Micropatterned Substrates - American Chemical Society

Intercellular Communication Pathways ... ment and physiology of the central nervous system (CNS). ... substrates as platforms for interrogating commun...
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Anal. Chem. 2002, 74, 4640-4646

Micropatterned Substrates: Approach to Probing Intercellular Communication Pathways Hajime Takano,†,‡ Jai-Yoon Sul,†,§,| Mary L. Mazzanti,†,§ Robert T. Doyle,†,§ Philip G. Haydon,*,†,| and Marc D. Porter*,†,‡

Roy J. Carver Laboratory for Ultrahigh-Resolution Biological Microscopy, Department of Chemistry, and Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50011

Intercellular signaling is critical for the normal development and physiology of the central nervous system (CNS). To study such signaling, it is vital to control where and when the cells make contact with one another. It is also important to determine whether the process used for cell localization has an impact on signaling. This paper describes a technique that controls the location for cell growth in vitro and demonstrates that the technique has minimal (if any) impact on intercellular signaling. By using photolithographic methods, poly(dimethylsiloxane) molds were fabricated to function as templates for micrometerlevel patterning of a nonadhesive agar (agarose) onto glass coverslips coated with a cell adhesive film (poly(L-lysine)). This process yields a surface composed of well-defined adhesive and nonadhesive microdomains. When endothelia or astrocytes are plated onto these substrates, confluent domains of endothelia or astrocytes grow on the poly(L-lysine) domains. Cocultures of astrocytes and neurons can also successfully be used to form interwoven networks on the adhesive domains. Moreover, studies of calcium signaling revealed that astrocytes grown on such patterns retain their native physiological activity. This conclusion is based on the observed propagation rate for calcium waves within individual astrocyte domains and across neighboring, but spatially disconnected, astrocyte domains. The potential to apply these micropatterned substrates as platforms for interrogating communication pathways in key components of the CNS is discussed. It is often difficult to identify the mechanistic pathways critical to intercellular signaling.1 The difficulties in unraveling the underpinnings in the propagation of intercellular calcium waves between mixed glial cells,2,3 astrocytes,4-12 endothelial cells,13,14 * To whom correspondence should be addressed: (e-mail) mporter@ porter1.ameslab.gov; (fax) 515-294-3254; (e-mail) [email protected], (fax) 215-746-6792.. † Roy J. Carver Laboratory for Ultrahigh-Resolution Biological Microscopy. ‡ Department of Chemistry. § Department of Zoology and Genetics. | Present address: Department of Neuroscience, 215 Stemmler Hall, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6074. (1) Haydon, P. G. Nat. Rev. Neurosci. 2001, 2, 185-193. (2) Charles, A. Glia 1998, 24, 39-49. (3) Charles, A. C.; Merrill, J. E.; Dirksen, E. R.; Sanderson, M. J. Neuron 1991, 6, 983-992.

