The authors would like to acknowledge Vertex Pharmaceuticals and the Cystic Fibrosis Research Innovation Award for funding R. Quinn and the NIH/NIAID for funding grant 1 U01 AI124316-01, a systems biology approach to treatment of multi-drug resistant pathogens. We would also like to thank the Department of Mechanical and Aerospace Engineering at UCSD's undergraduate mechanical engineering senior design course for facilitating the collaboration with the engineering aspects of this work.

1. Preparation of Stocks for the Artificial Sputum Media
<ol>
	<li>Create a 5% mucin solution. Add 1.0 g of dehydrated pig stomach mucin to 20 mL of deionized water. Autoclave the resulting solution. 
	NOTE: The mucin sterilization will destroy its inherent structure; other methods to sterilize the mucin in its dry form include UV sterilization and irradiation. These methods have not been extensively used for the WinCF system however.</li>
	<li>Add 2.2 g of KCl to 50 mL of deionized water and allow for dissolution. Add 5.0 g of NaCl to 50 mL of deionized water and allow for dissolution. Autoclave these two solutions.</li>
	<li>Add 100 mg of salmon sperm DNA to 10 mL of sterile deionized water. Heat this solution to about 85 &#176;C in a water bath for a few hours to ensure dissolution.</li>
	<li>Add 5.0 mg of ferritin to 5.0 mL of sterile deionized water.</li>
</ol>
2. Preparation of the Artificial Sputum Medium
<ol>
	<li>Combine the following components: 16 mL of mucin stock solution, 2.0 mL of KCl stock solution, 2.0 mL of NaCl stock solution, 200 &#181;L of egg yolk emulsion, 5.6 mL of DNA stock solution, 120 &#181;L of ferritin stock solution, 5.78 mL of essential amino acid solution, 5.78 mL of non-essential amino acid solution, and 2.44 mL of sterile water.
	<ol>
		<li>If small amounts of sediment appear, shake gently to mix.</li>
		<li>Pipet 5.0 mL of media into eight sterile 15 mL centrifuge tubes. 
		NOTE: The chemical conditions of each tube can be manipulated as desired. For example, buffer solutions and pH indicators can be added to each tube for the purpose of comparing microbial behavior at distinct pH levels. A demonstration of this is shown in the representative results section with 8 distinct pH levels, from 5.0 to 8.5 in 0.5 pH increments.</li>
	</ol>
	</li>
	<li>Once the chemical conditions of the media are successfully manipulated, freeze for later use. The media will remain stable frozen at -20 &#176;C for several months. Vortex upon thawing.</li>
</ol>
3. Preparation of a Control Run of the Capillary Tubes
<ol>
	<li>In a sterile biohood, fill eight sterile microcentrifuge tubes with 250 &#181;L of media each.</li>
	<li>Acquire eight more sterile 15 mL centrifuge tubes, each tube corresponding to a microcentrifuge tube mentioned in step 3.1.
	<ol>
		<li>Sterilize a paper towel with 70% ethanol solution and allow to dry. Once dry, tear the towel into pieces about four square inches each, and crumple each piece in the bottom of a 15 mL centrifuge tube. For additional tubes, spray and dry additional paper towels as needed.</li>
		<li>With one clump of paper at the bottom of each centrifuge tube, slightly dampen each paper clump with about 1.0 mL of sterile water to create a humid environment.</li>
	</ol>
	</li>
	<li>Obtain three glass capillary tubes for every microcentrifuge tube prepared in step 3.1 and a block of capillary tube putty sealant.</li>
	<li>For each microcentrifuge tube, fill three capillary tubes with media to about 5 mm away from the blue marker near the top of the tube.
	<ol>
		<li>Fill by holding one end of the tube in the microcentrifuge tube and tilting towards horizontal orientation, allowing capillary action to guide the medium into the tube (See Figure 1).</li>
