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Nature Biotechnology Nov 2023Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop...
Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method that does not require specialized microfluidic devices, expertise or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multiomics measurements and can accurately characterize cell types in human breast tissue compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible and scalable next-generation workflow that extends single-cell sequencing to new applications.
Topics: Humans; Animals; Mice; Microfluidics; High-Throughput Nucleotide Sequencing; Single-Cell Analysis; Genomics; Transcriptome
PubMed: 36879006
DOI: 10.1038/s41587-023-01685-z -
Frontiers in Bioengineering and... 2021Microbial resource mining of electroactive microorganism (EAM) is currently methodically hampered due to unavailable electrochemical screening tools. Here, we introduce...
Microbial resource mining of electroactive microorganism (EAM) is currently methodically hampered due to unavailable electrochemical screening tools. Here, we introduce an electrochemical microwell plate (ec-MP) composed of a 96 electrochemical deepwell plate and a recently developed 96-channel multipotentiostat. Using the ec-MP we investigated the electrochemical and metabolic properties of the EAM models and with acetate and lactate as electron donor combined with an individual genetic analysis of each well. Electrochemical cultivation of pure cultures achieved maximum current densities ( ) and coulombic efficiencies () that were well in line with literature data. The co-cultivation of and led to an increased current density of of 88.57 ± 14.04 µA cm (lactate) and of 99.36 ± 19.12 µA cm (lactate and acetate). Further, a decreased time period of reaching and biphasic current production was revealed and the microbial electrochemical performance could be linked to the shift in the relative abundance.
PubMed: 35242754
DOI: 10.3389/fbioe.2021.821734 -
International Journal of Biological... 2020Despite significant advances in parallel single-cell RNA sequencing revealing astonishing cellular heterogeneity in many tissue types, the spatial information in the... (Comparative Study)
Comparative Study Review
Despite significant advances in parallel single-cell RNA sequencing revealing astonishing cellular heterogeneity in many tissue types, the spatial information in the tissue context remains missing. Spatial transcriptome sequencing technology is designed to distinguish the gene expression of individual cells in their original location. The technology is important for the identification of tissue function, tracking developmental processes, and pathological and molecular detection. Encoding the position information is the key to spatial transcriptomics because different methods have different encoding efficiencies and application scenarios. In this review, we focus on the latest technologies of single-cell spatial transcriptomics, including technologies based on microwell plates, barcoded bead arrays, microdissection, hybridization, and barcode targeting, as well as mixed separation-based technologies. Moreover, we compare these encoding methods for use as a reference when choosing the appropriate technology.
Topics: Gene Expression Profiling; Genomics; Humans; Sequence Analysis, RNA; Single-Cell Analysis; Transcriptome
PubMed: 32792863
DOI: 10.7150/ijbs.43887 -
Pharmaceutics Jan 2021High-throughput light scattering instruments are widely used in screening of biopharmaceutical formulations and can be easily incorporated into processes by utilizing...
High-throughput light scattering instruments are widely used in screening of biopharmaceutical formulations and can be easily incorporated into processes by utilizing multi-well plate formats. High-throughput plate readers are helpful tools to assess the aggregation tendency and colloidal stability of biological drug candidates based on the diffusion self-interaction parameter (). However, plate readers evoke issues about the precision and variability of determined data. In this article, we report about the statistical evaluation of intra- and inter-plate variability (384-well plates) for the analysis of protein and peptide solutions. ANOVA revealed no significant differences between the runs. In conclusion, the reliability and precision of was dependent on the plate position of the sample replicates and value. Positive values (57.0 mL/g, coefficients of variation () 8.9%) showed a lower variability compared to negative values (-14.8 mL/g, 13.4%). The variability of was not reduced using more data points (120 vs. 30). A analysis exclusively based on center wells showed a lower (<2%) compared to edge wells (5-12%) or a combination of edge and center wells (2-5%). We present plate designs for analysis within the early formulation development, screening up to 20 formulations consuming less than 50 mg of active pharmaceutical ingredient (API).
