Sorting differentiated mammalian cells using deterministic lateral displacement microfluidic devices.

Separation of differentiated and undifferentiated cells without labeling is required for cell analyses and clinical application of cultured differentiated cells in vitro. To proceed with the passive separation of differentiated cells inside a clean bench, we developed a system of deterministic lateral displacement (DLD) microfluidic devices and applied this system to sort differentiated cells in vitro. The fluid flow is driven by compressed air to the buffer. Priming and sorting can be completed by air pressure control. We use this system to separate C2C12 mononuclear myocytes from multinuclear myotubes. Additionally, using a DLD microfluidic channel of Dc = 20 µm, multinuclear myotubes can be effectively sorted as larger particles. We prepared differentiated adipocytes from mouse embryonic fibroblast (MEF) cells and sorted those containing lipid droplets. The diameters of these sorted adipocytes considered larger particles, exceeded 20 µm, similar to the Dc of the DLD microfluidic channel. Differentiated cell sorting by cell size will contribute to single-cell analyses and in vitro tissue model preparation for drug discovery.

Microvortex-induced turbulent mixing for reconstitution of high-density lipoprotein-mimicking nanoparticles with aggregation-prone phosphatidylcholine.

Lipid nanoparticles often contain a phosphatidylcholine with a long chain fatty acid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). However, their preparation often encounters difficulties such as the inability to yield <20 nm nanoparticles due to the aggregation-prone behavior of DSPC. High-density lipoproteins (HDLs) are ∼10 nm protein-bound lipid nanoparticles in our body, and microfluidic preparations of HDL-mimicking nanoparticles (µHDL) have been reported. Herein, we report a new microfluidic mixing mode that enables preparation of µHDL with DSPC in high yield (≥90% on a protein basis). The critical mechanism of this mode is a spontaneous asymmetric distribution of the ethanol flow injected in a symmetric manner followed by turbulent mixing in a simple rectangular parallelepiped-shaped chip.

Peritoneal dialysis-associated infection caused by Mycobacterium abscessus in a pediatric patient on continuous peritoneal dialysis without switching to hemodialysis.

Most peritoneal dialysis (PD)-associated infections caused by Mycobacterium abscessus (M. abscessus) require a transfer from PD to hemodialysis (HD). Here, we report a pediatric case of exit-site and tunnel infections caused by M. abscessus, for whom PD was continued with catheter replacement, debridement of the infected site, and the administration of multiple antibacterial agents. A 10-year-old boy with end-stage kidney disease secondary to juvenile nephronophthisis with NPHP1 deletion, for whom PD was initiated at the age of 9 years, was admitted to the hospital with complaints of fever, pus at the exit-site of the PD catheter, and poor PD drainage. The dialysis effluent culture results were negative; however, M. abscessus was detected in the pus at the exit-site of the PD catheter. The management of HD was expected to be challenging owing to the presence of developmental disorders. Therefore, PD was continued with the simultaneous removal of the PD catheter, reinsertion of a new catheter at a new site, and debridement of the infected site. Multiple antibacterial therapies were administered for 2 months, and the patient was eventually discharged without switching to HD. To the best of our knowledge, this is the first pediatric case of a PD-associated infection caused by M. abscessus, for whom PD was continued without switching to HD. This treatment strategy is not generally recommended but may be an option for patients without peritonitis who have difficulty switching to HD.

Blood Cell Separation Using Polypropylene-Based Microfluidic Devices Based on Deterministic Lateral Displacement.

Mammalian blood cell separation methods contribute to improving the diagnosis and treatment of animal and human diseases. Microfluidic deterministic lateral displacement (DLD) devices can sort cells based on their particle diameter. We developed microfluidic DLD devices with poly(propylene)-based resin and used them to separate bovine and human red blood cells (RBCs) and white blood cells (WBCs) without electric devices. To determine the critical cut-off diameter (Dc) of these devices, we used immunobeads with a diameter of 1-20 µm. The Dc values of the microfluidic DLD devices for the immunobeads in the experiments were similar to the calculated Dc values (8-10 µm). Results from bovine blood cell separation experiments suggest that lymphocytes and neutrophils can be separated from diluted, whole blood. Human RBCs were occasionally observed in the left outlet where larger particles with diameters closer to the Dc value were collected. Based on the Dc values, human neutrophils were sorted to the left outlet, whereas lymphocytes were observed in both outlets. Although microfluidic channel optimization is required for the concentration of sorted cells, the microfluidic DLD device prepared with a poly(propylene)-based resin has the potential for clinical use.

