Benchtop isolation and characterization of functional exosomes by sequential filtration

Heinemann, Mitja L.; Ilmer, Matthias; Silva, Leslie P.; Hawke, David H.; Recio, Alejandro; Vorontsova, Maria A.; Alt, Eckhard; Vykoukal, Jody

doi:10.1016/j.chroma.2014.10.026

Cited by 226 publications

(181 citation statements)

References 39 publications

Supporting

Mentioning

180

Contrasting

Order By: Relevance

“…Due to limitations in rotor sizes and due to observations that ultracentrifugation might affect the integrity of EVs, ultracentrifugation-based methods might not be the methods of choice for the clinical grade production of EVs. Currently, several groups try to establish novel methods for the EV purification in closed systems such as tangential flow filtration (89,90).…”

Section: Discussionmentioning

confidence: 99%

Clinical potential of mesenchymal stem/stromal cell-derived extracellular vesicles

Giebel

Kordelas²,

Börger

2017

Stem Cell Investig.

126

115

View full text Add to dashboard Cite

Within the last two decades mesenchymal stem/stromal cells (MSCs) emerged after hematopoietic stem cells as the second most investigated and applied somatic stem cell entity so far. MSCs mediate immunosuppressive as well as pro-regenerative activities. Against the initial assumption, MSCs may not primarily exert their therapeutic functions in a cellular but rather in a paracrine manner. Here, extracellular vesicles (EVs), such as exosomes and microvesicles, have been identified as major mediators of these paracrine effects. Meanwhile, MSC-EVs have been applied to an increasing amount of different animal models and were tested in a patient suffering from steroid-refractory acute graft-versus-host disease (acute GvHD) as well as in a patient cohort with chronic kidney disease. So far, the MSC-EV administration appears to be safe in humans and all tested animal models. Improvements were reported in all settings. Thus, MSC-EVs appear as promising novel therapeutic agents which might help to improve disease associated symptoms in millions of patients. Here, we review some of the milestones in the field, briefly discuss challenges and highlight clinical aspects of acute GvHD and its treatment with MSCs and MSC-EVs.

show abstract

Section: Discussionmentioning

confidence: 99%

Clinical potential of mesenchymal stem/stromal cell-derived extracellular vesicles

Giebel

Kordelas²,

Börger

2017

Stem Cell Investig.

126

115

View full text Add to dashboard Cite

show abstract

“…Vesicles contain vast amounts of different cargo material, including functional RNA (Valadi et al 2007), DNA (Balaj et al 2011) and proteins (Skog et al 2008). By harvesting body fluids or cell culture supernatants, EVs can be isolated with a wide range of different methods, such as ultracentrifugation (UC) (Van Deun et al 2014), ultrafiltration (Heinemann et al 2014), commercial reagents (Schageman et al 2013), immuno-affinity capture (Tauro et al 2012) and size-exclusion chromatography (SEC) .…”

Section: Introductionmentioning

confidence: 99%

Systematic review of factors influencing extracellular vesicle yield from cell cultures

et al. 2015

View full text Add to dashboard Cite

The potential therapeutic utility of extracellular vesicles (EVs) has spawned an interest into a scalable production, where the quantity and purity of EV samples is sufficient for clinical applications. EVs can be isolated using several different protocols; however, these isolation protocols and the subsequent methods of quantifying the resulting EV yield have not been sufficiently standardized. Therefore, the possibility of comparing different studies with respect to these parameters is limited. In this review, we have presented factors that might influence the yield and function of EVs from cell culture supernatants. The methods of isolation, downstream quantification, and culture conditions of the EV producing cells have been discussed. In order to examine the inter-study coherency of EV yields, 259 studies were initially screened, and 46 studies were included for extensive downstream analysis of EV yields where information pertaining to the isolation protocols and quantification methods was obtained from each study. Several other factors influencing yield were compared, such as cell type producing EVs, cell confluence level, and cell stimulation. In conclusion, various factors may impact the resulting EV yield, including technical aspects such as EV isolation and quantification procedures, and biological aspects such as cell type and culture conditions. The reflections presented in this review might aid in future standardization of the workflow in EV research.

show abstract

“…To overcome the volume limitation of chromatography columns, centrifugal ultrafiltration was employed in some of these and other studies [28] to concentrate the starting material. Tangential flow filtration (TFF) may also be used as a method to process even larger volumes of conditioned media for EV purification [29]. TFF is a rapid processing method whereby sample flow is directed in parallel to a semipermeable membrane, allowing for buffer exchange by diafiltration as well as concentration of sample with simultaneous removal of non-EV components.…”

Section: Introductionmentioning

confidence: 99%

Scalable, cGMP‐compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL‐15/lactadherin complexes

Watson

Yung

Bergamaschi

et al. 2018

J of Extracellular Vesicle

123

102

View full text Add to dashboard Cite

The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches.

show abstract

Benchtop isolation and characterization of functional exosomes by sequential filtration

Cited by 226 publications

References 39 publications

Clinical potential of mesenchymal stem/stromal cell-derived extracellular vesicles

Clinical potential of mesenchymal stem/stromal cell-derived extracellular vesicles

Systematic review of factors influencing extracellular vesicle yield from cell cultures

Scalable, cGMP‐compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL‐15/lactadherin complexes

Contact Info

Product

Resources

About