Retinal Pigment Epithelial Cell Behavior is Modulated by Alterations in Focal Cell–Substrate Contacts

Abstract

purpose. To investigate how the cellular behavior of cultured retinal pigment epithelial (RPE) cells was affected by the manipulation of early focal contact.

methods. To manipulate early focal contact, a reduced focal cell–substrate contact area on the micropatterned surfaces was implemented by microfabrication with polydimethylsiloxane (PDMS). The micropatterned PDMS surfaces had a circular pillar with a diameter of 5 μm. The human retinal pigment epithelial cell line, ARPE-19, was seeded onto the fibronectin-coated PDMS surfaces. Cell adhesion, growth, cell cycle, morphology, and interleukin-6 (IL-6) expression were observed for 3 weeks.

results. The fluorescent images of localized vinculin and actin stress fibers appeared to be more prominent on smooth PDMS surfaces. Although there was no significant effect on cell adhesion, a statistically significant inhibition of cell cycle progression was observed for micropatterned PDMS surfaces. Similarly, micropatterned surfaces showed significantly less cell growth than that of smooth surfaces. Cultures over a period of 3 weeks showed a distinct cell–cell phenotype discrepancy. Furthermore, IL-6 mRNA and secreted protein induced by IL-1β in ARPE-19 were downregulated on micropatterned PDMS surfaces.

conclusions. Disturbed focal contact in ARPE-19 cells grown on micropatterned surfaces altered cell cycle, growth, morphology, and the expression of IL-6 in vitro.

Focal contact precedent by cellular adhesion to the extracellular matrix (ECM) is a fundamental phenomenon. ^{1 2 3 4} Virtually every cell in vertebrates adheres to ECM, and this cell–substrate contact, so-called focal contact, critically regulates cell survival, growth, and gene expression and function. ^{5 6 7} Focal contact is critical for the regulation of actin organization, as well as the mechanical signal transfer in intracellular signaling pathways. ^{8 9 10 11} The particular molecules that are necessary for the formation of focal contact are still under investigation, and the order with which the factors bind the integrins, actin, or each other is only partly understood. ¹² To date, almost all the reports of focal contact have noted the importance of focal contact on cellular behavior by manipulation of the molecules involved in its formation. ^{13 14 15 16 17}  

New microfabrication technology has produced the ability to exert precise control of two- and three-dimensional microstructures over a wide range of dimensions and shapes. ¹⁸ With the incorporation and adaptation of microfabrication technology into biology, it is now possible to design surfaces that reproduce some in vivo conditions. ^{19 20 21 22} Cells that adhere across several lines develop more focal adhesions and actin fibers in the areas of contact with the lines than in the areas between the lines. ^{23 24} The grooved surfaces are more potent in aligning and inducing elongated cells. ²⁵ Surface micropatterns can be used as a tool for reducing the inflammatory response at the implant tissue interface and in the design of biomaterials for tissue engineering. ^{26 27}  

Retinal pigment epithelial cells (RPEs) are closely associated with and play key roles in the development and maintenance of the retina. They form structurally tight intercellular junctions and basolateral adherence with Bruch’s membrane, which are crucial for normal regeneration, and secretion of growth factors that are essential for the proper differentiation of photoreceptors during development. ^{28 29 30 31 32} Previous studies have reported the pathologic role of RPE in the development of retinal degenerative diseases. ^{33 34 35} Considering the basolateral adherence of RPE with Bruch’s membrane, aspects of the RPE involved in the cellular response to the substrate may be significant for the development and pathogenesis of the retina. 

This study reports for the first time the influence of disturbed early focal contact made by micropatterned surfaces on the cellular behavior of cultured RPE cells in vitro. Microfabrication technology was implemented and used to generate micropatterned surfaces suitable for cell culture at the micrometer scale. 

Materials and Methods

Micropatterned Surface Preparation

Structure.

The initial step involved transfer of a two-dimensional pattern onto cylindrically symmetrical protruding pillars to disturb the early focal contact. To illustrate this concept, a planar pattern was used first to produce a structure with a cylindrical symmetry. For example, the 5 μm PDMS substrates were patterned with small, closely spaced circular islands so that early focal contact could be manipulated. The pillar with interspacing gaps of 5 μm for a 5 μm diameter was used to examine the topographical effect on the cell behavior. 

