Electro-, mechano-, chemotaxis and microfluidics

Exogenous and endogenous electric fields can play a role in cell physiology as a guiding mechanism. Electrotaxis of cells is a directional response found in several cell types (1, 2). It was described previously that Dictyostelium also exhibits electrotactic behavior in electric fields (3, 4, 5). We currently investigate the underlying electric sensing mechanisms of Dictyostelium in combination with microfluidics using single channel devices.

Although very heterogenous forest soil is the natural environment of Dictyostelium, all migration experiments are done on flat surfaces like cover slides or petri dishes. We are studying, in combination with microfluidics to exclude chemotactic effects, the mechanotactic behaviour of amebae to various other geometrical cues; our observations show that cells are contact-guided and we are currently identifying the underlying biomechanical processes.

Mainly however, we use microfluidic tools to expose Dictyostelium to directional chemotactic stimuli of cyclic adenosine 3',5' monophosphate (cAMP). We classify the migrational patterns of single cells and motility of cell populations in various gradients and microfluidic devices to quantify the accuracy of directional migration: in the following you find an overview of the most relevant microfluidic devices and techniques used in our group:

We use a microfluidic gradient mixer network that generates linear gradients of cAMP and have used it to study chemotaxis of Dicty-cells (6, 7) (see Figure 1). Depending on the interplay of flow speed and geometry with the diffusive time scale of the chemoattractant in the flow, the concentration distribution across the cell membrane may differ strongly from the optimal gradient in a perfectly smooth channel. We have also analyzed the underlying physics in a two-dimensional approximation and performed systematic numerical finite element simulations to characterize the three-dimensional case to identify optimal flow conditions (8).

Figure 1:

(a) The microfluidic gradient mixer used for the chemotaxis experiments. (b) Profile of the gradient visualized using fluorescein: the gradient is linear in the central part of the main channel, in a region 320 µm wide (shown by the blue rectangle). The scale bar is 90 µm.

Quantitative studies of cellular systems require experimental techniques that can expose single cells to well-controlled chemical stimuli with high spatial and temporal resolution. We have combined microfluidic techniques with the photochemical release of caged signaling molecules to generate tailored stimuli on the length scale of individual cells with subsecond switching times (9, 10, 11). The main idea of flow photolysis is as following: A flow is established in a microfluidic channel (of typical cross section 300 × 50 μm²) that carries a physiologically inert caged compound. A region in flow direction is illuminated with short wavelength light that is chosen as to cleave the photolysable bond between the caging group and the physiologically active part of the caged compound. For low uncaging light intensity, the time period a fluid element is exposed to the uncaging light determines the concentration. In other words, the shape of the illuminating light pattern directly gives the chemical gradient structure. Immediately downstream of the uncaging region, a living cell is placed. Taylor Dispersion that limits the temporal resolution of a concentration change is thus minimized. A detailed numerical and theoretical analysis shows that flow photolysis is superior to other switching technique (11).

Figure 2:

Flow photolysis. (top) Photo-uncaging of caged fluorescein in a triangular uncaging region (red) and switching time of photo-chemical release. (bottom) Gradient profiles with constant steepness Δc and changing midpoint c0 (left), constant relative gradient Δc/c0 (middle), and constant midpoint c0 and changing slope Δc (right).

Live cell flattening is valuable for improving microscopic observations, ranging from bright field, fluorescence and confocal microscopy to total internal reflection fluorescence (TIRF) microscopy. Overlay techniques (agar overlay or oil overlay) achieve cell flattening by the removal of the fluid between the glass floor and a confining top layer. The overlay methods provide little control on the degree of flattening. We have introduced a dobule layer microfluidic techniques to achieve greater control over the degree of flattening (12). The microfluidic approaches rely on applying a pressure difference across a deformable PDMS layer, which acts as a ceiling for the cell-flattening chamber. The height of this PDMS ceiling is adjusted by changing this pressure difference. In addition the actuation channel can be operated with oxygen rich gas and thus provides an oxygen rich environment to the cells. An example of flattened Dictyostelium-cells is presented in Figure 3 and in this movie (link). Experiments have shown that Dictyostelium-cells behave normally for compressions up to 4 μm. Flattened cells have not only the advantage of being restricted in the third dimension, but also the lateral cell size increases significantly. This in turn leads to a lengthening of the times for intracellular transport and allows the easier application of localized stimuli.

Figure 3:

Microfluidic double-layer flattening actuator (A) The cells are contained in the bottom channel. The actuation channel is open-ended allowing oxygen to flow through. The oxygen is supplied to the cells via the permeable PDMS membrane, which separates the two layers. (B) By adjusting the pressure of the oxygen supply at the inlet, the PDMS membrane is deformed and flattens the cells below. (C) Assembled double layer microfluidic channel with connected tubing. The actuation channel is filled with red and the lower channel with blue dye. (D, E) Flattening of Dictyostelium cells: confocal xy (top) and xz (bottom) sections are shown for a cAR1-GFP cell before flattening (D) and during flattening (E). The blue lines show the y and the z cuts. Scale bar: 10 µm.

Contact: Isabella Guido, Christoph Blum, Eberhard Bodenschatz

[1] R. Nuccitelli, T. Smart, J. Ferguson, Cell Motil. Cytoskeleton, 24, 54-66 (1993)
[2] C.D. McCaig, A.M. Rajnicek, B. Song, M. Zhao, Physiol. Rev., 85, 943-978 (2005)
[3] M. Zhao, T. Jin, C.D. McCaig, J.V. Forrester, P.N. Devreotes, J. Cell Biol., 157, 921-927 (2002)
[4] J.M. Sato, M. Ueda, H. Takagi, T.M. Watanabe, T. Yanagida, M. Ueda, BioSystems, 88, 261-272 (2007)
[5] R. Gao, X.D Zhang, Y.H. Sun, Y. Kamimura, A. Mogilner, P.N. Devreotes, M. Zhao, Eukaryotic Cell, 10, 1251-1256, (2011)
[6] L. Song, S.M. Nadkarni, H.U. Bödeker, C. Beta, A. Bae, C. Franck, W.-J. Rappel, W.F. Loomis, and E. Bodenschatz, Dictyostelium discoideum chemotaxis: threshold for directed motion, Eur. J. Cell Biol., 85, 981-989 (2006)
[7] Rhoads D. S., Nadkarni S. M., Song L., Voeltz C., Bodenschatz E., and Guan J. L., Using microfluidic channel networks to generate gradients for studying cell migration, Methods Mol. Biol. 294, 347 (2005)
[8] Beta C., Frohlich T., Bodeker H. U., and Bodenschatz E., Chemotaxis in microfluidic devices - a study of flow effects, Lab Chip 8, 1087 (2008)
[9] Beta C., Wyatt D., Rappel W. J., and Bodenschatz E., Flow photolysis for spatiotemporal stimulation of single cells, Analytical Chemistry 79, 3940 (2007)
[10] Beta C., Frohlich T., Bodeker H. U., and Bodenschatz E., Chemotaxis in microfluidic devices - a study of flow effects, Lab Chip 8, 1087 (2008)
[11] Bae A. J., Beta C., and Bodenschatz E., Rapid switching of chemical signals in microfluidic devices, Lab Chip 9, 3059 (2009)
[12] Westendorf C., Bae A. J., Erlenkamper C., Galland E., Franck C., Bodenschatz E., and Beta C., Live cell flattening - traditional and novel approaches, PMC Biophys. 3, 9 (2010)

Our lab protocols for fabrication of microfluidic devices can be downloaded here.