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Anal. Chem. 2010, 82, 2900–2906

Capillary Based Patterning of Cellular Communities in Laterally Open Channels Sung Hoon Lee,†,§ Austen James Heinz,†,§ Sunghwan Shin,†,§ Yong-Gyun Jung,‡ Sung-Eun Choi,†,§ Wook Park,†,§ Jung-Hye Roe,‡ and Sunghoon Kwon*,†,§ School of Electrical Engineering and Computer Science and School of Biological Sciences, Seoul National University, San 56-1, Daehak-dong, Gwanak-gu, Seoul, 151-744, South Korea, and Inter-university Semiconductor Research Center (ISRC), Seoul National University, South Korea In order to offer an easier way to study interactions between multiple cellular populations, we have developed a novel method to precisely place cells in a variety of nonoverlapping patterns using surface tension in laterally open microchannels. Our design is fundamentally different from previous strategies such as compartmentalization, stamping, stenciling, or mechanical approaches. It relies on capillary action or the propensity for liquid to move more readily through narrow spaces as a result of surface tension. Until now, capillary based patterning has been limited to coating chemically isolated areas. Here, we demonstrate, through use of surface tension and controlled flooding, that it is possible to pattern multiple cells and proteins using laterally open channels in a variety of designs. We demonstrate the relevance of the concept by coculturing different mammalian cell types and evaluating the behavior of engineered quorum sensing circuits in E. coli. In the future, we believe the laterally open channel designs shown here can be useful for rapidly creating and studying cellular ecologies using simple pipetting. Communication found in cellular communities such as those involved in tumor cell growth1 or in multispecies bacterial communities like the gut2 or soil3,4 have been understudied. This is largely due to the test tube or Petri-dish environment making coculturing enormously difficult.5,6 In most instances, without spatial division, one cellular population inevitably overtakes others. * To whom correspondence should be addressed. E-mail: [email protected]. Fax: 02-885-4459. † School of Electrical Engineering and Computer Science, Seoul National University. ‡ School of Biological Sciences, Seoul National University. § Inter-university Semiconductor Research Center (ISRC), Seoul National University. (1) Kaneda, A.; Wang, C. J.; Cheong, R.; Timp, W.; Onyango, P.; Wen, B.; Lacobuzio-Donahuel, C. A.; Ohlsson, R.; Andraos, R.; Pearson, M. A.; Sharov, A. A.; Longol, D. L.; Ko, M. S. H.; Levchenko, A.; Feinberg, A. P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 20926–20931. (2) Ley, R. E.; Hamady, M.; Lozupone, C.; Turnbaugh, P. J.; Ramey, R. R.; Bircher, J. S.; Schlegel, M. L.; Tucker, T. A.; Schrenzel, M. D.; Knight, R.; Gordon, J. I. Science 2008, 320, 1647–1651. (3) Ferrari, B. C.; Binnerup, S. J.; Gillings, M. Appl. Environ. Microbiol. 2005, 71, 8714–8720. (4) Weibel, D. B. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 18075–18076. (5) Barns, S. M.; Fundyga, R. E.; Jeffries, M. W.; Pace, N. R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1609–1613. (6) Kaeberlein, T.; Lewis, K.; Epstein, S. S. Science 2002, 296, 1127–1129.

