Abstract

Bacterial genome structure changes dynamically, and structural variants can change bacterial phenotype; However, obtaining the complete genome and analyzing genome structure of uncultured bacteria has been challenging. We aimed to develop a single-cell amplified genome long-read assembly (scALA) workflow to construct circular single-cell amplified genomes (cSAGs) from long-read single-cell sequencing data of targeted uncultured bacteria. In particular, scALA generated cSAGs from nanopore long-read sequencing data of SAGs by producing contiguous sequences with repeated bias reduction and assembly processes. From 12 human fecal samples, scALA generated 16 cSAGs of three specifically targeted bacterial species, Anaerostipes hadrus, Agathobacter rectalis, and Ruminococcus gnavus. A. hadrus cSAGs exhibited large, ten kbp-long, phage insertions, saccharide metabolic capacity, and frequent genomic recombination with related strains from cohabitant hosts. Noteworthy, cSAGs constructed using this method could expand bacterial genome databases and our understanding of within-species diversities in uncultured bacteria.

Culture-independent analysis with high-throughput sequencing has been widely used to characterize bacterial communities. However, signals derived from non-viable bacteria and non-cell DNA may inhibit its characterization. Here, we present a method for viable bacteria-targeted single-cell genome sequencing, called PMA-SAG-gel, to obtain comprehensive whole-genome sequences of surviving uncultured bacteria from microbial communities. PMA-SAG-gel uses gel matrixes that enable sequential enzymatic reactions for cell lysis and genome amplification of viable single cells from the microbial communities. PMA-SAG-gel removed the single-amplified genomes (SAGs) derived from dead bacteria and enabled selective sequencing of viable bacteria in the model samples of Escherichia coli and Bacillus subtilis. Next, we demonstrated the recovery of near-complete SAGs of eight oxygen-tolerant bacteria, including Bacteroides spp. and Phocaeicola spp., from 1331 human feces SAGs. We found the presence of two different strains in each species and identified their specific genes to investigate the metabolic functions. The survival profile of an entire population at the strain level will provide the information for understanding the characteristics of the surviving bacteria under the specific environments or sample processing and insights for quality assessment of live bacterial products or fecal microbiota transplantation and for understanding the effect of antimicrobial treatments.

Abstract

The production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. An example is defensive symbiosis that might account for much of the chemical richness of marine invertebrates including sponges (Porifera), 1 of the oldest metazoans. However, most bacterial members of sponge microbiomes have not been cultivated or sequenced, and therefore, remain unrecognized. Unequivocally linking metabolic functions to a cellular source in sponge microbiomes is, therefore, a challenge. Here, we report an analysis pipeline of microfluidic encapsulation, Raman microscopy, and integrated digital genomics (MERMAID) for an efficient identification of uncultivated producers. We applied this method to the chemically rich bacteriosponge (sponge that hosts a rich bacterial community) Theonella swinhoei, previously shown to contain ‘Entotheonella’ symbionts that produce most of the bioactive substances isolated from the sponge. As an exception, the antifungal aurantosides had remained unassigned to a source. Raman-guided single-bacterial analysis and sequencing revealed a cryptic, distinct multiproducer, ‘Candidatus Poriflexus aureus’ from a new Chloroflexi lineage as the aurantoside producer. Its exceptionally large genome contains numerous biosynthetic loci and suggested an even higher chemical richness of this sponge than previously appreciated. This study highlights the importance of complementary technologies to uncover microbiome functions, reveals remarkable parallels between distantly related symbionts of the same host, and adds functional support for diverse chemically prolific lineages being present in microbial dark matter.

BACKGROUND: Obtaining high-quality (HQ) reference genomes from microbial communities is crucial for understanding the phylogeny and function of uncultured microbes in complex microbial ecosystems. Despite improvements in bioinformatic approaches to generate curated metagenome-assembled genomes (MAGs), existing metagenome binners obtain population consensus genomes but they are nowhere comparable to genomes sequenced from isolates in terms of strain level resolution. Here, we present a framework for the integration of single-cell genomics and metagenomics, referred to as single-cell (sc) metagenomics, to reconstruct strain-resolved genomes from microbial communities at once. RESULTS: Our sc-metagenomics integration framework, termed SMAGLinker, uses single-cell amplified genomes (SAGs) generated using microfluidic technology as binning guides and integrates them with metagenome-assembled genomes (MAGs) to recover improved draft genomes. We compared sc-metagenomics with the metagenomics-alone approach using conventional metagenome binners. The sc-metagenomics approach showed precise contig binning and higher recovery rates (>97%) of rRNA and plasmids than conventional metagenomics in genome reconstruction from the cell mock community. In human microbiota samples, sc-metagenomics recovered the largest number of genomes with a total of 103 gut microbial genomes (21 HQ, with 65 showing >90% completeness) and 45 skin microbial genomes (10 HQ, with 40 showing >90% completeness), respectively. Conventional metagenomics recovered one Staphylococcus hominis genome, whereas sc-metagenomics recovered two S. hominis genomes from identical skin microbiota sample. Single-cell sequencing revealed that these S. hominis genomes were derived from two distinct strains harboring specifically different plasmids. We found that all conventional S. hominis MAGs had a substantial lack or excess of genome sequences and contamination from other Staphylococcus species (S. epidermidis). CONCLUSIONS: SMAGLinker enabled us to obtain strain-resolved genomes in the mock community and human microbiota samples by assigning metagenomic sequences correctly and covering both highly conserved genes such as rRNA genes and unique extrachromosomal elements, including plasmids. SMAGLinker will provide HQ genomes that are difficult to obtain using metagenomics alone and will facilitate the understanding of microbial ecosystems by elucidating detailed metabolic pathways and horizontal gene transfer networks. SMAGLinker is available at https://github.com/kojiari/smaglinker . Video abstract.

