TECHNICAL ARTICLE

A simple microfluidic strategy for cell migration assay in an in vitro wound-healing model Min Zhang, MD1; Hongjing Li, PhD2; Huipeng Ma, PhD3; Jianhua Qin, PhD1 1. Department of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Science, 2. Department of Orthopedics, The First Affiliated Hospital of Dalian Medical University, and 3. College of Medical Laboratory, Dalian Medical University, Dalian, Liaoning, China

Reprint requests: Dr. J. Qin, Department of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, 116023, China. Tel: +86 411 84379650; Fax: +86 411 84379650; Email: [email protected] Min Zhang and Hongjing Li contributed equally to this article. Manuscript received: August 13, 2012 Accepted in final form: July 25, 2013 DOI:10.1111/wrr.12106

ABSTRACT In vitro scratch wound assays are commonly used strategies to measure cell repair rate, facilitating the study of cell migration, tissue reorganization, and cell division. This work presented a simple and novel microfluidic device that allowed a quantitative investigation of the cell migration and cell proliferation behaviors in an in vitro wound-healing model, especially focused on the scratch assay. The microfluidic device is composed of four units, which include cell growth regions and cell-free regions created by micropillars. Using this device, we evaluated the proliferation and migration process of human gastric epithelial cells in the presence of different concentrations of the epidermal growth factor, and investigated the migration behavior of mesenchymal stem cells toward tumor cells as well. This approach has the unique capability to create localized cell-free regions in parallel, and facilitate quantitative research on cell migration in the wound-healing process, providing a powerful platform for elucidating the mechanism of cell migration in regeneration medicine.

Wound healing is a complex and dynamic process of repairing cellular structures and extracellular components after tissue damage. The conventional scratch assay provides a simple method in vitro to study the directional cell migration in wound-healing assay. Usually, it is performed by scratching the confluent cell monolayer to create an artificial injury area surrounding the cells, using a pipette tip or a syringe needle. The cell migration in the wound-healing process is monitored by capturing photographic images over time, from the point of wound creation until its closure with cell confluence.1–3 By assessing the recovery of the scratched area, it is possible to quantify the rate of wound closure and study the cell–cell and cell–matrix interactions.4 The rate of “wound” closure is the sum of numerous cell processes, including cell migration, proliferation, and morphological change, in response to both soluble and solid substrate influences.5 Although a simple and easy preparation, this classical method still has its own limitations. When it is used as a manual method, the depth and width of the “wound” area are mainly dependent on the user’s individual operation with more varieties. In addition, scratching a cell monolayer using a pipette tip or a syringe needle might destroy and damage cells on the initial wave front, which, in some cases, actually results in the transient contraction of the cell wave front.6 Moreover, in the scratching process, the underlying matrix of specific ligands might be removed, and the extracellular matrix secreted by the previously existing cell monolayer might remain. Obviously, all these factors might potentially influence the outcome of experiments under different operaWound Rep Reg (2013) 21 897–903 © 2013 by the Wound Healing Society

tors. Particularly, owing to the intrinsic limitations, the scratch assay is inappropriate for the quantitative assessment of cell migration rates in the wound-healing process. In other modified tests, agar block,7 silicone elastomer,8 or other types of barriers9 have been used to modify the surface to prevent the cells. However, the conclusions drawn from these studies are still controversial. Microfluidic technologies are emerging as an invaluable tool for cell biology research. They can provide precise cell patterns and controllable reagents distribution in a reproducible fashion, which is not easily achieved by standard tissue dish culture. In the past few years, microfluidic devices have been applied for use in wound-healing assay and artificial wound formations to evaluate the healing process.10–17 Keese et al. reported the use of a DC/AC electrical signal to damage the cells grown on the electrodes in order to form the wound edges,10 but this method has limitations, in which the debris and other substances produced by injured cells may produce a negative influence on the wound-healing process and affect the obtained results. Nie et al. attempted to generate wound edges easily by using a laminar flow of trypsin solution,11–13 but it is still limited by the uncontrollable shape and area of the wound sites. Recently, Yarrow et al. created same-sized wounds in a 384-well plate by mechanically scratching the cell substrate with a pin array for a high-throughput woundhealing assay.16 The other method was also reported for a high-throughput wound-healing assay, based on patterned cell cultivation in parallel channels.17 However, these methods are still limited by the tedious manual operations, which might 897