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epithelial cells,15 and glioma cells16 typify the many challenges in this area. One of the long-standing questions concerning the propagation of calcium waves is the route in which a molecular messenger moves between neighboring cells and triggers the wave.1,2,6 Intercellular diffusion of second messengers through the gap junctions that serve as interconnects between contacting cells is one possible route.5,6,17 Alternatively, messengers may be released from a stimulated cell into the extracellular fluid and diffuse toward neighboring cells.2,8-12,18 One approach to distinguish between these two possibilities examines whether calcium waves can propagate between cells that are physically separated from each other. This approach has been successfully tested by using a nonconfluent layer of cultured cells8 and by mechanically scoring cell-free areas in a confluent culture.2,4,18 Along these lines, noninvasive techniques that could provide a means to carefully control cell localization would be extremely valuable. Several laboratories have recently demonstrated that microfabricated poly(dimethylsiloxane) (PDMS) architectures, as either microcontact stamps,19-21 microfluidics channels,22,23 or stencils,24,25 (4) Hassinger, T. D.; Guthrie, P. B.; Atkinson, P. B.; Bennett, M. V. L.; Kater, S. B. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13268-13273. (5) Venance, L.; Stella, N.; Glowinski, J.; Giaume, C. J. Neurosci. 1997, 17, 1981-1992. (6) Giaume, C.; Venance, L. Glia 1998, 24, 50-64. (7) Scemes, E.; Dermietzel, R.; Spray, D. C. Glia 1998, 24, 65-73. (8) Guthrie, P. B.; Knappenberger, J.; Segal, M.; Bennett, M. V. L.; Charles, A. C.; Kater, S. B. J. Neurosci. 1999, 19, 520-528. (9) Innocenti, B.; Parpura, V.; Haydon, P. G. J. Neurosci. 2000, 20, 1800-1808. (10) Cotrina, M. L.; Lin, J. H. C.; Lopez-Garcia, J. C.; Naus, C. C. G.; Nedergaard, M. J. Neurosci. 2000, 20, 2835-2844. (11) Fam, S. R.; Gallagher, C. J.; Salter, M. W. J. Neurosci. 2000, 20, 28002808. (12) Newman, E. A. J. Neurosci. 2001, 21, 2215-2223. (13) Braet, K.; Paemeleire, K.; D’Herde, K.; Sanderson, M. J.; Leybaert, L. Eur. J. Neurosci. 2001, 13, 79-91. (14) Moerenhout, M.; Vereecke, J.; Himpens, B. Cell Calcium 2001, 29, 117123. (15) Homolya, L.; Steinberg, T. H.; Boucher, R. C. J. Cell Biol. 2000, 150, 13491359. (16) Fry, T.; Evans, J. H.; Sanderson, M. J. Microsc. Res. Tech. 2001, 52, 289300. (17) Reuss, B.; Unsicker, K. Glia 1998, 24, 32-38. (18) Wang, Z.; Haydon, P. G.; Yeung, E. S. Anal. Chem. 2000, 72, 2001-2007. (19) Mrksich, M. Cell. Mol. Life Sci. 1998, 54, 653-662. (20) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Biotechnol. Prog. 1998, 14, 356-363. (21) Kam, L.; Shain, W.; Turner, J. N.; Bizios, R. Biomaterials 2001, 22, 10491054. (22) Chiu, D. T.; Jeon, N. L.; Huang, S.; Kane, R. S.; Wargo, C. J.; Choi, I. S.; Ingber, D. E. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2408-2413. (23) Folch, A.; Toner, M. Biotechnol. Prog. 1998, 14, 388-392. 10.1021/ac0257400 CCC: $22.00

© 2002 American Chemical Society Published on Web 08/17/2002

can be employed to create micrometer-sized cellular patterns on various substrates.26 In this paper, we describe the use of microfabricated PDMS molds as templates for the facile patterning of adhesive and nonadhesive materials on glass substrates. These micrometer-sized patterns are formed by utilizing two standard cell culture materials: agarose as an adhesion inhibitor27-31 and poly(L-lysine) as an adhesion promoter.32 We demonstrate herein that these patterned substrates can be used to (1) form spatially separated domains of endothelia and astrocytes, (2) coculture interwoven domains of astrocytes and neurons, and (3) test for the existence of intercellular and extracellular signaling via the propagation rate of calcium waves and the affect of a purinergic receptor antagonist on wave propagation. The latter study also shows that this localization strategy has little, if any, detectable impact on the signaling pathway. The potential to apply these micropatterned platforms to interrogating the communication pathways between key components of the CNS is briefly discussed. EXPERIMENTAL SECTION Mold Fabrication. The glass substrates were coated with patterned domains of an adhesion promoter (poly(L-lysine); Sigma, MW 30 000-70 000) and an adhesion inhibitor (agarose, Fisher) by using microfabricated elastomeric molds that were constructed from PDMS. The PDMS molds were prepared through a mutistep process, starting with the preparation of photomasks and siliconsupported photoresist masters. Several types of photomasks were designed by developing patterns in photoemulsion plates, including black stripes that were 110 µm wide and separated from adjacent stripes by either 40- or 90-µm-wide gaps. A variety of square-shaped masters were also constructed and consisted of 53 × 53-µm squares surrounded by 47-µm gaps, 115 × 115 µm squares separated by 85-µm gaps, and 215 × 215 µm squares separated by 185-µm gaps. The first step in fabricating the photoresist master entailed spreading 3 mL of photoresist solution (SU8-5 or SU8-50, MicroChem Corp.) onto a 4-in. silicon substrate (Montco Silicon), which was subsequently spun at 2000-3000 rpm for 1 min. The coatings were then baked at 90 °C for 1 h, cooled to room temperature, irradiated with ultraviolet light through one of the photomasks, baked at 90 °C for another 15 min, and again cooled to room temperature. The pattern was developed by washing with propylene glycol methyl ether acetate (PMGEA, Aldrich), and the resulting photoresist master was baked at 90 °C for an additional 12 h. These architectures functioned as masters for casting molds of PDMS. (24) Folch, A.; Jo, B.-H.; Hurtado, O.; Beebe, D. J.; Toner, M. J. Biomed. Mater. Res. 2000, 52, 346-353. (25) Ostuni, E.; Kane, R.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2000, 16, 7811-7819. (26) Folch, A.; Toner, M. Annu. Rev. Biomed. Eng. 2000, 2, 227-256. (27) Parpura, V.; Haydon, P. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 86298634. (28) Bekkers, J. M.; Stevens, C. F. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 78347838. (29) Segal, M. M.; Furshpan, E. J. J. Neurophysiol. 1990, 64, 1390-1399. (30) Hammarback, J. A.; McCarthy, J. B.; Palm, S. L.; Furcht, L. T.; Letourneau, P. C. Dev. Biol. 1988, 126, 29-39. (31) Westermark, B. Exp. Cell. Res. 1978, 111, 295-299. (32) Banker, G.; Goslin, K. Culturing Nerve Cells; MIT Press: Cambridge, MA, 1991.

