This work was supported by NIH/NIAID (R21AI119042) as well as NIH U24 Sample Sparing assay program (U24-AI118656).

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 techniques15,16 were used to create the polydimethylsiloxane (PDMS) microchannel.
<ol>
	<li>Mix the PDMS...

<ol>
	<li>Hamid, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+sputum+processing+for+cell+counts,+immunocytochemistry+and+in+situ+hybridisation.">Methods of sputum processing for cell counts, immunocytochemistry and in situ hybridisation.</a> European Respiratory Journal. 20 (Supplement 37), 19S-23S (2002).</li><li>van Overveld, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+homogenization+of+induced+sputum+by+dithiothreitol+on+polymorphonuclear+cells.">Effects of homogenization of induced sputum by dithiothreitol on polymorphonuclear cells.</a> J Physiol Pharmacol. 56, 143-154 (2005).</li><li>Qiu, D., Tan, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dithiothreitol+has+a+dose-response+effect+on+cell+surface+antigen+expression.">Dithiothreitol has a dose-response effect on cell surface antigen expression.</a> J Allergy Clin Immunol. 103 (5 Pt 1), 873-876 (1999).</li><li>Usher, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Neutrophil+Apoptosis+by+the+Pseudomonas+aeruginosa+Exotoxin+Pyocyanin:+A+Potential+Mechanism+of+Persistent+Infection.">Induction of Neutrophil Apoptosis by the Pseudomonas aeruginosa Exotoxin Pyocyanin: A Potential Mechanism of Persistent Infection.</a> The Journal of Immunology. 168 (4), 1861-1868 (2002).</li><li>Di Carlo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inertial+microfluidics.">Inertial microfluidics.</a> Lab Chip. 9 (21), 3038-3046 (2009).</li><li>Martel, J. M., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inertial+focusing+dynamics+in+spiral+microchannels.">Inertial focusing dynamics in spiral microchannels.</a> Phys Fluids. 24 (3), 32001 (2012).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+and+applications+of+inertial+microfluidics:+a+review.">Fundamentals and applications of inertial microfluidics: a review.</a> Lab Chip. 16 (1), 10-34 (2016).</li><li>Hou, H. W., Bhattacharyya, R. P., Hung, D. T., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+and+drug-resistance+profiling+of+bacteremias+using+inertial+microfluidics.">Direct detection and drug-resistance profiling of bacteremias using inertial microfluidics.</a> Lab Chip. 15 (10), 2297-2307 (2015).</li><li>Warkiani, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-fast,+label-free+isolation+of+circulating+tumor+cells+from+blood+using+spiral+microfluidics.">Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics.</a> Nat Protoc. 11 (1), 134-148 (2016).</li><li>Warkiani, M. E., Tay, A. K., Guan, G., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-less+microfiltration+using+inertial+microfluidics.">Membrane-less microfiltration using inertial microfluidics.</a> Sci Rep. 5, 11018 (2015).</li><li>Warkiani, M. E., Wu, L., Tay, A. K., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-Volume+Microfluidic+Cell+Sorting+for+Biomedical+Applications.">Large-Volume Microfluidic Cell Sorting for Biomedical Applications.</a> Annu Rev Biomed Eng. 17, 1-34 (2015).</li><li>Kwon, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Cell+Retention+Device+for+Perfusion+of+Mammalian+Suspension+Culture.">Microfluidic Cell Retention Device for Perfusion of Mammalian Suspension Culture.</a> Sci Rep. 7 (1), 6703 (2017).</li><li>Wu, L., Guan, G., Hou, H. W., Bhagat, A. A., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+leukocytes+from+blood+using+spiral+channel+with+trapezoid+cross-section.">Separation of leukocytes from blood using spiral channel with trapezoid cross-section.</a> Anal Chem. 84 (21), 9324-9331 (2012).