Various spheroid formation techniques have been widely developed for efficient and reliable 3-D cell culture research. Although those efforts improved many aspects of spheroid generation, the ...procedures became complex and also required unusual laboratory equipment. Many recent techniques still involve laborious pipetting steps for spheroid manipulation such as collection, distribution and reseeding. In this report, we used a density-controlled polyethylene glycol and dextran aqueous two phase system to generate spheroids that are both consistent in size and precisely size-controllable. Moreover, by adding a few drops of fresh medium to the wells the contain spheroids, they can be simply settled and attached to the culture surface due to reduced densities of the phases. This unique attribute of the technique significantly reduces the numerous pipetting steps of spheroid manipulation to a single pipetting; therefore, the errors from those steps are eliminated and the reliability and efficiency of a research can be maximized.
Extracellular vesicles are categorized in subsets according to their biogenesis processes. To facilitate the investigation of subsets, an effective method is needed for isolating subpopulations. The ...efficacy of existing density and size-based isolation methods is limited, and as a result, the correlation of properties within separated subpopulations is modest. Here, we introduced size separation with ∼48 nm resolution that exploits Marangoni flow and the coffee-ring effect in microdroplets in which extracellular vesicles are spatially deposited at different location according to size of extracellular vesicle. Interestingly, the analysis of tetraspanin proteins of the extracellular vesicles facilitated by this method reveals that the size of extracellular vesicles is correlated with expression of tetraspanin proteins (CD9, CD63, CD81) that are associated with the size of extracellular vesicles. The findings show that CD9 and CD81 are uniformly expressed regardless of size, CD63 is highly expressed only in larger extracellular vesicles. This evidence indicates that extracellular vesicles can be classified based on size and expression of CD63.
We developed a new type of electroencephalogram (EEG) headset system with comb-shaped electrodes that enables the wearer to quickly don and utilize it in daily life. Two models that can measure EEG ...signals using up to eight channels have been implemented. The electrodes implemented in the headsets are similar to a comb and are placed quickly by wiping the hair (as done with a comb) using the headset. To verify this headset system, donning time was measured and three brain computer interface (BCI) application experiments were conducted. Alpha rhythm-based, steady-state visual evoked potential (SSVEP)-based, and auditory steady state response (ASSR)-based BCI systems were adopted for the validation experiments. Four subjects participated and ten trials were repeated in the donning experiment. The results of the validation experiments show that reliable EEG signal measurement is possible immediately after donning the headsets without any preparation. It took approximately 10 s for healthy subjects to don the headsets, including an earclip with reference and ground electrodes. The results of alpha rhythm-based BCI showed 100% accuracy. Furthermore, the results of SSVEP-based and ASSR-based BCI experiments indicate that performance is sufficient for BCI applications; 95.7% and 76.0% accuracies were obtained, respectively. The results of BCI paradigm experiments indicate that the new headset type is feasible for various BCI applications.
Extracellular vesicles (EVs) such as exosomes and microvesicles released from cells are potential biomarkers for blood-based diagnostic applications. To exploit EVs as diagnostic biomarkers, an ...effective pre-analytical process is necessary. However, recent studies performed with blood-borne EVs have been hindered by the lack of effective purification strategies. In this study, an efficient EV isolation method was developed by using polyethylene glycol/dextran aqueous two phase system (ATPS). This method provides high EV recovery efficiency (~70%) in a short time (~15 min). Consequently, it can significantly increase the diagnostic applicability of EVs.
Extracellular vesicles (EVs) are secreted nano‐sized vesicles that contain cellular proteins, lipids, and nucleic acids. Although EVs are expected to be biologically diverse, current analyses cannot ...adequately characterize this diversity because most are ensemble methods that inevitably average out information from diverse EVs. Here we describe a single vesicle analysis, which directly visualizes marker expressions of individual EVs using a total internal‐reflection microscopy and analyzes their co‐localization to investigate EV subpopulations. The single‐vesicle imaging and co‐localization analysis successfully illustrated the diversity of EVs and revealed distinct patterns of tetraspanin expressions. Application of the analysis demonstrated similarities and dissimilarities between the EV fractions that had been acquired from different conventional EV isolation methods. The analysis method developed in this study will provide a new and reliable tool for investigating characteristics of single EVs, and the findings of the analysis might increase understanding of the characteristics of EVs.
Ballistocardiographs (BCGs), which record the mechanical activity of the heart, have been a subject of interest for several years because of their advantages in providing unobtrusive physiological ...measurements. BCGs could also be useful for monitoring the biological signals of infants without the need for physical confinement. In this study, we describe a physiological signal monitoring bed based on load cells and assess an algorithm to extract the heart rate and breathing rate from the measured load-cell signals. Four infants participated in a total of 13 experiments. As a reference signal, electrocardiogram and respiration signals were simultaneously measured using a commercial device. The proposed automatic algorithm then selected the optimal sensor from which to estimate the heartbeat and respiration information. The results from the load-cell sensor signals were compared with those of the reference signals, and the heartbeat and respiration information were found to have average performance errors of 2.55% and 2.66%, respectively. The experimental results verify the positive feasibility of BCG-based measurements in infants.
