Impedimetric Gram-Positive Bacteria Biosensor Using Vancomycin-Coated Silica Nanoparticles with a Gold Nanocluster-Deposited Electrode.

We introduce a swift, label-free electrochemical biosensor designed for the precise on-site detection of Gram-positive bacteria via electrochemical impedance spectroscopy. The biosensor was prepared by electroplating the electrode surface with gold nanoclusters (AuNCs) on the gold-interdigitated wave-shaped electrode with a printed circuit board (Au-PCB) electrode, which plays a role in cost-effective and promising lab-on-a-chip microsystems and integrated biosensing systems. This was followed by the application of silica nanoparticle-modified vancomycin (SiNPs-VAN) that binds to Gram-positive bacteria and facilitates their detection on the AuNC-coated surface. The biosensor demonstrated remarkable sensitivity and specificity. It could detect as few as 102 colony-forming units (CFU)/mL of Staphylococcus aureus, 101 CFU/mL of Bacillus cereus, and 102 CFU/mL of Micrococcus luteus within 20 min. Additionally, SiNPs-VAN is also known for its high stability, low cost, and ease of preparation. It is effective in identifying Gram-positive bacteria in water samples across a concentration range of 102-105 CFU/mL and shows selective identification of Gram-positive bacteria with minimal interference from Gram-negative bacteria like Escherichia coli. The ability of the biosensor to quantify Gram-positive bacteria aligns well with the results obtained from the quantitative real-time polymerase chain reaction (qRT-PCR). These findings highlight the potential of electrochemical biosensors for the detection of pathogens and other biological entities, marking a significant advancement in this field.

Engineering Stem Cell Fate Controlling Biomaterials to Develop Muscle Connective Tissue Layered Myofibers.

Skeletal muscle connective tissue (MCT) surrounds myofiber bundles to provide structural support, produce force transduction from tendons, and regulate satellite cell differentiation during muscle regeneration. Engineered muscle tissue composed of myofibers layered within MCT has not yet been developed. Herein, a bioengineering strategy to create MCT-layered myofibers through the development of stem cell fate-controlling biomaterials that achieve both myogenesis and fibroblast differentiation in a locally controlled manner at the single construct is introduced. The reciprocal role of transforming growth factor-beta 1 (TGF-ß1) and its inhibitor as well as 3D matrix stiffness to achieve co-differentiation of MCT fibroblasts and myofibers from a human-induced pluripotent stem cell (hiPSC)-derived paraxial mesoderm is studied. To avoid myogenic inhibition, TGF-ß1 is conjugated on the gelatin-based hydrogel to control the fibroblasts' populations locally; the TGF-ß1 degrades after 2 weeks, resulting in increased MCT-specific extracellular matrix (ECM) production. The locations of myofibers and fibroblasts are precisely controlled by using photolithography and co-axial wet spinning techniques, which results in the formation of MCT-layered functional myofibers in 3D constructs. This advanced engineering strategy is envisioned as a possible method for obtaining biomimetic human muscle grafts for various biomedical applications.

A 3D hanging spheroid-filter plate for high-throughput drug testing and CAR T cell cytotoxicity assay.

Tumour spheroids are widely used in immune cell cytotoxicity assays and anticancer drug testing, providing a physiologically relevant model replicating the tumour microenvironment. However, co-culture of immune and tumour cells complicates quantification of immune cell killing efficiency. We present a novel 3D hanging spheroid-filter plate that efficiently facilitates spheroid formation and separates unbound/dead cells during cytotoxicity assays. Optical imaging directly measures the cytotoxic effects of anti-cancer drugs on tumour spheroids, eliminating the need for live/dead fluorescent staining. This approach enables cost-effective evaluation of T-cell cytotoxicity with specific chimeric antigen receptors (CARs), enhancing immune cell-based assays and drug testing in three-dimensional tumour models.

Efficient separation of large particles and giant cancer cells using an isosceles trapezoidal spiral microchannel.

