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Thrombin-generating procoagulant activity (TG-PCA) of cryopreserved platelets (CPPs) was 2-folds higher per PLT and 9-folds higher per mL as compared to liquid-stored platelets (LSPs). (a) Thrombin generation curve of LSPs and CPPs serially diluted in 2 folds. The first sample of LSPs, shown in solid line, was taken directly from the apheresis bag while CPPs, also shown in solid, were prediluted . (b) Thrombin peak height (TPH)/10 6 PLTs of LSP and CPP units. (c) TPH/1 mL of LSP and CPP units. Individual data points collected from n 012 donors are presented in scatter plots (mean); **p B0.01 and ***pB0.001.
Source publication
Background:
Freezing is promising for extended platelet (PLT) storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs) are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs). PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully el...
Citations
... Some authors have focused their research on low-temperature protective agents such as DMSO and trehalose. However, the protective effects vary among these agents [52][53][54]. In this study, our emphasis was on preserving EV miRNAs. ...
Extracellular vesicle (EV) miRNAs are promising biomarkers for clinical diagnosis. However, their stability is a crucial concern affecting reliability and accuracy. Factors such as sample collection, processing, storage conditions, and experimental procedures impact EV miRNA stability. Studying EV miRNA stability aims to find optimal handling and storage methods, ensuring integrity and functionality throughout research. In this study, we used RT-qPCR and GlyExo-Capture technology, which can specifically capture glycosylated EVs by lectin, to assess the stability of glycosylated EV miRNAs. We found that slow acceleration centrifugation and two-step centrifugation methods were suitable for subsequent experiments. To ensure uniformity, we recommend using the two-step centrifugation method. We also studied blood storage before serum separation and recommend separation within 2 h at 4 °C or 25 °C. For separated serum samples, higher temperatures accelerated miRNA degradation, and the storage duration should be adjusted based on laboratory conditions. Short-term storage at −20 °C is acceptable for up to 3 months while avoiding repeated freeze–thaw cycles. We developed protective agents to extend the storage time at 25 °C, meeting clinical requirements. Additionally, Lakebio’s cfRNA storage tubes effectively preserved the stability of miRNAs in plasma glycosylated EVs. Understanding EV miRNA stability provides insights into optimizing sample handling, storage strategies, and enhancing reliability in clinical applications.
... In CP with DMSO, GPIbα shedding and enhanced PS exposure may be associated with decreased adhesion and increased coagulation function respectively.101 PMP levels in CP are 10-15 folds higher than those at room temperature, which potentially promotes their coagulation.102 Recent RCTs also show that in patients with acute leukemia,103 thrombocytopenia 104 and high-risk cardiothoracic surgery, 105 CSP transfusions are safe and have hemostatic effects. ...
Background
Platelet concentrate (PC) transfusions are crucial in prevention and treatment of bleeding in infection, surgery, leukemia, and thrombocytopenia patients. Although the technology for platelet preparation and storage has evolved over the decades, there are still challenges in the demand for platelets in blood banks because the platelet shelf life is limited to 5 days due to bacterial contamination and platelet storage lesions (PSLs) at 20–24°C under constant horizontal agitation. In addition, the relations between some adverse effects of platelet transfusions and PSLs have also been considered. Therefore, understanding the mechanisms of PSLs is conducive to obtaining high quality platelets and facilitating safe and effective platelet transfusions.
Objective
This review summarizes developments in mechanistic research of PSLs and their relationship with clinical practice, providing insights for future research.
Methods
Authors conducted a search on PubMed and Web of Science using the professional terms “PSL” and “platelet transfusion.” The obtained literature was then roughly categorized based on their research content. Similar studies were grouped into the same sections, and further searches were conducted based on the keywords of each section.
Results
Different studies have explored PSLs from various perspectives, including changes in platelet morphology, surface molecules, biological response modifiers (BMRs), metabolism, and proteins and RNA, in an attempt to monitor PSLs and identify intervention targets that could alleviate PSLs. Moreover, novel platelet storage conditions, including platelet additive solutions (PAS) and reconsidered cold storage methods, are explored. There are two approaches to obtaining high‐quality platelets. One approach simulates the in vivo environment to maintain platelet activity, while the other keeps platelets at a low activity level in vitro under low temperatures.
Conclusion
Understanding PSLs helps us identify good intervention targets and assess the therapeutic effects of different PSLs stages for different patients.
