-
PloS One 2021To test the safety and efficacy of drugs via a high does drug heat map, a multi-spheroids array chip was developed by adopting a micropillar and microwell structure. In...
To test the safety and efficacy of drugs via a high does drug heat map, a multi-spheroids array chip was developed by adopting a micropillar and microwell structure. In the chip, patient-derived cells were encapsulated in alginate and grown to maturity for more than 7 days to form cancer multi-spheroids. Multi-spheroids grown in conventional well plates require many cells and are easily damaged as a result of multiple pipetting during maintenance culture or experimental procedures. To address these issues, we applied a micropillar and microwell structure to the multi-spheroids array. Patient-derived cells from patients with Glioblastoma (GBM), the most common and lethal form of central nervous system cancer, were used to validate the array chip performance. After forming multi-spheroids with a diameter greater than 100μm in a 12×36 pillar array chip (25mm × 75mm), we tested 70 drug compounds (6 replicates) using a high-dose to determine safety and efficacy for drug candidates. Comparing the drug response of multi-spheroids derived from normal cells and cancer cells, we found that four compounds (Dacomitinib, Cediranib, LY2835219, BGJ398) did not show toxicity to astrocyte cell and were efficacious to patient-derived GBM cells.
Topics: Antineoplastic Agents; Astrocytes; Cells, Cultured; Drug Screening Assays, Antitumor; Glioblastoma; High-Throughput Screening Assays; Humans; Primary Cell Culture; Spheroids, Cellular
PubMed: 34855773
DOI: 10.1371/journal.pone.0251998 -
Scientific Reports Jul 2021Recent statistics report that more than 3.7 million new cases of cancer occur in Europe yearly, and the disease accounts for approximately 20% of all deaths....
Recent statistics report that more than 3.7 million new cases of cancer occur in Europe yearly, and the disease accounts for approximately 20% of all deaths. High-throughput screening of cancer cell cultures has dominated the search for novel, effective anticancer therapies in the past decades. Recently, functional assays with patient-derived ex vivo 3D cell culture have gained importance for drug discovery and precision medicine. We recently evaluated the major advancements and needs for the 3D cell culture screening, and concluded that strictly standardized and robust sample preparation is the most desired development. Here we propose an artificial intelligence-guided low-cost 3D cell culture delivery system. It consists of a light microscope, a micromanipulator, a syringe pump, and a controller computer. The system performs morphology-based feature analysis on spheroids and can select uniform sized or shaped spheroids to transfer them between various sample holders. It can select the samples from standard sample holders, including Petri dishes and microwell plates, and then transfer them to a variety of holders up to 384 well plates. The device performs reliable semi- and fully automated spheroid transfer. This results in highly controlled experimental conditions and eliminates non-trivial side effects of sample variability that is a key aspect towards next-generation precision medicine.
Topics: Artificial Intelligence; Cell Culture Techniques; Cell Line, Tumor; Deep Learning; Drug Screening Assays, Antitumor; High-Throughput Screening Assays; Humans; Neoplasms; Precision Medicine; Spheroids, Cellular
PubMed: 34285291
DOI: 10.1038/s41598-021-94217-1 -
Life (Basel, Switzerland) Jun 2021Research in fields studying cellular response to surface tension and mechanical forces necessitate cell culture tools with tunability of substrate stiffness. We created...
Research in fields studying cellular response to surface tension and mechanical forces necessitate cell culture tools with tunability of substrate stiffness. We created a scalable hydrogel dish design to facilitate scaffold-free formation of multiple spheroids in a single dish. Our novel design features inner and outer walls, allowing efficient media changes and downstream experiments. The design is easily scalable, accommodating varying numbers of microwells per plate. We report that non-adherent hydrogel stiffness affects spheroid morphology and compaction. We found that spheroid morphology and viability in our hydrogel dishes were comparable to commercially available Aggrewell™800 plates, with improved tunability of surface stiffness and imaging area. Device function was demonstrated with a migration assay using two investigational inhibitors against EMT. We successfully maintained primary-derived spheroids from murine and porcine lungs in the hydrogel dish. These features increase the ability to produce highly consistent cell aggregates for biological research.
