Mayuree
Chanasakulniyom
,
Andrew
Glidle
and
Jonathan M.
Cooper
*
The Division of Biomedical Engineering, School of Engineering, The University of Glasgow, G12 8LT Glasgow, UK. E-mail: jon.cooper@glasgow.ac.uk; Fax: +44 (0)141 330 6002; Tel: +44 (0)141 330 4931
First published on 16th October 2014
Cell proliferation and migration are fundamental processes in determining cell and tissue behaviour. In this study we show the design and fabrication of a new single cell microfluidic structure, called a “vertically integrated array” or “VIA” trap to explore quantitative functional assays including single cell attachment, proliferation and migration studies. The chip can be used in a continuous (flow-through) manner, with a continuous supply of new media, as well as in a quiescent mode. We show the fabrication of the device, together with the flow characteristics inside the network of channels and the single cell traps. The flow patterns inside the device not only facilitate cell trapping, but also protect the cells from mechanical flow-induced stress. MDA-MB-231 human breast cancer cells were used to study attachment and detachment during the cell cycle as well as explore the influences of the chemokine SDF-1 (enabling the quantification of the role of chemokine gradients both on pseudopod formation and directional cell migration).
Typically such arrays have been made from PDMS and glass, functionalised either with extracellular matrix (ECM)5 or specific antibodies/cell-adhesive ligands.6 Although such devices are considered to be straightforward for high-throughput trapping and single-cell studies, they still have some limitations in use. The side walls of the traps not only impose restrictions on cell shape, but they also have a high surface area around the cell, which can result in a reduction in cell growth7 and a restriction in cell–cell communication. Thus, although attempts have been made to describe these structures in terms of the cell's natural environment, the reality is that the cell may be under stress, in situ.8
Photodefinable silicon elastomer (PDSE) is a photopatternable, spin-on polymer. The chemical composition of the PDSE is that of a silicone resin dissolved in a plasticising silicone matrix.9,10 PDSE has previously been extensively used for electronics and optical applications9,11–13 as its properties (which include low stress, low modulus, low temperature curing, low shrinkage, good moisture resistance, good dielectric properties, high thermal stability and high transparency) are of benefit to those applications. It has, however, also found various applications in biological studies, not least as it has demonstrated biocompatibility with various cell lines,14 providing an easy way to develop complex 2D and 3D structures. In this respect, the polymer has been shown to adhere well both to glass, as well as other elastomeric polymers such as polydimethylsiloxane (PDMS). In this study we use PDSE, PDMS and other photopatternable resist layers (such as AZ4562 photoresist) to create multilayer constructs providing a new geometry for the study of the chemokines and the response of individual cells.
Stromal cell-derived factor (SDF-1), also known as CXC chemokine ligand-12 is a small cytokine belonging to the CXC chemokine family. It binds exclusively to its receptor CXCR4, expressed on many hematopoietic cells such as CD34+ hematopoietic stem cells, T-lymphocytes, B-lymphocytes and neutrophils. It is also known as a co-receptor for HIV entry to the cell.15 CXCR4 is also expressed in various types of cancer, including those present in breast cancer.16–20 The expression of CXCR4 is undetectable in normal breast, ovarian, prostate epithelial cells. However, it is significantly up-regulated in cancer cells (it is the most common chemokine receptor expressed in most cancer cells).21–23
The interactions of SDF-1 and CXCR4 have been shown to play a critical role in regulating the metastatic destination of breast cancer cells.24 SDF1 has also been shown to increase the invasiveness and migration of breast cancer cells when present as concentration gradients (i.e. the cell responds to the presence of a change in concentration of the chemokine).24–26 As SDF-1 is highly expressed in lymph nodes, bone marrow, lung and liver, it may therefore account for the migration of breast cancer cells to these sites.22,24,27,28 Indeed, reduction of the CXCR4 expression or using CXCR4 antagonists can effectively inhibit the metastasis of breast cancer cells,22,29,30 indicating that the interaction between SDF-1 and CXCR4 is crucial for cancer metastasis.
In this paper, we are interested in the creation of a new chip structure that addresses the limitations of existing trapping geometries (which, it has been argued, can restrict cell growth and cell communication). To demonstrate the efficacy of the new geometry, we explore adhesion, proliferation and migration assays in the study of MDA-MB-231 in the presence of concentration gradients of SDF-1α.
The middle layer has an array of circular holes (40 μm in diameter) for cell trapping, aligned directly above the cavities. The array of holes is used to funnel the individual cells into the cavities of the bottom layer. The structures of both the bottom layer and middle layer were fabricated from a combination of PDSE and AZ4562 photoresist (see ESI†). The top layer was a PDMS chamber used for cell loading and perfusion with cell culture medium.
