Slope-Dependent Cell Motility Enhancements at the Walls of PEG

Aug 19, 2015 - In recent years, research utilizing micro- and nanoscale geometries and structures on biomaterials to manipulate cellular behaviors, su...
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Slope-Dependent Cell Motility Enhancements at the Walls of PEGHydrogel Microgroove Structures Keiichiro Kushiro,* Takamasa Sakai, and Madoka Takai* Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan S Supporting Information *

ABSTRACT: In recent years, research utilizing micro- and nanoscale geometries and structures on biomaterials to manipulate cellular behaviors, such as differentiation, proliferation, survival, and motility, have gained much popularity; however, how the surface microtopography of 3D objects, such as implantable devices, can affect these various cell behaviors still remains largely unknown. In this study, we discuss how the walls of microgroove topography can influence the morphology and the motility of unrestrained cells, in a different fashion from 2D line micropatterns. Here adhesive substrates made of tetra(polyethylene glycol) (tetra-PEG) hydrogels with microgroove structures or 2D line micropatterns were fabricated, and cell motility on these substrates was evaluated. Interestingly, despite being unconstrained, the cells exhibited drastically different migration behaviors at the edges of the 2D micropatterns and the walls of microgroove structures. In addition to acquiring a unilamellar morphology, the cells increased their motility by roughly 3fold on the microgroove structures, compared with the 2D counterpart or the nonpatterned surface. Immunostaining revealed that this behavior was dependent on the alignment and the aggregation of the actin filaments, and by varying the slope of the microgroove walls, it was found that relatively upright walls are necessary for this cell morphology alterations. Further progress in this research will not only deepen our understanding of topography-assisted biological phenomena like cancer metastasis but also enable precise, topography-guided manipulation of cell motility for applications such as cancer diagnosis and cell sorting.



vessel-like tissue formation.12 Thus, both 3D microstructures and 2D micropattern geometries have been shown to influence cell polarization and motility of some cell types, but because there have only been a limited number of studies, the comprehensiveness and the dependence on the type of material/surface of this topography- or geometry-induced phenomena remain largely unknown. As an example of such limitation, almost all of the microgroove systems, as a consequence of lithography-based fabrication protocols, have utilized only vertical walls. One interesting phenomenon arising from topography like microgrooves is the enhancement in cell motility, which is relevant for various biological phenomena such as wound healing and cancer metastasis. Also, such motility enhancements, which rely solely on the topography without the use of chemical or other forms of gradients, would have many useful applications, such as enhancing the rate of tissue formation on tissue engineering scaffolds or facilitating wound healing processes by utilizing microstructured biomaterials. Previous studies using either micro- (>10 μm wide and >75 μm high) or nanogroove (>700 nm wide and >800 nm high) structures have reported different degrees of cell motility enhancements, but such speed and persistence enhancements due to topographical

INTRODUCTION Cell motility plays important roles in various processes in the body, such as immune response, wound healing, and cancer metastasis,1−3 and it is becoming increasingly evident that topographies and microenvironments in the body affect these processes. For example, it has become more evident that cancer cells utilize or reorient the preexisting microstructures in the body, such as the blood or lymphatic vessels and collagen fibers, to facilitate metastasis.4,5 Although there has been some advancement in understanding the interactions between cells and their physical environments, there are still multitudes of unanswered questions. One way to prod such questions is to fabricate synthetic structures and topographies and observe their interactions with biological systems. In fact, there has recently been a rapid development in micropatterning technologies that have been used to modulate various cellular behaviors, in particular cell migration. For example, surfaces presenting 2D micropatterned lines of adhesion proteins that mimic the 1D topography of oriented matrix fibrils have been shown to turn cells into a uniaxial morphology.6 There has also been multitudes of studies in the past involving contact guidance of cells on microand nanogroove structures.7 These microgrooves made of various materials including polydimethylsiloxane (PDMS) or polystyrene have been used to orient and guide cell movement8−10 and aid axonal growth of neurons11 or blood © 2015 American Chemical Society