Fabrication Process.

The surface textured silicone specimens were produced by simple soft-lithography processes with PDMS (Polydimethylsiloxane, a commercially available silicone-based polymer Sylgard 182/184; Dow Corning, Midland, MI). PDMS is well known and widely used for prototyping of microfluidic systems. 

The micropatterned PDMS specimens were made by an impression of a PDMS cast on the wafers in collaboration with Digital BioTechnology Corp. (Seoul, Korea). Briefly, the wafer was cleaned and SU-8 (Negative near-UV photoresist; Microchem Corp, Newton, MA) was spin coated. The height of micropatterns was controlled by the time and speed of spin coating. The resist layer was soft baked to evaporate the solvent, and exposed to UV light by UV mask aligner (MA-6; Suss Microtec Lithography GmbH, Munich, Germany). After exposure, post expose bake and develop were performed to generate cross-links in the exposed portion of SU-8 to be the master patterns on the wafer. 

The PDMS mixture was prepared and spread over the master wafer, then cured in an oven at 150°C. To prepare the PDMS mixture, PDMS and the curing agent should be mixed and carefully evacuated to remove bubbles generated by mixing. See Figure 2 for an example of the resulting structure. The PDMS surfaces were treated by oxygen plasma to obtain a hydrophilic surface, and prepared individually in a 60-mm dish by autoclaving before being attached to the bottoms of the individual dishes. 

Cell Culture and Preparation of Co-Culture

The human retinal pigment epithelial cell line (CRL 2303; ARPE-19) was obtained from the American Type Culture Collection (ATCC) at passage 10. The cells were maintained in a 1:1 mixture of Dulbecco’s modified Eagles medium and Ham’s F12 medium (DMEM/F12; Gibco, Gaithersburg, MD) with HEPES buffer containing 10% fetal bovine serum (Gibco), a 56 mM final concentration of sodium bicarbonate (Sigma, St. Louis, MO) and 2 mM l-glutamine (Gibco), and incubated at 37°C in 5% CO₂. Cultures up to passage 20 were used for the experiments. No changes in growth characteristics or morphology were observed during this time. The cells in the routine culture had the typical appearance of epithelia that exhibit morphologic and functional polarity. 

The cells were harvested from a routine culture and seeded on PDMS substrates in the full-growth medium, which included the serum proteins. The seeding densities were 6 × 10⁵ cells/mL at 6.0 mL/well for the 60-mm dishes, and the incubation times ranged from 0.4 to 3 days. The medium was refreshed every 2 to 3 days. The samples were either uncoated or preadsorbed with human fibronectin for a minimum of 30 minutes at 37°C, which was then rinsed five times with the tissue culture medium. The bulk fibronectin concentration was 5 μg/mL at 3.2 mL/well, corresponding to 1.3 μg of fibronectin per square centimeter. Fluorescence microscopy was performed with an Olympus IX 51 microscope (Olympus Corp., Tokyo, Japan). 

Scanning Electron Microscopy

The three-dimensional pillar PDMS surfaces were examined before cell culture by scanning electron microscopy (SEM, XL30FEG; Philips Electronics, The Netherlands), at an accelerating voltage of 7 kV. 

Cell Adhesion Assay

The cell adhesion assay was performed as described previously. ³⁶ Briefly, the smooth and micropatterned PDMS were coated with fibronectin (5 μg/mL) in PBS at 4°C overnight. Then the PDMS surfaces were rinsed three times in PBS. Nonspecific binding was blocked with 2% BSA (Gibco) for 1 hour at 4°C. For preparation of cells, ARPE-19 cells were seeded onto the micropatterned surfaces at 2 × 10⁴ cells/100 μL. After incubation for 30 minutes at 37°C, unattached cells were removed by rinsing twice with PBS. Attached cells were incubated for 1 hour at 37°C in 50 mM citrate buffer, pH 5.0, containing 3.75 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide (hexosaminidase substrate) and 0.25% Triton X-100. The reaction was stopped by the addition of 50 mM glycine buffer, pH 10.4, containing 5 mM EDTA. The absorbance was measured at a wavelength of 405 nm (SPECTRA Max 250 Microplate Spectrophotometer; Molecular Devices Corps., Sunnyvale, CA). 

