385 research outputs found
A multi-chamber microfluidic intestinal barrier model using Caco-2 cells for drug transport studies
This paper presents the design and fabrication of a multi-layer and multi-chamber microchip system using thiol-ene 'click chemistry' aimed for drug transport studies across tissue barrier models. The fabrication process enables rapid prototyping of multi-layer microfluidic chips using different thiol-ene polymer mixtures, where porous Teflon membranes for cell monolayer growth were incorporated by masked sandwiching thiol-ene-based fluid layers. Electrodes for trans-epithelial electrical resistance (TEER) measurements were incorporated using low-melting soldering wires in combination with platinum wires, enabling parallel real-time monitoring of barrier integrity for the eight chambers. Additionally, the translucent porous Teflon membrane enabled optical monitoring of cell monolayers. The device was developed and tested with the Caco-2 intestinal model, and compared to the conventional Transwell system. Cell monolayer differentiation was assessed via in situ immunocytochemistry of tight junction and mucus proteins, P-glycoprotein 1 (P-gp) mediated efflux of Rhodamine 123, and brush border aminopeptidase activity. Monolayer tightness and relevance for drug delivery research was evaluated through permeability studies of mannitol, dextran and insulin, alone or in combination with the absorption enhancer tetradecylmaltoside (TDM). The thiol-ene-based microchip material and electrodes were highly compatible with cell growth. In fact, Caco-2 cells cultured in the device displayed differentiation, mucus production, directional transport and aminopeptidase activity within 9-10 days of cell culture, indicating robust barrier formation at a faster rate than in conventional Transwell models. The cell monolayer displayed high TEER and tightness towards hydrophilic compounds, whereas co-administration of an absorption enhancer elicited TEER-decrease and increased permeability similar to the Transwell cultures. The presented cell barrier microdevice constitutes a relevant tissue barrier model, enabling transport studies of drugs and chemicals under real-time optical and functional monitoring in eight parallel chambers, thereby increasing the throughput compared to previously reported microdevices
How to Characterize Individual Nano-Size Liposomes with Simple Self-Calibrating Fluorescence Microscopy
Nanosize
lipid vesicles are used extensively at the interface between
nanotechnology and biology, e.g., as containers for chemical reactions
at minute concentrations and vehicles for targeted delivery of pharmaceuticals.
Typically, vesicle samples are heterogeneous as regards vesicle size
and structural properties. Consequently, vesicles must be characterized
individually to ensure correct interpretation of experimental results.
Here we do that using dual-color fluorescence labeling of vesiclesof
their lipid bilayers and lumens, separately. A vesicle then images
as two spots, one in each color channel. A simple image analysis determines
the total intensity and width of each spot. These four data all depend
on the vesicle radius in a simple manner for vesicles that are spherical,
unilamellar, and optimal encapsulators of molecular cargo. This permits
identification of such <i>ideal</i> vesicles. They in turn
enable calibration of the dual-color fluorescence microscopy images
they appear in. Since this calibration is not a separate experiment
but an analysis of images of vesicles to be characterized, it eliminates
the potential source of error that a separate calibration experiment
would have been. Nonideal vesicles in the same images were characterized
by how their four data violate the calibrated relationship established
for ideal vesicles. In this way, our method yields size, shape, lamellarity,
and encapsulation efficiency of each imaged vesicle. Applying this
procedure to extruded samples of vesicles, we found that, contrary
to common assumptions, only a fraction of vesicles are ideal
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