Search Results (351)

Extracellular vesicles (EVs) have emerged as promising acellular tools for modulating immune responses for tissue engineering applications. This study explores the potential of human fibroblast-derived EVs delivered within a three-dimensional (3D) injectable scaffold composed of polycaprolactone (PCL) nanofibers and collagen (PNCOL) to reprogram macrophage behavior and support scaffold integrity under inflammatory conditions. EVs were successfully isolated from human fibroblasts using ultracentrifugation and characterized for purity, size distribution and surface markers (CD63 and CD9). Macrophage-laden PNCOL scaffolds were prepared under three conditions: macrophage-only (MP), fibroblast co-encapsulated (F-MP), and EV-encapsulated (EV-MP) groups. Structural integrity was assessed via scanning electron microscopy and Masson’s trichrome staining, while immunomodulatory effects were evaluated through metabolic assays, gene expression profiling, and immunohistochemistry for macrophage polarization markers (CD80, CD206). When co-encapsulated with pro-inflammatory (M1) macrophages in PNCOL scaffolds, fibroblast-derived EVs preserved scaffold structure and significantly enhanced macrophage metabolic activity compared to the control (MP) and other experimental group (F-MP). The gene expression and immunohistochemistry data demonstrated substantial upregulation of anti-inflammatory markers (TGF-β, CD163, and CCL18) and surface protein CD206, indicating a phenotypic shift toward M2-like macrophages for EV-encapsulated scaffolds relative to the other groups. The findings of this study demonstrate that fibroblast-derived EVs integrated into injectable PCL–collagen scaffolds offer a viable, cell-free approach to modulate inflammation, preserve scaffold structure, and support regenerative healing. This strategy holds significant promise for advancing immuno-instructive platforms in regenerative medicine, particularly in settings where conventional cell therapies face limitations in survival, cost, or safety. Full article

Beyond its immunological role, colostrum has emerged as a promising, non-invasive source of bioactive factors, including mesenchymal stem/stromal cells (MSCs). This study represents the first attempt to isolate and characterize MSCs from equine colostrum (C-MSCs) to assess their potential use in veterinary regenerative medicine. Colostrum (n = 6) was collected from mares immediately after their delivery and centrifuged, and the recovered cells were cultured under standard conditions. The C-MSCs displayed plastic adherence and a heterogeneous morphology, including spindle-shaped and epithelial-like cells. The population doubling time (PDT) values varied among the samples, and four out of six showed rapid proliferation (<2 days). Colony-forming unit (CFU) assays confirmed their clonogenic potential, though significant inter-sample variability was observed (p < 0.05). Spheroid formation assays revealed differences in cell–cell adhesion: four out of six samples formed stable spheroids within four days. A migration assay showed significant variability (p < 0.05): one out of six achieved complete wound closure within 72 h, whereas five out of six reached ~30% at 96 h. All samples were positive for adipogenic, chondrogenic, and osteogenic differentiation as shown via staining. RT-PCR confirmed MSC marker expression, while hematopoietic markers were absent. MHC-I expression was weak in five out of six samples, whereas MHC-II was consistently negative. These findings support equine colostrum as a viable MSC source, though its variability requires further validation with larger samples. Additional research is needed to investigate C-MSCs’ immunomodulatory properties and therapeutic potential. Full article

