many pioneering exosome explorations [3, 16]. We will next discuss the application and main features of three common ultracentrifugation methods to demonstrate the details of this \"gold standard” exosome isolation strategy. Table 1. Current strategies for exosome separation Isolation technique Principle Advantages Disadvantages Sequential ultracentrifugation Particles have different density and size show different sediment speed under centrifugal force • Low cost and • Low contamination risk with extra isolation reagents; •suitable for large volume preparation; • High equipment requirement • Time consuming • Labor intensive • Potential mechanical damage due to high speed centrifugation • Protein aggregation • Not suitable for small volume diagnosis • Low portability Gradient ultracentrifugation After centrifugation in a dense medium, objects in a tube could stay in the position of the medium with similar density • High purity of products • Allowing separation of subpopulation of exosomes • Lower volume processability • High equipment requirement • Time consuming • Labor intensive • Potential mechanical damage due to high speed centrifugation • Not suitable for small volume diagnosis • Low portability Ultrafiltration Utilizing filter membrane with defined size-exclusion limit or molecular weight cut-off • Low equipment cost • Fast procedure • good portability • Moderate purity • Potential deterioration induced by shear stress • Possible loss due to clogging and membrane trapping Size-exclusion chromatography After adding to porous materials, substances eluted out in accordance with their particle size, with big particles eluted earlier • High purity • Fast preparation • Keep native state of exosomes • Good reproducibility • Potential for both small and large sample capacity; • Capable of processing all type of samples • Relatively high device costs • Additional method for exosome enrichment is required Polymer Precipitation High hydrophilic water-excluding polymers can alternate the solubility of exosomes • Easy to use • Using ordinary equipment •Suitable for both small and large sample volume • High efficiency • Contaminants of protein aggregates, other extracellular vesicles and polymeric contaminants • Extended processing time • Require complicated clean-up steps • Affecting downstream analysis and quantification Immunoaffinity capture Based on specific binding between exosome markers and immobilized antibodies (ligands) • Suitable for separating exosomes of specific origin; • High-purity exosomes • Easy to use • No chemical contamination • High-cost antibodies; • Exosome markers must be optimized • Low processing volume and yields • Extra step for exosome elution may damage native exosome structure Microfluidics-based techniques Based on different principles including immunoaffinity, size and density • Highly efficient • Cost-effective • Portable • Easily automated integrated with diagnosis • Low sample capacity Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3687 2.1.1 Differential ultracentrifugation contributed to most pioneering exosome studies Differential ultracentrifugation, also referred to as simple ultracentrifugation or the pelleting method, is the most commonly reported strategy for exosome separation (45.7%) [17,18]. The principle of differential ultracentrifugation is quite simple – under certain centrifugal forces, different extracellular components of a fluidic sample can be sequentially separated based on density, size as well as shape. This method was first reported by Johnston in 1987 to isolate exosomes from the culture medium of reticulocyte tissue [16]. Later, in 2006, differential ultracentrifu-gation was further optimized by Thery and colleagues with a set of increasing centrifugal forces [19]. As demonstrated in Figure 1, depending on the nature of the tested samples, a cleaning step may be first conducted to eliminate large bio-particles by low- speed centrifugation (e.g., 300 ×g), followed by multiple cycles of centrifugation with centrifugal force from 2000 ×g up to 100,000 ×g, to sequentially remove contaminants such as cell derbies, apoptotic bodies and protein aggregates for purified exosome isolation. Importantly, this method easily scales up for large scale exosome preparation. Although commonly used ultracentrifugation tubes have a relatively low volume capacity (~5–20 mL), existing liquid concen-tration devices (e.g., Centricon® Plus-70 Centrifugal Filter Units) [6] can facilitate the process of volumes of up to 200 mL with a 5-mL loading capacity ultracentrifugation tube. Due to ease of use, little technical expertise requirement, and compatibility with large volume preparation without complicated sample pre- treatment, differential ultracentrifugation has been widely employed over the past 30 years to isolate exosomes from various sources such as cell culture medium, serum, saliva, urine, and cerebrospinal fluid [6, 20-22]. However, it should be noted that extra-cellular fluids feature high heterogeneity. Under a certain centrifugal force, all components (including exosomes, microvesicles, and non-vesicles such as protein aggregates and lipoproteins) with buoyant density, size, and mass reaching a certain threshold can be precipitated at the bottom of the tube [23]. Therefore, exosome samples prepared via differential ultracentrifugation often suffer from low purity, which potentially compromises many downstream applications, especially exosome-associated func-tional analysis [24]. For example, in a well-designed comparison study, Paolini and colleagues used several different strategies to separate exosomes from the blood of patients suffering from multiple mye-loma. In the subsequent functional study, they observed that exosomes prepared with differential ultracentrifugation (displayed high-amount contami-nation) demonstrated only poor and inconsistent biological functions compared to more purified exosome samples, which could induce prominent NF-κB nuclear translocation in endothelial cells [25]. Fortunately, to further improve the exosome isolation efficiency of this classical separation tech-nique, various types of centrifugation strategies have been developed during the past two centuries through the exploration of the different physical properties of objects. Among these strategies, a widely used method is density-gradient centrifugation, which separates particles by density [26-28]. 2.1.2 Isopycnic moving-zone density-gradient ultracentrifugation for high-quality exosome isolation In 1937, Linderstorm-Lang discovered that after centrifugation in a density-gradient tube, objects of a particular density would remain suspended in medium of a similar density [29]. Historically, the density-gradient-based centrifugation method has been commonly used in hematological study for the separation of subpopulations of blood cells, due to the differences in density of different cell types [30]. Simi-larly, due to the density differences between different extracellular components, purified exosomes can be obtained through this method [31, 32]. A typical density-gradient ultracentrifugation includes the following steps: First, layers of biocompatible med-ium with varying densities (e.g., iodoxinol or sucrose) covering the range of particle densities in the sample is placed into a tube, with gradually decreasing densities from bottom to top (Figure 2A). Next, the sample of interest is added onto the top of the density- gradient medium, followed by extended centrifuga-tion for a prolonged period (e.g., 100, 000 ×g for 16 h) [6, 33]. Eventually, the extracellular components, including exosomes, apoptotic bodies, and protein aggregates, gradually reach a static position (isopyc-nic position) in the layer of the same density. Through this method, components having different buoyant densities can be easily separated; protein aggregates concentrate at the bottom of the centrifugation tube while exosomes remain in the layer of medium bet-ween 1.10 and 1.18 g mL−1 [34]. Again, in reference to Paolini and colleagues’ comparison study [25], comp-ared with differential ultracentrifugation and popular one-step precipitation kits (to be discussed in Section 2.4); density-gradient ultracentrifugation achieves the purest exosome samples for downstream applica-tions. As a result, density-gradient ultracentrifugation has gained great popularity in recent years for exosome separation, representing around 11.6% of the currently used exosome strategies [17, 25]. Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3688 Figure 1. Schematic representation of differential ultracentrifugation-based exosome isolation. Differential ultracentrifugation is performed by multiple cycles of centrifugation with centrifugal forces from 300 ×g up to 100,000 ×g. After each centrifugation step, pellets including cells, cell debris as well as apoptotic bodies are removed while the supernatant was collected for further centrifugation. After the last centrifugation (i.e., 100,000 ×g), exosomes-containing pellets and contaminant proteins are collected by removing the supernatant. The centrifugation is performed at 4°C. Figure 2. Schematic representative of gradient density ultracentrifugation-based exosome isolation. (A) Isopycnic density-gradient ultracentrifugation is prepared by adding medium in layers of progressively decreased density from bottom to top. After prolonged centrifugation, extracellular components including exosomes, apoptotic bodies and protein aggregates reach a static position in medium of similar density to each component. However, because isopycnic gradient ultracentrifugation depends solely on the density difference between different solutes in samples, this method cannot separate substances (e.g., microvesicles) with similar buoyant density to exosomes. (B) The moving-zone gradient ultracentrifugation normally consists two gradient medium sections. The top layer is a medium with density lower than all of the solutes of the sample. The bottom is a high-density cushion. As the density of the solutes are all greater than that of the gradient medium, after centrifugation, all solutes will be sequentially separated based on not only density, but also mass/size, thereby allowing the separation of vesicles of comparable density but varying size. However, such commonly used isopycnic ultracentrifugation depends entirely on the density difference between different solutes in samples. Although this method effectively separates exosomes from common contaminants such as protein aggre-gates, this method cannot separate extracellular vesicles with similar buoyant density (but different size) to exosomes (e.g., microvesicles) [35]. To effectively address this technical issue, studies have used moving-zone density-gradient centrifugation (also termed as rate zonal centrifugation), which separates particles by both size and density [36]. As shown in Figure 2B, the moving-zone ultracentrifuge-tion features a medium with a density lower than that of all solutes in the sample. As the density of the solutes is greater than that of the gradient medium, after centrifugation, all solutes in the sample will be sequentially separated based on not only density, but also mass/size, thereby allowing the isolation of vesicles with comparable densities but varying sizes (e.g., exosomes, viruses and large microvesicles) [36]. However, unlike isopycnic ultracentrifugation, be-cause the concentration of the medium in this type of ultracentrifugation is lower than that of all sample components, all insoluble particles can be pelleted at the bottom of the tube after prolonged centrifugation (hence why it is called moving-zone centrifugation). Consequently, the centrifugation time must be care-fully determined for optimal exosome isolation. In order to minimize exosome pelleting, a high-density medium is normally loaded in the bottom of the centrifuge tube to serve as a cushion (Figure 2B). Despite various advantages and wide appli-cation, ultracentrifugation does have its short-comings. For instance, although gradient ultracentri-fugation is capable of purifying exosomes with minimal contamination, the processing volume of this method is limited by the thin loading zone [13]. Additionally, ultracentrifugation approaches require not only expensive equipment, but also highly trained technicians, especially for gradient ultracentrifu-gation. Furthermore, as has been emphasized by previous studies [37], the structure and biological function of the isolated exosomes can be detrimentally affected by prolonged periods of ultra-centrifugal force, which is very unfavorable for downstream applications such as exosome-based functional studies and drug development. In light of this issue, other size-based separation strategies such as ultra-filtration and size-exclusion chromatography have been introduced. As we discuss in the next sections, various simplified and highly efficient exosome Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3689 separation kits based on such techniques are now commercially available. 2.2 Ultrafiltration holds potential for industrial scale exosome preparation Similar to conventional filtration methods, ultrafiltration uses an ultrafine Nano-membrane with different MWCO (molecular weight cut-off) to isolate extracellular vesicles from clinical samples or cell culture medium and differentiate between exosomes and co-vesicles by size [38]. Compared with the ultra-centrifugation method, ultrafiltration-based exosome isolation dramatically shortens processing time and does not require special equipment, presenting an ideal substitute to the classical ultracentrifugation strategy [39]. Importantly, by easily adjusting filter size, ultrafiltration allows researchers to sort specific subsets of small extracellular vesicles (including exosomes) with defined particle sizes [40]. Based on this principle, several simplified and easy-to-use ultrafiltration devices have been recently developed to facilitate the fast preparation of exo-somes with yield comparable to that of the ultra-centrifugation method [40]. As demonstrated in Figure 3, there are two types of ultrafiltration devices that have been well-developed. The first is a tandem- configured microfilter (Figure 3A), which consists of two tandem-configured microfilters with defined size-exclusion limits around 20–200 nm [41]. When passed through the two membranes, large vesicles including apoptotic bodies, as well as the majority of microvesicles; are trapped in the 200-nm membrane whilst vesicles of 20–200 nm diameter remain on the bottom and smaller particles such as proteins pass through the 20-nm microfilter. On the other hand, sequential ultrafiltration is another popular method for exosome isolation (Figure 3B) [42]. In this mode, extracellular fluids are first passed through a 1000-nm filter to get rid of large particles including cell debris, cells, and apoptotic bodies. After that, the filtrate is then passed through a second filter with 500-kD MWCO to remove free proteins and other small particles. Finally, exosomes with diameters between 50–200 nm can be collected from the filtrate with a 200-nm filter. Based on this sequential ultrafiltration protocol, the Bio Scientific Corporation recently developed a kit called \"ExoMir™ exosome isolation” [43]. By leveraging a syringe filter-based adjustable fractionation process, this device enables large volume processing (10–25 mL/run), rapid isolation of small extracellular vesicles (including exosomes and microvesicles depending on filter size) from various types of fluids include serum, cerebrospinal fluid, and eukaryotic cell culture media. In addition, by including a second RNA isolation module, this kit allows real-time RNA isolation from the harvested small extracellular vesicles for further analysis. Over the past decade, due to high efficiency (minutes for ultrafiltration vs up to 16h for ultra-centrifugation) and simplicity (does not require special equipment), ultrafiltration gained increasing popularity, representing around 5.4% of the currently used exosome isolation methods [17]. However, this method has a few limitations. One of the most noticeable problems associated with the application