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Background: Detailed characterization of extracellular vesicles (EVs) is crucial for their application in medical diagnostics. However, the complexity of their chemical composition and the heterogeneity of EV populations make their characterization challenging. Here we describe two analytical procedures that can help overcome this challenge., Methods: Small EVs were isolated from conditioned cell culture media using ultracentrifugation and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Raman spectroscopy was used to assess the overall composition of the isolated samples and lipids extracted from them. Sulfophosphovanillin (SPV) colorimetric assay was used to quantify the contents of lipid., Results: Six samples of EVs were characterized. The lipid contents measured using SPV assay was in reasonable agreement with the quantitative estimates based on the particle size and concentration measured using NTA. The most peaks observed in the Raman spectra could be attributed to either proteins or lipids, and their origins was confirmed by lipid extraction. The protein-to-lipid ratio was estimated based on the Raman spectra., Conclusions: The experiential procedures described in this study will help to overcome the challenge of quick and highly informative characterization of the EVs., (© 2024 The Author(s). Published by IMR Press.)

Background: Wound healing represents a complex biological process, critically important in clinical practice due to its direct implication in a patient's recovery and quality of life. Conservative wound management frequently falls short in providing an ideal environment for the optimal tissue regeneration, often resulting in extended healing periods and elevated risk of infection and other complications. The emerging biomaterials, particularly hydrogels, have shown substantial promise in addressing these challenges by offering properties such as biocompatibility, biodegradability, and the ability to cure wound environment. Recent advancements have highlighted the therapeutic potential of integrating cell-derived conditioned medium (CM) into hydrogel matrices. Cell-derived CM represents a rich array of bioactive molecules, demonstrating significant efficacy in modulating cellular activities crucial for wound healing, including cellular proliferation, migration, and angiogenesis., Methods: The methodology of this review adheres to the standards set by the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines. The review includes a selection of studies published within the last five years, focusing on in vivo experiments involving various types of skin injuries treated with topically applied hydrogels loaded with CM (H-CM). The search strategy refers to the PICO framework and includes the assessment of study quality by CAMARADES tool., Results: The systematic review represents a detailed evaluation of H-CM dressings wound healing efficiency based on the experimental results of cell-based assays and animal wound models. The study targets to reveal wound healing capacity of H-CM dressings, and provides a comparative data analysis, limitations of methods and discussions of H-CM role in advancing the wound healing therapy., Conclusions: The data presented demonstrate that H-CM is a promising material for advanced wound healing and regenerative medicine. These dressings possess proved in vitro/in vivo efficacy that highlights their strong clinical potential and paves the way to further investigations of H-CM formulations within clinical trials., (© 2024. The Author(s).)

Small extracellular vesicles (EVs) play a pivotal role in intercellular communication across various physiological and pathological contexts. Despite their growing significance as disease biomarkers and therapeutic targets in biomedical research, the lack of reliable isolation techniques remains challenging. This study characterizes vesicles that were isolated from conditioned culture media (CCM) sourced from three myeloma cell lines (MM.1S, ANBL-6, and ALMC-1), and from the plasma of healthy donors and multiple myeloma patients. We compared the efficacy, reproducibility, and specificity of isolating small EVs using sucrose cushion ultracentrifugation (sUC) vs. ultrafiltration combined with size-exclusion chromatography (UF-SEC). Our results demonstrate that UF-SEC emerges as a more practical, efficient, and consistent method for EV isolation, outperforming sUC in the yield of EV recovery and exhibiting lower variability. Additionally, the comparison of EV characteristics among the three myeloma cell lines revealed distinct biomarker profiles. Finally, our results suggest that HBS associated with Tween 20 improves EV recovery and preservation over PBS. Standardization of small EV isolation methods is imperative, and our comparative evaluation represents a significant step toward achieving this goal.

The stimulation of mesenchymal stromal cells (MSCs) with inflammatory molecules is often used to boost their therapeutic effect. Prolonged exposure to inflammatory molecules has been explored to improve their action because MSCs therapies seem to be improved transiently with such stimuli. However, the possibility of cyclically stimulating MSCs to recover their optimized therapeutic potential is still to be elucidated, although the efficacy of cell-based therapies may be dependent on the ability to readapt to the relapse pathological conditions. Here, the response of MSCs, encapsulated in alginate hydrogels and cultured for 22 d, is explored using three different regimes: single, continuous, and intermittent stimulation with IFNγ. Exposure to IFNγ leads to a decrease in the secretion of IL-10, which is cyclically countered by IFNγ weaning. Conditioned media collected at different stages of pulsatile stimulation show an immunomodulatory potential toward macrophages, which directly correlates with IL-10 concentration in media. To understand whether the correlation between cyclic stimulation of MSCs and other biological actions can be observed, the effect on endothelial cells is studied, showcasing an overall modest influence on tube formation. Overall, the results describe the response of encapsulated MSCs to unusual pulsatile simulation regimens, exploring encapsulated MSCs as a living on-demand release system of tailored secretomes with recoverable immunomodulatory action., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