Polyploid giant cancer cells (PGCCs) contribute to the genetic heterogeneity and evolutionary dynamics of tumors. Their size, however, complicates their isolation from mainstream tumor cell populations. Standard techniques like fluorescence-activated cell sorting (FACS) rely on fluorescent labeling, introducing potential challenges in subsequent PGCC analyses. In response, we developed the Isosceles Trapezoidal Spiral Microchannel (ITSµC), a microfluidic device optimizing the Dean drag force (FD) and exploiting uniform vortices for enhanced separation. Numerical simulations highlighted ITSµC's advantage in producing robust FD compared to rectangular and standard trapezoidal channels. Empirical results confirmed its ability to segregate larger polystyrene (PS) particles (avg. diameter: 50 µm) toward the inner wall, while directing smaller ones (avg. diameter: 23 µm) outward. Utilizing ITSµC, we efficiently isolated PGCCs from doxorubicin-resistant triple-negative breast cancer (DOXR-TNBC) and patient-derived cancer (PDC) cells, achieving outstanding purity, yield, and viability rates (all greater than 90%). This precision was accomplished without fluorescent markers, and the versatility of ITSµC suggests its potential in differentiating a wide range of heterogeneous cell populations.

Subwavelength Terahertz Resonance Imaging (STRING) for Molecular Fingerprinting.

Subwavelength terahertz (THz) imaging methods are highly desirable for biochemical sensing as well as materials sciences, yet sensitive spectral fingerprinting is still challenging in the frequency domain due to weak light-matter interactions. Here, we demonstrate subwavelength THz resonance imaging (STRING) that overcomes this limitation to achieve ultrasensitive molecular fingerprinting. STRING combines individual ring-shaped coaxial single resonators with near-field spectroscopy, yielding considerable sensitivity gains from both local field enhancement and the near-field effect. As an initial demonstration, we obtained spectral fingerprints from isomers of α-lactose and maltose monohydrates, achieving sensitivity that was enhanced by up to 10 orders of magnitude compared to far-field THz measurements with pelletized samples. Our results show that the STRING platform could enable the development of THz spectroscopy as a practical and sensitive tool for the fingerprinting and spectral imaging of molecules and nanoparticles.

Vancomycin-conjugated polydopamine-coated magnetic nanoparticles for molecular diagnostics of Gram-positive bacteria in whole blood.

BACKGROUND: Sepsis is caused mainly by infection in the blood with a broad range of bacterial species. It can be diagnosed by molecular diagnostics once compounds in the blood that interfere with molecular diagnostics are removed. However, this removal relies on ultracentrifugation. Immunomagnetic separation (IMS), which typically uses antibody-conjugated silica-coated magnetic nanoparticles (Ab-SiO2-MNPs), has been widely applied to isolate specific pathogens in various types of samples, such as food and environmental samples. However, its direct use in blood samples containing bacteria is limited due to the aggregation of SiO2-MNPs in the blood and inability to isolate multiple species of bacteria causing sepsis. RESULTS: In this study, we report the synthesis of vancomycin-conjugated polydopamine-coated (van-PDA-MNPs) enabling preconcentration of multiple bacterial species from blood without aggregation. The presence of PDA and van on MNPs was verified using transmission electron microscopy, X-ray photoelectron spectroscopy, and energy disruptive spectroscopy. Unlike van-SiO2-MNPs, van-PDA-MNPs did not aggregate in the blood. Van-PDA-MNPs were able to preconcentrate several species of Gram-positive bacteria in the blood, lowering the limit of detection (LOD) to 10 colony forming units/mL by polymerase chain reaction (PCR) and quantitative PCR (qPCR). This is 10 times more sensitive than the LOD obtained by PCR and qPCR using van-SiO2-MNPs. CONCLUSION: These results suggest that PDA-MNPs can avoid aggregation in blood and be conjugated with receptors, thereby improving the sensitivity of molecular diagnostics of bacteria in blood samples.