Cell loading was performed by injecting a cell suspension into the top right, top layer inlet port of Fig. 1D using PEEK tubing (100 μm inner diameter) fitted to a gas-tight syringe (Hamilton) and microsyringe pump (Harvard Apparatus). Various cell densities were used to explore their influence on the microwell loading characteristics and the optimal one in terms of single cell occupancy and good overall filling was found to be 3 × 106 cell ml−1 (in culture media) The procedure was as follows: a flow rate of 0.5 μl min−1 was used for the initial introduction of the cells to prevent them sedimenting in the PEEK tubing; this was then reduced to 0.1 μl min−1 after the cells had entered the device (to reduce mechanical shear stress and potential cell damage during loading). The suspended cells were flowed with the flow rate of 0.1 μl min−1 for 30 s after which the flow was paused for 30 s. This procedure was repeated twice to achieve a maximum loading (estimated as 85%). Cells were perfused with culture media containing either 100 ng ml−1 of SDF-1α (Abcam) or free from added SDF-1α. After this was completed, the cell loading syringe was replaced, and SDF-1α containing media was introduced through the top left, top layer inlet (Fig. 1D). At the same time, SDF-1α free media being introduced through the bottom right, top layer inlet (Fig. 1D). For both inlets, the fluid flow rate was maintained at 0.05 μl min−1. Where the two fluid streams meet (in the centre of the microhole patterned region), a steep gradient of SDF-1α was generated by the diffusion of SDF-1α from the SDF-1α media, into the SDF-1α media. As in other studies, after a period of approximately 20 min, the spatial extent of this gradient reaches a steady state as a consequence of the continual removal and replenishment of material by the constantly flowing streams.31 Importantly, the reliability and pulse free nature of the syringe pumps used ensured that this gradient remained stable during the course of the experiments.
The MDA-MB-231 cell migration toward SDF-1α was analysed using ImageJ32 with two additional plug-in modules, namely Manual tracking (to collect movements of individual cells) and Chemotaxis tool (to analyse the records of cell movement off-line). Cells of interest were selected visually and image sequences were analysed frame by frame.
Fig. 2 An overview of the velocity contours for a part of the VIA device obtained from 3D CFD simulations. |
The velocity contours show that the flow rate inside the VIA is two orders of magnitude lower than the velocity in the top chamber, giving a greatly reduced shear stress (the velocity in the middle of the top layer chamber and the bottom layer linker are approximately 1 × 10−4 m s−1 and 4 × 10−6 m s−1, respectively). Calculations indicate that for an inlet flow rate of 1 × 10−4 m s−1, the shear stress experienced by a cell adhered to the base of the bottom channel in the device would be ~0.0002 Pa. This value is negligible compared to the 0.2 Pa that has been shown to influence T-lymphocyte migration in solutions containing SDF-1α.33 In that study it was shown that cells moved in the direction opposing the shear stress. Thus in studies such as that here, where we wish to explore the influence of chemical gradients alone, it is important to be able to eliminate possible influences from fluid flow, whilst still maintaining a means to deliver fresh nutrients and chemokines. Note, in this device, if desired, after seeding, the adhered cells can be subjected to high shear stress flows by using the bottom layer ports as inlets together with a high inlet flow rate.
As shown in Fig. 3, the majority of the fluid flow (streamlines) are in the top layer. This, combined with the top layer having a larger cross-section area than the bottom layer outlets, make it relatively easy to establish gradients of chemicals across the device (in x,y).
It was found that when using a loading density of 3 × 106 cells ml−1 and flow rate of 0.1 μl min−1 for 30 s, approximately 60% of the of the chambers occupied were occupied by single cells and ~50% of all of the available chambers were occupied by one or more cells. On repeating the procedure, the overall occupancy of cell loading increases to ~85% (Fig. 4). These numbers are comparable with those obtained using open microhole devices37 with an open hole of the same size as the top layer open hole here. However, as suggested by the data of ref. 37, the structure of the device employed here means that the single cell occupancy is proportionately higher than would be the case if top layer hole opening were the same size as the bottom layer cavity. Importantly, the larger sized bottom layer cavity enables the study of division and migration of cells that are within a semi-encapsulated volume defined in three dimensions.
As stated, cell attachment and proliferation inside the VIA devices was observed using time-lapse microscopy. Fig. 5(a–c) is representative of results and illustrates the attachment of MDA-MB-231 cells in three different positions under cell medium perfusion (at 0.05 μl min−1). Attachment occurred between 2.5–5 hours after loading (the process of attachment itself is important for the study of cell responses towards a stimulus, as the cells need to attach to the surface in order to migrate, and forms the basis of routine assays, per se).
Cell division in the VIA devices, Fig. 6, shows how the cell round-up and lose attachment to the surface. Cells take approximately 1 hour to divide after detachment from the surface. This behaviour and its duration corresponds to the typical timings for eukaryotic cells to complete the M (mitosis) phase where it is known cells become less adhesive and have the rounded morphology due to disassembly of focal adhesions.36,38,39 After mitosis, the cells started to spread again.
Attachment and detachment during the cell cycle is critical to cell proliferation, and the low fluid flow rates in the VIA serve to protect the dividing and daughter cells well from shear stress, see e.g.Fig. 3. Cell adhesion in Fig. 5 and cell division in Fig. 6 indicate that the natural environment in the device does not have a significant influence on cell survival, nor does it produce effects that lead to a quiescent cell state.