of ultrafiltration is vesicle clogging and trapping, which potentially reduces the lifetime of the expensive membranes and leads to low separation yield [44, 45]. Apart from pre-treatment with proteinase to reduce fluid sample viscosity [46, 47], tangential flow filtration techniques present an ideal solution to this problem [48]. As demonstrated in Figure 4, during tangential flow filtration, the feed stream flows parallel to the membrane [49]. Through manipulation of the hydrodynamic flow force, the pressure applied to the flow stream causes only part of the flow to cross the membrane. As the membrane is constantly under a parallel flow force, potential clogging can be efficiently minimized (via constant flushing). The remainder (retentate) can then be re-circulated back to the feed reservoir for repeated filtration during the tangential flow filtration procedure, thus allowing an automated procedure as well as high yield [50]. Tangential flow filtration-based exosome preparation has been applied to separate exosomes for various clinical trials due to these advantages [38, 51, 52]. In a recent clinical trial, dendritic cell-derived exosomes prepared by this method were able to effectively promote T-Cell response in a promising anti-cancer treatment [53]. In 2017, the University of Texas MD Anderson Cancer Centre developed a notable three- step sequential filtration device designed for process-ing large volumes of bio-fluids based on tangential flow technique [40]. The exosome isolation device consists of three separate modules where first, large particles such as cell debris are filtered out by a 100- nm filter which detains only inflexible solutes with sizes larger than 100 nm such as apoptosis bodies but allows flexible solutes to cross, even if they are larger than 100 nm in size. Next, tangential flow filtration is performed using 500-kDa MWCO hollow fibers to further deplete small contaminants such as proteins. Lastly, exosomes are isolated by filtering the retentate through a low-pressure filter with defined pore size (i.e., 100 nm). In addition to its fast processing, simplified procedure and isolation of exosomes with defined particle size, this semi-automated ultrafiltra-tion strategy allows the isolation of extracellular vesi-cles on an industrial scale with minimum structural damage (maintaining functional integrity) via careful Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3690 monitoring and maintenance of the transmembrane pressure, therefore holding great potential for exosome-based theranostic translations. Apart from vesicle clogging and trapping, the co-presence of nanoparticles with sizes comparable to those of exosomes presents another limitation of ultrafiltration [54]. The combination of two or more isolation methods (e.g., gradient ultracentrifugation) can address this problem [55, 56]. Importantly, the transmembrane pressure applied during ultra-filtration, if performed improperly, could detriment-ally affect the native state of the isolated exosomes, resulting in loss of function [42]. For this reason, caution needs to be taken during the whole ultrafil-tration procedure to avoid the collected exosomes from deformation and fragmentation [57, 58]. 2.3 Size-exclusion chromatography allows separation of exosomes with minimal structural damage In 1955, Grant H.L and Colin R.R invented a size-based separation technique termed size-exclusion chromatography (SEC) to isolate solutes of different molecular weights by passing aqueous solution through a column made of starch and water [59]. When passing a liquid sample through a stationary phase consisting of porous particles, molecules with different hydrodynamic radii submitted to different fates. While molecules smaller than the pores of the stationary phase are slowed because they enter into the pores, larger molecules, which cannot enter the pores are forced around the porous particles and are eluted earlier from the column (Figure 5). Over the past 50 years, this method was dramatically improved through the introduction of various fine, porous materials such as dextran polymer (Sephadex), aga-rose (Sepharose), and polyacrylamide (Sephacryl or BioGel) [60]. Long before the discovery of exosomes, SEC has been well-developed and widely applied to the high-resolution separation of large molecules or aggregates of macromolecules such as proteins, poly-mers, and various liposome particles [60-62]. The knowledge acquired from SEC-based liposome isola-tion translates readily to exosome separation, as exo-somes share many similar physical properties with liposomes. In merely 10 years of development, companies have developed various commercial SEC kits designed specifically for exosome isolation such as qEV (iZON) and PURE-EVs (Hansa Biomed). Figure 3. Schematic demonstration of ultrafiltration-based exosome separation. (A) Tandem- configured microfilter. Extracellular fluids are passed through tandem-configured microfilters with defined size-exclusion limits around 20–200 nm. When passing through the two membranes, large vesicles including cell debris, apoptotic bodies and the majority of microvesicles are trapped in the 200-nm membrane, while vesicles with diameter from 20 to 200 nm are retained on the lower 20 nm filter. (B) Sequential ultrafiltration. Extracellular fluids are first passed through a 1000-nm filter to get rid of larger particles (e.g., cells or cell debris); then the filtrate is passed through a second filter with 500-kD cut-off to remove small particles such as free proteins; finally, exosomes 200 nm are collected via a 200-nm filter. Figure 4. Tangential flow filtration ensures highly efficient ultrafiltration. During tangential flow filtration, the feed stream flows parallel to the membrane face. The applied pressure causes one portion of the flow stream to pass through the membrane according to the filter size. As the membrane is constantly under a parallel flow force, potential clogging can be efficiently minimized. During the tangential flow filtration procedure, the remainder is re-circulated back to the feed reservoir for repeated filtration, ensuring thorough filtration. Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3691 Figure 5. Principle for Size-exclusion chromatography-based exosome isolation. When passing a solution through a stationary phase consisting of porous resin particles, molecules can be separated according to size (A); While particles with hydrodynamic radii smaller than that of the pores of the stationary phase enter into the pores for longer traffic distance, larger particles, which cannot enter the pores move directly around the resin (B). This causes particles with different sizes to exhibit different retention times and therefore facilitate size-based separation. In terms of exosome-based therapeutic applica-tion and functional studies, perhaps the most appealing feature of SEC is its ability to preserve the natural biological activities of the separated exosomes [63]. Unlike ultracentrifugation and filtration, SEC is performed by passive gravity flow, which does not affect vesicle structure and integrity [58]. The natural state of exosomes can be further enhanced by the selection of elution buffers with physiological osmolarity and viscosity (e.g., PBS) [64]. Apart from maintaining exosome function, SEC has additional advantages. First, SEC requires minimal volumes. With commercially available SEC columns; volumes as small as 15 µL can be processed to achieve high-resolution, standardized, and reproducible exosome isolation suitable to exosome-based fingertip analysis [65]. Second, SEC-based exosome collection is simple, compatible with various types of fluids, and an extra pre-treatment step is generally not required [14]. Third, the SEC method saves time and labor. With selective porous materials and buffer systems, the whole process can be completed within a short and well-defined time period (e.g., 15 minutes) [66]. Fourth, similarly to the ultrafiltration method, fine adjustment of the pore size of the applied materials can yield a defined subpopulation of extracellular vesicles [66]. Lastly, compared to ultrafiltration-based separation, the contact-free manner of SEC (solutes do not interact with the stationary phases) ensures none or minimal sample loss and high yield [63]. Given all these merits, it is not surprising that in recent years’ SEC-based exosome isolation has becoming increa-singly popular for exosome-based basic and clinical investigations. Importantly, this method is not only suitable for processing trace amount liquid samples, but also easily scalable and automated for high- throughput exosome preparation. Recently, iZON developed an automatic exosome isolation system (qEV Automatic Fraction Collector) based on the established SEC platform and weight-dependent segment and sample collecting systems which allows fast, precise, scalable and automated exosome isolation [67]. Despite various advantages, the SEC method also faces several challenges. According to a recent comparison study, exosomes prepared via SEC column commonly displayed wider size distribution, especially in the lower size range, suggesting the existence of contaminants with sizes similar to those of exosomes such as proteins aggregates and lipo-proteins. To eliminate such contaminants, in the 2013 ISEV conference, Gardiner proposed an exosome iso-lation strategy by combining ultrafiltration and SEC [68]. Later, the combined use of ultrafiltration and SEC was practiced in cell culture medium by Shu and colleagues [69]. According to their assessment, in comparison with solely SEC or ultrafiltration, this combined strategy not only harvested exosomes with significantly improved purity, but also preserved exosome function. Similarly, functional exosomes were also prepared by Rood’s group via combined application of ultracentrifugation and SEC [70]. 2.4 High-yield polymer precipitation strategy coupling with issue of contamination Along with the size-dependent exosome isola-tion strategies discussed above, polymer-induced precipitation presents another commonly used strategy for exosome isolation. Analogous to ethanol- mediated nucleic acid precipitation, highly hydro-philic polymers interact with water molecules surrounding the exosomes to create a hydrophobic micro-environment, resulting in exosome precipita-tion [46]. Among various hydrophilic polymers, poly-ethylene glycol (PEG), a well-described, non-toxic polymer (a common excipient for pharmaceutical Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3692 products) with the ability to remodel the water solu-bility of surrounding materials has been commonly used [71], and constitutes the foundation for several popular commercial exosome isolation kits, such as ExoPrep (HansaBioMed, Estonia), Total Exosome Isolation Reagent (Invitrogen, USA), ExoQuick (System Biosciences, USA), Exosome Purification Kit (Norgen Biotek, Canada), miRCURY as well as Exosome Isolation Kit (Exiqon, Denmark). Existing polymer-based exosome precipitation methods generally employ PEG with molecular weights from 6000 to 20000 Da [71]. Firstly, a pre- treatment is required to remove big contaminant particles such as cell debris and apoptotic bodies, followed by incubation of the pre-treated samples with PEG solution at 4°C for overnight [72]. Next, the precipitated exosomes are collected via low-speed centrifugation (1500 ×g) (Figure 6). With such a straightforward protocol, this method has been widely used to isolate exosomes from various types of samples such as blood, cell culture medium, cerebro-spinal fluid, urine, and ascites [46, 73, 74]. Since polymer precipitation methods do not require sophis-ticated equipment, this method is easily scalable to large preparation volumes with high yield. This method also allows fast disease diagnosis through the integration of various detection platforms for exo-some (or protein/genetic material contents) analysis [75]. Typically, polymer precipitation-based exosome isolation is characterized by high yield. As shown in a recent comparison study using urinary samples, polymer precipitation achieved the highest yield of exosome and genetic contents (i.e., miRNAs and mRNAs) for subsequent profiling analysis, compared to differential ultracentrifugation and ultrafiltration methods [55]. However, water-excluding polymers can precipitate not only exosomes, but also various water-soluble materials such as nucleic acids, lipoproteins, protein, and even viruses [76, 77], there-fore the possibility of other extracellular contaminants could be very high. Indeed, after testing exosome samples collected via polymer precipitation through a mass spectrometry assay, noticeable detected protein contaminants included albumin and immunoglobu-lin, accompanied by residue polymer molecules [78, 79]. Currently, although various techniques (e.g., Nanosight particle tracking analysis, vesicle flow cytometry, tunable resistive pulse sensing, electron microscopy, and surface plasmon resonance) have been developed for exosome quantification, a gold standard exosome quantification strategy has not been developed. All of these strategies have their limitations. For instance, although Nanosight Nano-particle Tracking Analysis has been commonly employed, this method is expensive and restricted to a limited dynamic range for particle concentration measurements. In most current studies, exosome quantification relies on the measurement of the total protein content of the tested samples [6, 24]. For this reason, polymer precipitation inevitably causes false quantification of exosome preparations due to the existence of nonspecific protein contaminations (as well as protein from other non-exosome particles) [80]. In addition, the existence of such contaminants may also impair downstream analysis. In a recent comparison study, when Girijesh and colleagues treated human pancreatic cancer MiaPaCa cells with exosomes prepared via different methods, it was found that exosomes prepared by precipitation rather than other strategies resulted in unexpected cell toxicity [55]. To further improve the polymer-based exosome preparation, apart from applying extra pre- clean (i.e., centrifugation) and post-clean (i.e., via sephadex G-25 column) steps or the combined appli-cation of two or more techniques [71], the recently reported aqueous two-phase (layer) system (ATPSs) presents another option [81]. ATPS has been widely used to separate various substances including cells, proteins, and metal ions [82]. As shown in Figure 7, the principle of ATPS is very similar to that of the traditional organic-water solvent extraction system. When the relatively more hydrophobic solution (e.g., PEG) and more hydro-philic and denser solution (e.g., Dextran) are mixed together, a two-phase system occurs, where PEG con-sists of the upper phase while dextran forms the lower phase. Accordingly, after adding PEG and dextran to exosome-containing solutions, followed by a low- speed centrifugation, particles with different physico-chemical features separate into different phases. While exosomes preferentially accumulate in the dex-tran phase, proteins and other macromolecular com-plexes preferentially accumulate into the PEG phase. As reported, ordinary laboratory equipment and a mere 15-min incubation with the ATPS method yielded ~70% exosome recovery efficiency, about four times higher than the classical ultracentrifugation method [17]. Despite the observation on subsequent PCR of the adverse effect of high biopolymer concen-tration and high solution viscosity (e.g., up to 1.5% dextran) [17], this method presents a promising, inex-pensive and rapid exosome isolation strategy to simplify exosome-based various applications. 