In Vitro Model on Glass Surfaces for Complex Interactions between

Nov 1, 2010 - NanoTechnology, Beijing, China 100190, ‡Civil Aviation Medicine Center, Civil ... Chinese Academy of Sciences, Beijing, China 100049...
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In Vitro Model on Glass Surfaces for Complex Interactions between Different Types of Cells Zhenling Chen,†,‡ Wei Chen,† Bo Yuan,† Le Xiao,† Dingbin Liu,† Yu Jin,† Baogang Quan,† Jia-ou Wang,§ Kurash Ibrahim,§ Zhuo Wang,† Wei Zhang,*,† and Xingyu Jiang*,† †

CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety National Center for NanoScience and NanoTechnology, Beijing, China 100190, ‡Civil Aviation Medicine Center, Civil Aviation Administration of China, Beijing, China 100123, and §Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China 100049 Received August 6, 2010. Revised Manuscript Received October 2, 2010 This report establishes an in vitro model on glass surfaces for patterning multiple types of cells to simulate cell-cell interactions in vivo. The model employs a microfluidic system and poly(ethylene glycol)-terminated oxysilane (PEG-oxysilane) to modify glass surfaces in order to resist cell adhesion. The system allows the selective confinement of different types of cells to realize complete confinement, partial confinement, and no confinement of three types of cells on glass surfaces. The model was applied to study intercellular interactions among human umbilical vein endothelial cells (HUVEC), PLA 801 C and PLA801 D cells.

Introduction We report an in vitro model on glass surfaces for patterning multiple types of cells to simulate cell-cell interactions in vivo with a microfluidic system. Several reports outlined methods for patterning multiple types of cells on surfaces coated with a thin layer of gold1-9 because gold is biocompatible and can be easily modified on its surfaces using self-assambled monolayers (SAMs) of thiols. For example, our group employed microfluidic channels to modify gold surfaces selectively to pattern two types of cells and to simulate all three types of cell-cell interactions in vivo, which include the partial release of one type of cell and no release and complete release of both types of cells.1 These methods allow spatial and temporal control of the adhesion and motility of multiple types of cells. Existing systems, however, still have some drawbacks that retard their application to cell biology.10,11 Goldcoated substrates can severely quench and attenuate fluorescence signals, making it difficult to use fluorescence microscopy to observe cell structures and behavior in real time, and the surface properties of a gold film are unstable for extended periods of over *Corresponding authors. E-mail: [email protected], xingyujiang@ nanoctr.cn. (1) Chen, Z.; Li, Y.; Liu, W.; Zhang, D.; Zhao, Y.; Yuan, B.; Jiang, X. Angew. Chem., Int. Ed. 2009, 48, 8303–8305. (2) Li, Y.; Yuan, B.; Ji, H.; Han, D.; Chen, S.; Tian, F.; Jiang, X. Angew. Chem., Int. Ed. 2007, 46, 1094–1096. (3) Liu, D.; Xie, Y.; Shao, H.; Jiang, X. Angew. Chem., Int. Ed. 2009, 48, 4406– 4408. (4) Jiang, X.; Bruzewicz, D. A.; Wong, A. P.; Piel, M.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 975–978. (5) Barrett, D. G.; Yousaf, M. N. Angew. Chem., Int. Ed. 2007, 46, 7437–7439. (6) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X. Y.; Ingber, D. E. Annu. Rev. Biomed. Eng. 2001, 3, 335–373. (7) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363–2376. (8) Jiang, X. Surface Patterning for Controlling Cell-Substrate Interactions. In Micro and Nanoengineering of the Cell Microenviroment; Khademhosseini, A., Borenstein, J., Toner, M., Takayama, S., Eds.; Artech House: Boston, 2008; p 33. (9) Zhao, C.; Zawisza, I.; Nullmeier, M.; Burchardt, M.; Trauble, M.; Witte, I.; Wittstock, G. Langmuir 2008, 24, 7605–7613. (10) Lamb, B. M.; Westcott, N. P.; Yousaf, M. N. ChemBioChem 2008, 9, 2220– 2224. (11) Kandere-Grzybowska, K.; Campbell, C.; Komarova, Y.; Grzybowski, B.; Borisy, G. Nat. Methods 2005, 2, 739–741.

