Microfluidic platform for short-term chemotaxis assay.

<p>(<b>A</b>) Time-lapse images of MSCs migration under a PDGF-BB gradient for 24 hours. Images were taken every 15 minutes and individual color-coded cell tracks were assembled after 0, 8, 16, and 24 hours. A movie clip of the 24-hour cell migration data is available (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044995#pone.0044995.s008" target="_blank">Mov. S1</a>). (<b>B, C</b>) Migration traces of cells initially seeded in the lower PDGF-BB concentration region (cell no. 1-13) and in the higher PDGF-BB concentration region (cell no. 14-26), respectively. These cell traces (<b>B</b>) indicate that cells in the bottom half of the channel (0–50 ng/mL of PDGF-BB) exhibited directed migration, whereas (<b>C</b>) cells in the top half of the channel (50–100 ng/mL of PDGF-BB) exhibited random motion. Axes are in the units of 200 microns. (<b>D</b>) Chemotactic index, CI of MSCs in 0–50 ng/ml and 50–100 ng/ml PDGF-BB regions. Statistical significance was determined by Student's <i>t</i>-test comparing cells in the bottom and top parts of the channel (*p<0.05).</p

Microfluidic platform for long-term chemotaxis assay.

<p>(<b>A</b>) Long-term migration of MSCs (labeled with CFSE/Calcein AM) within a PDGF-BB gradient (0–100 ng/ml). The total number of cells present within the cell migration region was 105 at 0 hour and 107 at 72 hours. Limited by the visualization area of microscope, fluorescent images of adjacent areas were taken individually and spliced together. (<b>B</b>) Cell distribution within the cell migration region in the presence (0–100 ng/mL PDGF-BB) or absence (0-0 ng/mL or 100-100 ng/mL PDGF-BB) of a chemotactic gradient. Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044995#pone-0044995-g004" target="_blank">Figure 4A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044995#pone.0044995.s003" target="_blank">S3</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044995#pone.0044995.s005" target="_blank">S5</a> were represented as ratios of number of cells present in the upper half of the channel to that in the lower half of the channel. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044995#s2" target="_blank">Results</a> are means ± STD for n = 3. Statistical significance was determined by Student's <i>t</i>-test comparing results in the presence of a gradient from 0 and 72 hours (*p<0.05).</p

Gradient evolution inside the microfluidic gradient generator.

<p>(<b>A</b>) A macroscopic image of the gradient generator. (<b>B–C</b>) Gradient evolution inside the microfluidic gradient generator at different pumping rates powered by mechanical pump: (<b>B</b>) Visualization of the gradient at level 2 (Lv2), 5 (Lv5), 8 (Lv8), and 10 (Lv10)), as denoted by the dashed boxes. Fluorescence images were captured within each zone 2 hours after starting the pump. Fluorescence intensity was measured in the middle of the cell migration region denoted by the red dashed lines. Yellow dashed lines denote the upper and lower boundaries of the microchannel. (<b>C</b>) Normalized fluorescence intensity of the fluorescein gradients along the cell migration channel (red dashed line in b) at different pumping rates. (<b>D</b>) Fluorescein gradient evolution across the cell migration region (Lv10) inside the microfluidic gradient generator powered by ALZET® osmotic pumps (5 µL/hr) throughout a 9-day period. Normalized by taking the fluorescent intensity at 0 µm as 1. (<b>E</b>) Shear stress within the cell migration region modeled using COMSOL. Inset at bottom: a model cell (height 1.5 µm), experiences shear stresses in the range of 0.03–0.14 dynes/cm².</p