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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 -
Nature Protocols May 2023Human early development sets the stage for embryonic and adult life but remains difficult to investigate. A solution came from the ability of stem cells to organize into... (Review)
Review
Human early development sets the stage for embryonic and adult life but remains difficult to investigate. A solution came from the ability of stem cells to organize into structures resembling preimplantation embryos-blastocysts-that we termed blastoids. This embryo model is available in unlimited numbers and could thus support scientific and medical advances. However, its predictive power depends on how faithfully it recapitulates the blastocyst. Here, we describe how we formed human blastoids that (1) efficiently achieve the morphology of the blastocyst and (2) form lineages according to the pace and sequence of blastocyst development, (3) ultimately forming cells that transcriptionally reflect the blastocyst (preimplantation stage). We employ three different commercially available 96- and 24-well microwell plates with results similar to our custom-made ones, and show that blastoids form in clinical in vitro fertilization medium and can be cryopreserved for shipping. Finally, we explain how blastoids replicate the directional process of implantation into endometrial organoids, specifically when these are hormonally stimulated. It takes 4 d for human blastoids to form and 10 d to prepare the endometrial implantation assay, and we have cultured blastoids up to 6 d (time-equivalent of day 13). On the basis of our experience, we anticipate that a person with ~1 year of human pluripotent stem cell culture experience and of organoid culture should be able to perform the protocol. Altogether, blastoids offer an opportunity to establish scientific and biomedical discovery programs for early pregnancy, and an ethical alternative to the use of embryos.
Topics: Pregnancy; Adult; Female; Humans; Embryo Implantation; Blastocyst; Embryonic Development; Embryo, Mammalian; Cryopreservation
PubMed: 36792779
DOI: 10.1038/s41596-023-00802-1 -
Methods in Molecular Biology (Clifton,... 2021Multiplex immunoassays are important tools in basic research and diagnostics. The ability to accurately quantify the presence of several antigens within an individual...
Multiplex immunoassays are important tools in basic research and diagnostics. The ability to accurately quantify the presence of several antigens within an individual sample all at once has been useful in developing a proteomics view of biology. This in turn has enabled the development of disease-associated immunodiagnostic panels for better prognosis and well-being. Moreover, it is well understood that such multiplexing approaches lend themselves to automation, thereby reducing labor while providing the ability to dramatically conserve both reagent and sample all of which will reduce the cost per test. Here we describe various methods to create and use multiplex immunoassays in the wells of microtiter plates or similar formats.
Topics: Animals; Automation, Laboratory; Humans; Immunoenzyme Techniques; Immunologic Tests; Protein Array Analysis; Sensitivity and Specificity
PubMed: 33237405
DOI: 10.1007/978-1-0716-1064-0_2 -
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 -
Expert Review of Molecular Diagnostics Jul 2022Enzyme-linked immunosorbent assay (ELISA) is a key bio-analytical technique used for the detection of a large array of antigenic substances of scientific, clinical, food... (Review)
Review
INTRODUCTION
Enzyme-linked immunosorbent assay (ELISA) is a key bio-analytical technique used for the detection of a large array of antigenic substances of scientific, clinical, food safety, and environmental importance. The assay primarily involves capturing and detecting target analytes using specific antigen-antibody interactions. The wide usage of ELISA results from its high specificity and reproducibility. Notwithstanding, the conventional microwell plate-based format of ELISA has some major drawbacks, such as long assay time (4-18 h), large sample volumes requirement (100-200 μL), lack of multiplicity, and burdensome procedures that limit its utility in rapid and affordable diagnostics.
AREAS COVERED
Here, we reviewed microfluidic-ELISA, paper-ELISA, aptamer-ELISA, and those based on novel incubation such as heat-ELISA, pressure-ELISA, microwave-ELISA, and sound-ELISA. Further, the current trends and future prospects of these ELISA protocols in clinical diagnostics are discussed.
EXPERT OPINION
The reviewed non-conventional ELISA formats are relatively rapid, require low reagent volumes, are multiplexable, and could be performed in a low-cost setup. In our opinion, these non-conventional variants of ELISA are on a par with the conventional format for clinical diagnostics and fundamental biological research and hold added clinical translational potential for quick, inexpensive, and convenient measurements.
Topics: Enzyme-Linked Immunosorbent Assay; Humans; Microfluidics; Reproducibility of Results
PubMed: 36004453
DOI: 10.1080/14737159.2022.2117615 -
Small (Weinheim An Der Bergstrasse,... Mar 2020Single-cell analysis offers unprecedented resolution for the investigation of cellular heterogeneity and the capture of rare cells from large populations. Here,...
Single-cell analysis offers unprecedented resolution for the investigation of cellular heterogeneity and the capture of rare cells from large populations. Here, described is a simple method named interfacial nanoinjection (INJ), which can miniaturize various single-cell assays to be performed in nanoliter water-in-oil droplets on standard microwell plates. The INJ droplet handler can adjust droplet volumes for multistep reactions on demand with high precision and excellent monodispersity, and consequently enables a wide range of single-cell assays. Importantly, INJ can be coupled with fluorescence-activated cell sorting (FACS), which is currently the most effective and accurate single-cell sorting and isolation method. FACS-INJ pipelines for high-throughput plate well-based single-cell analyses, including single-cell proliferation, drug-resistance testing, polymerase chain reaction (PCR), reverse-transcription PCR, and whole-genome sequencing are introduced. This FACS-INJ pipeline is compatible with a wide range of samples and can be extended to various single-cell analysis applications in microbiology, cell biology, and biomedical diagnostics.
Topics: Cell Separation; Flow Cytometry; Miniaturization; Nanotechnology; Polymerase Chain Reaction; Single-Cell Analysis
PubMed: 31565845
DOI: 10.1002/smll.201903739 -
Journal of Visualized Experiments : JoVE Jun 2023Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment....
Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 µm diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.
Topics: Humans; Pluripotent Stem Cells; Islets of Langerhans; Cell Differentiation; Insulin-Secreting Cells; Insulins
PubMed: 37427943
DOI: 10.3791/64840 -
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 -
Small (Weinheim An Der Bergstrasse,... Nov 2023Droplet array is widely applied in single cell analysis, drug screening, protein crystallization, etc. This work proposes and validates a method for rapid formation of...
Droplet array is widely applied in single cell analysis, drug screening, protein crystallization, etc. This work proposes and validates a method for rapid formation of uniform droplet array based on microwell confined droplets electro-coalescence of screen-printed emulsion droplets, namely electro-coalescence droplet array (ECDA). The electro-coalescence of droplets is according to the polarization induced electrostatic and dielectrophoretic forces, and the dielectrowetting effect. The photolithographically fabricated microwells are highly regular and reproducible, ensuring identical volume and physical confinement to achieve uniform droplet array, and meanwhile the microwell isolation protects the paired water droplets from further fusion and broadens its feasibility to different fluidic systems. Under optimized conditions, a droplet array with an average diameter of 85 µm and a throughput of 10 in a 10 cm × 10 cm chip can be achieved within 5 s at 120 Vpp and 50 kHz. This ECDA chip is validated for various microwell geometries and functional materials. The optimized ECDA are successfully applied for digital viable bacteria counting, showing comparable results to the plate culture counting. Such an ECDA chip, as a digitizable and high-throughput platform, presents excellent potential for high-throughput screening, analysis, absolute quantification, etc.
PubMed: 37449335
DOI: 10.1002/smll.202302998