Microfabricated Environments to Study Collective Cell Behaviors

CHAPTER

Microfabricated Environments to Study Collective Cell Behaviors

16

Sri Ram Krishna Vedula*, Andrea Ravasio*, Ester Anon{,{, Tianchi Chen*, Gre´goire Peyret}, Mohammed Ashraf*, and Benoit Ladoux*,} *

Mechanobiology Institute, National University of Singapore, Singapore, Singapore Laboratoire Matie`re et Syste`mes Complexes (MSC), Universite´ Paris Diderot, and Unite´ Mixte de Recherche 7057 CNRS, Paris, France { Institute for Bioengineering of Catalonia, Barcelona, Spain } Institut Jacques Monod (IJM), CNRS UMR 7592 & Universite´ Paris Diderot, Paris, France

{

CHAPTER OUTLINE Introduction ............................................................................................................ 236 16.1 Microfabrication Processes for Studying Collective Cell Migration Under Geometrical Confinements ...............................................................................237 16.1.1 Fabrication of Photomasks and Silicon Wafers .............................. 237 16.1.1.1 Materials and Equipment ................................................... 237 16.1.1.2 Methods ............................................................................ 238 16.1.2 Fabrication of Micropatterned PDMS Stamps and Microcontact Printing ................................................................. 240 16.1.2.1 Materials and Equipment ................................................... 240 16.1.2.2 Methods ............................................................................ 240 16.1.3 Placing the PDMS Barrier and Cell Culture ................................... 242 16.1.3.1 Methods ............................................................................ 243 16.2 Microfabricated Substrates for Studying Epithelial Gap Closure .........................244 16.2.1 Fabrication of Photomasks and Silicon Wafers .............................. 244 16.2.1.1 Materials and Equipment ................................................... 244 16.2.1.2 Methods ............................................................................ 245 16.2.2 Fabrication of PDMS Master and Stencils ..................................... 247 16.2.2.1 Materials and Equipment ................................................... 247 16.2.2.2 Methods ............................................................................ 247 16.2.3 Placing the Stencils on the Culture Support and Cell Culture ......... 247 16.2.3.1 Materials and Equipment ................................................... 247 16.2.3.2 Methods ............................................................................ 248 General Conclusion ................................................................................................. 250 References ............................................................................................................. 251

Methods in Cell Biology, Volume 120 Copyright © 2014 Elsevier Inc. All rights reserved.

ISSN 0091-679X http://dx.doi.org/10.1016/B978-0-12-417136-7.00016-1

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Abstract Coordinated cell movements in epithelial layers are essential for proper tissue morphogenesis and homeostasis. Microfabrication techniques have proven to be very useful for studies of collective cell migration in vitro. In this chapter, we briefly review the use of microfabricated substrates in providing new insights into collective cell behaviors. We first describe the development of micropatterned substrates to study the influence of geometrical constraints on cell migration and coordinated movements. Then, we present an alternative method based on microfabricated pillar substrates to create well-defined gaps within cell sheets and study gap closure. We also provide a discussion that presents possible pitfalls and sheds light onto the important parameters that allow the study of long-term cell culture on substrates of well-defined geometries.

INTRODUCTION Microfabrication techniques (Xia & Whitesides, 1998) have been extensively used to study cell behaviors and the impact of geometrical, mechanical, and topographical constraints (le Digabel, Ghibaudo, Trichet, Richert, & Ladoux, 2010; Thery, 2010). At first, most of these techniques mainly focused on single cell behavior such as cell adhesion (Chen, Mrksich, Huang, Whitesides, & Ingber, 1997; Curtis & Wilkinson, 1997; Thery et al., 2005) and cell migration (Ghibaudo et al., 2009; Jiang, Bruzewicz, Wong, Piel, & Whitesides, 2005; Mahmud et al., 2009). However, besides the well-established mode of single cell adhesion and migration, detailed knowledge obtained over the past 30 years suggests that at least one additional major mechanism is relevant to cell translocation within tissues: the movement of cell groups, sheets, or strands consisting of multiple cells connected by cell–cell junctions (Ilina & Friedl, 2009). The regulation of such a migration mode, although ubiquitous in development, tissue repair, and tumor invasion, has been less explored and awaited experimental models to decipher the important steps during collective cell migration. In many biological processes that involve collective cell migration for tissue development (Behrndt et al., 2012; Lecaudey & Gilmour, 2006), cancer cell migration (Friedl, Locker, Sahai, & Segall, 2012), wound closure (Clark et al., 2009), cells are subjected to a broad range of geometrical constraints imposed by the extracellular environments. Microfabrication techniques offer the capabilities to modify the cellular microenvironments in vitro and thus, study collective cell behaviors in well-defined and reproducible conditions. Recently, various microfabrication methods have been developed to shed new light onto the regulation of collective cell migration. This includes the wound model assays (Anon et al., 2012; Murrell, Kamm, & Matsudaira, 2011; Nikolic, Boettiger, Bar-Sagi, Carbeck, & Shvartsman, 2006; Poujade et al., 2007), the measurements of mechanical tension within epithelial cell sheets (du Roure et al., 2005;