Figure 1. Illustration of the micropatterning process. A PDMS mold is placed on poly(L-lysine)-coated glass, and a drop of hot agarose solution (1%) is applied at its edge. The microchannels in the mold are then filled with agarose by pulling a moderate vacuum. After drying overnight, the mold is gently detached from the underlying substrate, revealing alternating lanes of agarose and poly(L-lysine).

The molds were constructed by pouring PDMS fluid (Sylgard 184, Edwards Adhesive) onto a photoresist master and curing for 12 h at 70 °C. The relief structure on the master therefore served as a negative for the structure of the PDMS mold. With this protocol, typical heights for the PDMS pads were 8 µm when a SU8-5 (2000 rpm) master was used and 24 µm when SU8-50 (3000 rpm) was used as a master. Substrate Patterning. Alternating domains of poly(L-lysine) and agarose were formed on glass substrates in two steps. These steps are illustrated in Figure 1. The first step completely coats the glass substrate with poly(L-lysine) (∼400 µL of a 1 mg/mL borate buffer (pH 8.4)) that is applied at room temperature and washed with deionized water. The PDMS mold is then carefully placed on the poly(L-lysine) coating, and a drop of boiling 1% aqueous agarose (gelation temperature 35-45 °C) solution is applied along one of the open edges of the mold. The channels are filled with agarose by pulling the solution through the channels with a glass pipet connected to moderate vacuum. The agarose solution in the channels is then allowed to dry for 12 h before the mold is carefully detached from the substrate. If not dried sufficiently, the agarose coating tended to detach from the underlying substrate upon removal of the PDMS mold. In all cases, the patterned substrates were immersed into 70% ethanol prior to cell plating. This step was employed to reduce potential complications caused by bacterial or fungal contamination. The substrates were then carefully dried to remove residual ethanol and used as described in the next section to grow various types of localized cell domains. Cell Cultures. A frozen endothelial cell line was purchased from American Type Cell Culture (CRL-2480). These cells were dissociated, placed into culture flasks containing media (Media 199, GibcoBRL) supplemented with 10% fetal bovine serum), and incubated in a humidified 5% CO2/95% air atmosphere for 8-10 days. The flasks were then rinsed twice with Earle’s balanced salt solution, and adherent cells were detached by trypsinization. The detached cells were spun at 750 rpm for 10 min, the supernant was discarded, and the cells were resuspended in media and plated onto the micropatterned substrates. These samples were subsequently incubated at 37 °C in a humidified 5% CO2/95% air atomosphere and allowed to grow to confluency (∼4 days). Analytical Chemistry, Vol. 74, No. 18, September 15, 2002