PubMed: 34204955
DOI: 10.3390/life11060517 -
Small (Weinheim An Der Bergstrasse,... Aug 2021Integrated microfluidic cellular phenotyping platforms provide a promising means of studying a variety of inflammatory diseases mediated by cell-secreted cytokines....
Integrated microfluidic cellular phenotyping platforms provide a promising means of studying a variety of inflammatory diseases mediated by cell-secreted cytokines. However, immunosensors integrated in previous microfluidic platforms lack the sensitivity to detect small signals in the cellular secretion of proinflammatory cytokines with high precision. This limitation prohibits researchers from studying cells secreting cytokines at low abundance or existing at a small population. Herein, the authors present an integrated platform named the "digital Phenoplate (dPP)," which integrates digital immunosensors into a microfluidic chip with on-chip cell assay chambers, and demonstrates ultrasensitive cellular cytokine secretory profile measurement. The integrated sensors yield a limit of detection as small as 0.25 pg mL for mouse tumor necrosis factor alpha (TNF-α). Each on-chip cell assay chamber confines cells whose population ranges from ≈20 to 600 in arrayed single-cell trapping microwells. Together, these microfluidic features of the dPP simultaneously permit precise counting and image-based cytometry of individual cells while performing parallel measurements of TNF-α released from rare cells under multiple stimulant conditions for multiple samples. The dPP platform is broadly applicable to the characterization of cellular phenotypes demanding high precision and high throughput.
Topics: Animals; Biosensing Techniques; Cytokines; Immunoassay; Mice; Microfluidic Analytical Techniques; Microfluidics; Tumor Necrosis Factor-alpha
PubMed: 34170616
DOI: 10.1002/smll.202101743 -
Methods in Molecular Biology (Clifton,... 2021Cells typically exist in a highly dynamic environment, which cannot easily be recreated in culture dishes or microwell plates. Microfluidic devices can provide precise...
Cells typically exist in a highly dynamic environment, which cannot easily be recreated in culture dishes or microwell plates. Microfluidic devices can provide precise control of the time, dose, and orientation of a stimulus, while simultaneously capturing quantitative single-cell data. The approach is particularly powerful when combined with the genetically tractable yeast model organism. The GPCR pathway in yeast is structurally conserved and functionally interchangeable with those in humans. We describe the implementation of a microfluidic device to investigate morphological and transcriptional responses of yeast to a gradient or pulse administration of a GPCR ligand, the peptide mating pheromone α-factor.
Topics: Ligands; Mating Factor; Microfluidics; Receptors, G-Protein-Coupled; Saccharomyces cerevisiae; Signal Transduction
PubMed: 34085275
DOI: 10.1007/978-1-0716-1221-7_18 -
International Journal of Nanomedicine 2021Nanomaterials for antimicrobial applications have gained interest in recent years due to the increasing bacteria resistance to conventional antibiotics. Wound...
PURPOSE
Nanomaterials for antimicrobial applications have gained interest in recent years due to the increasing bacteria resistance to conventional antibiotics. Wound sterilization, water treatment and surface decontamination all avail from multifunctional materials that also possess excellent antibacterial properties, eg zinc oxide (ZnO). Here, we assess and compare the effects of synthesized hedgehog-like ZnO structures and commercial ZnO particles with and without mixing on the inactivation of bacteria on surfaces and in liquid environments.
METHODS
Gram-positive () and Gram-negative () bacteria in microbial culture medium were added to reverse spin bioreactors that contained different concentrations of each ZnO type to enable dynamic mixing of the bacteria-ZnO suspensions. Optical density of the bacteria-ZnO suspensions was measured in real-time and the number of viable bacteria after 24 h exposure was determined using standard microbiological techniques. The concentration of zinc ion generated from ZnO dissolution in different liquid types was estimated from the dynamic interaction exposure. Static antibacterial tests without agitation in liquid media and on agar surface were performed for comparison.