Recent studies have shown that mutation at Ser522 causes inhibition of collapsin response mediator protein 2 (CRMP2) phosphorylation and induces axon elongation and partial recovery of the lost sensorimotor function after spinal cord injury (SCI). We aimed to reveal the intracellular mechanism in axotomized neurons in the CRMP2 knock-in (CRMP2KI) mouse model by performing transcriptome analysis in mouse sensorimotor cortex using micro-dissection punching system. Prior to that, we analyzed the structural pathophysiology in axotomized or neighboring neurons after SCI and found that somatic atrophy and dendritic spine reduction in sensorimotor cortex were suppressed in CRMP2KI mice. Further analysis of the transcriptome has aided in the identification of four hemoglobin genes Hba-a1, Hba-a2, Hbb-bs, and Hbb-bt that are significantly upregulated in wild-type mice with concomitant upregulation of genes involved in the oxidative phosphorylation and ribosomal pathways after SCI. However, we observed substantial upregulation in channel activity genes and downregulation of genes regulating vesicles, synaptic function, glial cell differentiation in CRMP2KI mice. Moreover, the transcriptome profile of CRMP2KI mice has been discussed wherein energy metabolism and neuronal pathways were found to be differentially regulated. Our results showed that CRMP2KI mice displayed improved SCI pathophysiology not only via microtubule stabilization in neurons, but also possibly via the whole metabolic system in the central nervous system, response changes in glial cells, and synapses. Taken together, we reveal new insights on SCI pathophysiology and the regenerative mechanism of central nervous system by the inhibition of CRMP2 phosphorylation at Ser522. All these experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Waseda University, Japan (2017-A027 approved on March 21, 2017; 2018-A003 approved on March 25, 2018; 2019-A026 approved on March 25, 2019).

Droplet microfluidic techniques have shown promising outcome to study single cells at high throughput. However, their adoption in laboratories studying "-omics" sciences is still irrelevant due to the complex and multidisciplinary nature of the field. To facilitate their use, here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput. First, a device encapsulating single cells in droplets at a rate of ∼250 Hz is described considering droplet size and cell growth. Then, we expand on previously reported fluorescence-activated droplet sorting systems to integrate the use of 4 independent fluorescence-exciting lasers (i.e., 405, 488, 561, and 637 nm) in a single platform to make it compatible with different fluorescence-emitting biosensors. For this sorter, both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz. Then, a passive droplet merger is also integrated into our pipeline to enable adding new reagents to already-made droplets at a rate of 200 Hz. Finally, we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools. Because of the overall integration and the technical details presented here, our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput capability (>50,000 cells/day) for mining and bioprospecting metagenomic data.

The central nucleus of the amygdala (CeA) and the lateral division of the bed nucleus of the stria terminalis (BNST) are the two major nuclei of the central extended amygdala that plays essential roles in threat processing, responsible for emotional states such as fear and anxiety. While some studies suggested functional differences between these nuclei, others showed anatomical and neurochemical similarities. Despite their complex subnuclear organization, subnuclei-specific functional impact on behavior and their underlying molecular profiles remain obscure. We here constitutively inhibited neurotransmission of protein kinase C-δ-positive (PKCδ+) neurons-a major cell type of the lateral subdivision of the CeA (CeL) and the oval nucleus of the BNST (BNSTov)-and found striking subnuclei-specific effects on fear- and anxiety-related behaviors, respectively. To obtain molecular clues for this dissociation, we conducted RNA sequencing in subnuclei-targeted micropunch samples. The CeL and the BNSTov displayed similar gene expression profiles at the basal level; however, both displayed differential gene expression when animals were exposed to fear-related stimuli, with a more robust expression change in the CeL. These findings provide novel insights into the molecular makeup and differential engagement of distinct subnuclei of the extended amygdala, critical for regulation of threat processing.

The genus Okeania is a globally distributed group of microorganisms that live in shallow seabed regions. These organisms play several environmentally important roles and are also known producers of several active secondary metabolites with potential human applications. Here, we present a draft genome of Okeania sp. strain KiyG1 (92.7% completeness) that was assembled from four single-amplified genomes.

Cancer chemoresistance is often attributed to the presence of cancer stem cell (CSC)-like cells, but whether they are homogeneously chemoresistant remains unclear. We previously showed that in colon tumors, a subpopulation of LGR5+ CSC-like cells driven by TCF1 (TCF7), a Wnt-responsive transcription factor, were responsible for tumorigenicity. Here we demonstrate that the tumorigenic subpopulation of mouse LGR5+ cells exists in a slow-cycling state and identify a unique 22-gene signature that characterizes these slow-cycling CSC. Seven of the signature genes are specifically expressed in slow-cycling LGR5+ cells from xenografted human colon tumors and are upregulated in colon cancer clinical specimens. Among these seven, four genes (APCDD1, NOTUM, PROX1, and SP5) are known to be direct Wnt target genes, and PROX1 was expressed in the invasive fronts of colon tumors. PROX1 was activated by TCF1 to induce CDKN1C and maintain a slow-cycling state in colon cancer organoids. Strikingly, PROX1 was required for recurrent growth after chemotherapeutic treatment, suggesting that inhibition of slow-cycling CSC by targeting the TCF1-PROX1-CDKN1C pathway is an effective strategy to combat refractory colon cancer in combination with conventional chemotherapy. SIGNIFICANCE: These findings illustrate the importance of a slow-cycling CSC subpopulation in colon cancer development and chemoresistance, with potential implications for the identified slow-cycling CSC signatures and the TCF1-PROX1-CDKN1C pathway as therapeutic targets.

Here, we present high-quality draft single-cell genome sequences of Gammaproteobacteria strains BBSC-SA01 and BBSC-SA02, obtained from uncultivated cells of soil in a strawberry farm using the single-cell sequencing platform bit-MAP. These draft genomes putatively represent novel species within Gammaproteobacteria and allow further investigation into the soil microbiome.