2.5 Immunoaffinity capture enables isolation of highly purified exosomes for in situ detection The observation that some proteins and recep-tors that are common in all exosomes, regardless of their origin [83], provides an opportunity to develop Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3693 immunoaffinity-based exosome isolation via the binding specificity between such protein markers and their corresponding antibodies (or exosome receptors and their ligands) (Figure 8). Theoretically, any pro-tein or cell membrane components solely or highly presented on the membrane of exosomes and lacking solvable counterparts in the extracellular fluids could be used for immunoaffinity-based exosome capture. During the past few decades, various exosome mark-ers have been recorded including lysosome associated membrane protein-2B, transmembrane proteins, heat shock proteins, platelet-derived growth factor recep-tors, fusion proteins (e.g., flotillins, annexins, and GTPases), lipid-related proteins, as well as phospho-lipases [84-87]. Among them, transmembrane proteins such as Rab5, CD81, CD63, CD9, CD82, annexin, and Alix have been extensively exploited for selective exosome isolation [88, 89], resulting in several popular exosome isolation products including the Exosome isolation and analysis kit (Abcam), Exosome-human CD63 isolation reagent (Thermofisher) and Exosome Isolation Kit CD81/CD63 (Miltenyi Biotec). Remark-ably, via specific biomarkers, immunoaffinity capture represents an ideal platform for isolating defined sub-populations of exosomes with specific origins. As demonstrated by a previous investigation, an EpCAM (overexpressed on tumor derived exosomes) anti-body-coated magnetic bead system allowed the specific isolation of tumor-originated exosomes from not only cell culture medium but also various types of clinical samples [90]. Recently, immunoaffinity separ-ation systems designed for the isolation of specific subpopulation of exosomes have become commer-cially available (e.g., Exosome-Human EpCAM Isolation Reagent, Thermofisher). Obviously, collec-ting exosomes of specific origin not only facilitates the study of their parental cells, but also provides important indicators for disease diagnosis (for exam-ple, via detecting EpCAM positive exosomes to assess the existence of EpCAM related cancers). 2.5.1 Solid matrices for antibody immobilization For effective immunoaffinity-based exosome iso-lation, antibodies need to be fixed on a solid surface for exosome separation. Over the past few years, matrices including chromatography, beads, plates, and various types of microfluidic apparatuses have been used [13]. Among these, submicron-sized magnetic particles (Figure 8), widely used for immuno-precipitation of recombinant proteins, have been most commonly used. This method not only yields high capture efficiency and sensitivity from its large surface and near-homogeneous processes, but also accommodates large starting sample volumes, therefore allowing upscaling or downscaling for specific applications [13]. Moreover, as reported, this method could be directly transformed to a diagnostic platform through the detection of disease-specific markers (e.g., EpCAM, CD133, EGFR for cancer cells) on the isolated exosomes, facilitated by disease- specific antibody and magnetically activated cell sort-ing [91]. Figure 6. Schematic of Polymer Precipitation Strategy. After the addition of highly hydrophilic polymers to an exosome-containing solution, water molecules surrounding the exosomes are tied up by the polymers, lowering the solubility of the exosomes and inducing their subsequent precipitation. The exosomes can be easily collected with low-speed centrifugation. Figure 7. Schematic of aqueous two-phase system-based exosome isolation. When the more hydrophobic polyethylene glycol (PEG) and more hydrophilic dextran solutions are mixed, a two-phase system could occur. After addition of PEG and dextran to exosome-containing solutions followed by incubation and low-speed centrifugation, proteins and other big molecular complexes preferentially accumulate into PEG while exosomes preferentially accumulate into the dextran phase. Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3694 Figure 8. Schematic of immunoaffinity-based exosome isolation. First, antibodies recognizing exosome-specific markers are immobilized onto solid matrices. After incubating exosome-containing fluids with antibody-conjugated solid matrices, exosomes can be enriched onto such solid matrices. Free exosomes can be collected via an additional elution step. Plates and microchips are also popular matrices on which to develop immunoaffinity-based exosome separation systems, in addition to the commonly used magnetic beads. For example, by using microplate, an anti-CD9 antibody-based system has been devised to capture and quantify exosomes from various types of mediums such as urine and blood [92]. Compared to the traditional ultracentrifugation method, this microplate-based immunoaffinity capturing device performs more efficiently in exosome isolation [92]. Requiring only 400 µL of initial plasma sample in a one-hour procedure, this method isolates a compar-able amount of exosome RNAs to that obtained by ultracentrifugation of 2.5 mL of plasma in a 16-hour procedure [92]. Despite multiple disadvantages such as low volume processing capacity and relatively lower capture efficiency, this microplate-based meth-od is suitable for the development of plate reader- based real-time diagnostic devices, especially for trace amount sample analysis [92]. 2.5.2 How to maintain the native state of exosomes? A major concern for immunoaffinity-based exosome isolation Even though immunoaffinity-based exosome isolation ensures high-purity exosome isolation with an easy procedure, the non-neutral pH and non- physiological elution buffers (to separate exosomes from antibodies) associated with this method could irreversibly affect the biological function of the collected exosomes. The denatured exosome samples, although generally acceptable for diagnosis purposes (via assessing genetic and protein contents of exo-some), are not favorable for exosome-based functional studies and various therapeutic applications [89, 93]. Great efforts have been made to prepare exosome samples with intact structures. In an ingenious study, rather than using antibodies, Nakai and colleagues designed an exosome isolation device using the Ca2+- dependent Tim4 protein, which specifically binds to phosphatidylserine, a protein highly expressed on the exosome surface [94]. By immobilizing Tim4 proteins onto magnetic beads, exosomes with high phospha-tidylserine expression can be specifically isolated. Importantly, since the binding between Tim4 and exosomes is strictly dependent on Ca2+ [95], exosomes can be easily separated from Tim4-coated beads by the removal of Ca2+ through the addition of elution buffers containing Ca2+ chelators such as EDTA. Under such a gentle Ca2+ chelator treatment, the nat-ural state of exosomes can be preserved. In another case, researchers from the Korea Advanced Institute of Science and Technology developed an Exosome- specific Dual-patterned Immune-filtration chip for specific exosome capture by introducing a chemically cleavable linker 3,3 -Dithiobis(sulfosuccinimidyl-propionate) (DTSSP) between the antibody (anti- CD63) and the solid immobilization surface [96]. With this method, a simple reduction step via tris-(2-carboxyethyl) phosphine (TCEP) or Dithiothreitol (DTT) cleaves the antibody link to release the exo-somes for reliable downstream analyses and applica-tions. Such cleavable link-based antibody immobiliza-tion methods would be equally eligible to be applied to other immobilization matrices such as magnetic beads or plates for functional exosome isolation. 2.5.3 How to isolate total exosomes rather than specific exosome groups? Another issue for immunoaffinity-based exosome isolation Although immunoaffinity allows separation of a specific subpopulation of exosomes, at the same time it raises concerns about isolation of only the specific populations of exosomes that possess the antibody- recognized proteins. Considering the vast number of heterogeneous properties of exosomes in body fluids, this would result in an analytical bias (underestima-tions and false negatives) [97]. This is especially true in cancer diagnosis, where protein expression under-goes constant modulation with the stage of cancer progress [98]. In addition, specific isolation of only a Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3695 subset of exosomes, although with higher purity, results in lower overall yield [39]. Taking this into account, apart from protein markers, other substances universally expressed on exosome membrane have been targeted, such as the saccharide chains (e.g., N-linked glycans, alpha-2,6 sialic acid, mannose, and polylactosamine) over-expressed on exosomal membranes [99]. In a recent exploration, Samsonov and colleagues efficiently iso-lated exosomes from urine samples via lectin, a type of sugar-binding protein displaying high affinity to saccharide residues. The composition of these exo-somes was further confirmed via miRNA profiling to be bulk exosomes rather than any particular exosome subpopulation [100]. Furthermore, heparin, a type of highly sulfated glycosaminoglycan, also holds poten-tial for total exosome isolation with its ability to non- specifically bind to a variety of proteins. As demon-strated in a recent study [101], heparin-affinity beads are capable of harvesting bulk exosomes from not only cell culture media but also human plasma, following an ultrafiltration step to remove free proteins. 2.5.4 Chemical antibody-based next generation immunoaffinity approach Even though antibody products possess distinct advantages, the high costs related to antibody development and production as well as their perisha-bility significantly compromises their application, especially for large scale exosome preparation. To counteract these problems, apart from combined application with other methods as previously suggested [102], another option is to employ cheaper and more stable antibody substitutes, such as aptamer technologies. Aptamers, which are short single- stranded DNA or RNA sequences, can specifically recognize and bind to their targets with high affinity and specificity in a manner similar to antibodies [103, 104]. However, unlike traditional antibodies, apta-mers can be produced by in vitro chemical synthesis and exhibit several advantages such as low batch- to-batch variation, easy scaling up and down for different applications, extended shelf life, low or no immunogenicity, low production cost and easy chemical modification to improve binding properties [105, 106]. Over the past years, several aptamer- mediated exosome isolation platforms have been developed [107, 108]. Importantly, in addition to presenting a practicable option for immunoaffinity- based exosome isolation, aptamers also allow the preparation of natural exosomes with relatively little effort. As known, the recognition of aptamers and their target is strictly determined by tertiary structure [109, 110] (Figure 9), which in turn is determined by various factors such as temperature, ionic strength, as well as buffering systems. By adjusting the salt species and key ions (e.g., Mg2+ and K2+) to the formation of specific three-dimensional structure of aptamers, the binding capacity of aptamers can be easily remodu-lated under mild conditions [109], thereby releasing the captured exosomes with native structure and intact biological function. 2.6 Integrated microfluidic technique facilitates combinatorial exosome isolation and analysis By exploring both the physiochemical and bio-chemical features of exosomes at microscale, the dramatic advances in microfabrication technologies have offered a valuable opportunity to develop lab- on-a-chip-type microfluidic systems for efficient exo-some isolation [111-113]. Facilitated by existing signal detecting platforms, these miniaturized microfluidic apparatuses allow for not only fast exosome isolation from fingertip amount of body fluids, but also real- time exosome characterization for in situ diagnosis (Figure 10). Indeed, microfluidic techniques are dramatically changing the landscape of exosome- based diagnosis by transferring the traditional two- step procedure (exosome isolation and characteriza-tion) to an integrated one-step process [113]. This is especially valuable for non-invasive disease detection, such as early-stage cancer screening [114, 115]. Figure 9. Aptamer-mediated immunoaffinity. Aptamers recognize and bind their target via conformational complementary. After adjusting key factors of the buffering system such as salt types and ionic strength, the shape of the aptamer undergoes change and releases the bound target molecules. Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3696 Figure 10. Integrated microfluidic technique allows combined exosome isolation and analysis. After adding exosome-containing fluids into the sheath medium, particles in the fluids including exosomes can be separated by different approaches based on the physical and biochemical properties of extracellular vesicles. Importantly, these miniaturized microfluidic apparatuses, facilitated by signal detecting platforms, allow for not only fast exosome isolation from small amount of body fluids, but also real-time exosome characterization for in situ diagnosis. 2.6.1 Immunoaffinity-based microfluidics During the past decade, various forms of microfluidics have been invented through the explo-ration of different physiochemical properties of exo-somes. Among them, the immuno-microfluidic tech-nique has been most commonly used, resulting in commercial microfluidic products (e.g., ExoChip [116]). Identical to the commonly used immuno-affinity-based exosome isolation method, the concept behind the immuno-microfluidic-based exosome sep-aration devices involves the specific recognition of exosome markers by corresponding antibodies immo-bilized on the chips. In 2010, Chen et al. pioneered a microfluidic immunoaffinity apparatus for quick exo-some isolation through the use of an anti-CD63 anti-body [117]. The resulting device was able to efficiently isolate exosomes from as small as 10 µL of cell culture medium and serum. Furthermore, by passing 300 µL of lysis buffer through the exosome-captured micro-channel followed by air flushing, the group could easily obtain total RNAs from the captured exosomes. Subsequent tests demonstrated that significantly higher amounts of RNA could be collected via this chip system than by directly extracting RNAs from an equal amount of serum [117]. Since then, high interest has been called toward either improving the efficiency or the specificity of such microfluidic-mediated exo-some isolation systems. 2.6.1.1 Efforts for immuno-microfluidic-based high-efficient exosome isolation For a certain channel volume, larger binding surface area means more antibody immobilization and therefore higher exosome isolation efficiency. With this in mind, in 2016, Zhang and colleagues developed a microfluidic system that featured a graphene oxide/polydopamine (GO/PDA) nano-interface [118]. The unique features of the GO-induced three-dimensional nano-porous struc-ture provided a higher amount of surface area for efficient antibody immobilization and exosome cap-turing. As demonstrated, the developed CD81 antibody-microfluidic system not only greatly impro-ved the efficacy of exosome isolation, but also the purity of the resulting exosome samples. Importantly, by encapsulating an ultrasensitive ELISA assay with both universal exosome biomarkers (CD81 and CD9) and cancer-specific biomarkers (EpCAM), this device allowed ultrasensitive in situ ovarian cancer detection in merely 2 µL of plasma [118]. In another case, to increase the capture efficiency of an anti-CD9 antibody-based immuno-microfluidic chip, Hisey et al. introduced the \"herringbone groove” (previously used to facilitate nanoparticle separation [119]) pattern on the ceiling of the microfluidic channels. As expected, this novel design ensured significantly increased total surface area for antibody immobiliza-tion, and greatly improved exosome yield. 