The development of bakeable foods supplemented with probiotics requires novel strategies to preserve the functionality of probiotic cells during thermal and gastrointestinal stress conditions. The objective of the present study was to evaluate the protective effect of multilayer double emulsions (W₁/O/W₂) stabilized with pectin-protein complexes on the viability of Limosilactobacillus reuteri (Lr) under thermal treatment (95 °C, 30 min), storage (4 °C, 28 d), and simulated gastrointestinal conditions. Emulsions were prepared with whey protein isolate (WPI) or sodium caseinate (Cas) as outer aqueous phase emulsifiers, followed by pectin coating and ionic gelation with calcium. All emulsions were stable and exhibited high encapsulation efficiency (>92%) with initial viable counts of 9 log CFU/mL. Double emulsions coated with ionically gelled pectin showed the highest protection against heat stress and gastrointestinal conditions due to the formation of a denser layer with lower permeability, regardless of the type of protein used as an emulsifier. At the end of storage, Lr viability exceeded 7 log CFU/mL in cross-linked pectin-coated microcapsules. These microcapsules maintained >6 log CFU/mL after thermal treatment, while viability remained >6.5 log CFU/mL during digestion and >5.0 log CFU/mL after consecutive heat treatment and simulated digestion. According to these results, the combination of double emulsion, multilayer formation and ionic crosslinking emerges as a promising microencapsulation technique. This approach offers enhanced protection for probiotics against extreme thermal and digestive conditions compared to previous studies that only use double emulsions. These findings support the potential application of this encapsulation method for the formulation of functional bakeable products. Full article

The domestic cat is the primary reservoir host of three flea-borne Bartonella species, one of which (Bartonella henselae) causes reduced fertility and reproductive failure in experimentally infected cats. Vertical transmission of Bartonella has been documented only in B-cell deficient mice, but not immunocompetent animals. As many free-roaming cats are chronically infected with Bartonella and may be immunocompromised by environmental stress or coinfection, we attempted to isolate Bartonella from the fetal and placental tissues of pregnant queens spayed during trap–neuter–release. Four samples from each tissue (ovary, uterus, fetus, and placenta) were split for direct DNA extraction, liquid culture, and culture on a blood agar plate. Samples from infected queens were inoculated into liquid media and sampled weekly for three weeks for DNA extraction and plating. Bartonella DNA was sequenced directly from 28% (5/18) of the free-roaming queens. For these five queens, liquid enrichment culture was attempted in duplicate for fetal and placental samples. Bartonella clarridgeiae DNA was amplified using qPCR liquid enrichment cultures from the placentas of two cats. These findings suggest that viable Bartonella organisms are present in feline reproductive tissue. Additional studies are needed to assess the transplacental transmission of Bartonella spp. and Bartonella’s influence on fetal development. Full article

The global chemical pigment industry faces environmental challenges despite its economic importance. This study investigates the potential of Kocuria sediminis AS04, an airborne isolate, for sustainable pigment and biomass production. Microbial kinetics were evaluated under Taguchi design conditions with temperature (30, 34, and 38 °C), stirring speed (110, 120, and 130), and pH (6.0, 6.5, and 7.0), measuring biomass through dry weight and viable cells, pigment production, and identification of its pigment using UPLC-MS/MS; structural and chemical characterization of biomass was conducted using SEM and FTIR. Among the tested conditions, the treatment at 30 °C, 130 rpm, and pH 6.5 resulted in the highest CFU count (5.7 × 10⁹ CFU mL⁻¹) and the greatest biomass yield (13.3 g L⁻¹). In contrast, the highest pigment yield (0.0016 mg g⁻¹) was obtained at 38 °C, 130 rpm, and pH 6.0. Cell extracts identified key carotenoid compounds such as β-cryptoxanthin, Rhodovibrin, and other precursors. These findings highlight the potential of Kocuria sediminis AS04 as a sustainable source of pigments and valuable bioproducts, offering promising alternatives for eco-friendly industrial applications. Full article

