Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures.

Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs - being small and non-living - are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.

Identification of storage conditions stabilizing extracellular vesicles preparations.

Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS-HAT). We report that PBS-HAT buffer facilitates clearly improved short-term and long-term EV preservation for samples stored at -80°C, stability throughout several freeze-thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications.

Quantification of extracellular vesicles in vitro and in vivo using sensitive bioluminescence imaging.

Extracellular vesicles (EVs) are naturally occurring nano-sized carriers that are secreted by cells and facilitate cell-to-cell communication by their unique ability to transfer biologically active cargo. Despite the pronounced increase in our understanding of EVs over the last decade, from disease pathophysiology to therapeutic drug delivery, improved molecular tools to track their therapeutic delivery are still needed. Unfortunately, the present catalogue of tools utilised for EV labelling lacks sensitivity or are not sufficiently specific. Here, we have explored the bioluminescent labelling of EVs using different luciferase enzymes tethered to CD63 to achieve a highly sensitive system for in vitro and in vivo tracking of EVs. Using tetraspanin fusions to either NanoLuc or ThermoLuc permits performing highly sensitive in vivo quantification of EVs or real-time imaging, respectively, at low cost and in a semi-high throughput manner. We find that the in vivo distribution pattern of EVs is determined by the route of injection, but that different EV subpopulations display differences in biodistribution patterns. By applying this technology for real-time non-invasive in vivo imaging of EVs, we show that their distribution to different internal organs occurs just minutes after administration.

Tangential Flow Filtration with or Without Subsequent Bind-Elute Size Exclusion Chromatography for Purification of Extracellular Vesicles.

Extracellular vesicles (EVs) have gained increased attention over the last decade due to their potential as biomarkers and therapeutic entities. However, the characterization and development of EV research has been hampered by the lack of sufficiently effective purification methods. Several concerns have been raised toward the gold standard purification method ultracentrifugation, such as operator-dependent yields, crushing and aggregation of vesicles, poor scalability, and relative lack of purity. Here, we describe, in details, the use of an alternative purification technique: tangential flow filtration with or without subsequent bind-elute size exclusion chromatography that we have previously shown to be reproducible and scalable for purification of EVs.

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures.

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

Assessment of in vitro cytotoxic and genotoxic activities of some trimethoprim conjugates.

Trimethoprim, a commonly used antibacterial agent, is widely applied in the treatment of variety of infections in human. A few studies have demonstrated an extensive exposure of man to antibiotics, but there is still a lack of data for cytotoxic effects including nephrotoxicity, gastrointestinal toxicity, hematotoxicity, neurotoxicity and ototoxicity. The main purpose behind this study was to determine cytotoxic and genotoxic activities of trimethoprim (1), trimethoprim with maleic acid (2) and trimethoprim in conjugation with oxalic acid dihydrate (3). The cytotoxic effects of these three conjugates were elucidated by employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoium bromide (MTT) assay using embryonic rat fibroblast-like cell line (F2408) and H-ras oncogene activated embryonic rat fibroblast-like cancer cell line (5RP7). Additionally, determination of genotoxic activity of these three compounds were studied by using cytokinesis blocked micronucleus assay (CBMN) in human lymphocytes. The results demonstrated that trimethoprim alone and its combination with other compounds are able to induce both cytotoxic and genotoxic damage on cultured cells (F2408, 5RP7, human lymphocytes).

Adhesion profile and differentiation capacity of human adipose tissue derived mesenchymal stem cells grown on metal ion (Zn, Ag and Cu) doped hydroxyapatite nano-coated surfaces.

Accelerated Mesenchymal Stem Cells (MSCs) condensation and robust MSC-matrix and MSC-MSC interactions on nano-surfaces may provide critical factors contributing to such events, likely through the orchestrated signal cascades and cellular events modulated by the extracellular matrix. In this study, human adipose tissue derived mesenchymal stem cells (hMSC)', were grown on metal ion (Zn, Ag and Cu) doped hydroxyapatite (HAP) nano-coated surfaces. These metal ions are known to have different chemical and surface properties; therefore we investigated their respective contributions to cell viability, cellular behavior, osteogenic differentiation capacity and substrate-cell interaction. Nano-powders were produced using a wet chemical process. Air spray deposition was used to accumulate the metal ion doped HAP films on a glass substrate. Cell viability was determined by MTT, LDH and DNA quantitation methods Osteogenic differentiation capacity of hMSCs was analyzed with Alizarin Red Staining and Alkaline Phosphatase Specific Activity. Adhesion of the hMSCs and the effect of cell adhesion on biomaterial biocompatibility were explored through cell adhesion assay, immunofluorescence staining for vinculin and f-actin cytoskeleton components, SEM and microarray including 84 known extracellular matrix proteins and cell adhesion pathway genes, since, adhesion is the first step for good biocompability. The results demonstrate that the viability and osteogenic differentiation of the hMSCs (in growth media without osteogenic stimulation) and cell adhesion capability are higher on nanocoated surfaces that include Zn, Ag and/or Cu metal ions than commercial HAP. These results reveal that Zn, Ag and Cu metal ions contribute to the biocompatibility of exogenous material.