The effects of concentrated fibroblast-conditioned media were tested to determine whether hepatocyte function can be maintained without direct contact between hepatocytes and fibroblasts. Primary rat ...hepatocytes cultured with a concentrated conditioned media of NIH-3T3 J2 cell line (final concentration of 55 mg/ml) showed significantly improved survival and functions (albumin and urea) compared to those of control groups. They also showed higher expression levels of mRNA, albumin and tyrosine aminotransferase compared to hepatocyte monoculture. The results suggest that culture with concentrated fibroblast-conditioned media could be an easy method for in vitro maintenance of primary hepatocytes. They also could be contribute to understand and analyze co-culture condition of hepatocyte with stroma cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Enveloped viruses pose a significant threat to human health, as evidenced by the recent COVID‐19 pandemic. Although current vaccine strategies have proven effective in preventing viral infections, ...the development of innovative vaccine technologies is crucial to fortify our defences against future pandemics. In this study, we introduce a novel platform called cell‐engineered virus‐mimetic nanovesicles (VNVs) and demonstrate their potential as a vaccine for targeting enveloped viruses. VNVs are generated by extruding plasma membrane‐derived blebs through nanoscale membrane filters. These VNVs closely resemble enveloped viruses and extracellular vesicles (EVs) in size and morphology, being densely packed with plasma membrane contents and devoid of materials from other membranous organelles. Due to these properties, VNVs express viral membrane antigens more extensively and homogeneously than EVs expressing the same antigen. In this study, we produced severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) VNVs expressing the SARS‐CoV‐2 Spike glycoprotein (S) on their surfaces and assessed their preclinical efficacy as a COVID‐19 vaccine in experimental animals. The administration of VNVs successfully stimulated the production of S‐specific antibodies both systemically and locally, and immune cells isolated from vaccinated mice displayed cytokine responses to S stimulation.
We demonstrate a fluorescence-based nanoparticle tracking analysis (NTA) system for the characterization of both the size and membrane protein expression of individual extracellular vesicles (EVs). A ...sheet of lasers with four different wavelengths was sequentially shone onto extracellular vesicles according to a preprogrammed schedule, providing scattering images intercalated by three fluorescent images. The presence of extracellular vesicles was tracked frame by frame from scattering images. Fluorescence-labeled membrane proteins on EVs were detected by comparing scattering and fluorescent images. The tetraspanins (CD9, CD63, and CD81) of individual HEK293 EVs analyzed by both NTA and total internal reflection fluorescence microscopy showed that the proposed NTA system can contribute to the understanding of individual extracellular vesicles.
Extracellular vesicles (EVs) are nano-sized membranous particles secreted by cells. EVs have been classified into subpopulations according to their presumed biogenesis pathway, but their detailed ...biogenesis mechanisms still need to be fully elucidated. Enveloped viruses are another type of cell-derived nano-vesicles, and their biogenesis processes are much better known than that of EVs. Recently, studies on the similarity between enveloped viruses and EVs have been increasingly reported. The biogenesis of EVs could be better understood if these similarities are adequately investigated. In this study, we utilized a single vesicle imaging technique to visualize the protein expressions of individual nano-sized vesicles and analyzed expression patterns within single vesicles. Using this technique, we identified unique tetraspanin expression patterns in single EVs and that these patterns were closely related to their subcellular origins. The expression of CD9 or CD81 in EVs implied that they originated from the plasma membrane, and the expression of CD63 in EVs implied that they originated from endosomal organelles. We further analyzed the tetraspanin expressions of two different types of virus-like particles (VLPs) and demonstrated that the HIV-Gag-induced VLPs were more similar to EVs than SARS-CoV-2-NP/M/E-induced VLPs. In addition, HIV-Gag-GFP-expressing VLPs were highly colocalized with CD9, CD63, and CD81 signals, whereas SARS-CoV-NP-GFP-expressing VLPs were not. Based on these observations, we could assume that tetraspanin-expressing EVs might be produced through a similar process by which HIV is produced.
•TIRF-based single-vesicle analysis visualized subpopulations in extracellular vesicles (EVs).•EV subpopulations showed different tetraspanin expression patterns.•Unique tetraspanin expression patterns in EVs are strongly correlated with subcellular tetraspanin expression localization.•CD9 and CD81 positive EVs might be microvesicles and CD63 positive EVs might be exosomes.•HIV-like particles showed a similarity in tetraspanin expressions with EVs but SARS-CoV-2-like particles did not.
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