In Japan, the imports of meat products have been increasing every year. Heat processing of meat is the current standard method for ensuring domestic animal health, particularly in case of meat products from areas where infectious diseases are known to have occurred in domestic animals. The Animal Quarantine Service needs to establish a method that detects the temperature at which the meat has been heat-processed (endpoint temperature) to ensure that the standard protocol is followed at the production location. Here, we developed a Raman spectroscopy and multivariate statistics (viz. multivariate curve resolution (MCR))-based simple and rapid method for accurately estimating the end point temperature. We showed that the temperature-dependent secondary structure modification of proteins can serve as an accurate indicator of the temperature of heat processing. This methodology can be easily automated for effective utilization by someone who is not an expert in spectroscopy. We envisage a wider application of this method in food analysis, although the present research investigated the application of this method in chicken meat heat processing analysis.

Bioprinting technology is expected to be applied in the fields of regenerative medicine and drug discovery. There are several types of bioprinters, especially inkjet-based bioprinter, which can be used not only as a printer for arranging cells but also as a precision cell-dispensing device with controlled cell numbers similar to a fluorescence activated cell sorter (FACS). Precise cell dispensers are expected to be useful in the fields of drug discovery and single-cell analysis. However, there are enduring concerns about the impacts of cell dispensers on cell integrity, particularly on sensitive cells, such as stem cells. In response to the concerns stated above, we developed a stress-free and media-direct-dispensing inkjet bioprinter. In the present study, in addition to conventional viability assessments, we evaluated the gene expression using RNA-seq to investigate whether the developed bioprinter influenced cell integrity in mouse embryonic stem cells. We evaluated the developed bioprinter based on three dispensing methods: manual operation using a micropipette, FACS and the developed inkjet bioprinter. According to the results, the developed inkjet bioprinter exhibited cell-friendly dispensing performance, which was similar to the manual dispensing operation, based not only on cell viability but also on gene expression levels.

Spatial transcriptomics is useful for understanding the molecular organization of a tissue and providing insights into cellular function in a morphological context. In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability during the image acquisition and biomolecule collection processes. Here, we developed a tissue processing method for robust and reproducible RNA-seq from tissue microdissection samples. In this method, we suppressed RNA degradation in fresh-frozen tissue specimens by dehydration fixation and effectively collected a small amount of RNA molecules from microdissection samples by magnetic beads. We demonstrated the spatial transcriptome analysis of the mouse liver and brain in serial microdissection samples (100 μm in a diameter and 10 μm in thickness) produced by a microdissection punching system. Using our method, we could prevent RNA degradation at room temperature and effectively produce a sequencing library with Smart-seq2. This resulted in reproducible sequence read mapping in exon regions and the detection of more than 2000 genes compared to non-fixed samples in the RNA-seq analysis. Our method would be applied to various transcriptome analyses, providing the information for region specific gene expression in tissue specimens.

How quiescent cells break dormancy is a key issue in eukaryotic cells including cancer. Fungal spores, for example, remain quiescent for long periods until nourished, although the mechanisms by which dormancy is broken remain enigmatic. Transcriptome analysis could provide a clue, but methods to synchronously germinate large numbers of spores are lacking, and thus it remains a challenge to analyse gene expression upon germination. Hence, we develop methods to assemble transcriptomes from individual, asynchronous spore cells of fission yeast undergoing germination to assess transcriptomic changes over time. The virtual time-lapse analyses highlights one of three copies of histone H3 genes whose transcription fluctuates during the initial stage of germination. Disruption of this temporal fluctuation causes defects in spore germination despite no visible defects in other stages of the life cycle. We conclude that modulation of histone H3 expression is a crucial 'wake-up' trigger at dormancy breaking.

<title>Abstract</title>To improve our understanding of the environmental microbiome, we developed a single-cell genome sequencing platform, named SAG-gel, which utilizes gel beads for single-cell isolation, cell lysis, and whole genome amplification (WGA) for sequencing. SAG-gel enables serial, parallel and independent reactions of &gt; 100,000 single cells in a single tube, delivering high-quality genome recovery with storable randomized single-cell genome libraries. From soil and marine environmental sources, we acquired 734 partial genomes that are recapitulated in 231 species, 35% of which were assigned as high-to-medium qualities. We found that each genome to be almost unique and 98.7% of them were newly identified, implying the complex genetic diversities across 44 phyla. The various metabolic capabilities including virulence factors and biosynthetic gene clusters were found across the lineages at single-cell resolution. This technology will accelerate the accumulation of reference genomes of uncharacterized environmental microbes and provide us new insights for their roles.

BACKGROUND: The gut microbiota can have dramatic effects on host metabolism; however, current genomic strategies for uncultured bacteria have several limitations that hinder their ability to identify responders to metabolic changes in the microbiota. In this study, we describe a novel single-cell genomic sequencing technique that can identify metabolic responders at the species level without the need for reference genomes, and apply this method to identify bacterial responders to an inulin-based diet in the mouse gut microbiota. RESULTS: Inulin-feeding changed the mouse fecal microbiome composition to increase Bacteroides spp., resulting in the production of abundant succinate in the mouse intestine. Using our massively parallel single-cell genome sequencing technique, named SAG-gel platform, we obtained 346 single-amplified genomes (SAGs) from mouse gut microbes before and after dietary inulin supplementation. After quality control, the SAGs were classified as 267 bacteria, spanning 2 phyla, 4 classes, 7 orders, and 14 families, and 31 different strains of SAGs were graded as high- and medium-quality draft genomes. From these, we have successfully obtained the genomes of the dominant inulin-responders, Bacteroides spp., and identified their polysaccharide utilization loci and their specific metabolic pathways for succinate production. CONCLUSIONS: Our single-cell genomics approach generated a massive amount of SAGs, enabling a functional analysis of uncultured bacteria in the intestinal microbiome. This enabled us to estimate metabolic lineages involved in the bacterial fermentation of dietary fiber and metabolic outcomes such as short-chain fatty acid production in the intestinal environment based on the fibers ingested. The technique allows the in-depth isolation and characterization of uncultured bacteria with specific functions in the microbiota and could be exploited to improve human and animal health. Video abstract.

