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With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24- and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health.

We discuss the current challenges and future prospects of flow-based organoid models and 3D self-assembling scaffolds. The existing paradigm of 3D culture suffers from a lack of control over organoid size and shape; can be an obstacle for cell harvesting and extended cellular and molecular analysis; and does not provide access to the function of exocrine glands. Moreover, existing organ-on-chip models are mostly composed of 2D extracellular matrix (ECM)-coated elastomeric membranes that do not mimic real organ architectures. A new comprehensive 3D toolbox for cell biology has emerged to address some of these issues. Advances in microfabrication and cell-culturing approaches enable the engineering of sophisticated models that mimic organ 3D architectures and physiological conditions, while supporting flow-based drug screening and secretomics-based diagnosis. Microfluidics and microfabrication have revolutionized the way in which cells can be studied and manipulated in systems that are starting to provide 3D models and organ-on-chip devices. Individual-organ models and multiple-organ interaction models address the issue of how microengineered approaches can faithfully reproduce key elements of physiologically relevant microenvironments. The innovative technical nature of such 3D systems opens up exciting possibilities of answering several important fundamental biological questions that cannot be addressed with standard culture conditions. In the race to closely mimic the structural and physiological functions of human tissues and organs, new possibilities have emerged in the form of 3D organ-level structures that integrate dynamic mechanical cues as well as chemical signals.

Despite microalgae recently receiving enormous attention as a potential source of biodiesel, their use is still not feasible as an alternative to fossil fuels. Recently, interest in microalgae has focused on the production of bioactive compounds such as polyunsaturated fatty acids (PUFA), which provide microalgae a high added value. Several considerations need to be assessed for optimizing PUFA production from microalgae. Firstly, a microalgae species that produces high PUFA concentrations should be selected, such as Nannochloropsis gaditana , Isochrysis galbana , Phaeodactylum tricornutum , and Crypthecodinium cohnii , with marine species gaining more attention than do freshwater species. Closed cultivation processes, e.g., photobioreactors, are the most appropriate since temperature, pH, and nutrients can be controlled. An airlift column with LEDs or optical fibers to distribute photons into the culture media can be used at small scale to produce inoculum, while tubular and flat panels are used at commercial scale. Depending on the microalgae, a temperature range from 15 to 28 °C and a pH from 7 to 8 can be employed. Relevant conditions for PUFA production are medium light irradiances (50–300 μmol photons m −2  s −1 ), air enriched with (0–1 % ( v / v ) CO 2 , as well as nitrogen and phosphorous limitation. For research purposes, the most appropriate medium for PUFA production is Bold’s Basal, whereas mixotrophic cultivation using sucrose or glucose as the carbon source has been reported for industrial processes. For cell harvesting, the use of tangential flow membrane filtration or disk stack centrifugation is advisable at commercial scale. Current researches on PUFA extraction have focused on the use of organic solvents assisted with ultrasound or microwaves, supercritical fluids, and electroporation or are enzyme assisted. Commercial-scale extraction involves mainly physical methods such as bead mills and expeller presses. All these factors should be taken into account when choosing a PUFA production system, as discussed in this review.