The creation of transgenic chickens holds significant promise for the agricultural and biotechnological sectors, offering potential improvements in disease resistance and production efficiency. The preferred method for generating gene-edited chickens involves the genetic manipulation of primordial germ cells (PGCs), making the identification and isolation of these cells a growing focus of research. PGCs are the precursors to sperm and oocytes, responsible for transmitting genetic material to the next generation. In humans, PGCs are characterized by their large size, round nuclei, and refractive lipids in the cytoplasm, and can be identified using periodic acid–Schiff (PAS) staining and the surface marker stage-specific embryonic antigen 1 (SSEA1). Similarly, chicken PGCs express SSEA1, but their most specific marker is the chicken vasa homologue (CVH), the avian equivalent of the RNA-binding factor gene vasa. However, SSEA1, along with other known surface markers, does not bind to all PGCs or lacks specificity, while CVH, although highly specific to PGCs, is intracellular and unsuitable for isolating viable cells. This study aims to develop an antibody targeting a PGC surface marker with the same specificity as CVH. Despite the importance of identifying surface markers for PGC characterization, to date, such reagents are limited. To address this, whole chicken PGCs were injected into mice, leading to the generation of a panel of monoclonal antibodies. One antibody was found to bind cultured chicken PGCs and showed reduced expression upon differentiation with retinoic acid, indicating its specificity to PGCs. Immunoprecipitation followed by mass spectrometry identified the antigen as myosin heavy chain-like (MYH9) protein. The antibody, αMYH9, was further characterized and shown to bind circulating PGCs and embryonic gonadal PGCs (Hamburger Hamilton (H-H) stage 30, embryonic day 6.5–7). Whilst our primary aim was to determine the binding to PGCs, further investigation is required to determine potential binding to somatic cells. In conclusion, this study provides the characterization of a surface marker for chicken PGCs, with significant implications for advancements in avian genetic preservation, agriculture, and biotechnology. Full article

Improved in vitro models are needed to reduce costs and delays in central nervous system (CNS) drug discovery. The FDA Modernization Acts 2.0 and 3.0 require human-centered alternative testing methods to mitigate animal-based experiments and discovery delays, and to ensure human safety. Developing cost-efficient, flexible microfluidic chips is essential to advance organ-on-chip (OoC) technology for drug discovery and disease modeling. While CNC micromilling shows promise for fabricating microfluidic devices, it remains underutilized due to limited accessibility. We present a simple CNC-milled flexible microfluidic chip fabricated from thermoplastic poly (methyl methacrylate) (PMMA). The structure of the microplate included drilled openings for connecting the wells. The chip’s biocompatibility was evaluated with isolated primary neuronal cultures from postnatal Wistar rat pups (p1). Primary cells cultured in the device showed high viability, differentiation, and 3D neurosphere formation, similar to conventional well-plate cultures. Neuronal cultures showed neurite growth and functional markers. Although cleanroom-based methods provide higher accuracy, the chip effectively promotes cell viability, differentiation, and alignment, offering an ideal platform for tissue modeling and OoC applications. It allows cell biologists to quickly create prototypes at lower cost and in less time than required for soft lithography and is a viable alternative to the current manufacturing methods. Full article

Osteoarthritis (OA) is a prevalent and debilitating joint disorder that affects a substantial proportion of the global population, underscoring the urgent need for therapeutic strategies that extend beyond symptomatic management. Although mesenchymal stem cells (MSCs) have emerged as a promising therapeutic modality, their clinical application remains constrained by several inherent limitations. This study explores a cell-free alternative by investigating the therapeutic potential of exosomes derived from bone marrow (BMSCs), adipose tissue (ADSCs), and umbilical cord (UMSCs) MSCs in mitigating OA pathogenesis, utilizing both in vitro and ex vivo models. Exosomes from each MSC source were isolated and characterized through nanoparticle tracking analysis, transmission electron microscopy, and Western blotting to confirm their identity and purity. Subsequently, their chondroprotective, anti-inflammatory, and regenerative properties were systematically assessed through evaluations of cell viability, expression profiles of inflammatory and chondroprotective markers, and chondrocyte migration assays. The results demonstrate that all three types of MSC-derived exosomes (MSC-Exos) exhibit low cytotoxicity while significantly suppressing proinflammatory markers and enhancing the expression of chondroprotective genes. Notably, BMSC-Exos and UMSC-Exos displayed superior efficacy in attenuating inflammation, promoting cartilage protection, and inhibiting chondrocyte apoptosis. Furthermore, all MSC-Exos markedly enhanced chondrocyte motility, a critical component of cartilage repair. Collectively, these findings support the therapeutic promise of MSC-Exos, particularly those derived from BMSCs and UMSCs, as a targeted, cell-free approach for the treatment of OA compared to ADSCs. By modulating inflammation, promoting cartilage regeneration, and preventing chondrocyte apoptosis, MSC-Exos may serve as a viable and scalable alternative to current MSC-based therapies for this widespread degenerative disease. Full article