</li><li>Guan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiral+microchannel+with+rectangular+and+trapezoidal+cross-sections+for+size+based+particle+separation.">Spiral microchannel with rectangular and trapezoidal cross-sections for size based particle separation.</a> Sci Rep. 3, 1475 (2013).</li><li>Kotz, K., Cheng, X., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+Device+Fabrication+and+Surface+Modification.">PDMS Device Fabrication and Surface Modification.</a> J Vis Exp. (8), e319 (2007).</li><li>Duffy, D. C., McDonald, J. C., Schueller, O. J. A., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Prototyping+of+Microfluidic+Systems+in+Poly(dimethylsiloxane).">Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane).</a> Analytical Chemistry. 70 (23), 4974-4984 (1998).</li><li>Ryu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-Derived+Airway+Secretion+Dissociation+Technique+To+Isolate+and+Concentrate+Immune+Cells+Using+Closed-Loop+Inertial+Microfluidics.">Patient-Derived Airway Secretion Dissociation Technique To Isolate and Concentrate Immune Cells Using Closed-Loop Inertial Microfluidics.</a> Anal Chem. 89 (10), 5549-5556 (2017).</li><li>Mach, A. J., Di Carlo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+scalable+blood+filtration+device+using+inertial+microfluidics.">Continuous scalable blood filtration device using inertial microfluidics.</a> Biotechnol Bioeng. 107 (2), 302-311 (2010).</li><li>Di Carlo, D., Irimia, D., Tompkins, R. G., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+inertial+focusing,+ordering,+and+separation+of+particles+in+microchannels.">Continuous inertial focusing, ordering, and separation of particles in microchannels.</a> Proc Natl Acad Sci U S A. 104 (48), 18892-18897 (2007).</li><li>Xiang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+elasto-inertial+particle+focusing+in+curved+microfluidic+channels.">Fundamentals of elasto-inertial particle focusing in curved microfluidic channels.</a> Lab Chip. 16 (14), 2626-2635 (2016).</li><li>Lotvall, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asthma+endotypes:+a+new+approach+to+classification+of+disease+entities+within+the+asthma+syndrome.">Asthma endotypes: a new approach to classification of disease entities within the asthma syndrome.</a> J Allergy Clin Immunol. 127 (2), 355-360 (2011).</li><li>Houston, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sputum+neutrophils+in+cystic+fibrosis+patients+display+a+reduced+respiratory+burst.">Sputum neutrophils in cystic fibrosis patients display a reduced respiratory burst.</a> J Cyst Fibros. 12 (4), 352-362 (2013).</li><li>Janoff, A., Scherer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediators+of+inflammation+in+leukocyte+lysosomes.+IX.+Elastinolytic+activity+in+granules+of+human+polymorphonuclear+leukocytes.">Mediators of inflammation in leukocyte lysosomes. IX. Elastinolytic activity in granules of human polymorphonuclear leukocytes.</a> J Exp Med. 128 (5), 1137-1155 (1968).</li><li>Kawabata, K., Hagio, T., Matsuoka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+neutrophil+elastase+in+acute+lung+injury.">The role of neutrophil elastase in acute lung injury.</a> Eur J Pharmacol. 451 (1), 1-10 (2002).</li><li>Rubin, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastic+Bronchitis.">Plastic Bronchitis.</a> Clin Chest Med. 37 (3), 405-408 (2016).</li><li>Kokot, K., Teschner, M., Schaefer, R. M., Heidland, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+and+inhibition+of+elastase+release+from+human+neutrophils+dependent+on+the+calcium+messenger+system.">Stimulation and inhibition of elastase release from human neutrophils dependent on the calcium messenger system.</a> Miner Electrolyte Metab. 13 (2), 133-140 (1987).</li></ol>

