High-Density Microcavity Array for Cell Detection: Single-Cell Analysis of Hematopoietic Stem Cells in Peripheral Blood Mononuclear Cells

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

Evaluation of amplification bias of droplet MDA.

<p>Distributions of sequencing coverage of MDA products from single <i>Escherichia coli</i> cells (n = 3) were compared between in-tube MDA (left column) and droplet MDA (right column). Each graph shows the results of independent reactions. The averaged sequencing coverages were calculated from raw sequencing reads that mapped to with <i>E</i>. <i>coli</i> reference genome within 1-kb windows. Sequencing reads were normalized to 60× sequencing effort in each experiment.</p

Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification

<div><p>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 <i>Escherichia coli</i> 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.</p></div

Droplet MDA of low-input lambda DNA.

<p>(a) Sequential fluorescent images of droplets encapsulating lambda DNA at a concentration of 265 ag/droplet (5 copies lambda DNA per droplet) with Evagreen dye. (b) Time-dependent appearance of the fluorescence signal during compartmentalized amplification of the denatured lambda DNA (input concentration 54 ag/droplet (1 copy lamda DNA per droplet) and 265 ag/droplet). All data are presented as averaged intensities of fluorescent positive droplets measured with SEM, and 100 droplets were analyzed at each time point.</p

Amplicon yields by in-tube MDA and droplet MDA.

<p>No template control (NTC), 1 and 10 <i>E</i>. <i>coli</i> cells were used as start material. In the droplet MDA, lysed cells were pumped into the droplet generators and genome DNA fragments were randomly encapsulated into picoliter droplets consist of MDA mixture (67 pL per droplet, total 1.5× 10⁵ droplets). After 180 min of MDA reaction, the yields were evaluated following droplet breaking and amplicon purification. A total of 10 μL of MDA mixture was used in both droplet MDA and in-tube MDA reactions.</p

Genome recovery from row sequence reads and <i>de novo</i> assembled contigs obtained from droplet MDA products of single and 10 <i>E</i>. <i>coli</i> cells.

<p>(a) Comparison of genome recovery from raw sequence reads as a function of sequencing effort. Each plot shows the averaged percentage of genome recovery with SD from raw sequence reads for single (n = 3) and 10 (n = 3) <i>Escherichia coli</i> cells at >1× or >10× sequencing coverage. (b) Comparison of genome recovery from <i>de novo</i> assembled contigs as a function of sequencing effort. Each plot shows <i>de novo</i> assembly result in in-tube MDA and the droplet MDA.</p

High-Density Microcavity Array for Cell Detection: Single-Cell Analysis of Hematopoietic Stem Cells in Peripheral Blood Mononuclear Cells

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

Digital Cell Counting Device Integrated with a Single-Cell Array

<div><p>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 mm² 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 (r² = 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.</p></div

Manipulation of a Single Circulating Tumor Cell Using Visualization of Hydrogel Encapsulation toward Single-Cell Whole-Genome Amplification

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

Assembly statistics of MDA products obtained from single <i>Escherichia coli</i> cells.

<p>A total of 10 μL of MDA product was evaluated for both droplet MDA and in-tube MDA. Sequencing reads were normalized to 0.8 M reads (60× sequencing effort) in each experiment.</p><p>Assembly statistics of MDA products obtained from single <i>Escherichia coli</i> cells.</p