... 21,22 For human neutrophilic granulocyte-derived EVs, the best storage temperature is −80°C to prevent significant changes in physical and functional properties compared to RT, +4 and −20°C, compared to the same period of time of 1 month. 23 To overcome damages associated with freezing, cryoprotectants can be added to EVs suspensions, being characterized by high water solubility and low toxicity, e.g., dimethyl sulfoxide 24 and trehalose. 14, 15,25 More recently, lyophilization has been proposed as an alternative method of cryopreservation for EVs storage. ...
Extracellular vesicles (EVs) have been studied for years for their role as effectors and mediators of cell-to-cell communication and their potential application to develop new and increasingly performing nanotechnological systems for the diagnosis and/or treatment of many diseases. Given all the EVs applications as just isolated, functionalized, or even engineered cellular-derived pharmaceuticals, the standardization of reliable and reproducible methods for their preservation is urgently needed. In this study, we isolated EVs from a healthy blood cell line, B lymphocytes, and compared the effectiveness of different storage methods and relative freeze-drying formulations to preserve some of the most important EVs' key features, i.e., concentration, mean size, protein content, and surface antigen's expression. To develop a preservation method that minimally affects the EVs' integrity and functionality, we applied the freeze-drying process in combination with different excipients. Since EVs are isolated not only from body fluids but also from culture media conditioned by the cells growing there, we decided to test both the effects of the traditional pharmaceutical excipient and of biological media to develop EVs solidified products with desirable appearance and performance properties. Results showed that some of the tested excipients, i.e., sugars in combination with dextran and glycine, successfully maintained the stability and integrity of EVs upon lyophilization. In addition, to evaluate the preservation of the EVs' biological activity, we assessed the cytotoxicity and internalization ability of the reconstituted EVs in healthy (B lymphocytes) and tumoral (Burkitt's lymphoma) cells. Reconstituted EVs demonstrated toxicity only toward the cancerous cells, opening new therapeutic opportunities for the oncological field. Furthermore, our study showed how some biological or cellular-conditioned fluids, commonly used in the field of cell cultures, can act not only as cryoprotectants but also as active pharmaceutical ingredients, significantly tuning the therapeutic effect of EVs, even increasing their cellular internalization.
... The storage of EVs is one such technology that critically affects their integrity, cargo molecule, and functions, influencing the subsequent therapeutic efficacy for clinical applications [27,28]. In this study, we evaluated the effect of short-term storage using buffers containing BSA [19] and trehalose [20] based on previous studies, which complemented the results of the PBS-HAT buffer reported by Görgens et al., who used HSA instead of BSA. Our study demonstrated that EV storage in PBS severely affects the cell culture media-derived bioactive EV integrity, cargo protection, and targeting functions following short-term storage at −80 • C. In addition, the formation of larger particles by particle fusion or aggregation started immediately after resuspension in PBS. ...
Extracellular vesicles (EVs), detectable in all bodily fluids, mediate intercellular communication by transporting molecules between cells. The capacity of EVs to transport molecules between distant organs has drawn interest for clinical applications in diagnostics and therapeutics. Although EVs hold potential for nucleic-acid-based and other molecular therapeutics, the lack of standardized technologies, including isolation, characterization, and storage, leaves many challenges for clinical applications, potentially resulting in misinterpretation of crucial findings. Previously, several groups demonstrated the problems of commonly used storage methods that distort EV integrity. This work aims to evaluate the process to optimize the storage conditions of EVs and then characterize them according to the experimental conditions and the models used previously. Our study reports a highly efficient EV storage condition, focusing on EV capacity to protect their molecular cargo from biological, chemical, and mechanical damage. Compared with commonly used EV storage conditions, our EV storage buffer leads to less size and particle number variation at both 4 °C and −80 °C, enhancing the ability to protect EVs while maintaining targeting functionality.
... When the cell is activated, the increase in cytosolic calcium also activates the enzyme floppase, which allows the movement of plasma membrane lipids to the outer membrane. In this way, phosphatidylserine is exposed on the outer leaflet of the phospholipid bilayer and can be easily detected as it binds to annexin V [24]. In a recent study, Peterson et al. described microvesicles containing multiple membrane compartments (referred to as multi-compartment microvesicles or MCMVs) that originate from MVB-containing cell protrusions at specialized sites on the surface of endothelial cells. ...