PubMed: 33514069
DOI: 10.3390/pharmaceutics13020172 -
Applied Microbiology and Biotechnology Jul 2023Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They...
Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They appear as by-product from meat production, but several challenges are related to their application: first, the high melting points (up to 60 °C); and second, the insolubility in the polar water phase of cultivations. This leads to film and clump formation in shake flasks and microwell plates, which inhibits microbial consumption. In this study, different flask and well designs were investigated to identify the most suitable experimental set-up and further to create an appropriate workflow to achieve the required reproducibility of growth and product synthesis. The dissolved oxygen concentration was measured in-line throughout experiments. It became obvious that the gas mass transfer differed strongly among the shake flask design variants in cultivations with the polyhydroxyalkanoate (PHA) accumulating organism Ralstonia eutropha. A high reproducibility was achieved for certain flask or well plate design variants together with tailored cultivation conditions. Best results were achieved with bottom baffled glass and bottom baffled single-use shake flasks with flat membranes, namely, >6 g L of cell dry weight (CDW) with >80 wt% polyhydroxybutyrate (PHB) from 1 wt% WAF. Improved pre-emulsification conditions for round microwell plates resulted in a production of 14 g L CDW with a PHA content of 70 wt% PHB from 3 wt% WAF. The proposed workflow allows the rapid examination of fat material as feedstock, in the microwell plate and shake flask scale, also beyond PHA production. KEY POINTS: • Evaluation of shake flask designs for cultivating with hydrophobic raw materials • Development of a workflow for microwell plate cultivations with hydrophobic raw materials • Production of polyhydroxyalkanoate in small scale experiments from waste animal fat.
Topics: Animals; Polyhydroxyalkanoates; Reproducibility of Results; Workflow; Bioreactors
PubMed: 37266584
DOI: 10.1007/s00253-023-12599-w -
Molecules (Basel, Switzerland) May 2023This study describes the development and validation of a new green and high-throughput microwell spectrophotometric assay (MW-SPA) for the determination of three...
Development and Validation of Green and High-Throughput Microwell Spectrophotometric Assay for the Determination of Selective Serotonin Reuptake Inhibitors in Their Pharmaceutical Dosage Forms.
This study describes the development and validation of a new green and high-throughput microwell spectrophotometric assay (MW-SPA) for the determination of three selective serotonin reuptake inhibitors (SSRIs) in their pharmaceutical dosage forms. These SSRIs are fluoxetine, fluvoxamine, and paroxetine, the most prescribed drugs for the treatment of depression. The proposed assay was based on the formation of orange-colored -substituted naphthoquinone derivatives upon the reaction of SSRIs with 1,2-naphthoquinone-4-sulphonate (NQS) in alkaline media. The assay was conducted in 96-microwell assay plates, and the absorbances of the reaction products were measured by a microplate reader at their maximum absorbance wavelengths. The optimum conditions of the reaction were refined and established. Under these conditions, calibration curves were generated, and linear regression equations were computed. The linear relations between the absorbances and drug concentrations were linear with good correlation coefficients (0.9992-0.9997) in the range of 2-80 µg/mL. The assay limits of detection were in the range of 1.5-4.2 µg/mL. The precision was satisfactory as the values of relative standard deviation did not exceed 1.70%. The accuracy of the assay was ≥98.2%. The proposed MW-SPA was successfully applied to the analysis of the SSRIs in their pharmaceutical dosage forms with acceptable accuracy and precision; the label claims were 99.2-100.5% (±0.96-1.35%). The results of the proposed MW-SPA were compared with those of the official/pre-validated assays by statistical analysis with respect to the accuracy (by -test) and precision (by F-test). No significant differences were found between the calculated and theoretical values of the t- and F-tests at the 95% confidence level, proving similar accuracy and precision in the determination of SSRIs by both assays. The greenness of the proposed assay was confirmed by two metric tools. In addition, the assay is characterized with a high-throughput property which enables the simultaneous analysis of many samples in a short time. Therefore, the assay is a valuable tool for rapid routine application in pharmaceutical quality control units for the determination of the investigated SSRIs.