In inertial microfluidics, particle and cells localize at a certain lateral position in a micro-channel based on their size5,18,19,20. Due to the combined effect of the Dean drag force and the inertial lift force in the curved microchannel, large particles or neutrophils (&#62;10 &#181;m) are located inside the channel and small particles, mucus aggregates, and debris smaller than 6 &#181;m are...

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 cells1,2. However, DTT can interfere with surface-bound antigens of airway neutrophils, resulting in the disruption of neutrophil function such as elastase and myeloperoxidase (MPO) release2,3.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 biomatrices5,6.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 cells8,9, pathogens in blood8, cells from a suspension culture10,11,12, and polymorphonuclear leukocytes (PMNs) from blood13,14.
Here, we introduce a protocol to prepare immune cells from a patient&#39;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.

<table>
	<tbody>
		<tr>
			<td>PDMS precursor</td>
			<td>Dow corning</td>
			<td>184 SIL ELAST KIT 3.9KG</td>
			<td>10:1 ratio of base and curing agent</td>
		</tr>
		<tr>
			<td>VWR gravity convection oven</td>
			<td>VWR</td>
			<td>414005-128</td>
			<td>PDMS precursor to be cured in 90 deg.</td>
		</tr>
		<tr>
			<td>100mm petri dish</td>
			<td>VWR</td>
			<td>89000-324</td>
			<td>Fabrication of PDMS Supporting layer</td>
		</tr>
		<tr>
			<td>Harris Uni-core puncher</td>
			<td>Sigma-aldrich</td>
			<td>WHAWB100076</td>
			<td>2mm diameter or other depending on the tubing size</td>
		</tr>
		<tr>
			<td>Air plasma machine</td>
			<td>Femto Science</td>
			<td>Cute</td>
			<td>Surface plasma treatment for PDMS device to bottom base.</td>
		</tr>
		<tr>
			<td>2&#8221; x 3&#8221; glass slide</td>
			<td>TED PELLA, INC.</td>
			<td>2195</td>
			<td>To support PDMS device</td>
		</tr>
		<tr>
			<td>Masterflex spooled platinum-cured silicone tubing, L/S 14</td>
			<td>Cole-Parmer</td>
			<td>EW-96410-14</td>
			<td>Tubing for microfluidics and peristlatic pump</td>
		</tr>
		<tr>
			<td>1/16 inch Luer connector, male</td>
			<td>Harvard apparatus</td>
			<td>PC2 72-1443</td>
			<td>Connector for fluid guide</td>
		</tr>
		<tr>
			<td>50mL Falcon tube</td>
			<td>Corning</td>
			<td>21008-940</td>
			<td>sample collection &#38; preparation</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline, 1X Without Calcium and Magnesium</td>
			<td>Corning</td>
			<td>45000-446&#160;</td>
			<td>Buffer solution to dilute sample</td>
		</tr>
		<tr>
			<td>Halyard Closed suction Catheter, Elbow, 14F/ channel 4.67mm</td>
			<td>HALYARD HEALTH</td>
			<td>22113</td>
			<td>Tracheal seceation suction catheter</td>
		</tr>
		<tr>
			<td>0.9% Sterile Normal saline, 10mL pre-filled syringe</td>
			<td>BD PosiFlush</td>
			<td>NHRIC: 8290-306547</td>
			<td>For tracheal seceation collection from the patients</td>
		</tr>
		<tr>
			<td>SecurTainer&#8482; III Specimen Containers, 20mL</td>
			<td>Simport</td>
			<td>1176R36</td>
			<td>Sterile sputum (airway secretion) collection container</td>
		</tr>
		<tr>
			<td>Syringe with Luer-Lok Tip, 10mL</td>
			<td>BD</td>
			<td>BD309604</td>
			<td>To pipette homogenize the mucus sample and reach the bottom of sample tube</td>
		</tr>
		<tr>
			<td>BD&#160; Blunt Fill Needle, with BD Luer-Lok&#160; Tip</td>
			<td>BD</td>
			<td></td>
			<td>To pipette homogenize the mucus sample and reach the bottom of sample tube</td>
		</tr>
		<tr>
			<td>40&#181;m nylon cell strainer&#160;</td>
			<td>Falcon</td>
			<td>21008-949</td>
			<td>To remove large chunk or blood clots, which can block the microfluidics access hole or the channel.</td>
		</tr>
		<tr>
			<td>Peristaltic pump (Masterflex L/S Digital Drive)</td>
			<td>Cole-Parmer</td>
			<td>HV-07522-30</td>
			<td>operation of microfluidics</td>
		</tr>
		<tr>
			<td>BD LSR II flow cytometer</td>
			<td>BD Bioscience</td>
			<td>LSR II flow cytometer</td>
			<td>Quantification of cell recovery ratio</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD66b monoclonal antibody</td>
			<td>BD Bioscience</td>
			<td>561927</td>
			<td>Immunostaining of neutrophils for Flow cytometer analysis</td>
		</tr>
		<tr>
			<td>Allophycocyanin (APC)-conjugated mouse anti-human CD45 monoclonal antibody</td>
			<td>BD Bioscience</td>
			<td>561864</td>
			<td>Immunostaining of neutrophils for Flow cytometer analysis</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Thermo Fisher scientific</td>
			<td>Varioskan</td>
			<td>Plate reader for neutrophil elastase assay, ex485/em525</td>
		</tr>
		<tr>
			<td>Neutrophil elastase assay kit</td>
			<td>Cayman Chemical</td>
			<td>600610</td>
			<td>Neutrophil functionality assessment</td>
		</tr>
		<tr>
			<td>Fluoresbrite YG Microspheres 10.0&#181;m</td>
			<td>PolyScience, Inc.</td>
			<td>18140-2</td>
			<td>Fluorescent particles to express white blood cell trajectory in microfluidics</td>
		</tr>
	</tbody>
</table>

label-free neutrophil enrichment from patient-derived airway secretion using closed-loop inertial microfluidics

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&#39;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.

