Abstract
When biologically interpretation of the data obtained from the single-cell RNA sequencing (scRNA-seq) analysis is attempted, additional information on the location of the single cells, ...behavior of the surrounding cells, and the microenvironment they generate, would be very important. We developed an inexpensive, high throughput application while preserving spatial organization, named “semibulk RNA-seq” (sbRNA-seq). We utilized a microfluidic device specifically designed for the experiments to encapsulate both a barcoded bead and a cell aggregate (a semibulk) into a single droplet. Using sbRNA-seq, we firstly analyzed mouse kidney specimens. In the mouse model, we could associate the pathological information with the gene expression information. We validated the results using spatial transcriptome analysis and found them highly consistent. When we applied the sbRNA-seq analysis to the human breast cancer specimens, we identified spatial interactions between a particular population of immune cells and that of cancer-associated fibroblast cells, which were not precisely represented solely by the single-cell analysis. Semibulk analysis may provide a convenient and versatile method, compared to a standard spatial transcriptome sequencing platform, to associate spatial information with transcriptome information.
Biological organisms use intricate networks of chemical reactions to control molecular processes and spatiotemporal organization. In turn, these living systems are embedded in self-organized ...structures of larger scales, for example, ecosystems. Synthetic in vitro efforts have reproduced the architectures and behaviors of simple cellular circuits. However, because all these systems share the same dynamic foundations, a generalized molecular programming strategy should also support complex collective behaviors, as seen, for example, in animal populations. We report here the bottom-up assembly of chemical systems that reproduce in vitro the specific dynamics of ecological communities. We experimentally observed unprecedented molecular behaviors, including predator–prey oscillations, competition-induced chaos, and symbiotic synchronization. These synthetic systems are tailored through a novel, compact, and versatile design strategy, leveraging the programmability of DNA interactions under the precise control of enzymatic catalysis. Such self-organizing assemblies will foster a better appreciation of the molecular origins of biological complexity and may also serve to orchestrate complex collective operations of molecular agents in technological applications.
The development of an in vitro functional liver zonation model is a major issue to reproduce physiological liver features. Oxygen concentration is one of the potential explanations of a primary ...regulating factor of zonation. In this frame, we investigated the oxygen gradient inside a microfluidic device containing rat hepatocyte cultures. The device integrated a platinum (Pt) (II) octaethylporphyrin sensor, allowing a 2D mapping of the oxygen concentration. After 3 hr adhesion of the hepatocytes, the sensor indicated an intense oxygen depletion, leading to an oxygen shortage in the center of the device. After a 30 min perfusion of the culture medium, we monitored the formation of the oxygen gradient along the culture due to cellular respiration. The profile of the oxygen gradient was modulated and controlled by increasing either the perfusion flow rate or the device thickness. In addition, the oxygen gradient was time dependent as far as it decreased with the time of culture. Perivenous and periportal liver patterns were characterized by the immunostaining of the hepatic markers. We put in evidence a spatio temporal hepatic organization. We observed the overexpression since 24 hr of perfusion of the APC and PCK1 proteins upstream in the oxygen‐rich area of the device. The overexpression of GS, GCK, CYP1A, and HIFα proteins were observed downstream in the oxygen‐poor area. Then, CYP3A2 and β‐catenin spatial reorganization was achieved after 48 hr of culture. The results presented a partial zonation‐like pattern that was superimposed with an oxygen gradient profile.
Biochemical systems in which multiple components take part in a given reaction are of increasing interest. Because the interactions between these different components are complex and difficult to ...predict from basic reaction kinetics, it is important to test for the effect of variations in the concentration for each reagent in a combinatorial manner. For example, in PCR, an increase in the concentration of primers initially increases template amplification, but large amounts of primers result in primer-dimer by-products that inhibit the amplification of the template. Manual titration of biochemical mixtures rapidly becomes costly and laborious, forcing scientists to settle for suboptimal concentrations. Here we present a droplet-based microfluidics platform for mapping of the concentration space of up to three reaction components followed by detection with a fluorescent readout. The concentration of each reaction component is read through its internal standard (barcode), which is fluorescent but chemically orthogonal. We describe in detail the workflow, which comprises the following: (i) production of the microfluidics chips, (ii) preparation of the biochemical mixes, (iii) their mixing and compartmentalization into water-in-oil emulsion droplets via microfluidics, (iv) incubation and imaging of the fluorescent barcode and reporter signals by fluorescence microscopy and (v) image processing and data analysis. We also provide recommendations for choosing the appropriate fluorescent markers, programming the pressure profiles and analyzing the generated data. Overall, this platform allows a researcher with a few weeks of training to acquire ∼10,000 data points (in a 1D, 2D or 3D concentration space) over the course of a day from as little as 100-1,000 μl of reaction mix.
