Performance and biocompatibility of OSTEMER 322 in cell-based microfluidic applications.

The Off-Stoichiometry Thiol-ene and Epoxy (OSTE+) polymer technology has been increasingly utilised in the field of microfluidics and lab-on-a-chip applications. However, the impact of OSTEMER polymers, specifically the OSTEMER 322 formulation, on cell viability has remained limited. In this work, we thoroughly explored the biocompatibility of this commercial OSTEMER formulation, along with various surface modifications, through a broad range of cell types, from fibroblasts to epithelial cells. We employed cell viability and confluence assays to evaluate the performance of the material and its modified variants in cell culturing. The properties of the pristine and modified OSTEMER were also investigated using surface characterization methods including contact angle, zeta potential, and X-ray photoelectron spectroscopy. Mass spectrometry analysis confirmed the absence of leaching constituents from OSTEMER, indicating its safety for cell-based applications. Our findings demonstrated that cell viability on OSTEMER surfaces is sufficient for typical cell culture experiments, suggesting OSTEMER 322 is a suitable material for a variety of cell-based assays in microfluidic devices.

Bridging the extracellular vesicle knowledge gap: insights from non-mammalian vertebrates, invertebrates, and early-diverging metazoans.

Extracellular vesicles (EVs) are lipid-enclosed structures that facilitate intercellular communication by transferring cargo between cells. Although predominantly studied in mammals, extracellular vesicles are ubiquitous across metazoans, and thus research in non-mammalian models is critical for fully elucidating extracellular vesicles biology. Recent advances demonstrate that extracellular vesicles mediate diverse physiological processes in non-mammalian vertebrates, including fish, amphibians, and reptiles. Piscine extracellular vesicles promote fin regeneration in zebrafish and carry heat shock proteins regulated by stress. Frog extracellular vesicles containing microRNAs modulate angiogenesis, while turtle extracellular vesicles coordinate reproductive functions. Venom from snakes contains extracellular vesicles that mirror the whole venom composition and interact with mammalian cells. Invertebrates also possess extracellular vesicles involved in immunity, development, and pathogenesis. Molluscan extracellular vesicles participate in shell formation and host interactions. Arthropod models, including Drosophila, genetically dissect conserved pathways controlling extracellular vesicles biogenesis and signalling. Nematode extracellular vesicles regulate larval development, animal communication, and ageing via conserved extracellular vesicles proteins. Ancient metazoan lineages utilise extracellular vesicles as well, with cnidarian extracellular vesicles regulating immunity and regeneration. Ultimately, expanding extracellular vesicles research beyond typical biomedical models to encompass phylogenetic diversity provides an unparalleled perspective on the conserved versus specialised aspects of metazoan extracellular vesicles roles over ∼500 million years. With a primary focus on the literature from the past 5 years, this review aims to reveal fundamental insights into EV-mediated intercellular communication mechanisms shaping animal physiology.

Biodistribution and toxicity assessment of methoxyphenyl phosphonium carbosilane dendrimers in 2D and 3D cell cultures of human cancer cells and zebrafish embryos.

The consideration of human and environmental exposure to dendrimers, including cytotoxicity, acute toxicity, and cell and tissue accumulation, is essential due to their significant potential for various biomedical applications. This study aimed to evaluate the biodistribution and toxicity of a novel methoxyphenyl phosphonium carbosilane dendrimer, a potential mitochondria-targeting vector for cancer therapeutics, in 2D and 3D cancer cell cultures and zebrafish embryos. We assessed its cytotoxicity (via MTT, ATP, and Spheroid growth inhibition assays) and cellular biodistribution. The dendrimer cytotoxicity was higher in cancer cells, likely due to its specific targeting to the mitochondrial compartment. In vivo studies using zebrafish demonstrated dendrimer distribution within the vascular and gastrointestinal systems, indicating a biodistribution profile that may be beneficial for systemic therapeutic delivery strategies. The methoxyphenyl phosphonium carbosilane dendrimer shows promise for applications in cancer cell delivery, but additional studies are required to confirm these findings using alternative labelling methods and more physiologically relevant models. Our results contribute to the growing body of evidence supporting the potential of carbosilane dendrimers as vectors for cancer therapeutics.

Gastropod Mucus: Interdisciplinary Perspectives on Biological Activities, Applications, and Strategic Priorities.