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affect the reliability and reproducibility of the test and increase the risk of contamination. In this work, we present a simple and novel microfluidic strategy that overcomes many of the shortcomings in previous assays. The two-layer microfluidic device is composed of four units, where cell growth regions and cell-free regions are created by micro-fabricated polydimethylsiloxane (PDMS) pillars. Cells are initially seeded on the glass substrate surrounding the pillars and grown until confluence. Cell-free regions are created by the occupancy of the PDMS pillars. Upon the removal of the top PDMS pillar, a series of cell-free regions with a uniform shape and area are produced simultaneously, which can be quantitatively evaluated for the multiple cell migration assays involved in wound healing. Particularly, this method offers a uniform initial area, and the potential capability for integration, which can be easily used to study the dynamic process of cell migration in the scratch assay.

MATERIALS AND METHODS Cell culture and cell tracker staining

Human gastric epithelial cells (GES-1) were obtained from the Cell Bank of Xiangya Medical School of Central South University (Changsha, China). Adenoid cystic carcinoma cells (ACCM) were established from salivary gland tumors, which were kindly offered by Dr. Wang (Guangzhou, China). The procedures were reviewed and approved by the institutional review board for human subjects. Mesenchymal stem cells (MSCs) line was isolated from mouse bone marrow originally and transfected with green fluorescent protein gene. More identification information about this cell line could be obtained from the reference.18 Cells were cultured in different mediums (GES-1/high-glucose Dulbecco’s modified Eagle medium [HG-DMEM], MSCs ACCM/ α-MEM, Sigma Chemical Company, St. Louis, MO) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 U/mL streptomycin, and maintained at 37°C with 5% CO2 and 95% relative humidity. For staining cells with celltracker dye (Life Technologies Corporation, Carlsbad, CA), medium was removed from the dish and prewarmed working solution (5 μM) was added in it when the cells had reached 70% confluence. Cells were incubated for 45 min under growth conditions and then the dye working solution was replaced with fresh prewarmed regular medium followed by incubating cells for additional 30 min at 37°C. Microfluidic device design and fabrication

The device is composed of two layers, the top PDMS (Sylgard 184, Dow Corning, Midland, MI) layer and the bottom glass substrate. As shown in Figure 1, the device is composed of four uniform units. Four units share the central common reservoir for cell loading and solution perfusion. Each unit contains three pillars to prevent cells adhesion and three baffles to ensure uniform cells deposition in cell loading. Pillars (diameter 0.8 mm) are designed to form the cell-free regions. Firstly, cells were initially seeded around the pillars and grown until confluence. Subsequently, after the removal of PDMS layer, 12 “cell-free” sites were created in the confluent cell monolayer. 898

Figure 1. Images and operation of the microfluidic device for cell migration assay. (A) Schematic diagram of the device which is composed of two layers, the top PDMS layer and bottom glass substrate. This device is composed of four uniform units, in which each unit contains three pillar structures (yellow dots). (B) Photograph of the real microfluidic device used in this experiment. Bright red ink is used to display the liquid flow network. (C) Enlargement of one functional unit in microfluidic device. Cells were initially seeded around this columnar barrier and grown until confluence. (D) Creation of cell-free sites in one unit of microfluidic device. After the removal of PDMS pillar layer, three uniform center regions were created in the confluent cell monolayer. PDMS, polydimethylsiloxane.

