Cell motility regulation on stepped micro pillar array device (SMPAD ...

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This goal is achieved through the use of 'stepped' micro pillar array device (SMPAD) whose top contact area with a cell is kept constant while the diameter of ...
CELL MOTILITY REGULATION ON STEPPED MICRO PILLAR ARRAY DEVICE (SMPAD) WITH DISCRETE STIFFNESS GRADIENT

Sujin Lee1, Biswajit Saha1,2, and Junghoon Lee1* 1 School of Mechanical & Aerospace Engineering, Seoul National University, Seoul, South Korea 2 BK21 PLUS Transformative Training Program for Creative Mechanical & Aerospace Engineers, Seoul National University, Seoul, South Korea case, the adhesion area of extra cellular matrix (ECM) also varied on different pillars because of the change in diameters. The variation of the adhesion area is one of the essential cues associated with the formation of focal adhesion that may affect cell migration and proliferation [1,14], thus needs to be decoupled from the effect of pure rigidity gradient. One extreme example would be the different modes of growth and movement of cells on patterned ECM vs. a flat substrate with a simple ECM coating [15].

ABSTRACT Here we have shown a new variation of the microfabricated pillar array detector (MPAD) that can decouple the stiffness gradient from the focal adhesion area of a cell. This goal is achieved through the use of “stepped” micro pillar array device (SMPAD) whose top contact area with a cell is kept constant while the diameter of pillar bodies vary for variable mechanical stiffness. We have observed manipulating cell behavior using this simple, artificial platform that produces a pure physical stimulus. This report includes a new discovery of gradientdependent cell motility enhancement as well as the “classical” demonstration of durotaxis on the SMPAD.

CONCEPT We present a stepped micro pillar array device (SMPAD) that offers the rigidity gradient in a discrete fashion without varying the top adhesion area. Figure 1 is the 3-D modeling view of a stepped micropillar array device. The inset of Figure 1 shows deformation of the pillars due to the attachment of focal adhesion and the force applied by the cell.

INTRODUCTION Cell migration guided by stiffness gradient The research outcomes of directional cell migration have direct implication on understanding many physiological processes such as morphogenesis [2], immune response, and wound healing [3]. It is well known that cell movements can be guided by gradients of various chemical [4,5], mechanical and topological signal [6]. These findings are critical in many biomedical applications including implantable medical devices, biomaterials, and tissue engineering [7]. Cell movement involves a number of related events, such as the protrusion of pseudopodia, the formation of new adhesions, the development of traction, and the release of old adhesions [8]. To achieve appropriate physiological outcomes, cell movement must maintain a distinct direction and speed in response to environment stimuli. Cell migration, which is controlled by the gradients of dissolved or substrate attached chemicals (chemotaxis and haptotaxis, respectively) has been investigated for many years. In addition, cells are known to orient and migrate in response to gradients of light intensity, electrostatic potential, and gravitational potential [9,10]. Cell migration guided by substrate rigidity is known as durotaxis. Durotaxis have been studied using soft and stiff 2D substrates with several materials in vitro [1,3,6,7]. These prior works utilized numerous methods to create polymer based substrates with varying rigidity. However, it was, challenging to maintain an accurate control over the arrangement of substrate stiffness such as the amount and direction of gradient.

Figure 1: 3-D view of stepped micro pillar array device. The black arrow represents the direction of increased stiffness gradient. The bottom layer of micro pillars have varying diameters (3 μm ~ 7 μm) while the diameter of the top layer is maintained identical (3 μm) by the double layers structure. Spring constant of the pillar is calculated by equation (1) and (2) 3π E 4 (1) k = r 4 L3 1 1 1 = + (2) k pi ka kb ,where kpi is the spring constant of a single pillar, ka and kb are the spring constants of top and bottom portions respectively. The variations in bottom layer diameters from 3 μm to 7 μm with a total height of 7 μm correspond to the stiffness variations from 0.067 to 0.76 μN/μm.

