BioChip J. DOI 10.1007/s13206-017-1301-1

Original Article

Design and Fabrication of Devices for Investigating Cell-sheet Stretch Yang Liu1, Yoshihiro Ojima2, Masanobu Horie3, Eiji Nagamori4 & Hideaki Fujita5,6,* Received: 29 September, 2016 / Accepted: 14 December, 2016 / Published online: 03 March, 2017 Ⓒ The Korean BioChip Society and Springer 2017

Abstract Recently, many efforts have been made

to investigate the cell stretch response through focal adhesions, which are usually utilized by cells cultured on elastic materials. However, not all stretch sensing is mediated through focal adhesions but from cell-cell contacts such as adherence junctions. To unveil the details of the stretch-sensing mechanism through cellcell contacts, we developed a cell-sheet extension device for visualizing dynamic changes of individual living cells induced by external mechanical stretch. A cell-sheet is an ideal observation object, as it can be comprised merely of cells without any mechanical influences from external matrices while maintaining normal cell-cell adhesions as in the in vivo situation. Two microfluidic extension devices were designed and fabricated using a silicone elastomer, which were capable of extending a cell-sheet made from Caco-2 cells up to 1.3- and 1.5-fold from its original state. Based on the obtained results, we expected that such cell-sheet 1Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, 560-8531 Osaka, Japan 2Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan 3Radioisotope Research Center, Division of Biochemical Engineering, Kyoto University, Yoshida, Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan 4Department of Biomedical Engineering, Osaka Institute of Technology, 5-16-1 Ohmiya, Asahi-ku, 535-8585, Osaka, Japan 5Quantitative Biology Center, Riken, 6-2-3 Furuedai, Suita, 565-0874 Osaka, Japan 6WPI, Immunology Frontier Research Center, Osaka University, 1-3 Yamadaoka, Suita, 565-0871 Osaka, Japan *Correspondence and requests for materials should be addressed to H. Fujita ( [email protected])

extension systems can be useful tools for understanding the mechanisms of cellular stretch sensing through cell-cell contacts in living cells. Keywords: Mechanobiology, Mechano-transduction, Caco-2, Cell-cell attachment, Cell stretch

Introduction Cells sense various mechanical stimuli from the surrounding environment such as the stiffness of the substrate, pressure, stretch, and shear stress. These stimuli are detected by mechano-sensors, for instance, by mechano-sensitive channels, through focal adhesions or adherence junctions. These mechanical signals are then transmitted to the nucleus, resulting in cell responses. Recent findings show that these mechanical stimuli are important in cell fate decision during differentiation. For example, a substrate with tissue-specific elasticity promotes the differentiation of mesenchymal stem cells (MSC) into a specific tissue lineage1. Shear stress induces osteogenic differentiation of MSC and endothelial differentiation of embryonic stem cells (ESC). Furthermore, mechanical stimuli are important in diseases such as heart failure2,3, cancer metastasis4,5, and fibrosis6,7. Among the various types of mechanical stimuli, stretch is one of the most studied because of its physiological importance. For instance, mechanical stretch of cells occurs in skeletal muscle during muscle contraction as well as in cardiac muscle during diastole. Cardiac diastole can also affect arteries, resulting in mechanical stretch of the various cells that constitute the artery wall, including vascular endothelial cells,

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vascular smooth muscle cells, and fibroblasts. Furthermore, such mechanical stretch is also present between lung alveolar cells during inspiration as well as intestinal epithelial cells during peristalsis. This mechanical stretch of cells triggers a wide variety of effects on these cells, such as activation in cardiac muscle8,9, enhancement of proliferation in epithelial cells10,11, or acceleration of wound closure in lung fibroblasts 12. Despite the importance of stretch on a wide variety of cell functions, mechanical stretch experiments on cultured cells have not been performed widely due to the difficulty in inducing mechanical stretch in cells. Stretching of a large tissue such as skeletal muscle can be realized easily; however, stretching cultured cells requires some effort and various methods have been proposed and utilized. Cells seeded on an elastic three-dimensional scaffold can be extended by stretching the scaffold13,14. Cells cultured on an extensible sheet such as silicone can also be stretched easily by the application of an external force15,16, and various devices have been designed so as to control the amplitude and direction of the stretch17,18. However, in these configurations, the cells are attached to a substrate; thus, the mechanical stimuli are sensed mostly through focal adhesions between the cells and matrices. To unveil the details of the stretch-sensing mechanism through cell-cell contacts, the consequences of cell-matrix (A)

contacts need to be restrained. In this study, we have designed two microdevices for stretching an integrated cell-sheet that is not attached to any substrate. Using these devices, a cell-sheet could be stretched up to 1.3- and 1.5-fold from the original state. The cell-sheet stretch devices introduced in this study are expected to be useful in studying the stretch-sensing mechanism of cells through cell-cell contacts.