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Microfluidic patterning offers a means of having precise control over cellular environments in ways that are not possible in a test tube or Petri dish. Such precision, particularly with respect to spatial definition, is necessary to allow multiple cell types or species to survive in the same coculture.7-14 For instance, in bacterial communities, this is the result of a number of factors such as differential growth rates, biofilm formation, and production of secondary toxic metabolites. In general, mixed cellular communities are highly complex nonlinear systems that can be nearly impossible to replicate using conventional technology. It is because of these complexities that some bacteria, such as certain gut and soil species, have been deemed “unculturable” in a laboratory setting. As a result, the majority of work done in biology has been limited to the study of isolated clonal populations,15,16 something rarely found in nature. While there have been a few successes at replicating synergistic communities using test tubes,17 the majority of work has been limited to the use of a conditioned medium to study soluble factor interactions. In simple artificial cell circuits, like the ones used here, variation of a single small molecule (in this case a homoserine lactone [HSL]) via the addition of conditioned medium at staggered time points, can partially replicate two population systems. However, in any study involving heterotypic “back and forth” reactions, the cell age, position in the cell cycle, or time in general are of interest or a study in which all the factors are unknown; then, conditioned medium is not a suitable replacement. Regard(7) Chiu, D. T.; Jeon, N. L.; Huang, S.; Kane, R. S.; Wargo, C. J.; Choi, I. S.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2408–2413. (8) Takayama, S.; McDonald, J. C.; Ostuni, E.; Liang, M. N.; Kenis, P. J. A.; Ismagilov, R. F.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5545–5548. (9) Abhyankar, V. V.; Beebe, D. J. Anal. Chem. 2007, 79, 4066–4073. (10) Khademhosseini, A.; Suh, K. Y.; Yang, J. M.; Eng, G.; Yeh, J.; Levenberg, S.; Langer, R. Biomaterials 2004, 25, 3583–3592. (11) Anderson, J. R.; Chiu, D. T.; Jackman, R. J.; Cherniavskaya, O.; McDonald, J. C.; Wu, H. K.; Whitesides, S. H.; Whitesides, G. M. Anal. Chem. 2000, 72, 3158–3164. (12) Chueh, B. H.; Huh, D.; Kyrtsos, C. R.; Houssin, T.; Futai, N.; Takayama, S. Anal. Chem. 2007, 79, 3504–3508. (13) Ismagilov, R. F.; Ng, J. M. K.; Kenis, P. J. A.; Whitesides, G. M. Anal. Chem. 2001, 73, 5207–5213. (14) Folch, A.; Toner, M. Annu. Rev. Biomed. Eng. 2000, 2, 227–256. (15) Araten, D. J.; Nafa, K.; Pakdeesuwan, K.; Luzzatto, L. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5209–5214. (16) Cillo, C.; Cantile, M.; Mortarini, R.; Barba, P.; Parmiani, G.; Anichini, A. Int. J. Cancer 1996, 66, 692–697. (17) Kato, S.; Haruta, S.; Cui, Z. J.; Ishii, M.; Igarashi, Y. Appl. Environ. Microbiol. 2005, 71, 7099–7106. 10.1021/ac902903q  2010 American Chemical Society Published on Web 03/08/2010

Figure 1. Multiple solutions patterned in a nonclosed channel wall PDMS device. (A) (i) Schematic diagram of patterning solution and (ii) cross sectional view cut at a-a’ from (A) (i). (B) (i) Top view of the PDMS microfluidic channel. (ii) Bright field image of red dashed line box. Blue and red food dyes are used for visualization. (Scale bar: 200 µm.)

less of whether the medium can be accurately replicated, cells react to and change their medium and other cells in a somewhat stochastic manner in real time. Therefore, in order to get the most accurate representation, coculturing is necessary. Previous work has been done in the laboratories of Beebe9 and Ismagilov18 to create synthetic heterogeneous cellular environments using physical separation. Distinct populations were kept apart using compartmentalization in a microfluidic chip. Two tissue culturing has been replicated in a somewhat different manner using a porous endothelial barrier set between two cellular populations patterned parallel to each other.19 A more recent design involves injecting cell-laden hydrogels in alternating hexagonal post array channels, polymerizing, and repeating for remaining channels. The posts prevent gel leakage during assembly, while allowing for cell to cell communication after polymerization.20 In some cases, it might be desirable to have a noncompartmental approach that allows for cells to be placed in immediate proximity to one another. The two most popular methods for doing this are microcontact printing21-23 in which a chemical stamp is used to define cell binding areas and stencil based approaches24-26 where cells are prevented from binding anywhere except for where there are holes in a membrane stencil. In a more recent approach, Bhatia27 created a device consisting of two interdigitated combs with cells patterned on top whose spacing could be altered using mechanical force. All of these methods: compartmentalization, stamp, stencil, or mechanical, require techniques that are not common to biology laboratories. Additionally, there is currently no method that could potentially be scaled to create several multipopulation communities with as many pipetting events as there are populations in a single community. Here, we report the first device in which surface tension and controlled flooding in laterally open channels allows for precise cuing of cellular signaling events between spatially defined cellular populations in a shared medium. The use of capillary action, as opposed to active pumping, is attractive because it does not require much time or precision in device loading. It does not require addition of tubing or use of a syringe pump and is, therefore, compatible with simple pipetting. Because of the simplicity of this new capillary based system, we expect biologists will be able use