16.1 Microfabrication Processes for Studying Cell Migration

Nelson et al., 2005; Serra-Picamal et al., 2012), the emergence of collective modes of migration (Vedula et al., 2012), and 3D tissue organization (Boghaert et al., 2012; Legant et al., 2009). Here we present two different techniques based on microfabrication methods to study collective cell behaviors. First, we present micropatterning techniques that allow us to confine epithelial cell sheets within defined geometries and study their dynamics over long periods of time. We focus on two examples where cells are confined either onto micropatterned fibronectin stripes of various widths (Vedula et al., 2012) or onto circular patterns of various diameters (Doxzen et al., 2013). Then, we present a simple procedure to create well-defined gaps within epithelial cell sheets (Anon et al., 2012). Such assays define new ways to examine how epithelial cells fill in empty spaces: a gap is created by growing cell monolayers around a column, which is later removed. Previous studies have typically used wound assays that involved ablating cells with a laser or scratching through tissue, resulting in possible cell death and debris (Garcia-Fernandez, Campos, Geiger, Santos, & Jacinto, 2009; Tamada, Perez, Nelson, & Sheetz, 2007). Instead, this model experiment keeps the cells intact as they encounter an empty space. Finally, we discuss the drawbacks and advantages of these techniques.

16.1 MICROFABRICATION PROCESSES FOR STUDYING COLLECTIVE CELL MIGRATION UNDER GEOMETRICAL CONFINEMENTS The micromechanical and physical cues within the cellular microenvironment (e.g., geometrical constraints, topography, and substrate stiffness) play a significant role in regulating cell migration. While the role of such physical cues has been well studied in the context of single cells, little is known about how epithelial monolayers and migrating multicellular clusters respond to such cues. Here we describe in detail a microfabrication-based approach that allows us to investigate how the migratory behavior of epithelial monolayers is altered when they are subjected to varying degrees of geometrical confinements.

16.1.1 Fabrication of photomasks and silicon wafers 16.1.1.1 Materials and equipment • • •

• • • •

L-Edit Layout editor (Tanner EDA) Laser writer (Heidelberg Instruments Mikrotechnik, DWL 66fs) 5 in. blank soda lime photomasks; size 5009, flatness: 4.3 mm, chrome type: LRC, ˚ ; OD: 3.02, reflectivity: 10.3%, resist/pre-bake: AZ1500, thickness: 1030 A ˚ ; grade: prime (BONDA TECHNOLOGY) thickness: 5003 A Photoresist developer for the mask developer (AZ Electronic Materials, AZ 400K) Cr etchant (TLG Technology) Photoresist stripper (AZ Electronic Materials, AZ300T) Mask Aligner (Suss Microtek, MJB4)

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• • • • • • • •

4 in. Silicon wafer (AZ Electronic Materials) Spin coater (Brewer Science, CEE 200X) Hotplates (Harry Gestigkeit, PZ44) SU8 3025 Negative Photoresist (Teltek Semiconductors Pacific, SU8 3025) SU8 developer (AZ Electronic Materials, Y020100) Acetone and isopropyl alcohol (IPA) MilliQ water Stylus-based profiler (Dektat XT)

