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Hydrodynamic guiding for addressing subsets of immobilized cells and molecules in microfluidic systems.

Brevig T, Krühne U, Kahn RA, Ahl T, Beyer M, Pedersen LH - BMC Biotechnol. (2003)

Bottom Line: The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible.The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further.The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Characterization, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark. tbr@bioteknologisk.dk

ABSTRACT

Background: The interest in microfluidics and surface patterning is increasing as the use of these technologies in diverse biomedical applications is substantiated. Controlled molecular and cellular surface patterning is a costly and time-consuming process. Methods for keeping multiple separate experimental conditions on a patterned area are, therefore, needed to amplify the amount of biological information that can be retrieved from a patterned surface area. We describe, in three examples of biomedical applications, how this can be achieved in an open microfluidic system, by hydrodynamically guiding sample fluid over biological molecules and living cells immobilized on a surface.

Results: A microfluidic format of a standard assay for cell-membrane integrity showed a fast and dose-dependent toxicity of saponin on mammalian cells. A model of the interactions of human mononuclear leukocytes and endothelial cells was established. By contrast to static adhesion assays, cell-cell adhesion in this dynamic model depended on cytokine-mediated activation of both endothelial and blood cells. The microfluidic system allowed the use of unprocessed blood as sample material, and a specific and fast immunoassay for measuring the concentration of C-reactive protein in whole blood was demonstrated.

Conclusion: The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible. The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further. The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale.

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Related in: MedlinePlus

Cell-membrane integrity of chinese hamster ovary cells on exposure to saponin. Immobilized cells were exposed for 30 s to saponin guided hydrodynamically into 200-μm-wide lanes (arrow indicate the direction of flow). Calcein AM and ethidium homodimer-1 were then applied in the same positions of the flow chamber. Calcein AM enters all cells, and is enzymatically converted to green-fluorescent calcein in the cytoplasm. Cells with an intact plasma membrane (viable cells) retain calcein, and thus fluoresce green. Only cells with a compromised plasma membrane (dead cells) take up ethidium homodimer-1. The red fluorescence of ethidium homodimer-1 is strongly enhanced once it interacts with the nucleic acids of the cell. The ratio of dead (red) to viable (green) CHO cells increased with increasing concentration of saponin. Scale bar, 100 μm.
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Figure 3: Cell-membrane integrity of chinese hamster ovary cells on exposure to saponin. Immobilized cells were exposed for 30 s to saponin guided hydrodynamically into 200-μm-wide lanes (arrow indicate the direction of flow). Calcein AM and ethidium homodimer-1 were then applied in the same positions of the flow chamber. Calcein AM enters all cells, and is enzymatically converted to green-fluorescent calcein in the cytoplasm. Cells with an intact plasma membrane (viable cells) retain calcein, and thus fluoresce green. Only cells with a compromised plasma membrane (dead cells) take up ethidium homodimer-1. The red fluorescence of ethidium homodimer-1 is strongly enhanced once it interacts with the nucleic acids of the cell. The ratio of dead (red) to viable (green) CHO cells increased with increasing concentration of saponin. Scale bar, 100 μm.

Mentions: Saponin was guided over chinese hamster ovary (CHO) cells, and the cells were immediately stained to determine their viability. The ratio of dead to viable cells increased with increasing concentration of saponin, as shown in Figure 3. On exposure to saponin at a concentration of 0.013% (w/v) for 30 s (shear stress of 15 dyn/cm2), 45% of the CHO cells died. Almost all CHO cells died when exposed to saponin at a concentration of 0.020% (w/v).


Hydrodynamic guiding for addressing subsets of immobilized cells and molecules in microfluidic systems.

Brevig T, Krühne U, Kahn RA, Ahl T, Beyer M, Pedersen LH - BMC Biotechnol. (2003)

Cell-membrane integrity of chinese hamster ovary cells on exposure to saponin. Immobilized cells were exposed for 30 s to saponin guided hydrodynamically into 200-μm-wide lanes (arrow indicate the direction of flow). Calcein AM and ethidium homodimer-1 were then applied in the same positions of the flow chamber. Calcein AM enters all cells, and is enzymatically converted to green-fluorescent calcein in the cytoplasm. Cells with an intact plasma membrane (viable cells) retain calcein, and thus fluoresce green. Only cells with a compromised plasma membrane (dead cells) take up ethidium homodimer-1. The red fluorescence of ethidium homodimer-1 is strongly enhanced once it interacts with the nucleic acids of the cell. The ratio of dead (red) to viable (green) CHO cells increased with increasing concentration of saponin. Scale bar, 100 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC183863&req=5

Figure 3: Cell-membrane integrity of chinese hamster ovary cells on exposure to saponin. Immobilized cells were exposed for 30 s to saponin guided hydrodynamically into 200-μm-wide lanes (arrow indicate the direction of flow). Calcein AM and ethidium homodimer-1 were then applied in the same positions of the flow chamber. Calcein AM enters all cells, and is enzymatically converted to green-fluorescent calcein in the cytoplasm. Cells with an intact plasma membrane (viable cells) retain calcein, and thus fluoresce green. Only cells with a compromised plasma membrane (dead cells) take up ethidium homodimer-1. The red fluorescence of ethidium homodimer-1 is strongly enhanced once it interacts with the nucleic acids of the cell. The ratio of dead (red) to viable (green) CHO cells increased with increasing concentration of saponin. Scale bar, 100 μm.
Mentions: Saponin was guided over chinese hamster ovary (CHO) cells, and the cells were immediately stained to determine their viability. The ratio of dead to viable cells increased with increasing concentration of saponin, as shown in Figure 3. On exposure to saponin at a concentration of 0.013% (w/v) for 30 s (shear stress of 15 dyn/cm2), 45% of the CHO cells died. Almost all CHO cells died when exposed to saponin at a concentration of 0.020% (w/v).

Bottom Line: The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible.The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further.The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Characterization, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark. tbr@bioteknologisk.dk

ABSTRACT

Background: The interest in microfluidics and surface patterning is increasing as the use of these technologies in diverse biomedical applications is substantiated. Controlled molecular and cellular surface patterning is a costly and time-consuming process. Methods for keeping multiple separate experimental conditions on a patterned area are, therefore, needed to amplify the amount of biological information that can be retrieved from a patterned surface area. We describe, in three examples of biomedical applications, how this can be achieved in an open microfluidic system, by hydrodynamically guiding sample fluid over biological molecules and living cells immobilized on a surface.

Results: A microfluidic format of a standard assay for cell-membrane integrity showed a fast and dose-dependent toxicity of saponin on mammalian cells. A model of the interactions of human mononuclear leukocytes and endothelial cells was established. By contrast to static adhesion assays, cell-cell adhesion in this dynamic model depended on cytokine-mediated activation of both endothelial and blood cells. The microfluidic system allowed the use of unprocessed blood as sample material, and a specific and fast immunoassay for measuring the concentration of C-reactive protein in whole blood was demonstrated.

Conclusion: The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible. The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further. The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale.

Show MeSH
Related in: MedlinePlus