The fabrication procedure of PDMS layer was described as studies previously.19,20 Briefly, glass wafers were spin-coated by SU-8 2035 photoresist (Microchem, Newton, MA) and patterned by photolithography. Then, masters were exposed to tridecafluoro-1,1,2,2-tetrahydrooctyl trichlorosilane vapor (Sigma Chemical Company). Sylgard 184 PDMS base and curing agent (Sylgard Silicone elastomer 184, Dow Corning Corp.) were mixed thoroughly (10:1 by mass) and degassed. The mixture was poured onto the master and solidified for 1 hour at 80°C. After cooling, the PDMS layer fabricated with pillar structure was peeled off from the master and trimmed to size. Holes were punched out of the PDMS slab to form reservoirs for introduction of liquid. The piece of PDMS was bonded to a glass slide reversibly. Before being used, the device was sterilized with UV light for 30 minutes.

Operation of the microfluidic device to create array wound sites

The operation of the microfluidic device for cell migration assay (shown in Figure 1) included several steps involving cell loading, cell culture, cell-free regions creation, and Wound Rep Reg (2013) 21 897–903 © 2013 by the Wound Healing Society

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migration assay. Simply, a suspension of cells at a density of 1 × 107 cells/mL was introduced into the central inlet reservoir. In co-culture migration assay, MSCs were cultured with GES-1 or ACCM both at a 1:1 ratio. The cells were infused into culture chambers quickly through the inlet under the observation of light microscopy (Leica DMIL, Danbury, CT). Then, the microfluidic device was maintained in a humidified atmosphere of 95% air, 5% CO2 at 37 °C overnight for appropriate cell attachment and spreading. The PDMS layer was removed by sterile tweezers after confluent cell monolayer formation, then the designated cell-free areas were created. Each microfluidic device created 12 similar cell-free sites (approximately 0.5 mm2). Cell proliferation analysis with Ki67 Fluorescence immunoassay

GES-1 cells were fixed by 95% ethanol and 0.1% Triton-X100 followed by rinsing with phosphate-buffered saline (PBS) solution three times, and then blocked with goat serum for 20 minutes. Then cells on the chip were incubated with antihuman Ki67 mouse monoclonal antibody (1:200) at 4 °C overnight. Negative controls were analyzed using PBS instead of primary antibody. After rinsing with PBS, cells were incubated with FITC-labeled anti-mouse IgG for 45 minutes at room temperature.21 To show cells’ proliferative activity, the number of Ki67-positive stained cells in defined area (approximately 1.7 mm2 surrounding the wound area) was shown. DAPI Staining Solution (diamidino-2-phenylindole, Sigma Chemical Company) is used to stain cell nuclei to represent cells position. For fluorescence immunoassay, the results were imaged by an inverse fluorescence microscope (Olympus IX 71, Olympus America, Center Valley, PA) and the statistical analysis was conducted by counting positive staining cells. p < 0.05 was considered as statistically significant.

Figure 2. The migration behavior of human gastric epithelial cell (GES-1) line under natural process without the addition of EGF. (A) Bright field images of cell migration at 0, 24, 48, and 64 hours. Fluorescence images at the same time points. Cellfree areas at 0 hour are about 0.5 mm2. Cells are stained with green live cell tracker. Scale bar = 0.4 mm. (B) The quantitative graph of central area under natural process in GES-1 at different time. Under natural conditions, the central areas were almost completely recovered after 64 hours from the starting point. EGF, epidermal growth factor.

Parallel wound healing assay on the microfluidic device

In this work, cell migration index (CMI) is used to identify the migration speed. Photographic images were captured every 8 hours upon the removal of PDMS layer by a video microscopy (Olympus IX 71), which enabled the recording of initial cell-free regions formation until the regions were covered by cells completely. According to the results, a linear regression equation was obtained, which indicated the present area/ initial cell-free area to time. Thus, CMI, which is equal to the absolute value of slope, was used to represent the migration speed of the tested cells. Briefly, the higher CMI value represents the faster cell migration speed. The data were expressed by mean ± SE and the difference between groups was analyzed by analysis of variance. p < 0.05 was considered as statistical significance.

