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Biochemical characterization of exosome preparations: exosome preparations were obtained from 1 mL MM pool with four different protocols. Samples were electrophoresed and analyzed by Western Blot (WB) for the presence of typical vesicular markers. (A) Exosome preparation obtained with differential centrifugation steps (P3). (B) Exosome preparation purified with a precipitation kit (Exo PK). (C) Exosome preparation obtained with discontinuous iodixanol gradient. Top numbers refer to the corresponding gradient fraction. (D) Exosome preparation obtained with discontinuous sucrose gradient. Top numbers refer to the corresponding gradient fraction.
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Exosomes are gaining a prominent role in research due to their intriguing biology and several therapeutic opportunities. However, their accurate purification from body fluids and detailed physicochemical characterization remain open issues. We isolated exosomes from serum of patients with Multiple Myeloma by four of the most popular purification me...
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Context 1
... serial centrifugation (P3) and the precipitation kit (Exo PK) were characterized by Western Blot (WB) for the presence of typical exosomal markers: the membrane fusion protein Annexin V, the tetraspannin CD63, the heat shock protein Hsp70 and TSG101, a protein which is involved in multivesicular body biogenesis 1,2,26 . Results are displayed ( Fig. 2A,B). P3 protein concentration was (6 ± 1) μg/μL as quantified by Bradford ...
Context 2
... was subsequently loaded on top of two different discontinuous density gradients: iodixanol and sucrose. Twelve fractions were collected from each gradient and exosomal proteins were visualized by WB in fractions from 6 to 9 in both gradients. The biomarkers were detected in the range of 1.077-1.17 g/mL, as expected 27 ( Fig. 2C,D). Overall, these data indicate that, according to biochemical parameters conventionally adopted to verify the presence of exosomes in preparations, we obtained comparable and consistent exosomes populations with all the four different isolation ...
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Citations
... Some commercial kits that are described as 'exosome isolation' kits in fact rely on such polymer precipitation and do not strictly 'isolate' EVs, much less subtypes of EVs. Precipitation methods may not achieve any appreciable separation of EPs (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). ...
... Precipitation-based kits in particular will concentrate all EPs in a mixture, even many free proteins, resulting in a highly impure preparation, especially from complex, NVEP-rich sources such as blood plasma and serum. Use of such kits is strongly discouraged unless for volume reduction alone (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). In contrast, affinity-based methods may isolate only subtypes of EVs, and the specificity of the affinity reagents may be difficult to assess if the exact reagents are not disclosed. ...
... Measurements can then be performed after either drying the sample or keeping it submerged in liquid, such as saline or cell culture media. AFM morphometry can be used to obtain EV size distribution and ultrastructural details and to check for the presence and relative amounts of contaminants (Cavallaro, Pevere, et al., 2021;Paolini et al., 2016Parisse et al., 2017). In addition, AFM is one of the very few techniques capable of measuring single-vesicle nanomechanical properties (Gautron et al., 2021;Piontek et al., 2021), which were found to correlate with EV identity and function (Bortot et al., 2021;LeClaire et al., 2021;Romanò et al., 2022;Sorkin et al., 2018;Vorselen et al., 2018;Whitehead et al., 2015;Ye et al., 2021). ...
... Density gradient ultracentrifugation enhances EV purity by layering the sample onto a density gradient medium by adding sucrose or iodixanol. 28,29 However, its thin loading layer make it unsuitable for large-scale applications. 30 Polymer-based precipitation method utilizes polymers (e.g. ...
Extracellular Vesicles (EVs) are nanoscale to microscale membranous vesicles released by cells. They are vital for intercellular communications in physiological and pathological conditions, offering immense potential in diagnostics and therapeutical applications due to their ability to transfer diverse biological cargos between cells. While research of EVs is continuously gaining popularity in academia, large-scale manufacturing aiming for clinical applications remains challenging. Herein, we introduce a novel large-scale EV production strategy, including an upstream process with fed-batch suspension HEK293 cell culture, and a downstream process with chromatographic purification for ultra-pure EVs. Such promising EV production strategy enables the potential capability of large scale GMP manufacture of EVs for clinical applications.