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3 to 4 weeks. Most importantly, gold slides are still expensive and inconvenient for most biological experiments. It is therefore necessary to explore new methods for patterning multiple types of cells on substrates that avoid fluorescence quenching and at the same time are stable and inexpensive.12-16 Herein, we report a strategy to pattern multiple types of cells directly on glass surfaces to simulate all three types of cell-cell interactions using a microfluidic system to assemble defect-free surfaces for resisting cell adhesion. Glass is one of the most commonly used substrates for culturing cells in biological laboratories because it is easy to sterilize and is compatible with most types of optical microscopy (because of its complete transparency in the range of wavelengths often employed). Glass surfaces modified with different terminal groups can promote or resist cell adhesion.17-25 In general, chlorosilanes or oxysilanes are employed to form polysiloxane, which is connected to surfaces by silanol groups (-SiOH) via Si-O-Si bonds with glass surfaces. In this work, we used (12) Myers, F. B; Lee, L. P. Lab Chip 2008, 8, 2015. (13) Zhang, Q.; Xu, J. J.; Chen, H. Y. Electrophoresis 2006, 27, 4943–4951. (14) Jamal, M.; Bassik, N.; Cho, J. H.; Randall, C. L.; Gracias, D. H. Biomaterials 2010, 31, 1683–1690. (15) Inaba, R.; Khademhosseini, A.; Suzuki, H.; Fukuda, J. Biomaterials 2009, 30, 3573–3579. (16) Kaji, H.; Sekine, S.; Hashimoto, M.; Kawashima, T.; Nishizawa, M. Biotechnol. Bioeng. 2007, 98, 919–925. (17) Faucheux, N.; Schweiss, R.; L€utzow, K.; Werner, C.; Groth, T. Biomaterials 2004, 25, 2721–2730. (18) Blummel, J.; Perschmann, N.; Aydin, D.; Drinjakovic, J.; Surrey, T.; Lopez-Garcia, M.; Kessler, H.; Spatz, J. Biomaterials 2007, 28, 4739–4747. (19) Yanker, D. M.; Maurer, J. A. Mol. BioSyst. 2008, 4, 502–504. (20) Revzin, A.; Lee, J. Y.; Shah., S. S.; Zimmer, C. C.; Liu, G. Langmuir 2008, 24, 2232–2239. (21) Kikuchi, Y.; Nakanishi, J.; Shimizu, T.; Nakayama, H.; Inoue, S.; Yamaguchi, K.; Iwai, H.; Yoshida, Y.; Horiike, Y.; Takarada, t.; Maeda, M. Langmuir 2008, 24, 13084–13095. (22) Kaji, H.; Yokoi, T.; Kawashima, T.; Nishizawa, M. Lab Chip 2009, 9, 427– 432. (23) Branch, D. W.; Wheeler, B. C.; Brewer, G. J.; Leckband, D. E. IEEE Trans. Biomed. Eng. 2000, 47, 290–300. (24) Wheeler, B. C.; Corey, J. M.; Brewer, G. J.; Branch, D. W. J. Biomech. Eng. 1999, 121, 73–78. (25) Nishizawa, M.; Takahashi, A.; Kaji, H.; Matsue, T. Chem. Lett. 2002, 30, 12–13.

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poly(ethylene glycol)-terminated oxysilanes (PEG-oxysilane) to modify parts of the glass surfaces to form areas covered with PEG-teminated layers in order to resist cell adhesion; we used fibronectin to modify parts of the glass surfaces to promote cell adhesion. Glass surfaces, however, are more difficult to modify homogeneously over large areas compared to gold surfaces because glass surfaces are amorphous and high-quality selfassembled monolayers are difficult to achieve.26,27 For example, PEG-terminated SAMs on the surfaces of microscopy cover slides still allowed a few cells to adhere.18 We employed a microfluidic system to create separated areas, and we modified glass surfaces with different properties within channels to achieve an in vitro model for cell-cell interaction on glass surfaces. Modifying glass surfaces within microchannels can be more effective than on bulk glass surfaces28-31 when forming modified surfaces without defects to resist cell adhesion. To the best of our knowledge, this report is the first to pattern multiple types of cells on glass surfaces directly to investigate complex intercellular interactions. This technique is simple, repeatable, biocompatible, cheap, and convenient enough to be a potential routine method for studying cell-cell interactions in biological laboratories.