2.6.1.2 Efforts for immuno-microfluidic-based highly specific exosome isolation Nonspecific binding is a big issue for micro-fluidic-based immunoaffinity isolation as the method is incompatible with extra blocking and washing steps. This is different than conventional bead- or plate- based immunoaffinity approaches, where non-specific binding between non-exosome vesicles and the exosome-specific antibodies (as well as the non-specific binding between vesicles and the immobili-zation matrices (e.g., bead or plate surface)) can be efficiently eliminated via stringent blocking and washing. In recent years, the advances in nano-technology have provided valuable opportunities to address this problem. For instance, Ramanathan et al. presented a powerful microfluidic system for high- specific exosome capturing and analysis, facilitated by the tunable alternating current electrohydrodynamic Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3697 (ac-EHD) mediated nanoscale lateral fluid flow (also known as nano-shearing fluid flow) technique [120]. As tested with three different antibodies, including anti-prostate specific antigen antibody, anti-CD9 anti-body, and anti-human epidermal growth factor receptor 2 (HER2) antibody, this technology enabled efficient elimination of nonspecific/weak bound nanoparticles from the immune-affinity sites. Conse-quently, a greater than three-time increase in the sensitivity of exosome detection was recorded comp-ared to that of traditional lateral flow assay [120]. In another example, via employing inertial lift forces to effectively and rapidly exchange the washing solution around the exosome-antibody binding sites, Dudani et.al developed a microfluidic chip featured by a \"spin-wash” procedure. A high signal-to-noise exo-some isolation was achieved according to subsequent assessment [121]. As discussed in Section 1.5, although immuno-affinity-based exosome isolation allows easy exosome isolation, this method is limited by isolation of only specific subset of exosomes, high cost, and difficulty in maintaining the natural structure of exosomes. Size-dependent microfluidic isolation and contact-free separation strategies are two types of the most successful instances to address this problem. Next, we will discuss these two types of instruments to demon-strate recently developed examples of microfluidic devices. 2.6.2 Size-based microfluidic separation techniques facilitate high-quality exosome isolation The first size-dependent microfluidic system discussed is the well-documented Exosome Total Isolation Chip (ExoTIC) [122]. First, up to 10-mL solutions were filtered through a 0.22 µm nano- porous filter using a syringe pumper. Then, through the same syringe pumper and inlet, PBS was applied through the nano-porous filter to thoroughly clean and recover small extracellular vesicles (including exosomes) in a small volume (e.g., 200 µL). In this way, ExoTIC can effectively separate exosomes from both cell culture media and various types of body fluids such as lung bronchoalveolar lavage fluid, plasma and urine with limited effect on the native structure of exosomes. Importantly, this system was able to isolate exosomes from very small sample volumes (10–100 μL) with the yield around 4–1000 times greater than that of ultracentrifugation [122], ideal for point-of-care clinical testing. The second example is the nanowire-based exosome trip system (Figure 11). As demonstrated in Wang’s study [123], following a similar principle with SEC, this device is characterized by nanowires (made of porous silicon) imprinted on the sidewalls of evenly separated micropillars to form a nanowire-on-micropillar hier-archy structure. According to the design, the interval between the nanowires can be adjusted from 30 to 200 nm to physically trap small extracellular vesicles, while the sub-micrometer micropillars, apart from offering support for nanowire anchoring, are effective for removing larger non-exosome particles such as cell debris and apoptotic bodies. Furthermore, the exosome isolation capacity of this device can be further enhanced by pre-loading exosome-specific antibodies onto the porous silicon nanowire to explore the immunoaffinity-based isolation. As tested, this microfluidic device can effectively isolate 40–100 nm exosome vesicles with a recovery rate of 60%, while allowing smaller (e.g., proteins) and large parti-cles (e.g., cell, cell debris) to pass by unhindered. Importantly, via simply incubating in PBS buffer for 10 min, the chemical etching of the nanowire surface could be dissolved, thereby releasing the intact and purified exosomes for subsequent applications. 2.6.3 Contact-free microfluidics–versatile tools for future exosome preparation In addition to antibody and size-dependent microfluidics, rapid developments in microfabrication technology have enabled researchers to explore contact-free particle sorting mechanisms (e.g., elastic lift force, acoustic, and dielectrophoresis), for efficient, scalable, and high-quality exosome isolation. Figure 11. Principle of the nanowire-based exosome trip system. (A) Similar to SEC-based separation, a nanowire-on-micropillar hierarchy structure could be created via imprinting of porous silicon-consisting nanowires on the walls of the evenly separated micropillars. After adding exosome-containing fluids to the nanowire-on-micro-pillar tiered structure, particles in fluids are subject to different fates: (1). Larger particles (e.g., cell) are directly excluded from the sub-micrometer micropillar array; (2). Particles with submicron sizes (e.g., cell debris) are able to enter the micropillar interval but are unable to enter the 30–200 nm nanowire interval; (3). Small molecules (e.g., proteins) move across the nanowire interval without being obstructed; (4). Particles of 30–200 nm (e.g., exosomes) are arrested by the nanowire forest. (B) Particles with different sizes present different retention time and therefore facilitates size-dependent separation. Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3698 Figure 12. Contact-free microfluidic enables simplified exosome separation procedure. (A) In the viscoelastic medium flow-based microfluidic system, the exosome-containing fluids (added from inlet 1) meet the sheath flow (added from inlet 2) and are first aligned along the microchannel wall. After exertion of the elastic lift force that arises from viscoelasticity of the fluid, exosomes, and other extracellular components are driven toward the centreline of the microchannel according to their sizes, with larger particles eventually reach the centreline. (B) Under the pressure of ultrasound waves, particles with different mechanical properties (e.g., compressibility, size and density) experience differential radiation forces and results in contact-free and size-dependent exosome separation in a continuous manner. In recent years, the unique migration pattern of particles in non-Newtonian viscoelastic fluids has attracted great interests. As documented, the elastic lift force created by a viscoelastic medium flow was able to control and manipulate the position of particles in a size-dependent manner (Figure 12A). Indeed, over the past few years, various viscoelastic flow-based microfluidic systems have been reported to isolate particles ranging from cancer cells, blood cells, bacteria, droplets to microspheres [124-126]. In 2017, a contact-free viscoelastic microfluidic device was developed for size-dependent, continuous, and label-free exosome separation via manipulation of the viscoelastic force applied on exosomes by sheath fluid consisting of low concentrated (0.1%) biocompatible poly-(oxyethylene) (PEO) [125]. After systematically optimizing key factors such as medium elasticity, microchannel geometry, and flow speed, this device allowed greater than 80% recovery rate and 90% purity, which is much higher than the 5%–25% recovery rate for ultracentrifugation. Although a size cut-off of 200 nm was demonstrated in this work, according to the authors, extracellular vesicles of defined sizes could be easily obtained by adjusting PEO concentration. Amazingly, with the capacity to process samples down to 100 μL in a mere 0.1s exosome passage time [125], this system holds potential to be used as a platform to separate exosomes from diverse biological samples for various types of theranostic applications. Importantly, without a sophisticated microfabrication structure or external force field, the contact-free feature of this viscoelastic exosome separation system can be conti-nuously performed, significantly streamlining the design and produce of microfluidic-based exosome separation systems [127]. Under the pressure of ultrasonic waves, particles with different mechanical properties (e.g., compressi-bility, size and density) experience differential radia-tion forces [128]. Based on this principle, in 2015, Lee and colleagues invented an acoustic nano-filter sys-tem allowing contact-free and size-specific exosome separation in a continuous manner [129]. As demon-strated in Figure 12B, under ultrasound standing waves, the larger the particle is, the stronger radiation forces it will exert, and therefore display faster migra-tion toward the pressure nodes, resulting in the sepa-ration of extracellular vesicles with defined particle sizes. When erythrocytes and cell culture medium were tested, such size-dependent acoustic technique could efficiently isolate purified exosomes [129]. Importantly, such acoustic-based device allowed real- time control of the \"size cut-off” via in situ electronic manipulation, which facilitated the isolation of exo-somes with preferred sizes [129]. In another case, Wu and colleagues reported a point-of-care device was capable of isolating exosomes from non-pre-treated raw blood samples in an automatic manner via the integration of microfluidic and acoustic techniques. This contact-free device provided the possibility to isolate intact, functional exosomes with high yield and purity for exosome-related therapeutics, disease diagnostics as well as health monitoring [130]. In addition to elastic lift force and acoustic force, the simplicity of electroactive strategies (without using instrumentation and specialized reagents) had also been explored for developing contact-free exosome isolation microfluidic systems [131]. For instance, Davies et al. designed a microfluidic device that was able to effectively drive exosomes through a membrane while filter out other extracellular vesicles via electrophoresis within a microchannel, [132]. To further improve electrode-based electrophoresis, dielectrophoresis had been employed by generating Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3699 nonuniform electric fields through inserting insu-lating posts into the microchannel. After introduction to this nonuniform electric field, particles with different radii are subject to differing dielectrophor-esis forces (inversely related to their radius) via pola-rization effects. Thus, under this electric field, smaller particles can be captured by greater gradients of the squared electric field (vice versa) and achieve size- dependent nanoparticle separation [133]. In 2018, Shi et al developed a device based on such a mechanism [133]. As reported, this microfluidic system could efficiently trap small extracellular vesicles near a glass nanopipette tip under 10 V/cm current [133]. In another case, glioblastoma-originated exosomes were successfully isolated from human plasma in less than 30 minutes [134]. These works were further improved in an effort led by Marczak for simultaneous isolation and concentration of exosomes [135]. First, the authors developed a transverse local electric field by applying an ion-selective membrane. Under this electric field, the exosomes in a microfluidic chip could be easily forced out of the cross flow. After directing the exosome samples to agarose gels to eliminate undesirable cell debris, purified exosomes with defined particle size could be trapped and concentrated by ion-selective membrane. When tested with cell culture media and serum, this device was able to consistently capture between 60% and 80% of exosomes, as assessed by both nanoparticle tracking analysis and fluorescence spectroscopy [135]. Importantly, with a concentration factor up to 15 ×, this device ensured efficient and reliable downstream exosome characterization [135]. However, in spite of recent progress, additional investigations are still needed to further improve the efficacy and reliability of this electroactive strategy, especially for the opti-mal current (alternative current or direct current) and biological conditions for different samples. 2.6.4 Microfluidic devices facilitate real-time exosome analysis Although the application of exosomes has been used in diverse therapeutic purposes such as drug delivery, novel cell-free vaccine development and regenerative medicine, there has been an emphasis on their potential for disease diagnosis, especially in non- invasive cancer liquid biopsy [91]. According to our statistics, diagnosis represents nearly half (54 in 127) of the currently registered exosome-related clinical trials (via Clinicaltrials.com). Apart from improving separation schedules to isolate high-quality exosomes with high yield, the establishment of simple and efficient detection techniques represents another major task in the development of microfluidic-based exosome separation devices. Indeed, in terms of exosome-based diagnostic applications, microfluidic devices possess multiple advantages for the develop-ment of low cost, reliable, real-time diagnostic devices to process fingertip amounts of easily attainable liquid samples such as serum, urine, breast milk, and saliva. To facilitate post-separation exosome imaging, Ashcroft and colleagues produced a novel immuno- microfluidic device featuring a mica channel surface [136]. Compare to commonly used glass or polymer materials, this antibody-bound mica surface, with a distinct atomically flat and hydrophilic surface, could be easily separated from the Polydimethylsiloxane (PDMS) fabricated flow cell base. This unique design thereby allows the attached exosomes and the mica surfaces to be directly imaged via ultrahigh-resolution atomic force microscopy. In another case, He and colleagues introduced an immuno-microchip integra-ted with ELISA assay as a method of quantitative detection. Unlike the traditional immunoaffinity- mediated exosome separation strategy, this approach allows the direct quantification of both surface and intra-vesicular markers of circular exosomes from 30 μL of plasma sample within 100 min [137]. Later, a simplified continuous-flow microfluidic system named ExoSearch was developed [138]. Facilitated by CD9 antibody (for exosome capture), CA-125 (for ELISA detection), EpCAM (for ELISA detection), and CD24 antibodies (for ELISA detection), this platform enabled rapid exosome isolation and in situ non- invasive cancer detection [138]. Later, an anti-CD63 antibody-based device named ExoChip became clinically available [116]. After isolation, exosomes collected by ExoChip were stained using a fluorescent carbocyanine dye (DiO) prior to plate reader-based quantification. Notably, ExoChip has been employed as a valuable exosome-mediated diagnostic system for various disease screening as it allows fast exosomal miRNA profiling [116]. In addition to developing miniaturized devices for potable detection, more sophisticated detection platforms have also been inte-grated with current microfluidic systems for advan-ced applications. For example, Ueda and colleagues constructed a simplified microtip device enabling rapid and automated exosome isolation from various body fluids via conjugation of CD9 antibodies with highly porous monolithic silica microtips [139]. By further combining this microtip device with a pro-teome-wide LC/MS/MS platform, the group establi-shed an exosomal biomarker discovery system that could simultaneously analyse up to 12 different sam-ples. Through this system, the group was able to identify a specific antigen of lung cancer-derived exo-somes, CD91 [139]. It should be noted that the analytical sensitivity of reported \"real-time on-chip exosome analysis” Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3700 (including detection limit and response time) prima-rily depends on the specificity and binding capacity of the selected antibodies (for ELISA assay) as well as the sensitivity and compatibility of the utilized equip-ment. Therefore, such features of the analytical mod-ule need to be carefully investigated when designing real-time exosome analysis microfluidic devices. 