3D hanging spheroid plate for high-throughput CAR T cell cytotoxicity assay.

BACKGROUND: Most high-throughput screening (HTS) systems studying the cytotoxic effect of chimeric antigen receptor (CAR) T cells on tumor cells rely on two-dimensional cell culture that does not recapitulate the tumor microenvironment (TME). Tumor spheroids, however, can recapitulate the TME and have been used for cytotoxicity assays of CAR T cells. But a major obstacle to the use of tumor spheroids for cytotoxicity assays is the difficulty in separating unbound CAR T and dead tumor cells from spheroids. Here, we present a three-dimensional hanging spheroid plate (3DHSP), which facilitates the formation of spheroids and the separation of unbound and dead cells from spheroids during cytotoxicity assays. RESULTS: The 3DHSP is a 24-well plate, with each well composed of a hanging dripper, spheroid wells, and waste wells. In the dripper, a tumor spheroid was formed and mixed with CAR T cells. In the 3DHSP, droplets containing the spheroids were deposited into the spheroid separation well, where unbound and dead T and tumor cells were separated from the spheroid through a gap into the waste well by tilting the 3DHSP by more than 20°. Human epidermal growth factor receptor 2 (HER2)-positive tumor cells (BT474 and SKOV3) formed spheroids of approximately 300-350 µm in diameter after 2 days in the 3DHSP. The cytotoxic effects of T cells engineered to express CAR recognizing HER2 (HER2-CAR T cells) on these spheroids were directly measured by optical imaging, without the use of live/dead fluorescent staining of the cells. Our results suggest that the 3DHSP could be incorporated into a HTS system to screen for CARs that enable T cells to kill spheroids formed from a specific tumor type with high efficacy or for spheroids consisting of tumor types that can be killed efficiently by T cells bearing a specific CAR. CONCLUSIONS: The results suggest that the 3DHSP could be incorporated into a HTS system for the cytotoxic effects of CAR T cells on tumor spheroids.

Palladium Probe Consisting of Naphthalimide and Ethylenediamine for Selective Turn-On Sensing of CO and Cell Imaging.

An assay to detect carbon monoxide (CO), one of the gaseous signaling molecules, has been prepared using a new palladium complex probe. The ethylenediamine group linked to the naphthalimide fluorophore coordinates to Pd(II) which intramolecularly quenches the emission. Upon treatment with CO, the absorbance of the turn-on fluorescent sensor changes due to the formation of a complex between Pd(II) and CO at room temperature in a phosphate buffer. As the concentration of CO increases, the probe peak emission intensity at 527 nm gradually increases. Other analyte controls, such as K+, Mg2+, Al3+, Zn2+, Cr3+, Hg2+, Fe3+, alanine, glycine, leucine, lysine, serine, threonine, tyrosine, F-, Cl-, Br-, NO, NO2-, NO3-, HCO3-, CH3COO-, H2O2, •OH, and tBuOO•, exhibit no significant effect on emission intensity. The response time of the probe to CO was quite fast because of the relatively weak coordination of Pd(II) to the pendent ethylenediamine group. The Pd probe is capable of detecting CO in aqueous buffer as well as in living cells with high selectivity and stability, providing a potential real-time indicator for studying CO-involved reactions in biological systems.

Decrease in membrane fluidity and traction force induced by silica-coated magnetic nanoparticles.