Experimental Section Synthesis of PEG-oxysilane. PEG-terminated triethoxysilanes were prepared by adding excess PEG (average molecular weight of 750) to a dry solution of 3-isocyanatopropyltriethoxysilane in toluene and refluxing overnight. The solvent was evaporated, and the raw products were purified by precipitation from petroleum ether to yield pure PEG-oxysilane as a colorless liquid. 1 H NMR (400 MHz, CDCl3): δ 0.59 (t, 2H, Si-CH2), 1.20 (t, 9H, O-CH2-CH3), 3.15 (dd, 2H, O-CH2-CH2), 3.37 (s, 3H, CH3-O-CH2), 3.72-3.54 (m, 60H, O-CH2-CH2-O), 3.83 (m, 6H, Si-O-CH2-CH3), 4.20 (t, 2H, CH2-OOC-NH). Molecular weights determined by MALDI: Mn (number average)=904, Mw(weight average)=932, Mw/Mn =1.01 Treatment of Glass Surfaces of Cover Slides. Cover slides were cleaned with piranha solution (strongly acidic and strongly oxidizing, handled with extreme caution) freshly prepared from concentrated H2SO4 and 30% H2O2 for 20 min at room temperature, washed with copious amount of purified water, and dried with N2. Fabrication of Microfluidic Stamps. Fabrication of the master for microchannels was accomplished using established methods.32 Patterns used in these experiments were designed by L-edit software and exported as a PS file. We printed the PS file on a transparency mask on a high-resolution printer (up to 3600 dpi). A photoresist film with a height of ∼60 μm was cast by spincoating SU 8-2100 (MicroChem) onto a silicon wafer at 2000 rpm for 30 s. The photoresist film on the wafer was baked at 65 °C for 3 min and at 95 °C for 9 min and exposed to a UV light source with the transparency mask directly on it. The wafer was postbaked at 65 °C for 1 min and at 95 °C for 7 min. The exposed photoresist film was developed using MicroChem’s SU-8 developer for 7 min. Microfeatures for these patterns were visible to the naked eye at this stage. The wafer was silanized with 1H,1H,2H,2H-perfluorodecyltriethoxysilane (Alfa Aesar, U.K.) overnight. To generate microfluidic channels, we replica molded polydimethylsiloxane (26) Ulman, A. Chem. Rev. 1996, 96, 1533–1554. (27) Yang, Z.; Galloway, J.; Yu, H. Langmuir 1999, 15, 8405–8411. (28) Kobayashi, J.; Mori, Y.; Okamoto, K.; Akiyama, R.; Ueno, M.; Kitamori, T.; Kobayashi, S. Science 2004, 304, 1305–1308. (29) Yoon, S. K.; Choban, E. R.; Kane, C.; Tzedakis, T.; Kenis, P. J. A. J. Am. Chem. Soc. 2005, 127, 10466–10467. (30) Janasek, D.; Franzke, J.; Manz, A. Nature 2006, 442, 374–380. (31) Campbell, C. J.; Fialkowski, M.; Bishop, K. J. M.; Grzybowski, B. A. Langmuir 2009, 25, 9–12. (32) Sun, K.; Wang, Z.; Jiang, X. Lab Chip 2008, 8, 1536–1543.

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Scheme 1. One-Step Functionalization of Microchannel Glass Surfaces by the Reactive Coupling of PEG-oxysilane to the Hydroxyl Moiety of Glass Surfaces (n = 12-18)