16.1.1.2 Methods PHOTOMASK WRITING 1. The pattern used here consists of a large rectangular “reservoir” (2000   800 mm) connected to rectangular strips with widths decreasing systematically from 400 to 20 mm. There are several considerations that need to be kept in mind when designing the pattern. Firstly, the spacing between the strips has to be large enough to prevent cells in neighboring strips from joining each other. A spacing of 100 mm appears to be sufficient for Madin-Darby canine kidney (MDCK) cells. Secondly, the size of the reservoir should be large enough to allow the placement of a Polydimethylsiloxane (PDMS) barrier (see below). 2. The patterns were first designed using L-Edit layout editor (version 15.0). Export the file in Graphic Database System (GDS) format which is a compatible format for the laser writer. 3. Copy the design file to the PC and convert to lic format and transferred to the machine through FTP transfer. 4. Load the blank photomask on the laser writing system (Heidelberg DWL66fs), initialize the stage and write using a 10 mm writing head, 405-nm semiconductor laser with a spot size correction of 700 nm, energy of 80 mW, and a compatible filter. 5. After laser writing (5 h), develop the mask for 1 min using a mixture of AZ400K and DI water in 1:4 ratio, rinse with DI water, and dry with nitrogen. 6. Etch the chrome for 1 min using the etchant CEP-200, rinse with DI water, and dry with nitrogen. 7. Strip the remaining resist for 2 min using AZ-300T, rinse with DI water, and dry with nitrogen. The photomask is ready to be used for UV lithography or deep reactive ion etching (DRIE) process (Fig. 16.1A). FABRICATION OF SU-8 WAFERS USING UV LITHOGRAPHY 1. Clean the silicon wafers by sonicating them in acetone for 20 min. Rinse in IPA and dry with nitrogen. Heat the wafer on a hotplate to 200  C for 20 min to remove organic impurities. 2. Cool the wafer for 2–3 min and place it at the center of a spin coater (CEE 200X). 3. Dispense 5 ml of the photoresist (SU-8 3025) on the wafer and spin first at 500 rpm for 10 s and then 4000 rpm for 30 s.

16.1 Microfabrication Processes for Studying Cell Migration

1A

Pattern design

Photomask writing UV lithography

Si wafer

Development of patterns on wafer

PDMS stamp

PDMS molding

Photoresist (SU8) deposition

1B PDMS stamp

Fn coating

PDMS stamp microcontact printing

Printed pattern

PDMS block

Migration into the pattern

Formation of monolayer within the reservoir

Cell seeding

Placing of block

Backfill with pluronics

1C

20 mm

FIGURE 16.1 Schematic showing procedure for studying collective cell migration under varying geometrical confinements. (A) Soft lithography to obtain silicon masters with the pattern of interest. PDMS stamps are prepared from these wafers and used subsequently for microcontact printing. (B) Placing of a PDMS barrier on the microcontact-printed pattern such that cells are confined to the “reservoir” region of the pattern. The barrier is released when cells reach confluence in the reservoir. (C) Cell monolayer migrates from the reservoir into the fibronectin strips of different widths upon removal of the barrier.

4. Prebake the wafer on a hotplate at 95  C for 12 min. Let it cool down for 1 min. 5. Place the wafer on a mask aligner system (MJB4) and expose it to UV light for 8 s at a lamp intensity of 22 mW/cm2. 6. Postbake the wafer on a hotplate at 65  C for 1 min and at 95  C for 4 min. Let it cool down for 1 min. 7. Develop the wafer in SU8 developer for 6 min. 8. Rinse the developed wafer in IPA and dry it using nitrogen. Measure the depth of the features using a stylus-based profiler. The typical height of the features with the above protocol was 25 mm. The wafers can now be used for molding PDMS after silanization (Fig. 16.1A).

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16.1.2 Fabrication of micropatterned PDMS stamps and microcontact printing 16.1.2.1 Materials and equipment • • • • • • • • • • • • • • • • • • • •

Oven (Memmert, E024) Vacuum bell and pump or desiccators Hood Plasma cleaner (Harrick, PDC-002) UV/ozone cleaner (Bioforce Nanoscience, ProCleaner) Spin coater (Laurell Technologies Corporation, WS-400-6NPP) Glass bottom petri dishes (Iwaki, 3930-035) Bacteriological petri dishes (Greiner) PDMS (Sylgard 184, Dow Corning) 0.2 mm filters (Sartorius Stedim) Pluronics F127 (Sigma, P2443) MilliQ water Phosphate-buffered saline (PBS) Lyophilized fibronectin (Sigma, F2006) CY3 protein conjugation kit (GE Healthcare, PA23001) 0.1 M Sodium carbonate, pH 9.3 Dialyzing cassette (Thermo Scientific Pierce, 87728) Chemical mixer Stainless steel metal blocks cut to 0.5 mm  0.5 mm. These are easily available in most engineering workshops Sharp scalpel and single edge razor, curved forceps, epifluorescent microscope

16.1.2.2 Methods PREPARING CY3-CONJUGATED FIBRONECTIN 1. Reconstitute the lyophilized fibronectin powder with sterile conjugation buffer to obtain a final concentration of 1 mg/ml. Care should be taken to prevent foaming of the protein solution. Try also to mix it as gently as possible to avoid formation of fibrils. 2. Add the protein solution (1 ml) in the Cy3 dye vial. Close it and incubate at room temperature for 30 min with additional mixing every 10 min. Protect the dye from light as much as possible during the conjugation process to avoid bleaching. 3. Dialysis is used to separate the excess of unconjugated dye from the labeled fibronectin solution. Add the protein solution in a dialysis cassette and immerse it in a beaker filled with the separation buffer (PBS). Use a chemical mixer at very low speed to improve the dialysis overnight at 4  C. On the morning, change the PBS in the beaker and continue the dialysis two more hours. The whole process should not exceed 18 h. COATING OF PDMS ON GLASS BOTTOM DISHES 1. Prepare a mixture of PDMS by mixing the curing agent and the base in a ratio of 1:10.