RESULTS The feasibility of the microdevice for cellular assay in natural wound healing process

The designed microfluidic device for cell migration assay and the procedures to produce the multiple “cell-free” sites are Wound Rep Reg (2013) 21 897–903 © 2013 by the Wound Healing Society

shown in Figure 1. Prior to the assay, the human gastric epithelial cell line GES-1 cells were initially seeded and attached on the bottom layer of glass substrate until confluent via cell inlet, and the multiple circular cell-free sites with uniform shape could be produced by peeling off the top PDMS pillar mold, Due to the good biocompatibility of the PDMS material, the cells were observed to grow and proliferate with good viability. As shown in Figure 2, the cells were stained by green cell tracker dye to reveal the cellular viability, and no obvious difference was found in the shape and size of circles between the bright field and green cell-tracker images within the specified period of time, indicating the feasibility of this approach to maintain good viability for cellular assay without damage to the cells. It was observed that the cells located in the circle edge intended to proliferate and migrated toward the circle center area without the addition of external factors, suggesting that a natural healing process appeared during this assay. Under natural conditions, the cell-free areas were almost completely covered after 64 hours, from the starting point. In this microfluidic cell migration assay, a series of “cell-free sites” could be created by the removal of top PDMS mold and enabled the multiple cell migration assays simultaneously in parallel. 899

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Figure 3. The effects of EGF on the proliferation of GES-1. (A) Cells were fixed and stained for nuclei with DAPI (blue) after incubation with or without EGF (90 ng/mL) for 0, 24, and 48 hours. (B) Cells were fixed and stained for Ki67 by FITC (green) labeled antibody after incubation with or without EGF for 0, 24, and 48 hours. (C) Statistic graph showed more Ki67 positive stained cells in defined area (approximately 1.7 mm2 surrounding the cell-free area) after EGF treatment. Scale bar = 0.4 mm. Error bars represented the SD from six different wound areas in one device. p < 0.05 vs. control treatment. EGF, epidermal growth factor; GES-1, human gastric epithelial cell.

Effects of EGF on the healing process of epithelial cells in multiple wound sites

Several growth factors have been extensively investigated in normal and pathological wound-healing processes. The role of epidermal growth factor (EGF) in wound healing has been studied, ranging from the treatment of acute wounds to its limited effect in chronic wounds. In the following work, we used GES-1 cells as a model and characterized the effects of EGF on the migration of gastric epithelial GES-1 cells in the constructed wound-healing model in parallel. After the formation of multiple cell-free sites on the GES-1 cells monolayer, the cells were incubated with the medium containing different concentration of exogenous EGF. The cell migration process was recorded by a video microscopy, and the migration area from each culture device was calculated by an Image Pro Plus software (Media Cybernetics, Rockville, MD). Cell proliferation is an important event during the process of wound healing and can be detected by the associated protein Ki-67, which is a nuclear protein necessary for cellular proliferation. Ki-67 antigen is principally expressed during late G1, S, G2, and M phase of the cell cycle. Meanwhile, noncycling cells (G0 phase) lack Ki-67 expression. Thus, in our experiment, Ki-67 was used as a proliferation marker and DAPI staining was used to reveal the nuclei and the positions of the visualized cells during cell migration process. The number of Ki67-positive cells in designated areas (approximately 1.7 mm2 surrounding the cell-free area) was used as an index for the quantitative evaluation of cell proliferation. As shown in Figure 3, the proliferation of GES-1 was significantly enhanced after incubation with EGF (90 ng/mL) for 24 hours and 48 hours, and this process exhibited a timedependent fashion. More Ki67-positive cells were observed to maintain around the front edges than other regions far from 900

