A Microfluidic Chip-Based Method for Rapid Neutrophil Chemotaxis Analysis Using Whole Blood

Overview

This video demonstrates neutrophil chemotaxis assay using an all-on-chip method. The method allows direct isolation of neutrophils from the whole blood by immunomagnetic negative selection followed by chemotaxis assay on a microfluidic chip.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Microfluidic device preparation.

1. Microfluidic Cell Migration Assay Preparation
   1. 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.
   2. 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).
   3. Remove the deionized water from the device.
   4. 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.
   5. 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.
   6. Incubate the device for another 1 h at room temperature; the device is then ready for the chemotaxis experiment.
2. Chemoattractant solution preparation for chemotaxis experiment.​
   1. 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.
3. 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.
   1. 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).
   2. Obtain the informed written consent forms from all subjects.
   3. Collect COPD patients' spontaneous sputum samples. Place 500 µL sputum sample in a 1.5 mL tube.
   4. 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.
   5. 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.
   6. 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.
4. Blood sample collection.
   1. 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).
   2. 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

1. On-chip cell isolation (Figure 1B).
2. 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.
3. 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.
4. Incubate the blood-Ab-MP mixture for 5 minutes at room temperature.
   NOTE: This will magnetically tag the antibody-labeled cells in the blood.
5. 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.
6. 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.
7. Wait a few minutes until enough neutrophils are trapped in the cell docking area.

3. Chemotaxis assay (Figure 1C).

1. Place the microfluidic device on the temperature-controlled microscope stage at 37 °C.
2. 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.
   1. For the medium control experiment, only add migration medium to both of the inlet reservoirs.
3. Acquire the fluorescence image of FITC-Dextran in the gradient channel.
4. Import the image to ImageJ software using the command "File|Open".
5. Measure the fluorescence intensity profile across the gradient channel using the command "Analyze|Plot Profile".
6. Export the measurement data to a spreadsheet for further plotting.
7. Incubate the device on the temperature-controlled microscope stage or in a conventional cell culture incubator for 15 min.
8. Image the gradient channel using a 10X objective to record the cells' final positions for data analysis.
9. If needed, record the cell migration in the device by time-lapse microscopy.

4. Cell Migration Data Analysis (Figure 1C)

1. Analyze the chemotaxis assay by calculating the cell migration distance from the docking structure as described below. See Figure 1C.
2. Import the image into NIH ImageJ software (ver. 1.45).
3. Select the center of each cell that moved into the gradient channel.
4. 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.
5. 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.
6. Calibrate the distance to a micrometer. Calculate the average and deviation of the migration distance of all cells as a measure of chemotaxis.
7. Compare the migration distance in the presence of a chemoattractant gradient to the medium control experiment using the Student's t-test.
8. 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.

Representative Results

Figure 1: Illustration of the all-on-chip method for neutrophil chemotaxis analysis. (A) Illustration of the microfluidic device. The device includes two layers. The first layer (4 µm high) defines the cell docking barrier channel to trap the cells beside the gradient channel. The second layer (60 µm high) defines the gradient generating channel, the port and channel for cell loading, the chemical inlet reservoirs, and the waste outlet. Alignment marks are designed for the two layers. For the second layer, the length and width of the upstream serpentine input channel are 60 mm and 200 µm, respectively; the length and width of the downstream serpentine input channel are 6 mm and 280 µm, respectively; (B) Illustration of the all-on-chip cell isolation method; (C) Illustration of the chemotaxis test.

Materials

Name	Company	Catalog Number	Comments
Device fabrication
Mask aligner	ABM	N/A
Spinner	Solitec	5000
Hotplate	VWR	11301-022
Plasma cleaner	Harrick Plasma	PDC-001
Vacuum dessicator	Fisher Scientific	08-594-15A
SU-8 2000 thinner	Microchem	SU-8 2000
SU-8 2025 photoresist	Microchem	SU-8 2025
(tridecafluoro-1,1,2,2- tetrahydrooctyl) trichlorosilane	Gelest	78560-45-9
Si wafer	Silicon, Inc	LG2065
Glass slides	Fisher Scientific	12-544-4
Cutting pad	N/A	N/A	Custom-made
Punchers
On-chip cell isolation and chemotaxis assay
RPMI 1640	Fisher Scientific	SH3025502
DPBS	Fisher Scientific	SH3002802
Bovine serum albumin (BSA)	Sigma-Aldrich	SH3057402
Fibronectin	VWR	CACB356008
fMLP	Sigma-Aldrich	F3506-10MG
Magnetic disks	Indigo Instruments	44202-1	5 mm in diameter, 1 mm thick
FITC-Dextran	Sigma-Aldrich	FD10S
Rhodamine	Sigma-Aldrich	R4127-5G
Giemsa stain solution	Rowley Biochemical Inc	G-472-1-8OZ
EasySep Direct Human Neutrophil Isolation Kit	STEMCELL Technologies Inc	19666
Dithiothreitol	Sigma-Aldrich	D0632
Nikon Ti-U inverted fluorescent microscope	Nikon	Ti-U
Microscope environmental chamber.	InVivo Scientific	N/A
CCD camera	Nikon	DS-Fi1