Cell Growth Assay

ARPE-19 cells were incubated at 2 × 10⁴ cells/100 μL onto each PDMS sample. After indicated days of culturing, the cells were washed three times in serum-free media, fixed with 4% paraformaldehyde (Sigma), and stained with Hoechst 33342 (Sigma). To establish the cell growth curve, cells were counted at 1, 3, 5, and 7 days. ARPE-19 cells were examined by fluorescence microscopy using an Olympus IX 51. The growth rate was counted using Image-Pro plus software (Media Cybernetics, Silver Spring, MD). The smooth PDMS surfaces were used as controls. The experiments were repeated in triplicate. Data are reported as the mean number at specific time points ± SD. 

Cell Cycle Analysis

ARPE-19 cells were plated at a density of 1 × 10⁵ cells/cm² in 1% FBS containing medium on tissue culture plates (TCP). For cell cycle synchronization, cells were incubated in serum-free medium for 16 hours and were then incubated for 2 hours with the specific inhibitor of DNA polymerase aphidicolin (20 μM; Sigma). The cells were seeded on the smooth and micropatterned PDMS in a 60-mm dish and incubated for 1 day as described above. Briefly, the cells were then washed in PBS by centrifugation and resuspended. A 1/10 volume of a BrdU labeling solution (Roche, Mannheim, Germany) was added to the culture medium in which the cells were growing. The cells were incubated for 30 to 60 minutes at 37°C in a humidified atmosphere (5% CO₂). The optimum incubation time in the presence of BrdU (labeling period) was selected for each individual experiments. The labeling culture medium was removed by either aspiration or tapping. The cells were washed three times in PBS. 

To fix the cells and perform immunostaining, the following procedure was adopted (In Situ Cell Proliferation Kit; Roche): The cells were washed in 0.5 mL PBS and incubated with 5 mL of the fixative. The cells were incubated for 30 minutes at 4°C and were resuspended in a 500 μL HCl-denaturation solution (Roche) and incubated for 10–20 minutes at room temperature. After denaturation, 2 mL PBS was added and washed for 10 minutes. To block unspecific binding, the cells were incubated with a 500 μL incubation buffer for 10 minutes at room temperature. The cells were incubated in 50 mL of an anti-BrdU-FLUOS antibody working solution (Roche). The cells were incubated for 45 minutes at 37°C in a humid chamber, washed twice in PBS, and then resuspended in 0.5–1 mL PBS. The resulting samples were subsequently analyzed by fluorescent microscopy (488 nm for excitation and a 515 nm bandpass filter for detection). 

Total RNA Preparation and Reverse Transcription–Polymerase Chain Reaction (RT-PCR)

The total cellular RNA was isolated by using a TRIzol reagent (Gibco). One microgram of the RNA was reversed-transcribed using a cDNA synthesis kit (Boehringer Mannheim, Indianapolis, IN). The cDNA was then amplified in a 20 μL reaction mixture by PCR using the following conditions: 0.4 μM each primer, 0.2 mM deoxynucleoside triphosphate mixture (Perkin Elmer Corp., Foster City, CA), 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, and 1 unit Taq polymerase (Perkin Elmer Corp.). The reaction mixtures were incubated in a thermal controller (Model PTC-100; MJ Research, Watertown, MA) for 35 cycles of denaturation for 45 seconds at 95°C, annealing for 1 minute at 58°C, and extension for 45 seconds at 72°C. The quantity of the amplified products was analyzed using an image documentation system (ImageMaster VDS; Pharmacia Biotech Inc., Uppsala, Sweden). The primer sequences specific to the genes examined and the predicted sizes for the human IL-6 cDNA were 5′-ACAGCCACTCACCTCTTCAG-3′ and 5′-GATGATTTTCACCAGGCAAG-3′, yielding a 214 bp amplification product. The primers used for the human β-actin cDNA were 5′-GTACAGTTGTTGGCGAGCA-3′ and 5′-TGCATCAGAAGTAAGCCTCTC-3′, yielding a 320 bp amplification product. All primer sequences were designed by using primer selection software offered through a web site (Primer 3; Center for Genome Research, the Whitehead Institute for Biomedical Research, Cambridge, MA; www.genome.wi.mit.edu). 