Since G-protein coupled receptors (GPCRs) are linked to various diseases, screening of functional ligands against GPCRs is vital for drug discovery. In the present study, we developed a high-throughput functional cell-based assay by combining human culture cells producing a GPCR, yeast cells secreting randomized peptide ligands, and a droplet microfluidic device. We constructed a reporter human cell line that emits fluorescence in response to the activation of human glucagon-like peptide-1 receptor (hGLP1R). We then constructed a yeast library secreting an agonist of hGLP1R or randomized peptide ligands. We demonstrated that high-throughput identification of functional ligands against hGLP1R could be performed by co-culturing the reporter cells and the yeast cells in droplets. We identified functional ligands, one of which had higher activity than that of an original sequence. The result suggests that our system could facilitate the discovery of functional peptide ligands of GPCRs.

After publication of the original article [1], the authors became aware of a miscalculation in the original Fig. 2d.

OBJECTIVE: Resting CD4+ T cells are major reservoirs of latent HIV-1 infection, and may be formed during the early phase of the infection. Although CCR5-tropic (R5) HIV-1 is highly transmissible during the early phase, newly infected individuals have usually been exposed to a mixture of R5 and CXCR4-tropic (X4) viruses, and X4 viral DNA is also detectable in the host. Our aim was to identify which subsets of resting CD4+ T cells contribute to forming the latent reservoir in the presence of both X4 and R5 viruses. RESULTS: Primary resting CD4+ naïve T (TN) cells, CCR5- memory T (TM) cells, and CCR5+ TM cells isolated by flow cytometry were infected simultaneously with X4 and R5 HIV-1, which harbored different reporter genes, and were cultured in the resting condition. Flow cytometry at 3 days post-infection demonstrated that X4 HIV-1+ cells were present in all three subsets of cells, whereas R5 HIV-1+ cells were present preferentially in CCR5+ TM cells, but not in TN cells. Following CD3/CD28-mediated activation at 3 days post-infection, numbers of R5 HIV-1+ cells and X4 HIV-1+ cells increased significantly only in the CCR5+ TM subset, suggesting that it provides a major reservoir of replication-competent, latently infected viruses.

Severe invasive group A Streptococcus (GAS) infection evades anti-bacterial immunity by attenuating the cellular components of innate immune responses. However, this loss of protection is compensated for by interferon (IFN)-γ-producing immature myeloid cells (γIMCs), which are selectively recruited upon severe invasive GAS infection in mice. Here, we demonstrate that γIMCs provide this IFN-γ-mediated protection by sequentially sensing GAS through two distinct pattern recognition receptors. In a mouse model, GAS is initially recognized by Toll-like receptor 2 (TLR2), which promptly induces interleukin (IL)-6 production in γIMCs. γIMC-derived IL-6 promotes the upregulation of a recently identified GAS-sensing receptor, macrophage-inducible C-type lectin (Mincle), in an autocrine or paracrine manner. Notably, blockade of γIMC-derived IL-6 abrogates Mincle expression, downstream IFN-γ production, and γIMC-mediated protection against severe invasive GAS infection. Thus, γIMCs regulate host protective immunity against severe invasive GAS infection via a TLR2-IL-6-Mincle axis.

Regular exercise reduces the risks for cardiovascular diseases. Although the gut microbiota has been associated with fitness level and cardiometabolic risk factors, the effects of exercise-induced gut microbiota changes in elderly individuals are unclear. This study evaluated whether endurance exercise modulates the gut microbiota in elderly subjects, and whether these changes are associated with host cardiometabolic phenotypes. In a randomized crossover trial, 33 elderly Japanese men participated in a 5-week endurance exercise program. 16S rRNA gene-based metagenomic analyses revealed that the effect of endurance exercise on gut microbiota diversity was not greater than interindividual differences, whereas changes in α-diversity indices during intervention were negatively correlated with changes in systolic and diastolic blood pressure, especially during exercise. Microbial composition analyses showed that the relative abundance of Clostridium difficile significantly decreased, whereas that of Oscillospira significantly increased during exercise as compared to the control period. The changes in these taxa were correlated with the changes in several cardiometabolic risk factors. The findings indicate that short-term endurance exercise has little effect on gut microbiota in elderly individuals, and that the changes in gut microbiota were associated with cardiometabolic risk factors, such as systolic and diastolic blood pressure, providing preliminary insight into the associations between the gut microbiota and cardiometabolic phenotypes.

Single-cell RNA-seq is a powerful tool for revealing heterogeneity in cancer cells. However, each of the current single-cell RNA-seq platforms has inherent advantages and disadvantages. Here, we show that combining the different single-cell RNA-seq platforms can be an effective approach to obtaining complete information about expression differences and a sufficient cellular population to understand transcriptional heterogeneity in cancers. We demonstrate that it is possible to estimate missing expression information. We further demonstrate that even in the cases where precise information for an individual gene cannot be inferred, the activity of given transcriptional modules can be analyzed. Interestingly, we found that two distinct transcriptional modules, one associated with the Aurora kinase gene and the other with the DUSP gene, are aberrantly regulated in a minor population of cells and may thus contribute to the possible emergence of dormancy or eventual drug resistance within the population.

Single-cell genomics is a straightforward approach to obtain genomes from uncultured microbes. However, sequence reads from a single-cell amplified genome (SAG) contain significant bias and chimeric sequences. Here, we describe Cleaning and Co-assembly of a Single-Cell Amplified Genome (ccSAG), a novel analytical workflow to obtain composite single-cell genomes with elimination of sequence errors. By the integration of ccSAG with a massively parallel single-cell genome amplification platform based on droplet microfluidics, we can generate multiple SAGs and effectively integrate them into the composite genomes quality equivalent to the data obtained from bulk DNA. We obtained two novel draft genomes from single gut microbial cells with high completeness (>96.6%) and extremely low contamination (<1.25%). Moreover, we revealed the presence of single nucleotide polymorphisms in the specific gene by sequence comparison at the single-cell level. Thus, the workflow yields near-complete genomes from uncultured microbes, and enables analyses of genetic heterogeneity within identical strains.