3. Efficient exosome/microvesicle separation is critical for exosome and microvesicle-related investigations As discussed in Section 1, the basic and applied researches of exosomes have been obstructed mainly by two issues [11]. One is how to simplify the extrac-tion procedure and improve the exosome yield; the other is how to effectively distinguish exosomes from other extracellular vesicles. In recent years, although standardized exosome extraction and qualitative/ quantitative protocols are still not available, the rapid development in separation technology has in a large extent solved the problem of exosome isolation. For example, in order to obtain a sufficient amount of exosomes from cerebrospinal fluid for proteomics and nucleic acid quantification studies, researchers prev-iously needed to collect 200–500 mL of cerebrospinal fluid to meet the requirements of ultracentrifugation [140]. Nowadays, with newly developed exosome separation techniques such as immunoaffinity, chro-matography and polymer precipitation, 6 mL of cere-brospinal fluid samples is sufficient to meet quan-titative requirements [141]. Improvements in the tra-ditional polymer-based precipitation method have also addressed the long-standing obstacle of hydro-phobic protein interference in urine exosome isolation [54]. Today, with commercial exosome isolation kits and commonly available molecular biology equip-ment, exosomes can be extracted from trace amounts of clinical samples for subsequent studies in a short period of time, which greatly facilitates the basic and applied exosome studies. However, the second technical problem – how to effectively distinguish exosomes from other extracellular vesicles, still pre-sents a major issue in exosome-related applications. We only have to consider the concept of the exosome to get an appreciation of what this means. The exo-some was first proposed in 1987 [3], denoting an extracellular vesicle originating from endosomes. It should be noted however that the concept of exosome is often not used properly in published articles or even clinical trials. As shown in Figure 13, apart from the endosome-originated exosomes, extracellular vesicles also contain a large number of microvesicles shed by the cell membrane. Unfortunately, due to their similar physicochemical properties and a large overlap in particle sizes, effective exosome/micro-vesicle separation still presents a very difficult task [11, 14]. Instead of being inert materials as previously assumed, growing evidence is suggesting that micro-vesicles also display important biological functions [142, 143], although many of the published observa-tions on \"exosomes” actually describe the combined effects of exosomes and microvesicles. Given our limited knowledge of the biofunction of micro-vesicles, the existence of microvesicles in the tested exosome samples inevitably affects the exosome- based basic and applied studies in an unpredictable manner. As demonstrated by several recent odd findings, even for the same cancer cell type, \"exo-somes” collected by different groups could display quantitative or even qualitative differences in bio-logical functions (either tumor promotion or inhibi-tion) [2]. As suggested, the different proportion of exosomes and microvesicles in the tested \"exosome” samples may be the primary culprit of such controversial phenomena. For more accurate and reliable exosome-based diagnosis and therapeutic applications, an efficient exosome/microvesicle separation is necessary. 3.1 Solely relying on high-specific exosome markers is not sufficient for purified exosome isolation Extracellular vesicles consist of mainly micro-vesicles and exosomes. In theory, the concentration of any component in the extracellular vesicle can be achieved by the isolation of another component. In practice, efforts aiming at efficient \"exosome and microvesicle separation” have relied mainly on obtaining purified exosomes via identifying highly specific exosome biomarkers. This is due to (1) the lack of understanding of microvesicles and (2) the great potential of exosomes displayed in both basic research and theranostic applications in recent years. Theoretically, this strategy is feasible as the exosome proteins (including nucleic acids) are not a random combination of cell fragments but are integrated by a strict protein sorting mechanism to maintain their stable protein expression [2]. Although current know-ledge cannot describe in detail this particular sorting mechanism, the existence of such a mechanism itself provides a basis for the search for exosome-specific markers. As mentioned previously, during the past decades, various exosome biomarkers such as TSG101, CD81, CD9, CD63, CD37, CD82, CHMP2A, ALIX, RAB11B, CHMP4B, RAB11A, and RAB5 have been tested for immunoaffinity-based exosome Isola-tion. However, according to experience collected over the past 50 years, whether normal cells, stem cells or tumor cells, 100% specific markers do not exist [144]; even classic exosome markers like CD63 and CD81 Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3701 show expression on other subcellular organs [88] or even microvesicles [145, 146]). Taking a step back, if the specificity of the employed exosome marker is not 100% specific (such as the currently used CD81 or CD63), then the proportion of microvesicles in the residue would not be calculated. Furthermore, under this circumstance it is even impossible to estimate the proportion of the exosome component. In the face of this reality, we believe it is necess-ary for all scientists engaged in extracellular vesicle- related work to consider the following questions: (1) Is it a mistake to rely solely on the identification of novel exosome markers for exosome and microvesicle separation? (2) Are 100% purified exosomes and microvesicle components necessary for current basic and clinical research? (3) What is the key speed- limiting factor for current exosome studies? 3.2 Impurification is not the real problem for exosome-related studies Is isolating 100% purified exosomes necessary for current exosome studies? In fact, the key obstacle facing current exosome studies is not the impurity of exosome samples, but the lack of information about the proportion of exosomes and microvesicles in the collected \"exosome” samples. This is understandable. From the perspective of exosome-based diagnosis and basic investigations, based on enriched exosome samples, rational experimental controls and optimiz-ed statistical models, as long as the composition of the studied samples could be accurately determined, the reliability of the assays could be ensured. For exam-ple, in functional studies conducted via siRNA-based gene regulation, it is unlikely, and not necessary, to completely inhibit the gene of interest. In general, an inhibition rate of around 80% is considered sufficient for most subsequent investigations. From the pers-pective of exosome-based drug development, accord-ing to the current drug approval system of most countries (including the United States, the European Union, Australia, China, and Japan), unlike the high purity required for chemical compounds, the require-ments for quality control and safety assessment of cell-derived compounds such as exosomes can be met as soon as (1) the exosome proportion is sufficiently high and (2) individual components of the prepara-tion can be quantified and described [11]. Therefore, the key question of current exosome studies is how to effectively quantify the individual components of the collected exosome samples. How-ever, we cannot achieve this goal by solely relying on exosome markers, as reflected in various immuno-affinity-based commercial exosome extraction rea-gents (using antibodies targeting exosome markers). Although they claim to be able to enrich exosomes, some crucial information, including the proportion (or purity) of exosomes in the extract, and the content of other components (e.g., microvesicle), is invariably absent. Surely, even if a perfect exosome marker with 100% specificity is available for exosome isolation (this would be very unlikely in practice), it still cannot guarantee that the remaining vesicles are all micro-vesicles, which may contain excess exosomes, apop-totic bodies or protein precipitates. Moreover, in the absence of understanding of the microvesicle, we run into issues in determining the specificity of the employed exosome marker. Figure 13. Extracellular vesicles consist of mainly two types of vesicles with similar physiochemical properties. Extracellular vesicles include exo-somes and microvesicles. The main differences between them lie in their subcellular origins. Microvesicles are 50–1000 nm shedding particles from cell membrane; exosomes are 30–150-nm extracellular vesicles originated from endosomes, they are secreted into body fluids through exocytosis after cell membrane and multivesicular body fusion. Due to a lack of effective strategy to separate microvesicle and exosome, it is still difficult to precisely assess their physiochemical properties and functions. Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3702 3.3 Combined application of exosome and microvesicle markers for quantitative exosome/microvesicle separation Since relying on biomarkers of only one compo-nent (i.e., exosome) cannot yield reliable quantitative information of individual components in the extra-cellular vesicle mixture, it seems that the only feasible method is to simultaneously employ both exosome and microvesicle markers. In fact, even if the applied biomarkers for exosome and microvesicle are not 100% specific, the respective markers of the two components combined with mathematical calculation (by Linear Equation in Two Unknowns, Figure 14) may present a reliable system to assess the proportion of exosomes and microvesicles. At the same time, by providing enriched microvesicles with quantitative information, the proposed separation system also provides opportunities for the investigation of micro-vesicle-based basic and clinical translations. Having specific biological functions [147, 148], microvesicles must have stable protein expression. In recent years, although several microvesicle markers such as annexin A1 [149], CD29 [150], and Sca1 [151] have been reported, bona fide markers for micro-vesicle separation are still not available [152]. Future developments in specific microvesicle marker identi-fication would promote quantitative exosome and microvesicle separation. 4. Perspective Over the past few decades, despite the dramatic advances made in deciphering the mysteries of exo-somes, the challenges in efficient exosome isolation have yet to be solved. This largely owes to the comp-lexity of biological fluids, the considerable overlap of the physicochemical and biochemical properties among the exosomes, lipoproteins, virus, and other extracellular vesicles, as well as the heterogeneity of exosomes themselves [35]. As a result, no specific exosome separation technique has currently been accepted as suitable for all studies [153]. Depending on the biology samples applied, even the gold stan-dard ultracentrifugation method often suffers from protein and lipoprotein contaminants. Under these circumstances, the combined application of two or more techniques presents a plausible strategy for efficient exosome isolation, as demonstrated by the previously reported combined use of immunoaffinity- based exosome capture (or ultrafiltration) and den-sity-gradient centrifugation [96, 154, 155]. Figure 14. Calculation of the proportion of exosome and microvesicle components of extracellular vesicles via exosome and microvesicle marker-specific antibodies. The extracellular vesicles A (A’) were concentrated via polymer precipitation. Then exosomes (B) or microvesicles (B’) were extracted using corresponding antibody-based immunoaffinity capture; after elution, exosomes (C’) and microvesicles (C) were extracted again from the elutes using antibody-based immunoaffinity method. Then, the extracted exosomes (B, C’) and microvesicles (B’, C) were quantified. The proportion of exosome and microvesicle in the original extracellular vesicles was calculated using the formulation as shown in D. Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3703 However, it should be noted that although combined isolation techniques result in higher exo-some purity, they often increase procedure cost and complexity, thus resulting in reduced overall yield and unreliable downstream analysis. Therefore, the nature of the samples as well as the purpose of the investigation needs to be carefully considered when a particular combination of techniques is selected. For example, when the immunoaffinity capture method was used to process large volume samples, a pre- treatment via polymer precipitation [156] may be beneficial, to both promote the efficacy of the anti-body-based exosome separation and avoid using excessive quantities of expensive antibodies. For both diagnosis and therapeutic applications, researchers should carefully consider the strengths and weaknesses of the accessible strategies. As dis-cussed previously, immunoaffinity capture promises selective isolation of highly purified exosomes of specific origins, or even subpopulations of exosomes from biological fluids. Therefore, in cases where diag-nosis is scheduled for the subsequent investigations, immunoaffinity may present the most sensitive and specific method. Unfortunately, the current immuno-affinity method is compromised heavily by the lack of reliable markers for exosome isolation [17]. Further-more, when considering the heterogeneity in antigen expression, especially in cancer cells, the possibility of underestimation and false negatives must be noted [153]. Even so, with the identification of more disease- specific exosome markers, coupled with recently developed microfabrication technologies (e.g., micro-fluidic), immunoaffinity-based exosome isolation may contribute greatly to future diseases diagnosis, espe-cially through non-invasive liquid biopsy. Further-more, as discussed in Section 3, isolation of specific exosome markers (as well as microvesicle markers) is also of great value to address the long-standing issue of quantitative exosome/microvesicle separation. On the other hand, the therapeutic applications of exosomes are limited by the lack of an effective method to isolate high-quality exosomes in bulk [157, 158]. In all likelihood, the ultrafiltration method may contribute most to this area due to its advantageous features, including ease of handling and analyzing large batches of biological samples, and capability of isolating exosomes with high purity and defined sizes. However, despite increasing popularity, ultra-filtration is not without its limitations, especially the problem of membrane clogging and vesicle trapping. This results in not only reduced lifetime of the expen-sive membranes, but also reduced isolation efficiency and erroneous interpretations of test results. Fortun-ately, this issue can be addressed by tangential flow filtration. Although current tangential flow filtration techniques are still limited by processing volume, given the ongoing progress in hydromechanics and material sciences, we believe the isolation efficiency of future ultrafiltration methods will be dramatically improved. Similarly, SEC, which features both high- quality exosome preparation and excellent repro-ducibility, also holds great potential for high- throughput industrial applications. This is especially true given the fact that the gravity flow used in SEC causes minimal damage to exosome structure and function. Collectively, we reckon that ultrafiltration and SEC may provide a basis for future standardiza-tion of clinical grade exosome samples. Conversely, no microfluidic device has been readily applied in clinical applications, in spite of the remarkable advances achieved in recent years. Major roadblocks to clinical applications include standardization, scalability, and validation [127, 159]. Furthermore, the relatively low isolation efficiency of such methods may pose detrimental effects on downstream assessments such as genomic and proteomic analysis, and result in compromised diag-nosis results. We believe further improvements in microfluidic processing capacity via multiple exo-some sorting mechanisms, as well as massively parallel microfluidic sets, represents plausible solu-tions. Importantly, most of the existing exosome isolation techniques are applied to basic research. For microfluidics to become more clinically relevant we suggest the design of techniques and devices for exosome isolation should take a more translational approach, by thoroughly evaluating a sufficient number of clinical samples for improved selectivity, robustness, and sensitivity. Furthermore, to facilitate the in-depth investiga-tion of exosomes and their related biological func-tions, more efforts need to be made for the develop-ment of simultaneous exosome separation/quanti-fication strategies and devices, to achieve not only efficient exosome isolation, but also real-time exo-some quantification and analysis. Although several real-time exosome isolation/detection apparatuses have been reported, standardizing the analysis mod-ule for comparable and reliable readouts still repre-sents a great challenge. In fact, compared to analysis, development of standardized exosome isolation methods constitutes an even harder task. Due to the heterogeneous features of biological samples, a reliable exosome separation technique suitable for every study is still not available. A bespoke selection of separation methods tailored for particular exosome-containing objects is imperative for high- quality exosome isolation and readout validation. As a result, future efforts may need to develop different exosome isolation standards to meet the particular Theranostics 2020, Vol. 10, Issue 8 http://www.thno.org 3704 properties of different types of biological samples and target particles (i.e., genetic or protein contents) to be screened. 