16.1 Microfabrication Processes for Studying Cell Migration

2. Degas in a vacuum bell. Wait until there are no bubbles at the surface. It should take around 30 min. 3. Add between 0.5 g and 0.1 g of PDMS on a glass bottom petri dish (Iwaki) or a glass coverslip. The initial amount is not critical and can be adjusted. 4. Spin coat in two steps: 1 min at 1000 rpm and 1 min at 4000 rpm. 5. Cure it for 2 h at 80  C in the oven. SILANIZING SILICON WAFERS 1. Because of its toxicity, a vacuum bell should be dedicated to silanization. 2. Put the silicon wafer inside the bell and place a drop of silane (1H,1H,2H,2Hperfluorooctyl-trichlorosilane 97%) in a chemical cup beside the wafer in the bell. 3. Degas for 15 min and close the valve of the bell to maintain the vacuum inside the bell. Wait for 2 h for the silane vapors to deposit on the wafer. Gently release the vacuum and let the vapors escape. This step should be carried out in a chemical fume hood. 4. Store it in a clean glass dish in a dry place. Silanization need not been done every time the wafer is used. However, it is a good practise to repeat the silanization after using the wafer several times (>20–30 times). PREPARING MICROPATTERNED PDMS STAMPS 1. PDMS is prepared by mixing the base and the curing agent in a ratio of 1:10. Usually, 5–10 g is enough to completely cover the wafer. 2. Pour the PDMS on the wafer and degas it in a vacuum bell for 30 min until there are no more air bubbles on the surface. The PDMS can also be degassed before being poured on the wafer but sometimes bubbles form when the PDMS flows in to fill the patterns. 3. Cure it at 80  C for 2 h. 4. After cooling down, the PDMS can be peeled off with a pair of tweezers, cut into pieces, and stored in clean dishes to prevent dust deposit. 5. Dilute the unconjugated fibronectin and Cy3-conjugated fibronectin solution such that the final working solution contains 5% of the former and 2.5% of the latter. The stamping works better if fibronectin is diluted into PBS for microcontact printing (mCP) on plastic petri dish and in MilliQ water for mCP on PDMS. INKING THE PDMS STAMPS WITH FIBRONECTIN MIXTURE The inking of the PDMS stamps is slightly different based on whether the stamps are to be mCP on plastic petri dishes or on PDMS (Fig. 16.1B). ON PLASTIC PETRI DISHES 1. Prepare a PDMS stamp (with the pattern) that is 1 cm2 in size and plasma clean it for 1 min to make its surface hydrophilic. 2. Add 50 ml of the fibronectin mixture on the stamp and let it spread to cover the complete pattern. Incubate for 30 min at room temperature protected from light.

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3. After the incubation, drain the fibronectin mixture and let it dry under a laminar flow hood. The drying time is very critical. Patterns are not completely transferred if the stamp is too dry. If the stamp is too wet, the patterns are distorted and can merge with each other. A rough guide is to wait till a “dewetting front” appears and reaches the pattern. ON PDMS 1. Cut small pieces of PDMS (0.25 cm2) stamps containing the pattern. 2. Add 30–50 ml of fibronectin mixture and make sure the droplet covers the pattern completely. Incubate at room temperature for 45 min protected from light. 3. Remove the protein mixture and wash the stamp once by adding and removing a drop of water (50 ml) on it. Completely dry the stamp under a hood or with an air gun. 4. Put the PDMS-coated petri dish in UV/ozone cleaner for 15 min before stamping. mCP THE SUBSTRATES 1. Place the stamp on the middle of the dish with a pair of tweezers. Make sure the PDMS is in contact with the surface by pressing it very gently with tweezers. Viewed against the light with slight degree of tilt, the patterns are dark and should be visible. Wait for 5 min for the protein to be transferred (Fig. 16.1B). 2. Remove the stamps carefully. Use fluorescence microscopy to check the quality of the transfer. At this stage the petri dish can be stored dry for several days at 4  C. Nevertheless, it is better to use it within 24–48 h after stamping. Storing it in PBS appears to remove the proteins and the pluronics with time. 3. Incubate the dishes in a 0.2% solution of Pluronics F127 diluted in MilliQ water and for 1 h at room temperature to prevent cell adhesion outside the fibronectin pattern. A stock of 10% pluronics solution can be prepared and diluted when needed. Pluronics takes time to dissolve, so it should be prepared in advance. It is also better to filter it with a 0.2 mm filter. 4. Wash the dishes 3–4 times with PBS to remove the excess Pluronics solution (until dewetting of the substrate is observed).

