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Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroi...
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Citations
... In particular, gel microcapsules composed of biological polymers have many advantages against the microdroplets, such as temperature-dependent hydrogel [24] and alginate, where gelation reaction proceeds with divalent cations such as Ca 2+ for cross-linking sodium alginate [25,26]. Several reports showed that alginate gel microcapsules could provide a suitable environment for encapsulated cells [27,28] and also for the single-cell polymerase chain reaction (PCR) platform as monoclonal, in analogy to a single-cellular clone [29,30]. One of the advantages of alginate gel microdroplets is their stability and capability to encapsulate. ...
Alginate microcapsules are one of the attractive non-invasive platforms for handling individual cells and clusters, maintaining their isolation for further applications such as imaging cell sorter and single capsule qPCR. However, the conventional cell encapsulation techniques provide huge numbers of unnecessary empty homogeneous alginate microcapsules, which spend an excessive majority of the machine time on observations and analysis. Here, we developed a simple alginate cell encapsulation method to form content size-dependent alginate microcapsules to eliminate empty microcapsules using microcapillary centrifugation and filtration. Using this method, the formed calcium alginate microcapsules containing the HeLa cells were larger than 20m, and the other empty microcapsules were less than 3m under 4000 rpm centrifugation condition. We collected cell-containing alginate microcapsules by eliminating empty microcapsules from the microcapsule mixture with simple one-step filtration of a 20 m cell strainer. The electrical surface charge density and optical permeability of those cell-encapsulated alginate microcapsules were also evaluated. We found that the surface charge density of cell-encapsulated alginate microbeads is more than double that of cells, indicating that less voltage is required for electrical cell handling with thin alginate gel encapsulation of samples. The permeability of the alginate microcapsule was not improved by changing the reflective index of the medium buffer, such as adding alginate ester. However, the minimized thickness of the alginate gel envelope surrounding cells in the microcapsules did not degrade the detailed shapes of encapsulated cells. Those results confirmed the advantage of alginate encapsulation of cells with the centrifugation method as one of the desirable tools for imaging cell sorting applications.
... AGMs or other reaction chambers containing single bacterial cells inevitably produce a proportion of chambers containing multiple bacterial cells 21 , and it is difficult to completely eliminate contaminating DNA in our experimental procedures, as in other previously developed methods 1 . Nevertheless, the application of a computer program for binning metagenomic fragments, such as metaWRAP 31 , enabled us to recover a considerable number of at least median-quality SAGs from mini-metagenome bins that resulted from multiple cells by eliminating contaminating sequences (Supplementary Table S8). ...
A novel type of agarose gel microcapsule (AGM), consisting of an alginate picolitre sol core and an agarose gel shell, was developed to obtain high-quality, single-cell, amplified genomic DNA of bacteria. The AGM is easy to prepare in a stable emulsion with oil of water-equivalent density, which prevents AGM aggregation, with only standard laboratory equipment. Single cells from a pure culture of Escherichia coli, a mock community comprising 15 strains of human gut bacteria, and a termite gut bacterial community were encapsulated within AGMs, and their genomic DNA samples were prepared with massively parallel amplifications in a tube. The genome sequencing did not need second-round amplification and showed an average genome completeness that was much higher than that obtained using a conventional amplification method on the microlitre scale, regardless of the genomic guanine–cytosine content. Our novel method using AGM will allow many researchers to perform single-cell genomics easily and effectively, and can accelerate genomic analysis of yet-uncultured microorganisms.
... In this sense, we used a fingertip spray bottle to eject the alginate/HA blend into a divalent ion crosslinking solution to instantly gel the droplets. To the best of our knowledge, there is only one study reporting the use of a fingertip spray bottle as well, generating pure alginate microspheres containing cells [24]. Our results demonstrated practically the same size distribution between triplicates with all alginate/HA blends tested, with a low coefficient of variation of the mean size (Figure 2), indicating high reproducibility of this method. ...