5. Conclusion The observation of exosome-mediated cell signaling provides a great opportunity for developing exosome-related basic and applied biomedical applications in various fields. Despite the revolution-ary progress in exosome-based theranostic applica-tions over the past few decades, there are still funda-mental unanswered questions in the field. These questions address some of the hotspots of current bio-medical research such as the secretory regulation mechanism of exosomes, exosomal content sorting mechanism and their intercellular transduction path-way. Various exosome separation strategies and devices have been suggested to facilitate the investi-gation of exosomes and their related biological func-tions. As comprehensively discussed in this work, standardization in exosome preparation such as specimen handling, isolation, and quantification has still not been established. Through studying the nature of particular samples and specific application settings, we believe careful selection of isolation tech-niques (or a combination of isolation techniques) will help investigators address many of the challenges faced in current exosome studies. In addition, we also believe that the exosome/microvesicle separation and quantification strategy (using both exosome and microvesicle markers) as suggested in this work can provide a plausible strategy to obtain accurate quantitative information for future exosome (and microvesicle)-related investigations. Acknowledgments D.Y, W.Z, H.Z, F.Z, C.C, T.W acknowledge the funding from the National Natural Science Foundation of China (No.81773175, No.11704343, and U1404814) and the China Postdoctoral Science Foundation (No. 2018M630839); T.W and R.N.V are supported by Murdoch University, and RNV also acknowledges the funding support from the McCusker foundation through Perron Institute of Neurological and Translational Sciences. T.W is also supported by Murdoch University commercial research funding and Perron early and mid-career researcher internal grants scheme; T. P is the recipient of Australian Research Council s Discovery Early Career Researcher Award (project number DE160100900). 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However, clogging and trapping of the particles leading to a low purity is a noticeable limitation [40]. Next is the PT-based methods which is based on the lowering the solubility of the exosomes by adding highly hydrophilic polymers to the solution. ...... Moreover, this method only requires lab-bench equipment. However, PT-based methods can cause exosome aggregation [40]. Another technique is the CT-based method which separates particles with different sizes by adding porous materials to the exosome containing solution [41]. ...Overcome the barriers of the skin: exosome therapyArticleFull-text availableDec 2021 Gi Hoon YangYoon Bum Lee Donggu KangHojun JeonExosomes are nano-sized cargos with a lipid bilayer structure carrying diverse biomolecules including lipids, proteins, and nucleic acids. These small vesicles are secreted by most types of cells to communicate with each other. Since exosomes circulate through bodily fluids, they can transfer information not only to local cells but also to remote cells. Therefore, exosomes are considered potential biomarkers for various treatments. Recently, studies have shown the efficacy of exosomes in skin defects such as aging, atopic dermatitis, and wounds. Also, exosomes are being studied to be used as ingredients in commercialized skin treatment products. In this review, we discussed the need for exosomes in skin therapy together with the current challenges. Moreover, the functional roles of exosomes in terms of skin treatment and regeneration are overviewed. Finally, we highlighted the major limitations and the future perspective in exosome engineering.ViewShow abstract... Despite challenges associated with their small size and low density, methods successfully applied in exosome isolation include ultracentifugation, size exclusion chromatography, microfluidic technologies, immunoaffinity capture-based techniques, and microchip-based techniques such as the exosome total isolation chip (ExoTIC) [52][53][54][55][56]. However, these techniques are not 100% efficient, with the co-isolation of other extracellular vesicles, such as microvesicles and ectosomes, often occurring during exosomal preparation [57]. Furthermore, exosomal isolates from human plasma samples have been reported to be contaminated with high-abundance plasma proteins such as albumin, which can hinder downstream proteomic applications such as mass spectrometry-based analysis via the suppression of peptide ion signals derived from exosomal proteins, thus complicating the proteomic profiling of exosomes [58,59]. ...Clinical Proteomics of Biofluids in Haematological MalignanciesArticleFull-text availableJul 2021INT J MOL SCI Katie DunphyKelly O’MahoneyPaul DowlingDespina BazouSince the emergence of high-throughput proteomic techniques and advances in clinical technologies, there has been a steady rise in the number of cancer-associated diagnostic, prognostic, and predictive biomarkers being identified and translated into clinical use. The characterisation of biofluids has become a core objective for many proteomic researchers in order to detect disease-associated protein biomarkers in a minimally invasive manner. The proteomes of biofluids, including serum, saliva, cerebrospinal fluid, and urine, are highly dynamic with protein abundance fluctuating depending on the physiological and/or pathophysiological context. Improvements in mass-spectrometric technologies have facilitated the in-depth characterisation of biofluid proteomes which are now considered hosts of a wide array of clinically relevant biomarkers. Promising efforts are being made in the field of biomarker diagnostics for haematologic malignancies. Several serum and urine-based biomarkers such as free light chains, β-microglobulin, and lactate dehydrogenase are quantified as part of the clinical assessment of haematological malignancies. However, novel, minimally invasive proteomic markers are required to aid diagnosis and prognosis and to monitor therapeutic response and minimal residual disease. This review focuses on biofluids as a promising source of proteomic biomarkers in haematologic malignancies and a key component of future diagnostic, prognostic, and disease-monitoring applications.ViewShow abstract... Existing isolation techniques lead to the inevitable mixing of nonexosomal components, such as lipoproteins, proteins, viruses, and bacteria, with exos isolated from different specimens [89][90][91]. In addition, the standards for separation are not uniform, and the different equipment used across different laboratories may lead to further differences and inconsistencies, which will eventually lead to different findings [92,93]. The techniques used for the characterization of exos are also different, and their accuracy varies too [94]. ...Role of Exosomal MicroRNAs and Their Crosstalk with Oxidative Stress in the Pathogenesis of OsteoporosisArticleFull-text availableJul 2021OXID MED CELL LONGEVJun LuYan ZhangJinqi Liang Zhao HongmouOsteoporosis (OP) is an aging-related disease involving permanent bone tissue atrophy. Most patients with OP show high levels of oxidative stress (OS), which destroys the microstructure of bone tissue and promotes disease progression. Exosomes (exos) help in the delivery of microRNAs (miRNAs) and allow intercellular communication. In OP, exosomal miRNAs modulate several physiological processes, including the OS response. In the present review, we aim to describe how exosomal miRNAs and OS contribute to OP. We first summarize the relationship of OS with OP and then detail the features of exos along with the functions of exo-related miRNAs. Further, we explore the interplay between exosomal miRNAs and OS in OP and summarize the functional role of exos in OP. Finally, we identify the advantages of exo-based miRNA delivery in treatment strategies for OP. Our review seeks to improve the current understanding of the mechanism underlying OP pathogenesis and lay the foundation for the development of novel theranostic approaches for OP.ViewShow abstract... Apoptotic bodies are relatively larger lipid vesicles released by dying cells which contain fragments of apoptotic cells such as micronuclei, chromatin remnants, and intact organelles [13]. EVs have traditionally been defined and sorted based on their different densities and sizes which enables separation by various methods such as differential centrifugation, filtration, and size exclusion chromatography [14]. It should be noted, however, that due to the overlapping size and density between EVs such as exosomes and microvesicles, current EV isolation techniques have limitation regarding precise purification without completely excluding other groups of EVs. ...Biodistribution of Exosomes and Engineering Strategies for Targeted Delivery of Therapeutic ExosomesArticleFull-text availableJul 2021TISSUE ENG REGEN MEDHojun ChoiYoorim ChoiHwa Young Yim C. H. ChoiExosomes are cell-secreted nano-sized vesicles which deliver diverse biological molecules for intercellular communication. Due to their therapeutic potential, exosomes have been engineered in numerous ways for efficient delivery of active pharmaceutical ingredients to various target organs, tissues, and cells. In vivo administered exosomes are normally delivered to the liver, spleen, kidney, lung, and gastrointestinal tract and show rapid clearance from the blood circulation after systemic injection. The biodistribution and pharmacokinetics (PK) of exosomes can be modulated by engineering various factors such as cellular origin and membrane protein composition of exosomes. Recent advances accentuate the potential of targeted delivery of engineered exosomes even to the most challenging organs including the central nervous system. Major breakthroughs have been made related to various imaging techniques for monitoring in vivo biodistribution and PK of exosomes, as well as exosomal surface engineering technologies for inducing targetability. For inducing targeted delivery, therapeutic exosomes can be engineered to express various targeting moieties via direct modification methods such as chemically modifying exosomal surfaces with covalent/non-covalent bonds, or via indirect modification methods by genetically engineering exosome-producing cells. In this review, we describe the current knowledge of biodistribution and PK of exosomes, factors determining the targetability and organotropism of exosomes, and imaging technologies to monitor in vivo administered exosomes. In addition, we highlight recent advances in strategies for inducing targeted delivery of exosomes to specific organs and cells.ViewShow abstract... phase is used to separate the exosomes based on their size. 28 The SEC results in highly pure exosomes; however, SEC reported to have low yield, time consuming and involves high cost. Besides these limitations, the SEC method has been applied for the analysis of clinical samples. ...Milk exosomes -A biogenic nanocarrier for small molecules and macromolecules to combat cancer Exploitation of Nano-bio interfaces for an alternative, accelerated, cost-effective therapeutic/health care product development process View project Formulation of Anthocyanins and Anthocyanidins View projectArticleFull-text availableJul 2021Raghuram Kandimalla Farrukh Aqil Neha Tyagi Ramesh GuptaViewHumanized Biomimetic Nanovesicles for Neuron TargetingArticleFull-text availableAug 2021 Assaf Zinger Caroline CvetkovicManuela Sushnitha Robert KrencikNanovesicles (NVs) are emerging as innovative, theranostic tools for cargo delivery. Recently, surface engineering of NVs with membrane proteins from specific cell types has been shown to improve the biocompatibility of NVs and enable the integration of functional attributes. However, this type of biomimetic approach has not yet been explored using human neural cells for applications within the nervous system. Here, this paper optimizes and validates the scalable and reproducible production of two types of neuron-targeting NVs, each with a distinct lipid formulation backbone suited to potential therapeutic cargo, by integrating membrane proteins that are unbiasedly sourced from human pluripotent stem-cell-derived neurons. The results establish that both endogenous and genetically engineered cell-derived proteins effectively transfer to NVs without disruption of their physicochemical properties. NVs with neuron-derived membrane proteins exhibit enhanced neuronal association and uptake compared to bare NVs. Viability of 3D neural sphere cultures is not disrupted by treatment, which verifies the utility of organoid-based approaches as NV testing platforms. Finally, these results confirm cellular association and uptake of the biomimetic humanized NVs to neurons within rodent cranial nerves. In summary, the customizable NVs reported here enable next-generation functionalized theranostics aimed to promote neuroregeneration.ViewShow abstractAdvances in Microfluidic Extracellular Vesicle Analysis for Cancer DiagnosticsArticleAug 2021LAB CHIPShibo ChengYutao LiHe YanYong ZengExtracellular vesicles (EVs) secreted by cells into the bloodstream and other bodily fluids, including exosomes, have been demonstrated to be a class of significant messengers that mediate intercellular communications. Tumor-derived extracellular vesicles are enriched in a selective set of biomolecules from original cells, including proteins, nucleic acids, and lipids, and thus offer a new perspective of liquid biopsy for cancer diagnosis and therapeutic monitoring. Owing to the heterogeneity of their biogenesis, physical properties, and molecular constituents, isolation and molecular characterization of EVs remain highly challenging. Microfluidics provides a disruptive platform for EV isolation and analysis owing to its inherent advantages to promote the development of new molecular and cellular sensing systems with improved sensitivity, specificity, spatial and temporal resolution, and throughput. This review summarizes the state-of-the-art advances in the development of microfluidic principles and devices for EV isolation and biophysical or biochemical