Background and Objectives: Extracellular vesicles (EVs) derived from mesenchymal stem cells have gained much attention as potential therapeutic agents in many diseases, including hearing disorders such as sensorineural hearing loss (SNHL). EVs inherit similar therapeutic effects, including the stimulation of tissue regeneration from the parental cells. The aim of our study was to isolate EVs produced by MSCs and use them to treat inner ear cells in culture to evaluate their protective potential against the damaging effect of an ototoxic drug. Materials and Methods: We isolated MSC-derived EVs by precipitation and characterized them by number, size, and morphology using nanoparticle tracking analysis and TEM, evaluated the protein concentration by BCA assay and the presence of EV markers CD9, CD63, and CD81 by the Dot Blot immunoblotting method. HEI-OC1 inner ear cell line was treated with EVs either alone or followed by Cisplatin. We assessed the uptake of EVs in HEI-OC1 cells by fluorescence microscopy after PKH26 labeling, ROS production by the DCFDA (dichlorfluorescein diacetate) assay, cellular viability by Alamar Blue assay, and apoptosis with the Annexin V/Propidium Iodide method. Results: The isolated EVs had mean dimensions of 184.4 nms and the concentration of the EV suspension was 180 × 10⁶ particles/mL. TEM analysis showed intact vesicular structures with lipid-bilayer membranes having similar sizes with those measured by NTA. The PKH26-labeled EVs were observed in the HEI-OC1 cells after 24 h incubation, the amount increasing with the concentration. EVs reduced ROS production and increased the number of viable cells both alone and as pretreatment before Cisplatin, dose-dependently. Cells in early apoptosis were inhibited by EVs, while those in late apoptosis were enhanced, both with and without Cisplatin. Conclusions: EVs secreted by MSC protected HEI-OC1 cells against Cisplatin toxicity, reduced ROS production, and stimulated cell viability and the elimination of damaged cells by apoptosis, protecting the HEI-OC1 cells against Cisplatin-induced damage. Full article

The low availability of phosphorus (P) in soil has become a critical factor limiting crop growth and agricultural productivity. This study aimed to isolate and evaluate a bacterial strain with high phosphate-solubilizing capacity to improve soil phosphorus utilization and promote crop growth. A phosphate-solubilizing bacterium, designated as YS-13, was isolated from farmland soil in Henan Province, China, and identified as Brevibacillus laterosporus based on morphological characteristics, physiological and biochemical traits, and 16S rDNA sequence analysis. Qualitative assessment using plate assays showed that strain YS-13 formed a prominent phosphate solubilization zone on organic and inorganic phosphorus media containing lecithin and calcium phosphate, with D/d ratios of 2.28 and 1.57, respectively. Quantitative evaluation using the molybdenum–antimony colorimetric method revealed soluble phosphorus concentrations of 21.24, 6.67, 11.73, and 17.05 mg·L⁻¹ when lecithin, ferric phosphate, calcium phosphate, and calcium phytate were used as phosphorus sources, respectively. The fermentation conditions for YS-13 were optimized through single-factor experiments combined with response surface methodology, using viable cell count as the response variable. The optimal conditions were determined as 34 °C, 8% inoculum volume, initial pH of 7.55, 48 h incubation, 5 g L⁻¹ NaCl, 8.96 g L⁻¹ glucose, and 8.86 g L⁻¹ peptone, under which the viable cell count reached 6.29 × 10⁸ CFU mL⁻¹, consistent with the predicted value (98.33%, p < 0.05). The plant growth-promoting effect of YS-13 was further validated through a pot experiment using Triticum aestivum cv. Jinchun 6. Growth parameters, including plant height, fresh biomass, root length, root surface area, root volume, and phosphorus content in roots and stems, were measured. The results demonstrated that YS-13 significantly enhanced wheat growth, with a positive correlation between bacterial concentration and growth indicators, although the growth-promoting effect plateaued at higher concentrations. This study successfully identified a high-efficiency phosphate-solubilizing strain, YS-13, and established optimal culture conditions and bioassay validation, laying a foundation for its potential application as a microbial inoculant and providing theoretical and technical support for reducing phosphorus fertilizer inputs and advancing sustainable agriculture. Full article