BACKGROUND: Nanoparticles are being increasingly used in biomedical applications owing to their unique physical and chemical properties and small size. However, their biophysical assessment and evaluation of side-effects remain challenging. We addressed this issue by investigating the effects of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate [MNPs@SiO2(RITC)] on biophysical aspects, such as membrane fluidity and traction force of human embryonic kidney 293 (HEK293) cells. We further extended our understanding on the biophysical effects of nanoparticles on cells using a combination of metabolic profiling and transcriptomic network analysis. RESULTS: Overdose (1.0 µg/µL) treatment with MNPs@SiO2(RITC) induced lipid peroxidation and decreased membrane fluidity in HEK293 cells. In addition, HEK293 cells were morphologically shrunk, and their aspect ratio was significantly decreased. We found that each traction force (measured in micropillar) was increased, thereby increasing the total traction force in MNPs@SiO2(RITC)-treated HEK293 cells. Due to the reduction in membrane fluidity and elevation of traction force, the velocity of cell movement was also significantly decreased. Moreover, intracellular level of adenosine triphosphate (ATP) was also decreased in a dose-dependent manner upon treatment with MNPs@SiO2(RITC). To understand these biophysical changes in cells, we analysed the transcriptome and metabolic profiles and generated a metabotranscriptomics network, which revealed relationships among peroxidation of lipids, focal adhesion, cell movement, and related genes and metabolites. Furthermore, in silico prediction of the network showed increment in the peroxidation of lipids and suppression of focal adhesion and cell movement. CONCLUSION: Taken together, our results demonstrated that overdose of MNPs@SiO2(RITC) impairs cellular movement, followed by changes in the biophysical properties of cells, thus highlighting the need for biophysical assessment of nanoparticle-induced side-effects.

Organ-on-a-Chip for Studying Gut-Brain Interaction Mediated by Extracellular Vesicles in the Gut Microenvironment.

Extracellular vesicles (EVs) are a group of membrane vesicles that play important roles in cell-to-cell and interspecies/interkingdom communications by modulating the pathophysiological conditions of recipient cells. Recent evidence has implied their potential roles in the gut-brain axis (GBA), which is a complex bidirectional communication system between the gut environment and brain pathophysiology. Despite the evidence, the roles of EVs in the gut microenvironment in the GBA are less highlighted. Moreover, there are critical challenges in the current GBA models and analyzing techniques for EVs, which may hinder the research. Currently, advances in organ-on-a-chip (OOC) technologies have provided a promising solution. Here, we review the potential effects of EVs occurring in the gut environment on brain physiology and behavior and discuss how to apply OOCs to research the GBA mediated by EVs in the gut microenvironment.

A Golgi Apparatus-Targeting, Naphthalimide-Based Fluorescent Molecular Probe for the Selective Sensing of Formaldehyde.

Formaldehyde (FA) is a colorless, flammable, foul-smelling chemical used in building materials and in the production of numerous household chemical goods. Herein, a fluorescent chemosensor for FA is designed and prepared using a selective organ-targeting probe containing naphthalimide as a fluorophore and hydrazine as a FA-binding site. The amine group of the hydrazine reacts with FA to form a double bond and this condensation reaction is accompanied by a shift in the absorption band of the probe from 438 nm to 443 nm upon the addition of FA. Further, the addition of FA is shown to enhance the emission band at 532 nm relative to the very weak fluorescent emission of the probe itself. Moreover, a high specificity is demonstrated towards FA over other competing analytes such as the calcium ion (Ca2+), magnesium ion (Mg2+), acetaldehyde, benzaldehyde, salicylaldehyde, glucose, glutathione, sodium sulfide (Na2S), sodium hydrosulfide (NaHS), hydrogen peroxide (H2O2), and the tert-butylhydroperoxide radical. A typical two-photon dye incorporated into the probe provides intense fluorescence upon excitation at 800 nm, thus demonstrating potential application as a two-photon fluorescent probe for FA sensing. Furthermore, the probe is shown to exhibit a fast response time for the sensing of FA at room temperature and to facilitate intense fluorescence imaging of breast cancer cells upon exposure to FA, thus demonstrating its potential application for the monitoring of FA in living cells. Moreover, the presence of the phenylsulfonamide group allows the probe to visualize dynamic changes in the targeted Golgi apparatus. Hence, the as-designed probe is expected to open up new possibilities for unique interactions with organ-specific biological molecules with potential application in early cancer cell diagnosis.

Noise exposure alters MMP9 and brevican expression in the rat primary auditory cortex.