In this paper, we propose a model that connects two standard inflammatory responses to viral infection, namely, elevation of fibrinogen and the lipid drop shower, to the initiation of non-thrombin-generated clot formation. In order to understand the molecular basis for the formation of non-thrombin-generated clots following viral infection, human epithelial and Madin-Darby Canine Kidney (MDCK, epithelial) cells were infected with H1N1, OC43, and adenovirus, and conditioned media was collected, which was later used to treat human umbilical vein endothelial cells and human lung microvascular endothelial cells. After direct infection or after exposure to conditioned media from infected cells, tissue surfaces of both epithelial and endothelial cells, exposed to 8 mg/mL fibrinogen, were observed to initiate fibrillogenesis in the absence of thrombin. No fibers were observed after direct viral exposure of the endothelium or when the epithelium cells were exposed to SARS-CoV-2 isolated spike proteins. Heating the conditioned media to 60 °C had no effect on fibrillogenesis, indicating that the effect was not enzymatic but rather associated with relatively thermally stable inflammatory factors released soon after viral infection. Spontaneous fibrillogenesis had previously been reported and interpreted as being due to the release of the alpha C domains due to strong interactions of the interior of the fibrinogen molecule in contact with hydrophobic material surfaces rather than cleavage of the fibrinopeptides. Contact angle goniometry and immunohistochemistry were used to demonstrate that the lipids produced within the epithelium and released in the conditioned media, probably after the death of infected epithelial cells, formed a hydrophobic residue responsible for fibrillogenesis. Hence, the standard inflammatory response constitutes the ideal conditions for surface-initiated clot formation.

Small extracellular vesicles (sEVs), a form of extracellular vesicles, are lipid bilayered structures released by all cells. Large-scale studies on sEVs from clinical samples are necessary, but a major obstacle is the lack of rapid, reproducible, efficient, and low-cost methods to enrich sEVs. Acoustic microfluidics have the advantage of being label-free and biocompatible, which have been reported to successfully enrich sEVs. In this paper, we present a highly efficient acoustic microfluidic trap that can offer low and large volume compatible ways of enriching sEVs from biological fluids by flexible structure design. It uses the idea of pre-loading larger seed particles in the acoustic trap to enable sub-micron particle capturing. The microfluidic chip is actuated using a piezoelectric plate transducer attached to a silicon-glass bonding plate with circular cavities. Each cavity works as a resonant unit, excited at the frequency of both the half wave resonance in the main plane and inverted quarter wave resonance in the depth direction, which has the ability to strongly trap seed particles at the center, thereby improving the subsequent nanoparticle capture efficiency. Mean trapping efficiencies of 35.62% and 64.27% were obtained using 60 nm and 100 nm nanobeads, respectively. By the use of this technology, we have successfully enriched sEVs from cell culture conditioned media and blood plasma at a flow rate of 10 μL min -1 . The isolated sEV subpopulations are characterized by NTA and TEM, and their protein cargo is determined by WB. This acoustic trapping chip provides a rapid and robust method to enrich sEVs from biofluids with high reproducibility and sufficient quantities. Therefore, it can serve as a new tool for biological and clinical research such as cancer diagnosis and drug delivery.

The widespread use of multipotent mesenchymal stromal cell-derived secretome (MSC-sec) requires optimal preservation methods. Lyophilization offers benefits like concentrating the secretome, reducing the storage volume, and making storage conditions more flexible. This study evaluated the influence of storage duration and temperature on lyophilized MSC-sec. The conditioned medium from Wharton's jelly MSCs was stored at - 80 °C or lyophilized with or without trehalose. Lyophilized formulations were kept at - 80 °C, - 20 °C, 4 °C, or room temperature (RT) for 3 and 30 months. After storage and reconstitution, the levels of growth factors and cytokines were assessed using multiplex assay. The storage of lyophilized MSC-sec at - 80 °C ensured biomolecule preservation for 3 and 30 months. Following 3 month storage at 4 °C and RT, a notable decrease occurred in BDNF, bNGF, and sVCAM-1 levels. Prolonged 30 month storage at the same temperatures significantly reduced BDNF, bNGF, VEGF-A, IL-6, and sVCAM-1, while storage at - 20 °C decreased BDNF, bNGF, and VEGF- A levels. Trehalose supplementation of MSC-sec improved the outcome during storage at 4 °C and RT. Proper storage conditions were crucial for the preservation of lyophilized MSC-sec composition. Short-term storage at various temperatures maintained over 60% of the studied growth factors and cytokines; long-term preservation was only adequate at -80 °C., (© 2024. The Author(s).)