Takanori Akagi

Cell electrophoresis on a chip: what can we know from the changes in electrophoretic mobility?

An overview of both experimental and theoretical studies of cell electrophoresis mobility (EPM) over the past fifty years and the relevance of cell EPM measurement are presented and discussed from the viewpoint of exploring the potential use of cell EPM as an index of the biological condition of cells. Physical measurements of the optical and/or electrical properties of cells have been attracting considerable attention as noninvasive cell-evaluation methods that are essential for the future of cell-based application technologies such as cell-based drug screening and cell therapy. Cell EPM, which can be measured in a noninvasive manner by cell electrophoresis, reflects the electrical and mechanical properties of the cell surface. Although the importance of cell EPM has been underestimated for a long time, mostly owing to the technical difficulties associated with its measurement, recent improvements in measurement technology using microcapillary chips have been changing the situation: cell EPM measurement has become more reliable and faster. Recent studies using the automated microcapillary cell electrophoresis system have revealed the close correlation between cell EPM and important biological phenomena including cell cycle, apoptosis, enzymatic treatment, and immune reaction. In particular, the converged EPM distribution observed for synchronized cells has altered the conventional belief that cell EPMs vary considerably. Finding a new significance of cell EPM is likely to lead to noninvasive cell evaluation methods essential for the next-generation of cell engineering.

Abnormal intrinsic dynamics of dendritic spines in a fragile X syndrome mouse model in vivo

Dendritic spine generation and elimination play an important role in learning and memory, the dynamics of which have been examined within the neocortex in vivo. Spine turnover has also been detected in the absence of specific learning tasks, and is frequently exaggerated in animal models of autistic spectrum disorder (ASD). The present study aimed to examine whether the baseline rate of spine turnover was activity-dependent. This was achieved using a microfluidic brain interface and open-dura surgery, with the goal of abolishing neuronal Ca2+ signaling in the visual cortex of wild-type mice and rodent models of fragile X syndrome (Fmr1 knockout [KO]). In wild-type and Fmr1 KO mice, the majority of baseline turnover was found to be activity-independent. Accordingly, the application of matrix metalloproteinase-9 inhibitors selectively restored the abnormal spine dynamics observed in Fmr1 KO mice, without affecting the intrinsic dynamics of spine turnover in wild-type mice. Such findings indicate that the baseline turnover of dendritic spines is mediated by activity-independent intrinsic dynamics. Furthermore, these results suggest that the targeting of abnormal intrinsic dynamics might pose a novel therapy for ASD.

On-Chip Immunoelectrophoresis of Extracellular Vesicles Released from Human Breast Cancer Cells

Extracellular vesicles (EVs) including exosomes and microvesicles have attracted considerable attention in the fields of cell biology and medicine. For a better understanding of EVs and further exploration of their applications, the development of analytical methods for biological nanovesicles has been required. In particular, considering the heterogeneity of EVs, methods capable of measuring individual vesicles are desired. Here, we report that on-chip immunoelectrophoresis can provide a useful method for the differential protein expression profiling of individual EVs. Electrophoresis experiments were performed on EVs collected from the culture supernatant of MDA-MB-231 human breast cancer cells using a measurement platform comprising a microcapillary electrophoresis chip and a laser dark-field microimaging system. The zeta potential distribution of EVs that reacted with an anti-human CD63 (exosome and microvesicle marker) antibody showed a marked positive shift as compared with that for the normal immunoglobulin G (IgG) isotype control. Thus, on-chip immunoelectrophoresis could sensitively detect the over-expression of CD63 glycoproteins on EVs. Moreover, to explore the applicability of on-chip immunoelectrophoresis to cancer diagnosis, EVs collected from the blood of a mouse tumor model were analyzed by this method. By comparing the zeta potential distributions of EVs after their immunochemical reaction with normal IgG, and the anti-human CD63 and anti-human CD44 (cancer stem cell marker) antibodies, EVs of tumor origin circulating in blood were differentially detected in the real sample. The result indicates that the present method is potentially applicable to liquid biopsy, a promising approach to the low-invasive diagnosis of cancer.

Anisotropic etching of amorphous perfluoropolymer films in oxygen-based inductively coupled plasmas

An amorphous perfluoropolymer, “Cytop™” (Asahi Glass Co., Ltd.), is a preferable material for the fabrication of micro total analysis system devices because of its superior optical transparency over a wide wavelength range and low refractive index of 1.34, which is almost the same as that of water, as well as excellent chemical stability. To establish the precise microfabrication technology for this unique resin, the dry etching of the amorphous perfluoropolymer in Ar/O2 low-pressure inductively coupled plasma has been studied. A relatively high etch rate of approximately 6.3 μm/min at maximum and highly anisotropic etched features was attained. Plasma measurements by a single Langmuir probe technique and actinometry revealed that etching is dominated by ion-assisted surface desorption above a 10% O2 mixing ratio, whereas the supply of active oxygen species is the rate-limiting process below 10%. Moreover, angled x-ray photoelectron spectroscopy measurements of an etched trench pattern revealed that a hig...

Lab-on-a-brain: Implantable micro-optical fluidic devices for neural cell analysis in vivo

The high-resolution imaging of neural cells in vivo has brought about great progress in neuroscience research. Here, we report a novel experimental platform, where the intact brain of a living mouse can be studied with the aid of a surgically implanted micro-optical fluidic device; acting as an interface between neurons and the outer world. The newly developed device provides the functions required for the long-term and high-resolution observation of the fine structures of neurons by two-photon laser scanning microscopy and the microfluidic delivery of chemicals or drugs directly into the brain. A proof-of-concept experiment of single-synapse stimulation by two-photon uncaging of caged glutamate and observation of dendritic spine shrinkage over subsequent days demonstrated a promising use for the present technology.