devices similar to the ones demonstrated here to more easily study complex multipopulation or even multispecies cellular ecologies.28 THEORY Previous work has been done to direct surface tension driven flow through chemically isolated areas, using an open channel approach.29 Here, UV lithography was used to alter the surface chemistry of the substrate in contact with a patterning laminar flow in order to create hydrophobic “virtual walls”. However, reliance on surface chemistry greatly limits what materials are patterned and, biologically speaking, what can be cultured. Here, we have developed a method that can pattern a wide variety of designs in an open channel not by creating differential surface chemistry but through use of an uneven channel topography. Capillary driven multipopulation cell patterning can be accomplished in laterally open channels through the use of deep and shallow channels (Figure 1). When fluid approaches a shallow channel, it enters and moves along the channel as a result of there being greater surface tension in the shallow channel (in front) than the deep channel adjacent to it. The forward pulling force in the shallow channels prevents the liquid from entering the deep channels. Our first and most basic device uses a molded polydimethylsiloxane (PDMS) layer that consists of several channels whose (18) Kim, H. J.; Boedicker, J. Q.; Choi, J. W.; Ismagilov, R. F. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 18188–18193. (19) Lee, P. J.; Gaige, T. A.; Ghorashian, N.; Hung, P. J. Biotechnol. Prog. 2007, 23, 946–951. (20) Huang, C. P.; Lu, J.; Seon, H.; Lee, A. P.; Flanagan, L. A.; Kim, H. Y.; Putnam, A. J.; Jeon, N. L. Lab Chip 2009, 9, 1740–1748. (21) Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Exp. Cell. Res. 1997, 235, 305–313. (22) Khademhosseini, A.; Suh, K. Y.; Jon, S.; Eng, G.; Yeh, J.; Chen, G. J.; Langer, R. Anal. Chem. 2004, 76, 3675–3681. (23) Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696–698. (24) Ostuni, E.; Kane, R.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2000, 16, 7811–7819. (25) Khetani, S. R.; Bhatia, S. N. Nat. Biotechnol. 2008, 26, 120–126. (26) Folch, A.; Jo, B. H.; Hurtado, O.; Beebe, D. J.; Toner, M. J. Biomed. Mater. Res. 2000, 52, 346–353. (27) Hui, E. E.; Bhatia, S. N. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5722– 5726. (28) Chung, S. E.; Park, W.; Shin, S.; Lee, S. A.; Kwon, S. Nat. Mater. 2008, 7, 581–587. (29) Zhao, B.; Moore, J. S.; Beebe, D. J. Science 2001, 291, 1023–1026.

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Figure 2. One solution injection into a shallow channel and success rates of injection without flooding. (A) (i) Schematic diagram of injecting solution into a shallow channel. (ii) Cross sectional diagram cut a-a’ from (A) (i) of solution confined in shallow channel of the width w and the height h with the contact angle θ. (B) Success rates of PBS injection with respect to the width and height of the shallow channel. When fluid is introduced through the shallow channel all the way through the outlet, we regard it as a successful injection. The exposure time of O2 plasma is 8 s. The solution is injected 15 min after O2 plasma treatment. The substrate is PDMS coated slide glass, and the height of the shallow channel is 20, 40, and 60 µm, respectively.