Single-cell genomics has enabled the exploration of cellular diversity in environmental microbes. However, current genome sequencing techniques, which utilizes next-generation sequencing (NGS), typically require nanogram to microgram levels of input DNA sample. Since single bacterial cells contain only a few femtograms of DNA, we have to amplify their genomes to adequate amount for sequencing. We aimed to develop a novel system for precise and high throughput single-cell genomics, to elucidate environmental microbial diversity. In this study, we have developed droplet-based microfluidic system to produce the compartmentalized reaction vessels for single-cell genome sequencing.

Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

Site-specific gene expression analyses are important for understanding tissue functions. Despite rapid developments in DNA-related technologies, the site-specific analysis of whole genome expression for a tissue remains challenging. Thus, a new tool is required for capturing multiple tissue micro-dissections or single cells while retaining the positional information. Here, we describe the development of such a system, which can pick up micro-dissections by punching a tissue repeatedly in a very short period, e.g., 5 s/sampling cycle. A photo of the punched tissue provides information on the dissected positions, allowing site-specific gene expression analysis. We demonstrate the site-specific analysis of a frozen tissue slice of mouse brain by analyzing many micro-dissections produced from the tissue at a 300-μm pitch. The site-specific analysis provided new insights into the gene expression profiles in a tissue and on tissue functions. The analysis of site-specific whole genome expression may therefore, open new avenues in life science.

A cell entrapment device consisting of a microcavity array was used to analyze the deformability of MCF-10 human breast epithelial and MCF-7 human breast cancer cell lines by confocal laser scanning microscopy. Entrapment of up to 8 × 103 cells was achieved within 3 min. Protrusions were formed at the bottom surface of the array with a pore size of 3 μm. Protrusion length increased at higher filtration pressures and could be used to distinguish between MCF-7 and MCF-10 cells. These results indicate that our system is useful for high-throughput deformability analysis of cancer cells, which can provide insight into the mechanisms underlying tumor cell malignancy.

Genetic characterization of circulating tumor cells (CTCs) could guide the choice of therapies for individual patients and also facilitate the development of new drugs. We previously developed a CTC recovery system using a microcavity array, which demonstrated highly efficient CTC recovery based on differences in cell size and deformability. However, the CTC recovery system lacked an efficient cell manipulation tool suitable for subsequent genetic analysis. Here, we resolve this issue and present a simple and rapid manipulation method for single CTCs using a photopolymerized hydrogel, polyethylene glycol diacrylate (PEGDA), which is useful for subsequent genetic analysis. First, PEGDA was introduced into the cells entrapped on the microcavity array. Then, excitation light was projected onto the target single cells for encapsulation of each CTC by confocal laser-scanning microscopy. The encapsulated single CTCs could be visualized by the naked eye and easily handled with tweezers. The single CTCs were only partially encapsulated on the PEGDA hydrogel, which allowed for sufficient whole-genome amplification and accurate genotyping. Our proposed methodology is a valuable tool for the rapid and simple manipulation of single CTCs and is expected to become widely utilized for analyses of mammalian cells and microorganisms in addition to CTCs.

In this study, a method involving polyamidoamine dendron-modified magnetic nanoparticles (PAMAM-MNPs) along with application of an alternating magnetic field (AMF) was developed for inactivation of bacteria in water samples. The PAMAM-MNPs efficiently bound to Escherichia coli cells, resulting in magnetic recovery of cells from aqueous solutions. By applying the AMF (5 kW, 250 kHz) to the cell suspension, E. coli cells were successfully inactivated within 10 min in the presence of the MNPs, while no effect was observed in the absence of the MNPs. The use of PAMAM-MNPs could increase the inactivation rate of E. coli under the applied AMF. E. coli cells with PAMAM-MNPs stained by propidium iodide (PI) exhibited apparent fluorescence after exposure to the AMF, suggesting the occurrence of membrane damage in the cells because of direct heat transfer from the PAMAM-MNPs. Our technique can be used to address bacterial contamination with wide varieties of microorganisms in water samples. (C) The Electrochemical Society of Japan, All rights reserved.

This study presents a novel method for CD4 testing based on one-shot large-field imaging. The large-field imaging system was fabricated by a microcavity array and a two-dimensional (2D) photosensor within the desk-top-sized instrument. The microcavity array was employed to separate leukocytes from whole blood based on differences in the size of leukocytes and other blood cells. The large-field imaging system with lower side irradiation enabled acquisition of cell signatures with high signal-to-noise ratio, because the metallic substrate of the microcavity array obstructed excessive excitation light. In this setting, dual-color imaging of CD4(+) and CD8(+) T cells was achieved within the entire image area (64 mm(2)) in 2s. The practical performance of the large-field imaging system was demonstrated by determining the CD4/CD8 ratio in a few microliter of control whole blood as small as those obtained by a finger prick. The CD4/CD8 ratios measured using the large-field imaging system correlated well with those measured by microscopic analysis. These results indicate that our proposed system provides a simple and rapid CD4 testing for the application of HIV/AIDS treatment.

This paper proposes a high-throughput, function-based screening approach of a metagenomic library for isolating novel microbial enzymes by droplet-based microfluidics. We used gel microdroplets (GMDs) dispersed in oil as picoliter-volume reaction vessels for lipolytic enzyme by encapsulating cells in individual GMDs. Using this approach, we monitored the growth of individual cells encapsulated in GMDs and assessed the enzyme reaction activities at the level of an individual GMD. We then applied this method to screen lipolytic enzyme genes from the metagenomic library constructed from soil collected from a quercus serrate forest of Mount Tsukuba, Ibaraki, Japan. In the workflow presented in this study, metagenomic library clones were encapsulated in 100-pL GMDs with a fluorogenic reporter substrate. A total of 67,000 metagenomic library clones can be screened in only 24 h with reduced consumption of reagents (i.e., <10 μL). As a result, we identified a novel lipolytic enzyme, EstT1, belonging to the EstD2 family of esterases and containing a putative signal peptide, which facilitates enzyme export and catalyzation of substrates in the periplasm. Our study demonstrates the potential of microfluidic GMDs as an efficient tool for metagenomic library screening of industrially relevant enzymes with the potential of significantly reducing the cost and time factors involved in successful practical application of microbial enzymes.