16.1.3 Placing the PDMS barrier and cell culture FABRICATION OF PDMS BARRIER AND CELL CULTURE PDMS blocks (1 cm  1 cm) prepared by casting and curing PDMS (as described in 16.1.2.2 Methods - Preparing Micropatterned PDMS Stamps) in a plastic petri dish can be used as barriers. However, we have found that simple PDMS blocks tend to float and shift from their original position either during addition of culture medium or when the dishes are being transferred to the incubator from the laminar flow cabinet. To overcome this, we suggest the preparation of PDMS blocks embedded with small stainless steel blocks to make them heavy. The procedure to prepare them and place them on the micropatterns is described below.

16.1 Microfabrication Processes for Studying Cell Migration

16.1.3.1 Methods 1. Prepare a mixture of PDMS (1:10 w/w) and pour it into a 10 cm plastic petri dish to a height of 1–2 mm. Cure the PDMS partially at 80  C for 30 min in an oven. 2. Take out the petri dish and place the stainless steel blocks over the PDMS and press gently such that they stick to the surface. Place the blocks sufficiently wide apart (2–3 cm) so that it is easy to cut the blocks later on. 3. Pour additional PDMS mixture into the petri dish such that the blocks are at least covered to half of their height. Degas for 10–15 min and cure at 80  C for 2 h. 4. After curing, with a sharp scalpel, cut a rectangular block of PDMS with the embedded steel block and remove it from the petri dish. Using a single edge razor, trim the edges of the block perpendicularly to make them straight and smooth. The blocks can be stored in a dust-free environment for weeks. Before placing the blocks on the patterns, they can be incubated in 0.2% Pluronics solution for about half hour, washed, and allowed to dry. 5. Place the microcontact-printed substrate (plastic petri dish or PDMS-coated glass bottom dish) on an epifluorescent microscope with the appropriate filter cube and center the field of view to the pattern of interest (typically using a 20  objective). Pin the dish firmly to the microscope stage with one hand and bring a PDMS block gently with a curve forceps over the pattern. Switch on the transmitted light and decrease the intensity such that both the edge of the PDMS block (seen as a dark shadow) and the fluorescent pattern are visible. When the edge of the PDMS block is over the reservoir (typically  100 mm or so into the reservoir), release the block (Fig. 16.1B). It is important to release the block carefully since repeated misplacement, removal, and adjustments of the block can disrupt the fibronectin pattern. 6. Gently tap the block to make sure it is firmly in contact with the substrate. Mark the underside of the dish to show the edge of the block that is partially covering the reservoir of the micropattern. 7. Cells can be seeded in two different ways. In the first method, after placing the blocks, the petri dish is filled with 2–3 ml of culture medium. Using a pipette tip, about 100–200 ml of cell suspension (4  106 cells/ml) is deposited at the edge (marked) of the PDMS barrier. Cells are allowed to settle down for about 10 min and the dish is transferred to the incubator for overnight incubation. In the second method, 50,000 cells in 100 ml of medium are first deposited as a drop of near the edge (marked) of the PDMS barrier. The dishes are transferred to the incubator and cells are allowed to settle and spread over the reservoir for 1 h. The incubation time in both cases is dependent on the ability of the cells to adhere and the type of ECM protein printed and hence has to be optimized by checking under a light microscope. 8. Once cells have filled the reservoir, the barrier has to be removed. Before removing the barrier, it is necessary to wash the dish gently 2–3 times with PBS or culture medium to remove the nonadherent cells. Many of these cells are still alive and hence can easily attach to the free fibronectin pattern after the removal of the barrier.

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9. After washing, gently lift the PDMS barrier using a forceps. Sometimes, the PDMS barrier can be very strongly stuck to the substrate in which case, small twisting movements can be used to dislodge the block. If not done carefully cells at the edge of the PDMS barrier or even the whole cell monolayer might be lifted off. 10. Dishes are washed again with culture medium before mounting them for imaging. The method described earlier provides a novel in vitro migration assay that combines a “model wound” with micropatterned substrates (Fig. 16.1C). By varying the design of the micropatterns, the method can be exploited to address a variety of biophysical questions regarding collective cell behavior.