walls are shorter in height than the layer’s edges. After being set, this layer is then placed on top of PDMS coated slide glass, which is treated to make a semihydrophilic surface, to form deep and shallow channels within the microfluidic device. In Figure 1, solution A and solution B are injected into opposite sides of the device into shallow channels. The interdigitated design is made possible by extending the shallow channels to meet the selected solution while terminating the shallow channels prior to the start of the nonselected solution. The movement of liquid in shallow channels preferentially over deep channels can allow for patterning of arbitrary designs in an open channel such as a spiral shape (see video S-1 in the Supporting Information). Prior to binding the molded PDMS layer to the substrate, both are O2 plasma treated to make both hydrophilic. It is worth stressing that device functionality is critically dependent on hydrophilic/hydrophobic properties. If it is too hydrophilic, then the device will flood with deep channels filling as easily as shallow channels. If the PDMS is too hydrophobic, then liquid will not enter either channel via capillary action. Trying to overcome excessive hydrophobicity through the application of outside force cannot rectify the problem since the device is based on capillary action. External pressure either has no effect or results in unintended flooding. Figure 2 shows that success of the device is dependent not only on the surface characteristics of the device materials but also on the height and width of the shallow channel. The shorter in height and wider the shallow channel is, the less likely it will flood during injection. A theoretical description of surface tension in confined microchannels has been described by Lam.30 Since shallow channels do not have closed walls, the aspect ratio and surface hydrophi(30) Lam, P.; Wynne, K. J.; Wnek, G. E. Langmuir 2002, 18, 948–951.

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licity of the channel determines the success rate of patterning. It is assumed that curvature at the liquid air interface and external forces such as hydrostatic pressure can be neglected. From these assumptions, the change in surface free energy per unit length of the hydrophilic shallow channel is drawn, ∆G1S, as a function of surface tension of the liquid, γ, the advancing contact angle, θ, of the solution-vapor on the hydrophilic surface, the width, w, and height, h, of the shallow channel as follows: ∆G1S ) γ(h - wcos θ). Since hydrophobic surfaces have a contact angle of θ g 90°, the above equation yields change in surface free energy that is always positive. For solution to move through a shallow channel by capillary action, this energy difference should be equal or less than 0. When this value is 0, the maximum height can be calculated as hmax ) 0.34w when θ ) 70° (measured by DSA100, KRUSS) after O2 plasma treatment of 8 s, followed by 15 min drying at room temperature. By inserting the experimentally determined values as well as the channel dimensions into the free energy equation, we were able to predict when injection would not occur. As shown in Figure 2, when the width of shallow channel is 50 µm, the maximum height where fluid injection is still possible given our experimental conditions is approximately 17 µm. Experimentally, we started with a minimum height of 20 µm, which failed as expected. Likewise, a shallow channel with a width of 100 µm requires a height of less than 34 µm for injection to occur; experimentally, we found that fluid injection was not possible at 40 µm, again confirming theoretical predictions. EXPERIMENTAL SECTION Device Fabrication. We fabricated multilevel PDMS based microfluidic devices using a soft lithography replica molding process. Photolithography for fabricating SU-8 (Microchem Corp.,