... Some commercial kits that are described as 'exosome isolation' kits in fact rely on such polymer precipitation and do not strictly 'isolate' EVs, much less subtypes of EVs. Precipitation methods may not achieve any appreciable separation of EPs (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). ...
... Precipitation-based kits in particular will concentrate all EPs in a mixture, even many free proteins, resulting in a highly impure preparation, especially from complex, NVEP-rich sources such as blood plasma and serum. Use of such kits is strongly discouraged unless for volume reduction alone (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). In contrast, affinity-based methods may isolate only subtypes of EVs, and the specificity of the affinity reagents may be difficult to assess if the exact reagents are not disclosed. ...
... Measurements can then be performed after either drying the sample or keeping it submerged in liquid, such as saline or cell culture media. AFM morphometry can be used to obtain EV size distribution and ultrastructural details and to check for the presence and relative amounts of contaminants (Cavallaro, Pevere, et al., 2021;Paolini et al., 2016Parisse et al., 2017). In addition, AFM is one of the very few techniques capable of measuring single-vesicle nanomechanical properties (Gautron et al., 2021;Piontek et al., 2021), which were found to correlate with EV identity and function (Bortot et al., 2021;LeClaire et al., 2021;Romanò et al., 2022;Sorkin et al., 2018;Vorselen et al., 2018;Whitehead et al., 2015;Ye et al., 2021). ...
... Some commercial kits that are described as 'exosome isolation' kits in fact rely on such polymer precipitation and do not strictly 'isolate' EVs, much less subtypes of EVs. Precipitation methods may not achieve any appreciable separation of EPs (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). ...
... Precipitation-based kits in particular will concentrate all EPs in a mixture, even many free proteins, resulting in a highly impure preparation, especially from complex, NVEP-rich sources such as blood plasma and serum. Use of such kits is strongly discouraged unless for volume reduction alone (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). In contrast, affinity-based methods may isolate only subtypes of EVs, and the specificity of the affinity reagents may be difficult to assess if the exact reagents are not disclosed. ...
... Measurements can then be performed after either drying the sample or keeping it submerged in liquid, such as saline or cell culture media. AFM morphometry can be used to obtain EV size distribution and ultrastructural details and to check for the presence and relative amounts of contaminants (Cavallaro, Pevere, et al., 2021;Paolini et al., 2016Parisse et al., 2017). In addition, AFM is one of the very few techniques capable of measuring single-vesicle nanomechanical properties (Gautron et al., 2021;Piontek et al., 2021), which were found to correlate with EV identity and function (Bortot et al., 2021;LeClaire et al., 2021;Romanò et al., 2022;Vorselen et al., 2018;Whitehead et al., 2015;Ye et al., 2021). ...
... Some commercial kits that are described as 'exosome isolation' kits in fact rely on such polymer precipitation and do not strictly 'isolate' EVs, much less subtypes of EVs. Precipitation methods may not achieve any appreciable separation of EPs (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). ...
... Precipitation-based kits in particular will concentrate all EPs in a mixture, even many free proteins, resulting in a highly impure preparation, especially from complex, NVEP-rich sources such as blood plasma and serum. Use of such kits is strongly discouraged unless for volume reduction alone (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). In contrast, affinity-based methods may isolate only subtypes of EVs, and the specificity of the affinity reagents may be difficult to assess if the exact reagents are not disclosed. ...
... Measurements can then be performed after either drying the sample or keeping it submerged in liquid, such as saline or cell culture media. AFM morphometry can be used to obtain EV size distribution and ultrastructural details and to check for the presence and relative amounts of contaminants (Cavallaro, Pevere, et al., 2021;Paolini et al., 2016Parisse et al., 2017). In addition, AFM is one of the very few techniques capable of measuring single-vesicle nanomechanical properties (Gautron et al., 2021;Piontek et al., 2021), which were found to correlate with EV identity and function (Bortot et al., 2021;LeClaire et al., 2021;Romanò et al., 2022;Vorselen et al., 2018;Whitehead et al., 2015;Ye et al., 2021). ...
Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its ‘Minimal Information for Studies of Extracellular Vesicles’, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.
... Some commercial kits that are described as 'exosome isolation' kits in fact rely on such polymer precipitation and do not strictly 'isolate' EVs, much less subtypes of EVs. Precipitation methods may not achieve any appreciable separation of EPs (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). ...
... Precipitation-based kits in particular will concentrate all EPs in a mixture, even many free proteins, resulting in a highly impure preparation, especially from complex, NVEP-rich sources such as blood plasma and serum. Use of such kits is strongly discouraged unless for volume reduction alone (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). In contrast, affinity-based methods may isolate only subtypes of EVs, and the specificity of the affinity reagents may be difficult to assess if the exact reagents are not disclosed. ...
... Measurements can then be performed after either drying the sample or keeping it submerged in liquid, such as saline or cell culture media. AFM morphometry can be used to obtain EV size distribution and ultrastructural details and to check for the presence and relative amounts of contaminants (Cavallaro, Pevere, et al., 2021;Paolini et al., 2016Paolini et al., , 2020Parisse et al., 2017). In addition, AFM is one of the very few techniques capable of measuring single-vesicle nanomechanical properties (Gautron et al., 2021;Piontek et al., 2021), which were found to correlate with EV identity and function (Bortot et al., 2021;LeClaire et al., 2021;Romanò et al., 2022;Sorkin et al., 2018;Vorselen et al., 2018;Whitehead et al., 2015;Ye et al., 2021). ...
... Some commercial kits that are described as 'exosome isolation' kits in fact rely on such polymer precipitation and do not strictly 'isolate' EVs, much less subtypes of EVs. Precipitation methods may not achieve any appreciable separation of EPs (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). ...
... Precipitation-based kits in particular will concentrate all EPs in a mixture, even many free proteins, resulting in a highly impure preparation, especially from complex, NVEP-rich sources such as blood plasma and serum. Use of such kits is strongly discouraged unless for volume reduction alone (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). In contrast, affinity-based methods may isolate only subtypes of EVs, and the specificity of the affinity reagents may be difficult to assess if the exact reagents are not disclosed. ...
... Measurements can then be performed after either drying the sample or keeping it submerged in liquid, such as saline or cell culture media. AFM morphometry can be used to obtain EV size distribution and ultrastructural details and to check for the presence and relative amounts of contaminants (Cavallaro, Pevere, et al., 2021;Paolini et al., 2016Parisse et al., 2017). In addition, AFM is one of the very few techniques capable of measuring single-vesicle nanomechanical properties (Gautron et al., 2021;Piontek et al., 2021), which were found to correlate with EV identity and function (Bortot et al., 2021;LeClaire et al., 2021;Romanò et al., 2022;Vorselen et al., 2018;Whitehead et al., 2015;Ye et al., 2021). ...
Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.
... Some commercial kits that are described as 'exosome isolation' kits in fact rely on such polymer precipitation and do not strictly 'isolate' EVs, much less subtypes of EVs. Precipitation methods may not achieve any appreciable separation of EPs (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). ...
... Precipitation-based kits in particular will concentrate all EPs in a mixture, even many free proteins, resulting in a highly impure preparation, especially from complex, NVEP-rich sources such as blood plasma and serum. Use of such kits is strongly discouraged unless for volume reduction alone (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). In contrast, affinity-based methods may isolate only subtypes of EVs, and the specificity of the affinity reagents may be difficult to assess if the exact reagents are not disclosed. ...
