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Development of a Single Cell Migration and Extravasation Platform through Selective Surface Modification Steven A. Roberts, Allen E. Waziri, and Nitin Agrawal Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04391 • Publication Date (Web): 02 Feb 2016 Downloaded from http://pubs.acs.org on February 4, 2016
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Analytical Chemistry
Development of a Single Cell Migration and Extravasation Platform through Selective Surface Modification Steven A. Roberts,a Allen E. Wazirib,c and Nitin Agrawala,c* a
Department of Bioengineering, George Mason University, Fairfax, VA, USA.
b
Department of Neurosurgery, Inova Fairfax Hospital, Fairfax, VA, USA.
c
Krasnow Institute, George Mason University, Fairfax, VA, USA *E-mail:
[email protected]. Phone +17039933970
Cell migration through three-dimensional tissue spaces is integral to many biological and pathological processes, including metastasis. Circulating tumor cells (CTCs) are phenotypically heterogeneous and in vitro analysis of their extravasation behavior is often impeded by the inability to establish complex tissue-like ECM environments and chemotactic gradients within microfluidic devices. We have developed a novel microfluidic strategy to manipulate surface properties of enclosed microchannels and create 3D ECM structures for real-time observation of individual migrating cells. The wettability of selective interconnected channels is controlled by a plasma pulse, enabling the incorporation of ECM exclusively within the transmigration regions. We applied this approach to collectively analyze CTC-endothelial adhesion, trans-endothelial migration, and subsequent motility of breast cancer cells (MDA-MB-231) through a 3D ECM under artificial gradients of SDF-1α. We observed migration velocities ranging from 5.12µm/hour to 12.8µm/hour, which closely correspond to single-cell migration in collagen blocks, but significantly faster than the migration of cell aggregates. The compartmentalized microchannels separated by the porous ECM makes this in vitro assay versatile and suitable for a variety of applications such as inflammation studies, drug screening, and co-culture interactions.
Chemotactic migration of cells plays an instrumental role in many physiological as well as pathological processes including embryogenesis1,2, wound healing3, immune response4, atherosclerosis5,6, and cancer progression7,8. The directional migration of cells is guided by complex gradients of signaling molecules present in the extracellular microenvironment. Particularly during metastatic progression or immune cell response, circulating cells in blood must adhere to the vascular endothelium, extravasate into the surrounding tissue and subsequently migrate through the three dimensional intercellular ECM to reach the target. To analyze the dynamic events and interactions that occur during homing and transmigration of motile cells across the endothelium, several in vitro approaches have been developed allowing directional migration under controlled conditions. For example, Boyden chambers provide a well-established technique for studying chemotactic responses of cells9–11. While commercially available and convenient to use, the amount of information obtained from Boyden chambers is limited to the number of cells that migrate across the porous membrane and intermediate events such as cell-cell and cellECM interactions and migratory patterns remain obscured. Alternatively, microfluidic platforms have been developed for real-time measurements of chemotactic behaviors. By generating on-demand chemical gradients, cells can interact with neighboring cells or the immobilized ECM in a precisely controlled microenvironment and migration behaviors can be observed in real-time. Our group as well as many others have
previously demonstrated motility analysis of cells on functionalized two dimensional (2D) surfaces12,13 and fluid filled narrow capillaries14 utilizing small volumes of biological samples. Although such microfluidic approaches allow both qualitative and quantitative analysis of cell motility, spatial extracellular ECM arrangements and three dimensional (3D) constraints found within the in vivo systems are often neglected. Three-dimensional migration platforms can be fabricated by infusing the channels with desired biosynthetic or natural ECM, such as Matrigel or collagen in which a chemokine gradient can be generated. However, the ability to create intricate systems containing interconnected threedimensional network of ECM (representing abluminal tissue) and hollow channels (representing vascular lumen) to investigate multiple extravasation events remains challenging. Additionally, circulating migratory cells (CTCs and leukocytes) are known to be phenotypically heterogeneous15– 18 , and therefore understanding their interactions as well as migration patterns at the single cell level is critical19,20. We have developed a novel and simplified strategy to modify surface characteristics of enclosed microchannels allowing fabrication of intricate ECM structures for extravasation and migration analyses of cells in a threedimensional microenvironment. This approach not only offers a more biomimetic experience, but also addresses current limitations of monitoring 3D cell migration of individual cells21–23. Without requiring any chemical treatment, the wettability of desired regions of the interconnected channel
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Analytical Chemistry
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subsequently incubated at 37 OC for 5 minutes or until the Matrigel was fully gelled. Empty channels were immediately filled with PBS to limit hydrogel evaporation.
network can be rapidly altered providing the ability for automatic surface tension driven infusion of ECM gel to recreate in vivo like conditions. We have demonstrated the applicability of this system by monitoring adhesion, extravasation and migration of breast cancer cells (MDA-MB231) through an endothelialized microvessel in response to a gradient of chemoattractant, stromal derived factor-1α (SDF1α)24,25. Nevertheless, this approach is not limited to extravasation events and can potentially be incorporated in studies pertaining to drug screening, drug delivery, immunology, atherosclerosis etc. The ability to analyze interactions and migration profiles of individual cells under precisely controlled conditions eliminates the inaccuracies arising from population based analyses.