characterization, in comparison to the conventional counterparts. We will also survey the progress in adapting the new microfluidic techniques to assess the emerging EV-associated biomarkers, mostly focused on proteins and nucleic acids, for clinical diagnosis and prognosis of cancer. Lastly, we will discuss the current challenges in the field of EV research and our outlook on future development of enabling microfluidic platforms for EV-based liquid biopsy.ViewShow abstractEmerging biosensing platforms for quantitative detection of exosomes as diagnostic biomarkersArticleNov 2021COORDIN CHEM REVJiayi TanYu WenMing LiExosomes are a class of extracellular vesicles secreted by all living cells. Exosomes carry abundant constituents from their parental cells that are proteins, nucleic acids, lipids, small biomolecules, and metabolites. Recent studies have implied that exosomes are promising diagnostic biomarkers for a wide range of diseases, owing to their prominent features superior to other circulating biomarkers. Despite of the clinical potential of circulating exosomes for disease diagnostics, their translation from the bench to the bed-side has met substantive hurdles stemming from the lack of high-performance analytical technologies. Current research has focused on developing biosensors for exosomes from various sources in biomedical fields and addressed challenges that need to be solved before their practical applications in the clinic and point-of-care (POC) fields. Functional nanostructures have been integrated into these emerging biosensing platforms with improved performances, allowing for molecular profiling and quantitative analysis of exosomes. This article provides a comprehensive overview regarding recent advances of new biosensing platforms for detection of exosomes. We will focus on the rational design, working principles and exosome-based diagnostic applications of these emerging biosensors. This review will expectedly provide valuable guidelines for the further development of biosensors for practical applications of exosomes in the clinic and POC diagnostics.ViewShow abstractExtracellular Vesicles under Oxidative Stress Conditions: Biological Properties and Physiological RolesArticleFull-text availableJul 2021 Elisabetta Chiaradia Brunella Tancini Carla Emiliani Sandra BurattaUnder physio-pathological conditions, cells release membrane-surrounded structures named Extracellular Vesicles (EVs), which convey their molecular cargo to neighboring or distant cells influencing their metabolism. Besides their involvement in the intercellular communication, EVs might represent a tool used by cells to eliminate unnecessary/toxic material. Here, we revised the literature exploring the link between EVs and redox biology. The first proof of this link derives from evidence demonstrating that EVs from healthy cells protect target cells from oxidative insults through the transfer of antioxidants. Oxidative stress conditions influence the release and the molecular cargo of EVs that, in turn, modulate the redox status of target cells. Oxidative stress-related EVs exert both beneficial or harmful effects, as they can carry antioxidants or ROS-generating enzymes and oxidized molecules. As mediators of cell-to-cell communication, EVs are also implicated in the pathophysiology of oxidative stress-related diseases. The review found evidence that numerous studies speculated on the role of EVs in redox signaling and oxidative stress-related pathologies, but few of them unraveled molecular mechanisms behind this complex link. Thus, the purpose of this review is to report and discuss this evidence, highlighting that the analysis of the molecular content of oxidative stress-released EVs (reminiscent of the redox status of originating cells), is a starting point for the use of EVs as diagnostic and therapeutic tools in oxidative stress-related diseases.ViewShow abstractPerspectives and Challenges in Extracellular Vesicles Untargeted Metabolomics AnalysisArticleJul 2021TRAC-TREND ANAL CHEM Danuta Dudzik Szymon Macioszek Wiktoria Struck Michal Jan MarkuszewskiThe discovery of extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, has opened a new frontier in the study of signal transduction and understanding cell-to-cell communication. EVs play a key role regulating various biological processes such as tissue regeneration, blood coagulation, immunomodulation or carcinogenesis. Therefore, they gain much attention as potentially therapeutic and non-invasive diagnostic targets. In this aspect, a comprehensive characterization of the EVs molecular content and understanding EVs functions are especially relevant. However, routine isolation of a usually small amount of high purity EVs is challenging, and analysis requires highly sensitive advanced analytical solutions. The utility of EVs in clinical settings is still limited due to the lack of standardization in existing protocols. This review focuses on the implication of the state-of-the-art mass-spectrometry based untargeted metabolomics approach for the molecular profiling of EVs, with the emphasis on the analytical methodologies, highlighting potential challenges and limitations.ViewShow abstractShow moreExtracellular vesicles: The next generation of biomarkers for liquid biopsy-based prostate cancer diagnosisArticleFull-text availableJan 2020thnoBairen Pang Ying Zhu Jie Ni Yong liProstate cancer (PCa) is a leading cause of cancer death for males in western countries. The current gold standard for PCa diagnosis - template needle biopsies - often does not convey a true representation of the molecular profile given sampling error and complex tumour heterogeneity. Presently available biomarker blood tests have limited accuracy. There is a growing demand for novel diagnostic approaches to reduce both the number of men with an abnormal PSA/ DRE who undergo invasive biopsy and the number of cores collected per biopsy. Liquid biopsy is a minimally invasive biofluid-based approach that has the potential to provide information and improve the accuracy of diagnosis for patients treatment selection, prognostic counselling and development of risk-adjusted follow-up protocols. Extracellular vesicles (EVs) are lipid bilayer-delimited particles released by tumour cells which may provide a real-time snapshot of the entire tumour in a non-invasive way. EVs can regulate physiological processes and mediate systemic dissemination of various types of cancers. Emerging evidence suggests that EVs have crucial roles in PCa development and metastasis. Most importantly, EVs are directly derived from their parent cells with their information. EVs contain components including proteins, mRNAs, DNA fragments, non-coding RNAs and lipids, and play a critical role in intercellular communication. Therefore, EVs hold promise for the discovery of liquid biopsy-based biomarkers for PCa diagnosis. Here, we review the current approaches for EV isolation and analysis, summarise the recent advances in EV protein biomarkers in PCa and focus on liquid biopsy-based EV biomarkers in PCa diagnosis for personalised medicine.ViewShow abstractSmooth muscle SIRT1 reprograms endothelial cells to suppress angiogenesis after ischemiaArticleFull-text availableJan 2020thnoYong-Qing Dou Peng KongChang-Lin LiMei HanObjective: Vascular smooth muscle cells (VSMCs) undergo the phenotypic changes from contractile to synthetic state during vascular remodeling after ischemia. SIRT1 protects against stress-induced vascular remodeling via maintaining VSMC differentiated phenotype. However, the effect of smooth muscle SIRT1 on the functions of endothelial cells (ECs) has not been well clarified. Here, we explored the role of smooth muscle SIRT1 in endothelial angiogenesis after ischemia and the underlying mechanisms. Methods: We performed a femoral artery ligation model using VSMC specific human SIRT1 transgenic (SIRT1-Tg) and knockout (KO) mice. Angiogenesis was assessed in in vivo by quantification of the total number of capillaries, wound healing and matrigel plug assays, and in vitro ECs by tube formation, proliferation and migration assays. The interaction of HIF1α with circRNA was examined by using RNA immunoprecipitation, RNA pull-down and in situ hybridization assays. Results: The blood flow recovery was significantly attenuated in SIRT1-Tg mice, and markedly improved in SIRT1-Tg mice treated with SIRT1 inhibitor EX527 and in SIRT1-KO mice. The density of capillaries significantly decreased in the ischemic gastrocnemius of SIRT1-Tg mice compared with SIRT1-KO and WT mice, with reduced expression of VEGFA, which resulted in decreased number of arterioles. We identified that the phenotypic switching of SIRT1-Tg VSMCs was attenuated in response to hypoxia, with high levels of contractile proteins and reduced expression of the synthetic markers and NG2, compared with SIRT1-KO and WT VSMCs. Mechanistically, SIRT1-Tg VSMCs inhibited endothelial angiogenic activity induced by hypoxia via the exosome cZFP609. The cZFP609 was delivered into ECs, and detained HIF1α in the cytoplasm via its interaction with HIF1α, thereby inhibiting VEGFA expression and endothelial angiogenic functions. Meantime, the high cZFP609 expression was observed in the plasma of the patients with atherosclerotic or diabetic lower extremity peripheral artery disease, associated with reduced ankle-brachial index. Knockdown of cZFP609 improved blood flow recovery after hindlimb ischemia in SIRT1-Tg mice. Conclusions: Our findings demonstrate that SIRT1 may impair the plasticity of VSMCs. cZFP609 mediates VSMCs to reprogram endothelial functions, and serves as a valuable indicator to assess the prognosis and clinical outcomes of ischemic diseases.ViewShow abstractOptimized Isolation of Extracellular Vesicles From Various Organic Sources Using Aqueous Two-Phase SystemArticleFull-text availableDec 2019Oğuz Kaan KIRBAŞ Batuhan Turhan Bozkurt Fikrettin Sahin Pakize Neslihan TaşlıFrom biomarkers to drug carriers, Extracellular Vesicles (EVs) are being used successfully in numerous applications. However, while the subject has been steadily rising in popularity, current methods of isolating EVs are lagging behind, incapable of isolating EVs at a high enough quantity or quality while also requiring expensive, specialized equipment. The \"isolation problem” is one of the major obstacles in the field of EV research - and even more so for their potential, widespread use for clinical diagnosis and therapeutic applications. Aqueous Two-Phase Systems (ATPS) has been reported previously as a promising method for isolating EVs quickly and efficiently, and with little contaminants - however, this method has not seen widespread use. In this study, an ATPS-based isolation protocol is used to isolate small EVs from plant, cell culture, and parasite culture sources. Isolated EVs were characterized in surface markers, size, and morphological manner. Additionally, the capacity of ATPS-based EV isolation in removing different contaminants was shown by measuring protein, fatty acid, acid, and phenol red levels of the final isolate. In conclusion, we have shown that EVs originating from different biological sources can be isolated successfully in a cost-effective and user-friendly manner with the use of aqueous two-phase systems.ViewShow abstractIsolation and Detection Technologies of Extracellular Vesicles and Application on Cancer DiagnosticArticleFull-text availableOct 2019Chunyan MaFan Jiang Yifan MaJingjing ZhangThe vast majority of cancers are treatable when diagnosed early. However, due to the elusive trace and the limitation of traditional biopsies, most cancers have already spread widely and are at advanced stages when they are first diagnosed, causing ever-increasing mortality in the past decades. Hence, developing reliable methods for early detection and diagnosis of cancer is indispensable. Recently, extracellular vesicles (EVs), as circulating phospholipid vesicles secreted by cells, are found to play significant roles in the intercellular communication as well as the setup of tumor microenvironments and have been identified as one of the key factors in the next-generation technique for cancer diagnosis. However, EVs present in complex biofluids that contain various contaminations such as nonvesicle proteins and nonspecific EVs, resulting in the interference of screening for desired biomarkers. Therefore, applicable isolation and enrichment methods that guarantee scale-up of sample volume, purity, speed, yield, and tumor specificity are necessary. In this review, we introduce current technologies for EV separation and summarize biomarkers toward EV-based cancer liquid biopsy. In conclusion, a novel systematic isolation method that guarantees high purity, recovery rate, and tumor specificity is still missing. Besides that, a dual-model EV-based clinical trial system includes isolation and detection is a hot trend in the future due to efficient point-of-care needs. In addition, cancer-related biomarkers discovery and biomarker database establishment are essential objectives in the research field for diagnostic settings.ViewShow abstractPurity and yield of melanoma exosomes are dependent on isolation methodArticleFull-text availableSep 2020 Shin La Shu Yunchen Yang Cheryl Allen Marc s ErnstoffBoth exosomes and soluble factors have been implicated in the generation of an immunosuppressive tumour microenvironment. Determining the contribution of each requires stringent control of purity of the isolated analytes. The present study compares several conventional exosome isolation methods for the presence of co-enriched soluble factors while isolating exosomes from human melanoma-derived cell lines. The resultant preparations were analysed by multiplex bead array analysis for cytokine profiles, and by electron microscopy and nanotracking analysis for exosome size distribution and concentration. It is demonstrated that the amount and repertoire of soluble factors in exosome preparations is dependent upon the isolation method used. A combination of ultrafiltration and size exclusion chromatography yielded up to 58-fold more exosomes than ultracentrifugation, up to 836-fold lower concentrations of co-purified soluble factors when adjusted for exosome yield, and a greater than two-fold increase in PD-L1 expressing exosomes. Mechanistically, in context of the immunomodulatory effects of exosomes, the exosome isolation method should be carefully considered in order to limit any effects due instead to co-eluted soluble factors.ViewShow abstractDevelopment of a novel DNA oligonucleotide targeting low-density lipoprotein receptorArticleFull-text availableNov 2019Tao Wang Kamal Rahimizadeh Rakesh N VeeduNon-alcoholic fatty liver disease (NAFLD) culminates in insulin resistance and metabolic syndrome. Because there are no approved pharmacological treatment agents for non-alcoholic steatohepatitis (NASH) and NAFLD, different signaling pathways are under investigation for drug development with the focus on metabolic pathways. Hepatocyte nuclear factor 4-alpha (HNF4A) is at the center of a complex transcriptional network where its disruption is directly linked to glucose and lipid metabolism. Resetting HNF4A expression in NAFLD is therefore crucial for re-establishing normal liver function. Here, small activating RNA (saRNA) specific for upregulating HNF4A was injected into rats fed a high-fat diet for 16 weeks. Intravenous delivery was carried out using 5-(G5)-triethanolamine-core polyamidoamine (PAMAM) dendrimers. We observed a significant reduction in liver triglyceride, increased high-density lipoprotein/low-density lipoprotein (HDL/LDL) ratio, and decreased white adipose tissue/body weight ratio, all parameters to suggest that HNF4A-saRNA treatment induced a favorable metabolic profile. Proteomic analysis showed significant regulation of genes involved in sphingolipid metabolism, fatty acid β-oxidation, ketogenesis, detoxification of reactive oxygen species, and lipid transport. We demonstrate that HNF4A activation by oligonucleotide therapy may represent a novel single agent for the treatment of NAFLD and insulin resistance.ViewShow abstractElectrokinetically Driven Exosome Separation and