A tapered channel microfluidic device for comprehensive cell adhesion analysis, using measurements of detachment kinetics and shear stress-dependent motion

Abstract

We have developed a method for studying cellular adhesion by using a custom-designed microfluidic device with parallel non-connected tapered channels. The design enables investigation of cellular responses to a large range of shear stress (ratio of 25) with a single input flow-rate. For each shear stress, a large number of cells are analyzed (500-1500 cells), providing statistically relevant data within a single experiment. Besides adhesion strength measurements, the microsystem presented in this paper enables in-depth analysis of cell detachment kinetics by real-time videomicroscopy. It offers the possibility to analyze adhesion-associated processes, such as migration or cell shape change, within the same experiment. To show the versatility of our device, we examined quantitatively cell adhesion by analyzing kinetics, adhesive strength and migration behaviour or cell shape modifications of the unicellular model cell organism Dictyostelium discoideum at 21 °C and of the human breast cancer cell line MDA-MB-231 at 37 °C. For both cell types, we found that the threshold stresses, which are necessary to detach the cells, follow lognormal distributions, and that the detachment process follows first order kinetics. In addition, for particular conditions' cells are found to exhibit similar adhesion threshold stresses, but very different detachment kinetics, revealing the importance of dynamics analysis to fully describe cell adhesion. With its rapid implementation and potential for parallel sample processing, such microsystem offers a highly controllable platform for exploring cell adhesion characteristics in a large set of environmental conditions and cell types, and could have wide applications across cell biology, tissue engineering, and cell screening.

Figure 1

Microfluidic device with four independent branches. (a) Photograph of the device, sealed on a 76 × 52 mm glass slide. The four channels are labeled with numbers. Inlet-outlet-distance is 50 mm. In- and outlet can be interchanged to avoid a high hydrodynamic pressure for large widths which occurs at the inlet site. (b) Photolithography mask (detail). The colored rectangles correspond to the visual field of different shear stress zones we used for our later discussed model experiment. (c) Shear stress (log-scale) related to the position in the tapered channel for a low flow rate Q = 5 ml/h. The corresponding letters are represented in (b). The square on the top left shows the highest possible shear stress that could be attained at this flow rate.

Figure 2

Detachment curves with first order kinetic fits. (a) Cell detachment curves for D. Discoideum DH1 in fresh medium, fitted with first order kinetics. The letters on the right show the channel position (cf. Fig. 1) which is used for the corresponding detachment curve. Higher shear stresses correspond to greater detachment. The detachment curve for 0.50 Pa (letter G) is highlighted by larger dots, the detachment curve for DH1 in HCM at the same shear stress is drawn in green open circles. This shows that the final detachment is very similar for both conditions, whereas the kinetics are much faster for HCM. (b) Detachment curves for MDA-MB-231 human breast cancer cells. The more significant fluctuations are due to a lower cell concentration and image treatment.

Figure 3

Detachment efficiency and detachment rate for different experiments. (a) The cumulative lognormal threshold-stress distribution function (cdf), fitted with Eq. 4 for four datasets, listed from left to right: Dictyostelium on glass substrate in highly conditioned medium (green, downward pointing triangles) and in fresh medium (blue, upward pointing triangles), in fresh medium on Aptes-coated substrate (red, crosses), and MDA-MB-231 on collagen-coated substrate (black, circles). Every datapoint for the Dictyostelium data in fresh medium corresponds to one position in Fig. 1. Error bars are shown for one condition each for Dictyostelium and MDA-MB-231 to give an idea of magnitude. σ_1/2 can be read off at the point where the dashed line intersects the fits. (b) Detachment rates k, obtained by first order kinetics fits. The color/symbol code is the same as in (a). Stress errors are the same as for (a), fit errors for k are of the size of the symbols. Data points without significant detachment (Aptes for σ < 0.5 Pa) could not be fitted properly and were left out. Data points are connected as a guide for the eye.

Figure 4

Dictyostelium cells motility during a detachment experiment on APTES coated substrate. (a) Detachment of a single Dictyostelium cell at 20× magnitude, at 2.6 Pa: despite the very high stress, the cell extends also lateral pseudopods (arrows) and migrates. Finally, the cell rounds and detaches. The arrow in the last frame indicates the flow direction. The migrative behavior comes out better in a time-lapse movie. (b) (c) Centered trajectories of at least 150 cells submitted to a shear stress of 0.25 Pa (b) and 0.70 Pa (c), respectively, over 52 min. Cell migration is almost random at low shear stresses, whereas it is strongly biased in flow direction for higher shear stresses. Flow direction is given by arrows. (d) Mean directionality of cell movement as a function of applied shear stress, indicating to which extent migration is aligned with flow direction. Directionality is defined as the angle between the flow direction and the cell movement direction over a 52 min interval. Therefore, cos(θ) = 1 indicates fully biased cell movement in flow direction, cos(θ) = 0 indicates random direction of migration. The chosen positions (b) and (c) are represented by black arrows, σ_1/2 = 0.65 Pa by the red arrow (enhanced online) .

Figure 5

Detachment mode of MDA-MB-231 cancer cells. (a) Example of cell alignment in response to the imposed shear stress (see the time-lapse movie online for the different phases of detachment). (b) Single cell drop formation at 4×. The black arrow indicates flow direction. (c) The cell (its center of mass) moves after a short acceleration phase with constant velocity in flow direction. Leaps (marked by dotted lines) occur, when adhesion points break off. (d) Cell deformability is analysed by computing the solidity parameter s = A/A_ch, which uses the cell area A and its convex hull A_ch (shown in the 5th frame). A sudden increase of s corresponds to a rupture of an anchor point (see dashed lines) (enhanced online) .