Search Results (302)

Extracellular vesicles (EVs) from ovarian cancer cells are valuable sources for candidate biomarker studies, but conventional static flask culture yields limited material and is difficult to scale reproducibly. We evaluated a serum-free CELLine AD 1000 bioreactor workflow for producing EVs from four ovarian cancer-related (OC-related) cell lines (OVCAR4, CaOV3, PA1, SW626) and human dermal fibroblasts (HDFa) as a non-cancer control. Cells were adapted to CDM-HD serum-free medium and maintained for eight weeks with twice-weekly conditioned-medium collection. EVs were isolated by differential ultracentrifugation followed by size-exclusion chromatography and characterized by nanoparticle tracking analysis, imaging flow cytometry, Western blotting, and transmission and scanning electron microscopy. Across longitudinal harvests, OC-related cultures generally produced higher EV particle concentrations and A280-based bulk protein estimates than HDFa, while individual cell lines showed distinct production profiles and membrane-associated growth patterns. A parallel OVCAR4 T-175 flask, maintained in its original serum-containing medium, provided a contextual reference indicating higher per-collection EV particle recovery with the bioreactor, although this was not a matched culture-format comparison. EV-enriched preparations contained vesicle-like particles, with modal diameters of approximately 96–128 nm. Using imaging flow cytometry, the CD9 signal was higher in OC-related EVs and CD63 was most prominent in HDFa; CD9 and CD63 were also detected in OC-related EV lysates by Western blotting. Because one bioreactor was operated per cell line, these findings should be interpreted as preliminary and descriptive rather than statistically comparative. Overall, this study provides a practical serum-free CELLine AD 1000 workflow for generating characterized OC-related EV material for downstream analytical studies. Full article

Nowadays, most cancer research still depends on traditional cell culture in Petri dishes or cell culture flasks which do not have the ability to mimic physiological-like conditions in vitro. However, the behavior of cancer cells strongly relies on the interaction with their extracellular microenvironment. Consequently, current advanced approaches focus on three-dimensional (3D) cell culture to overcome such limitations and to enable a better understanding of fundamental processes including cancer development, progression, apoptosis and invasion. However, transcriptional adaptation to and temporal stability within an in vitro 3D microenvironment still appear to be remarkably understudied. In our study, we compared the cellular behavior and whole transcriptome gene expression of three frequently used non-invasive cancer cell lines (HCT-116, A549 and T47D), embedded within a collagen I (Coll I)-based 3D microenvironment to its counterparts grown as simple monolayers in a time-dependent manner. Thereby, changes in morphology and doubling time became apparent between both cultivation systems, and RNA sequencing-based transcriptome-wide analysis revealed a remarkable increase in transcriptional complexity under 3D conditions. In line with the 3D-dependent phenotype, unidirectional shifts for genes involved in cell cycle regulation (e.g., CCNB1, CCNB2), cell–matrix interaction (e.g., ADAM8, ITGA2) and metabolic signaling (e.g., HK2, ENO2) were identified over time, being either activated or repressed. Interestingly, all three cell lines cultured in Coll I matrices displayed a highly distinct RNA content and composition, along with a significantly increased number of expressed protein-coding genes (increase of 3–6%) as well as long non-coding RNAs (increase of 26–48%), suggesting a more multifaceted transcription profile under 3D conditions. Our work clearly highlights that an in vitro 3D Coll I-based cell culture system has an incisive cell-specific impact on the whole transcriptome on a qualitative and quantitative level. This tremendous transcriptional reprogramming implies essential changes in gene regulatory networks and affects phenotypic cancer cell behavior, which should be considered when focusing on downstream applications. Full article