This study on the isolation, identification, and characterization of the probiotic properties of lactic acid bacterial strains from the rose blossom of Rosa damascena Mill. (R. damascena) is crucial for discovering novel, plant-derived probiotics with potential health benefits and applications in food, medicine, and cosmetics. Nine lactic acid bacterial (LAB) strains were isolated from rose blossom of R. damascena, and they were identified to the species level by applying physiological and biochemical (API 50 CHL), and molecular genetic (16S rRNA gene sequencing) methods. The isolates were identified as belonging to the Lactobacillus helveticus, Lactobacillus acidophilus, and Lactiplantibacillus plantarum species. Some probiotic properties of the newly isolated and identified LAB strains were examined: their antibacterial activity against pathogens by the agar well diffusion method, and their antibiotic resistance profile by the agar paper disc diffusion method. The LAB strains studied demonstrated significant antibacterial activity against the Escherichia coli, Staphylococcus aureus, Salmonella Abony, Proteus vulgaris, Listeria monocytogenes, and Enterococcus faecalis pathogens and were resistant to most of the antibiotics used in clinical practice, which in turn suggested the possibility of their joint inclusion in therapy, in the composition of probiotic preparations. A batch fermentation process was conducted with Lactiplantibacillus plantarum 5/20, and the kinetic parameters of the batch fermentation process were determined in order to obtain a concentrate with a high viable cell count (10¹³CFU/cm³). The resultant concentrate was freeze-dried, and freeze-dried preparations with a high viable cell count (over 10¹² CFU/g) were obtained. Research on LAB strains isolated from R. damascena could reveal valuable LAB strains with significant probiotic properties. These strains will be suitable for various applications in the composition of starter cultures for functional beverages and foods, as well as probiotic preparations, showcasing the untapped potential of plant-associated microbiota. Full article

Isolation of neurons and glia from the enteric nervous system (ENS) enables ex vivo studies, including the analysis of genomic and transcriptomic profiles. While we previously reported a fluorescence-activated cell sorting (FACS)-based isolation protocol for human ENS cells, no equivalent exists for mice. As directly applying the human protocol to mouse tissue resulted in low recovery of live ENS cells, we optimized tissue dissociation using mouse colons. A 30 min Liberase-based digestion showed optimal recovery of viable ENS cells, with CD56 and CD24 emerging as the most reliable markers to select and subdivide these cells. ENS’ identity was further validated by FACS, using neuronal (TUBB3) and glial (SOX10) markers and reverse transcriptase quantitative PCR on sorted fractions. Overall, the mouse ENS expression profile significantly overlapped with the human one, showing that current dissociation protocols yield a mixed population of enteric neurons and glia. Nonetheless, using the imaging flow cytometer BD S8 FACS Discover and ELAVL4 as a neuronal soma-associated marker, we observed enrichment of neurons in a CD56/CD24^TIP population. In conclusion, we present here a protocol for high-purity FACS-based isolation of viable mouse ENS cells, suitable for downstream applications. Full article