Micro pillar array Micro-scale polymeric pillar arrays were normally used as cell force detectors [11,12]. This polymeric micro pillar array has been recently used to study cell movement and shape with various methods including the gradient of rigidity provided by varying pillar diameters [13]. In this

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MEMS 2015, Estoril, PORTUGAL, 18 - 22 January, 2015

FABRICATION

EXPERIMENT AND RESULTS

Figure 2 shows the fabrication process to implement this design via “stepped” molding. Fibronectin was “stamped” on the top area as an extracellular matrix that facilitates biological contacts with cells. The fabrication of the SMPAD begins with a double-step patterning of SU-8 photoresist (MicroChem, USA). After developing and hard baking the first patterned SU-8 layer with the thickness of 2 μm, additional layer was coated and patterned again as a second mold layer with the thickness of 5 μm. Degassed polydimethylsiloxane PDMS (Dow Corning, USA) was poured and cured at 60 ˚C overnight. The PDMS substrate was peeled off from the mold to complete the device. Oxygen plasma was used to render the surface hydrophilic for enhancing the adhesion of the ECM (Sigma Aldrich, USA). The ECM was placed on the top pillar surface by stamping on a flat PDMS substrate with ECM coating [11]. It should be emphasized that the size and arrangement of the stamped ECM (FITC labeled), thus the adhesion area and the pattern, was kept uniform while the size of the pillar body varies (Figure 2(f)). The remaining area of the pillar and the bottom substrate were blocked by 2% Pluronic F127 (BASF, USA) to prevent the interference of cell behavior due to unnecessary attachment. Some non-specific adhesion of cells occurred on the pluronic treated surface. The efficiency of the non-specific adhesion, however, was negligible especially when a single cell was used in the experiment.

NIH3T3 cells were used for migration experiments. NIH3T3 cell is a mouse fibroblast with high motility, frequently used for such experiments [16,17]. A live imaging instrument (Nikon, Japan) was used to monitor the trajectory of migration. In order to minimize cell-to-cell communication, small number of cells (1×103 cells/mL) was plated sparsely on the SMPAD contained in a culture dish. Then we focused on the migration of an individual cell on the array.

Figure 3: Values for stiffness gradient of three type of platforms (a), Cell test results on platforms with Δk = 0.02 (b), 0.06 (c), and 0.1 (d) μN/μm. Cells randomly distributed initially, ended up with the vicinity of indicated locations after 18 hours of migration. White arrows indicate the stiffness of the final locations. (h: hard, m: medium, s: soft area).

Figure 2: Process flow of SMPAD substrate fabrication (a-d). The result of process is shown in SEM image (e). Fluorescence-labeled FN image of pillar top shows constant contact area (f).

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This stiffness-guided migration was more consistent on the pillar array with the stiffness gradient of 0.1 μN/μm. Figure 4 shows that the motility of cells depending on the stiffness gradient. After 6 hours, cells moved much faster on the pillar array with a stiffness gradient than on controls (Figure 4 (a), n=15). This motility enhancement became more pronounced on the larger stiffness gradient as illustrated by Figure 4 (b). This is an important finding that verifies some physiological behaviors in-vivo [6], possibly leading to the design of effective scaffolds.

In a control experiment a stepped micro pillar array without variation in diameter was tested. It was found that the cells migrated randomly in this case. Figure 3, in contrast, indicates that the cell on the SMPAD substrate migrates toward the direction of stiffer pillars. Figures 3 (b), (c), and (d) show the locations of the enhanced green fluorescence protein (eGFP) expressed NIH3T3 cells 60 hours after launching the experiment. When the rigidity gradient was 0.02 μN/μm, the cells wandered almost randomly without a specific orientation. The cells started showing directional migration toward higher stiffness region when the gradient became 0.06 μN/μm. This stiffness guided migration was highly repeatable and consistent on the pillar array with the stiffness gradient of 0.1 μN/μm. The final locations of cells in Figures 3 (b), (c), and (d) manifests these behavior. It was hard to design the gradient higher than 0.1 μN/μm because the inter-pillar distance became too small to be fabricated.