Gradient Generation and Characterization. The diffusion coefficient (Dc) of the Matrigel was determined using a range of molecular weight dextran standards (70-10 kDa). The dextrans (Life Technologies) were functionalized with amine groups and conjugated to a Cy5 fluorophore through an NHS-amine reaction. The reaction was allowed to occur for 16 hours in the dark at room temperature. The excess dye was removed via centrifugation using a spin column (Pierce, Rockford, IL). Fluorescein (Sigma) was also used as a low weight diffusion standard. Diffusion was monitored via time-lapsed epifluorescent microscopy. Time-lapsed images were taken every 3 seconds due to high diffusivity of the dye through porous matrigel. The images were imported into ImageJ (NIH, Bethesda, Maryland) and the individual color channels were extracted.
EXPERIMENTAL SECTION Device Fabrication. Microfluidic features were designed using AutoCAD software (Autodesk Inc.) (Figure 1A) and were written on a chrome mask using a DWL 66+ 405 nm laser writer (Heidelberg, Germany). Standard soft lithography procedures were used to create a silicon master wafer with SU-8 photoresist (Microchem, MA). Devices were fabricated by pouring Sylgard 184 (Dow Corning, MI) onto the mold, degassed, and thermally cured at 70 OC for two hours26. The cured PDMS sheet was peeled off of the master, cut apart, and inlets and outlets were made using an 18 gauge flat end sharpened needle. PDMS devices and glass slides were cleaned by sonication in 70% isopropanol for 20 seconds and rinsed with deionized water to remove debris and fine PDMS particles generated during punching (Figure S1 A). Once dry, the surface of the slides and devices were irradiated for 60 seconds (pattern side up) with oxygen plasma (Plasma Etch, Carson City, NV) and irreversibly bonded to obtain a finished device.
On-chip Cell Culture. All cell lines were obtained from ATCC (Manassas, VA). Fetal Bovine Serum (FBS), Dubelcco’s Modified Eagles Medium (DMEM), 1% Penicillin-Streptamyacin, CellTracker-CMFDA, and 0.5% Trypsin/EDTA were purchased from Life Technologies (Grand Island, NY). HUVECs were grown to 80% confluence in Endothelial Growth Media (Lonza, Walkersville, MD) and trypsinized for one minute with 0.05% trypsin-EDTA solution. Trypsin was neutralized using 20% FBS in PBS. HUVECs were collected via centrifugation and resuspended to a final concentration of 1.6e7 cells/mL. Following infusion of the Matrigel, the central channel of each device was coated with 0.2% gelatin from porcine skin (Sigma) for 30 minutes followed by 2 µg/mL fibronectin (Biomedical Technologies, Stoughton, MA) for 2 hours, both at room temperature. Once seeded in the device, HUVECs were allowed to attach for 30 minutes and non-adhered cells were flushed out with fresh media. Media was refreshed every 24 hours. HUVECs were grown in the device for 2 days prior to their interaction with cancer cells. MDA-MB-231 metastatic breast cancer cells (TCs) were grown to 80% confluence in DMEM containing 10% Fetal Bovine Serum and 1% Penicillin-Streptamyacin. The cancer cells were removed from their flask utilizing similar protocol as HUVECs. Following collection via centrifugation, cancer cells were stained with CellTracker for 30 minutes. The cells were then washed twice with endothelial basal media to remove excess dye. Prior to their addition to the devices containing confluent HUVECs, cancer cells were washed three times via centrifugation with basal media to remove residual growth factors that may influence migration. Confluent devices were washed with endothelial basal media and incubated for at least 3 hours in basal media to remove residual growth factors. TCs were introduced into the lumen channel of the device and allowed to attach to the underlying endothelium for two hours. Non-adhered cells were flushed out with fresh media. 300 ng/mL SDF-1α (Peprotech, Rock Hill, NJ) was added into one of the side channels and fresh media to the other side channel as a control.
Selective Surface Modification and ECM Infusion. Completed devices were rendered hydrophobic by an overnight bake at 70 OC. Prior to hydrogel infusion, devices were sterilized by exposing them to a 40 watt UV lamp for 10 minutes. By exposing the gel loading channel to a pulse of low intensity plasma from a handheld corona gun (Figure 2A), the migration channels of the initially hydrophobic device were selectively activated. Surface tension and hydrophobic forces keep the hydrogel within the migration channel. A 4.5MHz BD-20AC handheld corona treater (Electro-Technic Products, Chicago, IL) was used to produce a short pulse of plasma (