them, indicating that cell proliferation occurred in the front area. The phenomena probably results from the release of specific cell factors from the cells surrounding the edge area. We further investigated the migration behavior of GES-1 cells under EGF treatment at different concentrations (60, 90, and 120 ng/mL). As shown in Figure 4, the migration ability of GES-1 was obviously enhanced after the addition of EGF with different concentrations for 8 hours. The cells were found to occupy the central circular area after 48 hours after the addition of 120 ng/mL EGF in culture medium. Actually, as we can see from Figure 3, the cells that proliferate appear somewhat confined to the nonmigrating cells at the time point of 24 and 48 hours; therefore, we assume that the cells presented in the cell-free regions are mainly due to the cells that migrated from the surroundings. According to the results, the concentration of growth factor EGF has a close relationship with the CMI, and this can be used to represent the migration speed of the tested cells. At the concentration of 120 ng/mL EGF, the CMI value is 0.237, which is much higher than that of 60 and 90 ng/mL EGF, respectively. However, the CMI value is very close to each other at the EGF concentration of 60 and 90 ng/mL, which are both higher than that from control group. Migration of mesenchymal stem cells toward cancer cells

MSCs are bone marrow-derived mesenchymal cells, which have the capacity to differentiate into cells with mesodermal, ectodermal, and endodermal characteristics. In some cases, tumor tissue had the ability to become enriched in MSCs and guide them to migrate to cancer nest. The migrated MSCs usually differentiate into different type cells to circumscribe the infiltration of cancer cells and “repair” wound-like tissue defect. In this work, we used the established approach to evaluate the MSCs migrations behavior in co-culturing with cancer cells. The mixer of MSCs and adenoid cystic carcinoma cells were seeded and co-cultured on the micro-device for 0 to 48 hours, followed by the creation of circular cell-free spaces on the device. As seen in Figure 5, MSCs showed a higher percentage in the circle zone when co-culturing with ACCM cells instead of control group. Actually, the increased numbers in the center area included the migrated and proliferated MSCs as well. It is assumed that the physical contact between MSCs and ACCM cells was necessary for MSCs migration, indicating the possible recruiting role of ACCM cells by secreting autocykine or paracine factors.

DISCUSSION The motivation of this study is to create a simple and efficient cell migration assay in the in vitro model of wound healing by using the microfluidic approach. In a traditional experiment, the scratch assay is a widely adopted technique to investigate wound-healing processes,22,23 but it still has some drawbacks in reproducibility due to the lack of quality control and capability for a high-throughput assay. The traditional scratching assay might remove not only the cells, but also the matrix or coating from the surface. In contrast, the proposed approach provides a convenient and simple strategy to create multiple “cell-free” site arrays upon the removal of the PDMS pillars, and parallel cell migration assays could be performed simulWound Rep Reg (2013) 21 897–903 © 2013 by the Wound Healing Society

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Figure 4. The cell migration process of GES-1 in response to different concentration of EGF. (A) GES-1 cells were treated with 0, 60, 90, 120 ng/mL EGF after peeling off the PDMS layer. The enhanced migration of the GES-1 after the addition of EGF at different concentrations for 8 hours, and the complete coverage of central area were observed after 48 hours, in addition to 120 ng/mL EGF in the medium. Scale bar = 0.48 mm. (B) Statistic graph of each group in the coverage of the central areas. A linear regression equation was obtained to represent the ratio of central area to time. Error bars represented the SD from six different areas in one device. (C) Cell migration index (CMI) (equal to the absolute value of slope) was used to represent the migration speed of cells. EGF treatment (60, 90, 120 ng/mL) markedly increased the migration speed of cells. Error bars represented the SD from six different areas in one device. p < 0.05 vs. control treatment. EGF, epidermal growth factor; GES-1, human gastric epithelial cell; PDMS, polydimethylsiloxane.