Circulating tumor cells (CTCs) are well recognized as useful biomarker for cancer diagnosis and potential target of drug discovery for metastatic cancer. Efficient and precise recovery of extremely low concentrations of CTCs from blood has been required to increase the detection sensitivity. Here, an automated system equipped with a microcavity array (MCA) was demonstrated for highly efficient and reproducible CTC recovery. The use of MCA allows selective recovery of cancer cells from whole blood on the basis of differences in size between tumor and blood cells. Intra- and inter-assays revealed that the automated system achieved high efficiency and reproducibility equal to the assay manually performed by well-trained operator. Under optimized assay workflow, the automated system allows efficient and precise cell recovery for non-small cell lung cancer cells spiked in whole blood. The automated CTC recovery system will contribute to high-throughput analysis in the further clinical studies on large cohort of cancer patients.

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.

For high-throughput screening of novel cosmetic preservatives, a rapid and simple assay to evaluate the antimicrobial activities should be developed because the conventional agar dilution method is time-consuming and labor-intensive. To address this issue, we evaluated a microbial sensor as a tool for rapid antimicrobial activity testing. The sensor consists of an oxygen electrode and a filter membrane that holds the test microorganisms, Staphylococcus aureus and Candida albicans. The antimicrobial activity of the tested cosmetic preservative was evaluated by measuring the current increases corresponding to the decreases in oxygen consumption in the microbial respiration. The current increases detected by the sensor showed positive correlation to the concentrations of two commercially used preservatives, chlorphenesin and 2-phenoxyethanol. The same tendency was also observed when a model cosmetic product was used as a preservative solvent, indicating the feasibility in practical use. Furthermore, the microbial sensor and microfluidic flow-cell was assembled to achieve sequential measurements. The sensor system presented in this study could be useful in large-scale screening experiments.

A straightforward in vivo monitoring technique for biomolecules would be an advantageous approach for understanding their spatiotemporal dynamics in living cells. However, the lack of adequate probes has hampered the quantitative determination of the chemical composition and metabolomics of cellular lipids at single-cell resolution. Here, we describe a method for the rapid, direct, and quantitative determination of lipid molecules from living cells using single-cell Raman imaging. In vivo localization of lipids in the form of triacylglycerol (TAG) within oleaginous microalga and their molecular compositions are monitored with high spatial resolution in a nondestructive and label-free manner. This method can provide quantitative and real-time information on compositions, chain lengths, and degree of unsaturation of fatty acids in living cells for improving the cultivating parameters or for determining the harvest timing during large-scale cultivations for microalgal lipid accumulation toward biodiesel production. Therefore, this technique is a potential tool for in vivo lipidomics for understanding the dynamics of lipid metabolisms in various organisms.

The study of spatial distribution of secondary metabolites within microbial cells facilitates the screening of candidate strains from marine environments for functional metabolites and allows for the subsequent assessment of the production of metabolites, such as antibiotics. This paper demonstrates the first application of Raman microspectroscopy for in situ detection of the antifungal antibiotic amphotericin B (AmB) produced by actinomycetes-Streptomyces nodosus. Raman spectra measured from hyphae of S. nodosus show the specific Raman bands, caused by resonance enhancement, corresponding to the polyene chain of AmB. In addition, Raman microspectroscopy enabled us to monitor the time-dependent change of AmB production corresponding to the growth of mycelia. The Raman images of S. nodosus reveal the heterogeneous distribution of AmB within the mycelia and individual hyphae. Moreover, the molecular association state of AmB in the mycelia was directly identified by observed Raman spectral shifts. These findings suggest that Raman microspectroscopy could be used for in situ monitoring of antibiotic production directly in marine microorganisms with a method that is non-destructive and does not require labeling.

In this study, we describe a less invasive and rapid single-cell patterning technique for monitoring of cellular behaviors. To form a high-density grid pattern of living cells, single cells were firstly captured on a geometry-controlled array pattern of 100,000 microcavities by applying negative pressure. The captured cells on the microcavities were immersed in an agarose solution and embedded in agarose gels. The high efficiency transfer of individual yeast cells (Saccharomyces cerevisiae) and diatom cells (Fistulifera sp.) onto agarose gels was successfully achieved in 20 min. The patterning process had no effect on the cell proliferation or division. These results indicate that this technique shows a dramatic increase in patterning efficiency compared to previous patterning technologies. Furthermore, it allows the long-term monitoring of diatom cell divisions for 24 h. Continuous long-term observation of single cells provides technological advantages for the successful acquisition of information to better understand cellular activities.

In this paper, we present a novel cell counting method accomplished using a single-cell array fabricated on an image sensor, complementary metal oxide semiconductor sensor. The single-cell array was constructed using a microcavity array, which can trap up to 7,500 single cells on microcavities periodically arranged on a plane metallic substrate via the application of a negative pressure. The proposed method for cell counting is based on shadow imaging, which uses a light diffraction pattern generated by the microcavity array and trapped cells. Under illumination, the cell-occupied microcavities are visualized as shadow patterns in an image recorded by the complementary metal oxide semiconductor sensor due to light attenuation. The cell count is determined by enumerating the uniform shadow patterns created from one-on-one relationships with single cells trapped on the microcavities in digital format. In the experiment, all cell counting processes including entrapment of non-labeled HeLa cells from suspensions on the array and image acquisition of a wide-field-of-view of 30 mm(2) in 1/60 seconds were implemented in a single integrated device. As a result, the results from the digital cell counting had a linear relationship with those obtained from microscopic observation (r(2)  = 0.99). This platform could be used at extremely low cell concentrations, i.e., 25-15,000 cells/mL. Our proposed system provides a simple and rapid miniaturized cell counting device for routine laboratory use.