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...

Watch this Scientific Journal Video about Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics at JoVE.com

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

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.

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

This method can help answer key questions in the pulmonary diseases research such as pneumonia, asthma, cystic fibrosis, and chronic obstructive pulmonary disease. The main advantage of this technique is to isolate and enrich patient neutrophil from patient airway secretion without vertical chemical disturbance. Demonstrating the procedure will be Kyungyong, a PhD student from my laboratory.To start with, maintain a ratio of 10 to one and mix the PDMS precursor with the base and curing agent. Next, add 30 grams of the PDMS mixture on the micro machine aluminum mold. Then add another 20 grams of PDMS mixture on a 100 millimeter Petri dish.For the purpose of degas, put the mold and the Petri dish in the vacuum desiccator. After degassing, release the vacuum and place the mold and the Petri dish in an oven at 90 degrees Celsius for an hour. After one hour, remove the mold and the Petri dish from the oven and let it cool at room temperature for 10 minutes.Next, use a knife to cut the outline and punch the fluid access holes with two millimeter biopsy punch on the device. After punching the holes, secure a tape and adhere it to the channel side of the device and on the thick film of PDMS. Use forceps to peel the tape carefully to remove the dust.Then start treating the channel side of the device and the plain PDMS with air plasma. Then bond both the channel side and the plain PDMS with the prepared supporting layer of plain PDMS. Next, leave the chip on a glass slide with dimensions of 102 by 76 millimeters.Place the glass slide in an oven at 70 degrees Celsius for at least 30 minutes to increase the bond strength. Use a 10 milliliter syringe to collect one milliliter of airway secretion samples in nine 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 micro channels. 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 the 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 five 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 one milliliter per minute. After placing the sample in the sample reservoir, switch on the peristaltic pump and set the flow rate at four milliliters per minute.After the sample volume reaches the desired volume of approximately one to two milliliters, stop the operation and disconnect the tubing. Clinical sample consisting of patient tracheal secretion is used for the microfluidic dissociation method in order to obtain the PMN rich population of cells. On subjecting the microfluidic dissociation method, highly purified CD45, CD66B plus rich PMN population of cells are obtained.Here, the samples obtained from two different patients represented as case one and two are subjected to microfluidic dissociation to obtain the resulting cell suspension. Next, on subjecting the patient airway secretion sample to microfluidics, 94%of PMN are recovered consistently from all six samples. On the other hand, subjecting the patient sample to DTT mucolysis method resulted in recovering only 53.5%of PMNs with wide variation among the samples.The neutrophils isolated by the microfluidics and DTT mucolytic methods are treated with PMA to trigger the neutrophil elastase release. Interestingly, patient samples subjected to microfluidics show intact functional cells with increased release of elastase by PMA stimulation. Whereas cells obtained after mucolytic dissociation show no significant increase in the elastase release from six different patient samples studied.This is due to a chemically disturbed neutrophil surface. Once mastered, this technique can be done in 50 minutes if it is performed properly. By providing a dissociation protocol to capture neutrophils from clinical airway secretions, the method presented in this study is expected to expand the scope of clinical research on respiratory diseases such as pneumonia, asthma, cystic fibrosis, and chronic obstructive pulmonary disease.The implications of this technique extend towards therapy of immune related pulmonary diseases such as asthma and chronic obstructive pulmonary disease because the proposed methods provides a way to capturing patient immune cell in noninvasive and label-free manner. Though this method can provide insight into respiratory research, it can also be applied to other patient biofluids with challenging biometrics such as nasal fluid, cervical fluid, and intestinal fluid. After watching this video, you should have a good understanding of how to separate neutrophils from patient airway secretion using close-looped spiral inertia microfluidics.Don't forget that working with clinical samples can be extremely hazardous. All experiments must be performed under a biosafety cabinet with the personal protective equipments.

label-free-neutrophil-enrichment-from-patient-derived-airway

Research

JoVE Journal

Immunology and Infection

6.3K visualizações.  Massachusetts Institute of Technology. This method can help answer key questions in the pulmonary diseases research such as pneumonia, asthma, cystic fibrosis, and chronic obstructive pulmonary disease. The main advantage of this technique is to isolate and enrich patient neutrophil from patient airway secretion without vertical chemical disturbance. Demonstrating the procedure will be Kyungyong, a PhD student from my laboratory.To start with, maintain a ratio of 10 to one and mix the PDMS precursor with the base and curing...