Label-Free Neutrophil Separation from Tracheal Secretion Using Spiral Microfluidics Channel

Abstract

Source: Ryu, H., et al., Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics. J. Vis. Exp. (2018).

This video demonstrates separating neutrophils from tracheal secretions using a microfluidic device with spiral channels. The device achieves efficient separation based on cell size by utilizing varying thicknesses and trapezoidal-shaped channels. The interplay between forces results in large neutrophils accumulating near the inner wall while erythrocytes and mucin aggregates remain near the outer wall, ensuring effective neutrophil separation.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Device Fabrication and Soft Lithography

NOTE: Standard soft lithography techniques were used to create the polydimethylsiloxane (PDMS) microchannel.

1. Mix the PDMS precursor in a 10:1 ratio of base and curing agent.
2. Pour 30 g of the PDMS precursor mixture into the micro-machined aluminum mold (see Figure 1 for dimensions)....

Results

Figure 1: Spiral microfluidic device and experimental settings. Photograph images of (A) spiral microfluidics and (B) the device assembled with the fluidic adaptor. (C) Schematic diagram of closed-loop separation using spiral inertial microfluidics. By recirculating the par.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PDMS precursor	Dow corning	184 SIL ELAST KIT 3.9KG	10:1 ratio of base and curing agent
VWR gravity convection oven	VWR	414005-128	PDMS precursor to be cured in 90 deg.
100mm petri dish	VWR	89000-324	Fabrication of PDMS Supporting layer
Harris Uni-core puncher	Sigma-aldrich	WHAWB100076	2mm diameter or other depending on the tubing size
Air plasma machine	Femto Science	Cute	Surface plasma treatment for PDMS device to bottom base.
2" x 3" glass slide	TED PELLA, INC.	2195	To support PDMS device
Masterflex spooled platinum-cured silicone tubing, L/S 14	Cole-Parmer	EW-96410-14	Tubing for microfluidics and peristlatic pump
1/16 inch Luer connector, male	Harvard apparatus	PC2 72-1443	Connector for fluid guide
50mL Falcon tube	Corning	21008-940	Sample collection & preparation
Phosphate-Buffered Saline, 1X Without Calcium and Magnesium	Corning	45000-446 	Buffer solution to dilute sample
Halyard Closed suction Catheter, Elbow, 14F/ channel 4.67mm	HALYARD HEALTH	22113	Tracheal seceation suction catheter
0.9% Sterile Normal saline, 10mL pre-filled syringe	BD PosiFlush	NHRIC: 8290-306547	For tracheal seceation collection from the patients
SecurTainer™ III Specimen Containers, 20mL	Simport	1176R36	Sterile sputum (airway secretion) collection container
Syringe with Luer-Lok Tip, 10mL	BD	BD309604	To pipette homogenize the mucus sample and reach the bottom of sample tube
BD  Blunt Fill Needle, with BD Luer-Lok  Tip	BD		To pipette homogenize the mucus sample and reach the bottom of sample tube
40µm nylon cell strainer 	Falcon	21008-949	To remove large chunk or blood clots, which can block the microfluidics access hole or the channel.
Peristaltic pump (Masterflex L/S Digital Drive)	Cole-Parmer	HV-07522-30	 Operation of microfluidics