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Purified cortical astrocyte cultures were prepared as previously described.9 Briefly, cortexes of 1-4-day postnatal mice pups were dissected, dissociated, and placed into culture flasks containing phenyl red-free modified minimal essential medium (MMEM) (Eagle’s minimum essential medium, 2 mM glutamine, 1 mM sodium pyruvate, 100 units/mL penicillin, and 100 mg/mL streptomycin) supplemented with 10% fetal bovine serum). These flasks were placed in a humidified 5% CO2/95% air atmosphere for 8-10 days to produce astrocyte-enriched cultures. The flasks were then rinsed twice with ice-cold MMEM, tapped abruptly to dislodge any neuronal cells, and then placed on an orbital shaker for 1.5 h at 260 rpm. The flasks were again rinsed twice with icecold MMEM, tapped, and returned to the shaker for an additional 18 h. The adherent cells were detached by trypsinization. The detached cells were spun at 750 rpm for 10 min, the supernant was discarded, and the cells were resuspended in MMEM and plated onto the micropatterned substrates. The samples were subsequently incubated at 37 °C in a humidified 5% CO2/95% air atomosphere and allowed to grow to confluency (∼4 days). The cortical astrocyte and neuron cocultures were prepared as previously described,33 plated on the micropatterned substrates, and allowed to grow for ∼7 days. Membrane Staining. The endothelial cell membranes were stained using a multistep process. First, endothelial cells cultured on micropatterned substrates were washed with 0.1 M phosphatebuffered (pH 7.4) saline solution (PBS), placed in 2 mL of the saline containing 10 µL of a 2.5 mg/mL solution of a membranestaining fluorescent dye, 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindodicarbocyanine perchlorate (DiD, Molecular Probes). After incubating for 10 min at 37 °C, the cells were washed and fixed with 4% paraformaldehyde in PBS. The coverslips were then mounted on microscope slides with a 1-µL droplet of n-propyl gallate glycol solution and sealed with transparent nail polish. Immunocytochemistry. The astrocyte-neuron cocultures were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min, rinsed with PBS, and permeabilized with Triton X-100 (0.25% in PBS) for 10 min. The culture was then exposed for 30 min to PBS which also contained 5% BSA, 5% normal goat serum, 0.25% Triton X-100, and 0.02% NaN3 in order to minimize nonspecific binding. Next, the culture was incubated for ∼12 h at 4 °C with mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (Sigma) to selectively label the astrocytes and with rabbit anti-microtubule-associated protein 2 (MAP2) polyclonal antibody (Chemicon International) to specifically identify the neurons. After washing three times with PBS containing 5% normal goat serum, 0.5% BSA, 0.25% Triton X-100, and 0.02% NaN3, the culture was incubated with fluorescently labeled secondary polyclonal antibodies (Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 568 goat anti-rabbit IgG, Molecular Probes) for 2 h in the dark. The coverslips were then mounted onto glass microscope slides for imaging. Imaging Instrumentation. An inverted microscope (Nikon, Eclipse TE-200), which was equipped with epifluorescence, differential interference contrast (DIC) hardware, and a high sensitivity CCD camera (Hamamatsu), was used for imaging cells. An inverted microscope (Nikon), configured with phase contrast filters, was employed to image the patterned substrates. (33) Basarsky, T. A.; Parpura, V.; Haydon, P. G. J. Neurosci. 1994, 14, 640211.

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Figure 2. Phase contrast images of poly(L-lysine)/agarose domains supported on microscope coverslips. The brighter regions are the poly(L-lysine) domains, and the darker regions are the agarose domains: (a) 110-µm lanes of poly(L-lysine) separated by 90-µm agarose lanes; (b) 53 × 53 µm poly(L-lysine) wells separated by 47µm-wide walls of agarose. The topographic differences between domains are ∼80 nm for both (a) and (b).

