Search Results (126)

Solid–liquid stirred tanks are widely used in multiphase processes, including bioreactors for mesenchymal stem cell (MSC) culture, yet simultaneous experimental data for both dispersed and carrier phases remain limited. Here, a refractive index-matched (RIM) suspension of PMMA microparticles ($d_p=168\mathrm{\mu m}$, ${\rho }_p/{\rho }_l\approx 0.96$) in an NH₄SCN solution is studied at an intermediate Reynolds number ($Re\approx 5000$), low Stokes number ($St=0.078$), and particle volume fractions $0.1\le {\alpha }_p\le 0.5$ v%. This system was previously established and studied for the effect of addition of particles on the carrier phase. In this work, a dual-camera PIV set-up provides simultaneous velocity fields of the liquid and particle phases in a stirred tank equipped with a three-blade down-pumping HTPGD impeller. The liquid mean flow and circulation loop remained essentially unchanged with particle loading, whereas particle mean velocities were lower than single-phase and liquid-phase values in the impeller discharge. Turbulence levels diverged between phases: liquid-phase turbulent kinetic energy (TKE) in the impeller region increased modestly with ${\alpha }_p$, while solid-phase TKE was attenuated. Slip velocity maps showed that particles lagged the fluid in the impeller jet and deviated faster from the wall in the upward flow, with slip magnitudes increasing with ${\alpha }_p$. An approximate axial force balance indicated that drag dominates over lift in the impeller and wall regions, while the balance is approximately satisfied in the tank bulk, providing an experimental benchmark for refining drag and lift models in this class of stirred tanks. Full article

Washingtonia filifera is important for its ecological, economic, cultural, horticultural, ornamental, and medicinal potential. Elicitation of in vitro cultures presents a promising and efficient method for the large-scale production of valuable bioactive compounds. This study assessed the effect of chitosan concentration (0, 20, 40, 60, 80, 100 mg L⁻¹) on biomass growth [fresh weight (FW), dry weight (DW)] and phytochemical profile [total phenolic content (TPC), total flavonoid content (TFC), DPPH antioxidant activity, total phenolic productivity (TPP), total flavonoid productivity (TFP)] in W. filifera callus and cell suspension cultures. Among different plant growth regulator combinations tested, 3 mg L⁻¹ 2,4-D + 0.5 mg L⁻¹ 2ip gave higher callus induction (90%) (MS medium, 12 weeks). A maximum growth curve (FW: 180 mg) of cell suspension culture was achieved 7 weeks after initiation (shaker at 90 rpm for 24 h). Cell suspension exhibited higher FW, DW, TPC, TFC, DPPH, TPP, and TFP than callus, while flavonoid production was higher than phenolic production. FW and DW were higher in both systems, with 40 mg L⁻¹ chitosan. Chitosan at 60 mg L⁻¹ best enhanced the phytochemical profile of both the 4-week solidified callus and the 7-week liquid cell suspension (TPC: 29.9 and 32.1 mg GAE g⁻¹ DW; TFC: 40.5 and 56.1 mg QE g⁻¹ DW; TPP: 969.2 and 1122.6 mg L⁻¹; TFP: 1313.9 and 1521.7 mg L⁻¹; DPPH: 87.4 and 92.3%), respectively, while 40 mg L⁻¹ chitosan was equally effective regarding DW, TFC, and TFP in cell suspension. Chitosan elicitation provides a powerful strategy to upregulate phenolic and flavonoid biosynthesis in W. filifera in vitro systems, conferring superior antioxidant potential. The identification of peak elicitation parameters (chitosan concentration, exposure time) allows for the targeted enhancement of bioactive compound yields, suggesting a viable path for industrial bioproduction and commercialization in pharmaceuticals, nutraceuticals, and functional foods, leveraging bioreactor technology for efficient scale-up. Full article