High expression of carcinoembryonic antigen and telomerase reverse transcriptase in circulating tumor cells is associated with poor clinical response to the immune checkpoint inhibitor nivolumab.

The present study aimed to enrich circulating tumor cells (CTCs) from blood samples using a new size-sorting CTC chip. The present study also set out to identify a blood sensitivity marker for the immune checkpoint inhibitor nivolumab in patients with advanced, pre-treatment lung cancer. The CTC sorting efficacy of the chip was investigated and the large cell fraction of blood samples from 15 patients with pre-treatment lung cancer who were later administered nivolumab were purified. The expression levels of carcinoembryonic antigen (CEA), human Telomerase Reverse Transcriptase (hTERT), cytokeratin19 (CK19), and programmed death ligand-1 (PD-L1) were investigated to clarify the association between these CTC markers and the clinical response to nivolumab. The CTC chip effectively enriched cells from lung cancer cell line PC-9. The large cell fraction had a high expression of CEA and hTERT, with the former being significantly associated with the clinical response to nivolumab. The expression of CEA and hTERT in CTCs derived from the blood of a patient with lung cancer were also validated. The evaluation of CEA and possibly hTERT in CTCs collected by the CTC chip may represent a promising predictive blood marker for sensitivity to nivolumab. To the best of our knowledge this is the first report to describe the predictive CTC marker for nivolumab in pre-treatment patients.

Capture of esophageal and breast cancer cells with polymeric microfluidic devices for CTC isolation.

The present study evaluated the capture efficiency of esophageal and breast cancer cells with a modified 'polymeric circulating tumor cells (CTC)-chip' microfluidic device, which was developed for the isolation of circulating tumor cells. Esophageal cancer cell lines KYSE150, KYSE220 and KYSE510, and breast cancer cell lines MCF7, SKBR3 and MDA-MB-231 were used for evaluation. The capture efficiencies of the esophageal cancer cell lines in phosphate-buffered saline (PBS) were ~0.9, irrespective of epithelial cell adhesion molecule (EpCAM) expression, which was represented as the mean fluorescent intensity from 528 to 76. In the breast cancer cell lines, efficient capture was observed for MCF7 and SKBR3 in PBS; however, a low value of ~0.1 was obtained for MDA-MB-231. Fluorescent imaging of immunolabeled cells revealed marginal EpCAM expression in MDA-MB-231. Using whole blood, no clogging occurred in the microstructure-modified CTC-chip and efficiency of capture was successfully evaluated. Capture efficiencies for KYSE220 and MCF7 in whole blood were >0.7, but were of either equal or lesser efficiency in comparison to PBS. Therefore, the modified CTC-chip appears useful for clinical application due to its cost, practicality of use, and efficient cancer cell capture.

Polymeric microfluidic devices exhibiting sufficient capture of cancer cell line for isolation of circulating tumor cells.

Here, we developed polymeric microfluidic devices for the isolation of circulating tumor cells. The devices, with more than 30,000 microposts in the channel, were produced successfully by a UV light-curing process lasting 3 min. The device surface was coated with anti-epithelial cell adhesion molecule antibody by just contacting the antibody solution, and a flow system including the device was established to send a cell suspension through it. We carried out flow tests for evaluation of the device's ability to capture tumor cells using an esophageal cancer cell line, KYSE220, dispersed in phosphate-buffered saline or mononuclear cell separation from whole blood. After the suspension flowed through the chip, many cells were seen to be captured on the microposts coated with the antibody, whereas there were few cells in the device without the antibody. Owing to the transparency of the device, we could observe the intact and the stained cells captured on the microposts by transmitted light microscopy and phase contrast microscopy, in addition to fluorescent microscopy, which required fluorescence labeling. Cell capture efficiencies (i.e., recovery rates of the flowing cancer cells by capture with the microfluidic device) were measured. The resulting values were 0.88 and 0.95 for cell suspension in phosphate-buffered saline, and 0.85 for the suspension in the mononuclear cell separation, suggesting the sufficiency of this device for the isolation of circulating tumor cells. Therefore, our device may be useful for research and treatments that rely on investigation of circulating tumor cells in the blood of cancer patients.