Topics: Selective Serotonin Reuptake Inhibitors; Spectrophotometry; Fluoxetine; Fluvoxamine; Pharmaceutical Preparations
PubMed: 37241961
DOI: 10.3390/molecules28104221 -
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 -
Scientific Reports Mar 2022Pluripotent stem-cell derived cells can be used for type I diabetes treatment, but we require at least 10-10 islet-like clusters per patient. Although thousands of...
Pluripotent stem-cell derived cells can be used for type I diabetes treatment, but we require at least 10-10 islet-like clusters per patient. Although thousands of uniform cell clusters can be produced using a conventional microwell plate, numerous obstacles need to be overcome for its clinical use. In this study, we aimed to develop a novel bag culture method for the production of uniform cell clusters on a large scale (10-10 clusters). We prepared small-scale culture bags (< 10 clusters) with microwells at the bottom and optimized the conditions for producing uniform-sized clusters in the bag using undifferentiated induced pluripotent stem cells (iPSCs). Subsequently, we verified the suitability of the bag culture method using iPSC-derived pancreatic islet cells (iPICs) and successfully demonstrate the production of 6.5 × 10 uniform iPIC clusters using a large-scale bag. In addition, we simplified the pre- and post-process of the culture-a degassing process before cell seeding and a cluster harvesting process. In conclusion, compared with conventional methods, the cluster production method using bags exhibits improved scalability, sterility, and operability for both clinical and research use.
Topics: Cell Differentiation; Diabetes Mellitus, Type 1; Humans; Induced Pluripotent Stem Cells; Pluripotent Stem Cells
PubMed: 35338209
DOI: 10.1038/s41598-022-09124-w -
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 -
Molecules (Basel, Switzerland) Mar 2023This study describes the development of a one-step microwell spectrofluorimetric assay (MW-SFA) with high sensitivity and throughput for the determination of four...
One-Step Microwell Plate-Based Spectrofluorimetric Assay for Direct Determination of Statins in Bulk Forms and Pharmaceutical Formulations: A Green Eco-Friendly and High-Throughput Analytical Approach.
This study describes the development of a one-step microwell spectrofluorimetric assay (MW-SFA) with high sensitivity and throughput for the determination of four statins in their pharmaceutical and formulations (tablets). These statins were pitavastatin (PIT), fluvastatin (FLU), rosuvastatin (ROS) and atorvastatin (ATO). The MW-SFA involves the measurement of the native fluorescence of the statin aqueous solutions. The assay was conducted in white opaque 96-microwell plates, and the fluorescence intensities of the solutions were measured by using a fluorescence microplate reader. The optimum conditions of the assay were established; under which, linear relationships with good correlation coefficients (0.9991-0.9996) were found between the fluorescence intensity and the concentration of the statin drug in a range of 0.2-200 µg mL with limits of detection in a range of 0.1-4.1 µg mL. The proposed MW-SFA showed high precision, as the values of the relative standard deviations did not exceed 2.5%. The accuracy of the assay was proven by recovery studies, as the recovery values were 99.5-101.4% (±1.4-2.1%). The assay was applied to the determination of the investigated statins in their tablets. The results were statistically compared with those obtained by a reference method and the results proved to have comparable accuracy and precision of both methods, as evidenced by the t- and F-tests, respectively. The green and eco-friendly feature of the proposed assay was assessed by four different metric tools, and all the results proved that the assay meets the requirements of green and eco-friendly analytical approaches. In addition, ever-increasing miniaturization as handling of large numbers of micro-volume samples simultaneously in the proposed assay gave it a high-throughput feature. Therefore, the assay is a valuable tool for the rapid routine application in the pharmaceutical quality control units for the determination of statins.
Topics: Hydroxymethylglutaryl-CoA Reductase Inhibitors; Drug Compounding; Spectrometry, Fluorescence; Tablets
PubMed: 36985779
DOI: 10.3390/molecules28062808