Botrytis cinerea, the causal agent of gray mold disease, is a major phytopathogen responsible for substantial losses in horticultural crops. In this study, cultivation conditions for Bacillus velezensis R22 were optimized to maximize overall antifungal activity against B. cinerea. A Plackett–Burman design was used to identify medium components affecting antifungal activity in flask cultures, followed by response surface methodology based on a central composite design (CCD) to optimize sucrose concentration, temperature, and agitation speed in a stirred bioreactor. Maximum antifungal activity was obtained at 17.45 g/L initial sucrose, 31.8 °C, and 293 rpm. The biological relevance of the optimized culture was confirmed in a tomato infection model, in which gray mold severity was reduced by 85.3% relative to the untreated control and by 59.9% relative to the non-optimized R22 culture. The same CCD approach was subsequently applied to determine cultivation conditions that maximize the concentration of R22 viable cells. The optimal parameters for 24-h growth (35.46 g/L sucrose, 36.5 °C, and 454 rpm) differed markedly from those identified for maximal antifungal activity. When evaluated on uninfected tomato plants, cultures produced under conditions favoring higher cell density showed enhanced plant growth-promoting activity compared to the non-optimized culture. Mass spectrometric analysis of lipopeptide extracts revealed that the enhanced antifungal activity was accompanied by an increased abundance of long-chain homologs across all major lipopeptide families, particularly surfactins. Thus, our results indicate that maximizing overall antifungal activity may be of greater practical significance than optimization of the individual fungicidal agent. Full article

A series of 7-aminopyrazolo[1,5-d][1,2,4]triazin-4(5H)-ones was synthesized via a cascade condensation of methyl aroylpyruvates with 1,3-diaminoguanidine hydrochloride. The scope and limitations of this approach were investigated. Methyl mesitoylpyruvate bearing a sterically hindered mesityl substituent diverted the reaction pathway, affording a 1,2,4-triazine derivative. Diethyl 2,4,6-trioxoheptanedioate resulted in an unexpected pyrazolo[1,5-d][1,2,4]triazepine scaffold. All synthesized compounds were evaluated for growth-regulating activity using the green microalga Chlorella vulgaris as a model organism. 7-Amino-2-(4-methoxyphenyl)pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one has shown the best results in the initial microplate screening, showing increased cell density at 10 μmol/L. However, subsequent validation in 50 mL flask cultures revealed no significant effect on biomass accumulation, photosynthetic pigment content, carbohydrate levels, or neutral lipid production compared to the negative control. Only a modest increase in protein content was observed at the concentration of 100 μmol/L. Full article

The isolation of fibroblast-like cells from crocodile skeletal muscle provides a valuable platform for studies in comparative physiology, reptilian biology, regenerative medicine, and tissue engineering. In this article, we present an optimized protocol for isolating and characterizing fibroblast-like cells derived from the embryonic skeletal muscle of the Siamese crocodile (Crocodylus siamensis). The procedure improved cell yields and viability while maintaining phenotypic and genetic stability. Dorsal and tail skeletal muscle tissue was cultured in flasks pre-coated with collagen. The cells attached and began migrating from the explants within one day. Optimal culture conditions were achieved using Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 or Minimum Essential Medium Alpha supplemented with 10% fetal bovine serum, 2% crocodile serum, and growth-promoting factors, incubated at 28 °C without CO₂. These conditions supported a shorter population doubling time and enhanced cell proliferation. The established cells displayed a spindle-shaped fibroblastic morphology, expressed the fibroblast-specific marker fibronectin, and maintained a stable karyotype through passage 20. The cell line remained stable and proliferative for at least 30 passages under standard culture conditions. Our study established the first standardized fibroblast-like cell line derived from C. siamensis, thus providing a foundation for future studies in reptilian physiology, cytogenetics, and regenerative biotechnology. Full article