Results and Discussion Device with a Single Balloon Structure

First, we designed a simple device with a single balloon structure (Figure 1). This device consisted of a gap-patterned polydimethylsiloxane (PDMS) layer where the cell-sheet was to be attached, a thin PDMS membrane layer, and a chamber layer. The gap-patterned PDMS layer was settled above the center of the chamber, and the cell-sheet was placed bridging the gap patterns. The chamber layer and thin PDMS membrane layer composed the balloon structure, and when positive pressure was applied to the chamber, the patterned PDMS membrane was lifted so that the gap between the top parts of the thick PDMS membrane increased, which would result in stretching of the cell(B)

(C)

Figure 1. Design of the cell-sheet stretch device with a single balloon structure. (A) Schematic view. (B) Schematic illustration of the cell-sheet stretch procedure. Cross-sectional view along the line x-y in (A) is shown. (C) Photograph of the fabricated device (upper left) and cross-sectional image (lower left), and micrograph of the top view before (upper right) and after (lower right) applying pressure to the chamber. Scale bar, 1 mm.

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sheet attached to the thick PDMS membrane. When positive pressure was applied to the pressure chamber, inflation of the thin PDMS membrane pushed up the thick PDMS membrane, resulting in an increased gap between the PDMS membranes. Approximately 1.3 × stretch was obtained with this device.

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(A)

Cell-sheet Stretch Using the Single Balloon Device

To test whether the fabricated device can actually stretch a cell-sheet, a Caco-2 cell-sheet was placed on the device and incubated overnight for cell attachment. For cell-sheet stretch, a microfluidic flow control system (OB1; Elveflow, Paris, France) was connected to the device with a single balloon structure to allow the pressure for injecting air into extension balloon to increase by 30 mbar per step until 750 mbar. When positive pressure was applied to the chamber, cell-sheet stretch was observed (Figure 2B). By applying 300 mbar to the chamber, the cell-sheet was stretched up to 1.3 × of its original length; however, increasing the pressure resulted in disruption of the cell-sheet.

(B)

Device with a Through-hole Array

Although the device with a single balloon structure successfully stretched the cell-sheet, the magnitude of extension was only up to ~1.3 × . In addition, expanding the balloon structure resulted in a large shift of the Z position of the cell-sheet, resulting in difficulties in observations during stretch. To overcome these shortfalls, a device with a through-hole array was designed and fabricated. The device consisted of a thin PDMS mesh with a through-hole having a diameter of 300 μm, and two vacuum chambers (Figure 3A & B). Both vacuum chambers had a thin flexible wall that formed two footholds under the PDMS mesh and over the cellsheet. The shape of the flexible walls was changed by applying negative pressure, and consequently stretched the through-hole (Figure 3C). The diameter of the through-hole increased by 1.9 × after application of negative pressure. Cell-sheet Stretch Using the Device with a Through-hole Array

For cell-sheet stretch, a Caco-2 cell-sheet was placed on the device and incubated overnight for cell attachment. To assist cell attachment to the PDMS surface, the device was coated with an ECM solution containing 300 μg/mL Matrigel and 50 μg/mL type I collagen. Cells were present both on the alginic acid gel surface and PDMS surface (Figure 4A). A vacuum pump (DA41D; ULVAC KIKO, Inc., Miyazaki, Japan) was con-

Figure 2. Cell-sheet stretch using the single balloon device. (A) Phase contrast image of the single balloon device with a Caco-2 cell-sheet. Scale bar, 500 μm. (B) Phase contrast image of the single balloon device before (left) and after cell-sheet stretch; 300 mbar was applied to the pressure chamber. Scale bar, 500 μm.

nected to the device to remove the air in the vacuum channels on the bottom layer, and to tilt the PDMS walls to the outer sides so as to stretch the porous PDMS membrane on the top layer. When negative pressure was applied to the chamber, the through-hole was stretched, resulting in cell-sheet stretch. The cell-sheet was stretched up to 1.5 × from its original size (Figure 4B). Vertical displacement was small compared to that of the device with a single balloon, enabling us to observe the process of stretch. Thus, although the structure of the device was more complicated than the device with a single balloon, the device with a throughhole array is considered to be a more ideal cell-sheet stretch device. To evaluate the effect of change in dimension of the

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(A)

(B)

(C)

Figure 3. Design of the cell-sheet stretch device with a through-hole array. (A) Schematic view. (B) Schematic illustration of the cell-sheet stretch procedure. Cross-sectional view along the line x-y in (A) is shown. (C) Photograph of the fabricated device (left) and micrograph of the top view before (upper right) and after (lower right) applying negative pressure. Scale bar, 500 μm.

device structure on extension capability, we fabricated devices with varying wall height, wall thickness, center chamber width and vacuum chamber width, and the extension capability was assessed (Figure 5A). As expected, change in these sizes altered the extension capability (Figure 5B), and the most effective structure was determined (Table 1). Using these devices, we stretched Caco-2 cell-sheet, which resulted in the improvement of stretch capability (Figure 5C).