Bone has the intrinsic capacity to regenerate itself, as long as the damage is small, through the sequential stimulation of specific phases, such as angiogenesis followed by osteogenesis. However, when the damage is extensive it is unable to regenerate and bone tissue engineering is used as an alternative. In this study, we developed a platform to allow the triple ion delivery with sequential delivery capacity to potentially stimulate antibacterial, angiogenic and osteogenic processes. The scaffold-based platform consisted of alginate/hydroxyapatite (HA) microparticles embedded in alginate fibers. Firstly, microparticles were developed using different ratios of alginate:HA using the spraying method, resulting in a high reproducibility of the technique. Microparticle size between 100–300 µm and ratio 1:40 resulted in a more spherical morphology and were selected for their incorporation into alginate fiber. Different amounts of copper and cobalt were added with the microparticles and alginate fiber, respectively, were used as model ions which could eventually modulate and mimic antimicrobial and angiogenic processes. Moreover, calcium ion was also incorporated in both, in order to provide the system with potential osteogenic properties together with HA. The multiple delivery of copper, cobalt and calcium released were in the therapeutic range as measured by induced coupled plasma (ICP), providing a promising delivery strategy for tissue engineering.
... However, these techniques are not available for many microbiologists, and the amount of the ampli cation product is often too small for the direct genome sequencing and a second-round MDA 14 may be required, which ultimately enhances the ampli cation bias. ...
... However, MDA reagents are sequentially introduced into the droplets in the microchannel, which complexi es the microchannel structure. Single-cell encapsulating gel beads are easily prepared by gelating cell-containing solated gel droplets [14][15][16] . The MDA of the single-cell genome in the bead is done by exchanging reagents through gel. ...
A novel type of agarose gel microcapsule (AGM), consisting of an alginate picolitre sol core and an agarose gel shell, was developed to obtain high-quality single-cell amplified genomic DNA of bacteria. The AGM is easy to prepare in a stable emulsion with oil of water-equivalent density which prevents AGM aggregation, with only standard laboratory equipment. Single cells from a pure culture of Escherichia coli , a mock community comprising 15 strains of human gut bacteria, and a termite gut bacterial community were encapsulated within AGMs, and their genomic DNAs were obtained with massively parallel amplifications in a tube. The genome sequencing did not need second-round amplification, and showed an average genome completeness that was much higher than that obtained by the conventional amplification method in microlitre scale, regardless of the genomic guanine-cytosine contents. Our novel method using AGMs allows many researchers to perform single-cell genomics easily and effectively, and can enhance the genome analysis of yet-uncultured microorganisms.
... Cells were encapsulated into alginate beads following the work of Bigdeli et al. [25] Sodium alginate (Sigma Aldrich) was mixed into deionized water at 2% (w/v) and stirred for 4 h at room temperature. A 2% (w/v) calcium chloride solution was prepared by dissolving powder into deionized water and stirring for 1 h at room temperature. ...
Improving the physiological relevance of drug testing for drug-loaded nanoparticles using 3D tumor cell cultures - Volume 9 Issue 3 - Priya Nimbalkar, Peter Tabada, Anuja Bokare, Jeffrey Chung, Marzieh Mousavi, Melinda Simon, Folarin Erogbogbo
... Reagents. Sodium alginate from brown algae (Sigma-Aldrich: 71238) composed of approximately 70% guluronic acid 18,19 was purchased from Sigma-Aldrich. Calcium carbonate (light, precipitated powder, particle size ca. 1 μm) was provided by Magnesia GmbH (Germany). ...
The first evidence of solvent spillage under subcritical conditions during aerogel production is presented. The main objective was to understand the underlying phenomena controlling the solvent extraction kinetics during autoclave pressurisation. Alginate, silica and polyurethane as gels and ethanol, methyl ethyl ketone and ethanol/water as solvents were investigated. When CO2 diffuses in the gel solvent, there is a relative volume expansion of the liquid solvent. This expanded liquid mixture spills out of the gel and accumulates as a separate liquid phase at the bottom of the autoclave. A lag time was observed between the start of the autoclave pressurisation and the moment in which liquid starts to accumulate in the autoclave. The time needed for the solvent to start accumulating at the bottom of the autoclave is controlled by capillary forces and the saturation of the CO2-gas phase, on which temperature has an important effect. Low operating temperature and thereby low solubility of the solvent in the gas phase is suggested as the factor that enhances the kinetics of solvent removal at subcritical conditions.
... 26,27) Several reports showed that alginate gel microdroplets can provide a suitable environment for encapsulated cells, 28,29) and also for the single-cell polymerase chain reaction (PCR) platform as "monoclonal", in analogy to a single-cellular clone. 30,31) One of the advantages of alginate gel microdroplets is their stability and capability to encapsulate. For example, alginate gel microdroplet PCR is a miniature scale of biological operation with fast heat transfer in a contaminationfree environment with the microdroplet structural stability and even it is capable to capture the PCR products within the microdroplets. ...
