Keiko Sumitomo

High-throughput single-cell manipulation system for a large number of target cells

A sequential and high-throughput single-cell manipulation system for a large volume of cells was developed and the successive manipulation for single cell involving single-cell isolation, individual labeling, and individual rupture was realized in a microhydrodynamic flow channel fabricated by using two-dimensional simple flow channels. This microfluidic system consisted of the successive single-cell handlings of single-cell isolation from a large number of cells in cell suspension, labeling each isolated single cell and the lysate extraction from each labeled single cell. This microfluidic system was composed of main channels, cell-trapping pockets, drain channels, and single-cell content collection channels which were fabricated by polydimethylsiloxane. We demonstrated two kinds of prototypes for sequential single-cell manipulations, one was equipped with 16 single-cell isolation pockets in microchannel and the other was constructed of 512 single-cell isolation pockets. In this study, we demonstrated high-throughput and high-volume single-cell isolation with 512 pocket type device. The total number of isolated single cells in each isolation pocket from the cell suspension at a time was 426 for the cell line of African green monkey kidney, COS-1, and 360 for the rat primary brown preadipocytes, BAT. All isolated cells were stained with fluorescence dye injected into the same microchannel successfully. In addition, the extraction and collection of the cell contents was demonstrated using isolated stained COS-1 cells. The cell contents extracted from each captured cell were individually collected within each collection channel by local hydrodynamic flow. The sequential trapping, labeling, and content extraction with 512 pocket type devices realized high-throughput single-cell manipulations for innovative single-cell handling, feasible staining, and accurate cell rupture.

Acetic acid denaturing pulsed field capillary electrophoresis for RNA separation

Based on our previous work of in‐capillary denaturing polymer electrophoresis, we present a study of RNA molecular separation up to 6.0 kilo nucleotide by pulsed field CE. This is the first systematic investigation of electrophoresis of a larger molecular mass RNA in linear hydroxyethylcellulose (HEC) under pulsed field conditions. The parameters that may influence the separation performance, e.g. gel polymer concentration, modulation depth and pulse frequency, are analyzed in terms of resolution and mobility. For denaturing and separating RNA in the capillary simultaneously, 2 M acetic acid was added into the HEC polymer to serve as separation buffer. Result shows that (i) in pulsed field conditions, RNA separation can be achieved in a wide range of concentration of HEC polymer, and RNA fragments between 0.3 and 0.6 kilo nucleotide are sensitive to the polymer concentration; (ii) under certain pulsed field conditions, RNA fragments move linearly as the modulation depth increases; (iii) 12.5 Hz is the resonance frequency for RNA reorientation time and applied frequency.

Acetic acid denaturing for RNA capillary polymer electrophoresis.

A strong denaturant to cleave intramolecular hydrogen bonds in RNA is required for RNA size separation in a small sample volume (<10 nL). We found that carboxylic acids were strong denaturants for RNA and the RNA separation performance was dramatically improved by capillary electrophoresis with a sieving matrix containing acetic acid. We revealed that the denaturing ability of 2.0 M acetic acid was stronger than that of either 2.5 M formaldehyde or 7.0 M urea by estimating DNA melting temperature. Consequently, we suggested “in‐capillary denaturing polymer electrophoresis” as the RNA size separation methodology to simultaneously denature and separate RNA in a small sample volume without conventional in vitro sample preparation before electrophoresis. The baseline separation of RNA with a size of 100–10 000 nt was achieved in 25 min by “in‐capillary denaturing polymer electrophoresis” with the running buffer containing 2.0 M acetic acid. The resolution and the theoretical plates of RNA separation peaks were larger than those of the RNA separation in a conventional CGE with in vitro sample preparation by 7.0 M urea. In addition, we detected RNA peaks from the nucleic acids extracted from NIH 3T3 cells without DNase enzyme treatment.

The influence of polymer concentration, applied voltage, modulation depth and pulse frequency on DNA separation by pulsed field CE.

DNA fragments (0.1-10 kbp (kbp, kilo base pair)) separation by square-wave pulsed field CE in hydroxyethylcellulose (HEC, 1300 K) polymer was performed in this work. The effects of polymer concentration, pulse field strength, pulse frequency and modulation depth were investigated. We found that low HEC (about 0.1%) concentration is suitable for the separation of small DNA fragments (<1 kbp), whereas higher HEC concentration (>0.5%) is appropriated for high-mass DNA molecular (>1 kbp) separation. The mobility of DNA fragments is nearly linearly related to average separation voltage under pulsed field conditions. Higher modulation depth is suited to separate the longer DNA fragments and lower modulation depth favors the resolution of short DNA fragments. Thus, the intermediate modulation depth (100%) and pulse frequency (about 31.3 Hz) are prerequisite for high-resolution DNA separation.

Dynamic light-scattering measurement of sieving polymer solutions for protein separation on SDS CE

We evaluated the mesh size and homogeneity of polymer network by dynamic light scattering and discussed the relationship between the physical properties of polymer network and the protein separation behavior by capillary polymer electrophoresis. We compared three kinds of sieving polymers in solutions with a wide range of molecular weights and concentrations: polyacrylamide and polyethylene oxide as flexible polymers, and hydroxyethyl cellulose as a semiflexible polymer. We found that the mobility of protein was dominated primarily by the mesh size ξ, irrespective of the type of sieving polymers, and the peak spacing between protein peaks increased drastically in the range of ξ<10 nm, where the mobility also decreased. And the peak widths were dependent on the molecular species of sieving polymers and their homogeneity of polymer network. We proposed that a polymer network with a homogenous mesh size of less than 10 nm is the best sieving medium for separation of the proteins in the molecular weight range 14 300–97 200 Da from the view point of the resolution in protein separation.

Buffers to suppress sodium dodecyl sulfate adsorption to polyethylene oxide for protein separation on capillary polymer electrophoresis

Although polyethylene oxide (PEO) offers several advantages as a sieving polymer in SDS capillary polymer electrophoresis (SDS‐CPE), solution properties of PEO cause deterioration in the electrophoresis because PEO in solution aggregates itself, degrades into smaller pieces, and forms polymer–micelle complexes with SDS. We examined protein separation on SDS‐CPE with PEO as a sieving matrix in four individual buffer solutions: Tris‐CHES, Tris‐Gly, Tris‐Tricine, and Tris‐HCl buffers. The solution properties of PEO as a sieving matrix in those buffers were examined by dynamic light scattering (DLS) and by surface tension. Preferential SDS adsorption onto PEO disturbed protein–SDS complexation and impaired the protein separation efficiency. Substantial adsorption of SDS to PEO was particularly observed in Tris‐Gly buffer. The Tris‐CHES buffer prevented SDS from adsorbing onto the PEO. Only Tris‐CHES buffer achieved separation of six proteins. This study demonstrated efficient protein separation on SDS‐CPE with PEO.

Large scale microfluidic systems for sequential trapping, labeling and content extraction of single cells

Parallel and sequential manipulation of trapping, labeling and content extraction of single cell was realized using a 2-D simple flow channels. The proposed system consists of main channels, cell trapping pockets, drain channels and single cell content collection channels. We fabricated prototypes having 16 cell trapping pockets and 512 cell trapping pockets. Sequential all single cell handling for COS-1 cell was confirmed in 16 pockets type [1]. The sequential trapping and labeling were achieved in 512 pockets type. Capture rate for COS-1 cell was 83.2% and that for BAT cell was 70.3%. These devices are useful for high throughput single cell analysis.