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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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Adhesion of mononuclear leukocytes to activated human umbillical vein endothelial cells. (A): HUVEC were incubated with TNF-α for 6 hr and then stained for CD54, CD62E and CD106 by secondary immunofluorescence (FITC-conjugated secondary antibody). CD54, CD62E and CD106 were all induced by TNF-α, and were present in the plasma membrane and around the nuclei (stained blue by DAPI). IgG1 with an irrelevant specificity (isotype control) did not give staining of the cells. Scale bar, 50 μm. (B): Mononuclear leukocytes from two individuals (black bars: individual 1; white bars: individual 2) were stained with BCECF-AM, given 10 or 100 ng/ml of both MCP-1 and MIP-1α, and then hydrodynamically guided in 500-μm-wide lanes at 10 nl/s (0.75 dyn/cm2) for 10 min over a confluent monolayer of TNF-α-activated HUVEC. HUVEC were washed (15 dyn/cm2), and the number of adherent blood cells was counted. Chemokine-activated mononuclear leukocytes did not adhere to non-activated HUVEC or to the supporting polystyrene slides (not shown). (C): In three different experiments, mononuclear leukocytes from individual 1 were mixed with MCP-1 and MIP-1α (both at 10 ng/ml), and aliquots of this leukocyte-chemokine mixture were applied to TNF-α-activated HUVEC immediately or after a 10, 20, 30 or 40 min preincubation at 37°C. The leukocytes were guided over HUVEC in 200-μm-wide lanes at 4.0 nl/s (0.75 dyn/cm2) for 5 min, and had thus been in contact with the chemokines for 0–5, 10–15, 20–25, 30–35 or 40–45 min, as indicated on the x-axis. HUVEC were washed (15 dyn/cm2), and the number of adherent blood cells was counted (counts are normalized because different counting frames were used). Mononuclear leukocytes activated with chemokines for any of the exposure times did not adhere to non-activated HUVEC (not shown).
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Figure 4: Adhesion of mononuclear leukocytes to activated human umbillical vein endothelial cells. (A): HUVEC were incubated with TNF-α for 6 hr and then stained for CD54, CD62E and CD106 by secondary immunofluorescence (FITC-conjugated secondary antibody). CD54, CD62E and CD106 were all induced by TNF-α, and were present in the plasma membrane and around the nuclei (stained blue by DAPI). IgG1 with an irrelevant specificity (isotype control) did not give staining of the cells. Scale bar, 50 μm. (B): Mononuclear leukocytes from two individuals (black bars: individual 1; white bars: individual 2) were stained with BCECF-AM, given 10 or 100 ng/ml of both MCP-1 and MIP-1α, and then hydrodynamically guided in 500-μm-wide lanes at 10 nl/s (0.75 dyn/cm2) for 10 min over a confluent monolayer of TNF-α-activated HUVEC. HUVEC were washed (15 dyn/cm2), and the number of adherent blood cells was counted. Chemokine-activated mononuclear leukocytes did not adhere to non-activated HUVEC or to the supporting polystyrene slides (not shown). (C): In three different experiments, mononuclear leukocytes from individual 1 were mixed with MCP-1 and MIP-1α (both at 10 ng/ml), and aliquots of this leukocyte-chemokine mixture were applied to TNF-α-activated HUVEC immediately or after a 10, 20, 30 or 40 min preincubation at 37°C. The leukocytes were guided over HUVEC in 200-μm-wide lanes at 4.0 nl/s (0.75 dyn/cm2) for 5 min, and had thus been in contact with the chemokines for 0–5, 10–15, 20–25, 30–35 or 40–45 min, as indicated on the x-axis. HUVEC were washed (15 dyn/cm2), and the number of adherent blood cells was counted (counts are normalized because different counting frames were used). Mononuclear leukocytes activated with chemokines for any of the exposure times did not adhere to non-activated HUVEC (not shown).

Mentions: Human umbilical vein endothelial cells (HUVEC) were incubated with tumor necrosis factor (TNF)-α for 6 hr and then stained for CD54 (ICAM-1), CD62E (E-selectin) and CD106 (VCAM-1) by secondary immunofluorescence. CD54, CD62E and CD106 were all induced by TNF-α, and were present in the plasma membrane and around the nuclei (Figure 4A). A primary antibody with an irrelevant specificity (isotype control) did not give staining of the cells. Mononuclear leukocytes from two individuals were stained to fluoresce green, given macrophage inflammatory protein (MIP)-1α and monocyte chemoattractant protein (MCP)-1 chemokines, and then guided over a confluent monolayer of TNF-α-activated HUVEC (shear stress of 0.75 dyn/cm2). Mononuclear leukocytes not activated with MCP-1 and MIP-1α showed only very limited adhesion to TNF-α-activated HUVEC, whereas activation with these chemokines increased the number of adherent leukocytes 14–25 fold (Figure 4B). Adhesion of the leukocytes decreased with time of preincubation with the chemokines, and no or very few leukocytes adhered when preincubated for more than 40 min with the chemokines (Figure 4C). Chemokine-activated mononuclear leukocytes from either of the individuals neither adhered to non-activated HUVEC nor to the supporting polystyrene slides (not shown).