16.2 MICROFABRICATED SUBSTRATES FOR STUDYING EPITHELIAL GAP CLOSURE Closure of gaps is fundamental in several morphogenetic processes such as dorsal closure in Drosophila, ventral enclosure in Caenorhabditis elegans, during the last stages of neurulation and tracheal tube closure among others. Furthermore, efficient closure of gaps and wounds is essential to maintain the integrity of mature epithelial monolayer, which in turn is essential for survival of the organism. Here we present a method that allows forming and controlling gaps in a monolayer. This is done by placing PDMS stencils on a coverslip and culturing cells around them. Upon removal of the stencils, cells can be visualized and manipulated while they move to close the gap.

16.2.1 Fabrication of photomasks and silicon wafers Pillar arrays typically included 10  10 features, separated 150–200 mm between them to avoid cross-correlation between gaps. It is important to note that pillars should be around 100 mm in height in order to allow fluid transport beneath the pillars grid. Shorter pillars would result in a static culture, where cells do not grow at normal pace.

16.2.1.1 Materials and equipment •

• • • • • •

5 in. blank sodalime photomasks; size 5009, flatness: 4.3 mm, chrome type: LRC, ˚ ; OD: 3.02, reflectivity: 10.3%, resist/pre-bake: AZ1500, thickness: 1030 A ˚ ; grade: prime (BONDA TECHNOLOGY) thickness: 5003 A HMDS 379212 (SIGMA-ALDRICH PTE LTD) Spin coater (Brewer Science, CEE 200X) AZ9260 positive tone resist (AZ Electronic Materials, AZ9260) Hot plate (Harry Gestigkeit, PZ44) Mask aligner MJB4 (Suss Microtek, MJB4) AZ 400K Developer (AZ Electronic Materials, AZ 400K)

16.2 Microfabricated Substrates for Studying Epithelial Gap Closure

• • • • • •

MilliQ water Acetone/IPA Photoresist stripper AZ300T (AZ Electronic Materials, AZ300T) ICP tool for DRIE-Bosch process (Surface Technology Systems, STS ICP RIE) Stylus-based profiler (Dektat XT) Sonication bath (Elma Hans Schmidbauer)

16.2.1.2 Methods PHOTOMASK WRITING 1. Photomasks were designed as described in 16.1.1.2 Methods - Photomask Writing. FABRICATION OF SILICON WAFERS USING DRIE Silicon molds were fabricated using DRIE-Bosch process. Etching of silicon was used to meet two main prerequisites for the experiment: (a) very flat tops of the features and (b) high aspect ratio of the features and better height control. 1. Clean the silicon wafers by sonicating them in acetone for 20 min. Rinse in IPA and dry with nitrogen. Heat the wafer on a hotplate to 200  C for 20 min to remove organic impurities. 2. Dispense 2–3 drops of HMDS on silicon substrate and spin 3000 rpm for 30 s. 3. Dispense 5 ml of the photoresist (AZ9260) on the wafer and spin first at 500 rpm for 10 s and then 3000 rpm for 35 s. 4. Prebake the wafer on a hotplate at 110  C for 80 s. Let it cool down for 1 min. 5. Place the wafer on a mask aligner system (MJB4) and expose it to UV light for 35 s at a lamp intensity of 22 mW/cm2. 6. Develop the wafer in AZ 400K developer for 2 min 20 s. 7. Rinse the developed wafer in IPA and dry it using nitrogen. Measure the depth of the features using a stylus-based profiler. The typical height of the photoresist pattern with this protocol should be 5.6 mm. This height is good for the photoresist to act as a mask for deep etching of silicon substrate. 8. Load the patterned wafers on the chamber of STS ICP tool for etching (Bosch process). 9. Etch using the following parameters: etching cycle for 12 s (SF6 130 sccm; O2 13 sccm), passivation cycle for 8 s (C4F8 100 sccm), coil power of 800 W, platen power of 14 W, and substrate temperature at 20  C. 10. The total etching time is calculated based on the etching rate and the required depth of the feature. With the above protocol, etching rate was found to be 1.8 mm/min. Etch for 55 min to obtain features that are 100 mm deep. 11. Strip the remaining photoresist using AZ300T for 20 min in an ultrasonic bath. 12. Rinse the wafer in DI water and dry with nitrogen. Confirm the height of the feature using a stylus-based profiler. The wafer is now ready for silanization and preparation of PDMS masters (Fig. 16.2A). 13. Silanize the wafer as described in 16.2.2.2 Methods - Fabrication of PDMS Stencils Step 1&2.