The genus Plukenetia includes lianas or vines with oleaginous seeds rich in omega-3 and omega-6 fatty acids, proteins, and vitamin E, and the presence of flavonoids, steroids, and terpenoids has also been reported in leaves. Several species of Plukenetia have traditionally been cultivated in their native distribution areas, and their propagation is usually by seed. The aim of this work was to establish callus and cell suspension cultures of P. carabiaseae, an endemic species of Mexico, for the evaluation of the in vitro antioxidant and anti-inflammatory potential of its extracts. Three light conditions were evaluated for the establishment of P. carabiaseae callus lines from leaf explants. Friable calluses obtained under constant light were used to initiate a cell suspension cultures in Gamborg basal (B5) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (CIN), as growth regulators. After 35 days of cultivation, different polarity extracts from biomass were obtained, showing that the acetone extract had the highest antioxidant activity and a high total phenolic content (30.57 mg of gallic acid equivalent (GAE)/g dry weight). The anti-inflammatory activity of the methanolic extract, evaluated in murine macrophages induced with bacterial lipopolysaccharides, was dose-dependent, without cytotoxic effects. This is the first report of the establishment of P. carabiasiae cell suspension culture and demonstrates its potential as a biotechnological source of antioxidant and anti-inflammatory metabolites. Full article

Background: Inflammatory bowel diseases include Crohn’s disease (CD) and ulcerative colitis (UC). These diseases are characterized by periods of exacerbated inflammation of the gastrointestinal mucosa, interspersed with periods of remission. Current pharmacological interventions are only partially effective. There is a need for effective dietary therapies and interventions involving plant substances that can alleviate the course of this disease. Objectives: This study aimed to determine the effects of a 28-day dietary intervention involving a 3% solution of chemically pure, low-molar-mass oat beta-glucan (OBG) in patients diagnosed with de novo UC. Similar-aged men and women were compared. Methods: The OBG was isolated and prepared for consumption as a sterile aqueous suspension. This solution had previously been evaluated for in vitro toxicity using 3D intestinal co-cultures comprising Caco-2, HT29-MTX and THP-1 cells. Before and after the dietary intervention, endoscopic colon examinations were performed and blood hematological, biochemical and immunological parameters, as well as stool calprotectin concentrations, were analyzed. The Disease Activity Index (DAI), endoscopic Mayo score, the Lichtiger Colitis Activity Index (LCAI) and the neutrophil-to-lymphocyte ratio (NLR) were also determined. Following dietary intervention, the Mayo score, DAI, fecal calprotectin levels, and indices of peripheral blood white cells, CRP, and pro-inflammatory cytokine concentrations were decreased. Results/Conclusions: The obtained results demonstrated the beneficial effect of dietary intervention with OBG in accelerating the achievement of clinical remission in patients with UC. Full article

The purpose of this study was to present a chemical analysis of the metabolome of cell aggregates of the tree fern Cyathea smithii (J.D. Hooker) cell suspension culture. The LC/MS and GC/MS techniques were used for identification of metabolites. The kinetics of fresh weight, dry weight, and ash content showed 3.5-fold increases during 15-day-long culture. The analysis demonstrated high metabolic activity of cultured cells. In total, 160 metabolites from primary and secondary metabolism and almost 2000 compounds of unknown identity were identified. Three flavonoids—the chalcone isookanin [(2S)-2-(3,4-dihydroxyphenyl)-7,8-dihydroxy-2,3-dihydrochromen-4-one], a methoxy derivative of the flavone gardenin B (5-Hydroxy-2-(4-methoxyphenyl)-6,7,8-trimethoxy-4H-1-benzopyran-4-one), and the isoflavone tectoridin (4′,5-Dihydro-6-methoxy-7-(O-glucoside)isoflavone)—had not been previously detected in the cell culture of C. smithii. Principal component analysis revealed five distinct groups of samples; groups 4 and 5 showed the greatest similarity and corresponded to cultures on days 12 and 15, respectively. The number of differentiating compounds was 75, indicated by a heatmap showing positive and negative correlations between the days of culture. The studies described in this paper are crucial for further identification of metabolites and establishing the relationship between the metabolic composition of tree fern cells in culture and their biological activity, assessed by physiological parameters. By determining the relationship between the chemical composition of cells and their growth from culture initiation to senescence, we will provide a more complete picture of the potential for environmental factors to regulate this relationship. Based on previous studies, environmental stimuli such as electromagnetic fields or light of different wavelengths can result in altered growth physiology and cell mass, as well as metabolite diversification and accumulation. The research results presented in this paper provide a foundation for further studies aimed at predicting and regulating the productivity of C. smithii cells in suspension culture and elucidating the significance of tree fern-derived metabolic products in human cell biology, particularly in thyroid cells. Full article