taneously. Particularly, it is possible to produce highthroughput cell-free sites with the same initial area. The shape, position, and area of scratch sites are perfectly controlled without cell damage and contraction. The localized cell-free region is beneficial for a dynamic observation and a quantitative measurement of cell migration. The compatible PDMS and glass substrate make a low-cost cell culture and assay possible, and display great potentials for practical use by the evaluation of multiple types of cells during the cell migration process. Generally, epithelial cell migration from the wound margin and the following proliferation indicate the early process of gastrointestinal ulcer healing. In this study, GES-1 is used as a model to mainly show the cell migration and proliferation in the wound-healing process with this device. The gastric mucosa injury usually occurs with the presence of alcohol and acid products, and the imbalance between aggressive and defensive factors determines ulcer formation. The ulcerhealing process requires restoration of epithelial continuity (i.e., reepithelialization) and reconstruction of the underlying tissues, such as blood vessels and muscle layers. According to the result, GES-1 cells can grow and proliferate in the central area to facilitate the cell migration and cover the whole central region gradually. An important application of cell migration assay is in assessing the effects of drugs or the growth factor on the wound-healing process. In this work, the effects of different concentrations of the exogenous EGF on the migration of GES-1 cells were analyzed, and adequate reproducibility could be achieved by evaluating multifactor effects on this process simultaneously. These results validate the repeatability of the microfluidic approach for evaluation in the cell migration process, and the potential for application in other Wound Rep Reg (2013) 21 897–903 © 2013 by the Wound Healing Society

cell types and models. In addition, this device provides a dynamic, parallel, and quantitative approach for a highthroughput cell migration assay. In this article, we also try to investigate the migration ability of mesenchymal stem cells when co-cultured with tumor cells. Clinical and experimental data have shown that MSCs could selectively migrate to the sites of injury and tissue regeneration. In a sense, the tumor microenvironment can be considered as the site of tissue injury or a “wound that never heals.”24 The MSCs have been previously observed to influence the morphology and proliferation of cells within their vicinity through a combination of cell–cell interactions and the secretion of chemo-attractant cytokines.25 In this work, we explored the interaction between MSCs and ACCM cells using the established in vitro wound-healing model. As shown in Figure 5, a higher number of MSCs could obviously migrate into the central area when co-cultivated with ACCM cells instead of GES-1 cells, indicating the enhanced migration ability of MSCs in the presence of tumor cells. This novel and user-friendly approach can also offer the potential to investigate more complicated cell migration processes in vitro. Further work is proceeding to reconstruct a 3D wound model by culturing cells in a multiple-component extracellular matrix, such as collagen, basement membrane, or fibronectin, and this will mimic the physiologically relevant cell behaviors in vivo.26,27 Meanwhile, related cells, such as inflammatory and other cells, will be introduced to this device for the creation of new applications.28,29 Using this device, many cues of cell migration can be further tested for fundamental investigation and clinical application. Cell migration is an important biological procedure in the process of wound healing, which involves many cell types, growth 901

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REFERENCES

A MSCs +GES-1

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Figure 5. (A) The migration process of MSCs when co-cultivated with GES-1 and ACCM, respectively. MSCs were cultured with GES-1 and ACCM both at a 1:1 ratio. ACCM, GES-1 (CellTracker Red); MSCs (CellTracker Green). Scale bar = 0.2 mm. (B) The comparison of the numbers of MSCs migrated to central area in co-culturing with GES-1 and ACCM cells. MSCs showed a higher tendency to migrate into the central area after co-culturing with ACCMs. Error bars represented the SD from six different areas in one device. p < 0.05 vs. control treatment. ACCM, adenoid cystic carcinoma cell; GES-1, human gastric epithelial cell; MSC, mesenchymal stem cell.

factors, and other proteins. We believe that our proposed approach is a reasonable alternative for cell migration study, with its simplicity and low cost, which might provide a quantitative method for a high-throughput assay in the applications of regeneration medicine.

ACKNOWLEDGMENTS Source of Funding: This research was supported by Knowledge Innovation Program of the Chinese Academy of Sciences (KJCX2-YW-H18), the National Nature Science Foundation of China (No.91227123, No.11161160552), Key Projects in the National Science & Technology Pillar Program in the Twelfth Five-year Plan Period of China (No.2012BAK02B00, 2012BAK02B03), Special Fund for Agro-scientific Research in the Public Interest (No.201303045). Conflicts of Interest: None of the authors have any conflict of interest. 902

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A simple microfluidic strategy for cell migration assay in an in vitro wound-healing model.

In vitro scratch wound assays are commonly used strategies to measure cell repair rate, facilitating the study of cell migration, tissue reorganizatio...
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