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2A

Pattern design

Photomask writing UV lithography and development

Si wafer

1st PDMS molding

2nd PDMS molding

PDMS master mold

Photoresist (AZ9260) deposition

PDMS stencils

PDMS stencils

2B

Fn coating

Multiple steps of DRIE (Bosch process)

Coated substrate

Placing of stencils

Backfill with pluronics

Gap closure

2C

Cell seeding

Formation of monolayer within the stencils

FIGURE 16.2 Schematic showing the procedure to regulate the size of wounds within an epithelial monolayer using microfabricated stencils. (A) Various steps in fabrication of PDMS stencils consisting of micropillars. (B) Placing the micropillar stencils on cell culture substrate and seeding of cells. Stencils are removed when cells reach confluence. (C) Microphotographs of an array of circular stencils (white arrows) interrupting the continuity of a confluent monolayer of cells. Right image, enlarged scale of region in the box (scale bars 50 mm).

16.2 Microfabricated Substrates for Studying Epithelial Gap Closure

16.2.2 Fabrication of PDMS master and stencils 16.2.2.1 Materials and equipment • • • • •

Oven (Memmert, E024) Vacuum bell and pump or desiccators Hood PDMS (Dow Corning, Sylgard 184) Sharp scalpel and single edge razor, curved forceps.

16.2.2.2 Methods FABRICATION OF PDMS MASTERS 1. Weigh 20 g PDMS base for a 10 cm diameter wafer. 2. Add the crosslinking agent at a ratio of 1 part per 10 parts of base and mix thoroughly. 3. Degas the mixture in a vacuum jar for 30 min. 4. Pour PDMS mixture over the wafer and degas again until all bubbles have disappeared. Cure at 80  C for 3 h or at 60  C overnight. 5. Carefully peel off the PDMS master from the wafer with tweezers and store it in a clean petri dish. 6. Check the PDMS master under the microscope to ensure that the shapes of the patterns are well formed. Possible causes of the deformation of masters include unclean wafers, insufficient silanization, uneven mixing of PDMS base, and cross linker or insufficient curing. FABRICATION OF PDMS STENCILS 1. Place the PDMS masters in a vacuum jar with the micropatterned surface facing up. Add a few drops of silane (1H,1H,2H,2H-perfluorooctyl-trichlorosilane 97%) on a coverslip placed beside the master. Apply vacuum for 30 min to let the air out and then seal the jar to let it incubate for at least 2 h. A short high power plasma treatment of the master (5 min) before silanization is useful especially when the patterns contain very small features. 2. Carefully dispose the rest of the silane and transfer the PDMS master to a clean petri dish. 3. Repeat 1–6 steps above using the PDMS master instead of the wafer as the mold to produce PDMS stencils with micropillars. The PDMS stencils can be stored in a clean petri dish for a long time. Each master can be reused several times though we suggest discarding the master after using it for five times (Fig. 16.2A).

16.2.3 Placing the stencils on the culture support and cell culture 16.2.3.1 Materials and equipment • • • •

Hood Plasma cleaner (Harrick, PDC-002) Glass bottom petri dishes (MatTek) Pluronic F127 (Sigma, P2443)

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• • • •

MilliQ water PBS Lyophilized fibronectin (Sigma, F2006) Sharp scalpel and single edge razor, curved forceps, epifluorescent microscope.

16.2.3.2 Methods PLACING THE MICROPILLAR STENCILS ON CULTURE SUPPORT 1. Use a razor to cut the PDMS stencils containing a small patch of pillars. Typically, the PDMS stencil will have an area of 25–100 mm2. In order to facilitate cell seeding, cut the PDMS as close to the pillar array as possible. Avoid flat PDMS margins. 2. Incubate glass bottom petri dishes or glass coverslips with 100 ml of 20 mg/ml fibronectin in PBS for 1 h at room temperature or 20 min at 37  C. Rinse with PBS and let dry. Glass coverslips are more convenient for immunostaining experiments. In this case, glass coverslips must be thoroughly cleaned. 3. Treat the PDMS stencils (with pillars facing up) and glass bottom petri dishes with oxygen plasma at medium power for 30 s (Fig. 16.2B). 4. The power and time of oxygen plasma treatment depends highly on the plasma cleaner machine itself. The strength of the plasma can be roughly determined by its color. Violet color plasma is generally adequate. White plasma (pure oxygen) would result in too strong attachment and pink plasma (air) would result in weak attachment of the stencils to the substrate. Thus, an optimization step is required when using different plasma machines. 5. Carefully place the PDMS stencil with micropillars (facing down) on the glass substrate with tweezers. To ensure proper attachment of pillars to glass substrate, place the PDMS stencil in contact with glass, wait for 30 s, remove the stencil, and place it again. If the PDMS stencil cannot be removed, it indicates that the stencil is too firmly attached. Discard the sample. 6. Let the effect of oxygen plasma fade away from the PDMS for 1 h. 7. Incubate with 0.2% Pluronics in PBS solution for 1 h at room temperature. The time must be strictly controlled. Shorter Pluronics incubation time will not properly prevent cell attachment to the pillar walls resulting in cell damage when the PDMS stencil is peeled off. Longer incubation times will passivate all surfaces including the glass substrate. Thus, cells will not be able to attach to the fibronectin-coated glass. 8. Rinse carefully three times with PBS. During the last time, aspirate completely any liquid beneath the stencils with a yellow pipette tip and let it dry. 9. Sterilize with UV light in biosafety cabinet for 10 min and store the dish in the incubator before seeding of cells. CELL CULTURE AND CELL SEEDING 1. Prepare a flask of MDCK cells that is 80% confluent. 2. Trypsinize cells, centrifuge at 100  g for 3 min, and resuspend in small volume of medium (100–200 ml per flask). The concentration of cells will determine the final seeding density as the volume is kept constant by the height of the pillars.