		<li>Stop the filling by gently placing a gloved finger over the open end of the tube, and then seal the other end of the tube by pressing it down into the sealant block.</li>
	</ol>
	</li>
	<li></li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55532/55532fig1.jpg" /> 
Figure 1: Example pH Gradient, Filling Capillary Tube with Artificial Sputum Medium. The medium is added by inserting one end of the tube into the liquid and tilting to facilitate capillary action. The medium coloration in this example is due to pH indicator added to help demonstrate potential changes in acidity after incubation. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Place each set of three capillary tubes into 15 mL centrifuge tubes filled with paper towels completely moistened with sterile water, putty-sealed side down. Cap the tube and label. These three capillary tubes are for replication of each control condition.</li>
	<li>When all 15 mL centrifuge tubes are filled with their designated capillary tubes, place the tubes in a holding rack. Place the rack such that the tubes will be incubated horizontally (to catch gas bubbles) (See Figure 2). Incubate the capillary tubes inside the centrifuge tubes (containing the moistened paper clumps) at 37 &#176;C for 48 h.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/55532/55532fig2.jpg" /> 
Figure 2: Example pH Gradient, Capillary Tubes Ready for Incubation. Once three capillary tubes have been filled and sealed, they are placed in a centrifuge tube with damp paper towel at the bottom. This tube is then capped and put into a rack. The rack must be oriented sideways during incubation, as pictured here, so that gas production can be observed once incubation is complete. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Imaging of the Control Capillary Tubes after Incubation
<ol>
	<li>Remove the rack of centrifuge tubes from the incubator, making sure to keep the tubes horizontal. Carefully slide the capillary tubes out of the centrifuge tubes, keeping each set of three separate from other sets.</li>
	<li>Arrange the capillary tubes next to each other on a lightbox, all lined up so that the contents of the tubes are visible and illuminated. Leave a gap every three tubes to separate different chemical conditions.</li>
	<li>With the tubes aligned and lightbox turned on, photograph from directly above. (See Figure 3)</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/55532/55532fig3.jpg" /> 
Figure 3: Example pH Gradient, Control Run, Pre-incubation, No Sputum Added. Artificial sputum medium after being added to capillary tubes in sets of three, increasing in pH from left to right. The combination of indicators added to the medium result in more acidic tubes appearing more yellow, while less acidic tubes become more purple. The tubes are arranged horizontally and are illuminated from below, photographed from above. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Dispose of the control materials in biohazardous waste.</li>
</ol>
5. Inoculating the WinCF Capillary Tubes with a Sputum Sample
<ol>
	<li>In a sterile biohood, fill eight sterile microcentrifuge tubes with 225 &#181;L of media each.</li>
	<li>Homogenize the sputum sample by withdrawing and ejecting the sputum repeatedly with a 3 mL syringe (a plastic syringe without the needle). Do this until the sputum is of smooth consistency.</li>
	<li>Add 25 &#181;L of homogenized sputum to each microcentrifuge tube (1/10 dilution in ASM media) prepared in step 5.1. Then vortex the tubes for 30 s to mix sufficiently.</li>
	<li>Add the media to capillary tubes following the same procedure as steps 3.2 through 3.5.</li>
</ol>
6. Imaging of Sample Capillary Tubes after Incubation
<ol>
	<li>After the 48-h incubation time, remove and image the capillary tubes following the same procedure as steps 4.1 through 4.3.</li>