BACKGROUND: This study aimed to investigate the changes in molecules related to perineuronal nets (PNNs) and synaptic transporters in the primary auditory cortices of rats with noise-induced hearing loss. Female Sprague-Dawley rats at postnatal day 7 were divided into the noise and control groups. Four hours of 115 dB SPL white noise was delivered for 10 days to the noise group. Thirty days after noise exposure, the primary auditory cortex and the inferior colliculus were harvested. The expression levels of vesicular glutamatergic transporter (VGLUT)1, VGLUT2, vesicular GABA transporter (VGAT), glutamate decarboxylase (GAD)67, brevican, aggrecan, MMP9, and MMP14 were evaluated using real-time reverse transcription polymerase chain reaction or western blot. An immunofluorescence assay was conducted to assess parvalbumin (PV), Wisteria floribunda agglutinin (WFA), and brevican. The immune-positive cells were counted in the primary auditory cortex. RESULTS: The expression level of VGLUT1 in the primary auditory cortex was decreased in the noise group. The expression level of VGLUT2 in the inferior colliculus was elevated in the noise group. The expression levels of brevican and PV + WFA in the primary auditory cortex were decreased in the noise group. The expression level of MMP9 in the primary auditory cortex was increased in the noise group. CONCLUSION: Noise-induced hearing loss during the precritical period impacted PNN expression in the primary auditory cortex. Increased MMP9 expression may have contributed to the decrease in brevican expression. These changes were accompanied by the attenuation of glutamatergic synaptic transporters.

Effect of silica-coated magnetic nanoparticles on rigidity sensing of human embryonic kidney cells.

BACKGROUND: Nanoparticles (NPs) can enter cells and cause cellular dysfunction. For example, reactive oxygen species generated by NPs can damage the cytoskeleton and impair cellular adhesion properties. Previously, we reported that cell spreading and protrusion structures such as lamellipodia and filopodia was reduced when cells are treated with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate (MNPs@SiO2(RITC)), even at 0.1 µg/µL. These protruded structures are involved in a cell's rigidity sensing, but how these NPs affect rigidity sensing is unknown. RESULTS: Here, we report that the rigidity sensing of human embryonic kidney (HEK293) cells was impaired even at 0.1 µg/µL of MNPs@SiO2(RITC). At this concentration, cells were unable to discern the stiffness difference between soft (5 kPa) and rigid (2 MPa) flat surfaces. The impairment of rigidity sensing was further supported by observing the disappearance of locally contracted elastomeric submicron pillars (900 nm in diameter, 2 µm in height, 24.21 nN/µm in stiffness k) under MNPs@SiO2(RITC) treated cells. A decrease in the phosphorylation of paxillin, which is involved in focal adhesion dynamics, may cause cells to be insensitive to stiffness differences when they are treated with MNPs@SiO2(RITC). CONCLUSIONS: Our results suggest that NPs may impair the rigidity sensing of cells even at low concentrations, thereby affecting cell adhesion and spreading.

3D-Printed Modular Microfluidic Device Enabling Preconcentrating Bacteria and Purifying Bacterial DNA in Blood for Improving the Sensitivity of Molecular Diagnostics.

Molecular diagnostics for sepsis is still a challenge due to the presence of compounds that interfere with gene amplification and bacteria at concentrations lower than the limit of detection (LOD). Here, we report on the development of a 3D printed modular microfluidic device (3DpmµFD) that preconcentrates bacteria of interest in whole blood and purifies their genomic DNA (gDNA). It is composed of a W-shaped microchannel and a conical microchamber. Bacteria of interest are magnetically captured from blood in the device with antibody conjugated magnetic nanoparticles (Ab-MNPs) at 5 mL/min in the W-shaped microchannel, while purified gDNA of the preconcentrated bacteria is obtained with magnetic silica beads (MSBs) at 2 mL/min in the conical microchamber. The conical microchamber was designed to be connected to the microchannel after the capturing process using a 3D-printed rotary valve to minimize the exposure of the MSBs to interfering compounds in blood. The pretreatment process of spiked blood (2.5 mL) can be effectively completed within about 50 min. With the 3DpmµFD, the LOD for the target microorganism Escherichia coli O157:H7 measured by both polymerase chain reaction (PCR) with electrophoresis and quantitative PCR was 10 colony forming unit (CFU) per mL of whole blood. The results suggest that our method lowers the LOD of molecular diagnostics for pathogens in blood by providing bacterial gDNA at high purity and concentration.