Terrestrial gastropod mucus exhibits multifunctional attributes, enabling diverse applications. This comprehensive review integrates insights across biomedicine, biotechnology, and intellectual property to elucidate the bioactivities, physicochemical properties, and ecological roles of snail and slug mucus. Following an overview of mucus functional roles in gastropods, promising applications are highlighted in wound healing, antimicrobials, biomaterials, and cosmetics, alongside key challenges. An analysis of global patent trends reveals surging innovation efforts to leverage gastropod mucus. Strategic priorities include bioprospecting natural diversity, optimizing stabilization systems, recombinant biosynthesis, and fostering collaboration to translate promising potentials sustainably into impactful technologies. Ultimately, harnessing the remarkable multifunctionality of gastropod mucus holds immense opportunities for transformative innovations in biomedicine, biotechnology, and beyond.

Immunocapturing rare cells from blood: A simple and robust microsystem approach.

Cell immunocapture microsystems are a fast-emerging field with several potential medical diagnostic applications. Isolation and quantification of circulating rare cells (CRCs) show great importance in the early stages of disease diagnostics and prognostics. Here, we present a simple and robust stop-flow microsystem (fabricated by a combination of glass microblasting and 3D printing) based on a planar antibody-coated surface that is effective in the immunocapture of the model as well as naturally occurring rare cells. A chip with a planar immunocapture channel working in the so-called stop-flow dynamic regime was designed to enable monitoring the efficiency of the cell capture by fluorescence microscopy. Up to 90% immunocapture efficiency of MCF-7 cells spiked into whole blood on CD326 antibody-coated planar surfaces was achieved. We discuss the role of the planar surface modifications, the influence of the set stop-flow dynamic conditions, and medium complexity on the efficiency of cell immunocapture. The presented results could be further employed in the design of microsystems for cell-size-independent isolation and identification of rare cells from blood.

Mucus-derived exosome-like vesicles from the Spanish slug (Arion vulgaris): taking advantage of invasive pest species in biotechnology.

The slug Arion vulgaris has attracted major attention as one of the worst invasive herbivore pests in Europe and is renowned for the stiff mucus it secretes for locomotion. In this study we focused on the isolation and characterisation of extracellular vesicles, specifically exosomes and exosome-like vesicles, from Arion secretions. We developed a method for slug mucus collection and subsequent vesicle isolation by ultracentrifugation. The isolated vesicles with an average diameter of ~ 100 nm carry abundant proteins and short RNAs, as well as adhesion molecules similar to mammalian galectins. We demonstrated that the slug extracellular vesicles are internalised by plant cells and human cancer cells in in vitro assays and are loadable by bioactive compounds, which makes them an interesting tool for utilisation in biotechnology.

Cerium-Bismuth Oxides/Oxynitrates with Low Toxicity for the Removal and Degradation of Organophosphates and Bisphenols.

Nanoscale cerium-bismuth oxides/oxynitrates were prepared by a scalable low-temperature method at ambient pressure using water as the sole solvent. Solid solutions were formed up to a 1:1 Ce/Bi molar ratio, while at higher doping levels, bismuth oxynitrate photocatalysts with a pronounced layered structure were formed. Bismuth caused significant changes in the structure and surface properties of nanoceria, such as the formation of defects, oxygen-containing surface groups, and Lewis and Brønsted acid sites. The prepared bifunctional adsorbents/photocatalysts were efficient in the removal of toxic organophosphate (methyl paraoxon) from water by reactive adsorption followed by photocatalytic decomposition of the parent compound and its degradation product (p-nitrophenol). Bi-doped ceria also effectively adsorbed and photodegraded the endocrine disruptors bisphenols A and S and outperformed pure ceria and the P25 photocatalyst in terms of efficiency, durability, and long-term stability. The very low toxicity of Bi-nanoceria to mammalian cells, aquatic organisms, and bacteria has been demonstrated by comprehensive in vivo/in vitro testing, which, in addition to its simple "green" synthesis, high activity, and durability, makes Bi-doped ceria promising for safe use in abatement of toxic chemicals.

Conventional and Nonconventional Sources of Exosomes-Isolation Methods and Influence on Their Downstream Biomedical Application.

Despite extensive study of extracellular vesicles (EVs), specifically exosomes (EXs) as biomarkers, important modulators of physiological or pathological processes, or therapeutic agents, relatively little is known about nonconventional sources of EXs, such as invertebrate or plant EXs, and their uses. Likewise, there is no clear information on the overview of storage conditions and currently used isolation methods, including new ones, such as microfluidics, which fundamentally affect the characterization of EXs and their other biomedical applications. The purpose of this review is to briefly summarize conventional and nonconventional sources of EXs, storage conditions and typical isolation methods, widely used kits and new "smart" technologies with emphasis on the influence of isolation techniques on EX content, protein detection, RNA, mRNA and others. At the same time, attention is paid to a brief overview of the direction of biomedical application of EXs, especially in diagnostics, therapy, senescence and aging and, with regard to the current situation, in issues related to Covid-19.

