A triple-unit microfluidic device (D3-chip) for cell migration research

Chemotaxis is an important research field and many research labs are studying the mechanisms of chemotaxis of different cell types and its biomedical applications. During the past 20 years, microfluidic devices have been extensively used for chemotaxis research mainly owing to their advantage in cellular microenvironmental control. However, microfluidic chemotaxis experiments are not easy to set up and often limited by lengthy data analysis and the low-throughout. To address these issues, we developed a new triple-unit gradient-generating microfluidic device (i.e. D3-Chip). The main features of this D3-Chip include standalone stable gradient generation, docking structure for cell alignment, parallel chemotaxis experiments on a single chip and rapid chemotaxis distance measurement without time-lapse imaging and single cell tracking. This D3-Chip was successfully applied to two recently published chemotaxis studies including the effect of fibroblast growth factor 23 (FGF23) on neutrophil chemotaxis and the effect of activin A on neutrophil and human breast cancer cell chemotaxis. We believe this D3-Chip can be broadly useful to other cell migration researchers. In this protocol, we describe detailed fabrication and operation of the D3-Chip for cell migration experiments.


Cell migration and microfluidic assays
Cell migration plays an important role in many physiological processes such as inflammation [1,2], wound healing [3], and cancer metastasis [4]. Cell migration directed by chemical concentration gradient, termed chemotaxis, is one of the most important guiding mechanisms [1,5,6]. Conventional chemotaxis assays such as Boyden chamber [7], Dunn chamber [8], Zigmond chamber [9], under agarose assay [10] and micropipette-based assay [11] are typically limited by the lack of ability for precise chemical gradient generation. Microfluidic devices offer useful features in gradient control, reduced reagent and sample consumption and quantitative single cell analysis [5,12]. During the past 20 years, various microfluidic devices have been extensively applied to cell migration research [13][14][15]. On the other hand, current microfluidic chemotaxis experiments are not easy to set up and often limited by lengthy data analysis and the low-throughout. To address these issues, we have previously 3 developed a microfluidic device with the standalone gradient generation and cell docking features that allow easier and more accurate chemotaxis analysis [16]. More recently, we further developed this device to configure three parallel independently controlled test units for high throughput chemotaxis test (i.e. D3-Chip) ( Fig. 1) [17,18].

Overview
The D3-Chip was fabricated by the standard multi-layer photo-lithography for the SU-8 master and soft-lithography for the final PDMS replica. Then the PDMS D3-Chip was functionalized with the substrate coating for chemotaxis experiments preparation. Parallel chemotaxis experiments on a single D3-Chip were performed by cell loading and alignment in each test unit and chemoattractant gradient application. Cells in the channels were imaged for dynamic cell tracking analysis or end-point cell migration distance measurement for rapid chemotaxis assessment.

Key features of the technique
This developed D3-Chip has the following key features to enable advanced chemotaxis experiment. 1) Standalone stable gradient generation without requiring external pumping instrument. The flowbased gradient generation strategy allows rapid gradient generation within minutes.
2) The docking structure aligns cells by the thin barrier channel before gradient application. The identical initial cell positions enable rapid and accurate data analysis without time-lapse imaging and cell tracking.
3) The triple-unit design allows parallel independently-controlled chemotaxis experiments on a single chip.

Applications and target users
In one of our recent studies, we applied the D3-Chip to study the effect of fibroblast growth factor 23 (FGF23) on neutrophil chemotaxis with relevance to chronic kidney disease (CKD) [18]. In another recent study, we employed the D3-Chip to study the effect of activin A on neutrophil and human breast cancer cell chemotaxis [17]. In the same study, we also used the D3-Chip for transendothelial migration studies by patterning an endothelial cell layer to mimic the blood vessel wall. In addition to 4 neutrophils and breast cancer cells, this D3-Chip can be broadly useful to study chemotaxis of other cell types of interest. We have successfully used the D3-Chip for studying chemotaxis of human blood lymphocytes, mouse NK cells, human and rat adipose-derived stem cells and mouse myoblasts (unpublished data). Researchers from the cell migration community are the natural target users of this D3-Chip. With the growing interest of applying chemotaxis as a disease evaluation tool, we envision many medical scientists and professionals will be interested in this D3-Chip for potential clinical applications.

Advantages, limitations and adaptations
Compared to existing microfluidic chemotaxis devices, the key advantage of this D3-Chip is its integrated functions to permit standalone stable gradient generation, cell alignment, parallel chemotaxis experiments and rapid chemotaxis measurement. These features will allow researchers to explore cell migration questions by sophisticated and reliable microfluidic cell migration analysis. We believe this D3-Chip can be easily adapted in any cell migration research lab. Further integration of on-chip cell isolation and portable imaging system that we have developed will facilitate clinical applications of the D3-Chip16,19. More parallel test units can be added to the device design to further improve the experimental throughput. On the other hand, the flow-based gradient-generation method limits the ability of the current D3-Chip to study chemotaxis in 3D extracellular matrix.

Reagents
Chemicals and reagents

5.
Put the microfluidic device on the stage of an inverted fluorescence microscope.

6.
Image the fluorescence intensity of FITC-Dextran inside the channel to verify gradient generation.

7.
Record cell migration images at 6 frames/min for 15 min.
8. Import the images to "ImageJ"to track cells.

9.
Use "Chemotaxis and Migration Tool" to further analyze the tracking data.

10.
Export the tracking data to Excel for further calculation of migration parameters.

11.
Some migration parameters can be calculated from the tracking data such as chemotactic index (CI), flowtactic index (FI), and migration speed (V) [18].

12.
Alternatively, chemotaxis assay using the D3-Chip can be done without taking timelapse images. After cell loading and gradient generation, put the device in the incubator for 15 min.

13.
At the end of the assay, capture the final cell images in the channel for data analysis.
14. Import the images to ImageJ.

15.
Calculate the cell migration distance along the gradient direction (across the gradient relative to the thin barrier channel). Fig. 3)
Add FITC-Dextran to the chemoattractant solution as the gradient indicator.

4.
As cancer cells migrate more slowly comparing to neutrophils, it is an advantage of the D3-Chip that chemotaxis assay can be done without time-lapse microscopy. After cell loading and gradient application, put the device in an incubator for several hrs.

5.
At the end of the assay, capture the final cell images in the channels for data analysis.
6. Import the images to ImageJ.

7.
Analyze cell migration distance along the gradient direction (across the gradient relative to the thin barrier channel).

Timing
See the TIMING notes in the Procedures section.

Troubleshooting
See the CAUTION notes in the Procedures section.

Anticipated Results
In one of our recent studies, we applied the D3-Chip to study the effect of FGF23 on neutrophil chemotaxis with relevance to CKD [18]. Our results showed that FGF23 weakens neutrophil chemotaxis (Fig. 4). This study demonstrated the effect of FGF23 on neutrophil chemotaxis with relevance to CKD. In another study, we employed the D3-Chip to study the effect of activin A on chemotaxis of both neutrophils and human breast cancer cells [17]. Our results showed that activin A reduced neutrophil chemotaxis to a fMLP gradient (Fig. 5). We also found that activin A promotes MDA-MB-231 cell migration but reduces EGF-induced migration of MDA-MB-231 cells (Fig. 6). These results were supported by both cell tracking analysis and end-point migration distance analysis.   Procedures for chemotaxis assay using the D3-chip.