PurposeDendritic cell (DC) is a spearhead responsible for immune response and surrounded by extracellular matrix in three-dimensional (3D) tissue. Nevertheless, conventional DC culture has relied on suspension or two-dimensional (2D) tissue culture plate (TCP)-based culture system. This culture condition often fails to recapitulate the physiological behavior of DC in real tissue. In this work, the effect of culture condition on DC physiology was explored with varying 3D hydrogel property (i.e., degradability, adhesion, and stiffness). In particular, DC differentiation and maturation in 3D were evaluated comparing the conventional TCP-based culture condition.MethodTHP-1 cells were encapsulated in poly(ethylene glycol) (PEG) hydrogel via thiol-ene photocrosslinking with non-degradable or proteolytically degradable peptide crosslinker. Hydrogel stiffness was manipulated by controlling the concentration of crosslinker. The metabolic activities and cytotoxicity of the encapsulated cells were measured by resazurin and Live/Dead assays, respectively. Cell harvesting was conducted via enzymatic degradation using α-chymotrypsin, and differentiation and maturation of the liberated DCs were evaluated by quantitative polymerase chain reaction and flow cytometry.ResultsTHP-1 cells well proliferated in the soft degradable hydrogel with a higher metabolic activity. However, the stiff matrix inhibited cell growth in 3D. The gene expression assay indicated that the 3D hydrogel condition was superior to 2D culture in terms of differentiation and maturation of DC. Interestingly, the stiffness of matrix was important factor in DC function. In the stiff hydrogel, the expression levels of differentiation and maturation markers were higher compared to the low stiffness hydrogel. The mature DCs caged in the hydrogel matrix were harvested after short enzymatic digestion of hydrogel and the liberated cells had over 90% viability. The flow cytometric result revealed that the proportion of CD80 + /CD86 + cells from the stiff hydrogel was relatively higher than cells either from 2D or soft hydrogel in 3D.ConclusionThe collected evidence indicated that the proteolytically degradable PEG hydrogel matrix promoted DC differentiation and maturation. In addition, the matrix stiffness control could manipulate the marker expressions of differentiation and maturation. Particularly, the mature DC was successfully collected from the hydrogel matrix. These results highlighted the PEG hydrogel-based DC culture might be a useful tool for potential DC-based immunotherapies.

MS-based metabolite profiling of adherent mammalian cells comprises several challenging steps such as metabolism quenching, cell detachment, cell disruption, metabolome extraction, and metabolite measurement. In LC-MS, the final metabolome coverage is strongly determined by the separation technique and the MS conditions used. Human liver-derived cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (−) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids. Graphical abstract A novel analytical workflow for the LC-MS-based metabolomic analysis of adherent cultured hepatic cells was developed. Three key steps were evaluated: a) cell harvesting, which includes cell detachment and metabolism quenching; b) metabolite extraction; and c) LC-MS analysis, assessing the use of RP and HILIC chromatographies. The final protocol allowed us to extend the metabolome coverage and enabled the detection of a wide range of metabolites from highly polar compounds to a wide range of lipids

Annexin V and propidium iodide (PI) dual staining is commonly applied in bioscience as a method to detect apoptosis. However, excessive handling of adherent cells may interfere with the integrity of plasma membrane and hence impede the accuracy of this method. Here, we exploited PI uptake as an indicator of cell integrity and investigated how cell harvesting methods and solutions involved in common apoptosis detection techniques affected measurement results. Different cell harvesting techniques, staining with PI and flow cytometry were performed. Non-fixed scrapped cells revealed significantly higher fractions of PI-positive staining compared to non-fixed trypsinized cells. In the case of harvesting cells by scrapping, samples stained in binding buffer (68.30±3.55%) showed consistently higher PI-positive staining than samples stained in PBS (36.37±5.90%) in a significant manner (p=0.015). Enzymatic harvesting using 0.25% trypsin instead of mechanical harvesting by rubber scraper caused less damage of cell integrity. Furthermore, the binding buffer used in the apoptosis detection protocol aggravated the existing plasma membrane damage caused by the rubber scraper.

Pichia pastoris expression system was introduced with post-translation process similar to higher eukaryotes. Preliminary studies were performed toward process intensification and magnetic immobilization of this system. In this experiment, effects of magnetic immobilization on the structure of recombinant protein were evaluated. P. pastoris cell which express human serum albumin (HSA) was used as a model. The cells were immobilized with various concentrations of APTES coated magnetite nanoparticles. HSA production was done over 5 days induction and structure of the product was analyzed by UV–vis, fluorescence, and ATR-FTIR spectroscopy. Second derivative deconvolution method was used to analyze the secondary structure of HSA. P. pastoris cell that were immobilized with 0.5 and 1 mg/mL of nanoparticles were produced HSA with intact structure. But immobilization with 2 mg/mL of nanoparticles resulted in some modifications in the secondary structures (i.e., α-helixes and β-turns) of produced HSA. Based on these data, immobilization of P. pastoris cells with 0.5 or 1 mg/mL of nanoparticles is completely efficient for cell harvesting and has any effect on the structure of recombinant product. These findings revealed that decoration of microbial cells with high concentrations of nanoparticles has some impacts on the structure of secretory proteins.