A millifluidic chip for cultivation of fish embryos and toxicity testing fabricated by 3D printing technology.

Zebrafish (Danio rerio) serves as a popular animal model for in vivo acute toxicity evaluation with the Fish embryo test (FET). Over the last few years there has been an effort to develop various systems for a high-throughput zebrafish embryo cultivation and FET. In this paper, we present a novel design of a millifluidic system fabricated by 3D printing technology and we evaluate its functional properties on Danio rerio embryos cultivation and toxicity testing. The development and the optimization of the millifluidic chip was performed by experimental measurements supported by numerical simulations of mass and momentum transport. The cultivation chip with two inlets and one outlet consisted of two individual channels placed on top of each other and separated by a partition with cultivation chambers. An individual embryo removal functionality, which can be used during the cultivation experiments for selective unloading of any of the cultivated embryos out of the chip, was added to the chip design. This unique property raises the possibility of detailed studies of the selected embryos by additional methods. Long-term (96 hours) perfusion cultivation experiments showed a normal development of zebrafish embryos in the chip. Model toxicity tests were further performed with diluted ethanol as a teratogen. Compared to the FET assays, an increased toxic effect of the ethanol on the embryos cultivated in the chip was observed when the median lethal dose and the percentage of the morphological end-points were evaluated. We conclude that the presented 3D printed chip is suitable for long-term zebrafish embryo cultivations and toxicity testing and can be further developed for the automated assays.

Class II biocompatible E-Shell 300 3D printing material causes severe developmental toxicity in Danio rerio embryos and reduced cell proliferation in vitro - implications for 3D printed microfluidics.

Additive manufacturing is a new technology that represents a highly promising, cheap, and efficient solution for the production of various tools in the biomedicine field. In our study, the toxicity of the commercially available E-Shell 300 series photopolymer, which is used in the manufacture of hearing aids and other implants and which could be potentially exploited in microfluidic device fabrication, was tested using in vivo and in vitro biological models. We examined B14 cell proliferation in direct contact with the three-dimensional (3D)-printed material as well as in water extracts to evaluate in vitro cytotoxicity. Similarly, in vivo tests were performed using an OECD-standardized fish embryo acute toxicity (FET) test on Danio rerio embryos in direct contact with the material and in extracts as well. Despite E-Shell 300 3D-printed material being declared as class-IIa biocompatible, in the case of direct contact with both biological models, the results demonstrated a considerable negative impact on cell proliferation and severe developmental toxicity. In this study, up to 84% reduced cell proliferation in vitro and 79% mortality of in vivo models were observed. In contrast, a negligible toxic influence of E-Shell 300 water extracts was present. Four different post-processing treatments to reduce the toxicity were also tested. We observed that post-printing treatment of 3D-printed material in 96% ethanol can reduce embryonic mortality in the FET test by 71% and also completely eliminate negative effects on cell proliferation. We analyzed leachates from the polymeric structures by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, and we discovered the presence of surfactant residues. In summary, our results indicate the importance of biocompatibility testing of the 3D printing photopolymer material in direct contact with the given biological model. On the other hand, the possibility of eliminating toxic effects by an appropriate post-processing strategy opens the door for broader applications of E-Shell 300 photopolymers in the development of complex microfluidic devices for various biological applications.

Carbosilane dendrimers with phosphonium terminal groups are low toxic non-viral transfection vectors for siRNA cell delivery.

Non-viral gene delivery vectors studied in the gene therapy applications are often designed with the cationic nitrogen containing groups necessary for binding and cell release of nucleic acids. Disadvantage is a relatively high toxicity which restricts the in vivo use of such nanoparticles. Here we show, that the 3rd generation carbosilane dendrimers possessing (trimethyl)phosphonium (PMe3) groups on their periphery were able to effectively deliver the functional siRNA into the cells (B14, Cricetulus griseus), release it into the cytosol and finally to achieve up to 40% gene silencing of targeted gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) with the comparable or, in some cases, even better effectivity as their ammonium counterparts. Moreover, such cationic dendrimers show relatively low in vivo toxicity as compared to their ammonium analogues when analyzed by standard fish embryo test (FET) on Danio rerio in vivo model, with LD50 = 6.26 µM after 48 h of incubation. This is more than 10-fold improvement as compared to published values for various other types of cationic dendrimers. We discuss the potential of further increase of the transfection efficiency, endosomal escape and decrease of toxicity of such non-viral vectors, based on the systematic screening of different types of substituents on central phosphonium atom.