Received: March 17, 2015 Published: August 19, 2015 10215

DOI: 10.1021/acs.langmuir.5b02511 Langmuir 2015, 31, 10215−10222

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The University of Tokyo) onto a layer of SU-8 negative photoresist (MicroChem) to make a mold, onto which PDMS (Sylgard 184; Dow Corning) was cast to make the stamp. For the microgroove structures, the unreacted hydrogel components were thinly coated on the 35 mm culture dish (Iwaki), stamped with the PDMS microgroove structures from the top, and allowed to form the hydrogel for 2 h, followed by the RGDC peptide incubation. For the microgroove structures with slanted walls, the nickel mold was provided by Optnics Precision. The structure of the slanted walls on the mold was confirmed by scanning electron microscopy (Figure S1, Supporting Information). For the 2D line pattern substrates, the thinly coated hydrogel components were allowed to react for 30 min, on top of which a similarly semiformed hydrogel microgroove structure (with holes punched) with the opposite composition to that of the other hydrogel (i.e., it has excess thiol-containing tetra-PEG) was attached, so that it would react with the hydrogel surface for 1.5 h to form hydrogel microchannels. RGDC peptide solution (1 mg/mL) was ran through this channel and allowed to react for 4 h. Afterward, the top microgroove structures were gently peeled off from the microchannels to leave the 2D line pattern hydrogel substrate. Fabrication of Fluorescent Tetra-PEG Hydrogel Microgroove Structures and RGDC Distribution Assessment. RGDC peptides (1 mg/mL) were tagged with fluorescence by reacting with Atto488 NHS ester (Sigma) at a 2-fold molar excess for 2 h at room temperature. The tetra-PEG hydrogel microgroove structures incorporating these fluorescence-tagged RGDC peptides were prepared in the same manner as the previous sections. The fluorescent structures were visualized using a confocal microscope (LSM510; Carl Zeiss) at 20× magnification. MCF-10A Cell Culture. MCF-10A human epithelial cells were cultured in growth medium composed of Dulbecco’s modified Eagle’s medium/Ham’s F-12 containing HEPES and L-glutamine (DMEM/ F12, Invitrogen) supplemented with 5% horse serum (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 10 μg/mL insulin (Sigma), 0.5 μg/mL hydrocortizone (Sigma), 20 ng/mL EGF (Peprotech), and 0.1 μg/mL cholera toxin (Sigma) and maintained under humidified conditions at 37 °C and 5% CO2. Cells were passaged regularly by dissociating confluent monolayers with 0.05% trypsin-EDTA (Invitrogen) and suspending cells in DMEM/F12 supplemented with 20% horse serum and 1% penicillin/streptomycin. Cells were passaged at 1:4 ratio in growth medium. Time-Lapse Microscopy and Motility Quantification. Cells were seeded at 2 × 104 cells/mL in growth medium for 2 h on the micropatterned substrates. After several washes to remove nonadherent cells, the culture was incubated with fresh growth medium for 4 h and imaged at 10× magnification every 5 min using CCM-1.4Z Cell Culture Monitoring System (ASTEC). In this system, the cells were maintained at 37 °C and 5% CO2 in a heated chamber with temperature and CO2 controller during time-lapse imaging. CCM Software 2.4.5.2 (ASTEC) was used for image processing. To track cell motility on micropatterned surfaces, the positions of the lamellipodial edges were tracked using CCM and ImageJ softwares. Speed was calculated as distance/time, and persistence was measured as the distance for cell migration runs before they changed their direction by >90°. Only isolated cells that were not in contact with other cells were analyzed. The unpaired, two-tailed Student’s t test was used for statistical analysis. Differences were considered significant at p < 0.05. All statistical analysis was performed using corresponding functions in Microsoft Excel. Immunostaining of Actin, Vinculin, and Paxillin. Cells were fixed on 35 mm glass-bottomed dish (Iwaki) using 4% formaldehyde solution (Sigma) for 20 min in room temperature, permealized using 0.2% Triton X-100 (Amersham Biosciences) for 10 min at 4 °C, blocked with 0.1% bovine serum albumin (Sigma) for 2 h at room temperature, and incubated with dyes and antibodies. 4′,6-Diamidino2-phenylindole, dihydrochloride (DAPI; Invitrogen) was incubated for 10 min to visualize the cell nucleus, Alexa Fluor 594 phalloidin (Invitrogen) was incubated for 20 min to visualize the actin filaments, and primary antivinculin antibody (H-300; Santa Cruz Biotechnology) or antipaxillin antibody (Y113; Abcam) was incubated overnight then

effects achieved thus far have not exceeded more than 2fold.13−15 Moreover, for microgroove structures that are roughly the width of a single cell, it has been reported that high degrees of confinement (