	<li>If bubbles are present in the tubes, make sure the photographs taken clearly depict the delineation between the bubbles and media in the tubes. If biofilm is present, make sure the photographs can clearly depict its presence as well.</li>
</ol>
7. Removal of Media for Downstream Applications
<ol>
	<li>To facilitate downstream analysis, remove the media from the capillary tubes after imaging. Potential applications include culturing and DNA/RNA sequencing and metabolomic profiling. 
	Caution: The glass capillary tubes filled with pathogens are a significant biohazard, thus, these steps must be done very carefully with specific equipment. If capillary tubes break, dispose in proper biohazard sharps container.</li>
	<li>Use blunt ended needles of 25 gauge and 0.5 inch in length to remove the media. Insert the blunt ended needle into the plugged end of the tube to break the seal.</li>
	<li>After breaking the seal, turn the capillary tube upside down and the media will drip out from the top portion. If the media does not drip out easily, use a pipette with a 200 &#181;L tip and expel the media out of the tube by pressing down on the pipette plunger when inserted into the end of the capillary tube. Collect the expelled tube media in an appropriate container (1.5 mL centrifuge tube). 
	NOTE: For transcriptomic or other RNA analysis, the media can be expelled directly into RNA stabilizing buffers.</li>
</ol>
8. The WinCF FLUD System
NOTE: The WinCF Fluid Loading Utility Device (FLUD) System is an optional suite of complementary devices designed to optimize the throughput of the WinCF System. The WinCF FLUD System is comprised primarily of 3D printable materials. 3D printed manufacturing allows for quick and easy replacement of materials to ensure minimal downtime for researchers as well as minimal manufacturing requirements. Designs, stl files, 3D printing instructions and the WinCF FLUD manual are available in the online supplement.
<ol>
	<li>Preparing media for capillary tube loading
	<ol>
		<li>In a sterile biohood, fill eight sterile 2 mL microcentrifuge tubes with 900 &#181;L of medium each.</li>
		<li>Homogenize any sputum samples by withdrawing and ejecting the sputum repeatedly with a 3 mL syringe (a plastic syringe without the needle). Do this until the sputum is of smooth consistency.</li>
		<li>Add 100 &#181;L of homogenized sputum to each microcentrifuge tube (1/10 dilution in medium) prepared in step 8.1.1. Then vortex the tubes for 30 s to mix sufficiently.</li>
		<li>Slot the filled and open microcentrifuge tubes into the rotator tube holder oriented so that the tubes are vertical.</li>
	</ol>
	</li>
	<li>Placement of capillary tubes
	<ol>
		<li>Retrieve three capillary tubes for every microcentrifuge tube in step 8.1.</li>
		<li>Slot three tubes into the rubber cradle so that they are aligned with the microcentrifuge tubes at the other end of the apparatus. Make sure the marked ends of the tubes face away from the microcentrifuge tubes. Fit the three holes on the bottom of the cradle properly over the three studs on the cradle stand.</li>
		<li>With the placed capillary tubes resting in their guide channels on the apparatus, place the rubber tamp over their midsections securely to prevent shifting. (See Figure 4)</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/55532/55532fig4.jpg" /> 
Figure 4: The FLUD System Fully Loaded with Capillary Tubes Secured by the Rubber Tamp Over Their Midsections. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Loading media into the capillary tubes
	<ol>
		<li>Carefully use one hand to grasp the end of the apparatus where the capillary tubes are loaded, and use the other hand to hold the rotator rack in which the microcentrifuge tubes are loaded.</li>