Platelet shortage poses a significant barrier to research and transfusion therapies because native megakaryocytes (MKs) are scarce in blood. To overcome this limitation, pluripotent stem cell–derived MKs (PSC-MKs) offer a standardized, donor-independent platform for research and therapeutic development, including disease modeling and ex vivo platelet production. Here, we report a chemically defined, feeder-free protocol to generate MKs from human pluripotent stem cells (hPSCs). The protocol combines the small molecule MPL agonist Butyzamide, macrophage colony-stimulating factor (M-CSF), and three-dimensional (3D) suspension culture, achieving high efficiency and reproducibility. Butyzamide replaced recombinant thrombopoietin (TPO), yielding comparable CD41⁺/CD42b⁺ populations and enhanced polyploidization. M-CSF accelerated nuclear lobulation and induced 4N MKs, while 3D culture increased yield, cell size, and substrate detachment. Multiple independent assays confirmed mature MK hallmarks, multi-nuclei, demarcation membranes, granules, and elevated mitochondrial respiration. Single-cell RNA sequencing outlined a continuous trajectory from early progenitors to functionally specialized MK subsets. This platform enables reliable MK supply for mechanistic studies and in vitro platelet production, advancing both basic research and therapeutic development. Full article

Background: Bletilla striata is a medicinal orchid, whose bioactive constituent militarine has therapeutic interest but limited natural availability. Suspension culture coupled with transcriptomics offers a scalable production route and a means to uncover biosynthetic regulators. Methods: Four B. striata landraces were evaluated. Single-factor experiments and response surface methodology optimized sucrose, NH₄NO₃, and agitation to maximize biomass and militarine yield. Militarine and four related metabolites were quantified by HPLC-UV. For transcriptomics, RNA from high- and low-producing landraces was sequenced on Illumina HiSeq, assembled de novo, and analyzed with RSEM (FPKM) and DESeq2 to identify DEGs. Results: The landrace SMPF-NL achieved the highest militarine yield (33.06 mg/g) under optimized conditions (sucrose, 35 g/L; NH₄NO₃, 625 mg/L; agitation, 135 rpm; and half-strength MS medium with 1.0 mg/L of 6-BA, 3.0 mg/L of 2,4-D, and 0.5 mg/L of NAA). Transcriptomic profiling highlighted candidate biosynthetic and regulatory genes, including SuSy2, SUS, ALDO, AOC3, Comt, GOT2, MAOB, BGLU20, and BGLU22. Conclusions: We present an optimized suspension culture system and transcriptomic leads that lay the groundwork for the functional validation and scale-up of controlled militarine production. Full article

Head and neck cancer (HNC) is highly heterogeneous and difficult to treat, underscoring the need for rapid, patient-specific models. Standard three-dimensional (3D) cultures often lose stromal partners that influence therapy response. We developed a patient-derived system maintaining tumor cells, cancer-associated fibroblasts (CAFs), and cells undergoing partial epithelial–mesenchymal transition (pEMT) for drug sensitivity testing. Biopsies from four HNC patients were enzymatically dissociated. CAFs were directly cultured, and their conditioned medium (CAF-CM) was collected. Cryopreserved primary tumor cell suspensions were later revived, screened in five different growth media under 2D conditions, and the most heterogeneous cultures were re-embedded in 3D hydrogels with varied gel mixtures, media, and seeding geometries. Tumoroid morphology was quantified using a perimeter-based complexity index. Viability after treatment with cisplatin or Notch modulators (RIN-1, recombination signal-binding protein for immunoglobulin κ J region (RBPJ) inhibitor; FLI-06, inhibitor) was assessed by live imaging and the water-soluble tetrazolium-8 (WST-8) assay. Endothelial Cell Growth Medium 2 (ECM-2) medium alone produced compact CAF-free spheroids, whereas ECM-2 supplemented with CAF-CM generated invasive aggregates that deposited endogenous matrix. Matrigel with this medium and single-point seeding gave the highest complexity scores. Two of the three patient tumoroids were cisplatin-sensitive, and all showed significant growth inhibition with the FLI-06 Notch inhibitor, while the RBPJ inhibitor RIN-1 induced minimal change. The optimized scaffold retains tumor–stroma crosstalk and provides patient-specific drug response data within days after operation, supporting personalized treatment selection in HNC. Full article