Conclusions We designed two cell-sheet-stretch devices in this study: a device with a single balloon structure and a device with a through-hole array. Even though similar devices for cell stretching has previously reported, these devices are designed to stretch cells attached to elastic surface. The devices presented in this study are designed to stretch cell-sheets, which are not attached to substrate, which will be useful in studying mechanotransduction through cell-cell interface. Although the device with a single balloon is simple and easy to fabricate, the device with a through-hole array is suitable for the study of mechano-transduction during cell-sheet stretch as vertical displacement is minimal, which enables microscopic observations during stretch. The use

of these devices will facilitate simple and controlled studies of mechano-transduction through cell-cell adhesions where the effects of cell-substrate interactions are minimal.

Materials and Methods Fabrication of Devices for Cell-sheet Stretch

Both devices for cell-sheet stretch used in this study were fabricated using a soft-lithographic technique that was reported previously19. The bottom chamber layers and the top membrane layers (Figures 1 & 3) of both devices were prepared by demolding cured PDMS (Sylgard-184; Dow Corning, Auburn, MI, USA) from master molds containing positive patterns. The master molds were made of SU-8 3050 and SU-8 2075 photoresist (Microchem, Newton, MA, USA) and followed a typical photolithography process, including substrate preparation, photoresist spin coat, prebake, exposure, post-exposure bake, development, and post-bake. The photomasks were created by AutoCAD (Autodesk, San Rafael, CA, USA) and plotted on clear films. The ratio of PDMS for preparing all parts of both devices was generated by casting PDMS polymer and a curing agent at 10 : 1 (w/w). To bind the upper layer of a PDMS membrane to the lower PDMS parts, the surfaces of

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nel patterns for 10 s at 1,000 rotations per min, and then cured at 70°C for 1 h. The device was sterilized by autoclaving. To assist cell attachment to the PDMS surface, the device was coated with an ECM solution containing 300 μg/mL Matrigel (BD Biosciences, Bedford, MA, USA) and 50 μg/mL type I collagen (BD Biosciences).

(A)

Fabrication of a Device with a Through-hole Array

(B)

(a)

(b)

(c)

(d)

Figure 4. Cell-sheet stretch using the device with a throughhole array. (A) Phase contrast image of the device with a through-hole array seeded with Caco-2 cells. Scale bar, 500 μm. (B) Phase contrast image of the device with a throughhole array before (a) and after (b) cell-sheet stretch. Scale bar, 100 μm. (c, d) Magnified view of the cell-sheet area shown in black rectangle in (a). Brightness and contrast is linearly enhanced to visualize the cells.

each layer were treated with a corona plasma device (PIB-10 Ion Bombarder; Shinku Device Co., Ltd., Ibaraki, Japan), and immediately attached together. Fabrication of a Device with a Single Balloon Structure

For fabrication of a device with a single balloon structure, a 5 mm (length) × 5 mm (width) × 0.3 mm (height) chamber sealed with a thin PDMS membrane was prepared on the bottom layer for fabrication of an extension balloon, and then the balloon structure was covered by a PDMS membrane with spatial gap-patterns of parallel gaps at 0.3 mm height and 0.6 mm width with 0.8 mm spacing on top. The PDMS membrane was fabricated by spin-coating pre-cured PDMS polymers on a slide glass for 60 s at 1,000 rotations per min, and then cured at 70°C for 1 h. The gap-patterned PDMS membrane was fabricated by spin-coating pre-cured PDMS polymers on a master mold containing chan-

For fabrication of a device with a through-hole array, three parallel channels were fabricated on the bottom layer. Two vacuum channels (0.5 mm width × 0.5 mm height) were prepared on both sides of the extension channel (1 mm width × 0.5 mm height). The PDMS walls for separating these channels were 0.2 mm thick. The vacuum channels were sealed by 0.08 mm thick PDMS membranes, while the extension channel was kept open. Then, a porous PDMS membrane (0.08 mm thick) was adhered to the top of all channels. The PDMS membrane was fabricated by spin-coating precured PDMS polymers on a slide glass for 60 s at 1,000 rotations per min, and then cured at 70°C for 1 h. To fabricate the porous PDMS membrane, pre-cured PDMS polymer was spin coated on a pre-silanized (Trichloro(1H,1H,2H,2H-perfluorooctyl)silane; SigmaAldrich, St. Louis, MO, USA) PDMS slab for 60 s at 1,000 rotations per min. Then, the surface of the PDMS slab with pre-cured PDMS polymer was overlaid on a master mold containing post arrays of circular pillars (0.3 mm diameter × 0.3 mm height with 0.3 mm spacing). A 500 g weight was added to the PDMS slab. After PDMS curing overnight at room temperature, and an additional 1 h at 70°C, the resultant porous membrane was immediately bonded with the lower layer following corona plasma treatment on both surfaces. The fabricated device was sterilized by autoclaving. Cell Culture and Cell-sheet Fabrication