		<li>Delicately rotate the microcentrifuge rack so that the microcentrifuge tubes are nearly horizontal and proceed to slowly push the rack towards the capillary tubes. (See Figure 5)</li>
		<li>When the ends of the capillary tubes make contact with the media in the microcentrifuge tubes, ensure that capillary action immediately begins filling the capillary tubes. To adjust fill rate and fill level, gently rotate the apparatus as a whole. While doing this, be careful not to spill media out of the microcentrifuge tubes. (See Figure 6)</li>
		<li>When the capillary tubes are filled to desired levels, place the apparatus level on a surface and carefully yet quickly pull the rack of microcentrifuge tubes off the ends of the capillary tubes to cease filling. The microcentrifuge tubes can now be retracted all the way back to vertical position and closed.</li>
		<li>Seal the protruding ends of the capillary tubes by pressing a sealant block onto each triplicate set, sealing one set at a time until all sets are sealed. To reduce risk of contamination, press a different part of the sealant block onto each triplicate set (See Figure 7).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" src="/files/ftp_upload/55532/55532fig5.jpg" /> 
Figure 5: The FLUD System with Medium Tubes Deployed to a Horizontal Orientation, Ready to Make Contact with Capillary Tubes. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55532/55532fig6.jpg" /> 
Figure 6: The FLUD System with Capillary Tubes Loading with Media via Capillary Action. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/55532/55532fig7.jpg" /> 
Figure 7: Sealing Filled Capillary Tubes on the FLUD System One Triplicate Set at a Time Using a Sealant Block. This sealant block had plastic along the edges that was cut off to prevent contact with neighboring triplicate sets during sealing. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Incubation
	<ol>
		<li>Remove the rubber tamp over the tube midsections and lift the rubber cradle off the main apparatus. This should take all the capillary tubes with it. Now set the cradle and the tubes being held onto the imaging rack. This rack has three stubs that fit into the cradle, as well as small guide channels to set the tubes into.</li>
		<li>Set the imaging rack as a whole into the clear plastic incubation box. Soak small amounts of sterile paper towels in sterile water and place along the two shorter sides of the box to provide humidity during incubation. (See Figure 8)</li>
		<li>Close the box completely and set inside a 37 &#176;C incubator, making sure to keep the tubes horizontal. Incubate for 48 h.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 8" src="/files/ftp_upload/55532/55532fig8.jpg" /> 
Figure 8: Capillary Tubes in Rubber Cradle Transferred from FLUD System to an Imaging Rack, Which has been Placed in a Clear Incubation Box Alongside Damp Paper Towels to Provide Humidity. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Imaging and extraction
	<ol>
		<li>Remove the imaging rack holding the tubes from the incubation box and set it on a lightbox, illuminated from below. With the tubes already in the imaging rack, the triplicate sets will be properly spaced and ready to image immediately.</li>
		<li>Focus the camera on the tubes such that all are visible in the field of view and light from the light box provides sufficient contrast and visualization of the color in the tube dyes. Photograph from directly above.</li>
		<li>To extract the contents of the tubes, remove the capillary tubes from the rubber cradle one set of triplicates at a time. Gently lift the tubes up and out of the cradle or slide them out.</li>
		<li>For each set of triplicates, follow the extraction procedure detailed in steps 7.1 through 7.3.</li>
	</ol>
	</li>
</ol>

