A low resistance microfluidic system for the creation of stable concentration gradients in a defined 3D microenvironment

Abstract

The advent of microfluidic technology allows control and interrogation of cell behavior by defining the local microenvironment with an assortment of biochemical and biophysical stimuli. Many approaches have been developed to create gradients of soluble factors, but the complexity of such systems or their inability to create defined and controllable chemical gradients has limited their widespread implementation. Here we describe a new microfluidic device which employs a parallel arrangement of wells and channels to create stable, linear concentration gradients in a gel region between a source and a sink well. Pressure gradients between the source and sink wells are dissipated through low resistance channels in parallel with the gel channel, thus minimizing the convection of solute in this region. We demonstrate the ability of the new device to quantitate chemotactic responses in a variety of cell types, yielding a complete profile of the migratory response and representing the total number of migrating cells and the distance each cell has migrated. Additionally we show the effect of concentration gradients of the morphogen Sonic hedgehog on the specification of differentiating neural progenitors in a 3-dimensional matrix.

Fig. 1

RC Device design and stable gradient formation. (a) A schematic representation of the RC device layout in 2D. The unshaded channels represent the sink and sink reservoir connected by the sink channel. The dark shaded region represents the source and source reservoir/channel as well as a channel connecting the source and sink reservoirs. The lightly shaded region is the mirror image of the dark shaded source region and serves as an internal control for each experiment. The blue region is the location of the hydrogel in which the relevant concentration gradient forms, and the red region is the analogous control hydrogel. The chemical species of interest is placed in the source well and source reservoir. The remainder of the device is filled with a control solution. During cell migration experiments, cells are loaded into the cell/sink well and after an incubation period the distribution of cells in the experimental and control gels is analyzed. (b) A 3D representation of the RC device constructed from PDMS and bonded to a glass coverslip. The diameter of the source, sink, and control wells is 3 mm, and the diameter of the source, sink, and control reservoirs is 4 mm. The hydrogel channels are 1.5 mm long, 0.6 mm wide, and 0.120 mm tall. The remaining channels are 20 mm long, 2 mm wide, and 0.120 mm tall. (c) An analogous RC electrical circuit can be used to depict how the low resistance channels dissipate pressure gradients more rapidly than the high resistance gel region. (d) A 10 kDa FITC-dextran molecule was used to model the diffusion of a similarly sized protein such as SDF-1. The dextran solution (25 μg/ml) was loaded into the source well and source reservoir. The sink well, control well, sink reservoir, and control reservoir were filled with PBS. The device was placed on a fluorescent microscope and images were acquired every hour (representative time points shown) for 24 h. To prevent evaporation the device was covered with a glass cover slip during image acquisition. Within 2 h a linear concentration profile is achieved in the gel region and maintained for the duration of the experiment. (e) The dextran gradient was tracked over the course of 6 days and the average slope of the concentration profile is shown for each day. The slope remains constant for the first 48 h and on day 3 the slope has decayed by 10%. **P<0.01 (f) By changing the geometry of the gel region (as shown in the inset) a non-linear concentration profile with an exponential dependency was achieved in the RC device. Fluorescent images were acquired every 2 h for 12 h

Fig. 2

Dissipation of pressure gradients in RC device without the low resistance bypass channels. (a) Initially the gel region and the sink well did not contain any solute. The high concentration source well was loaded with a 1 mm hydrostatic pressure difference relative to the sink well. Without the low resistance circuit the pressure difference must be dissipated via fluid flow through gel region. This fluid flow is accompanied by convection of the solute into the gel. The solute is modeled as SDF-1 with a diffusivity of 1.6×10⁻⁶ cm²/s and the permeability of the gel region is 1×10⁻¹² m². The simulation was executed for 1 h. (b) A gradient was allowed to develop across the experimental gel region for 6 h by placing a 25 μg/ml FITC-dextran solution in the source well of a device without the bypass channels. Then 10 μl of the dextran solution was added to the source well and the gradient was imaged every 10 min for an additional hour. The gradient is disrupted and after 6 h does not return to its original profile. (c) In the RC device with the bypass channels although the concentration profile changed slightly, within 10 min the gradient returned to a linear profile after the introduction of a 10 μl bolus. Images were acquired every 10 min for 1 h after the introduction of the bolus (d) The source well and reservoir were filled with a 50 μg/ml solution of FITC-dextran and the concentrations in all of the wells and reservoirs were measured before and after the addition of a 10 μl injection into the source well. The sink reservoir (location 3) experiences the greatest concentration change while the concentrations in the sink well, control well, and control reservoir remain relatively unchanged