Newton, MA) mold onto a wafer was used. Two layers of SU-8 mold were fabricated using double coating and exposure steps with a single developing step. PDMS (Sylgard 184, Dow Corning) was poured on the mold and baked at 150 °C for 10 min before it was peeled off the mold. Microfluidic channels and substrate made of PDMS were treated with plasma cleaning (PDC-32G, Harrick Plasma) for 8 s with 750 mtorr of high level radio frequency. For the polystyrene dish, 4 s of cleaning was applied under the same conditions. After surface treatment, the molded PDMS and substrate were bonded to each other and dried for 25-30 min at room temperature. Mammalian Cell Cultures and Preparation. HeLa and NIH3T3 fibroblast cells, used in all experiments, were cultured on polystyrene in Dulbecco’s modified Eagle medium (DMEM with 4.5 g L-1 glucose and 10% FBS, Sigma-Aldrich) and incubated at 37 °C in 5% CO2 and 95% air until near confluence. Cells were detached from the culture dish via trypsinization with 0.25% trypsin and 0.13% EDTA in phosphate-buffered saline. Mammalian cells were stained with red and green colors using the CellTracker kit (Invitrogen) for visualization. Protein Coating for Absorption in PDMS Coated Substrate. Fibronectin (Sigma) and poly L-lysine (PLL, FITC-labeled, MW 30 000-70 000, Sigma) were dissolved in PBS (pH 7.4) at a concentration of 0.02, 0.5 mg/mL, respectively.10,22,31 Two microliters of diluted fibronectin or PLL were added to inlets to fill the shallow channel, followed by a 30 min storage at room temperature. For the solution not to dry out in the shallow channel, the solution needs to be stored under sealed conditions. After storing, we dried the microfluidic channel using a vacuum pump for 10 min and then injected PBS and allowed passive flow to occur for 20 min for rinsing. When the PDMS layer was bonded to a polystyrene dish, instead of PDMS, no protein coating was done. Mammalian Cell Seeding and Culture Inside Microfluidic Channels. Two to four microliters (1-5 × 107 cells/mL) of HeLa and NIH3T3 fibroblast cells were seeded at inlets which fed shallow channels by capillary force. For cell attachment, cells needed to be stored stably under sealed conditions in a 37 °C incubator for 45 min. Fresh cell medium was added into inlets and outlets at different volumes to generate gravity-driven flow. For high density patterning, mammalian cells should be incubated overnight. The survival rate of mammalian cells was measured using a Live/Dead cytotoxicity kit (Invitrogen). All PDMS chips were autoclaved prior to use.7 E.coli Transformation and Culturing. Escherichia coli strain DH5alpha (Invitrogen) competent cells were transformed and used for all experiments.32 Cells were transformed with the plasmids k084012 (sender), t9002 (receiver), and k137019 (inverse receiver) available from the Standard Registry of Biological Parts (partsregistry.org). E.coli cells were inoculated into LB with 100 µg/mL ampicillin, incubated for 15 h, and inoculated again in fresh LB with 100 µg/mL ampicillin, followed by incubation for 3 h the following day at 37 °C while shaking. Making Polymerized Hydrogel and E.coli Cell Circuit Evaluation. We used poly(ethylene glycol) diacrylate (PEG-DA, Sigma-Aldrich, Mn 575) with 20 wt % of photoinitiator (2,2(31) Rhee, S. W.; Taylor, A. M.; Tu, C. H.; Cribbs, D. H.; Cotman, C. W.; Jeon, N. L. Lab Chip 2005, 5, 102–107. (32) Sambrook, J.; Russell, D. W. Molecular Cloning: A Laboratory Manual, 3rd ed.;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2001.

dimethoxy-2-phenylacetophenone) as the photocurable resin. Soon after introducing resin through a shallow channel, a high intensity mercury-xenon lamp (200W bulb) was used for ultraviolet polymerization. We applied the UV light with intensity of 100% for 10 s to give the hydrogel a uniform mesh size. Before seeding, cells were centrifuged with 5000 rpm to get rid of the conditioned medium so that all results could be attributed to on chip behavior. For optimal cell activity, fresh cell medium including 100 µg/mL ampicillin was added into each inlet every 30 min. We set the same exposure time with an intensity of zero in the sender region when fluorescence images were taken. We used a customized software program (Adobe Photoshop) to measure fluorescence intensity from pixel values of the same area in each picture. RESULTS AND DISCUSSION While our results showed that a shallow channel with a height of 20 µm was optimal to prevent channel flooding, such a low channel height is not practical for culturing mammalian cells that are on the order of 10 µm. To overcome this hurdle, we had to invent a different method that would not overly confine the cells and would ideally give flexible control of the 3D environment. We accomplished this using a new design that works via controlled flooding between two closely spaced shallow channels. By adding additional pressure, we were able to convert shallow channels into “channel banks” on either side of a deep channel containing most of the patterned material. Therefore, instead of materials such as mammalian cells being confined to the 20 µm height of the shallow channel, they could be contained within the deep channel. Using this approach, cells can be cultured in a channel whose height is not restricted by the need to maintain sufficient surface tension. Using the channel bank approach, a spiral shaped design was fabricated using a plasma treated PDMS chip bonded to a plasma treated PDMS coated glass slide. In Figure 3, PLL containing solution was added to the inlet where it successfully migrated via capillary action through the seeding area to the outlet. Before cell seeding, enough PBS solution was flowed through the shallow channel so that the surface of the substrate was coated evenly without leaving behind undesirable remnants. Fibroblast cells were then patterned on the spiral design using the same method. Precell patterning capabilities using this method are quite versatile and are not limited to PLL. Fibronectin, collagen, and a wide range of other proteins or compounds can be patterned in the device and used to form an extra cellular matrix. As a word of caution, it is important to note that ventilation channels were used in this design so that after mammalian cells were attached to the substrate they would have time to grow overnight within the channel to create a high density pattern. If ventilation channels are not used to dry the chip, then humidity due to steady evaporation will increase the chip’s hydrophilicity resulting in unintended flooding if the liquid is left in the device for several hours prior to whole device flooding. A “Live/Dead” assay was run in order to assess whether or not cells were surviving within the confines of the microcapillary channel bank design. As shown in Figure 4A, over 95% of cells display green fluorescence, indicating cells patterned in this open microfluidic channel provides proper conditions for mammalian cell culturing. Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