Figure 5: The cell initially moves to the direction of stiffer pillars (Δk = 0.1) (a). When the cell reaches the edge of the stiff region (c), it changes the direction of motion, moving along the boundary of the stiff region (d)-(f). The trajectory of the cell is marked by the yellow arrow (f). The cell migrates for about 180 μm within 5 hours in this case. Due to design constraint several rows of pillars with the same diameter were arranged at the end of the rigidity gradient. The behavior of the cell in this constant rigidity region is of particular interest. As shown in Figure 5, the cell in the constant stiffness area may change the direction. However, when the cell approached the edge of the high stiffness area, its protrusion of pseudopodia started touching the soft area as a way of probing, and the cell instantly turned back and migrated parallel to the boundary, staying in the original area. We believe this interesting behavior is attributed to the step rigidity gradient along the boundary.

Figure 4: Motility of cells depending on the stiffness gradient. Cells moved faster on the pillar array with stiffness gradient after 6 hours of observation (n=15) (a). Average velocity of each cell is traced for 24 hours (n~20) and error bar represents Vmax and Vmin of each platform (b). It is clearly shown that the strength of stiffness gradient highly affects cell motility.

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[7] C. M. Lo, H. B. Wang, M. Dembo, and Y. l. Wang, “Cell Movement Is Guided by the Rigidity of the Substrate”, J. Biophy., vol. 79, pp. 144-152, 2000. [8] D. A. Lauffenburger, and A. F. Horwitz, “Cell migration: a physically integrated molecular process”, Cell, vol. 84(3), pp. 359-369, 1996. [9] J. Saranak, and K. W. Foster, “Rhodopsin guides fungal phototaxis”, Nature, vol. 387, pp. 465-466, 1997. [10] C. A. Erickson, and R. Nuccitelli, “Embryonic cell motility can be guided by weak electric fields”, J. Cell Biol., vol. 95, pp. 314a, 1982. [11] N. Q. Balaban, “Force and adhesion assembly: a close relationship between studies using elastic micropattemed substrates”, Nat. Cell Biol., vol. 3, pp. 466-473, 2001. [12] J. L. Tan, J. Tien, D. M. Pirone, D. S. Gray, K. Bhadriraju, and C. S. Chen, “Cells lying on a bed of microneedles: An approach to isolate mechanical force”, Proceedings of the National Academy of Science, vol. 100 (4), pp. 1484-1489, 2003. [13] R. Sochol, and L. Lin, “Microscale Control of Micropost Stiffness to Induce Cellular Durotaxis”, Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences, pp. 1335-1337, 2008. [14] M. A. Wozniak, K. Modzelewska, L. Kwong, P. J. Keely, “Focal adhesion regulation of cell behavior”, Biochim Biophys Acta., vol. 1692(2-3), pp. 103-119, 2004. [15] D. A. Rubenstein and M. D. Frame, “Micro-stamped ECM proteins enhance endothelial cell adhesion and directed growth”, The FASEB Journal, vol. 21, pp. 897.1, 2007. [16] T. J. Bos, P. Margiotta, L. Bush, and W. Wasilenko, “Enhanced cell motility and invasion of chicken embryo fibroblasts in response to JUN over-expression”, Int. J. Cancer, vol. 81, pp. 404-410, 1999.

CONCLUSION In conclusion, cell migration was demonstrated along the discrete rigidity gradient created by pillars with varying diameters. By maintaining the top area of the pillars constant we could eliminate the concern for the size effect of adhesion area. Double step fabrication technique of the double-layered pillar enabled this special configuration. The SMPAD introduced can be further used to study the cell migration for many other biological applications such as artificial tissue engineering, plastic surgery, and studying the migrational behavior of cancerous cells.

ACKNOWLEDGEMENTS This work was supported by the ICT & Future Planning as Global Frontier Project (CISS-2012M3A6A 6054193). And the fabrication was performed at the Interuniversity Semiconductor Research Center (ISRC) in Seoul National University.

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CONTACT *J. Lee, tel: +82-2-880-9104; [email protected]

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