Fig. 7A1, B1 and C1 show cell paths during 20 hours with the final positions of cells at the end time point from the top, middle and bottom position in the device (defined as in Fig. 7D), respectively (n = 30 cells). Cell paths and final cell positions are shown relative to their starting point, as the origin (0,0).
Fig. 7A1 shows migration pathways in a constant concentration of SDF-1α (no gradient) whereas Fig. 7C1 shows the migration in a region of very low SDF-1α concentration (<5 ng ml−1). Fig. 7B1 shows that the greatest number of migration paths are in the region of the steepest concentration gradient.
Fig. 7A2, B2 and C2 show the trajectories of growth of pseudopods analysed from the top, middle and bottom position in the device, respectively. In the middle, Fig. 7B2, and the bottom, Fig. 7C2, the distribution of the pseudopod direction was biased toward the direction of the SDF-1α source, whereas the distribution of pseudopod directions in the top position is random, Fig. 7A2.
To confirm that this directional bias was due to the SDF-1α gradient, the flow direction of the SDF-1α was switched by introducing culture media either containing 100 ng ml−1 of SDF-1α or without the SDF-1α through the bottom layer inlets at the same flow rate, 0.05 μl min−1, Fig. 8. The cell paths and the final position of cells in the middle position having both SDF-1α and cell culture medium were recorded, Fig. 8B1. Again, the controls were observations recorded in regions of uniform SDF-1α concentration, Fig. 8A1 – where cells move randomly. In regions of low SDF-1α concentration, Fig. 8C1 cells again move toward the higher concentration of SDF-1α. Similarly, the distribution of pseudopod directions was biased toward the direction of the SDF-1α source for cells in both this region and the region of the steepest concentration gradient, Fig. 8B2. In contrast, a random distribution of pseudopod directions was observed from the cells in the uniform SDF-1α region, Fig. 8A2.
An average chemotaxis index40 was calculated for each of the six groupings of cells in Fig. 7 and 8 by evaluating the cosine of the angle that each cell moves with respect to the average direction of the particular group in which the cell is (from the rose diagrams, it can be seen that this average direction is qualitatively similar to the average direction of the fluorescein gradients shown in Fig. 7E and 8E). These chemotaxis index calculations show that the chemotaxis index is low (~0.05) for cells in regions of high SDF-1α concentration (~100 ng ml−1, Fig. 7A and 8A), consistent with a hypothesis that when the cells are surrounded by a moderate or high concentration of SDF-1α, they do not respond to gradients in that concentration. In contrast, for the cells in the regions of steepest gradient (the central parts of the flow field, Fig. 7B and 8B, SDF-1α concentration 25–75 ng ml−1), the migration is strongly directed towards regions of higher SDF-1α concentration (chemotaxis indices of 0.75 and 0.65 respectively).
As a consequence of the VIA traps being arranged on a well defined, regular array, it is easy to compare in more detail the chemotactic movements of individual cells with the concentration profile of SDF-1α. Thus, as Fig. 9 shows, the migration of the cells is clearly towards the higher concentration of SDF-1α (i.e. in the direction right to left, Fig. 9). Furthermore, the extent of movement is generally largest in the region where the gradient is steepest i.e. along the diagonal, top left to bottom right. It is noted that the numbers of cells that experience a particular gradient in any given experiment with a single VIA device are relatively small, but, as a consequence of the well defined, regular array format, chemotactic responses from experiments performed on a series of different devices can be reliably grouped together to improve the statistical quality of the data.
Fig. 9 Left: distribution of fluorescein-dextran (MW. 10 kDa, a proxy for SDF-1α) in the top layer of the device of Fig. 8 (the bright area corresponds to the fluorescein-dextran region, the dark area corresponds to the media only region). Right: displacement of individual cells during the course of the 20 h experiment of Fig. 8. |
Finally, it is also seen that in areas where the SDF-1α concentration is low (0–25 ng ml−1, Fig. 7C and 8C and top right of Fig. 9), but nevertheless there is still a small gradient in concentration (see ESI† Fig. S3), the cells migrate towards the higher SDF-1α concentration (chemotaxis indices of 0.6 and 0.65 respectively). These results are consistent with the observations that many eukaryotic cells are able to interpret differences in concentration of a chemo-attractant, which may be as little as 2% differences over the length of the cell,41 and the results here support this as a mechanism, however this migration along a concentration gradient may only occur when the stimulant concentration is below a certain threshold value.
In order to migrate, cells will produce pseudopods in response to chemo-attractive signals and which ultimately guide them toward chemoattractants.42Fig. 10 shows such events, namely that the cell extends its pseudopods to sense the surroundings and it maintains only the pseudopod toward the direction of the SDF-1α gradient. This result together with the results above (Fig. 7 and 8) indicate the efficacy of the VIA device in the study of MDA-MB-231 migration in the presence of SDF-1α gradients.
Footnote |
† Electronic supplementary information (ESI) available: Fabrication of VIA trap and experiment set-up. See DOI: 10.1039/c4lc00774c |
This journal is © The Royal Society of Chemistry 2015 |