Due to the increasing interest in probiotic components to improve quality of life, this study aimed to investigate the bioactive potential of a paraprobiotic derived from a selected strain of probiotic lactic acid bacteria (Lacticaseibacillus paracasei MIUG BL80) on Saccharomyces cerevisiae MIUG D129, used as a cellular model organism. The paraprobiotics (inactivated cells) were obtained through a combination of ultrasonic and conventional heat treatments. It was observed that adding more than 10 % of the paraprobiotic suspension to the cultivation medium of yeast had a positive influence on the metabolic activity of the starter culture (S. cerevisiae). The specific growth rate increased from 0.227 in the control sample to 0.507 in the sample with 15% paraprobiotic supplementation (S3), while the generation time decreased from 4.403 h to 1.972 h. This suggests that adding probiotics to the cultivation medium enhances the metabolic performance of S. cerevisiae cells. Additionally, an improvement in yeast cell viability during wet biomass storage (from 48 h to 14 days at 4 °C) was observed. Full article

Organoids allow to model healthy and diseased human tissues. and have applications in developmental biology, drug discovery, and cell therapy. Traditionally cultured in immersion/suspension, organoids face issues like lack of standardization, fusion, hypoxia-induced necrosis, continuous agitation, and high media volume requirements. To address these issues, we developed an air–liquid interface (ALi) technology for culturing organoids, termed AirLiwell. It uses non-adhesive microwells for generating and maintaining individualized organoids on an air–liquid interface. This method ensures high standardization, prevents organoid fusion, eliminates the need for agitation, simplifies media changes, reduces media volume, and is compatible with Good Manufacturing Practices. We compared the ALi method to standard immersion culture for midbrain organoids, detailing the process from human pluripotent stem cell (hPSC) culture to organoid maturation and analysis. Air–liquid interface organoids (3D-ALi) showed optimized size and shape standardization. RNA sequencing and immunostaining confirmed neural/dopaminergic specification. Single-cell RNA sequencing revealed that immersion organoids (3D-i) contained 16% fibroblast-like, 23% myeloid-like, and 61% neural cells (49% neurons), whereas 3D-ALi organoids comprised 99% neural cells (86% neurons). Functionally, 3D-ALi organoids showed a striking electrophysiological synchronization, unlike the heterogeneous activity of 3D-i organoids. This standardized organoid platform improves reproducibility and scalability, demonstrated here with midbrain organoids. The use of midbrain organoids is particularly relevant for neuroscience and neurodegenerative diseases, such as Parkinson’s disease, due to their high incidence, opening new perspectives in disease modeling and cell therapy. In addition to hPSC-derived organoids, the method’s versatility extends to cancer organoids and 3D cultures from primary human cells. Full article

Three-dimensional (3D) spheroid models have revolutionized in vitro cancer research by offering more physiologically relevant alternatives to traditional two-dimensional (2D) cultures. A systematic search identifies English-language studies on patient-derived cancer spheroids for drug screening, using defined inclusion and exclusion criteria, with data extracted on cancer type, culture methods, spheroid characteristics, and therapeutic responses. This manuscript evaluates the methods for spheroid formation and the cellular sources used, highlighting the diverse applications and preferences in this field. The five most investigated cancer origins for spheroid seeding are breast, colon, lung, ovary, and brain cancers, reflecting their clinical importance and research focus. Among seeding methodologies, forced-floating and scaffold-based methods predominate, demonstrating reliability and versatility in spheroid generation. Other techniques, including microfluidics, bioprinting, hanging drop, and suspension culture also play significant roles, each with distinct advantages and limitations. This review underscores the increasing use of spheroid models and the need for standardization in methodologies to enhance the reproducibility and translational potential in cancer research. Full article

Castanea mollissima Bl. is rich in nutrition and strong in stress resistance, and has nutritional, economic, and ecological values. A protoplast is impactful in somatic fusion and germplasm creation. Here, we propose an effective scheme for the construction of an embryonic suspension cell, protoplast isolation, and fusion. Studies have shown that when 1.0 g yellow loose embryonic callus was inoculated into MS + 1.5 mg∙L⁻¹ 6-BA + 0.2 mg∙L⁻¹ NAA + 0.5 mg∙L⁻¹ 2, 4-D liquid medium, a stable suspension cell line can be obtained. After further culturing for 2–4 days, protoplast isolation was performed. First, single-factor screening was conducted on the four enzymes, and then a two-factor random block was further set up to screen the enzyme combinations based on the results. We found that 1.0%cellulase R-10 + 0.5%pectolase Y-23 led to the highest protoplast yield (9.27 × 10⁶/g FW) and the highest activity (92.49%). Furthermore, the protoplast yield could be increased to 9.47 × 10⁶/g FW by adding 0.4 M mannitol and shaking for 8 h. The protoplasts were purified by centrifuging at 40× g for 4 min and then mixed with 30% PEG 6000 at a volume ratio of 1.5:1 for 25 min. The fusion rate could reach 70.00%. This study laid a foundation for the creation of new germplasm by Castanea mollissima Bl. Full article

