Selective cell retrieval method using light-responsive gas-generating polymer-based microarrays.

Selective cell retrieval from base material is necessary for developing and improving cell analyzing technologies as well as regenerative medicine. Many conventional technologies, such as micromanipulators, are developed for selective cell retrieval. However, selective cell retrieval at the single-cell level remains challenging because it is quite difficult to retrieve adhered single cells from base material with ease, rapidity, and no damage. Here, we propose a novel selective cell retrieval method using microarrays made of a light-responsive gas-generating polymer (LGP microarray). The convex LGP microarray was fabricated by a molding process using polystyrene microarray chips. LGP microarrays generate N2 gas when exposed to a specific light used for fluorescence microscopy. A human cervical cancer cell (HeLa) suspension was spread on the LGP microarray coated by fibronectin. After these HeLa cells were adhered to the surface of the LGP microarray structure, light at a wavelength of 365 nm was used to irradiate the LGP microarray. All the target HeLa cells were selectively released from the light-irradiated surface area of the LGP microarray by the generated N2 gas. The LGP microarray system was also applied to single-cell retrieval, and we easily and rapidly retrieved 100% of the single HeLa cells from the microarrays. In addition, approximately 90% of single HeLa cells retrieved from the LGP microarray proliferated on a chamber of a 96-well plate. Therefore, the LGP microarray system enables easy and selective retrieval of adhered cell groups or single cells with only harmless light irradiation.

Novel Quick Cell Patterning Using Light-Responsive Gas-Generating Polymer and Fluorescence Microscope.

Conventional cell patterning methods are mainly based on hydrophilic/hydrophobic differences or chemical coating for cell adhesion/non-adhesion with wavering strength as it varies with the substrate surface conditions, including the cell type and the extracellular matrix components (ECMs) coating; thus, the versatility and stability of cell patterning methods must be improved. In this study, we propose a new cell patterning method using a light-responsive gas-generating polymer (LGP) and a conventional fluorescence microscope. Herein, cells and cellular tissues are easily released from the substrate surface by the nitrogen gas bubbles generated from LGP by the excitation light for fluorescence observation without harming the cells. The LGP-implanted chip was fabricated by packing LGP into a polystyrene (PS) microarray chip with a concave pattern. HeLa cells were spread on the LGP-implanted chips coated with three different ECMs (fibronectin, collagen, and poly-D-lysine), and all HeLa cells on the three LGP patterns were released. The pattern error between the LGP pattern and the remaining HeLa cells was 8.81 ± 4.24 µm, less than single-cell size. In addition, the LGP-implanted chip method can be applied to millimeter-scale patterns, with less than 30 s required for cell patterning. Therefore, the proposed method is a simple and rapid cell patterning method with high cell patterning accuracy of less than the cell size error, high scalability, versatility, and stability unaffected by the cell type or the ECM coating.

Ellagic acid glycosides with hepatoprotective activity from traditional Tibetan medicine Potentilla anserina.

Two new gallic acid glycosides, potentillanosides G (1) and H (2), were newly isolated from the methanol extract of the tuberous roots of Potentilla anserina (Rosaceae), together with a known compound, ellagic acid 3-O-α-L-rhamnopyranoside (3). Their structures were elucidated on the basis of chemical and physicochemical evidence. Among the constituents, potentillanoside H (2, IC50 = 99.5 µM) was found to show hepatoprotective activity.

Hepatoprotective triterpenes from traditional Tibetan medicine Potentilla anserina.

A methanol extract from the tuberous roots of Potentilla anserina (Rosaceae) exhibited hepatoprotective effects against d-galactosamine (d-GalN)/lipopolysaccharide-induced liver injuries in mice. Six triterpene 28-O-monoglucopyranosyl esters, potentillanosides A-F, were isolated from the extract along with 32 known compounds, including 15 triterpenes. The structures of potentillanosides A-F were determined on the basis of spectroscopic properties and chemical evidence. Four ursane-type triterpene 28-O-monoglycosyl esters, potentillanoside A (IC50=46.7µM), 28-O-ß-d-glucopyranosyl pomolic acid (IC50=9.5µM), rosamutin (IC50=35.5µM), and kaji-ichigoside F1 (IC50=14.1µM), inhibited d-GalN-induced cytotoxicity in primary cultured mouse hepatocytes. Among these four triterpenes, potentillanoside A, rosamutin, and kaji-ichigoside F1 exhibited in vivo hepatoprotective effects at doses of 50-100mg/kg, p.o. The mode of action was ascribable to the reduction in cytotoxicity caused by d-GalN.

Enzyme-linked sensitive fluorometric imaging of glutamate release from cerebral neurons of chick embryos.

This paper describes a method for imaging the endogenous release of glutamate from cerebral neurons. This method is based on the reactions of glutamate oxidase and peroxidase, and on the detection of hydrogen peroxide by a fluorescent substrate of peroxidase. Glutamate has been sensitively measured in vitro in the range of 20 nM to 1 microM. We used two types of Ca(2+) channel inhibitors, MK-801 and omega-Conotoxin GVIA, which act to suppress Ca(2+) transport at postsynaptic and presynaptic neurons, respectively. MK-801 did not inhibit the increase in glutamate release after KCl stimulation, while there was no increase in glutamate release after KCl stimulation when omega-Conotoxin GVIA was used, probably due to the inhibition of voltage-activated Ca(2+) channels in the presynapse. Glutamate release and Ca(2+) flow in the synaptic regions were imaged using a laser confocal fluorescence microscope. KCl-evoked glutamate release was localized around cell bodies linked to axon terminals. This procedure allows imaging that can be sensitively detected by the fluorometric enzymatic assay of endogenous glutamate release in synapses.