Biology

Hepatocyte spheroid arrays inside microwells connected with microchannels

Abstract

Spheroid culture is a preferable cell culture approach for some cell types, including hepatocytes, as this type of culture often allows maintenance of organ-specific functions. In this study, we describe a spheroid microarray chip (SM chip) that allows stable immobilization of hepatocyte spheroids in microwells and that can be used to evaluate drug metabolism with high efficiency. The SM chip consists of 300-μm-diameter cylindrical wells with chemically modified bottom faces that form a 100-μm-diameter cell adhesion region surrounded by a nonadhesion region. Primary hepatocytes seeded onto this chip spontaneously formed spheroids of uniform diameter on the cell adhesion region in each microwell and these could be used for cytochrome P-450 fluorescence assays. A row of microwells could also be connected to a microchannel for simultaneous detection of different cytochrome P-450 enzyme activities on a single chip. The miniaturized features of this SM chip reduce the numbers of cells and the amounts of reagents required for assays. The detection of four cytochrome P-450 enzyme activities was demonstrated following induction by 3-methylcholantlene, with a sensitivity significantly higher than that in conventional monolayer culture. This microfabricated chip could therefore serve as a novel culture platform for various cell-based assays, including those used in drug screening, basic biological studies, and tissue engineering applications.

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Hepatocyte spheroid arrays inside microwells connected with microchannels

Method of seeking at least one analyte in a medium likely to contain it

Abstract

A new method of seeking the presence of an analyte bound to a probe, wherein a periodic geometric pattern (24), constituting a diffractive system (2), is formed by alternating areas including a probe A, and areas not including the probe A. The diffractive system (2) is made to be diffractive before a sensitization step, i.e. a step during which a probe is temporarily brought into contact with a medium likely to contain an analyte and during which the possible analyte binds to the probe. The method includes at least the following steps: measurement of a power P1 of a first-order diffraction beam of a diffraction field produced by the diffractive system, with the probe unsensitized, sensitizing the probe A, measurement of a power P1a of a first-order diffraction beam of a diffraction field produced by the diffractive system, and comparison of the measured powers P1 and P1a.

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Method of seeking at least one analyte in a medium likely to contain it

Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors

Abstract

Pallet arrays enable cells to be separated while they remain adherent to a surface and provide a much greater range of cell selection criteria relative to that of current technologies. However there remains a need to further broaden cell selection criteria to include dynamic intracellular signaling events. To demonstrate the feasibility of measuring cellular protein behavior on the arrays using highresolution microscopy, the surfaces of individual pallets were modified to minimize the impact of scattered light at the pallet edges. The surfaces of the three-dimensional pallets on an array were patterned with a coating such as fibronectin using a customized stamping tool. Micropatterns of varying shape and size were printed in designated regions on the pallets in single or multiple steps to demonstrate the reliability and precision of patterning molecules on the pallet surface. Use of a fibronectin matrix stamped at the center of each pallet permitted the localization of H1299 and mouse embryonic fibroblast (MEF) cells to the pallet centers and away from the edges. Compared to palletarrays with fibronectin coating the entire top surface, arrays with a central fibronectin pattern increased the percentage of cells localized to the pallet center by 3–4-fold. Localization of cells to the pallet center also enabled the physical separation of cells from optical artifacts created by the rough pallet side walls. To demonstrate the measurement of dynamic intracellular signaling on the arrays, fluorescence measurements of high spatial resolution were performed using a RhoA GTPase biosensor. This biosensor utilized fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure localized RhoA activity in cellular ruffles at the cell periphery. These results demonstrated the ability to perform spatially resolved measurements of fluorescence-based sensors on the pallet arrays. Thus, the patterned pallet arrays should enable novel cell separations in which cell selection is based on complex cellular signaling properties.

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Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors

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