<ol><li>Quinn, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Winogradsky-based+culture+system+shows+an+association+between+microbial+fermentation+and+cystic+fibrosis+exacerbation%5BTitle%5D)+AND+%22ISME+J+%22%5BJournal%5D)">A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation.</a> ISME J . 9, 1024-1038 (2015).</li>
<li>Quinton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cystic+fibrosis%3A+impaired+bicarbonate+secretion+and+mucoviscidosis%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Cystic fibrosis: impaired bicarbonate secretion and mucoviscidosis.</a> Lancet. 372 (9636), 415-417 (2008).</li>
<li>Harrison, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+ecology+of+the+cystic+fibrosis+lung%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Microbial ecology of the cystic fibrosis lung.</a> Microbiology. 153 (Pt 4), 917-923 (2007).</li>
<li>Caverly, L. J., Zhao, J., LiPuma, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cystic+fibrosis+lung+microbiome%3A+Opportunities+to+reconsider+management+of+airway+infection%5BTitle%5D)+AND+%22Pediatr+pulmonol%22%5BJournal%5D)">Cystic fibrosis lung microbiome: Opportunities to reconsider management of airway infection.</a> Pediatr pulmonol. 50, Suppl 4. S31-S38 (2015).</li>
<li>Blainey, P. C., Milla, C. E., Cornfield, D. N., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+the+human+airway+microbial+ecology+reveals+a+pervasive+signature+for+cystic+fibrosis%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">Quantitative analysis of the human airway microbial ecology reveals a pervasive signature for cystic fibrosis.</a> Sci Transl Med. 4 (153), 153ra130(2012).</li>
<li>Willner, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+distribution+of+microbial+communities+in+the+cystic+fibrosis+lung%5BTitle%5D)+AND+%22ISME+J%22%5BJournal%5D)">Spatial distribution of microbial communities in the cystic fibrosis lung.</a> ISME J. 6 (2), 471-474 (2012).</li>
<li>Delhaes, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+airway+microbiota+in+cystic+fibrosis%3A+a+complex+fungal+and+bacterial+community--implications+for+therapeutic+management%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">The airway microbiota in cystic fibrosis: a complex fungal and bacterial community--implications for therapeutic management.</a> PloS one. 7 (4), e36313(2012).</li>
<li>Rogers, G. B., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC179861/">D. Bacterial diversity in cases of lung infection in cystic fibrosis patients: 16S ribosomal DNA (rDNA) length heterogeneity PCR and 16S rDNA terminal restriction fragment length polymorphism profiling.</a> J clin microbiol. 41 (8), 3548-3558 (2003).</li>
<li>Stenbit, A. E., Flume, P. A. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/21881509">Pulmonary exacerbations in cystic fibrosis.</a> Curr Opin Pulm Med. 17 (6), 442-447 (2011).</li>
<li>Twomey, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbiota+and+metabolite+profiling+reveal+specific+alterations+in+bacterial+community+structure+and+environment+in+the+cystic+fibrosis+airway+during+exacerbation%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Microbiota and metabolite profiling reveal specific alterations in bacterial community structure and environment in the cystic fibrosis airway during exacerbation.</a> PloS one. 8 (12), e82432(2013).</li>
<li>Carmody, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+cystic+fibrosis+airway+microbiota+at+pulmonary+exacerbation%5BTitle%5D)+AND+%22Ann.+Am.+Thorac.+Soc%22%5BJournal%5D)">Changes in cystic fibrosis airway microbiota at pulmonary exacerbation.</a> Ann. Am. Thorac. Soc. 10 (3), 179-187 (2013).</li>
<li>Worlitzsch, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+reduced+mucus+oxygen+concentration+in+airway+Pseudomonas+infections+of+cystic+fibrosis+patients%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients.</a> J. Clin. Invest. 109 (3), 317-325 (2002).</li>
<li>Cowley, E. S., Kopf, S. H., LaRiviere, A., Ziebis, W., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pediatric+Cystic+Fibrosis+Sputum+Can+Be+Chemically+Dynamic%2C+Anoxic%2C+and+Extremely+Reduced+Due+to+Hydrogen+Sulfide+Formation%5BTitle%5D)+AND+%22mBio%22%5BJournal%5D)">Pediatric Cystic Fibrosis Sputum Can Be Chemically Dynamic, Anoxic, and Extremely Reduced Due to Hydrogen Sulfide Formation.</a> mBio. 6 (4), e00767-e00715 (2015).</li>
<li>Sriramulu, D. D., Lünsdorf, H., Lam, J. S., Römling, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microcolony+formation%3A+a+novel+biofilm+model+of+Pseudomonas+aeruginosa+for+the+cystic+fibrosis+lung%5BTitle%5D)+AND+%22J.+Med.+Microbiol%22%5BJournal%5D)">Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung.</a> J. Med. Microbiol. 54 (Pt 7), 667-676 (2005).</li>
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</ol>