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Figure 3. Patterned cell culture on PDMS coated slide glass via controlled flooding using channel banks. (A) (i) Schematic illustration of patterned cell culture and (ii) cross sectional view of medium flow. The flow occurs as a result of the volume difference between inlet and outlet. (B, C) (i) Cross sectional view of the channel coated with PLL on which cells are cultured. (B) (ii) Fluorescent image showing spiral of PLL coated on the surface. (C) (ii) DIC image of the patterned fibroblast located in the deep channel between channel banks. (Scale bar: 200 µm.)

Figure 4. Single and multipopulation patterning in a laterally open microfluidic device. Fluorescent image showing fibroblast (A) (i) a spiral patterning on PDMS coated slide glass and (ii) a winding patterning on polystyrene dish. Live cells emit green fluorescence and dead cells emit red fluorescence. (B) (i, ii) Various shaped two population patterning on a polystyrene dish. Red and green cells indicate fibroblast and HeLa cells, respectively. (Scale bar: 200 µm.)

After running a Live/Dead viability test for single cell types on different substrates, we created a device for patterning multiple mammalian cell types: afterward, we patterned red stained fibroblast alongside green stained HeLa cells (Figure 4B). Since the cell loading for different cell types is done at different ports, separate cell lines can be patterned as close as can be allowed given current photolithographic technology. While this does not allow for different cell populations to physically touch, except in cases of random migration, it allows for proximity under 50 µm as shown in Figure 4. The maximum limit for proximity between two populations (using our facilities) was not tested. 2904

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In order to demonstrate capillary based community coculturing with a measurable interaction between populations, we used engineered quorum sensing circuits in bacteria. Specifically, we used a commonly studied sender-receiver circuit taken and modified from a naturally occurring system in V. fischeri and made into standard biological parts.33 In the modified system, the sender and receiver components are decoupled and transformed into separate E. coli populations. Sender cells produce autoinducer, 3-oxo-hexanoyl-HSL that turns ON GFP production in the receiver through promoter activation. We also used a less well-known inverse receiver circuit designed to express a GFP repressor in the presence of HSL, turning the circuit OFF from its default ON state. Since E.coli cells are sized on a scale of less than 5 µm, we used a shallow channel design for these studies. To evaluate the circuits, we used two approaches. In the first approach, we used a barrier-less system which was designed in this case to show the response of the receiver cells to the diffusion gradient of 3-oxo-hexanoyl-HSL (see Figure S-1 in the Supporting Information). We also used the same capillary based approach to create a barrier system to allow for simultaneous evaluation of sender, receiver, and inverse receiver circuits. In order to pattern the barrier, we injected hydrogel in a forked shallow channel, which separated three other shallow channels. We then polymerized the hydrogel using UV curing. To evaluate cell circuits, we introduced sender, receiver, and inverse receiver populations into the three shallow channels of each inlet (Figure 5). In the design, a deep channel separates the bacteria and polymer stored in the shallow channels. Therefore, diffusion between populations through the polymerized barrier does not occur until additional fresh medium is added to the deep channel. In this way, we can allow the growth of cells in the device for a set period of time before we activate the ecosystem via flooding (giving it the same community cuing capability as our other (33) Canton, B.; Labno, A.; Endy, D. Nat. Biotechnol. 2008, 26, 787–793.