3D Bioprinted Vascularized Tumour for Drug Testing.

An in vitro screening system for anti-cancer drugs cannot exactly reflect the efficacy of drugs in vivo, without mimicking the tumour microenvironment (TME), which comprises cancer cells interacting with blood vessels and fibroblasts. Additionally, the tumour size should be controlled to obtain reliable and quantitative drug responses. Herein, we report a bioprinting method for recapitulating the TME with a controllable spheroid size. The TME was constructed by printing a blood vessel layer consisting of fibroblasts and endothelial cells in gelatine, alginate, and fibrinogen, followed by seeding multicellular tumour spheroids (MCTSs) of glioblastoma cells (U87 MG) onto the blood vessel layer. Under MCTSs, sprouts of blood vessels were generated and surrounding MCTSs thereby increasing the spheroid size. The combined treatment involving the anti-cancer drug temozolomide (TMZ) and the angiogenic inhibitor sunitinib was more effective than TMZ alone for MCTSs surrounded by blood vessels, which indicates the feasibility of the TME for in vitro testing of drug efficacy. These results suggest that the bioprinted vascularized tumour is highly useful for understanding tumour biology, as well as for in vitro drug testing.

WT1 Measurable Residual Disease Assay in Patients With Acute Myeloid Leukemia Who Underwent Allogeneic Hematopoietic Stem Cell Transplantation: Optimal Time Points, Thresholds, and Candidates.

The absence of relevant guidelines for Wilms tumor 1 (WT1) gene quantification as a measurable residual disease (MRD) assessment for patients with acute myeloid leukemia (AML) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) has limited the widespread use in practice. We investigated optimal time points, thresholds, and candidates for the bone marrow WT1 MRD assay in 425 consecutive patients with AML who underwent allo-HSCT. WT1 expression kinetics before allo-HSCT and at 1 or 3 months after allo-HSCT were determined by real-time PCR using the European LeukemiaNet (ELN) normalized method. Relapsed patients had significantly higher WT1 levels before allo-HSCT and at 3 months after allo-HSCT. The best time point for the WT1 MRD assay was before allo-HSCT by the receiver operating characteristic curve. Among various thresholds, 250 copies recommended from ELN researchers were mostly predictive of post-transplant relapse. In multivariate analysis, WT1 MRD positivity independently predicted relapse, resulting in inferior survival. In subgroup analyses, pretransplant WT1 MRD positivity was predictive of post-transplant relapse in the intermediate group, whereas WT1 MRD positivity occurred at 3 months after allo-HSCT in favorable and adverse risk groups. Among MRD-positive patients before allo-HSCT, all patients who were MRD positive at 3 months relapsed within 6 months. The WT1 MRD assay before allo-HSCT or 3 months after allo-HSCT is useful for predicting post-transplant relapse with a different significance in each risk group by time points, showing the benefit of multiple tests over time. Such monitoring is particularly available in patients with AML without specific molecular targets.

Rapid emergence and mechanisms of resistance by U87 glioblastoma cells to doxorubicin in an in vitro tumor microfluidic ecology.