Atomic Force Microscopy. A Dimension 3000 AFM (Digital Instruments) with a Nanosope IIIa control system and a 100-µm tube scanner was utilized for thickness determinations of the dried agarose films. These images were collected in tapping mode with 100-µm silicon cantilevers (Nanoprobes). Calcium Imaging. Astrocytes cultured on patterned glass coverslips were incubated with 10 µM fluorescent Ca2+ indicator, Fluo-3AM (Molecular Probes), in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered saline solution for 45 min at 37 °C. The coverslips were then rinsed with HEPESbuffered saline and mounted on the imaging chamber of the microscope. A glass micropipet was used to mechanically stimulate the cells. A 1-µL aliquot of 50 mM pyridoxal phosphate-6azophenyl-2′,4′-disulfonate (PPADS) was added to ∼1 mL of HEPES buffer to test the effect of purinergic receptor antagonist on the calcium waves. RESULTS AND DISCUSSION Substrate Patterning. Figure 2 shows a set of phase contrast images for two of the patterned glass substrates made using molds cast against SU8-50 masters. In both cases, the regions of darker contrast correspond to the agarose domains. Figure 2a is an image of a substrate composed of 90-µm-wide lanes of agarose that are separated by 110-µm-wide lanes of poly(L-lysine). The example shown in Figure 2b is a patterned substrate consisting of 53 × 53 µm square-shaped wells of poly(L-lysine) that are surrounded by agarose domains with widths of 47 µm. The topographic difference between the two materials is fairly small but is dependent on the architecture of the master and the concentration of the boiling agarose solution (e.g., ∼80 nm when a freshly prepared, 1% agarose solution and a mold fabricated against a SU8-50 master was used). Larger, similarly shaped patterns were also formed (e.g., 215 × 215 µm square microwells deposited on glass substrates of varied sizes (22 × 22 mm coverslips, 12-mm-diameter coverslips, and 1 × 3 in. glass microscope slides)). Occasionally, we found that the smaller sized grid patterns had an irregular shape, which is attributed to an ineffective filling of the PDMS mold by the agarose solution. Other types of cell-adherent materials have been fabricated and tested. For instance, a polystyrene culture dish was utilized as a cell-adherent substrate and patterned with nonadherent agarose domains. We were also able to reverse the coating process by first depositing agarose directly on a glass substrate, which,

Figure 3. DIC (a, c) and fluorescence (b, d) micrographs of fixed endothelial cells cultured on two different poly(L-lysine)/agarose patterns: (a, b) 110-µm-wide poly(L-lysine) lanes separated by 90µm-wide agarose lanes; (c, d) 53 × 53 µm poly(L-lysine) wells surrounded by 47-µm-wide agarose walls. In both cases, the cell membranes were stained with DiD. A Cy5 filter cube was used in the epifluorescence images.

after drying, was patterned by flowing poly(L-lysine) solution through the PDMS channels. However, poly(L-lysine) weakly adheres to agarose and often remained attached to the master when an attempt was made to remove the master from the poly(L-lysine) coated glass substrate. We believe that the ability to more reliably fabricate patterns when using poly(L-lysine) as the material to coat the glass substrate results from the strong electrostatic interaction between the positively charged poly(Llysine) and the negatively charged surface of the glass substrate. We have not, however, tested the validity of this assertion. Cell Growth and Immunocytochemistry. As a starting point for evaluating the utility of our patterned substrates for cell localization, we examined the growth and immunocytochemistry of three different systems: endothelia, purified astrocytes, and a mixture of astrocytes and neurons. This section describes the results of experiments with endothelial cells and with astrocyteneuron cocultures. The findings from the astrocyte study, which included an examination of calcium wave propagation, are presented in a later section. Panels a and b of Figure 3 present a DIC and corresponding fluorescence image for endothelial cells that were cultured for ∼4 days on a substrate patterned, stained with DiD, and then fixed with paraformaldehyde. The substrate was patterned with 110µm-wide “cell-adherent” lanes (i.e., poly(L-lysine)-coated lanes) separated by 90-µm-wide “cell-free” lanes (i.e., agarose-coated lanes). Upon plating, the cells were randomly distributed across both portions of the patterned substrate and had a spherical shape. The cells, however, began to adhere preferentially to the poly(Llysine) lanes and to adopt a more elongated appearance as culture time increased. Within 4 days, the cells formed confluent layers on the poly(L-lysine) lanes. This development is evident in the images in Figure 3, with the DiD image more readily revealing