(PDMS, Dow Corning) from the masters. The microchannels used here had the following dimensions: width, 200 or 500 μm; depth, 100 μm; and length, 1.5 cm. Holes at the inlets and outlets of the microfluidic channels (∼800 μm in diameter) were generated by a sharpened needle tip. Cell Culture. NIH 3T3, 3T3-L1, and 3T6 cells (mouse embryonic fibroblasts) were cultured in a DMEM medium containing 10% fetal bovine serum supplemented with penicillin/ streptomycin. NIH 3T3 cells were stained with a green fluorescent dye (C3099, Invitrogen), 3T6 cells were stained with a red fluorescent dye (C34551, Invitrogen), and 3T3-L1 cells were not stained with any fluorescent dye before being introduced into the microchannels. HUVEC (human umbilical vein endothelial cell line), PLA801 C, and PLA801 D cells (a pair of homological sublines from lung-giant-cell line PLA801) were cultured in an RPM 1640 medium containing 10% fetal bovine serum supplemented with penicillin/streptomycin. Cell Patterning. We placed a 5 μL drop of a suspension of cells into the inlets of each channel. The density of the suspension of cells was about 3 million/mL for NIH 3T3, 3T3-L1, and 3T6 cells and about 5 million/mL for HUVEC, PLA801 C, and PLA801 D. After 60-120 min, cells were allowed to attach and spread onto the substrate. We peeled off the PDMS from the glass substrate and left the substrate in the culture medium. Cells were allowed to grow for 48 h at 37 °C in a 5% CO2 atmosphere to form confluent layers in confined patterns.

Results and Discussion Our general strategy for patterning multiple types of cells on glass cover slides is described as follows (Scheme 1): PEGoxysilane was used to modify glass surfaces to form inert surfaces that resist cell adhesion (“inert” areas), and extracellular matrix protein fibronectin (FN) was used to form surfaces that promote cell adhesion (“permissive” areas). Microchannels in our experiments have two functions: (i) to create areas with different surface properties for cell adhesion (either permissive or inert) and (ii) to transport different types of cells to different areas on the surface. The material for constructing microchannels is a PDMS stamp with embedded microfluidic channels and a flat piece of a glass cover slide. In vivo, for three types of nearby cells, considering relative migration, there are three types of interactions among them in 2D space: no relative migration (three types of cells fixed in places without relative migration, which is called complete confinement); partial relative migration (one or two types of cells migrate and the other two or one types of cells are fixed, which is called partial confinement); and relative migration (all three types DOI: 10.1021/la103132m

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Figure 2. SEM images of modified glass surfaces with PEGoxysilane concentrations of 0.2, 0.5, and 1.0% (v/v).

Figure 1. Strategy for patterning multiple types of cells on the same glass surface. A PDMS stamp with an embedded microfluidic system formed enclosed microchannels with a glass substrate precleaned with freshly prepared piranha solution. Different microchannels introduced aqueous solutions of 0.2% PEG-oxysilane (v/v, yellow to indicate the inert surfaces) and PBS. Solutions of FN introduced into microchannels replaced PBS. Different types of cells adhered in different channels, all of which were modified with FN. After the removal of the PDMS stamp, patterns of different types of cells formed. (A) In complete confinement, all three types of cells were confined to separate areas after the PDMS stamp was removed. (B) In partial confinement, one type of cell was well confined while the other two types of cells moved freely after the PDMS stamp was removed. (C) In no confinement, all three types of cells moved freely after the PDMS stamp was removed.

of cells migrate in relation to each other, which is called no confinement). To achieve all three types of cell-cell interactions, we used one overall approach with a slightly different experimental protocol for each specific control (Figure 1). We employed PEG-trioxysilane to modify glass surfaces because it was soluble in water and reacted with the glass surfaces in an aqueous phase with hydrochloride acting as a catalyst (Scheme 1). We used different concentrations of hydrochloride (120, 12, 1.2 μM) to optimize the conditions for the modification of areas that resist cell adhesion and at the same time allow cells in neighboring channels to survive (Figure 1A,B). Modified glass surfaces at all three concentrations of hydrochloride effectively resist cell adhesion, but cells survived only with 1.2 μM hydrochloride after ample rinsing. When the concentration of hydrochloride was 120 μM, we found that most cells were dying, even after every microchannel was rinsed with PBS many times. We speculated that residue HCl might spread into PDMS stamps and lead to cell death.33 We completed the modification in microchannels (via the silane-based chemistry) and the sterilization of the substrate by placing them in a oven for 40 min at 80 °C. We determined the ability of modified glass surfaces to resist the adhesion of 3T6 cells (mouse embryonic fibroblasts). Modified glass surfaces could resist cell adhesion for at least 2 days at all three concentrations of 0.2, 0.5, and 1.0% (v/v). Moreover, the modified glass surfaces with PEG-oxysilane at 0.2% (v/v) could resist cell adhesion for more than 3 days. To investigate the (33) Regehr, K. J.; Domenech, M.; Koepsel, J. T.; Carver, K. C.; Ellison-Zelshi, S. J.; Murphy, W. L.; Schuler, L. A.; Alarid, E. T.; Beebe, D. J. Lab Chip 2009, 9, 2132–2139.