... Measurements can then be performed after either drying the sample or keeping it submerged in liquid, such as saline or cell culture media. AFM morphometry can be used to obtain EV size distribution and ultrastructural details and to check for the presence and relative amounts of contaminants (Cavallaro, Pevere, et al., 2021;Paolini et al., 2016Parisse et al., 2017). In addition, AFM is one of the very few techniques capable of measuring single-vesicle nanomechanical properties (Gautron et al., 2021;Piontek et al., 2021), which were found to correlate with EV identity and function (Bortot et al., 2021;LeClaire et al., 2021;Romanò et al., 2022;Sorkin et al., 2018;Vorselen et al., 2018;Whitehead et al., 2015;Ye et al., 2021). ...
... Some commercial kits that are described as 'exosome isolation' kits in fact rely on such polymer precipitation and do not strictly 'isolate' EVs, much less subtypes of EVs. Precipitation methods may not achieve any appreciable separation of EPs (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). ...
... Precipitation-based kits in particular will concentrate all EPs in a mixture, even many free proteins, resulting in a highly impure preparation, especially from complex, NVEP-rich sources such as blood plasma and serum. Use of such kits is strongly discouraged unless for volume reduction alone (Gámez-Valero et al., 2016;Karttunen et al., 2019;Lobb et al., 2015;Paolini et al., 2016). In contrast, affinity-based methods may isolate only subtypes of EVs, and the specificity of the affinity reagents may be difficult to assess if the exact reagents are not disclosed. ...
... Measurements can then be performed after either drying the sample or keeping it submerged in liquid, such as saline or cell culture media. AFM morphometry can be used to obtain EV size distribution and ultrastructural details and to check for the presence and relative amounts of contaminants (Cavallaro, Pevere, et al., 2021;Paolini et al., 2016Paolini et al., , 2020Parisse et al., 2017). In addition, AFM is one of the very few techniques capable of measuring single-vesicle nanomechanical properties (Gautron et al., 2021;Piontek et al., 2021), which were found to correlate with EV identity and function (Bortot et al., 2021;LeClaire et al., 2021;Romanò et al., 2022;Sorkin et al., 2018;Vorselen et al., 2018;Whitehead et al., 2015;Ye et al., 2021). ...
Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their
cell of origin and change the functions and phenotypes of other cells. These features
indicate strong biomarker and therapeutic potential and have generated broad interest,
as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and
in understanding and applying EV biology. However, hurdles remain to realising the
potential of EVs in domains ranging frombasic biology to clinical applications due to
challenges in EVnomenclature, separation fromnon-vesicular extracellular particles,
characterisation and functional studies. To address the challenges and opportunities
in this rapidly evolving field, the International Society for Extracellular Vesicles
(ISEV) updates its ‘Minimal Information for Studies of Extracellular Vesicles’, which
was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively.
The goal of the current document, MISEV2023, is to provide researchers with
an updated snapshot of available approaches and their advantages and limitations for
production, separation and characterisation of EVs from multiple sources, including
cell culture, body fluids and solid tissues. In addition to presenting the latest state
of the art in basic principles of EV research, this document also covers advanced
techniques and approaches that are currently expanding the boundaries of the field.
MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.
... Mechanistically, size-based methods can produce purer EVs compared to a higher yield of less pure EV fractions obtained by precipitation 46 . Impurity of EV preparations can impede EV uptake and/or function 47 . Several additional less prominent but significant associations between isolation method and cargo or function were identified (Fig. 2B). ...
The evolution of extracellular vesicle (EV) research has introduced nanotechnology into biomedical cell communication science while recognizing what was formerly considered cell “dust” as constituting an entirely new universe of cell signaling particles. To display the global EV research landscape, we performed a systematic review of 20,364 original research articles selected from all 40,684 EV‐related records identified in PubMed 2013 – 2022. We used machine‐learning to categorize the high‐dimensional data and further dissected significant associations between EV source, isolation method, cargo and function. Unexpected correlations between these four categories indicate prevalent experimental strategies based on cargo connectivity with function of interest being associated with certain EV sources or isolation strategies. Conceptually relevant association of size‐based EV isolation with protein cargo and uptake function will guide strategic conclusions enhancing future EV research and product development. Based on this study, we built an open‐source database to facilitate further analysis with conventional or AI tools to identify additional causative associations of interest.
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