A membrane-based microfluidic device for mechano-chemical cell manipulation

We introduce a microfluidic device for chemical manipulation and mechanical investigation of circulating cells. The device consists of two crossing microfluidic channels separated by a porous membrane. A chemical compound is flown through the upper “stimulus channel”, which diffuses through the membrane into the lower “cell analysis channel”, in which cells are mechanically deformed in two sequential narrow constrictions, one before and one after crossing the stimulus channel. Thus, this system permits to measure cell deformability before and after chemical cues are delivered to the cells within one single chip. The validity of the device was tested with monocytic cells stimulated with an actin-disrupting agent (Cytochalasin-D). Furthermore, as proof of principle of the device application, the effect of an anti-inflammatory drug (Pentoxifylline) was tested on monocytic cells activated with Lipopolysaccharides and on monocytes from patients affected by atherosclerosis. The results show that the system can detect differences in cell mechanical deformation after chemical cues are delivered to the cells through the porous membrane. Diffusion of Cytochalasin-D resulted in a considerable decrease in entry time in the narrow constriction and an evident increase in the velocity within the constriction. Pentoxifylline showed to decrease the entry time but not to affect the transit time within the constriction for monocytic cells. Monocytes from patients affected by atherosclerosis were difficult to test in the device due to increased adhesion to the walls of the microfluidic channel. Overall, this analysis shows that the device has potential applications as a cellular assay for analyzing cell-drug interaction

A membrane-based microfluidic device for mechano-chemical cell manipulation

We introduce a microfluidic device for chemical manipulation and mechanical investigation of circulating cells. The device consists of two crossing microfluidic channels separated by a porous membrane. A chemical compound is flown through the upper "stimulus channel", which diffuses through the membrane into the lower "cell analysis channel", in which cells are mechanically deformed in two sequential narrow constrictions, one before and one after crossing the stimulus channel. Thus, this system permits to measure cell deformability before and after chemical cues are delivered to the cells within one single chip. The validity of the device was tested with monocytic cells stimulated with an actin-disrupting agent (Cytochalasin-D). Furthermore, as proof of principle of the device application, the effect of an anti-inflammatory drug (Pentoxifylline) was tested on monocytic cells activated with Lipopolysaccharides and on monocytes from patients affected by atherosclerosis. The results show that the system can detect differences in cell mechanical deformation after chemical cues are delivered to the cells through the porous membrane. Diffusion of Cytochalasin-D resulted in a considerable decrease in entry time in the narrow constriction and an evident increase in the velocity within the constriction. Pentoxifylline showed to decrease the entry time but not to affect the transit time within the constriction for monocytic cells. Monocytes from patients affected by atherosclerosis were difficult to test in the device due to increased adhesion to the walls of the microfluidic channel. Overall, this analysis shows that the device has potential applications as a cellular assay for analyzing cell-drug interaction

Shear modulus.

<p>Scatterplot of the characteristic differential stress (p_wall – p)/2 as a function of strain ε_r – ε_z for not-treated (GREEN), activated LPS-treated (RED) and cytoD-treated (BLUE) cells. In the insert of the figure, the quantification of the shear modulus for not-treated (cell n = 7) and activated cells (cell n = 5) is shown. The shear modulus was calculated by two linear fits, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092814#pone-0092814-g002" target="_blank">Fig.2</a>.</p

Capillary micromechanics setup.

<p>Capillary micromechanics; schematic of experimental setup. In brief: The tip of a horizontally stabilized tapered capillary (3-μm-wide) leads into a liquid drop of buffer solution (PBS). A cell is lodged in the tapered part of the capillary. A flexible tube is attached at the backside of the capillary. The filling height h of PBS buffer in the tubing determines the pressure difference in the system (1 mm H2O is 10 Pa, changed in steps of 10 Pa) and thereby the externally applied stress acting on the cell. Cells are imaged in real-time (shape and volume) using an inverted microscope (40x objective) at each pressure step after a cell lodges in the capillary.</p

Actin analysis: immunohistochemistry and FACS.

<p>A) Representative immunofluorescent images of HL60 cells showing structural organization of F-actin (green) and G-actin (red). B) Typical intensity distribution of F-actin for NT cells (red), LPS-treated cells (green) and CytoD-treated cells (blue) as measured with flow cytometry.</p

Compressive modulus.

<p>Scatterplot of the compressive stress versus the volumetric strain for non-treated (NT, GREEN), activated LPS-treated (RED) and cyto-D treated (BLUE) cells. To quantify the effect of activation, the compressive moduli K1 and K2 for the NT (cell n = 7) and LPS-treated cells (cell n = 5) are shown (insert). The compressive moduli were calculated by two linear fits to the data, as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092814#pone-0092814-g002" target="_blank">Fig.2</a>.</p

Monocytic cells become less compressible but more deformable upon activation

Aims Monocytes play a significant role in the development of atherosclerosis. During the process of inflammation, circulating monocytes become activated in the blood stream. The consequent interactions of the activated monocytes with the blood flow and endothelial cells result in reorganization of cytoskeletal proteins, in particular of the microfilament structure, and concomitant changes in cell shape and mechanical behavior. Here we investigate the full elastic behavior of activated monocytes in relation to their cytoskeletal structure to obtain a better understanding of cell behavior during the progression of inflammatory diseases such as atherosclerosis. Methods and Results The recently developed Capillary Micromechanics technique, based on exposing a cell to a pressure difference in a tapered glass microcapillary, was used to measure the deformation of activated and non-activated monocytic cells. Monitoring the elastic response of individual cells up to large deformations allowed us to obtain both the compressive and the shear modulus of a cell from a single experiment. Activation by inflammatory chemokines affected the cytoskeletal organization and increased the elastic compressive modulus of monocytes with 73–340%, while their resistance to shape deformation decreased, as indicated by a 25–88% drop in the cell’s shear modulus. This decrease in deformability is particularly pronounced at high strains, such as those that occur during diapedesis through the vascular wall. Conclusion Overall, monocytic cells become less compressible but more deformable upon activation. This change in mechanical response under different modes of deformation could be important in understanding the interplay between the mechanics and function of these cells. In addition, our data are of direct relevance for computational modeling and analysis of the distinct monocytic behavior in the circulation and the extravascular space. Lastly, an understanding of the changes of monocyte mechanical properties will be important in the development of diagnostic tools and therapies concentrating on circulating cells