The authors have nothing to disclose.

The microbiological makeup of a lung with CF contains a great variety of organisms, but the conditions within the lung likely have a significant influence on what kinds of microbes can survive and thrive13,15. Specific mechanisms through which these conditions change and the exact effects they have on the lung microbiome are generally unclear at present. In this experimental method, we present an analysis of microbiological changes based upon manipulated chemical...

The method described in this manuscript is called the WinCF system1. The overall goal of WinCF is to provide an experimental setup capable of simulating the environment of a mucus-filled lung bronchiole. This will allow for a tractable system to study microbial pathogens of lung diseases with a mucus hypersecretion phenotype including cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), asthma and others. The procedure was designed specifically for the study of CF, which is characterized by mutations that cause lung secretions to become thick and hard to clear, eventually filling bronchioles and other small passageways with mucus2. Such blockages in the lung inhibit gas exchange because inhaled air is no longer able to reach many alveoli and also provide a habitat for bacterial colonization3,4. The inability to prevent microbial growth in the excessive lung mucus eventually leads to the development of complex chronic infections of the airway. These communities contain a variety of organisms, including viruses, fungi, and bacteria like Pseudomonas aeruginosa, all interacting with one another5,6,7,8. The activity of the CF lung microbiome is believed to be involved in flares of symptoms called pulmonary exacerbations1,9,10,11. WinCF enables study of the microbial community behavior around these exacerbations and is now being expanded to act as a base experimental system to study lung microbial ecology. Traditionally, exacerbations have been studied through direct analysis of samples taken from the lung. Many confounding factors make direct analysis of microbial behavior in lungs challenging, with the WinCF system, many of these factors are removed and the behavior of the lung microbiome can be studied more directly, allowing for finer analysis of bacterial activity in a mucus-plugged bronchiole.
The WinCF system provides a method to grow and analyze bacteria in a manner that effectively mimics the lung environment. Traditional methods for growing lung bacteria often involved culturing samples on traditional agar plates. These methods leave the samples open to atmospheric oxygen, neglecting to account for the hypoxic and often anoxic conditions found in lung bronchioles plugged with mucus12,13. Culturing on agar under aerobic conditions is nothing like the environment of the CF lung and can mislead clinicians and researchers concerning the behavior of the pathogens that they are trying to treat. Additionally, the nutrients available to bacteria on agar plates are dissimilar to those available in actual sputum, which is accounted for in WinCF by utilizing artificial sputum media (ASM). As shown by the Pseudomonas cultures in Sriramulu et al.14, ASM includes a specific set of components that mimics the resources available to sputum microbes and also replicates the physical consistency of sputum. Because a diseased lung has a specific microbiome, the study of such microorganisms should ideally take place in the specific conditions of the lung as well.
The WinCF system enables rapid analysis and easy manipulation of the experimental conditions to observe microbial changes similar to how they would occur in an actual lung bronchiole. This technique allows for the inoculation of a myriad of related sample types including sputum, saliva, other body secretions and pure or mixed bacterial cultures. The nature of the experimental setup allows for immediate visual interpretation of the microbial community behavior and is designed to enable easy downstream application of a multitude of microbiological and omics procedures. Such studies are important because bacterial community composition changes based upon the physiochemical conditions of their environment. With WinCF the chemical conditions of the media can be manipulated to analyze the effects on bacterial activity. For example, the acidity of the media can be altered prior to inoculation with a sample. After incubation, the bacterial activity in each of these conditions can be directly compared, and conclusions can be drawn about how bacteria in those sputum samples behave in response to varying pH. Here, we outline the procedures for applying the WinCF system and examples of how the media chemistry can be manipulated to study the effects on the lung microbiome.

<table><tbody><tr><td>Color-Coded Capillary Tubes</td><td>Fisher Scientific</td><td>22-260943</td><td></td></tr><tr><td>Cha-seal Tube Sealing Compound</td><td>Kimble-Chase</td><td>43510</td><td></td></tr><tr><td>Mucin from porcine stomach</td><td>Sigma</td><td>M1778</td><td></td></tr><tr><td>Ferritin, cationized from horse spleen</td><td>Sigma</td><td>F7879</td><td></td></tr><tr><td>Salmon sperm DNA Sodium salt (sonified)</td><td>AppliChem Panreac</td><td>A2159</td><td></td></tr><tr><td>MEM Nonessential Amino Acids</td><td>Corning cellgro</td><td>25-025-CI</td><td></td></tr><tr><td>MEM Amino Acids</td><td>Cellgro</td><td>25-030-CI</td><td></td></tr><tr><td>Egg Yolk Emulsion, 50%</td><td>Dalynn Biologicals</td><td>VE30-100</td><td></td></tr><tr><td>Potassium Chloride</td><td>Fisher Scientific</td><td>P2157500</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>S271500</td><td></td></tr><tr><td>15 mL centriguge tubes with Printed Graduations and Flat Caps</td><td>VWR</td><td>89039-666</td><td></td></tr><tr><td>50 mL centrifuge tubes with Printed Graduations and Flat Caps</td><td>VWR</td><td>89039-656</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>Corning</td><td>MCT-150-R</td><td></td></tr><tr><td>2.0 mL microcentrifuge tubes</td><td>Corning</td><td>MCT-200-C</td><td></td></tr></tbody></table>