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Figure 3. Phase-contrast micrograph of modified glass surfaces resisting cell adhesion. After the modification of glass surfaces within a microchannel of a glass cover slide and the peeling off of the PDMS stamp, 3T6 cells adhered and covered the whole glass surfaces of the slide except in areas modified with PEG-oxysilane (the middle strip without cell adhesion, marked with a white arrow).

difference in duration under varied concentrations of PEGoxysilane, we analyzed the morphology of modified glass surfaces with scanning electron microscopy (SEM). The images (Figure 2) show that the surface morphology was uniform at 0.2% (v/v). Many white points appeared on the surfaces, which destroyed their uniform structure at the higher concentration of 0.5% (v/v). The density and size of white points increased at the highest concentration of 1.0% (v/v). We further analyzed elements of the white points with energy dispersive X-ray spectrometry (EDXS) and found Si and C contents similar to those of other areas. We therefore speculated that trioxysilane polymerized and formed polymers as white points with an increase in PEG-oxysilane concentration and that these white points broke the uniform structure of modified surfaces and might provide adhesive sites for cells. However, the uniform structure of 0.2% PEG was well maintained with PEG-terminal groups to prevent cell adhesion. We used PEG-oxysilane to modify glass surfaces directly within microchannels at 0.2% (v/v) and successfully prevented cell adhesion completely (Figure 3). This method of modifying glass surfaces to prevent cell adhesion was also simple, stable, and repeatable. Langmuir 2010, 26(23), 17790–17794

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Figure 4. Time-lapse micrographs (phase-contrast and fluorescence) for the three types of cell-cell interactions between NIH3T3, 3T3-L1, and 3T6 cells. The numbers at the upper left of the images indicate the time (in hours) after the PDMS stamp was peeled off. The areas between two neighboring dashed lines are the channels that deliver PEG-oxysilane to the surfaces. The areas between two neighboring solid lines are the channels that introduce cells. The areas between neighboring solid lines and dashed lines are parts of the surfaces between the channels that introduce FN and the channels that introduce PEG-oxysilane (originally directly in contact with PDMS). (A) Complete confinement: all cells are well confined. (B) Partial confinement: 3T3-L1 cells are confined in the stripe while NIH 3T3 and 3T6 cells move freely. (C) No confinement: all three types of cells move freely on the glass surfaces.

To achieve the model for complete confinement (Figure 1A), we first introduced solutions of phosphate-buffered saline (PBS, pH 7.4) into the microchannels before introducing the solution of PEG-oxysilane into flanking microchannels. The sequential introduction of PBS and PEG-oxysilane avoids the spread of HCl into the microchannels that introduce PBS. After incubation at 80 °C for 40 min and allowing PEG-oxysilane to render the glass surfaces inert, we rinsed the microchannels with PBS and Langmuir 2010, 26(23), 17790–17794

