Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Summary

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

Abstract

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient's immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

Introduction

Since cells are encapsulated in a large amount of mucus in airway secretions, the functional assessment of leukocytes by an in vitro assay has been hindered. Dithiothreitol (DTT) is the most common lysis buffer to dissociate and homogenize the sputum for cytological analysis and detection of mediators while providing tolerable viability of isolated cells^1,2. However, DTT can interfere with surface-bound antigens of airway neutrophils, resulting in the disruption of neutrophil function such as elastase and myeloperoxidase (MPO) release^2,3.Therefore, few studies of human airway neutrophil function have been conducted with peripheral blood neutrophils, which may not reveal the physiological characteristics of pulmonary⁴. Meanwhile, inertial microfluidics has made advances in isolating cells from various patient biomatrices^5,6.The equilibrium between inertial lift forces and Dean drag aligns the particle/cell according to their size, which allows label-free particle separation⁷. Our group previously introduced a sample preparation method for circulating tumor cells^8,9, pathogens in blood⁸, cells from a suspension culture^10,11,12, and polymorphonuclear leukocytes (PMNs) from blood^13,14.

Here, we introduce a protocol to prepare immune cells from a patient's airway secretions using closed-loop inertial microfluidics for a downstream in vitro assay, such as the neutrophil elastase (NE) assay. This method provides both high concentration and recovery, especially when there is a significant overlap in the lateral direction of the cell/particle from which the cell/particle-of-interest is to be removed, which is commonly observed in clinical samples. By recirculating the Inner wall (IW)-focused large particles or cells back to the input sample tube, the particle or cell-of-interest concentrates in the original reservoir, while background fluids with small mucin aggregates pass through the waste reservoir. Despite the heterogeneous fluidic properties of clinical samples, the proposed method recovers consistently above 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with a clean saline solution (~1 mL). By contrast, the lysis method presents a wide range of PMNs recovery rates depending on the sample condition. The proposed protocol captures leukocytes in a label-free manner with no physical or chemical disruption, which provides the possibility to harvest delicate cells from clinically challenging biometrics with minimally invasive procedures.

Protocol

The sample collection was approved by the University of Pittsburgh Institutional Review Board (IRB# PRO16060443, PRO10110387). All experiments are performed under a biosafety cabinet with the proper personal protective equipment.

1. Device Fabrication and Soft Lithography

NOTE: Standard soft lithography techniques^15,16 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 on the micro-machined aluminum mold (see Figure 1 for dimensions) and 20 g of the PDMS precursor mixture on 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 sized-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 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 half-way 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 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 at 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).

5. Flow Cytometry Analysis

NOTE: To compare the dissociation methods (microfluidics and lysis), flow cytometry with immunofluorescence was used.

1. Add fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD66b monoclonal antibody and allophycocyanin (APC)-conjugated mouse anti-human CD45 monoclonal antibody to the initial suspensions (step 3.3) and resulting suspensions (step 4.9) (200 µL of each antibody) in the ratio of 1:25 v/v.
2. Incubate the samples at 4 °C in the dark for 30 min.
3. Analyze both samples on a flow cytometer to quantify the PMNs.
   NOTE: The population that is FITC-APC double positive, CD66b positive and CD45 positive was considered to be neutrophils. Recovery was calculated by the ratio of the total cell numbers of the input and the resulting suspension.

6. NE Release Analysis

NOTE: To compare the functionality of the isolated neutrophil by the microfluidics and lysis method, an NE assay kit was used.

1. Add PMA (provided in the assay kit) to each resulting suspension (obtained at step 4.9) to a final concentration of 50 nM.
2. Incubate the samples at 37 °C for 2 h.
3. Centrifuge the suspensions at 300 x g for 10 min.
4. Transfer 10 µL of each supernatant to a 96-well plate (provided in the assay kit).
   NOTE: A 96-well microplate with a flat bottom and black polystyrene can be used as well.
5. Add 90 µL of diluted assay buffer (provided in the assay kit) to each well.
6. Add 10 µL of the elastase substrate (Z-Ala-Ala-Ala-Ala 2Rh110, provided in the assay kit).
7. Incubate for 1.5 h at 37 °C.
8. Read the 96-well plate using a fluorometer at an excitation wavelength of 485 nm and an emission wavelength of 525 nm.
   NOTE: The level of NE was divided by the PMN count from the flow cytometry analysis.

Results

We achieved transparent immune-cell suspensions with both DTT mucolysis and microfluidics dissociation (Figure 3A). Microfluidics dissociation collected 4.40 x 10⁵ PMNs on average (2.1 x 10⁴ to 5.60 x 10⁵ PMNs, n = 6) from airway secretion samples diluted 1,000-fold (50 mL total volume) in 1 mL of clean suspension. Compared to the initial diluent, 94.0% PMNs (CD66b⁺/CD45⁺) were recovered in a small volume...

Discussion

In inertial microfluidics, particle and cells localize at a certain lateral position in a micro-channel based on their size^5,18,19,20. Due to the combined effect of the Dean drag force and the inertial lift force in the curved microchannel, large particles or neutrophils (>10 µm) are located inside the channel and small particles, mucus aggregates, and debris smaller than 6 µm are...

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
BD LSR II flow cytometer	BD Bioscience	LSR II flow cytometer	Quantification of cell recovery ratio
Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD66b monoclonal antibody	BD Bioscience	561927	Immunostaining of neutrophils for Flow cytometer analysis
Allophycocyanin (APC)-conjugated mouse anti-human CD45 monoclonal antibody	BD Bioscience	561864	Immunostaining of neutrophils for Flow cytometer analysis
Plate reader	Thermo Fisher scientific	Varioskan	Plate reader for neutrophil elastase assay, ex485/em525
Neutrophil elastase assay kit	Cayman Chemical	600610	Neutrophil functionality assessment
Fluoresbrite YG Microspheres 10.0µm	PolyScience, Inc.	18140-2	Fluorescent particles to express white blood cell trajectory in microfluidics