Figure 5. E.coli cell circuit evaluation chip. The sender E.coli population continuously produces homoserine lactone [HSL] by LuxI protein. In receiver cells, the HSL activator molecule forms a complex with the LuxR protein that leads to the transcription of the GFP gene, resulting in the receiver cells turning ON (glowing). The inverse receiver circuit also processes HSL from the sender population. In this case, LuxR-HSL increases transcription of TetR, repressing transcription of the GFP gene. The end result is that the receiver turns OFF (no glowing) in the presence of HSL. (Scale bar: 200 µm.)

Figure 6. Fluorescent intensity measurements for the receiver and inverse receiver. (A) (i-iv) The sender produces HSL to turn the receiver ON and inverse receiver OFF. The brightness of the receiver increases continuously while the inverse receiver decreases in brightness. (B) Time plots measured fluorescent intensity for (i) receiver and control and (ii) inverse receiver and control. The controls show the fluorescent intensity when the sender population is not present. Error bar indicates standard deviation. (Scale bar: 200 µm.)

capillary based devices). Since a hydrogel barrier was used, prepatterning using PLL was not necessary to confine the cells to their respective areas. A volume difference between inlets was made to generate flow from the sender to the receiver populations. Since the mesh size of hydrogel is small enough to block E.coli cells, only HSL can pass through the barrier to activate the cell circuits. To provide a quantitative cell circuit evaluation, we measured the fluorescent intensity of both the receiver and the inverse receiver with controls (see Table S-1 in the Supporting Information). While the receiver increases in brightness via HSL activating the transcription of the GFP gene, the inverse receiver decreases

in brightness as a result of HSL induced synthesis of TetR, which represses transcription of the GFP gene. Time plots indicate the receiver turns ON while the inverse receiver turns OFF (Figure 6). We found that both control populations got brighter, which we attributed to cell population growth inside the device (see Figure S-2 in the Supporting Information). It is worth mentioning that in nature cells assemble and conglomerate into populations that are not always separated from other cell types or species of cells due to premade barriers, and while the use of barriers does allow for small molecule communication, they also prevent direct physical interaction between populations and the cross mixing of larger protein conglomeraAnalytical Chemistry, Vol. 82, No. 7, April 1, 2010

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tions and cellular debris. However, we found that in cases where the measured signal is the same and the growth rate of the cells rapidly outpaces the signal time scale; then, a barrier system can be useful. By forcing neighboring populations to respect fixed boundaries, cell crossover, which can potentially lead to signal mixing, is impossible. Furthermore, by changing the mesh size of the hydrogel via differential curing time, we were able to observe different response rates (data not shown), which might be useful for systematically altering the speed of natural small molecule communication systems. CONCLUSION Existing microfluidic patterning devices used for coculturing are not easy enough for biologists who do not have a background in microfluidics to use. To solve this problem, we have developed a capillary based method to place multiple populations of cells in a variety of 3-dimensional nonoverlapping patterns via simple pipetting. In doing so, we also demonstrated that our system could be used to control the extracellular matrix of each cellular population via prepatterning with small molecules or proteins. We also showed our capillary based system could be used to culture isolated populations on chip before cuing community behavior via flooding. Finally, we demonstrated that our method could also be applied to creating coculturing systems with diffusible barriers. Further development of similar capillary based devices should allow for expanded studies of complex cellular ecologies, such as

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those that occur during human disease or in bacterial environments found in nature, with minimal effort required by biologists. ACKNOWLEDGMENT This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (2008-05446), the Technology Innovation Program (2009F-020-01), and System IC 2010 project of Ministry of Knowledge Economy funded by the Ministry of Knowledge Economy (MKE, Korea). We thank NANO Square for allowing us to use their facilities. We would also like to thank the Registry of Standard Biological Parts for providing the vectors used in this study as well as B. Canton and D. Tischer for creating the receiver and the inverted receiver devices, respectively. NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web March 8, 2010 with an error in the symbol for the surface tension of the liquid. The corrected version was reposted on March 11, 2010. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 19, 2009. Accepted February 18, 2010. AC902903Q