Concentration Using Dielectrophoretic-Enhanced PDMS-Based MicrofluidicsArticleFull-text availableNov 2019ANAL CHEMSergio Ayala-Mar Victor H. Perez-Gonzalez Marco Mata José González-ValdezExosomes are a specific subpopulation of extracellular vesicles that have gained interest because of their many potential biomedical applications. However, exosome isolation and characterization are the first steps toward designing novel applications. This work presents a direct current-insulator-based dielectrophoretic (DC-iDEP) approach to simultaneously capture and separate exosomes by size. To do so, a microdevice consisting of a channel with two electrically insulating post sections was designed. Each section was tailored to generate different nonuniform spatial distributions of the electric field and, therefore, different dielectrophoretic forces acting on exosomes suspended in solution. Side channels were placed adjacent to each section to allow sample recovery. By applying an electric potential difference of 2000 V across the length of the main channel, dielectrophoretic size-based separation of exosomes was observed in the device. Analysis of particle size in each recovered fraction served to assess exosome separation efficiency. These findings show that iDEP can represent a first step toward designing a high-throughput, fast, and robust microdevice capable of capturing and discriminating different subpopulations of exosomes based on their size.ViewShow abstractDevelopment of a Novel DNA Oligonucleotide Targeting Low-Density Lipoprotein ReceptorArticleFull-text availableNov 2019 Tao Wang Kamal Rahimizadeh Rakesh N VeeduLow-density lipoprotein receptor (LDL-R) is a cell surface receptor protein expressed in a variety of solid cancers, including lung, colon, breast, brain, and liver, and therefore it opens up opportunities to deliver lysosome-sensitive anti-cancer agents, especially synthetic nucleic acid-based therapeutic molecules. In this study, we focused on developing novel nucleic acid molecules specific to LDL-R. For this purpose, we performed in vitro selection procedure via systematic evolution of ligands by exponential enrichment (SELEX) methodologies using mammalian cell-expressed human recombinant LDL-R protein as a target. After 10 rounds of selections, we identified a novel DNA oligonucleotide aptamer, RNV-L7, that can bind specifically to LDL-R protein with high affinity and specificity (KD = 19.6 nM). Furthermore, flow cytometry and fluorescence imaging assays demonstrated efficient binding to LDL-R overexpressed human cancer cells, including Huh-7 liver cancer cells and MDA-MB-231 breast cancer cells, with a binding affinity of ∼200 nM. Furthermore, we evaluated the functional potential of the developed LDL-R aptamer RNV-L7 by conjugating with a previously reported miR-21 targeting DNAzyme for inhibiting miR-21 expression. The results showed that the miR-21 DNAzyme-RNV-L7 aptamer chimera efficiently reduced the expression of miR-21 in Huh-7 liver cancer cells. As currently there are no reports on LDL-R aptamer development, we think that RNV-L7 could be beneficial toward the development of targeted cancer therapeutics.ViewShow abstractCardio-renal Exosomes in Myocardial Infarction Serum Regulate Proangiogenic Paracrine Signaling in Adipose Mesenchymal Stem CellsArticleJan 2020thnoLei GaoShuya MeiShuning ZhangHongming ZhuRationale: Mesenchymal stem cells (MSCs) play important roles in tissue repair and regeneration. However, the molecular mechanisms underlying MSCs activation remain largely unknown, thus hindering their clinical translation. Exosomes are small vesicles that act as intercellular messengers, and their potential for stem cell activation in pathological conditions has not been fully characterized yet. Here, we aim to investigate whether serum exosomes are involved in the remote activation of MSCs after myocardial infarction (MI). Methods: We established MI mouse model by ligating the left anterior descending branch of the coronary artery. Afterwards, serum exosomes were isolated from control (Con Exo) and MI mice (MI Exo) by differential centrifugation. Exosomes were characterized through transmission electron microscopy and nanoparticle tracking analysis. The cell proliferation rate was evaluated by CCK-8 and EdU incorporation assays. Exosomal miRNA and protein levels were assessed using qRT-PCR and western blotting, respectively. VEGF levels in the supernatant and serum were quantified by ELISA. Matrigel plug and tube formation assays were used to evaluate angiogenesis. To explore miR-1956 roles, overexpression and knock-down experiments were performed using mimic and inhibitor, respectively. Finally, miR-1956 target genes were confirmed using the luciferase reporter assay. Results: Both types of exosomes exhibited typical characteristics and could be internalized by adipose-derived MSCs (ADMSCs). MI Exo enhanced ADMSCs proliferation through the activation of ERK1/2. Gain- and loss-of-function studies allowed the validation of miR-1956 (enriched in MI Exo) as the functional messenger that stimulates ADMSCs-mediated angiogenesis and paracrine VEGF signaling, by downregulating Notch-1. Finally, we found that the ischemic myocardium and kidney may be the main sources that release serum exosomes after MI. Conclusions: Cardio-renal exosomes deliver miR-1956 and activate paracrine proangiogenic VEGF signaling in ADMSCs after MI; this process also involves Notch-1, which functions as the core mediator.ViewShow abstractMononuclear phagocyte system blockade improves therapeutic exosome delivery to the myocardiumArticleJan 2020thnoZhuo WanLianbi ZhaoFan Lu Li LiuRationale: Exosomes are emerging as a promising drug delivery carrier. However, rapid uptake of exosomes by the mononuclear phagocyte system (MPS) remains an obstacle for drug delivery into other targeted organs, including the heart. We hypothesized that prior blocking of uptake of exosomes by the MPS would improve their delivery to the targeted organs. Methods: Exosomes were isolated from the cell culture medium. Fluorescence-labeled exosomes were tracked in vitro and in vivo by fluorescence imaging. The expression of clathrin heavy chain (Cltc), cavolin1, Pak1 and Rhoa, known genes for endocytosis, were profiled in various cell lines and organs by qPCR. The knockdown efficiency of siRNA against Cltc was analyzed by Western blotting. Exosomecontrol and exosomeblocking were constructed by encapsulating isolated exosomes with siControl or siClathrin via electroporation, while exosometherapeutic was constructed by encapsulating isolated exosomes with miR-21a. Doxorubicin-induced cardiotoxicity model was used to verify the therapeutic efficiency of the exosome-based miR-21a delivery by echocardiography. Results: Exosomes were preferentially accumulated in the liver and spleen, mainly due to the presence of abundant macrophages. Besides the well-known phagocytic effect, efficient endocytosis also contributes to the uptake of exosomes by macrophages. Cltc was found to be highly expressed in the macrophages compared with other endocytosis-associated genes. Accordingly, knockdown of Cltc significantly decreased the uptake of exosomes by macrophages in vitro and in vivo. Moreover, prior injection of exosomeblocking strikingly improved the delivery efficiency of exosomes to organs other than spleen and liver. Consistently, compared with the direct injection of exosometherapeutic, prior injection of exosomeblocking produced a much better therapeutic effect on cardiac function in the doxorubicin-induced cardiotoxicity mouse model. Conclusions: Prior blocking of endocytosis of exosomes by macrophages with exosomeblocking successfully and efficiently improves the distribution of following exosometherapeutic in targeted organs, like the heart. The established two-step exosome delivery strategy (blocking the uptake of exosomes first followed by delivery of therapeutic exosomes) would be a promising method for gene therapy.ViewShow abstractShow moreAdvertisementRecommendationsDiscover more about: ExosomesProjectAptamer development and applicaitons Tao WangAptamer development and applicaitons View projectProjectTargeting cancer stem cells with aptamer-guided therapeutics Wang Yin Sarah Shigdar Tao Wang[...] Wei DuanView projectArticleCan exosomal micro-RNAS be as biomarkers of diseases?January 2016Y. LiP.-F. TangBACKGROUND: Exosome, a kind of cystic vesicle with bilayer structure, is widely distributed in the body fluids. Exosomes are involved in various cellular communications, and its contents including proteins, short chain peptides, DNA, RNA, phospholipids, and miRNA are resistant to degradation. OBJECTIVE: To clarify the characters of exosomes, and to investigate the possibility of exosomal miRNAs ... [Show full abstract] as biomarkers for different diseases to provide a new strategy for clinical diagnosis. METHODS: A computer-based search of CNKI and PubMed databases was performed by the first author for articles related to exosomal miRNAs. The keywords were \"exosome, microvesicles, extracellular vesicles, miRNA, biomarker, early diagnosis, progrosis” in Chinese and English, respectively. Totally 50 eligible articles were included in result analysis. RESULTS AND CONCLUSION: After reviewing researches of exosomes in different diseases, we can confirm that exosomes broadly participant in physiological and pathological process of various system diseases. The abnormal expression of exosomal micro-RNAs has been identified in many studies, indicating the exosomal micro-RNAs have a great potential to be biomarkers for disease diagnosis. Further studies should focus on extracting the contents of exosomes, the pathogenesis of exosomes is involved in and screening the appropriate exosomal miRNAs for early diagnosis. © 2016, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.Read moreArticleFull-text availableDevelopment of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome...November 2020 · Molecules Tao WangZhenguo SongJun Mao[...] Roberto A BarreroCD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of various types of cancers such as melanoma and breast cancer and serves as a potential marker for cancer detection. Recently, its important role as a classic exosome marker was also emphasized. In this work, via using a magnetic ... [Show full abstract] bead-based competitive SELEX (systematic evolution of ligands by exponential enrichment) procedure and introducing a 0.5M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein (Kd = 38.71nM and 78.43, respectively). Furthermore, CD63-1 was found to be efficient in binding CD63 positive cells, including breast cancer MDA-MB-231 cells and CD63-overexpressed HEK293T cells, with a medium binding affinity (Kd~ 100 nM) as assessed by flow cytometry. When immunostaining assay was performed using clinical breast cancer biopsy, the CD63-1 aptamer demonstrated a comparable diagnostic efficacy for CD63 positive breast cancer with commercial antibodies. After developing a magnetic bead-based exosome immunoaffinity separation system using CD63-1 aptamer, it was found that this bead-based system could effectively isolate exosomes from both MDA-MB-231 and HT29 cell culture medium. Importantly, the introduction of the NaCl elution in this work enabled the isolation of native exosomes via a simple 0.5M NaCl incubation step. Based on these results, we firmly believe that the developed aptamers could be useful towards efficient isolation of native state exosomes from clinical samples and various theranostic applications for CD63-positive cancers.View full-textArticleFull-text availableDevelopment of nucleic acid aptamer-based lateral flow assays: A robust platform for cost-effective...March 2021 · Theranostics Tao Wang Rakesh N VeeduLanmei Chen[...] Arpitha ChikkannaLateral flow assay (LFA) has made a paradigm shift in the in vitro diagnosis field due to its rapid turnaround time, ease of operation and exceptional affordability. Currently used LFAs predominantly use antibodies. However, the high inter-batch variations, error margin and storage requirements of the conventional antibody-based LFAs significantly impede its applications. The recent progress in ... [Show full abstract] aptamer technology provides an opportunity to combine the potential of aptamer and LFA towards building a promising platform for highly efficient point-of-care device development. Over the past decades, different forms of aptamer-based LFAs have been introduced for broad applications ranging from disease diagnosis, agricultural industry to environmental sciences, especially for the detection of antibody-inaccessible small molecules such as toxins and heavy metals. But commercial aptamer-based LFAs are still not used widely compared with antibodies. In this work, by analysing the key issues of aptamer-based LFA design, including immobilization strategies, signalling methods, and target capturing approaches, we provide a comprehensive overview about aptamer-based LFA design strategies to facilitate researchers to develop optimised aptamer-based LFAs.View full-textArticleFull-text availableA systematic investigation of key factors of nucleic acid precipitation toward optimized DNA/RNA iso...February 2020 · BioTechniques Yalin Li Suxiang ChenNan Liu[...]Xumiao JingNucleic acid precipitation is important for virtually all molecular biology investigations. However, despite its crucial role, a systematic study of the influence factors of nucleic acid precipitation has not been reported. In the present work, via rational experimental design, key factors of nucleic acid precipitation, including the type of nucleic acid, temperature and time of incubation, speed ... [Show full abstract] and time of centrifugation, volume ratio of ethanol/isopropanol to nucleic acid solution, type of cation-containing salt solution and type of coprecipitator, were comprehensively evaluated in an attempt to maximize the efficiency of nucleic acid precipitation. Our results indicate that the optimal conditions of each influence factor of nucleic acid precipitation may vary in accordance with the chemistry, structure and length of nucleic acids.View full-textPreprintFull-text availableHigh-grade Extracellular Vesicles Preparation by Combined Size- exclusion and Affinity Chromatograph...February 2021Cristina BellottiKristina Lang Nataliya Kuplennik[...]Robert SteinfeldExtracellular vesicles (EVs) have recently gained growing interest for their diagnostic and therapeutic potential. Despite this, few protocols have been reported for the isolation of EVs with preserved biological function. Most EV purification methods include a precipitation step that results in aggregation of vesicles and most available techniques do not efficiently separate the various types of ... [Show full abstract] EVs such as exosomes, microvesicles, microparticles, and ectosomes which are involved in distinct biological processes. For this reason, we developed a new two-step fast performance liquid chromatography (FPLC) protocol for purification of large numbers of EVs. The method comprises size exclusion chromatography followed by immobilized metal affinity chromatography, which is enabled by expression of poly-histidine tagged folate receptor α in the parental cells. Characterisation and comparison of the EVs obtained by this method to EVs purified by differential centrifugation, currently the most common method to isolate EVs, demonstrated higher purity and more selective enrichment of exosomes in EV preparations using our FPLC method, as assessed by comparison of marker proteins and density distribution. Our studies reveal new possibilities for the isolation of defined subpopulations of EVs with preserved biological function that can easily be upscaled for production of larger amounts of EVs.View full-textInterested in research on Exosomes?Join ResearchGate to discover and stay up-to-date with the latest research from leading experts in Exosomes and many other scientific topics.Join for free ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! Please log in.Email · HintTip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleNo account? 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