Ergothioneine (EGT), a naturally occurring amino acid derivative with potent antioxidant and cytoprotective properties, is widely applied in the food, cosmetic, and medical industries. Traditional production methods are limited by high costs, low efficiency, and environmental concerns, so microbial fermentation serves as a sustainable alternative for EGT production. In this study, Escherichia coli BL21 (DE3) was employed as the chassis strain. First, a basic EGT-producing engineered strain was constructed by heterologously expressing the egtB gene from Methylobacterium pseudosasicola along with the egtD and egtE genes from Mycobacterium smegmatis. This initial strain achieved a yield of 84.84 ± 1.64 mg/L of EGT in shake-flask cultures. To enhance production, solubility-enhancing tags were introduced to improve the soluble expression of the key enzymes, and metabolic pathways were rationally engineered to strengthen the supply of essential precursor amino acids. These modifications led to the development of a high-yield EGT strain. After optimizing the fermentation process, the best results were achieved using a medium with glycerol as the carbon source, 0.5 g/L of histidine, 1.5 g/L of methionine, and 1.0 g/L of cysteine, along with induction at 25 °C using 0.2 mM IPTG for 120 h. Under these conditions, the final EGT yield reached 385.70 ± 4.86 mg/L. The engineered strain for EGT synthesis and optimized fermentation strategy developed in this study offer a useful basis for further process development. Full article

Class II biological safety cabinets (BSCs) are designed to protect the user, the product, and the laboratory environment by maintaining HEPA-filtered airflow; however, their fans, alarms, and structural resonances introduce acoustic and vibrational stimuli that may confound mechanosensitive cell-culture assays. In this study, we characterized the vibroacoustic environment of a cell-culture laboratory and a Class II BSC, selected representative tray locations based on measured and modeled stimuli, and evaluated in vitro wound closure in HaCaT keratinocytes using a scratch assay under alarm-induced acoustic exposure. Wound closure after 24 h was quantified using a relative area-closure metric defined as one minus the ratio of wound area at 24 h to wound area at 0 h. For each biological replicate (one flask and one scratch), two non-overlapping image regions were treated as technical subsamples and averaged to obtain a single flask-level value. Three independent experimental runs were performed, each including one flask per tray point, yielding n equals 3 independent flasks per tray point. Mean wound closure values were 73.7 percent plus or minus 15.6 percent, 75.6 percent plus or minus 7.2 percent, and 79.4 percent plus or minus 14.8 percent for tray points P1, P5, and P6, respectively (mean plus or minus standard deviation). No statistically significant differences were detected among points (one-way ANOVA on flask-level values, F equals 0.15, p equals 0.86). These findings highlight that BSC-associated acoustic and vibration stimuli should be documented when interpreting scratch-assay outcomes and motivate larger, sham-controlled studies to resolve small effect sizes relevant for assay reproducibility. Full article

Twenty strains of Schizophyllum commune from the BIOTEC culture collection were selected for this study. S. commune is characterized by white to gray fan-shaped caps with lobed margins and distinctive split gills. Phylogenetic analysis of combined LSU rDNA and ITS rDNA sequences data using maximum parsimony placed the fungi in a strongly supported clade with S. commune. All strains were primarily screened for exopolysaccharide (EPS) and biomass production using potato dextrose broth (PDB) and peptone yeast glucose medium (PYGM) in 250 mL flasks shaken at 200 rpm for 7 days. The results revealed three strains with high EPS production, each exceeding 2.3 g/L, namely MMCR00487, MMCR00474 and MMCR00256. These strains were selected for media optimization using a Plackett–Burman design. Among them, MMCR00256 exhibited the highest EPS yield of 8.34 ± 1.47 g/L, followed by MMCR00487 and MMCR00474. Therefore, the strain MMCR00256 was further optimized by central composite design. The results revealed that the optimized medium for MMCR00256 increased the production of EPS to 10.39 ± 1.69 g/L, with a biomass yield of 26.28 ± 1.63 g/L (395 mg/g). The 5 L bioreactor optimization tested two inoculum types (mycelial and pellet) and two media (CCD and estimated) using strain MMCR00256. The mycelial inoculum grown in the estimated medium produced the highest EPS yield of 8.37 ± 0.26 g/L after 3 days, with 13.56 ± 2.94 g/L biomass. In conclusion, this study demonstrates that S. commune MMCR00256, when cultivated using the estimated medium and mycelial inoculum, can achieve enhanced exopolysaccharide production with improved efficiency, highlighting its significant potential for the development of efficient and scalable schizophyllan production processes at the industrial scale. Furthermore, this study provides essential insights into the cultivation and optimization of schizophyllan in S. commune. Full article