the wincf model - an inexpensive and tractable microcosm of a mucus plugged bronchiole to study the microbiology of lung infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

Microbiological growth across the various chemical conditions induced within the samples varied dramatically in some cases and more subtly in others. Many changes in activity were visual in nature, being readily apparent as soon as the incubation period ended. In the example of pH manipulation, the samples across the pH spectrum varied greatly as shown by multiple factors that became apparent after incubation. When no sputum samples were added to the media, the only change exhibited acros...

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The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

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Pharmacology

Immunology and Infection

Unit 1

Lower Respiratory Disorders

SCIENTISTS IN ACTION

9.2K Views.  University of California, San Diego.  De mucuspluggen van cystische fibrose (CF) patiënten zijn een ideale omgeving om microbiële pathogenen te bloeien. Het manuscript beschrijft een nieuwe methode om de CF-longmikrobiome te bestuderen in een omgeving die naboots waar ze ziekte veroorzaken en hoe veranderingen van chemische omstandigheden microbiële dynamiek kunnen veroorzaken. 

Video: The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards optimizing drug inhalation techniques as well as predicting deposition patterns of potentially toxic airborne particles in the pulmonary alveoli. Here, soft-lithography techniques are used to fabricate complex acinar-like airway structures at the truthful anatomical length-scales that reproduce physiological acinar flow phenomena in an optically accessible system. The microfluidic device features 5 generations of bifurcating alveolated ducts with periodically expanding and contracting walls. Wall actuation is achieved by altering the pressure inside water-filled chambers surrounding the thin PDMS acinar channel walls both from the sides and the top of the device. In contrast to common multilayer microfluidic devices, where the stacking of several PDMS molds is required, a simple method is presented to fabricate the top chamber by embedding the barrel section of a syringe into the PDMS mold. This novel microfluidic setup delivers physiological breathing motions which in turn give rise to characteristic acinar air-flows. In the current study, micro particle image velocimetry (&#181;PIV) with liquid suspended particles was used to quantify such air flows based on hydrodynamic similarity matching. The good agreement between &#181;PIV results and expected acinar flow phenomena suggest that the microfluidic platform may serve in the near future as an attractive in vitro tool to investigate directly airborne representative particle transport and deposition in the acinar regions of the lungs.

Soft-lithography was utilized to produce a representative true-scale model of pulmonary alveolated airways that expand and contract periodically, mimicking physiological breathing motion. This platform recreates respiratory acinar flows on a chip, and is anticipated to facilitate experimental investigation of inhaled aerosol dynamics and deposition in the pulmonary acinus.

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards ...

Bioengineering

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Experimental Model to Evaluate Resolution of Pneumonia

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.

This manuscript describes the establishment of an infectious model of pneumonia in mice and the respective characterization of injury resolution along with methods for growing bacteria and intratracheal instillation. A novel approach using high-dimensional flow cytometry to evaluate the immune landscape is also described.

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to ...

Establishing a Lung-on-Chip Model for Respiratory Infection Studies

Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.

Author Spotlight: Developing a Microfluidic Lung-on-Chip Model for In-Depth Study of Human Immune Response and Infection Mechanisms

Lung-on-chip models surpass traditional 2D cultures by mimicking the air-liquid interface and endothelial cell perfusion, simulating blood flow and nutrient exchange crucial for lung physiology studies. This enhances lung research relevance, offering a dynamic, physiologically accurate environment to advance the understanding and treatment of respiratory infections.