the presence of individual cells.34 We rarely observed the adherence of cells to the agarose domains after 4 days of culturing, which is consistent with the long-standing use of this material as a nonadherent substrate.27-31 Panels c and d of Figure 3 show a pair of images of endothelial cells prepared the same way as the sample in (a) and (b), but on a substrate patterned with 53 × 53 µm square microwells of poly(L-lysine). Cells of differing numbers are evident in the different wells. In some of the wells, the cells have grown to confluency, whereas only a few cells are present in others. There are also a few wells devoid of cells, indicative of the stochastic nature of cell plating. Together, these images demonstrate that these substrates can be used to control the localization of cell growth. The feasibility of coculturing astrocytes and neurons to form interwoven networks on the patterned domains was also tested. Figure 4 shows a series of fluorescent micrographs for an astrocyte-neuron coculture after plating on a substrate composed of 110-µm-wide lanes of poly(L-lysine) separated by 90-µm-wide lanes of agarose and then fixing and processing the coculture for immunocytochemistry. The astrocytes were labeled with antibodies raised against glial fibrillary acidic protein. Anti-GFAP antibodies is used to selectively label astrocytes in tissue of the central nervous system and in cell cultures.32,35 The microtubule-associated protein 2 localizes in cell bodies and dendrites of neurons,32,36 and antibodies against MAP2 are selective for neurons. The astrocytes and neurons can therefore be identified by using mouse anti-GFAP antibody and rabbit anti-MAP2 antibody as primary antibodies and Alexa Fluor 488 conjugated goat anti-mouse antibody (green fluorescence) and Alexa Fluor 568 goat anti-rabbit antibody (red fluorescence) as secondary antibodies to visualize the primary antibodies. Figure 4 presents three different images after labeling a cocultured sample. Figure 4a displays the patterned sample with the epifluorescence microscope set to detect the green emission from Alexa Fluor 488, and the image for the same sample is shown in Figure 4b with the microscope set to detect the red emission of Alexa Fluor 568. Figure 4c is an overlay of the two images. As evident, the astrocytes are tightly packed in an elongated arrangement along the long axis of the lanes (Figure 4a). We can also clearly observe several neurons with neurites interconnected along the same lanes (Figure 4b). Neurites are unique structural feature of neurons, and the image shows that neurites have formed throughout each of the poly(L-lysine) lanes. Together, these results show that our substrates can be used to form highly organized, interwoven cellular networks, which could then potentially be used to unravel communication pathways in higher order systems. Calcium Waves at Micropatterned Astrocyte Arrays. Although once thought to play only a supportive role in the neurological processes of the brain, it has become clear that astrocytes actively respond to many different messengers.1 Along these lines, delineating the mechanistic origin and propagation pathways of calcium waves in astrocytes has emerged as an important topic.1,2,6 Intercellular diffusion of messengers through (34) Servant, G.; Weiner, O. D.; Neptune, E. R.; Bourne, H. R. Mol. Biol. Cell 1999, 10, 1163-1178. (35) Eng, L. F.; Ghirnikar, R. S.; Lee, Y. L. Neurochem Res 2000, 25, 14391451. (36) Sa´nchez, C.; Dı´az-Nido, J.; Avila, J. Prog. Neurobiol. 2000, 61, 133-168.

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Figure 4. Fluoresence micrographs of an astrocyte-neuron coculture grown on a glass substrate patterned with 110-µm-wide poly(L-lysine) lanes that are separated by 90-µm-wide agarose lanes. The images were obtained using the following: (a) FITC filter cube to identify the portions of the coculture labeled with GFAP (AlexaFluor488); (b) rhodamine filter cube to identify the portions of the coculture labeled with MAP2 (AlexaFluor568); and (c) an overlay of images (a) and (b).

Figure 5. Two sets of fluorescence (∆F/F0) image sequences for confluent lanes of astrocytes formed on 110-µm-wide poly(L-lysine) lanes that are separated by 40-µm-wide agarose films. (a) Sequence of images for astrocytes loaded with Fluo-3AM immediately after mechanical stimulation. (b) Sequence of images for astrocytes loaded with Fluo-3AM and with 50 µM PPADS present in the extracellular saline.