Background: In the Republic of Korea, a bivalent foot-and-mouth disease (FMD) vaccine covering serotypes O and A is administered to livestock, while antigens for the other serotypes are stockpiled in overseas antigen banks. To achieve self-reliance in FMD vaccine production, various vaccine strains have been developed using in-house technology. Although SAT 3 has historically been confined largely to Africa, preparedness against this serotype remains necessary, as the possibility of its introduction into Korea cannot be completely excluded. Methods: In this regard, we evaluated the commercial potential of the SAT 3 ZIM-R vaccine strain by assessing antigen productivity, scalability, inactivation kinetics, and immunogenicity. Results: Supplementation with 3 mM Ca²⁺ markedly increased antigen yield compared with that obtained in the absence of calcium. Further optimization showed that antigen yield was highest at pH 8.0–8.5. During scale-up, antigen yield was maintained at 9.2–9.8 μg/mL in flask cultures and remained high at approximately 7.8 μg/mL in a bioreactor, demonstrating robust scalability. Treatment with 2 mM binary ethylenimine at 26 °C achieved complete inactivation within 24 h. Vaccines formulated with the SAT 3 ZIM-R antigen produced either in flasks or in a bioreactor induced comparable neutralizing antibody responses in pigs following both the primary and booster immunizations. Conclusions: Collectively, these findings indicate that SAT 3 ZIM-R is a promising vaccine candidate for large-scale vaccine antigen production and the future establishment of a domestic FMD antigen bank in Korea. Full article

Entomopathogenic nematodes (EPNs) are crucial biocontrol agents, yet optimizing the yield and quality of infective juveniles (IJs) during commercial liquid production remains challenging. This study utilized a central composite rotatable design to optimize liquid culture parameters (ascaroside, dimethyl sulfoxide, medium volume, IJ inocula) for Heterorhabditis bacteriophora H06 and Steinernema carpocapsae All. The results demonstrated that improving aeration (inferred from reduced media volume), combined with ascr#11 regulation, synergistically enhanced IJ yield and quality. Under optimized conditions, yields reached 3.35 × 10⁵ IJs/mL for H. bacteriophora H06 and 2.67 × 10⁵ IJs/mL for S. carpocapsae All. Crucially, the IJs from the high-yield flask exhibited significantly superior infectivity (24–26% single-IJ infection rate) compared to solid-culture controls (13–14%). Targeted metabolomics profiling of sugar, energy and fatty acids of H. bacteriophora H06 revealed upregulated tricarboxylic acid (TCA) cycle intermediates (citrate, pyruvate) and the significant accumulation of stress-protectant trehalose and immune-modulating polyunsaturated fatty acids (eicosapentaenoic acid, arachidonic acid). These findings establish a fermentation strategy that simultaneously enhances IJ yield and biological quality by reducing media volume (used as a proxy for improved aeration) and supplementing ascr#11. Furthermore, the distinct metabolic profile enriched in energy, stress, and immune-modulating metabolites identified in H. bacteriophora provides a plausible explanatory framework for the parallel phenotypic improvements observed across both species. Full article