... A spray method was used for the controlled gelation of alginate microdroplets (Fig. 2). 31) We used sodium alginate (Kanto Chemical); barium chloride dihydrate and calcium chloride dihydrate (BaCl 2 1 2H 2 O and CaCl 2 1 2H 2 O, Wako Pure Chemical Industries); phosphate-buffered salt (PBS) tablets (Takara Bio); and ethylenediamine-N,N,NA,NA-tetraacetic acid, disodium salt, and dihydrate (EDTA 1 2NA; Dojindo Laboratories). ...
Cells encapsuled by polymer microdroplets are an effective platform for the identification and separation of individual cells for single-cell-based analysis. However, a key challenge is to maintain and release the captured cells in the microdroplets selectively, nondestructively, and noninvasively. We developed a simple method of encapsulating cells in alginate microdroplets having different digestion characteristics. Cells were diluted with an alginate polymer of sol state and encapsulated into microdroplets with Ba²⁺ and Ca²⁺ by a spray method. When a chelating buffer was applied, alginate gel microdroplets were digested according to the difference in chelating efficiency of linkage-divalent cations; hence, two types of alginate microdroplets were formed. Moreover, we examined the capability of the alginate gel to exchange linkage-divalent cations and found that both Ca²⁺ exchange in Ba-alginate microdroplets and Ba²⁺ exchange in Ca-alginate microdroplets occurred. These results indicate that the potential applications of a mixture of alginate microdroplets with different divalent cations control the selective digestion of microdroplets to improve the high-throughput, high-content microdroplet-based separation, analysis, or storage of single cells.
... In particular, ionic crosslinking of alginate using divalent ions has been widely used for the generation of single-cell-laden microgels [46][47][48][49][50][51]. Crosby and coworkers leveraged the reversible nature of physical interactions for the controlled release and facile analysis of single-cell components by dissolving barium-crosslinked alginate microgels using sulfate as a barium chelator [52]. Reversible physical crosslinking can also endow hydrogels with self-healing properties that are, for example, key to provide a hydrogel with stress relaxation properties, which have recently been proven essential to trigger physiological cell responses in cell-laden bulk constructs [30]. ...
... This strategy enabled facile RNA quantification by counting the number of fluorescently labeled rolling circle amplification products (I.e., 'RNA colonies') in the microgel. More recently, single-cell-laden microgels were combined with next-generation sequencing for single-cell whole-genome analysis [52]. Following similar affinity bead strategies, single-cell microdroplet and microgel technologies were used to isolate and/or detect specific compounds secreted by individual cells, including antibodies [2], lipids [77], cytokines [78], and proteases [79]. ...
Single-cell-laden microgels effectively act as the engineered counterpart of the smallest living building block of life: a cell within its pericellular matrix. Recent breakthroughs have enabled the encapsulation of single cells in sub-100-μm microgels to provide physiologically relevant microniches with minimal mass transport limitations and favorable pharmacokinetic properties. Single-cell-laden microgels offer additional unprecedented advantages, including facile manipulation, culture, and analysis of individual cell within 3D microenvironments. Therefore, single-cell microgel technology is expected to be instrumental in many life science applications, including pharmacological screenings, regenerative medicine, and fundamental biological research. In this review, we discuss the latest trends, technical challenges, and breakthroughs, and present our vision of the future of single-cell microgel technology and its applications.
... 25,26 Biochemical reactions, like an abridged polymerase chain reaction (PCR) protocol for whole genome amplification (WGA), were completed on single-cells trapped inside these microspheres. 27 The microspheres are capable of being generated easily in large numbers using a Na-alginate precursor with a simple crosslinking reaction using established droplet microfluidics systems, and are therefore an ideal candidate system for preparing high molecular weight single-cell genomes. Alginate polymerization and depolymerisation is quickly catalyzed and unaffected by temperature, making it suitable to withstand sample preparation protocols outside of room temperature. ...
The preparation and handling of mammalian single-cell genomic DNA is limited by the complexity bottleneck inherent to performing multi-step, multi-reagent operations in a microfluidic environment. We have developed a method for benchtop preparation of high-molecular weight, intact, single-cell genomes and demonstrate the extraction of long nucleic acid molecules in a microfluidic system. Lymphoblasts are encapsulated inside of alginate microparticles using a droplet microfluidics, and cells are lysed in bulk. The purified genomes are then delivered to and imaged on a dedicated microfluidic device. High-molecular weight DNA is protected from shear and retains its original cellular identity. Using this encapsulation protocol, we were able to extract individual nucleic acid strands on the millimeter scale inside of a microfluidic channel.