Extracellular vesicles (EVs) are a heterogeneous class of cell-derived membrane vesicles released by various cell types that serve as mediators of intercellular signaling. When released into circulation, EVs may convey their cargo and serve as intermediaries for intracellular communication, reaching nearby cells and possibly also distant organs. In cardiovascular biology, EVs released by activated or apoptotic endothelial cells (EC-EVs) disseminate biological information at short and long distances, contributing to the development and progression of cardiovascular disease and related disorders. The significance of EC-EVs as mediators of cell-cell communication has advanced, but a thorough knowledge of the role that intercommunication plays in healthy and vascular disease is still lacking. Most data on EVs derive from in vitro studies, but there are still little reliable data available on biodistribution and specific homing EVs in vivo tissues. Molecular imaging techniques for EVs are crucial to monitoring in vivo biodistribution and the homing of EVs and their communication networks both in basal and pathological circumstances. This narrative review provides an overview of EC-EVs, trying to highlight their role as messengers of cell-cell interaction in vascular homeostasis and disease, and describes emerging applications of various imaging modalities for EVs visualization in vivo.
... This increase in size could mean a possible platelet aggregation or morphological change, which has been previously observed by electron microscopy. 17 In fact, there are previous studies where it is reported that, after freezing, platelets suffer alterations in terms of morphology and biochemistry. 18 Increased platelet volume has also been previously associated with swelling and, thus, with a possible initiation of platelet activation. ...
Plain Language Summary
What is the context? Platelet-Rich Plasma (PRP) is a biological treatment widely used in regenerative medicine as a result of its high content of growth factors.
In its routine use, PRP has autologous character, since it is obtained from the same patient and its infiltration in the affected area takes place immediately after it is obtained.
Its storability would give PRP versatility in use, which could enable a potential allogeneic use and even significantly reduce the number of blood draws for each patient.
It is necessary to establish a PRP storage protocol where its properties are not affected.
What is new? PRP was stored at two time points (1 and 3 months) and temperatures (−20ºC and −80ºC) in activated and unactivated states. Afterwards, platelet number, size and activation were measured. Furthermore, the biomechanical properties of the resulting clot, the growth factor content and PRP’s impact on cell viability were analyzed.
The limiting factor was not having used aggregometry or other techniques that measure other cellular processes, as well as the limited sample size.
The results showed that freezing affected platelet size, the levels of platelet-derived GFs and the biomechanical properties of the clot. However, plasmatic levels of growth factors or its capacity to boost cellular proliferation were not affected.
What is the impact?The clinical impact of this work is the ability to preserve PRP by freezing. This is especially relevant as it allows a possible use of PRP as an allogeneic treatment. Moreover, its preservation significantly reduces the number of blood draws for each patient, especially in those with puncture difficulties or with apprehension.
... structure. SEM primarily allows observation of the morphology and has a stereoscopic sense; however, it is limited to the surface structure of exosomes (Pascucci et al., 2014;Tegegn et al., 2016). The advantage of AFM is that the samples are intact, and the requirements for sample analysis can be readily met compared with those of SEM and TEM, as exosomes can be directly analyzed in atmospheric and liquid environments. ...
Ischemic stroke is one of the major contributors to death and disability worldwide. Thus, there is an urgent need to develop early brain tissue perfusion therapies following acute stroke and to enhance functional recovery in stroke survivors. The morbidity, therapy, and recovery processes are highly orchestrated interactions involving the brain with other tissues. Exosomes are natural and ideal mediators of intercellular information transfer and recognized as biomarkers for disease diagnosis and prognosis. Changes in exosome contents express throughout the physiological process. Accumulating evidence demonstrates the use of exosomes in exploring unknown cellular and molecular mechanisms of intercellular communication and organ homeostasis and indicates their potential role in ischemic stroke. Inspired by the unique properties of exosomes, this review focuses on the communication, diagnosis, and therapeutic role of various derived exosomes, and their development and challenges for the treatment of cerebral ischemic stroke.
... It has been shown previously that the 2000 G supernatant of DTC contain mostly platelet fragments and microparticles. 44 Although the supernatant platelets do not contribute to clot strength, they do contribute to the platelet count, which may explain the low MA values we observe in our diluted DTC samples. PPL activity was very high and PPL clotting time was F I G U R E 4 Effect of À80°C storage duration and fresh or cold-stored thawed plasma on TEG and PPL. ...