Mesenchymal stem cells (MSCs) are multipotent cells that have the ability to mediate cellular repair through a combination of soluble paracrine factors, as well as bioactive cargo packaged within extracellular vesicles (EVs). Although MSC-derived EVs have been widely investigated for their regenerative potential, progress toward translational evaluation has been limited in part by challenges in scalable and reproducible manufacturing. We recently reported that human telomerase reverse transcriptase (hTERT)-immortalized MSCs reproducibly produce EVs that retain key characteristics of EVs derived from primary MSCs. Building on this work, three-dimensional (3D) culture systems have emerged as promising platforms for large-scale manufacturing. In this study, we compared the yield, molecular composition, and functional activity of EVs produced from hTERT-immortalized MSCs cultured in either a fixed-bed bioreactor or conventional two-dimensional (2D) flasks. Our data demonstrate that bioreactor culture results in increased EV yield as compared to an equivalent production from 2D cultures. Molecular analyses indicated that bioreactor-derived EVs were associated with a broader spectrum of cargo and were enriched with molecules that may contribute to enhanced reparative function. Importantly, bioreactor-derived EVs also exerted a more pronounced effect in cellular repair assays in vitro. Collectively, these results highlight the potential of fixed-bed bioreactors as scalable platforms for EV production, offering higher yields while preserving molecular composition and functional activity. This approach represents an important step toward achieving the reproducible, high-quality EV production required for research and future translational applications. Full article

The microbial production of 1,5-pentanediol (1,5-PDO), a versatile platform chemical with extensive industrial applications, remains limited by suboptimal fermentation titers and incomplete understanding of metabolic bottlenecks. To address these challenges, this study employed comparative transcriptomics to systematically identify novel genetic targets capable of enhancing 1,5-PDO biosynthesis in engineered Escherichia coli. Transcriptomic profiling of the 1,5-PDO-producing strain relative to the parental E. coli W3110, conducted at both exponential (24 h) and stationary (96 h) growth phases, revealed 1384 significantly differentially expressed genes, including 851 upregulated and 533 downregulated genes. From these, 20 candidate metabolic genes associated with 1,5-PDO synthesis were selected for functional validation through plasmid-based overexpression or CRISPR interference (CRISPRi)-mediated repression. Reverse engineering confirmed that overexpression of fecA (encoding an iron(III)-citrate transporter) and deletion of gadA (encoding glutamate decarboxylase) significantly enhanced 1,5-PDO production. Subsequent chromosomal integration of fecA at the neutral ilvG locus and deletion of gadA generated the optimized strain S7, which achieved a 1,5-PDO titer of 1.7 g/L in shake flask cultures, representing a 13.3% increase over the parental strain, with a concomitant 50% improvement in glucose yield (0.18 mol/mol). In fed-batch fermentation at the 5 L bioreactor scale, strain S7 attained a titer of 12.45 g/L and a glucose yield of 0.26 mol/mol, marking a 15.6% enhancement in carbon conversion efficiency relative to the parental strain (0.225 mol/mol), while concurrently improving biomass accumulation by 7.6%. These findings demonstrate that transcriptomics-guided reverse engineering constitutes an effective strategy for elucidating nonobvious metabolic determinants and optimizing microbial cell factories for efficient 1,5-PDO production. The identification of fecA and gadA as beneficial targets provides valuable insights into the metabolic rewiring underlying enhanced 1,5-PDO biosynthesis and establishes a foundation for further strain improvement through systems metabolic engineering. Full article

Dried okara (DOK), a lignocellulosic byproduct from tofu production, was evaluated as both a carbon source and culture medium to enable cost-effective polyhydroxybutyrate (PHB) production. Hydrolysis with either HCl or H₂SO₄ generated 48–51 g/L reducing sugars with peak values reaching 60.2 g/L using 3% acid at 121 °C. Analysis of monosaccharides indicated pentoses, especially xylose, as the main sugars present. A novel strain, Burkholderia sp. EP10 exhibited direct growth and PHB accumulation in DOK hydrolysate without requiring detoxification, tolerating inhibitory compounds such as furfural and 5-hydroxymethylfurfural. In shake flask experiments, the strain achieved 6.9 g/L biomass and 26.3 wt% PHB, while in fermentor studies, biomass reached 10.9 g/L and PHB content was 29.3 wt% at a C/N ratio of 5.7. Notably, these outcomes were achieved without pH control, constituting a key benefit for operational simplification and cost minimization. The biopolymer was verified as PHB using gas chromatography, Fourier transform infrared spectroscopy, and proton nuclear magnetic resonance spectroscopy. The PHB displayed melting transitions at 163.5 and 172.4 °C, a degradation onset at 268 °C, and high molecular weight (4.66 × 10⁵ Da). Burkholderia sp. EP10 for sustainable PHB production via direct bioconversion of lignocellulosic hydrolysates, without the need for pH adjustment, detoxification, or complex medium development. Full article