An electrochemical disinfection system employing a honeycombed platinum coated titanium electrode was developed for the disinfection of seawater. Cell suspensions (2 l, 10³ cells/ml) of the fish pathogens, Vibrio alginolyticus, Edwardsiella tarda, Lactococcus garvieae and Vibrio anguillarum were circulated in a reactor equipped with 10 sets of these electrodes at a flow rate of 200 ml/min with an applied potential of 1.0 V vs. Ag/AgCl reference electrode. The circulated cells were completely disinfected after 3 h of treatment, whereas free residual chlorine generated due to seawater electrolysis was below 0.1 ppm. In addition, a diphenyl-1-pyrenylphosphine fluorescent assay revealed that lipid peroxidation in the cell membranes of disinfected bacteria was induced probably by reactive oxygen species generated during electrochemical treatment.

In this study, we present a method for efficient enrichment of small-sized circulating tumor cells (CTCs) such as those found in the blood of small-cell lung cancer (SCLC) patients using a microcavity array (MCA) system. To enrich CTCs from whole blood, a microfabricated nickel filter with a rectangular MCA (10(4) cavities/filter) was integrated with a miniaturized device, allowing for the isolation of tumor cells based on differences in size and deformability between tumor and blood cells. The shape and porosity of the MCA were optimized to efficiently capture small tumor cells on the microcavities under low flow resistance conditions, while allowing other blood cells to effectively pass through. Under optimized conditions, approximately 80% of SCLC (NCI-H69 and NCI-H82) cells spiked in 1 mL of whole blood were successfully recovered. In clinical samples, CTCs were detectable in 16 of 16 SCLC patients. In addition, the number of leukocytes captured on the rectangular MCA was significantly lower than that on the circular MCA (p < 0.001), suggesting that the use of the rectangular MCA diminishes a considerable number of carryover leukocytes. Therefore, our system has potential as a tool for the detection of CTCs in small cell-type tumors and detailed molecular analyses of CTCs.

Monitoring of hematotoxicity, which requires serial blood collection, is difficult to carry out in small animals due to a lack of non-invasive, individual animal-appropriate techniques that enable enumeration of leukocyte subsets from limited amounts of whole blood. In this study, a microfluidic device equipped with a microcavity array that enables highly efficient separation of leukocytes from submicroliters of whole blood was applied for hematotoxicity monitoring in mice. The microcavity array can specifically separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Mouse leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered almost 100% of mouse leukocytes in 0.1 μL of whole blood without the effect of serial blood collection such as changes in body weight and total leukocyte count. We assessed benzene-associated hematotoxicity in mice using this system. Mice were administered with benzene once daily and the depression of leukocyte numbers induced in individual mice was successfully monitored from tail vein blood collected every other day for 2 weeks. Serial monitoring of the leukocyte number in individual mice will contribute to the understanding of hematotoxicity and reduction of the number of animal experiment trials.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM)-based enumeration of circulating tumor cells (CTC) has prognostic value in patients with solid tumors, such as advanced breast, colon, and prostate cancer. However, poor sensitivity has been reported for non-small cell lung cancer (NSCLC). To address this problem, we developed a microcavity array (MCA) system integrated with a miniaturized device for CTC isolation without relying on EpCAM expression. Here, we report the results of a clinical study on CTCs of advanced lung cancer patients in which we compared the MCA system with the CellSearch system, which employs the conventional EpCAM-based method. METHODS: Paired peripheral blood samples were collected from 43 metastatic lung cancer patients to enumerate CTCs using the CellSearch system according to the manufacturer's protocol and the MCA system by immunolabeling and cytomorphological analysis. The presence of CTCs was assessed blindly and independently by both systems. RESULTS: CTCs were detected in 17 of 22 NSCLC patients using the MCA system versus 7 of 22 patients using the CellSearch system. On the other hand, CTCs were detected in 20 of 21 small cell lung cancer (SCLC) patients using the MCA system versus 12 of 21 patients using the CellSearch system. Significantly more CTCs in NSCLC patients were detected by the MCA system (median 13, range 0-291 cells/7.5 mL) than by the CellSearch system (median 0, range 0-37 cells/7.5 ml) demonstrating statistical superiority (p = 0.0015). Statistical significance was not reached in SCLC though the trend favoring the MCA system over the CellSearch system was observed (p = 0.2888). The MCA system also isolated CTC clusters from patients who had been identified as CTC negative using the CellSearch system. CONCLUSIONS: The MCA system has a potential to isolate significantly more CTCs and CTC clusters in advanced lung cancer patients compared to the CellSearch system.

Absolute counting of total leukocytes or specific subsets within small amounts of whole blood is difficult due to a lack of techniques that enable separation of all leukocytes from limited amounts of whole blood. In this study, a microfluidic device equipped with a size-controlled microcavity array for highly efficient separation of leukocytes from submicroliters of whole blood was developed. The microcavity array can separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered over 90% of leukocytes in 1 µL of whole blood without pretreatment such as density gradient centrifugation or erythrocyte lysis. In addition, the proposed system successfully performed absolute enumeration of human CD4(+) and CD8(+) leukocytes from 1 µL of whole blood, and the obtained data showed good correlation with conventional flow cytometric analysis. Our microfluidic device has great potential as a tool for a point-of-care leukocyte analysis system.

Magnetic nanoparticles (MNPs) modified with the thiol functionalized polyamidoamine (PAMAM) dendron were synthesized to estimate their DNA recovery capabilities. Aminosilane-modified MNPs and MNPs surrounded by a phospholipid (distearoylphosphatidylethanolamine (DSPE)) bilayer were used as core particles. Cystamine-core PAMAM dendrimers were reduced by dithiothreitol to dendron thiols and chemically conjugated to the core particles. Characterization of the synthesis revealed an increase of the surface amine charge from generation 1 (G1) to G6, starting with an aminosilane initiator. Particle size distribution analysis indicated that G6 PAMAM-modified MNPs exhibited monodispersity in an aqueous solution. G6 PAMAM-MNPs and G6 PAMAM-PE-MNPs synthesized by the proposed method have equivalent DNA recovery abilities to PAMAM-MNPs prepared by the conventional divergent synthesis method. In optimized conditions, 96% of λDNA was recovered using G6 PAMAM-PE-MNPs. Therefore, the method for preparing PAMAM-MNPs and PAMAM-PE-MNPs proposed in this study will be a novel approach for producing DNA carriers for efficient DNA purification by magnetic separation.