Enzyme-Linked Immunosorbent Assay (ELISA)

The method reported by Hirano et al. ^{37 38} was used for the ELISA measurements (Human IL-6 Immunoassay; R&D Systems, Minneapolis, MN). Briefly, the ARPE-19 cells were seeded at a 6 × 10⁵ cells/mL density on micropatterned and smooth PDMS, as well as TCP in a 60-mm dish (6.0-mL suspension) and incubated for 1, 2, or 3 days. After incubation, the harvest procedure from the samples was performed as follows: The supernatant medium was transferred to the centrifuge tubes. Fifty μL of the medium from each sample was pipetted into the wells of the 96-well plastic plates and incubated at room temperature overnight. All samples were assayed in triplicate. After incubation, the plates were washed with PBS-Tween (0.5%, v/v, Tween 20) and incubated with the same IL-6 conjugate (R&D Systems) that were added for 2 hours at room temperature. The plates were washed and incubated with an alkaline phosphatase-conjugated substrate for 1 hour at room temperature. After washing with PBS-Tween, 100 μL of the diethanolamine buffer with p-nitrophenylphosphate was added and the absorbance of each well was read at 490 nm using Micro ELISA reader (SPECTRAMax 250 Microplate Spectrophotometer; Molecular Devices Corps., Sunnyvale, CA). 

Immuncytochemistry

The cells were first rinsed in a 1 M phosphate-buffered saline (PBS) solution at pH 7.4. A 20-minute fixation step in 4% paraformaldehyde (Sigma) in PBS was followed by further washing in PBS. The cells were then blocked with 2% bovine serum albumin (Gibco) in PBS for 45 minutes at 37°C. A 1:100 dilution of the primary antibodies (vinculin; mouse monoclonal anti-human vinculin; Sigma) was incubated overnight at 4°C. The samples were then rinsed with PBS and incubated for 1 hour and 20 minutes at 4°C in Texas Red-conjugated goat anti-mouse secondary antibodies (Molecular Probe, Eugene, OR) and rhodamine-phalloidin (Molecular Probe). The control samples were treated in the same way except that the primary antibodies were omitted. After several washes in PBS, the cells were mounted on glass slides, the coverslips were sealed at the edges, and the cells were examined with an Olympus IX51 fluorescence microscopy. 

Results

Structure of Patterned Microstructure PDMS Surfaces

A schematic diagram of the micropatterned surfaces is shown in Figure 1A . Briefly, considering the typical size of RPE (∼10 μm), the diameter of circular 5 μm pillar PDMS surfaces were separated by the 5 μm interdistance gaps. A schematic of the fabrication process is given in Figure 1B . To fabricate the PDMS specimen, the wafer was cleaned and evacuated; SU-8 was spin-coated onto the surface. After UV light exposure and development, the fabricated silicon master was cured for PDMS and the micropatterned PDMS surfaces were made. 

SEM of Patterned Microstructure PDMS Surfaces

A surface inspection by SEM showed that the pattern of micropatterned PDMS surfaces had no significant defects or irregularities (Fig. 2) . The micropatterned PDMS surfaces did not exhibit a significant geometrical variation with respect to the silicone master. However, occasional minor defects at the tip of the pillar might have been induced during the release of the PDMS from the silicon wafer. The circular pillars were similar to the specified dimensions (5 μm). 

Cell–Substrate Contact Area as Regulator of Early Focal Contact

By labeling cells for vinculin and F-actin, the intimate association of cell-to-substrate focal contact and actin stress fibers was demonstrated. As shown in Figure 3 , immunofluorescent labeling of the focal contact protein, vinculin, revealed a characteristic streaked pattern for the cells cultured on the smooth surfaces (Fig. 3G) . Arrowheads indicate vinculin at focal contact assembly. By contrast, the immunostained pattern of vinculin on the micropatterned surfaces showed less apparent distribution (Fig. 3H) . Intense F-actin staining was concentrated at the focal contact edge of the cells on the smooth surfaces (Fig. 3E) , whereas there was less notable staining on the micropatterned surfaces (Fig. 3F) . These results clearly showed that a disturbed early focal contact on the micropatterned surfaces gave way to a less distinct and diffuse distribution of vinculin and F-actin compared to the cells on the smooth surfaces. Corresponding phase contrast images were shown in Figures 3A and 3B . 

