This study focuses on the separation of neutrophils from human tracheal secretion using a spiral microfluidic channel. The method utilizes varying channel thicknesses to achieve efficient cell separation based on size.
Neutrophils — immune cells with multilobed nuclei, regulate tracheal immunity by engulfing invading pathogens.
To separate neutrophils from tracheal secretion, begin with a spiral microfluidic channel.
The spiral channel originates from the central inlet, progressively extending and bifurcating into inner and outer wall outlets. The bifurcation channel has varying thicknesses, ensuring efficient separation. Each curved channel resembles a trapezoid, with the outer wall height greater than the inner wall's, generating a variable flow pattern.
Fill the inlet with a pre-filtered, diluted human tracheal secretion comprising mucin aggregates, neutrophils, and erythrocytes — as incidental contaminants. Flow the sample at a specific flow rate through the spiral microchannel leading to the dispersion of the cells and aggregates.
As cells travel through the trapezoidal channel, they experience Dean drag and inertial lift forces. The interplay between these two forces creates an equilibrium, separating cells by their sizes. As a result, larger-sized neutrophils accumulate near the channel's inner wall, while erythrocytes and mucin aggregates remain near the channel's outer wall.
Finally, neutrophils move through the broader channel and get collected in the inner wall outlet.
In contrast, the narrow channel collects erythrocytes and mucin aggregates in the outer wall outlet, ensuring efficient neutrophil separation.
Use a 10-milliliter syringe to collect 1 milliliter of airway secretion samples in 9 milliliters of phosphate-buffered saline. Then, with the help of a blunt pipette, homogenize the mucus sample. Next, use a 40-micrometer nylon cell strainer to strain the diluent, and remove large chunks of tissue or blood clots. After the straining is complete, put the sample on ice.
Then, add 50 milliliters of phosphate-buffered saline to the 0.5-milliliter sample diluent to achieve a final concentration of 1,000 times diluted sample. Use the fluid guide to assemble the PDMS chip in order to apply uniform flow to each of the four spiral microchannels. Next, use a male luer connector with 1/16-inch diameter to connect to the inlet, the inner wall outlet, and the outer wall outlet ports of the fluid guide.
Then, connect a silicone tubing to the sample suspension. Next, connect the peristaltic pump to the inlet tubing. Place the outer wall outlet tubing in the waste reservoir. Then position the end of the inlet tubing and the inner wall outlet tubing in the sample reservoir.
After positioning the tubing, leave a 50-milliliter tube filled with 5 milliliters of phosphate-buffered saline without calcium and magnesium in the sample reservoir. Then, in order to prime the device, start pumping at a flow rate of approximately 1 milliliter per minute.
After placing the sample in the sample reservoir, switch on the peristaltic pump and set the flow rate at 4 milliliters per minute. After the sample volume reaches the desired volume of approximately 1 to 2 milliliters, stop the operation and disconnect the tubing.
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