In vitro prediction of the probable rapid emergence of resistance to a drug in tumors could act to winnow out potential candidates for further costly development. We have developed a microfluidic device consisting of ∼500 hexagonal microcompartments that provides a complex ecology with wide ranges of drug and nutrient gradients and local populations. This ecology of a fragmented metapopulation induced the drug resistance in stage IV U87 glioblastoma cells to doxorubicin in 7 d. Exome and transcriptome sequencing of the resistant cells identified mutations and differentially expressed genes. Gene ontology and pathway analyses of the genes identified showed that they were functionally relevant to the established mechanisms of doxorubicin action. Specifically, we identified (i) a frame-shift insertion in the filamin-A gene, which regulates the influx and efflux of topoisomerase II poisons; (ii) the overexpression of aldo-keto reductase enzymes, which convert doxorubicin into doxorubicinol; and (iii) activation of NF-κB via alterations in the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway from mutations in three genes (CARD6, NSD1, and NLRP13) and the overexpression of inflammatory cytokines. Functional experiments support the in silico analyses and, together, demonstrate the effects of these genetic changes. Our findings suggest that, given the rapid evolution of resistance and the focused response, this technology could act as a rapid screening modality for genetic aberrations leading to resistance to chemotherapy as well as counter selection of drugs unlikely to be successful ultimately.

A Microbore Tubing Based Spiral for Multistep Cell Fractionation.

Cells were separated with the aid of a multistep spiral fractionation device, utilizing hydrodynamic forces in a spiral tubing. The spiral was fabricated using "off-the-shelf" microbore tubing, allowing for cheap and fast prototyping to achieve optimal cell separation. As a first step, a model system with 20 and 40 µm beads was used to demonstrate the effectiveness of the multistep separation device. With an initial purity of 5%, a separation purity of 83% was achieved after a two-step separation with the addition of 0.1% polyethylene glycol (PEG)-8000. Next, doxorubicin-resistant polyploid giant breast cancer cells (MDA-MB-231) were separated from doxorubicin-sensitive monoploid small breast cancer cells in the same fashion as the beads, resulting in a purity of around 40%, while maintaining a cell viability of more than 90%. Combined with basic cell analytical methods, the hydrodynamic separation principle of the device could be envisaged to be useful for a variety of cell fractionation needs in cell biology and in clinical applications.

Development of bufferless gel electrophoresis chip for easy preparation and rapid DNA separation.

This work presents a handy, fast, and compact bufferless gel electrophoresis chip (BGEC), which consists of precast agarose gel confined in a disposable plastic body with electrodes. It does not require large volumes of buffer to fill reservoirs, or the process of immersing the gel in the buffer. It withstands voltages up to 28.4 V/cm, thereby allowing DNA separation within 10 min with a similar separation capability to the standard gel electrophoresis. The results suggest that our BGEC is highly suitable for in situ gel electrophoresis in forensic, epidemiological settings and crime scenes where standard gel electrophoresis equipment cannot be brought in while quick results are needed.

Formation of size-controllable tumour spheroids using a microfluidic pillar array (µFPA) device.

Spheroids are recognized for replicating the physiological microenvironment of tumours. However, because of the lack of controllability of the spheroid size, the response to anticancer drugs is variable in conventional spheroid culture methods. In this paper, we describe a method to generate several hundreds of spheroids of various types of cancer cells including patient derived cancer cells (PDCs) using a microfluidic device with pillars (diameter: 40 µm, height: 70 µm, center-to-center distance: 140 µm), called a microfluidic pillar array (µFPA) device. About three hundred glioma (U87) spheroids were obtained in the µFPA device within 3 days, and about 90% of them ranged from 175 to 225 µm. These spheroids were more resistant to doxorubicin at 10 µM than U87 cells in a monolayer. The former showed higher expression of CD133, a cancer stem cell marker, than the latter. Hypoxia inducible factor-1α (HIF-1α), another cancer stem cell marker, was found in the nucleus of the former, but found in the cytoplasm of the cells in a monolayer. Drug responses of spheroids of another glioma cell line (U251) and triple negative breast cancer (TNBC) primary cells were also easily quantified by measuring changes in spheroid size at different concentrations of their respective drug on the µFPA device. The µFPA device can be a powerful platform for obtaining uniform spheroids and monitoring the drug response of cancer cells including PDCs.