Separation and separation-based analysis of biomolecules are fundamentally important techniques in the field of biotechnology. These techniques, however, depend on stochastic processes that ...intrinsically involve uncertainty, and thus it is not possible to achieve 100% separation accuracy. Theoretically, the ultimate resolution and sensitivity should be realized in a single-molecule system because of the deterministic nature of single-molecule manipulation. Here, we have proposed and experimentally demonstrated the concept of a 'single-molecule sorter' that detects and correctly identifies individual single molecules, realizing the ultimate level of resolution and sensitivity for any separation-based technology. The single-molecule sorter was created using a nanofluidic network consisting of a single inlet channel that branches off into multiple outlet channels. It includes two major functional elements, namely a single-molecule detection and identification element and a flow path switching element to accurately separate single molecules. With this system we have successfully demonstrated the world's first single-molecule sorting using DNA as a sample molecule. In the future, we hope to expand the application of such devices to comprehensive sorting of single-proteins from a single cell. We also believe that in addition to the single-molecule sorting method reported here, other types of single-molecule based processes will emerge and find use in a wide variety of applications.
Total ATP (adenosine triphosphate) concentration is a useful biochemical parameter for detecting microbial biomass or biogeochemical activity anomalies in the natural environment. In this study, we ...describe the development and evaluation of a new version of in situ ATP analyzer improved for the continuous and quantitative determination of ATP in submarine environments. We integrated a transparent microfluidic device containing a microchannel for cell lysis and a channel for the bioluminescence L⁻L (luciferin⁻luciferase) assay with a miniature pumping unit and a photometry module for the measurement of the bioluminescence intensity. A heater and a temperature sensor were also included in the system to maintain an optimal temperature for the L⁻L reaction. In this study, the analyzer was evaluated in deep sea environments, reaching a depth of 200 m using a remotely operated underwater vehicle. We show that the ATP analyzer successfully operated in the deep-sea environment and accurately quantified total ATP within the concentration lower than 5 × 10
M.
We have compared the transcriptomic profiles of human induced pluripotent stem cells after their differentiation in hepatocytes like cells in plates and microfluidic biochips. The biochips provided a ...3D and dynamic support during the cell differentiation when compared to the 2D static cultures in plates. The microarray have demonstrated the up regulation of important pathway related to liver development and maturation during the culture in biochips. Furthermore, the results of the transcriptomic profile, coupled with immunostaining, and RTqPCR analysis have shown typical biomarkers illustrating the presence of responders of biliary like cells, hepatocytes like cells, and endothelial like cells. However, the overall tissue still presented characteristic of immature and foetal patterns. Nevertheless, the biochip culture provided a specific micro-environment in which a complex multicellular differentiation toward liver could be oriented.
•We performed iPS hepatic like cells culture in a microfluidic biochip.•We compared the transcriptomic profiles from biochips and plated cultures.•We found higher hepatic differentiation patterns in biochips.•We measured higher functional liver like activities in biochips.•Although encouraging results, the biochip cultures remained a liver fœtal stage.
An efficient method for cell trapping onto a designated location and reduction of the sample volume is required to reduce the risk of sample losses during the discrimination and molecular ...characterization of low-abundant cells suspended in a large volume of the sample. In this study, we propose a novel microfluidic device that allows for highly efficient trapping of the cancer cells onto a designated location and correction of the trapped cells with a significantly reduced sample volume. The microfluidic device, which utilizes dielectrophoresis cell trapping, consists of an attraction-zone to control the vertical position of the cells and a trap-zone to capture the target cells by drastically decreasing the flow velocity around the cells with a sharply increased channel height. The device showed that 92 ± 9% of the introduced cancer cells were trapped on a designated location (500-μm behind the step) with a high inlet flow rate of 100 μl min−1. The trapped cells were corrected with a one-hundredth volume by introducing reagents for the downstream analysis to the outlet of the device. The feasibility of the system with regard to the localization and recovery of target cells was successfully demonstrated by trapping a mixture of cancer cells and white blood cells and detecting single nucleotide variants from the collected samples. We believe that our system is suitable for the sample preparation of low-abundant cells suspended in a large sample volume, owing to its ability to perform the localization and recovery of trapped cells with minimal target cell loss.
We have developed and tested a functionally integrated in situ analyzer, the IISA-ATP system, for microbial activity assays based on a quantitative determination of the total (particulate and ...dissolved) ATP in ocean environments. The IISA-ATP utilizes a PDMS-glass hybrid microfluidic device as its core functional element, which can perform cell lysis and total ATP quantification by a luciferin-luciferase bioluminescence assay in situ. Transparent heaters and a temperature sensor fabricated on a glass substrate provide temperature control. As a result of the evaluation using the microfluidic device with ATP standard solutions, the bioluminescence intensity was linearly correlated with 2 × 10(-12) to 2 × 10(-8) M of ATP. A detection limit of 1.1 × 10(-11) M was determined using the completed IISA-ATP system, which includes a miniature pumping module and a control module. As a result of the evaluation using the environmental seawater sample collected from Tokyo Bay, Japan, 2.7 × 10(-10) M of total ATP was successfully determined in the laboratory by the IISA-ATP. The system was operated at a shallow submarine hot spring area in Okinawa, Japan for an in situ trial. The result shows the system was successfully operated in situ and the total ATP was determined to be 3.4 × 10(-10) M.
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