Summary
Splenectomised β‐thalassaemia/haemoglobin E (HbE) patients have increased levels of circulating microparticles or medium extra‐cellular vesicles (mEVs). The splenectomised mEVs play important ...roles in thromboembolic complications in patients since they can induce platelet activation and endothelial cell dysfunction. However, a comprehensive understanding of the mechanism of mEV generation in thalassaemia disease has still not been reached. Thalassaemic mEVs are hypothesised to be generated from cellular oxidative stress in red blood cells (RBCs) and platelets. Therefore, a proteomic analysis of mEVs from splenectomised and non‐splenectomised β‐thalassaemia/HbE patients was performed by liquid chromatography with tandem mass spectrometry. A total of 171 proteins were identified among mEVs. Interestingly, 72 proteins were uniquely found in splenectomised mEVs including immunoglobulin subunits and cytoskeleton proteins. Immunoglobulin G (IgG)‐bearing mEVs in splenectomised patients were significantly increased. Furthermore, complement C1q was detected in both mEVs with IgG binding and mEVs without IgG binding. Interestingly, the percentage of mEVs generated from RBCs with IgG binding was approximately 15–20 times higher than the percentage of RBCs binding with IgG. This suggested that the vesiculation of thalassaemia mEVs could be a mechanism of RBCs to eliminate membrane patches harbouring immune complex and may consequently prevent cells from phagocytosis and lysis.
Medium extra‐cellular vesicles (mEVs) from splenectomised β‐thalassaemia/haemoglobin E (HbE) patients play important roles in thromboembolic complications. A proteomic analysis identified 72 proteins unique to splenectomised mEVs, including immunoglobulin subunits and cytoskeleton proteins. Immunoglobulin G (IgG)‐ and C1q‐bearing mEVs were significantly increased in splenectomised patients. The IgG‐binding red blood cell (RBC)‐mEVs were markedly higher than the IgG‐binding RBCs. This suggests that the vesiculation of thalassaemia mEVs could be a mechanism to eliminate immune complex‐harbouring membrane patches and potentially prevent RBC phagocytosis and lysis.
Exosomes are nanovesicles secreted by most cellular types that carry important biochemical compounds throughout the body with different purposes, playing a preponderant role in cellular ...communication. Because of their structure, physicochemical properties and stability, recent studies are focusing in their use as nanocarriers for different therapeutic compounds for the treatment of different diseases ranging from cancer to Parkinson's disease. However, current bioseparation protocols and methodologies are selected based on the final exosome application or intended use and present both advantages and disadvantages when compared among them. In this context, this review aims to present the most important technologies available for exosome isolation while discussing their advantages and disadvantages and the possibilities of being combined with other strategies. This is critical since the development of novel exosome‐based therapeutic strategies will be constrained to the effectiveness and yield of the selected downstream purification methodologies for which a thorough understanding of the available technological resources is needed.
Exosomes are a subpopulation of cell membrane-derived vesicles which play an essential role in cellular communication. In recent years, several studies have exploited the natural properties of ...exosomes as nanocarriers for several applications such as immunotherapy or drug delivery. Consequently, numerous techniques have been developed to improve their immunogenicity, drug loading efficiency, or targeting. Nonetheless, to date, there is no consensus on which technique results in more advantages for this purpose. In this context, this review discusses the currently used methodologies regarding traditional and engineered exosome loading and targeting techniques. Here, we focus on the advantages and disadvantages of each method while discussing some results obtained in relevant reports. Although there is a lack of evidence regarding the effects of exogenous exosomes in humans and several limitations in exosome isolation and purification techniques at the large-scale exist, the formulation of new exosome-based therapeutics is in the spotlight. Therefore, the development of more efficient functionalization techniques is required to reduce the potential risks associated with the clinical use of these vesicles.
Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on ...these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4-10), osmolarity (50-1000 mOsm/L), temperature (15-60 °C), and surfactant Triton X-100 (10-500 μM)). Their stability was evaluated by LS by considering the hydrodynamic radius (
), intensity of scattered light (
), and the shape parameter (
). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS-citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50-1000 mOsm/L, pH 4-10) had no significant effect on the
(=100-130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in
of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.
Small extracellular vesicles (EVs) in the last 20 years are demonstrated to possess promising properties as potential new drug delivery systems, biomarkers, and therapeutic targets. Moreover, EVs are ...described to be involved in the most important steps of tumor development and progression including drug resistance. The acquired or intrinsic capacity of cancer cells to resist chemotherapies is one of the greatest obstacles to overcome to improve the prognosis of many patients. EVs are involved in this mechanism by exporting the drugs outside the cells and transferring the drug efflux pumps and miRNAs in recipient cells, in turn inducing drug resistance. In this mini-review, the main mechanisms by which EVs are involved in drug resistance are described, giving a rapid and clear overview of the field to the readers.
Unlike humans and many other mammalian species, conventional in vitro fertilization (IVF) in equine species is not successful. To mimic in vitro equine spermatozoon-oviduct interaction as close as ...possible to that which occurs in vivo, extracellular vesicles (EVs) secreted by the female genital tract were used.