Fig. 3

The RC Device demonstrates migration of three different cell types: vascular smooth muscle cells, Jurkat T-lymphocytes, and bovine aortic endothelial cells. (a) Vascular smooth muscle cells were loaded into the sink well of the RC Device and media supplemented with 4 nM of PDGF-BB was added to the source well and reservoir. The sink and control wells and reservoirs were filled with control media. (Left) Migration of vascular smooth muscle cells from left to right toward 4 nM PDGF-BB and the matching control from a sample device. (Right) Comparison of migration toward PDGF-BB vs control. The number of cells at each location decreases with increasing distance into the gel. A particularly marked difference between the PDGF and control gels is observed in the first 200 μm of the gel region. n=4. (b) (a) Left. Migration of Jurkat T lymphocytes from left to right toward a 1 nM solution of SDF-1 and a 10 nM solution of SDF-1. Right. Dose response of Jurkat T lymphocyte migration toward SDF-1 concentrations. (c) Left. Migration of bovine aortic endothelial cells from left to right toward a 0.1 nM solution of VEGF and a 1 nM solution of VEGF. Right. Dose response of migration of bovine aortic endothelial cells toward VEGF concentrations. 10X. White = DAPI. **P<0.01, arrows indicate direction of migration

Fig. 4

Sonic hedgehog gradients in the RC device produce graded expression of neural transcription factors. (a) ES cell reporter lines (Olig2-GFP and Nkx2.2-tdTomato) were mixed and differentiated in hanging drops. Two days later (day 0 of Shh exposure), embryoid bodies we seeded in collagen gels and exposed to a concentration gradient ranging from 25 nM to 0 nM along the length of the gel. On day 3, Olig2 and Nkx2.2 expression were evident by fluorescent signal at the higher concentration region in the gel. On day 4 this expression had increased in intensity and penetration into the gel as regions of lower Shh concentration received sufficient temporal exposure to induce a response. Measured day 4 expression for Olig2 (b) and Nkx2.2 (c) compared to untreated gels shows the extent of differentiation at different points in the gradient by plotting reporter fluorescent intensity vs. distance from the Shh source. Expression is highest at the locations in the gel closest to the Shh source and eventually falls to the baseline level present in the gels that contained embryoid bodies that were not simulated with Shh. This indicates the location in the gel where the Shh concentration is equal to the threshold level necessary to induce Olig2 and Nkx2.2 expression

Fig. 5

Gradients formed by different concentrations of Sonic hedgehog produced a dose response in the expression of neural transcription factors. Two day old embryioid bodies were seeded in the gel regions of the RC device and exposed to Shh gradients produced by 12.5 nM (blue), 25 nM (green), 50 nM (pink), and 125 nM (black) Shh concentrations in the source well. After 4 days of Shh exposure Olig2 (a) and Nkx2.2 (b) expression in the gel region as judged by fluorescent reporter intensity was plotted against distance from the source well. The expression levels at all four of the Shh doses were highest in the in the regions adjacent to the source wells and decreased to the baseline levels present in the gels with untreated embryoid bodies (red dashed line). Increasing Shh doses increased the Shh concentration at each location in the gel. Thus at higher concentrations the location of the expression threshold moves farther form the source well and the Olig2 and Nkx2.2 expression extends farther into the gel. (c) The location of the threshold concentration as a function of the Shh source concentration exhibits a linear relationship for both Olig2 and Nkx2.2 confirming a constant threshold concentration of approximately 10 nM Shh