A Portable Chemotaxis Platform for Short and Long Term Analysis

Flow-based microfluidic systems have been widely utilized for cell migration studies given their ability to generate versatile and precisely defined chemical gradients and to permit direct visualization of migrating cells. Nonetheless, the general need for bulky peripherals such as mechanical pumps and tubing and the complicated setup procedures significantly limit the widespread use of these microfluidic systems for cell migration studies. Here we present a simple method to power microfluidic devices for chemotaxis assays using the commercially available ALZET® osmotic pumps. Specifically, we developed a standalone chemotaxis platform that has the same footprint as a multiwell plate and can generate well-defined, stable chemical gradients continuously for up to 7 days. Using this platform, we validated the short-term (24 hours) and long-term (72 hours) concentration dependent PDGF-BB chemotaxis response of human bone marrow derived mesenchymal stem cells.Harvard Stem Cell InstituteNational Institutes of Health (U.S.) (grant HL095722)National Institutes of Health (U.S.) (grant HL097172)Massachusetts Institute of Technology (MIT-UROP program)Massachusetts Institute of Technology (John Reed Fund)National Institute of Biomedical Imaging and Bioengineering (U.S.) (BioMEMS Resource Center (P41 EB 002503)

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

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

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

Tracking Mesenchymal Stem Cells with Iron Oxide Nanoparticle Loaded Poly(lactide-co-glycolide) Microparticles

Monitoring the location, distribution and long-term engraftment of administered cells is critical for demonstrating the success of a cell therapy. Among available imaging-based cell tracking tools, magnetic resonance imaging (MRI) is advantageous due to its noninvasiveness, deep penetration, and high spatial resolution. While tracking cells in preclinical models via internalized MRI contrast agents (iron oxide nanoparticles, IO-NPs) is a widely used method, IO-NPs suffer from low iron content per particle, low uptake in nonphagocytotic cell types (e.g., mesenchymal stem cells, MSCs), weak negative contrast, and decreased MRI signal due to cell proliferation and cellular exocytosis. Herein, we demonstrate that internalization of IO-NP (10 nm) loaded biodegradable poly­(lactide-co-glycolide) microparticles (IO/PLGA-MPs, 0.4–3 μm) in MSCs enhances MR parameters such as the <i>r</i>₂ relaxivity (5-fold), residence time inside the cells (3-fold) and <i>R</i>₂ signal (2-fold) compared to IO-NPs alone. Intriguingly, in vitro and in vivo experiments demonstrate that internalization of IO/PLGA-MPs in MSCs does not compromise inherent cell properties such as viability, proliferation, migration and their ability to home to sites of inflammation