A human colorectal adenocarcinoma cell line (Caco-2; Riken Cell Bank, Ibaraki, Japan) was purchased from Riken Cell Bank (Tsukuba, Japan) and cultured in Dulbecco’s Modified Eagle’s Medium (Nacalai Tesque, Inc., Kyoto, Japan) containing 4.5 g/L D-glucose and 10% fetal bovine serum (Gibco, Waltham, MA, USA). The cells were incubated at 37°C in humidified air containing 5% CO2. To fabricate integrated cell-sheets and transfer them to the top of the devices without any change in shape, we created an original strategy using a hydrogel sheet as a sacrificial template. We employed alginate (MW 70,000; Kimica, Tokyo, Japan) to form the hydrogel sheet and gelatin (type A, from porcine

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(A) (a)

(b)

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Figure 5. Effect of changing various dimensions of the structure on the device with a through-hole array. (A) Images of chamber layer with various dimensions. Top view (upper panel) and side view (lower panel) are shown. Sizes of wall height, wall thickness, center chamber width and vacuum chamber width of these devices are indicated in Table 1. Scale bar, 1 mm. (B) Images of devices after application of negative pressure. Extension rate of these devices are indicated in Table 1. Scale bar, 1 mm. (C) Examples of Caco-2 cell-sheet stretch using devices of various dimensions. Device (a) and (b) are shown. Scale bar, 200 μm. Table 1. Dimensions of fabricated device for cell-sheet stretch. Device

Wall thickness

Wall height

Vacuum chamber width

Center chamber width

a b c d e

180 280 380 280 380

540 480 460 540 560

560 610 610 580 580

1000 1020 1020 460 450

Extension ( × ) 2.9 2.7 1.9 3.2 2.3

Values for wall thickness, wall height, vacuum chamber width and center camber width are in μm. Positions where dimensions were measured are indicated in Figure 5A.

skin, 300 Bloom; Sigma-Aldrich) to provide the cell adhesion surface. Both polymers were modified by tyramine using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimidecatalyzed reactions20,21. The phenol moieties from tyramine on the side chains of alginate or gelatin can be crosslinked through a horseradish peroxidase (HRP)catalyzed reaction consuming H2O2. The brief procedure of hydrogel sheet formation is described below.

Firstly, we put a small droplet of a saline solution containing 2% alginate derivative on the bottom of a cell culture dish. A mixed cellulose ester membrane with 0.2-μm-diameter pores (Advantec MFS, Inc., Tokyo, Japan) was soaked in a saline solution containing 100 mM CaCl2 in advance, and laid on the droplet of alginate derivative to extend the droplet into a sheet shape. Meanwhile, Ca2+ ions on the membrane induced crosslinking between the alginate chains, resulting in hy-

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drogel formation. Then, the hydrogel sheet was soaked in a saline solution containing 10 U/mL HRP, and a saline solution containing 0.5% gelatin derivative and 0.5 mM H2O2, subsequently. The residual H2O2 was removed by catalase (from bovine liver; Wako, Osaka, Japan). After triple rinsing with cell culture medium, 1.25 × 105 Caco-2 cells/cm2 were seeded on the hydrogel sheets. Caco-2 cells were cultured on the hydrogel sheet for 3 days to form a cell-sheet. A 6-mm-diameter biopsy punch was used to cut a small hydrogel sheet with a cell-sheet surface. Tweezers were used to move the small piece of hydrogel sheet to the microdevices. The cell-sheet surface attached closely to the top layer of the microdevices and then cultured overnight. The cell-sheet extension experiments were performed immediately following treatment with 0.4 mg/mL alginate lyase (Sigma-Aldrich) for 2 h so as to remove the hydrogel sheet template. Conflict of Interest The authors declare that there are

no conflict of interests regarding the publication of this paper.

Acknowledgements This work was supported by the Program for Creating Future Wisdom, Osaka University (2014-2016). This work was also supported by Nanotechnology Platform Project (Nanotechnology Open Facilities in Osaka University) (No. F-16-OS-0012) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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