16.2 Microfabricated Substrates for Studying Epithelial Gap Closure

3. Place a drop of medium with high concentration of cells at one side of the PDMS stencil. 4. Cells will go underneath the stencils by capillary force. This can be observed by the naked eye. The concentration of cells should be very high to ensure even distribution of cells. If the cells form aggregates during seeding, we find it helpful to seed cells in calcium ion free medium. Switch to normal culture medium after 1 h when the cells have attached to the fibronectin-coated surface. 5. Let cells sit and attach to the substrate in the incubator. After 30 min, add 2 ml of growth medium and incubate overnight. The time of incubation is important because the confluency of the monolayer will affect the result of the gap closure. Generally, for MDCK cells, we incubate for 12–16 h until the monolayer reaches confluence (Fig. 16.2C). Too short incubation will not produce a confluent monolayer whereas too long incubation will produce aggregates and dead cells. 6. On the following day, peel off the PDMS stencil by holding the PDMS stencil between the tweezers and pulling it up perpendicular to the glass substrate. Try not to move the stencil horizontally to avoid damaging the cells. This step is critical as the physical forces exerted on the cells may damage them. We find it helpful to attach a hook made from staples to the top of the PDMS stencils beforehand. A thin fishing wire (can be produced by stretching and rolling a piece of Parafilm) can then be used to pluck the stencil out. This will introduce a purely vertical movement of the pillars and prevent the horizontal movement that may damage the cells (Fig. 16.2B). 7. Wash the cells with medium for three times. If fluorescent microscopy is to be performed, change to phenol red-free (low autofluorescence) culturing media. 8. Proceed to time-lapse microscopy. It is essential to start the imaging immediately after the removal of the stencil as the closure process starts very quickly. We recommend removing the stencil on stage of the microscope after focusing and software setup. This will also help stabilize the temperate and reduce drifting during imaging. This method is designed to gain precise control over the experimental conditions under which cells close a gap. This is essential to closely mimic in vivo scenarios. For example, using microstencils we can control whether or not cells will suffer damage. Damage to cells can drastically alter cell signaling, cell mechanics and, consequently, mode of closure. The method as described earlier is a typical “model” wound experiment where cells are left intact as passivation with pluronics prevents cell attachment to the stencils (Fig. 16.3A, left). Conversely, cell damage may be inflicted by allowing adhesive proteins to be adsorbed onto the stencils. In this scenario, cells attached to PDMS are pulled and ruptured during removal of the stencil (Fig. 16.3A, middle). An alternative method to induce cell damage is pressing the stencils onto the cell monolayer (Fig 16.3A, right). This mechanical compression will inflict damage on the cells underneath the stencils and produce necrotic regions within the monolayer. Another example of the flexibility of this method is the possibility to vary the size and shape of the gaps (Fig. 16.3B and C). In nature, gaps display a large variation in size and shapes and are very often irregular. The method

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3A

Undamaged gap

Cells damaged by attachment to stencil

Cell necrosis caused by poking

Small features

Different geometry

Combination of parameters

3B

3C

50mm

50mm 50mm

FIGURE 16.3 Microstencils can be used to determine the initial conditions of gap closure. (A) Cell damage can be avoided or inflicted to different degrees and (B, C) the initial size and/or geometry of the gaps can be varied.

described earlier allows us to control the size and shape of the gap by altering the geometry of the stencils. Finally, it is important to note that this method is based on an array of stencils. Thus, it allows simultaneous analysis of multiple gaps. This not only increases the throughput of the experiment (Figs. 16.2C and 16.3B), but also allows direct comparison of different gaps (Fig. 16.3B and C).

GENERAL CONCLUSION The two methods described here use microfabrication approaches to provide welldefined initial conditions as well as precise spatial control of the size and geometry of the free space that epithelial cell sheets invade. Such methods not only allow us to

References

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