Analyses of the modulatory effects of antibacterial silver doped calcium phosphate-based ceramic nano-powder on proliferation, survival, and angiogenic capacity of different mammalian cells in vitro.

In this study, the antibacterial, cytotoxic, and angiogenic activities of silver doped calcium phosphate-based inorganic powder (ABT or PAG) were systematically investigated. ABT powders containing varying silver content were fabricated using a wet chemical manufacturing method. Antibacterial efficiencies of the ABT powders were investigated using a standard test with indicator bacteria and yeast. The cytotoxic effects of ABT on three different fibroblast cells and human umbilical vein endothelial cells (HUVECs) were assessed using MTT assay. ABT powder exhibits concentration-related cytotoxicity characteristics. Apoptotic activity, attachment capability, and wound healing effects were examined on fibroblasts. The angiogenic activity of ABT was investigated by tube formation assay in HUVECs; 10 µg ml(-1) and 100 µg ml(-1) concentrations of the highest metal ion content of ABT did not disrupt the tube formation of HUVECs. All these tests showed that ABT does not compromise the survival of the cells and might impose regeneration ability to various cell types. These results indicate that silver doped calcium phosphate-based inorganic powder with an optimal silver content has good potential for developing new biomaterials for implant applications.

Synthesis and characterization of Bodipy functionalized magnetic iron oxide nanoparticles for potential bioimaging applications.

Multifunctional magnetic nanoparticles were synthesized for potential bio-imaging applications. Uniform PEI coated magnetic Fe3O4 (PEI-Fe3O4) nanoparticles were prepared by a modified co-precipitation method and then covalently conjugated with a fluorophore molecule, Bodipy-5 by the DCC/DMAP coupling reaction. The covalent binding of Bodipy-5 to the PEI coated magnetic Fe3O4 nanoparticles were confirmed by means of FTIR and XPS measurements. The imaging ability of the Bodipy coated magnetic nanoparticles was determined on two human cancer cells, A549 (human lung adenocarcinoma epithelial) and Ishikawa (endometrial adenocarcinoma), for the first time. Cytotoxicity of BOD-MNPs was evaluated in both cancer cells and healthy human umbilical vein endothelial cell line (HUVEC) by standard MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. In vitro activities of the nanoparticles were also investigated.

Investigation of the pharmacological profiles of dinuclear metal complexes as novel, potent and selective cytotoxic agents against ras-transformed cells.

Around the world scientists try to design successful cures against still incurable diseases, especially cancers. New targets for prevention and new agents for therapy need to be identified. We synthesized novel metal complexes [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2(L2)Pt]Cl2 for determining their cytotoxic and apoptotic effects. The complexes are synthesized by using 1,8-diaminonaphthalene (L1), and bis-1,4-di[([1,10]phenanthroline-5-il)aminomethyl]cyclohexane (L2) as ligands. This is the first study to examine these metals and these molecules in cancer treatment. We elucidated the effects of test compounds with embryonic rat fibroblast-like cells (F2408) and H-ras oncogene activated embryonic rat fibroblast-like cancer cells (5RP7). Results showed that our complexes are more effective than cisplatin to kill ras-transformed cells. Although the [Au(L1)(L2)Pt]Cl2 compound showed a cytotoxic potency higher than [Ru(L1)2(L2)Pt]Cl2 against cancer cells, it proved to be almost five times less effective in decreasing cell viability over healthy cells. Au(III) compound selectively targets the cancer cells but not the healthy cells.

Studies on the cytotoxic, apoptotic and antitumoral effects of Au(III) and Pt(II) complexes of 1, 10-phenanthroline on V79 379A and A549 cell lines.

In the present study, Au(III) and Pt(II) complexes of 1, 10-phenanthroline (phen) were synthesized and used as the test compounds. The structure elucidation of the synthesized compounds was performed by IR, (1)H-NMR and MASS spectroscopic data and the results of elemental analyses. The cytotoxic and apoptotic effects of test compounds were elucidated on V79 379A (Chinese hamster lung fibroblast like) and A549 (human lung carcinoma epithelial like) cell lines. Cytotoxicity was measured with MTT assay and antitumoral effect was determined by colony forming ability methods. In addition, nuclear fragmentation and activation of apoptotic enzyme (caspase-3) and DAPI staining were used to detect the apoptotic effect of the compounds. All the test compounds induced time and concentration-dependent cytotoxic and antitumoral effects. Significant increases in the levels of apoptosis were observed with increasing exposure concentration.