gap junctions represents one possible route.6,7,17 Alternatively, messengers may be released into the extracellular fluid and diffusively move toward neighboring cells.2,4,8-12,18 One way to distinguish between these two possibilities examines whether a calcium wave can be passed between cells that are physically separated from each other. Such a series of experiments would also provide a basis in which to assess whether the process of localization on our patterned substrates impairs cellular function. This section explores the utility of our micropatterned substrates in addressing these questions by determining the following: (1) whether astrocytes cultured on our patterned substrates can propagate calcium waves; (2) whether calcium waves can propagate from an array of astrocytes physically separated from neighboring arrays; and (3) whether the release of a diffusible extracellular transmitter mediates wave propagation. These experiments were conducted by plating astrocytes on 110-µm-wide lanes of poly(L-lysine) that were separated by 40µm-wide lanes of agarose. After maintaining the cultures for ∼4 days, which allowed the astrocytes to proliferate and grow to confluence within the poly(L-lysine) lanes, the cells were loaded with the calcium indicator Fluo-3AM. This dye is a cell-permeant, calcium-selective indicator that becomes compartmentalized in the 4644

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cytoplasma upon nonspecific cleavage of pendant acetoxymethyl ester groups.37 Thus, increases in intracellular Ca2+ levels can be determined by the increase in the fluorescence of the complex formed between the indicator and metal ion. After loading, the immobilized cells are stimulated by briefly bringing a micropipet into light physical contact with the surface of an astrocyte, triggering a calcium wave.3 The results, which are shown by the images in Figure 5, are presented as comparisons of the fluorescent intensity of all subsequent images (Ft) to that of the image prior to stimulation (F0), where ∆F/F0 represents the percent change in fluorescence via eq 1.

∆F/F0 ) ((Ft - F0)/F0) × 100

(1)

Figure 5a shows a calcium wave at a substrate patterned with 110-µm-wide astrocyte lanes that are separated by 40-µm-wide cellfree lanes. Upon mechanical stimulation, a calcium wave initiates at the point of contact and quickly propagates to neighboring cells within the same lane, i.e., the “primary” lane. Propagation within (37) Kao, J. P.; Hartootunian, A. T.; Tsien, R. Y. J. Biol. Chem. 1989, 14, 81798184.

the primary lane continues for several seconds, spanning a distance of more than 50 µm. At 8-10 s, the wave appears in the two adjacent astrocyte lanes, i.e., the “secondary” lanes. The waves continue to expand within their respective lanes for another 1214 s, whereupon waves in the “tertiary” lanes become evident. The ability of the wave to propagate across the cell-free lanes supports the existence of an extracellular communication pathway via a messenger like ATP, which is generally viewed to be one of the messengers that mediates calcium waves in glial cells.8,10,12,18 To evaluate further the efficacy of our astrocyte arrays, the effects of a purinergic receptor antagonist on wave propagation was examined by adding PPADS to the extracellular saline solution. Recent findings have shown that the addition of ATP to the extracellular fluid induces an increase in intracellular Ca2+, whereas the introduction of purinergic receptor antagonists such as suramin and PPADS into the extracellular fluid suppresses wave propagation by blocking ATP receptor sites on the external cell membrane.10,12 Figure 5b presents the resulting sequence of fluorescence images for the array after addition of PPADS. As in the case when PPADS was absent, wave propagation is evident after introduction of PPADS into the extracellular fluid; however, the wave now propagates at a much slower rate. For example, the wave in Figure 5a (no PPADS) has propagated across both the secondary and tertiary lanes within 20-24 s after stimulation. The wave in Figure 5b (PPADS present), in contrast, has yet to detectably move across the gap that separates the primary and secondary lanes in the same time interval. Furthermore, the wave observed with PPADS present spans a much smaller fraction of the area in the primary lane than that for the wave prior to addition of PPADS. This set of results qualitatively differentiates the relative signaling rates for the intercellular and gap junction pathways, further demonstrating the utility of our substrates as platforms for investigations of cellular signaling. The images in Figure 5 can also be used to more quantitatively determine the propagation rate of the calcium wave across the array with and without the presence of PPADS. As discussed in earlier reports,7,16 the propagation rate can be assessed by monitoring the fluorescence intensity as a function of time, with the arrival of the wave at a given position defined by a 30% increase in intensity with respect to that before stimulation.38 Figure 6 summarizes the results from this analysis and includes an inset of intensity versus time data from three different locations on the primary lane as examples of the data used to construct the plots. Each of the profiles in the inset is sigmodially shaped. As expected, the arrival time of the wave increases as the distance from the stimulation point increases. For example, the response profile ∼40 µm from the stimulation point undergoes a nearinstantaneous increase after cell stimulation, reaching its maximum in just under 5 s. The profiles for the other two locations (100 and 160 µm) are nevertheless offset to longer times by virtue of being farther from the stimulation point. These profiles also show that the rate at which the fluorescence intensity increases slows with increased distance from the stimulation point. The basis of this decrease, which is the subject of extensive speculation, reflects a complex mixing of dilution phenomena in both the extraand intracellular signaling pathways.2,6 (38) Araque, A.; Parpura, V.; Sanzgiri, R. P.; Haydon, P. G. Eur. J. Neurosci. 1998, 10, 2129.