Three female genital tracts were collected at slaughterhouse from mares in late estrus. Ipsilateral proximal and apical horn endometrial explants were digested with collagenase and trypsin and cells obtained were cultured on insert system to allow their polarization. Ipsilateral oviducts were squeezed out to obtain spheroids. To produce EVs, proximal and apical horn endometrial cells and oviductal spheroids were cultured for three days in serum free medium. To trace interaction between spermatozoa and EVs by fluorescence microscopy, EVs were differently labeled. Pooled samples of ejaculated spermatozoa from three stallions were incubated in capacitating medium (CM) for 6 h and to induce hyperactivation for other 6 h in CM supplemented with different kind of EVs alone or in combination. A control was performed in absence of EVs.
Sperm were assessed for motility by CASA system, EV incorporation by confocal microscopy and acrosomal reaction (AR) by staining with FITC-PNA/PI. In vitro fertilization was performed, and presumed zygotes were subjected to chromatin configuration. The results show that incorporation of EVs of the proximal horn does not take place, while apical horn EVs are incorporated in the head of the spermatozoon in 4 h. The EVs of oviductal spheroids are incorporated in the middle tract in 1 h. The rate of AR with EVs of the apical horn and oviductal spheroids were respectively 50.25% and 57.14%. When these EVs were added in combination, the rate of AR was 71.42%. In the control, the rate of AR was of 15%. After in vitro fertilization, 44% of oocytes showed male and female pronuclei, whereas no fertilization is obtained in the control. In conclusion, EVs from apical horn and oviduct could be involved in cell trafficking during equine semen hyperactivation, and their possible use in vitro could facilitate the development of equine reproductive biotechnologies.
•Oviduct secretions have significant effect on capacitation in stallion sperm.•Extracellular vesicles (EVs) were obtained by endometrial cells and oviductal spheroids.•Spermatozoa incorporate EVs from endometrial cells in 4 h in the head.•Spermatozoa incorporate EVs from oviductal spheroids in 1 h in head and middle tract.•After EV incorporation, spermatozoa are able to capacitate and produce zygotes.
Mesenchymal stem cells (MSCs) are the most frequently used stem cells in clinical trials due to their easy isolation from various adult tissues, their ability of homing to injury sites and their ...potential to differentiate into multiple cell types. However, the realization that the beneficial effect of MSCs relies mainly on their paracrine action, rather than on their engraftment in the recipient tissue and subsequent differentiation, has opened the way to cell-free therapeutic strategies in regenerative medicine. All the soluble factors and vesicles secreted by MSCs are commonly known as secretome. MSCs secretome has a key role in cell-to-cell communication and has been proven to be an active mediator of immune-modulation and regeneration both
and
. Moreover, the use of secretome has key advantages over cell-based therapies, such as a lower immunogenicity and easy production, handling and storage. Importantly, MSCs can be modulated to alter their secretome composition to better suit specific therapeutic goals, thus, opening a large number of possibilities. Altogether these advantages now place MSCs secretome at the center of an important number of investigations in different clinical contexts, enabling rapid scientific progress in this field.
The multitude of clinical trials using mesenchymal stromal cells (MSCs) has underscored their significance as a promising cell source for regenerative therapies. Most studies have however shown that ...MSCs get entrapped into the microvasculature of lungs, liver and spleen. In addition to intercellular communication, MSCs exert their effects in a paracrine manner by secretion of extracellular vesicles (EVs). The therapeutic effects of MSC-derived EVs have been examined in several diseases such as hepatic failure, liver injury, hematopoiesis etc. Therefore, optimization of cryopreservation strategies for the long-term storage of functional EVs could help in the development of off-the-shelf biologics. The aim of this study was to develop an optimal cryopreservation strategy for the efficient storage of both types of EVs – Microvesicles (MVs) and exosomes, independently, and to further examine the effect of the cryopreserved EVs on the ex vivo expansion of HSCs. MVs and exosomes were separately cryopreserved at different temperatures using PBS or PBS supplemented with trehalose (pTRE), and these cryopreserved EVs were then assessed for their functionality after revival. We found that addition of trehalose during cryopreservation helped in maintaining the morphology and functionality of the EVs, as assessed by their HSC-supportive potential, ability to expand phenotypically defined HSCs and ability to maintain the chemotactic migration potential of the HSCs co-cultured with them. This strategy could prove to be beneficial for facilitating the use of EVs as cell-free ready-to-use biologics for the ex vivo expansion of HSCs and in regenerative medicine.
In order to prepare optimal platelet and extracellular vesicle (EV)-rich plasma for the treatment of chronic temporal bone inflammation, we studied effects of centrifugation parameters on ...redistribution of blood constituents in blood samples of 23 patients and 20 volunteers with no record of disease. Concentrations of blood cells and EVs were measured by flow cytometry. Sample content was inspected by scanning electron microscopy. A mathematical model was constructed to interpret the experimental results. The observed enrichment of plasma in platelets and EVs after a single spin of blood depended on the erythrocyte sedimentation rate, thereby indicating the presence of a flow of plasma that carried platelets and EVs in the direction opposite to settling of erythrocytes. Prolonged handling time correlated with the decrease of concentration of platelets and larger EVs in platelet and EV-rich plasma (PVRP), R = −0.538, p = 0.003, indicating cell fragmentation during the processing of samples. In further centrifugation of the obtained plasma, platelet and EV enrichment depended on the average distance of the sample from the centrifuge rotor axis. Based on the agreement of the model predictions with observations, we propose the centrifugation protocol optimal for platelet and EV enrichment and recovery in an individual sample, adjusted to the dimensions of the centrifuge rotor, volume of blood and erythrocyte sedimentation rate.
PDF