Synthesis and characterization of high-quality skin-cooling sheets containing thermosensitive poly(N-isopropylacrylamid).

Poly(N-isopropylacrylamide) (PNIPAAm) is the most popular thermosensitive polymer, and exhibits a low critical solution temperature of approximately 32°C. This study aimed to examine the usefulness of new cooling sheets, which are manufactured using a thermosensitive poly(N-isopropylacrylamide) (PNIPAAm) material. We prepared cooling-hydrogel sheets containing PNIPAAm (PNIPAAm sheet). We measured the skin temperature on the arms of the subjects using a thermograph and compared the usefulness of the PNIPAAm sheet and a control cooling-hydrogel sheet that did not contain the PNIPAAm mixture. Thermographic measurements obtained 40 min after the treatment with the cooling sheets showed the skin temperature of the subjects treated with the 3.% (w/w) PNIPAAm sheets to be significantly lower than that of the subjects treated with the control cooling-hydrogel sheet (p < 0.005). Compared with the control sheet, the cooling effect of the new PNIPAAm sheet also persisted for a longer duration (up to 100 min). The PNIPAAm sheets exhibited excellent cooling effects. This sheet may therefore be useful for lowering the body temperature of patients with high-grade fever, such as fever due to influenza infection.

Intrageneric relationships of members of the genus Rhodopseudomonas.

The intrageneric structure of the genus Rhodopseudomonas was evaluated by studying sequence information on 16S rRNA genes, 16S-23S rRNA gene internal transcribed spacer (ITS) regions, and puf genes using 33 test strains. The topology of phylogenetic trees based on these sequences was similar to those of every other independent method for tree construction. These phylogenetic data indicated that the test strains were grouped into at least 7 clusters possibly at the species level. This was supported by genomic DNA-DNA similarities among 12 representative test strains selected from these clusters. Our molecular data confirmed that the currently available strains of Rhodopseudomonas (Rps.) palustris are genetically quite heterogeneous within the genus. For example, Rps. palustris strains DSM 123(T) and ATCC 17001(T) are different from each other at the species level despite their status as the type strain of the species. Rps. palustris strain ATCC 17005 and the full genome-sequenced strains BisA53, BisB18, BisB5, and HaA2 should be re-classified into different species from Rps. palustris or as novel species of the genus Rhodopseudomonas.

Excited states of porphyrin isomers and porphycene derivatives: a SAC-CI study.

Excited states of free-base porphyrin isomers, porphycene (Pc), corrphycene (Cor), and hemiporphycene (hPc), were studied by the Symmetry-Adapted Cluster (SAC)/SAC-Configuration Interaction (CI) method. The absorption peaks of the porphyrin isomers were assigned on the basis of the SAC-CI spectra. The X, Y, X', and Y' bands of the porphyrin isomers, which have weak intensities, are identified. The differences in the Q-band absorptions among the isomers were clearly explained by the four-orbital model. In Cor and hPc, the wave function of the B-band corresponds to the mixture of the four-orbital excitations and the optically forbidden excitation of free-base porphin (P), due to the molecular symmetry lowering in the isomers. The B-band character is described by the five-orbital model in Pc and the six-orbital model in Cor and hPc. Two tetrazaporphycenes and two ring-extended (dibenzo) porphycenes were designed, and the Q-band transition moment was successfully controlled. These examples show that the control of the four-orbital energy levels is the guiding principle for pigment design in porphyrin compounds.