RESULTS
A correlation between increasing ZnO particle concentration and reduction in viable bacteria was not monotonous. The lowest concentration tested (10 µg/mL) even stimulated bacteria growth. The hedgehog ZnO was significantly more antibacterial than commercial ZnO particles at higher concentrations (up to 1000 µg/mL tested), more against than . Minimum inhibitory concentration in microwell plates was correlated with those results. No inhibition was detected for any ZnO type deposited on agar surface. Zinc ion release was greatly suppressed in cultivation media. Scanning electron microscopy images revealed that ZnO needles can pierce membrane of bacteria whereas the commercial ZnO nanoparticles rather agglomerate on the cell surface.
CONCLUSION
The inhibition effects are thus mainly controlled by the interaction dynamics between bacteria and ZnO, where mixing greatly enhances antibacterial efficacy of all ZnO particles. The efficacy is modulated also by ZnO particle shapes, where hedgehog ZnO has superior effect, in particular at lower concentrations. However, at too low concentrations, ZnO can stimulate bacteria growth and must be thus used with caution.
Topics: Animals; Anti-Bacterial Agents; Biomimetic Materials; Escherichia coli; Hedgehogs; Microbial Sensitivity Tests; Staphylococcus aureus; Zinc Oxide
PubMed: 34079247
DOI: 10.2147/IJN.S300428 -
STAR Protocols Jun 2021This protocol describes how to produce human liver spheres from pluripotent stem cell-derived hepatic progenitors, endothelial cells, and hepatic stellate cells. Liver...
This protocol describes how to produce human liver spheres from pluripotent stem cell-derived hepatic progenitors, endothelial cells, and hepatic stellate cells. Liver spheres form by self-assembly in microwells, generating up to 73 spheres per well of a 96-well plate. This process was automated using liquid handling and pipetting systems, permitting cost-effective scale-up and reducing sphere variability. In its current form, this system provides a powerful tool to generate human liver tissue for disease modeling and drug screening. For complete details on the use and execution of this protocol, please refer to Lucendo-Villarin et al. (2020) (https://doi.org/10.1088/1758-5090/abbdb2).
Topics: Automation, Laboratory; Cell Culture Techniques; Cell Differentiation; Humans; Liver; Pluripotent Stem Cells; Spheroids, Cellular
PubMed: 33997816
DOI: 10.1016/j.xpro.2021.100502 -
Journal of Lasers in Medical Sciences 2020Photodynamic therapy (PDT) is a process that uses a light source (e.g. laser), oxygen molecules and a photosensitizing agent. PDT aims to act against pathogens,...
Photodynamic therapy (PDT) is a process that uses a light source (e.g. laser), oxygen molecules and a photosensitizing agent. PDT aims to act against pathogens, including those resistant to antimicrobials. The association of PDT with natural drugs, such as Propolis, has not been widely studied. Therefore, this study aimed to evaluate the antimicrobial effect of PDT in vitro by using Propolis as a photosensitizing agent. For this purpose, the dry Propolis extract was used as a photosensitizer and a low-power laser (Photon Laser III model) was irradiated onto the microwells for 90 seconds. Gram-positive and Gram-negative bacterial strains were used in the tests at a concentration of 5 × 10 CFU/mL. Initially, the antibacterial activity of the photosensitizers without laser action was determined by using a serial microdilution method before the experiment with a laser. After the incubation of the plates in a bacteriological oven, resazurin (0.1%) was added and the minimum inhibitory concentration (MIC) was determined. Alterations in the morphology of the bacteria were analysed by using atomic force microscopy (AFM). Bacteria were sensitive to Propolis with MICs ranging from 13.75 to 0.85 mg/mL, but no susceptibility was observed for methylene blue without laser application. A change was observed for MIC values of Propolis against after irradiation, which decreased from 1.71 mg/mL to 0.85 mg/mL. However, this behaviour was not observed in , the only gram-negative strain used. In addition, AFM images revealed alterations in the size of one of the bacteria tested. The Propolis is more active against gram-positive bacteria and PDT improved its activity against one of the strains tested.