Figure 6. Arrival time for calcium wave as a function of distance from point of mechanical stimulation: Primary lane (b), secondary lanes (O), and primary lane with PPADS (2). Each data point is the average of an analysis of the intensity profiles for six equidistant points from the stimulation point; the error bars represent the standard deviation of each data set. Inset: plots of ∆F/F0 as a function of time at various distances from the stimulation point. Each plot was fit to a sigmoid curve in order to estimate the calcium wave arrival time, which was defined as the time when the fluorescent intensity exceeds its original intensity by 30%.

Figure 6 provides a broader picture of the findings by plotting the arrival time of the wave as a function of distance from the stimulation point for the primary and secondary lanes in the absence of PPADS and of the primary lane in the presence of PPADS. In all three instances, the arrival time undergoes a nonlinear increase with distance from the stimulation point. These dependencies indicate that the rate of wave propagation slows as the distance from the point of stimulation increases. In the absence of PPADS, the propagation rate in the primary lane evolves from ∼15 µm/s at locations less than 80 µm from the stimulation point to ∼7 µm/s between 140 and 200 µm from the stimulation point. The evolution of the wave in the secondary lane roughly parallels that of the primary lane. These rates are comparable to those (530 µm/s) reported by other laboratories, indicating that our approach to patterning does not notably impact wave propagation.2,6 Another intriguing aspect of the data is the marked difference in the rate of wave propagation in the primary lane with and without PPADS. The rate with PPADS present is ∼5 µm/s. Because PPADS blocks ATP receptor sites, the rate of propagation with PPADS present is attributed primarily to signaling through gap junctions. Following this line of reasoning, we attribute the evolution of the propagation rate close to the stimulation point to a mixing of gap junction and extracellular signaling pathways, with the effects of dilution reducing the importance of the extracellular pathway as the distance from the stimulation point increases. CONCLUSIONS This paper has demonstrated that micropatterning a cell adhesive inhibitor, agarose, over a uniform cell adherent layer, poly(L-lysine), is an effective method for the controlled localization and growth of cells in culture. Our results have shown that these Analytical Chemistry, Vol. 74, No. 18, September 15, 2002

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substrates can be used to localize cell arrays and to culture interwoven, higher level architectures (i.e., astrocyte-neuron networks). That is, our micropatterned substrates maintain the effective environment of traditional cell culture, which is supported by the fact that several cell types, known to grow on poly(L-lysine), readily attach to the substrate and proliferate. Moreover, the localized cells display characteristics normally found in traditional cell culture by expressing cell-specific proteins (GFAP for astrocytes, MAP-2 for neurons) and propagating calcium waves when stimulated. The combined weight of these results argues that our patterning strategy does not detectably alter cell function. We believe that this approach will have utility in a wide range of investigations, taking advantage of the fact that once a PDMS mold has been created in the microfabrication facility, the rest of the process can be routinely performed in a standard biological laboratory. Along these lines, we are currently designing experiments to apply these substrates to further unravel key aspects of the signaling pathway in glia, including whether ATP is released

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only from the stimulated cell or is regenerated at neighboring cells after the wave propagates to these cells. We are also examining ways to incorporate extracellular matrix39 proteins into the patterning process in our effort to extend the scope of our strategy. ACKNOWLEDGMENT H.T. gratefully acknowledges the support from the Institute for Physical Research and Technology, Iowa State University. The research was supported in part by the Roy J. Carver Charitable Trust and by IPRT Microanalytical Instrumentation Center. This work was also supported by grants from the NIH to P.G.H. (NS44007 and NS43142). Received for review April 29, 2002. Revised manuscript received July 17, 2002. Accepted July 23, 2002. AC0257400 (39) Mrksich, M. Chem. Soc. Rev. 2000, 29, 267-273.