DNA recovery using solid-phase extraction is a fundamental technique in molecular biology and biotechnology. Our research group developed a novel DNA recovery method using amine-modified magnetic nanoparticles (MNPs) as a solid support. The use of MNPs simplifies the DNA recovery processes and permits their use in automated systems. In this study, we prepared polyamidoamine-modified superparamagnetic particles (PAMAM-SpMNPs) with 10-nm magnetite cores and used them for DNA recovery. To improve the DNA-release efficiency, the surface amine numbers on the particles were evaluated to modify each generation of PAMAM. With this optimization, the PAMAM-SpMNPs maintained a high DNA adsorption capacity and high dispersivity in solution. As a result, the DNA release from the PAMAM-SpMNPs of every generation was highly efficient. In particular, the release of DNA from the G4 to G6 PAMAM-SpMNPs was greater than 95%. Furthermore, an alternating magnetic field (AMF) was applied to expedite the DNA release from the PAMAM-SpMNPs. Complete DNA release was achieved using AMF treatment for 10 min. The DNA recovery method using the PAMAM-SpMNPs will permit various types of testing using DNA from a low volume sample, such as in a micro total analytical system. Polymer Journal (2012) 44, 672-677; doi:10.1038/pj.2012.32; published online 4 April 2012

Here, we report the fabrication of a chemical gradient microfluidic device for single-cell cytotoxicity assays. This device consists of a microfluidic chemical gradient generator and a microcavity array that enables entrapment of cells with high efficiency at 88 ± 6% of the loaded cells. A 2-fold logarithmic chemical gradient generator that is capable of generating a serial 2-fold gradient was designed and then integrated with the microcavity array. High density single-cell entrapment was demonstrated in the device without cell damage, which was performed in 30 s. Finally, we validated the feasibility of this device to perform cytotoxicity assays by exposing cells to potassium cyanide (0-100 μM KCN). The device captured images of 4000 single cells affected by 6 concentrations of KCN and determined cell viability by counting the effected cells. Image scanning of the microcavity array was completed within 10 min using a 10× objective lens and a motorized stage. Aligning cells on the microcavity array eases cell counting, observation, imaging, and evaluation of singular cells. Thus, this platform was able to determine the cytotoxicity of chemicals at a single-cell level, as well as trace the cytotoxicity over time. This device and method will be useful for cytotoxicity analysis and basic biomedical research.

This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader™) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader™ consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader™ with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader™ for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis.

Circulating tumor cells (CTCs) are tumor cells circulating in the peripheral blood of patients with metastatic cancer. Detection of CTCs has clinical significance in cancer therapy because it would enable earlier diagnosis of metastasis. In this research, a microfluidic device equipped with a size-selective microcavity array for highly efficient and rapid detection of tumor cells from whole blood was developed. The microcavity array can specifically separate tumor cells from whole blood on the basis of differences in the size and deformability between tumor and hematologic cells. Furthermore, the cells recovered on the microcavity array were continuously processed for image-based immunophenotypic analysis using a fluorescence microscope. Our device successfully detected approximately 97% of lung carcinoma NCI-H358 cells in 1 mL whole blood spiked with 10-100 NCI-H358 cells. In addition, breast, gastric, and colon tumor cells lines that include EpCAM-negative tumor cells, which cannot be isolated by conventional immunomagnetic separation, were successfully recovered on the microcavity array with high efficiency (more than 80%). On an average, approximately 98% of recovered cells were viable. Our microfluidic device has high potential as a tool for the rapid detection of CTCs and can be used to study CTCs in detail.

Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using phi29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, "Magnetospirillum magneticum AMB-1," whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.

Detection and isolation of specific cell types from limited biological samples have become a major challenge in clinical diagnosis and cell biology research. Here, we report a high-density microcavity array for target cell detection in which thousands of single cells were neatly arrayed onto 10,000 microcavities with high efficiency at approximately 90% of the loaded cells. Cell-specific immunophenotypes were exclusively identified at the single-cell level by measuring fluorescence intensities of cells labeled with antibodies targeting cell surface markers, and the purity of hematopoietic stem cells (HSCs) within human peripheral blood analyzed by this system was correlated with those obtained by conventional flow cytometry. Furthermore, gene expression of the stem cell marker, CD34, was determined from HSCs by isolating single cells using a micromanipulator. This technology has proven to be an effective tool for target cell detection and subsequent cellular analytical research at the single-cell level.

Here, we report a high-efficiency single-cell entrapment system with a poly(dimethylsiloxane) (PDMS) microfluidic device integrated with a micromesh, and its application to single-cell fluorescence in situ hybridization (FISH) analysis. A micromesh comprising of 10 x 10 microcavities was fabricated on a black poly(ethylene terephthalate) (PET) substrate by laser ablation. The cavity was approximately 2 microm in diameter. Mammalian cells were driven and trapped onto the microcavities by applying negative pressure. Trapped cells were uniformly arrayed on the micromesh, enabling high-throughput microscopic analysis. Furthermore, we developed a method of PDMS surface modification by using air plasma and the copolymer Pluronic F-127 to prevent nonspecific adsorption on the PDMS microchannel. This method decreased the nonspecific adsorption of cells onto the microchannel to less than 1%. When cells were introduced into the microfluidic device integrated with the black PET micromesh, approximately 70-80% of the introduced cells were successfully trapped. Moreover, for mRNA expression analysis, on-chip fluorescence in situ hybridization (e.g., membrane permeabilization, hybridization, washing) can be performed in a microfluidic assay on an integrated device. This microfluidic device has been employed for the detection of beta-actin mRNA expression in individual Raji cells. Differences in the levels of beta-actin mRNA expression were observed in serum-supplied or serum-starved cell populations.