Oleaginous yeasts are promising microbial platforms for lipid production from non-conventional carbon sources; however, acetate utilization is frequently constrained by physiological limitations associated with culture pH. In this study, acetate utilization, biomass formation, and lipid production by Lipomyces starkeyi were investigated under flask and fed-batch cultivation to evaluate the influence of culture pH and pH control strategy. Statistically supported flask-scale experiments demonstrated that acetate concentration and cultivation time significantly affected acetate consumption, biomass formation, volumetric lipid concentration, and culture pH, with excessive acetate loading resulting in culture alkalization, incomplete substrate utilization, and reduced process performance. Although volumetric lipid concentration increased with increasing acetate concentration, lipid content and fatty acid composition remained unchanged, indicating that enhanced lipid production was primarily attributable to increased biomass formation rather than to changes in lipid biosynthesis. Fed-batch cultivation under different pH-control strategies provided qualitative insights into the relationships among pH regulation, acetate availability, and lipid accumulation under controlled fermentation conditions. While lipid accumulation was observed under both HCl-based and acetic acid-based pH control, differences in pH stability and cumulative acetate availability were associated with distinct patterns of lipid production. Collectively, these results identify culture pH as a critical physiological parameter influencing acetate utilization and lipid accumulation in L. starkeyi and suggest that coordinated pH control and carbon feeding strategies may improve the robustness of acetate-based lipid production processes. Further replicated fed-batch studies will be required to quantitatively validate these trends and support industrial applications. Full article

The present study investigates the potential of olive mill wastewater (OMW), supplemented with expired commercial glucose syrup, as a sustainable substrate for the submerged cultivation of Tuber spp. wild mushrooms. OMW contains considerable quantities of phenolic compounds, making it both a challenging pollutant and a promising nutrient source. To assess fungal performance under increasing phenolic stress, culture media were prepared with varying OMW concentrations (0–75% v/v on agar; 0–50% v/v in liquid media), while glucose was adjusted to ~30 g/L using expired glucose syrup. A sequential experimental approach was followed, beginning with Petri dish screenings on substrate/strain selection (measuring the mycelial growth rate; Kr, mm/day), progressing to 25-day shake flask fermentations and subsequently scaling up the most promising strain (Tuber mesentericum) in a controlled stirred-tank bioreactor. Throughout cultivation, substrate consumption (glucose, phenolics), pH evolution and decolorization were evaluated, while the resulting biomass was analyzed for polysaccharides, β-glucans, proteins, lipids, fatty acids, antioxidants, phenolic acids and triterpenoids content. Results showed that increasing OMW concentration enhanced tolerance and metabolic activity in selected Tuber species, with T. mesentericum exhibiting the highest resilience and achieving comparable or higher biomass yields in OMW-based media than in glucose (control). Phenolic removal exceeded 60% in flasks and 50% in the bioreactor, confirming simultaneous bioremediation capacity. Bioreactor cultivation demonstrated efficient substrate utilization and biomass production, while OMW-grown biomass presented high lipid content, enriched with unsaturated fatty acids, high β-glucan levels and increased antioxidant and phenolic profiles. Overall, this study demonstrates that OMW (supplemented with expired glucose syrup) can serve as a cost-effective and environmentally beneficial substrate for Tuber biomass production with dietary and antioxidant properties, offering an alternative source to mushroom carposomes, as well as supporting the circular bioeconomy strategies within olive oil processing industries. Full article