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introduced FN into microchannels previous filled with PBS. After incubation at room temperature for about 1 h, we rinsed the microchannels modified with FN with a cell culture medium and introduced three types of cells (mouse embryonic fibroblasts NIH 3T3, 3T3-L1, and 3T6) into FN-modified microchannels. After the attachment of cells for 1 to 2 h, we peeled off the PDMS stamp and immersed the substrate with attached cells in a normal culture medium (in a Petri dish). After releasing the PDMS stamp from the glass slide, cells invaded areas on parts of the surfaces between the channels that introduced FN and the channels that introduced PEG-oxysilane (originally directly in contact with PDMS, as shown by areas between neighboring solid and dashed lines in Figure 4A). This result implied that the modification of glass surfaces occurred only in microchannel areas. This protocol realized complete confinement for the patterned cells (Figure 4A). We illustrated partial confinement (Figure 1B) by confining one type of cell (3T3-L1) to certain areas on the surfaces and allowing the other two types of cells (NIH 3T3 and 3T6) to move freely. We employed essentially the same protocols as those used in the complete confinement model, except that PEG-oxysilane was not introduced into the flanking microchannels that introduced NIH 3T3 and 3T6 cells (to allow these cells to move freely once the PDMS stamp was removed). We introduced NIH 3T3, 3T3-L1, and 3T6 cells into their respective microchannels. After cell attachment, we peeled the PDMS stamp off of the glass slide to immerse the substrate in the cell culture medium. Doing so confined cells flanked by PEG-oxysilane to their original areas and allowed other cells to move freely. NIH 3T3 and 3T6 cells freely migrated while 3T3-L1 cells were confined after being cocultured for at least 48 h (Figure 4B). A partial confinement model was hence achieved. To achieve the model for no confinement (Figure 1C), we introduced only FN into microchannels to form a permissive surface for promoting cell adhesion. After incubation at room temperature for about 1 h, we introduced NIH 3T3, 3T3-L1, and 3T6 cells into their respective microchannels. After the attachment of cells, we peeled off the PDMS stamp and immersed the substrate in the cell culture medium. All three types of cells migrated freely on the glass surfaces (Figure 4C). This method, therefore, achieved the no confinement model. We applied the model to the study of intercellular interactions between blood vascular cells and tumor cells. Angiogenesis, or the development of new blood vessels, is required for the development of many types of tumors. Most tumors cannot grow beyond 1 to 2 mm and may remain dormant without the onset of angiogenesis. Tumor cells recruit endothelial cells for angiogenesis in order to construct blood vessels that deliver nutrients to meet requirements for growth.34-36 PLA801 C and PLA801 D cells are a pair of sublines that are established from the same parental human lung giant cell carcinoma cell line PLA801 (originating from the surgical lung tumor tissue of a male patient suffering from lung giant cell carcinoma).37,38 Experimental results of spontaneous tumorigenicity and metastasis potential in vivo show that PLA801 D cells spontaneously metastasize much more easily than PLA801 C. PLA801 D cells also significantly up-regulate VEGF mRNA (vascular endothelial growth factor mRNA) compared with lowly metastatic PLA801 C cells.39 It has not (34) Heath, V. L.; Bicknell, R. Nat. Rev. Clin. Oncol. 2009, 6, 395–404. (35) Sun, S.; Schiller, J. H. Crit. Rev. Oncol. Hemat. 2007, 62, 93–104. (36) Gimbrone, M. A.; Leapman, S. B., Jr.; Cotran, R. S.; Folkman, J. J. Exp. Med. 1972, 136, 261–276. (37) Zhang, J.; Liu, G.; Meng, Y.; Lin, H.; Lu, Y. Oncol. Rep. 2009, 21, 697–706. (38) Ling, X.; Lu, Y.; Du, Z. J. Tumor Marker Oncol. 2003, 18, 312–320. (39) Jiang, D.; Ying, W.; Lu, Y.; Wan, J.; Zhai, Y.; Liu, W.; Zhu, Y.; Qiu, Z.; Qian, X.; He, F. Proteomics 2003, 3, 724–737.

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to promote vascular cells growth and migration toward cancer cells. And PLA801 D cells significantly up-regulated VEGF mRNA compared to PLA801 C cells. It was speculated that PLA801 D cells secreted significantly more amount of VEGF than PLA801 C cells. PLA801 D cells, therefore, significantly attracted HUVEC cells compared with PLA801 C cells. This type of side-by-side comparison among multiple types of cells would have been impossible or difficult without this new model.

