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

Overview

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) and 20 g of the PDMS precursor mixture into the 100 mm Petri dish.  
   NOTE: The Petri dish is used to make the thick film of PDMS (~3 mm) for the supporting layer. The supporting layer of PDMS provides uniform physical surface properties throughout the microfluidics. Alternatively, a thin PDMS film on the glass slide can be used. The master mold with specific channel dimensions was designed and fabricated by micro-milling on the aluminum sheet. The spiral channel used in this study was an 8-loop spiral microchannel with one inlet and two outlets, with the radius increasing from 8 mm to 24 mm for efficient size-based separation.
3. Place the mold and the Petri dish into a vacuum desiccator to degas until no bubbles are visible on the surface, typically 5 - 10 min. Use a house vacuum for the desiccator.
4. Release the vacuum and place the mold and the Petri dish in a 90 °C oven for 1 h.
   NOTE: A hotplate or other heating tools can be used instead of an oven.
5. Remove the mold and the Petri dish from the oven and allow it to cool at room temperature for 10 min.
6. Carefully cut out the outline and punch the fluid access holes on the device using a 2 mm puncher.         
   NOTE: The size of the punch can vary depending on the size of the tubing or fluid guide.
7. Adhere a tape to the channel side of the device and a thick film of PDMS, and peel carefully with forceps to remove dust.
8. Treat both the channel side of the device and plain PDMS with air plasma and bond with the prepared supporting layer of plain PDMS (Figure 1A).
9. Place the chip on the 50 mm x 70 mm glass slide and place it in a 70 °C oven for at least 30 min to enhance the bonding strength.

2. Tracheal Secretion Collection from Mechanically Ventilated Patients

NOTE: Tracheal secretions can be obtained from mechanically ventilated patients during normal routine airway care by using a protocol modified from conventional methods to accommodate the standard, adult ventilated patient. Samples were de-identified and sent immediately for processing.

1. If tracheal aspiration is indicated, use a catheter to extract airway secretions.
2. Advance the catheter carefully halfway into the endotracheal tube and instill 5 mL of 0.9% sterile normal saline from a pre-filled 10 mL syringe.
3. When the catheter is advanced fully, or resistance is met, aspirate the secretions and collect them into a sterile sputum collection container.
4. Collect 10 mL of tracheal secretions in a sterile sputum container and place it on ice. Send samples immediately for processing.

3. Tracheal Secretion Sample Preparation

NOTE: All experiments must be performed under a biosafety cabinet with the proper personal protective equipment.

1. Disperse 1 mL of airway secretion samples in 9 mL of phosphate-buffered saline (PBS) using a plastic 10 mL syringe (for a 10x dilution). Use a blunt pipette to homogenize the mucus sample.
2. Strain the diluent with a 40 µm nylon cell strainer to remove large chunks or blood clots, which can block the microfluidics access hole or the channel. Place the sample on the ice after processing.
3. Disperse the diluent of each sample using a 100x volume of PBS buffer, resulting in a 1,000x diluted suspension. Place the sample on the ice during the entire dissociation operation.

4. Experimental Setup

NOTE: All experiments must be performed under a biosafety cabinet with the proper personal protective equipment.

1. Assemble the PDMS chip with the fluid guide to apply uniform flow to each of the four spiral microchannels (Figure 1B).    
   NOTE: Four channels were used to increase the throughput by parallelization, as well as optimization of the volume and cell density of the resulting suspension. The fluid guide is designed and made with a stereolithography-type 3D printer with clear resin. The inlet and the outlet port of the fluid guide use female Luer connectors for ease of connection and stable sealing during the operation.
2. Connect a 1/16-inch male Luer connector to the inlet, the inner wall (IW) outlet, and the outer wall (OW) outlet port of the fluid guide and connect a silicone tubing to the sample suspension.
3. Insert blunt tips to each end of the inlet and outlets to reach the bottom of the sample reservoir.
4. Connect the peristaltic pump with the inlet tubing.
5. Place the end of the inlet tubing and IW outlet tubing in the sample reservoir and place the end of the OW outlet tubing in the waste reservoir (Figure 1C, D).
6. Place the 50 mL tube of filtered PBS without calcium and magnesium in the sample reservoir.
7. Start pumping at a low flow rate (~1 mL/min) to prime the device.
   NOTE: When bubbles are captured in the channel, push the top of the channel with the tweezer to destabilize and eliminate air bubbles. When the device is fully filled with buffer solution, change the PBS tube to prepare the airway secretion sample tube.
8. When the sample is placed in the sample reservoir, switch on the peristaltic pump and set the flow rate at 4 mL/min (Figure 1C, D).
9. When the sample volume reaches the designated volume (~1 mL), stop the operation and disconnect the silicone tubing.   
   NOTE: The proposed method is more efficient at reducing a large volume of diluent (>50 mL) into a micro-centrifuge tube volume (~1 mL) (Figure 2).

Representative 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 particle/cell concentrated stream (IW outlet) back into the original sample reservoir, immune-related cells were concentrated in a small volume while the background fluid containing mucin aggregates and minor RBCs were continuously removed through the waste tube connected to the OW outlet. (D) Experimental setup and the fluorescent image of a 10 µm particle, the trajectory (green), and the outlet of the spiral channel (upper right).

Figure 2: Schematic diagram of the airway secretion sample preparation using closed-loop inertial microfluidics. Clinically induced airway secretion dispersed mechanically in 1,000x volume of buffer solution. The diluent is concentrated by microfluidics, resulting in ~1 mL of cell suspension with a clear background. Adapted with permission from Ryu et al., Copyright (2017) American Chemical Society.

Materials

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