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)

Adhesion of mononuclear leukocytes to activated human umbillical vein endothelial cells. (A): HUVEC were incubated with TNF-α for 6 hr and then stained for CD54, CD62E and CD106 by secondary immunofluorescence (FITC-conjugated secondary antibody). CD54, CD62E and CD106 were all induced by TNF-α, and were present in the plasma membrane and around the nuclei (stained blue by DAPI). IgG1 with an irrelevant specificity (isotype control) did not give staining of the cells. Scale bar, 50 μm. (B): Mononuclear leukocytes from two individuals (black bars: individual 1; white bars: individual 2) were stained with BCECF-AM, given 10 or 100 ng/ml of both MCP-1 and MIP-1α, and then hydrodynamically guided in 500-μm-wide lanes at 10 nl/s (0.75 dyn/cm2) for 10 min over a confluent monolayer of TNF-α-activated HUVEC. HUVEC were washed (15 dyn/cm2), and the number of adherent blood cells was counted. Chemokine-activated mononuclear leukocytes did not adhere to non-activated HUVEC or to the supporting polystyrene slides (not shown). (C): In three different experiments, mononuclear leukocytes from individual 1 were mixed with MCP-1 and MIP-1α (both at 10 ng/ml), and aliquots of this leukocyte-chemokine mixture were applied to TNF-α-activated HUVEC immediately or after a 10, 20, 30 or 40 min preincubation at 37°C. The leukocytes were guided over HUVEC in 200-μm-wide lanes at 4.0 nl/s (0.75 dyn/cm2) for 5 min, and had thus been in contact with the chemokines for 0–5, 10–15, 20–25, 30–35 or 40–45 min, as indicated on the x-axis. HUVEC were washed (15 dyn/cm2), and the number of adherent blood cells was counted (counts are normalized because different counting frames were used). Mononuclear leukocytes activated with chemokines for any of the exposure times did not adhere to non-activated HUVEC (not shown).
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Related In: Results  -  Collection

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Figure 4: Adhesion of mononuclear leukocytes to activated human umbillical vein endothelial cells. (A): HUVEC were incubated with TNF-α for 6 hr and then stained for CD54, CD62E and CD106 by secondary immunofluorescence (FITC-conjugated secondary antibody). CD54, CD62E and CD106 were all induced by TNF-α, and were present in the plasma membrane and around the nuclei (stained blue by DAPI). IgG1 with an irrelevant specificity (isotype control) did not give staining of the cells. Scale bar, 50 μm. (B): Mononuclear leukocytes from two individuals (black bars: individual 1; white bars: individual 2) were stained with BCECF-AM, given 10 or 100 ng/ml of both MCP-1 and MIP-1α, and then hydrodynamically guided in 500-μm-wide lanes at 10 nl/s (0.75 dyn/cm2) for 10 min over a confluent monolayer of TNF-α-activated HUVEC. HUVEC were washed (15 dyn/cm2), and the number of adherent blood cells was counted. Chemokine-activated mononuclear leukocytes did not adhere to non-activated HUVEC or to the supporting polystyrene slides (not shown). (C): In three different experiments, mononuclear leukocytes from individual 1 were mixed with MCP-1 and MIP-1α (both at 10 ng/ml), and aliquots of this leukocyte-chemokine mixture were applied to TNF-α-activated HUVEC immediately or after a 10, 20, 30 or 40 min preincubation at 37°C. The leukocytes were guided over HUVEC in 200-μm-wide lanes at 4.0 nl/s (0.75 dyn/cm2) for 5 min, and had thus been in contact with the chemokines for 0–5, 10–15, 20–25, 30–35 or 40–45 min, as indicated on the x-axis. HUVEC were washed (15 dyn/cm2), and the number of adherent blood cells was counted (counts are normalized because different counting frames were used). Mononuclear leukocytes activated with chemokines for any of the exposure times did not adhere to non-activated HUVEC (not shown).
Mentions: Human umbilical vein endothelial cells (HUVEC) were incubated with tumor necrosis factor (TNF)-α for 6 hr and then stained for CD54 (ICAM-1), CD62E (E-selectin) and CD106 (VCAM-1) by secondary immunofluorescence. CD54, CD62E and CD106 were all induced by TNF-α, and were present in the plasma membrane and around the nuclei (Figure 4A). A primary antibody with an irrelevant specificity (isotype control) did not give staining of the cells. Mononuclear leukocytes from two individuals were stained to fluoresce green, given macrophage inflammatory protein (MIP)-1α and monocyte chemoattractant protein (MCP)-1 chemokines, and then guided over a confluent monolayer of TNF-α-activated HUVEC (shear stress of 0.75 dyn/cm2). Mononuclear leukocytes not activated with MCP-1 and MIP-1α showed only very limited adhesion to TNF-α-activated HUVEC, whereas activation with these chemokines increased the number of adherent leukocytes 14–25 fold (Figure 4B). Adhesion of the leukocytes decreased with time of preincubation with the chemokines, and no or very few leukocytes adhered when preincubated for more than 40 min with the chemokines (Figure 4C). Chemokine-activated mononuclear leukocytes from either of the individuals neither adhered to non-activated HUVEC nor to the supporting polystyrene slides (not shown).

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