Conclusions

Figure 5. Results of intercellular interactions among HUVEC, PLA801 C, and PLA801 D cells. (A) The numbers at the upper left of the images indicate the time (in hours) after the PDMS stamp peel off. Areas between two neighboring white solid lines were parts of channels introducing HUVEC cells. HUVEC cells (middle strip) migrated under the influence of PLA801 C (left strip) and PLA801 D (right strip). (B) HUVEC cellular densities near PLA801 C cells indicated as black columns and the densities near PLA801 D cells indicated as white columns. The densities of HUVEC cells near PLA801 D cells were obviously higher than those near PLA801 C cells.

been demonstrated, however, whether PLA801 D cells had a greater ability to recruit endothelial cells than PLA801C. We applied our new model to answer this question. We plated and confined PLA801 D cells on the left strip and PLA801 C cells on the right strip. HUVEC cells (endothelial cells) were plated on the middle strip without confinement (Figure 5A). The distance between two neighboring types of cells was designed to be 700 μm. After the PDMS stamp was peeled off, HUVEC cells would spread to the right and left sides under the influence of PLA801 C and PLA801 D cells. Given that the possibility was equal for HUVEC cells to spread to the right or to the left without the influence of other type of cells, there should be no difference between the right and left sides of the cellular distribution of HUVEC. The difference observed between two sides has to be a result of interactions among three types of cells. After coculturing the three types of cells for 40 h, we analyzed the density of HUVEC cells spreading toward PLA801C and PLA801 D cells. PLA801 C and PLA801 D cells grew, spread, and filled the confined strips at the first 20 h. HUVEC cells also grew and spread in the middle under the influence of two neighboring types of cells (Figure 5A). The density of HUVEC on the right side (attracted by PLA801 D cells) became greater than that on the left side (attracted by PLA801 C cells) with increasing coculture time. The difference has statistical significance for coculturing for 40 h (Figure 5B). The HUVEC cellular density increased on the areas between HUVEC and PLA801 C/D cells with increasing coculture time. The HUVEC cellular density close to PLA801 D cells was obviously higher than that close to PLA801 C cells. PLA801 D cells, therefore, had a significantly greater ability to attract HUVEC cells than PLA801 C cells. Cancer cells secreted VEGF

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We have established a versatile technique for patterning multiple types of cells directly on glass surfaces to simulate three types of cell-cell interactions using surface modification with oxysilane and microfluidic systems. This approach is fully compatible with optical microscopy. The method uses glass substrates, making it much easier to observe and analyze intercellular interactions accurately with various fluorescence techniques. The manipulation required in this system is quite straightforward, simple, and stable and enables us to obtain time lapse data for intercellular interactions. The method is potentially adaptable to many types of experiments in cell biology, such as investigations of the pathogenesis of complex diseases and complex processes in developmental biology, as well as to screening drugs for diseases. The combination of surface chemistry within microfluidic channels on glass surfaces may also be useful for work ranging from immunoassays to basic investigations on surfaces.40-42 Acknowledgment. We thank Prof. Mian Long (Institute of Mechanics, CAS) and Prof. Xiyun Yan and Prof. Wei Liang (Institute of Biophysics, CAS) for generously providing cells. We thank the Institute of Basic Medical Science, Academy of Military Medical Science for kindly providing human lung giant cell line PLA801 C and D cells. The XPS measurements were performed with synchrotron radiation at the Beijing Synchrotron Radiation Facility at the Institute of High Energy Physics, Chinese Academy of Sciences. The Human Frontier Science Program, the Chinese Academy of Sciences (KJCX2-YW-M15), the National Science Foundation of China (90813032 and 20890023), and the Ministry of Science and Technology (2008CB617505, 2009CB930001, 2007CB714502 and 2011CB933201.) provided financial support. This work was partially supported by a fellowship from Corning, Inc., the China Postdoctoral Science Foundation, and the K. C. Wong Education Foundation (Hong Kong). Supporting Information Available: Additional information on the PEG-oxysilane mass spectrum and X-ray photoelectron spectroscopic (XPS) spectra of modifications to glass surfaces. This material is available free of charge via the Internet at http://pubs.acs.org. (40) Liu, Y.; Yang, D.; Yu, T.; Jiang, X. Electrophoresis 2009, 30, 3269–3275. (41) Yang, D.; Niu, X.; Liu, Y.; Wang, Y.; Gu, X.; Song, L.; Zhao, R.; Ma, L.; Shao, Y.; Jiang, X. Adv. Mater. 2008, 20, 4770–4775. (42) Sudibya, H.; Ma, J.; Dong, X.; Ng, S.; Li, L.; Liu, X.; Chen, P. Angew. Chem., Int. Ed. 2009, 48, 2723–2726.

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