Protoplast-based transformation is a vital tool for genetic studies in fungi, yet no protoplast method existed for P. sclerotiorum-scaumcx01 before this study. Here, we optimized protoplast isolation, regeneration, and transformation efficiency. The highest protoplast yield (6.72 × 10⁶ cells/mL) was obtained from liquid mycelium after 12 h of enzymatic digestion at 28 °C using Lysing Enzymes, Yatalase, cellulase, and pectinase. Among osmotic stabilizers, 1 M MgSO₄ yielded the most viable protoplasts. Regeneration occurred via direct mycelial outgrowth and new protoplast formation, with a 1.02% regeneration rate. PEG-mediated transformation with a hygromycin resistance gene and GFP tagging resulted in stable GFP expression in fungal spores and mycelium over five generations. LC/MS-based metabolomic analysis revealed significant changes in glycerophospholipid metabolism, indicating lipid-related dynamics influenced by GFP tagging. Microscopy confirmed successful colonization of tomato roots by GFP-tagged scaumcx01, with GFP fluorescence observed in cortical tissues. Enzymatic (cellulase) seed pretreatment enhanced fungal colonization by modifying root surface properties, promoting plant–fungal interaction. This study establishes an efficient protoplast transformation system, reveals the metabolic impacts of genetic modifications, and demonstrates the potential of enzymatic seed treatment for enhancing plant–fungal interactions. Full article

Circulating tumor cells (CTCs) hold recognized prognostic value in various cancers, including breast cancer, where their presence correlates with survival outcomes. However, the typical 24 h window for blood processing and CTC isolation poses a logistical challenge, particularly for multicenter studies. This study aimed to evaluate cryopreservation at different stages of CTC isolation and immunocytological detection to extend the blood sample processing period. Using spiked peripheral blood samples with MDA-MB-231, SKBR3, and MCF7 breast cancer cell lines, four distinct cryopreservation points were assessed: following Ficoll gradient separation, immunomagnetic separation, cytocentrifugation, and cytokeratin labeling. Our findings demonstrated that cryopreservation of the mononuclear and granulocytic cell fraction after double-density Ficoll gradient separation was the only viable method for subsequent CTC detection. This approach allowed for consistent recovery of CK+ CTCs, with an average recovery rate of over 81% after one year of cryopreservation. In contrast, cryopreservation at later stages resulted in undetectable CTCs or only cellular debris. In conclusion, cryopreservation following density gradient centrifugation is a feasible strategy for delaying CTC isolation and immunocytological analysis in breast cancer research, facilitating its application in multicenter clinical trials. Full article

Lactic acid bacteria (LAB) isolated from plant sources are gaining increasing attention due to their potential probiotic and postbiotic functionalities. In the present study, Limosilactobacillus fermentum isolated from Prunus padus (bird cherry) was evaluated for its physiological, functional, and technological attributes for application in fermented dairy products. The strain was isolated through anaerobic fermentation and identified using API 50 CHL and 16S rRNA sequencing. Its acid tolerance, antioxidant capacity, antibacterial effects, and hemolytic activity were assessed. The cell-free supernatant (CFS) was evaluated for thermal and pH stability. Fermentation trials were conducted using both mono- and co-culture combinations with the commercial yogurt starter strain YC-380. Physicochemical properties, viable cell counts, and viscosity were monitored throughout fermentation and refrigerated storage. The L. fermentum isolate exhibited strong acid resistance (48.28% viability at pH 2.0), non-hemolytic safety, and notable DPPH radical scavenging activity. Its CFS showed significant antibacterial activity against five Escherichia coli strains, which remained stable after heat treatment. Co-cultivation with YC-380 enhanced fermentation efficiency and improved yogurt viscosity (from 800 to 1200 CP) compared to YC-380 alone. During 24 days of cold storage, co-cultured samples maintained superior pH and microbial stability. Additionally, the moderate acidification profile and near-neutral pH of L. fermentum created favorable conditions for postbiotic compound production. These results indicate that L. fermentum derived from P. padus holds considerable promise as a functional adjunct culture in yogurt production. Its postbiotic potential, technological compatibility, and heat-stable bioactivity suggest valuable applications in the development of safe, stable, and health-promoting fermented dairy products. Full article