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Cell Biology
Patterning Mammalian Cells
Soft lithography offers the ability to generate patterns and structures on the micron scale that are useful in examining cells. Using soft lithography, we have demonstrated the ability to control the molecular structure of surfaces, pattern the complex molecules relevant to biology, fabricate channels for the examination of cells, and pattern and manipulate cells. Poly(dimethylsiloxane) is a useful material for the study of cells because it is compatible with many cell types, it is optically transparent, and it is permeable to many gases.
Using microcontact printing, we form patterns of self-assembled monolayers (SAMs) on gold or palladium (SAMs). We can generate patterns of molecules that either promote or resist the binding of cells to the surface, which results in the patterning of cells to selected geometrical regions. We found that the adherent area available to cells determines the viability of cells. SAMs can be electrochemically desorbed from the gold surface, releasing cells from patterned regions. Using microcontact printing, we have recently created asymmetric, tear-drop-shaped patterned SAMs for cell adsorption. When cells adhere to these patterns, the cytoskeleton of the cells are polarized (Figure 1). When cells are electrochemically released from the surface, the initial polarization determines the direction of cell migration.
We have also patterned cells using elastomeric membranes fabricated using soft lithography. We have generated patterns and gradients within microfluidic channels and examined the behavior of cells to solution and surface gradient. We have partially treated cells in laminar flow to study the subcellular movement of microchondria and changes in cytoskeletal structure. Using a microfluidic gradient generator, we created substrate-bound gradients of laminin and found the neurons preferentially extended their axon towards increasing laminin concentration.
Bacterial Swarming
We are interested in using soft lithography techniques to study the behavior of microorganisms. Microchannels and microwells allow for the examination of single microorganisms in a chemically and mechanically controlled environment. We are interested in developing microdevices that utilize the motion of swimming microorganisms.
We have examined the behavior of E. coli swarmer cells in composite hydrogel/PDMS channels shown in Figure 2. We found that the agar surface affects the hydrodynamics of swimming cells more than the PDMS surfaces do. These different interactions bias the motion of cells in microchannels causing E. coli cells to "drive on the right" in rectangular microchannels. Movie 3 shows this traffic-like behavior of cells. This preferential movement could be used as a new strategy for directing cells in microdevices that would not require external pumping.
We have confined single bacterial cells in small, agarose microchambers, which allow for the continued growth of cells in a confined but nutritive environment. Movie 4 shows motile E. coli cells confined in these chambers. We have grown multinucleate, non-septate, filamentous cells in these microchambers (Figure 5). Filamentous cells still maintain the shape imposed by the chann8el after their release. We have observed that even when molded into a long, spiral shapes, the cells are still capable of swimming. (Movie 6)
Patterning Bacteria
We have recently developed a technique for microcontact printing patterns of bacteria on growth media using topographically-patterned agarose stamps. This method produces patterns of multiple bacteria with feature sizes as small as 200 um over areas as large as 50 cm^2. Figure 7 shows different patterns of the luminescent bacteria, Vibrio fischeri, produced using this technique. Micropatterned agarose stamps inked once with bacteria can be used to create hundreds of replica patterns (Figure 8). The cells of bacteria thrive on the surface of agarose stamps containing media, making it possible to prepare stamps that "regenerate" their own ink. This technique can be used to pattern several different strains of bacteria using a single stamp (Figure 9). We are now using patterns of bacteria to explore organism-organism, organism-small molecule, and organism-surface interactions.
"Microoxen"
We have developed a conceptually new approach for harnessing the transduction of energy by microorganisms. We use the power produced by eukaryotic flagella in intact cells of the unicellular, photosynthetic algae Chlamydomonas reinhardtii to transport loads in microfluidic networks. These motile microorganisms -- which we refer to in this context as "microoxen" -- move microscale objects (1-3 um diameter beads) at velocities of ~100-200 um/sec and over distances as large as 20 cm. Cells carrying loads are steered using phototaxis (Movie 10). Controlling the surface chemistry of the loads allows us to attach them to cells; loads are detached from cells using photochemistry (Movie 11).
Select Publications:
1. Singhvi, R. et al. "Engineering cell shape and function". Science 264, 696-698 (1994).
2. Takayama, S. et al. "Patterning cells and their environments using multiple laminar fluid flows in capillary networks". PNAS 96, 5545-5548 (1999).
3. Ostuni, E. et al. "Patterning mammalian cells using elastomeric membranes". Langmuir 16, 7811-7819 (2000).
4. Takayama, S. et al. "Laminar flows: Subcellular positioning of small molecules". Nature 411, 1016 (2001).
5. Whitesides, G. M. et al "Soft lithography in biology and biochemistry." Annual Review of Biomedical Engineering 3, 335-373 (2001).
6. Dertinger, S. K. et al "Gradients of substrate-bound laminin orient axonal specification of neurons". PNAS 99, 12542-12547 (2002).
7. Takeuchi, S. et al. "Controlling the Shape of Filamentous Cells of Escherichia coli" Nano Letters; 2005; 5(9); 1819-1823.
8. Weibel, D. et al. "Bacterial Printing Press that Regenerates Its Ink: Contact-Printing Bacteria Using Hydrogel Stamps" Langmuir; 2005; 21(14); 6436-6442
9. Weibel, D. et al. "Microoxen: Microorganisms To Move Microscale Loads" PNAS 2005 102: 11963-11967.
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