Early Focal Contact Disruption as Regulator of Cell Adhesion, Cell Cycle Progression, Cell Growth, and Cell Morphology

Cell adhesion was determined as a function of incubation period. Kinetic studies of cell adhesion at different times (15, 30, and 60 minutes) showed the similar rates of attachment (Fig. 4A) . Our data demonstrated that at the end of the 60-minute seeding period, most of the seeded cells had attached to either surface regardless of surface topography. For cell cycle analysis, ARPE-19 cells were synchronized by treatment with aphidicolin. ³⁹ The use of BrdU enabled the cell cycle to be readily assessed either on the smooth or micropatterned PDMS surfaces. RPE cell cycle progression was assessed after 1 day of culture. As shown in Figure 4B , significant cell cycle inhibition was observed on the micropatterned PDMS when compared to the smooth surface profile. To directly assess RPE growth rate, cells were stained with Hoechst 33342, as described in Materials and Methods. Similar to the cell cycle process, the growth rate on micropatterned surfaces was significantly inhibited (Fig. 4C) . 

Finally, to see the effect of micropatterned substrates on cell morphology, ARPE-19 cells were cultured for 3 weeks. To characterize cell morphology, actin stress fibers were visualized by rhodamine-phalloidin staining. Overall, the integrity of the actin on the micropatterned surfaces was disturbed throughout the culture period. After 1 day of culture, cells started to spread on the smooth surfaces (Fig. 4DA) , whereas cells on the micropatterned surfaces were mostly rounded (Fig. 4DC) . At 3 days, cells on the smooth surfaces were well spread (Fig. 4DE) , whereas cells on the micropatterned were mostly disturbed (Fig. 4DG) . By 7 days, cells were mostly confluent and morphologically transformed, showing a fibroblast-like phenotype (Fig. 4DI) , whereas a network of monolayered stress fibers maintained on the micropatterned surfaces (Fig. 4DK) . At 3 weeks, cells became fibroblastic with a fibrillar network of multilayered actin stress fibers on the smooth surface (Figs. 4DM ; 4DN) , whereas a fibrillar network of cytoskeleton was not observed on the micropatterned surfaces (Fig. 4DO) . In other words, cells had a multilayered fibroblastic appearance on smooth surfaces, whereas cells did not show multilayered fibroblast-like phenotypes on micropatterned surfaces. 

Effect of Early Focal Contact on Gene Expression of IL-6 mRNA and Secretion on ARPE-19 Cells by IL-β₁

The ARPE-19 cells were incubated in either the presence or absence of IL-1β (1 ng/mL), and the accumulated culture media were collected at 24 hours after treatment. The quantity of total RNA, on a per cell basis, was similar in the cultures on the smooth and micropatterned surfaces. 

Although the total RNA level and protein secreted did not change appreciably between the smooth and micropatterned surfaces on a per cell basis, the IL-6 mRNA level was statistically lower in RPE cells cultured on the micropatterned surfaces (Fig. 5A , left). The micropatterned surfaces reduced the IL-6 mRNA level by ∼ 56% at 24 hours, while the β-actin mRNA levels were essentially unaltered. (Fig. 5A , right). 

Secreted IL-6 was examined using a human IL-6 immunoassay from equal samples of collected media. The basal level of untreated cultured medium was subtracted from the IL-1β-treated medium. The quantity of IL-6 secretion from the micropatterned surfaces was significantly lower than that secreted from the smooth surfaces. A representative of several experiments is shown in Figure 5B . 