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs ...

Preserving Fungal Spore Localization in Lungs for Fluorescent Microscopy Analysis

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and bronchioles, branch until terminating in the alveolar airspace where gas exchange occurs. Infections originating in the bronchioles or alveoli elicit distinct host responses and disease manifestations. Therefore, understanding precisely where spores naturally localize in the lungs, particularly soon after infection, expands opportunities for investigation of host-pathogen interactions. Herein, we detail an in-situ analysis of lungs from mice infected with Coccidioides posadasii cts2/ard1/cts3&#916; arthroconidia. Conventional methods for histological preservation involve liquid inflation of the airways with a fixative solution, which displaces the natural location of aspirated fungal particles, pushing spores from proximal bronchioles to terminal airspaces.
Conversely, this method of air-inflation with blood vasculature perfusion-fixation preserves the physiologic position of fungal spores within the bronchioles. Moreover, we describe a simple approach to cryopreserving, embedding, and imaging lung specimens. We also share high-throughput computational techniques via the open-source QuPath program to analyze the spatial distribution of fungal spores within the lung. The method presented here is simple and quick, requires minimal equipment to perform, and can be easily adapted for use with many respiratory fungal infection models.

We present a method for a fungal pathogenesis model that preserves the natural positioning of fungal spores in the lung airways for analysis via fluorescent microscopy.

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and ...

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G&#39;, G&#34;, G*, and tan &#948;) and gel point characteristics (&#947;c&#160;and &#963;c) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is &#60;10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more &#34;liquid-like&#34; behavior overall (e.g., higher tan &#948;). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.

The viscoelastic properties of mucus play a critical role in mucociliary clearance. However, traditional mucus rheological techniques require complex and time-consuming approaches. This study provides a detailed protocol for the use of a benchtop rheometer that can rapidly and reliably perform viscoelastic measurements.

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., ...

Medicine

In Vitro Polymicrobial Interactions in Cystic Fibrosis Lung Infections

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat, chronic, and polymicrobial biofilm-based infections detected in the airways of individuals living with cystic fibrosis (CF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of people with CF (pwCF), no in vitro models thus far have fully integrated critical CF-relevant lung features. Therefore, a significant knowledge gap exists in the capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections. Here, we describe a recently developed four-species microbial community model, including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial&#160;recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

This protocol describes a cystic fibrosis (CF) lung-relevant four-species polymicrobial biofilm model that can be used to explore the impact of bacterial interspecies interactions.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This ...

Biology

Assessing Biophysical Properties of Differentiated Airway Epithelium 

Determining Ciliary Function and Membrane Impermeability of the Pseudostratified Lung Airway Epithelium

Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified airway epithelium that mimics the apical side of the lung airway. While the differentiation of primary lung airway cells is expected, the assessment of biophysical properties like ciliary function and membrane impermeability provides a quality assessment of the airway epithelium and ensures the reliability of the experiment. Here, we describe a straightforward protocol for the development of multi-cellular pseudostratified airway epithelium in ALI culture and assess two important biophysical properties: ciliary function and membrane impermeability. To determine ciliary function, we captured the ciliary movement of a 4-week differentiated airway epithelium using a high-speed camera attached to an inverted microscope, followed by quantifying ciliary beat frequency (CBF) using the Simon Amon Video Analysis (SAVA) system. We also measured transepithelial electrical resistance (TEER) using a volt-ohm meter to determine the epithelial barrier integrity of the airway epithelium.

Air-liquid interface culture is commonly used to develop pseudostratified airway epithelium by differentiating primary normal human bronchial epithelial cells that mimic the apical side of the lung airway. Here, we describe an easy protocol for determining its quality by monitoring its biophysical properties, such as ciliary function and membrane integrity.

Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

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