Evaluation of desialylation effect on zeta potential of extracellular vesicles secreted from human prostate cancer cells by on-chip microcapillary electrophoresis

Extracellular vesicles including exosomes have the potential to be used in therapeutic and diagnostic applications. Towards the noninvasive diagnosis of prostate cancer using urinary exosomes, technical challenges lie in developing precise methods of analyzing exosomes. Previously, we developed an on-chip microcapillary electrophoresis (µCE) system equipped with a laser dark-field microscope. The zeta potential of exosomes of cancer cells was found to be larger than that of normal cells. In this study, the zeta potential of exosomes of normal and cancer prostate cells was evaluated using this system after treating with sialidase. The large negative charge of cancer exosomes was found to be due to the large amount of sialic acids. These results suggest that an on-chip µCE system is useful for the accurate evaluation of events that occur on the exosome surfaces at the single-particle level and promising for the prescreening of prostate cancer exosomes without the need for labeling.

Electrokinetic evaluation of individual exosomes by on-chip microcapillary electrophoresis with laser dark-field microscopy

Cell-secreted nanovesicles called exosomes are expected as a promising candidate biomarker of various diseases. Toward the future application of exosomes as a disease biomarker for low-invasive diagnostics, challenges remain in the development of sensitive and precise analysis methods for exosomes. In this study, we performed the electrokinetic evaluation of individual exosomes by the combined use of on-chip microcapillary electrophoresis and laser dark-field microscopy. We extracted exosomes from six types of human cell cultured in a serum-free medium by differential ultracentrifugation and their zeta potential (electrophoretic mobility) were evaluated. We demonstrated that the proposed electrophoresis apparatus is particularly suitable for the tracking analysis of the electrophoretic migration of individual exosomes and enables the accurate evaluation of the zeta potential distribution of exosomes, for the first time. From the experimental results, we found that there is a strong correlation between the average zeta potentials of exosomes and their cells of origin.

Statistical fluctuation in zeta potential distribution of nanoliposomes measured by on-chip microcapillary electrophoresis.

The zeta potential of nanoliposomes with a diameter below 100 nm has been studied by the combined use of on‐chip microcapillary electrophoresis (μCE) and sensitive fluorescence imaging. Tracking the electrophoretic migration of individual nanoliposomes has enabled the accurate evaluation of the zeta potential distribution of nanoliposomes and the first observation of its abnormal broadening due to a statistical fluctuation phenomenon specific to the “nanoscale world.” The materials used for liposome preparation were phosphocholine as the neutral lipid, phosphatidylserine as the anionic lipid, and cholesterol. The size of the liposomes encapsulating calcein, a fluorescent dye used for imaging convenience, was tailored by extrusion through polycarbonate membrane filters of different pore sizes ranging from 50 to 1000 nm. The on‐chip μCE system comprised a μCE chip, a laser source, an inverted microscope, and an electron‐multiplying charge‐coupled device camera. The electrophoresis experiment using this system revealed that the relative standard deviation of the zeta potential distribution of nanoliposomes is inversely proportional to their diameter and apparently increases below 100 nm. This abnormal broadening of zeta potential distribution of nanoliposomes is explained by prominent discreteness effect of the number of anionic lipid molecules in nanoliposomes.

Implementation of tetra-poly(ethylene glycol) hydrogel with high mechanical strength into microfluidic device technology

Hydrogels have several excellent characteristics suitable for biomedical use such as softness, biological inertness and solute permeability. Hence, integrating hydrogels into microfluidic devices is a promising approach for providing additional functions such as biocompatibility and porosity, to microfluidic devices. However, the poor mechanical strength of hydrogels has severely limited device design and fabrication. A tetra-poly(ethylene glycol) (tetra-PEG) hydrogel synthesized recently has high mechanical strength and is expected to overcome such a limitation. In this research, we have comprehensively studied the implementation of tetra-PEG gel into microfluidic device technology. First, the fabrication of tetra-PEG gel/PDMS hybrid microchannels was established by developing a simple and robust bonding technique. Second, some fundamental features of tetra-PEG gel/PDMS hybrid microchannels, particularly fluid flow and mass transfer, were studied. Finally, to demonstrate the unique application of tetra-PEG-gel-integrated microfluidic devices, the generation of patterned chemical modulation with the maximum concentration gradient: 10% per 20 μm in a hydrogel was performed. The techniques developed in this study are expected to provide fundamental and beneficial methods of developing various microfluidic devices for life science and biomedical applications.

Application of on-chip electrophoresis of cell to evaluation of cell cycle stages of HL-60 cells

In this article, we report a new method of cell cycle stage typing using a microcapillary electrophoresis (µCE) chip. Cell electrophoretic mobility (EPM) is considered to be useful information for noninvasive cell state analysis because EPM reflects the surface properties of cells. Human leukemic cells (HL-60) were synchronized at certain phases of the cell cycle using various synchronizing drugs, and their EPMs were measured using a µCE chip. It was found that the EPM distribution of the synchronized cells is much narrower than that of the nonsynchronized cells, and that the peak value of the EPM distribution differs between all cell cycle stages. These results suggest that on-chip EPM measurement is a promising method of cell cycle stage typing.