Discussion

The data presented in this study support the hypothesis that the amount of surface area with which the cell is in contact alters their cellular responses. Our reason for manipulating focal cell–substrate contact area was to create micropatterned surfaces which would prevent a normal cell–substrate focal contact assembly but would maintain cell–cell contact. Inhibited cell growth, cell cycle, and disturbed cell morphology of ARPE-19 were observed in the presence of disturbed early focal contact. In addition, the amount of IL-6 mRNA and secreted protein in ARPE-19 cells after IL-1β treatment were downregulated in the presence of disturbed focal contact. 

With respect to cell cycle progression, it was demonstrated that BrdU labeling is a sensitive and simple method for analyzing cell cycle progression on PDMS surfaces. It should be noted that cell cycle synchronization is a prerequisite for the proper assessment of the cell cycle. Cell cycle and growth results concur with the previous findings of cell–substrate contact area for proliferation in that human skin fibroblasts cultured on a surfaces with 10 μm wide grooves exhibited lower proliferation rates than those on 2 μm and 5 μm wide grooves. ^{40 41} It is not clear whether the effect on cell cycle and growth is via direct cell–substrate adhesion receptors such as integrins or through the associated integrin-dependent changes in cytoskeleton tension. 

Prior studies demonstrated that cell morphology is an important regulator of many processes that occur on a time scale of hours to days, including cell growth, differentiation, and fibrosis. ^{42 43 44} In this study, actin stress fibers were assessed to demonstrate the effect on cell morphology of micropatterned surfaces. Interestingly, cells showed a multilayered phenotype on smooth surfaces, whereas cells displayed a monolayer distribution on micropatterned surfaces in long-term cultures. This result indirectly suggested that cell morphology is closely related to the maintenance of focal cell–substrate contact. However, further studies are required to elucidate the mechanism for morphology dependence on focal cell–substrate contact. 

The effect of disturbed focal contact on IL-6 mRNA and secreted protein was measured because it was previously shown to be modulated by IL-1β. It is unlikely that these effects are just restricted to IL-6. Our unpublished results indicated that the extracellular matrix enzymes are also involved. The mechanism by which the RPE cells undergo changes in focal contact assembly into different biochemical responses remains unclear and requires further investigation. ^{45 46} However, these results provided strong evidence to support the previous findings that substrate surface topography alters cell shape and modulates gene expression at both the transcriptional and post-transcriptional levels, as well as determining the amount of secreted protein released into the extracellular matrix. ^{47 48}  

The experimental system used in this study may also find use in applied cell culture, including the development of support substrates for tissue engineering. In the future, detailed investigation of RPE on microfabricated surfaces will be needed so that control of desired cell responses can be obtained at the micro level. ^{49 50} In conclusion, a disturbance in early focal contact by manipulation of the cell–substrate contact area is a good model for the investigation of RPE cell behavior in vitro. 

 Supported in part by the Nano Bioelectronics and System Research Center (ERC-NBS) of the Korea Science and Engineering Foundation at Seoul National University; by the Intelligent Microsystem Center, Korea Institute of Science and Technology, and by the BK 21 Project, Seoul National University.

 Submitted for publication September 20, 2003; revised February 25, and May 17, 2004; accepted June 3, 2004.

 Disclosure: J.-M. Lim, None; S. Byun, None; S. Chung, None; T.H. Park, None; J.-M. Seo, None; C.-K. Joo, None; H. Chung, None; D. Cho, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 2.

 

Scanning electron micrograph (SEM) pictures of the micropatterned PDMS surfaces. An SEM of the micropatterned PDMS shows the circular diameter of the 5 μm pillars. The higher-magnification images illustrate the characteristics of a part of the pillar arrays (A): w = 5 μm, g = 5 μm, ×500; (B): w = 5 μm, g = 5 μm, ×2000. Scale bar, 10 μm.

Figure 1.

