Isolation of Small Extracellular Vesicles from Human Sera

Małys, Małgorzata S Sabina; Aigner, Christof; Schulz, Stefan M Martin; Schachner, Helga; Rees, Andrew J.; Kain, Renate

doi:10.3390/ijms22094653

Cited by 17 publications

(13 citation statements)

References 39 publications

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“…Little is known about the functions of CD62p+ exosomes in severe injury. These exosomes were shown to be released from endothelial cells, but also from platelets and megakaryocytes ( 31 ). Experimental studies in vivo and in vitro have shown that the release of particles from endothelial cells can be caused by chest trauma or as a result of endothelial cell incubation with a polytrauma cytokine cocktail ( 19 ).…”

Section: Discussionmentioning

confidence: 99%

Release of exosomes in polytraumatized patients: The injury pattern is reflected by the surface epitopes

Weber

Henrich²,

Schindler³

et al. 2023

Front. Immunol.

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BackgroundTrauma is still a leading cause of morbidity and mortality, especially in the younger population. Trauma patients need a precise, early diagnostic to avoid complications like multiorgan failure and sepsis. Exosomes were described as markers and mediators in trauma. The aim of the present study was to analyze, whether the surface epitopes of plasma-exosomes can reflect the injury pattern in polytrauma.Material and MethodsPolytraumatized patients (Injury Severity Score = ISS ≥16, n = 38) were subdivided according to the predominant injury in either abdominal trauma, chest trauma or traumatic brain injury (TBI). Plasma exosomes were isolated via size exclusion chromatography. The concentration and size distribution of the plasma exosomes from emergency room samples were measured by nanoparticle tracking analysis. The exosomal surface antigens were investigated by bead-based multiplex flow cytometry and compared with healthy controls (n=10).ResultsIn contrast to other studies, we did not observe an increase in the total amount of plasma exosomes in polytrauma patients (1,15x109 vs. 1,13x109 particles/ml), but found changes in the exosomal surface epitopes. We found a significant reduction of CD42a+ (platelet-derived) exosomes in polytrauma patients, CD209+ (dendritic cell-derived) exosomes in the patients with predominant abdominal trauma, and CD11+ (monocyte-derived) exosomes in the patients with chest trauma. The group of patients with TBI was characterized in contrast by an increase of CD62p+ (endothelial/platelet-derived) exosomes (*p<0.05).ConclusionOur data showed that the polytrauma injury pattern might be reflected by the cellular origin/surface epitopes of plasma-released exosomes immediately after trauma. The observed reduction of CD42+ exosomes in polytrauma patients was not associated with a reduction of total platelets in polytrauma patients.

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Section: Discussionmentioning

confidence: 99%

Release of exosomes in polytraumatized patients: The injury pattern is reflected by the surface epitopes

Weber

Henrich²,

Schindler³

et al. 2023

Front. Immunol.

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“…By contrast, the CD81 abundance was similar in all three sample types. The sEV purified by Exo-spin gave qualitatively similar results, albeit with lower abundance, reflecting the paucity of sEV obtained by this method (Figure 1b, d) (Małys et al, 2021). Indeed, CD9 and CD63 were only detectable in serum-derived sEV and CD81 was uniformly low (Figure 1d).…”

Section:  Differences Between Sev Preparations From Various Sample ...mentioning

confidence: 72%

“…Afterwards, the sEV pellet was collected into an Eppendorf tube, in which the volume was adjusted to a total of 200 μL by addition of cold PBS and down-stream analysis was performed immediately or within the next hours of 4 • C storage. This method was previously validated by us showing purity and quality of the sEV preparations according to MISEV standards (Małys et al, 2021;Théry et al, 2018).…”

Section:  Isolation Of Sev By Differential Ultracentrifugationmentioning

confidence: 99%

Small extracellular vesicles are released ex vivo from platelets into serum and from residual blood cells into stored plasma

Małys

Köller

Papp

et al. 2023

J of Extracellular Bio

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Small extracellular vesicles (sEV) purified from blood have great potential clinically as biomarkers for systemic disease; however interpretation is complicated by release of sEV ex vivo after blood taking. To quantify the problem and devise ways to minimise it, we characterised sEV in paired serum, plasma and platelet poor plasma (PPP) samples from healthy donors. Immunoblotting showed twofold greater abundance of CD9 in sEV fractions from fresh serum than from fresh plasma or PPP. MACSPlex confirmed this, and showed that proteins expressed on platelet sEV, either exclusively (CD41b, CD42a and CD62P) or more widely (HLA‐ABC, CD24, CD29 and CD31) were also twofold more abundant; by contrast non‐platelet proteins (including CD81) were no different. Storage of plasma (but not serum) increased abundance of platelet and selected leukocyte sEV proteins to at least that of serum, and this could be recapitulated by activating cells in fresh plasma by Ca2+, an effect abrogated in PPP. This suggests that a substantial proportion of sEV in serum and stored plasma were generated ex vivo, which is not the case for fresh plasma or PPP. Thus we provide strategies to minimise ex vivo sEV generation and criteria for identifying those that were present in vivo.

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“…The presence of TSG101, CD81, and CD9 on the MV surface was determined by Western blotting [ 44 – 46 ]. The protein concentration was measured using the BCA assay following the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Microvesicle-camouflaged biomimetic nanoparticles encapsulating a metal-organic framework for targeted rheumatoid arthritis therapy

et al. 2022

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Background Methotrexate (MTX) has been highlighted for Rheumatoid arthritis (RA) treatment, however, MTX does not accumulate well at inflamed sites, and long-term administration in high doses leads to severe side effects. In this study, a novel anti-RA nanoparticle complex was designed and constructed, which could improve the targeted accumulation in inflamed joints and reduce side effects. Results Here, we prepared a pH-sensitive biomimetic drug delivery system based on macrophage-derived microvesicle (MV)-coated zeolitic imidazolate framework-8 nanoparticles that encapsulated the drug methotrexate (hereafter MV/MTX@ZIF-8). The MV/MTX@ZIF-8 nanoparticles were further modified with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate (polyethylene glycol)-2000] (hereafter FPD/MV/MTX@ZIF-8) to exploit the high affinity of folate receptor β for folic acid on the surface of activated macrophages in RA. MTX@ZIF-8 nanoparticles showed high DLE (~ 70%) and EE (~ 82%). In vitro study showed that effective drug release in an acidic environment could be achieved. Further, we confirmed the activated macrophage could uptake much more FPD/MV/MTX@ZIF-8 than inactivated cells. In vivo biodistribution experiment displayed FPD/MV/MTX@ZIF-8 nanoparticles showed the longest circulation time and best joint targeting. Furthermore, pharmacodynamic experiments confirmed that FPD/MV/MTX@ZIF-8 showed sufficient therapeutic efficacy and safety to explore clinical applications. Conclusions This study provides a novel approach for the development of biocompatible drug-encapsulating nanomaterials based on MV-coated metal-organic frameworks for effective RA treatment. Graphical Abstract

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Isolation of Small Extracellular Vesicles from Human Sera

Cited by 17 publications

References 39 publications

Release of exosomes in polytraumatized patients: The injury pattern is reflected by the surface epitopes

Release of exosomes in polytraumatized patients: The injury pattern is reflected by the surface epitopes

Small extracellular vesicles are released ex vivo from platelets into serum and from residual blood cells into stored plasma

Microvesicle-camouflaged biomimetic nanoparticles encapsulating a metal-organic framework for targeted rheumatoid arthritis therapy

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