Advanced cell-based therapies, including immunotherapy, regenerative medicine, and other biotechnological applications, require large quantities of viable mammalian cells for research and clinical use. Conventional enzymatic harvesting methods, such as trypsini-zation, can compromise cell integrity and reduce viability. This study investigates an al-ternative temperature-responsive approach using alginate beads incorporated with poly(N-isopropylacrylamide) (PNIPAAm), a polymer exhibiting a lower critical solution temperature (LCST) of approximately 32 °C. This system enables temperature-controlled cell detachment while preserving cellular structure and extracellular matrix components, thereby potentially improving post-harvest viability compared to trypsin treatment. Ho-mogeneous alginate hydrogel beads were synthesized using a standard infusion pump and ionically crosslinked with calcium cations. The beads were characterized by scanning electron microscopy (SEM) for morphology and by Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and micro-computed tomography (µ-CT) for compositional and thermal analysis. Mouse fibroblast cells (L929 cell line) were cultured on the beads, and their proliferation and viability were assessed using CCK-8 and Live/Dead assays, demonstrating significant cell growth over seven days. The results suggest that PNIPAAm-modified alginate beads provide a promising, enzyme-free platform for efficient mammalian cell harvesting and delivery, with potential applications across advanced cell manufacturing and therapeutic technologies.

During the heterotrophic cultivation of microalgae, a cooled process against temperature rise caused by the metabolism of exogenous organic carbon sources greatly increases cultivation cost. Furthermore, microalgae harvesting is also a cost-consuming process. Cell harvesting efficiency is closely related to the characteristics of the algal cells. It may be possible to change cell characteristics through controlling culture conditions to make harvesting easier. In this study, the mesophilic Chlorella pyrenoidosa was found to be a thermal-tolerant species in the heterotrophic mode. The cells could maintain their maximal specific growth rate at 40°C and reached 1.45 day −1 , which is equivalent to that of cultures at 35°C but significantly higher than those cultured at lower temperatures. Interestingly, the cells cultured at 40°C were much easier to be harvested than those at lower temperatures. The harvesting efficiency of the cells cultured at 40°C reached 96.83% after sedimentation for 240 min, while the cells cultured at lower temperatures were reluctant to settle. Likely, the same circumstance occurred when cells were harvested by centrifugation or flocculation. The promotion of cell harvesting for cells cultured at high temperatures was mainly attributed to increased cell size and decreased cell surface charge. To the best of our knowledge, this is the first report that cells cultured at high temperatures can promote microalgae harvesting. This study explores a new approach to simplify the cultivation and harvesting of microalgae, which effectively reduces the microalgae production cost.

The present study reports the optimization of a green method for the synthesis of silver nanoparticles (AgNPs) via reduction of Ag+ ions using cell-free supernatant of mutant Bacillus licheniformis M09. UV–Visible spectroscopy showing an absorption peak at ~ 430 nm confirmed the synthesis of AgNPs. Transmission electron microscope (TEM) analysis exhibited spherical AgNPs within the size range of 10–30 nm. Fourier transform infrared (FTIR) measurements assured the presence of effective functional molecules which could be responsible for stabilizing the AgNPs. X-ray diffraction (XRD) pattern verified the crystalline nature of AgNPs. Furthermore, the synthesized AgNPs showed an excellent photocatalytic degradation of methylene blue dye in less than 3 h under visible light proving their potential as a catalytic agent for bioremediation for next-generation dye degradation in effluent treatment. The AgNPs demonstrated antimicrobial activity against Gram-positive and Gram-negative foodborne pathogens which endorsed its suitability as agents to extend shelf-life in food packaging and food safety applications. The results also revealed a strong concentration-dependent cytotoxicity of AgNPs against human breast adenocarcinoma cells (MCF-7), while 15.07 µg/mL of IC50 was attained. The outcome suggests the possible application of these AgNPs in nanomedicine formulations. Thus, these findings propose promising ways for the valorization of the waste fermentation supernatant left after cell harvesting and desired metabolite extraction.Graphical abstract