 

Schematic and fabrication diagram of the micropatterned surfaces exposed to cultured ARPE-19 cells. (A) Schematic diagram of the micropatterned PDMS substrates. The planar pattern of a 5 μm PDMS pillar was patterned to disturb the early focal contact. Closely spaced islands were designed to examine the effect on cell behavior. (B) Schematic illustration of the procedure used to fabricate the PDMS specimen from a master and having microstructures made of SU-8 on its surface. (1) The wafer was cleaned in H₂SO₄ to get rid of organics and salts. (2) Evacuated SU-8 was poured onto the wafer and evacuated again to remove the bubbles in SU-8. After evacuation, the wafer should be kept for several hours on a flat surface to enhance the surface profile of SU-8. (3) UV light was used to cross-link the exposed region, while developing removed the unexposed region. Post exposure baking was then used to vaporize any remaining solvent in the SU-8. (4) Fabricated master for PDMS curing. (5) Mixed and evacuated PDMS was applied to the fabricated master and baked at 150°C for 20 minutes. (6) Baked and stripped PDMS chips can then be used as a micropatterned substrate.

Figure 3.

 

Disturbed focal contact formation by reduced cell–substrate contact area. The focal assembled complexes were disturbed on the micropatterned surfaces after culturing for 3 hours. The pattern of vinculin on smooth PDMS indicated the formation of strong focal contact compared with the 5 μm micropattern. Note that the integrity of the actin stress fibers on the micropatterned surfaces was disrupted. (A, C, E, and G): phase contrast and immunostained images of the smooth surfaces; (B, D, F, and H): 5 μm micropatterned surfaces; (A and B): phase contrast images of ARPE-19 cells; (C, D, E, and F): fluorescence images of the nucleus and F-actin; (G and H): formation of actin stress fibers. All the images were recorded using an Olympus IX 51 (Olympus).

Figure 4.

 

Early focal contact as a regulator of cell adhesion, cell cycle progression, and cell growth. (A) Similar rates of cell adhesion were determined. For quantitative cell adhesion assay, unattached cells were removed from the surfaces. Cells at different times (15, 30, and 60 minutes) were measured and incubated in hexosaminidase substrate. The absorbance was determined at a wavelength of 405 nm. The data are expressed as the mean absorbance at 405 nm ± SD and represent one of three experiments performed in triplicate. (B) Inhibited cell cycle progression by a disturbed focal contact. Top: BrdU-labeled synchronized ARPE-19 cells on the (A) smooth and (B) 5 μm PDMS surfaces (×200). Bottom: A percentage plot of the BrdU-labeled ARPE-19 cells indicated that the cell cycles were significantly inhibited on the micropatterned surfaces. P < 0.05 for the difference between the smooth and micropatterned surfaces. (C) Inhibited cell growth by disturbed focal contact. ARPE-19 cells were adhered to the smooth and micropatterned surfaces for 1, 2, 3, 5, and 7 days of incubation. Cells were washed, fixed with 4% paraformaldehyde, and stained with Hoechst 33342. Data represent the mean ± SD of results. Experiments were performed in triplicate. Statistically significant deviations (P < 0.05) were observed from the respective smooth controls. (D) Cell phenotype variation due to disturbed focal contact RPE were cultured to 3 weeks postconfluency. The cells were fixed and double-stained with Hoechst 33342 (nucleus) and rhodamine-phalloidin (F-actin). Cells that had just reached confluence displayed a fibroblastic appearance on the smooth surfaces (Fig. 4D-M ), whereas a disturbed fibrillar network in the cytoskeleton was observed on the micropatterned surfaces (Fig. 4D-O). Left and right panels are magnified ×200.

Figure 5.

 

Downregulated expression of IL-6 mRNA and secretion by a disturbed focal contact. (A) RNA samples extracted from ARPE-19 cells attached to the smooth and micropatterned surfaces were used. Total RNA aliquots of cells were analyzed by semi-quantitative RT-PCR. PCR reactions in the presence of primers for IL-6 and β-actin as an internal standard were performed. The cells were cultured on fibronectin (FN)-coated smooth PDMS, and FN-coated 5 μm PDMS patterned surfaces for 24 hours; M, 1 Kb marker; C, smooth surfaces; 5D, 5 μm micropatterned surfaces; IL-1β, 1 ng/mL treatment. (B) ELISA analysis of the IL-6 secretion by ARPE-19 on the smooth and micropatterned substrates as described in Figure 5A. The absorbance was determined at a wavelength of 490 nm. The data represent one of three experiments in triplicate. A significant difference (P < 0.05) was observed between the smooth and micropatterned surfaces.