Bioreactors enable scalable cell cultivation by providing controlled environments for temperature, oxygen, and nutrient regulation, maintaining viability and enhancing expansion efficiency. Automated systems improve reproducibility and minimize contamination risks, making them ideal for high-density cultures. While fed-batch bioreactors dominate biologics production, continuous systems like perfusion cultures offer superior resource efficiency and productivity. The Quantum hollow-fiber perfusion bioreactor supports cell expansion via semi-permeable capillary membranes and a closed modular design, allowing continuous media exchange while retaining key molecules. We developed a multiple-harvest protocol for suspension cells in the Quantum system, yielding 2.5 × 10¹⁰ MEL-745A cells within 29 days, with peak densities of 4 × 10⁷ cells/mL—a 15-fold increase over static cultures. Viability averaged 91.3%, with biweekly harvests yielding 3.1 × 10⁹ viable cells per harvest. Continuous media exchange required more basal media to maintain glucose and lactate levels but meaningfully less growth supplement than the 2D culture. Stable transgene expression suggested phenotypic stability. Automated processing reduced hands-on time by one-third, achieving target cell numbers 12 days earlier than 2D culture. Despite higher media use, total costs for the automated were lower compared to the manual process. Quantum enables high-density suspension cell expansion with cost advantages over conventional methods. Full article

Uniform electric field distribution in electroporation is crucial for achieving the effective localized delivery of drug molecules. Currently, in vitro electroporation studies on adherent cells lack a standardized test setup for obtaining consistent and repeatable results, unlike in vitro electroporation studies on cell suspensions that use electroporation cuvettes, which provide uniform electric field distribution. Considering this, we designed, built, and tested a novel inset design for low-volume round well plates, such as the 24- and 96-well plates which are most commonly used in cell culture labs. The inset design was realized using 3D printing and experimentally tested using potato phantoms and HeLa cells. Finite element analysis (FEA) was used to compute the electric field distribution in the round well plates with and without the inset. The FEA indicated that the electric field contour map at the bottom of the well with the inset had a more uniform electric field distribution, with an average value close to the expected 1000 V/cm. In contrast, it was only 840 V/cm without the inset, indicating non-uniform electric field distribution. Uniform electric fields were also obtained using the inset for the potato phantoms and the HeLa cells, indicating the merit of the inset and its usability with low-volume cell culture well plates, which enable the transfer of cells for various assays without additional steps, as well as its cost-effectiveness. Full article

Enterococcus faecalis (E. faecalis) has been associated with the specific production of l-(+)-lactic acid. However, in this study, d-(−)-lactic acid production by E. faecalis was observed under specific pH conditions. E. faecalis PR31 exhibited a significant amount of d-(−)-lactic acid under a stirring culture in MRS broth at pH 4.5, 5.8, and 6.0, and the contents of d-(−)-lactic acid were 45.1, 35.9, and 36.2%, respectively. When the cell suspension prepared at a pH of 6.0 was reacted with l-(+)- or d-(−)-lactic acid, d-(−)- or l-(+)-lactic acid was produced, respectively, in a time- and dose-dependent manner. Therefore, this phenomenon of d-(−)-lactic acid production in PR31 was suggested to be due to the activation of the larA gene encoding lactate racemase that is present in PR31. However, even in the E. faecalis-type strain NBRC 100480, which contains neither larA nor vanH, encoding d-(−)-lactate dehydrogenase VanH, d-(−)-lactic acid was also produced at specific pH values. Therefore, the production of d-(−)-lactic acid in NBRC 100480 was thought to occur not via the activation of larA. The biological significance of d-(−)-lactic acid production in E. faecalis depending on the pH and the detailed underlying mechanism, including whether it is the same in PR31 and NBRC 100480, remain to be elucidated in future studies. Full article