Background:
The Netherlands Armed Forces (NLAF) are using -80°C deep-frozen thrombocyte concentrate (DTC) since 2001. The aim of this study is to investigate the effect of storage duration and alterations in production/measurement techniques on DTC quality. It is expected that DTC quality is unaffected by storage duration and in compliance with the European guidelines for fresh and cryopreserved platelets.
Study design and methods:
Pre-freeze and post-thaw product platelet content and recovery were collected to analyze the effects of dimethyl sulfoxide (DMSO) type, duration of frozen storage (DMSO-1 max 12 years and DMSO-2 frozen DTC max 4 years at -80°C) and type of plasma used to suspend DTC. Coagulation characteristics of thawed DTC, plasma and supernatant of DTC (2× 2500 G) were measured with Kaolin thromboelastography (TEG) and phospholipid (PPL) activity assay.
Results:
Platelet content and recovery of DTC is ±10%-15% lower in short-stored products and remained stable when stored beyond 0.5 years. Thawed DTC (n = 1724) were compliant to the European guidelines (98.1% post-thaw product recovery ≥50% from original product, 98.3% ≥200 × 109 platelets/unit). Compared to DMSO-1, products frozen with DMSO-2 showed ±8% reduced thaw-freeze recovery, a higher TEG clot strength (MA 58 [6] vs. 64 [8] mm) and same ±11 s PPL clotting time. The use of cold-stored thawed plasma instead of fresh thawed plasma did not influence product recovery or TEG-MA.
Discussion:
Regardless of alterations, product quality was in compliance with European guidelines and unaffected by storage duration up to 12 years of -80°C frozen storage.
... However, prior studies have shown that CPP transfusion results in minimal platelet increments following transfusion. Cryopreservation and thawing results in activation and fragmentation of platelets leading to an increased number of microparticles, expression of activation markers such as P-selectin and externalization of phosphatidylserine 3,4 . While RT-stored platelets increase clot firmness, CPP have minimal impact, but do increase thrombin potential and reduce the time to initiation of clot 5 . ...
Background:
Platelet concentrates have a limited shelf life due to room temperature storage and therefore, are not kept in regional centres where turnover is low. Cryopreserved platelets have been proposed as an alternative to platelet transfusion in austere circumstances and fibrinogen concentrate has improved thromboelastometry parameters in thrombocytopenia. This study compared the ability of stored haemostatic products and platelets to correct thromboelastometry parameters in thrombocytopenia.
Materials and methods:
Blood from eight patients with severe thrombocytopenia was combined with platelet concentrates, cryoprecipitate, fibrinogen concentrate, factor VIII, factor XIII and cryopreserved platelets in ratios equivalent to transfusion. Tissue factor initiated thromboelastometry (EXTEM) was compared between the products.
Results:
EXTEM amplitude at 20 minutes (A20) improved by 13.1 mm with platelets (p<0.01). The 5mm increase in A20 seen with cryoprecipitate (p=0.06) was not statistically different from platelets (p=0.19). No improvement in A20 was observed with cryopreserved platelets or factor concentrates. EXTEM clotting times (CT) improved with cryopreserved platelets (19.4 s, p=0.001) and cryoprecipitate (24.1 s, p<0.05), but not fibrinogen, and both were superior to platelets (9.9 s, p<0.05). Clotting concentrates did not improve EXTEM parameters although further studies suggested the improvement in A20 was largely driven by higher fibrinogen concentrations in cryoprecipitate.
Discussion:
These results suggest that cryopreserved platelets enhance clot initiation but do not contribute to clot strength in thrombocytopenia. When platelets are not available for transfusion, cryoprecipitate may be of value, however this requires further clinical studies.
... The recovery rate of EVs could be improved in PBS supplemented with human albumin and trehalose at -80 °C [194]. Another practical method includes dimethyl sulfoxide (DMSO) cryopreservation [195]. To sum up, flexible adjustments of additives may generate surprisingly positive outcomes. ...
Extracellular vesicles (EVs), of which exosomes are a representative subgroup, are naturally secreted nanoparticles with a variety of payloads. With the intrinsic merits of stability, biocompatibility, low immunogenicity, and large capacity, EVs are widely regarded as effective carriers of drug delivery. However, disadvantages, such as low yield, complicated isolation procedures, and low loading efficiency, hinder its clinical translation. In this review, we systematically summarize the advances in EV (especially exosomes) engineering for clinical application, focusing on strategies toward high yield, facile isolation, efficient cargo loading, improved delivery, and optimized manufacturing, which might unleash the infinite power of EVs in clinical translation.
