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All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Microfluidic device preparation.
- Microfluidic Cell Migration Assay Preparation
- Prepare 50 µg/mL fibronectin solution by diluting 50 µL of stock fibronectin solution (1 mg/mL) to 950 µL Dulbecco's phosphate-buffered saline (DPBS) inside a biosafety cabinet.
- Prepare the migration medium by mixing 9 mL of Roswell Park Memorial Institute medium (RPMI-1640) and 1 mL of RPMI-1640 with 4% bovine serum albumin (BSA).
- Remove the deionized water from the device.
- Add 100 µL fibronectin solution to the device from the outlet. Wait 3 min to ensure that all the channels are filled with fibronectin solution. Place the microfluidic device in a covered Petri dish for 1 h at room temperature.
- Remove the fibronectin solution from the device. Add 100 µL migration medium from the outlet. Wait 3 min to ensure that all the channels are filled with migration medium.
- Incubate the device for another 1 h at room temperature; the device is then ready for the chemotaxis experiment.
- Chemoattractant solution preparation for chemotaxis experiment.
- Prepare 100 nM fMLP solution in a total of 1 mL migration medium. Mix 5 µL of stock FITC-Dextran (10 kDa, 1 mM) with the fMLP solution in a 1.5 mL tube.
NOTE: FITC-Dextran is used for gradient measurement. Alternatively, use Rhodamine as the gradient indicator. The fMLP chemoattractant solution is then ready for the chemotaxis experiment.
- Sputum sample preparation.
NOTE: Neutrophil chemotaxis induced by a gradient of sputum samples from COPD patients was tested as a clinical diagnostic application of this all-on-chip method.
- Obtain a human ethics protocol to collect sputum samples from COPD patients.
NOTE: We obtained approvals to collect samples at the Seven Oaks General Hospital in Winnipeg (approved by the University of Manitoba).
- Obtain the informed written consent forms from all subjects.
- Collect COPD patients' spontaneous sputum samples. Place 500 µL sputum sample in a 1.5 mL tube.
- Add 500 µL 0.1% dithiothreitol in the 1.5 mL tube and gently mix. Place the tube in a water bath at 37 °C for 15 min.
- Centrifuge the sample at 753 x g for 10 min and then collect the supernatant. Centrifuge the supernatant at 865 x g for 5 min and then collect the final supernatant. Store the collected supernatant inside a -80 °C freezer before use.
- When ready for the chemotaxis experiment, thaw the sputum solution; transfer 900 µL migration medium to a 1.5 mL tube and mix with 100 µL sputum solution inside a biosafety cabinet; the sputum solution is then ready for the chemotaxis experiment.
- Blood sample collection.
- Obtain a human ethics protocol to collect blood samples from healthy donors. Obtain the informed written consent forms from all blood donors.
NOTE: Here samples were obtained at the Victoria General Hospital in Winnipeg (approved by the Joint-Faculty Research Ethics Board at the University of Manitoba).
- Collect the blood sample by venipuncture and put the sample into an EDTA-coated tube. Keep the tube in a biosafety cabinet before the experiment.
2. All-on-chip Chemotaxis Assay Operation
- On-chip cell isolation (Figure 1B).
- Place 10 µL whole blood in a 1.5 mL tube inside a biosafety cabinet.
NOTE: Details of blood sample collection are in section 1.4.
- Add 2 µL antibody cocktail (Ab) and 2 µL magnetic particles (MP) from the neutrophil isolation kit (see the table of materials) into the 1.5 mL tube and gently mix; this will label cells in the blood except for the neutrophils.
- Incubate the blood-Ab-MP mixture for 5 minutes at room temperature.
NOTE: This will magnetically tag the antibody-labeled cells in the blood.
- Attach two small magnetic disks to the two sides of the cell loading port of the device. Aspirate the medium from all ports of the device.
- Slowly pipette 2 µL blood-Ab-MP mixture into the microfluidic device from the cell loading port.
NOTE: The magnetically labeled cells are trapped to the side walls of the cell loading port while neutrophils will flow into the device and become trapped at the cell docking structure.
- Wait a few minutes until enough neutrophils are trapped in the cell docking area.
3. Chemotaxis assay (Figure 1C).
- Place the microfluidic device on the temperature-controlled microscope stage at 37 °C.
- Add 100 µL chemoattractant solution (fMLP or sputum solution) and 100 µL migration medium to their designated inlet reservoirs using two pipettors; this will generate a chemoattractant gradient in the gradient channel by continuous laminar flow-based chemical mixing assisted by a pressure balancing structure.
NOTE: Details of the sputum collection from COPD patients are in section 1.3.
- For the medium control experiment, only add migration medium to both of the inlet reservoirs.
- Acquire the fluorescence image of FITC-Dextran in the gradient channel.
- Import the image to ImageJ software using the command "File|Open".
- Measure the fluorescence intensity profile across the gradient channel using the command "Analyze|Plot Profile".
- Export the measurement data to a spreadsheet for further plotting.
- Incubate the device on the temperature-controlled microscope stage or in a conventional cell culture incubator for 15 min.
- Image the gradient channel using a 10X objective to record the cells' final positions for data analysis.
- If needed, record the cell migration in the device by time-lapse microscopy.
4. Cell Migration Data Analysis (Figure 1C)
- Analyze the chemotaxis assay by calculating the cell migration distance from the docking structure as described below. See Figure 1C.
- Import the image into NIH ImageJ software (ver. 1.45).
- Select the center of each cell that moved into the gradient channel.
- Measure the coordinates of the selected cells for their final positions. Measure the coordinate of a point at the edge of the docking structure as the initial reference position.
- Export the measured coordinate data to spreadsheet software (e.g. Excel). Calculate the migration distance of the cells as the difference between a cell's final position and the initial reference position along the gradient direction.
- Calibrate the distance to a micrometer. Calculate the average and deviation of the migration distance of all cells as a measure of chemotaxis.
- Compare the migration distance in the presence of a chemoattractant gradient to the medium control experiment using the Student's t-test.
- If the time-lapse images of the cell migration are recorded, the cell migration and chemotaxis can be further analyzed by cell tracking analysis.
NOTE: The materials required to construct and perform the all-on-chip chemotaxis assay are detailed in the table of materials.