Evaluation of toxicological and teratogenic effects of carbosilane glucose glycodendrimers in zebrafish embryos and model rodent cell lines.

Glycodendrimers (Glyco-DDMs) represent a rapidly growing class of nanoparticles with promising properties for biomedical applications but concerns regarding the impact on human health and environment are still justified. Here we report, for the first time, the comparative study of in vivo developmental toxicity of carbosilane Glyco-DDMs and their cytotoxicity in vitro. Carbosilane Glyco-DDMs (generation 1-3) containing 4, 8, and 16 ß-d-glucopyranosyl units at the periphery (DDM1Glu, DDM2Glu, and DDM3Glu) were synthesized and characterized by 1H, 13C and 29Si NMR, mass spectrometry, dynamic light scattering, atomic force microscopy (AFM), and computer modeling. In vitro cytotoxicity assay (MTT) of DDM1-3Glu was performed on three different rodent cell lines (Cricetulus griseus) - B14 (ATCC, CCL-14.1), BRL 3A (ATCC, CRL-1442), and NRK 52E (ATCC, CRL-1571). Overall, very low cytotoxicity was observed with calculated IC50 in mM range with slight difference between each cell line and DDM generation investigated. Modified fish embryo test (FET) was further used for DDM3Glu developmental toxicity testing on zebrafish (Danio rerio) embryos. While seemingly harmless to intact embryos, adverse effects of DDMs on the embryonic development become evident after chorion removal (LD50=2.78 µM at 96 hpe). We summarized that the modified FET test showed a two to three orders of magnitude difference between the in vitro cytotoxicity and in vivo developmental toxicity of DDM3Glu. While, in general, the Glyco-DDMs show great promises as efficient vectors in targeted drug delivery or as therapeutic molecules itself, we suggest that their developmental toxicity should be thoroughly investigated to exclude safety risks associated with their potential biomedical use.

Cubozoan genome illuminates functional diversification of opsins and photoreceptor evolution.

Animals sense light primarily by an opsin-based photopigment present in a photoreceptor cell. Cnidaria are arguably the most basal phylum containing a well-developed visual system. The evolutionary history of opsins in the animal kingdom has not yet been resolved. Here, we study the evolution of animal opsins by genome-wide analysis of the cubozoan jellyfish Tripedalia cystophora, a cnidarian possessing complex lens-containing eyes and minor photoreceptors. A large number of opsin genes with distinct tissue- and stage-specific expression were identified. Our phylogenetic analysis unequivocally classifies cubozoan opsins as a sister group to c-opsins and documents lineage-specific expansion of the opsin gene repertoire in the cubozoan genome. Functional analyses provided evidence for the use of the Gs-cAMP signaling pathway in a small set of cubozoan opsins, indicating the possibility that the majority of other cubozoan opsins signal via distinct pathways. Additionally, these tests uncovered subtle differences among individual opsins, suggesting possible fine-tuning for specific photoreceptor tasks. Based on phylogenetic, expression and biochemical analysis we propose that rapid lineage- and species-specific duplications of the intron-less opsin genes and their subsequent functional diversification promoted evolution of a large repertoire of both visual and extraocular photoreceptors in cubozoans.

Molecular analysis of the amphioxus frontal eye unravels the evolutionary origin of the retina and pigment cells of the vertebrate eye.

The origin of vertebrate eyes is still enigmatic. The "frontal eye" of amphioxus, our most primitive chordate relative, has long been recognized as a candidate precursor to the vertebrate eyes. However, the amphioxus frontal eye is composed of simple ciliated cells, unlike vertebrate rods and cones, which display more elaborate, surface-extended cilia. So far, the only evidence that the frontal eye indeed might be sensitive to light has been the presence of a ciliated putative sensory cell in the close vicinity of dark pigment cells. We set out to characterize the cell types of the amphioxus frontal eye molecularly, to test their possible relatedness to the cell types of vertebrate eyes. We show that the cells of the frontal eye specifically coexpress a combination of transcription factors and opsins typical of the vertebrate eye photoreceptors and an inhibitory Gi-type alpha subunit of the G protein, indicating an off-responding phototransductory cascade. Furthermore, the pigmented cells match the retinal pigmented epithelium in melanin content and regulatory signature. Finally, we reveal axonal projections of the frontal eye that resemble the basic photosensory-motor circuit of the vertebrate forebrain. These results support homology of the amphioxus frontal eye and the vertebrate eyes and yield insights into their evolutionary origin.