PubMed: 33995978
DOI: 10.34172/jlms.2020.S17 -
Journal of Personalized Medicine Apr 2021Altered miRNA expression and DNA methylation have highly active and diverse roles in carcinogenesis. Simultaneous detection of the molecular aberrations may have a...
Altered miRNA expression and DNA methylation have highly active and diverse roles in carcinogenesis. Simultaneous detection of the molecular aberrations may have a synergistic effect on the diagnosis of malignancies. Herein, we develop a high-throughput assay for detecting multiple miRNAs and DNA methylation using droplet digital PCR (ddPCR) coupled with a 96-microwell plate. The microplate-based ddPCR could absolutely and reproducibly quantify 15 miRNAs and 14 DNA methylation sites with a high sensitivity (one copy/µL and 0.1%, respectively). Analyzing sputum and plasma of 40 lung cancer patients and 36 cancer-free smokers by this approach identified an integrated biomarker panel consisting of two sputum miRNAs (miRs-31-5p and 210-3p), one sputum DNA methylation (RASSF1A), and two plasma miRNAs (miR-21-5p and 126) for the diagnosis of lung cancer with higher sensitivity and specificity compared with a single type of biomarker. The diagnostic value of the integrated biomarker panel for the early detection of lung cancer was confirmed in a different cohort of 36 lung cancer patients and 39 cancer-free smokers. The high-throughput assay for quantification of multiple molecular aberrations across sputum and plasma could improve the early detection of lung cancer.
PubMed: 33946992
DOI: 10.3390/jpm11050359 -
ELife Apr 2021Powered by flagella, many bacterial species exhibit collective motion on a solid surface commonly known as swarming. As a natural example of active matter, swarming is...
Powered by flagella, many bacterial species exhibit collective motion on a solid surface commonly known as swarming. As a natural example of active matter, swarming is also an essential biological phenotype associated with virulence, chemotaxis, and host pathogenesis. Physical changes like cell elongation and hyper-flagellation have been shown to accompany the swarming phenotype. Less studied, however, are the contrasts of collective motion between the swarming cells and their counterpart planktonic cells of comparable cell density. Here, we show that confining bacterial movement in circular microwells allows distinguishing bacterial swarming from collective swimming. On a soft agar plate, a novel bacterial strain sp. SM3 in swarming and planktonic states exhibited different motion patterns when confined to circular microwells of a specific range of sizes. When the confinement diameter was between 40 μm and 90 μm, swarming SM3 formed a single-swirl motion pattern in the microwells whereas planktonic SM3 formed multiple swirls. Similar differential behavior is observed across several other species of gram-negative bacteria. We also observed 'rafting behavior' of swarming bacteria upon dilution. We hypothesize that the rafting behavior might account for the motion pattern difference. We were able to predict these experimental features via numerical simulations where swarming cells are modeled with stronger cell-cell alignment interaction. Our experimental design using PDMS microchip disk arrays enabled us to observe bacterial swarming on murine intestinal surface, suggesting a new method for characterizing bacterial swarming under complex environments, such as in polymicrobial niches, and for in vivo swarming exploration.
Topics: Animals; Bacterial Load; Cluster Analysis; Colitis; Computer Simulation; Dextran Sulfate; Disease Models, Animal; Enterobacter; Female; Flagella; Gastrointestinal Microbiome; Host-Pathogen Interactions; Intestinal Mucosa; Mice, Inbred C57BL; Models, Theoretical; Movement; Numerical Analysis, Computer-Assisted; Phenotype; Mice
PubMed: 33884952
DOI: 10.7554/eLife.64176