Part of this work was performed at the Marine Biological Lab with support from the Marine Biological Lab, DOE (DE-SC0016127), NSF (MCB1822263), HHMI (grant number 5600373), and a gift from the Simons Foundation.

This study received approval from the Partners Institutional Review Board (Protocol # 2018P002934). Inclusion criterion included adult patients with cystic fibrosis who provided written informed consent for the study. There was no exclusion criterion. According to protocol guidelines, all sputum samples were collected from patients with cystic fibrosis during a scheduled outpatient visit with their clinical provider.
1. Artificial Sputum Medium Preparation
NOTE: Quantities listed here are for the production of 1 L of final artificial sputum medium, and assume the specific reagents listed in the Materials Table. Numbers must be adjusted for other volumes or for the use of different reagents to ensure the same final product. See Table 1 for target concentrations.
<ol>
	<li>Artificial sputum chemical mix (ASCM) 
	NOTE: ASCM makes up 25% of the final medium volume. It is shelf-stable and can be prepared in bulk or in advance. If being prepared for later use, autoclave the chemical mix and safely store it at room temperature.
	<ol>
		<li>Mix the constituent chemical stock solutions.
		<ol>
			<li>Prepare 1 M NaCl stock: Add 58.44 g of NaCl per liter of sterile water.</li>
			<li>Prepare 1 M KCl stock: Add 74.55 g of KCl per liter of sterile water.</li>
			<li>Prepare 1 M MgSO4 stock: Add 246.47 g of MgSO4&#183;7H2O per liter of sterile water, or 120.37 g of anhydrous MgSO4 per liter of sterile water.</li>
			<li>Prepare 1 M glucose stock: Add 180.16 g of glucose per liter of sterile water.</li>
		</ol>
		</li>
		<li>Autoclave sterilize the chemical stock solutions, as well as an empty 250 mL bottle. Perform the autoclaving steps to at least standard values of 121 &#176;C and 15 PSI for 30 min.</li>
		<li>Add 80.59 mL of sterile water to the empty 250 mL bottle.</li>
		<li>Add 152.30 mL of 1 M NaCl stock to the mix.</li>
		<li>Add 15.8 mL of 1 M KCl stock to the mix.</li>
		<li>Add 610 &#181;L of 1 M MgSO4 stock to the mix.</li>
		<li>Add 700 &#181;L of 1 M glucose stock to the mix.</li>
	</ol>
	</li>
	<li>Artificial sputum mucin mix (ASMM) 
	NOTE: ASMM makes up 50% of the final medium volume. Ensure that it is prepared on the same day as the final medium batch.
	<ol>
		<li>Add 450 mL of sterile water to an empty 1 L bottle.</li>
		<li>Add 50 mL of 10x Phosphate-Buffered Saline (PBS) to the bottle.</li>
		<li>Add a disposable magnetic stir bar to the bottle.</li>
		<li>Autoclave the bottle containing PBS and the stir bar.</li>
		<li>Measure out 10 g of mucin powder and add it to the PBS.</li>
		<li>Shake the bottle vigorously for preliminary mixing.</li>
		<li>Place the bottle onto a hot plate with a magnetic stirrer. Set heat to medium-high targeting 50 &#176;C and stirring speed to 1100 rpm. Ramp up the speed gradually so that the bar does not fly off the magnet.
		<ol>
			<li>Allow to heat and stir for 15 min.</li>
			<li>Pick up the bottle with heat-resistant gloves. Observe to check if mucin powder settles out of the solution.</li>
			<li>If mucin powder is not fully dissolved, return the bottle to heat/stirrer for 5-min intervals until it is completely dissolved.</li>
		</ol>
		</li>
		<li>Allow the mucin mix to cool to room temperature.</li>
	</ol>
	</li>
	<li>Artificial sputum biological mix (ASBM) 
	&#8203;NOTE: ASBM is 25% of the final medium volume. Prepare it on the same day as the final medium batch, and unlike the other mixes, do not expose its components to any heat.
	<ol>
		<li>Thaw the 100x vitamin stock in a 4 &#176;C fridge or on ice. 
		&#8203;NOTE: Pre-portion the vitamin stock into 10 mL aliquots to minimize the number of freeze/thaw cycles.</li>
		<li>Add 124.24 mL of sterile water to the empty autoclaved 250 mL bottle.</li>
		<li>Add 25.76 mL of 50x essential amino acid stock to the mix.</li>
		<li>Add 80.14 mL of 100x non-essential amino acid stock to the mix.</li>
		<li>Add 10 mL of (thawed) 100x vitamin stock to the mix.</li>
		<li>Add 1 mL of 1000x trace metals stock to the mix.</li>
		<li>Add 8.33 mL of 30% egg yolk emulsion to the mix.</li>
		<li>Add 400 &#181;L of 10 g/L ferritin stock to the mix.</li>
		<li>Mix the solution well via manual shaking.</li>
	</ol>
	</li>
	<li>Artificial sputum medium (ASM)
	<ol>
		<li>Add 250 mL of ASCM to the 1 L bottle containing ASMM.</li>
		<li>Add 250 mL of ASBM to the medium bottle.</li>
		<li>Titrate the medium with basic MOPS buffer (1 M) to reach a pH of 6.3 on a narrow range pH paper. Prior to titration, the medium mix will be too acidic.</li>
		<li>Refrigerate the resulting artificial sputum medium at 4 &#176;C until it is ready for filtration.</li>
		<li>To start the filtration process, transfer 200 mL of unfiltered artificial sputum medium to a vacuum filtration system with a 0.22 &#181;m pore size filter.</li>
		<li>Connect the filtration system to the vacuum pump, turn on the vacuum pump, set it to 70 mbar, and then place the chamber on an orbital shaker shaking at 90 rpm in a cold room at 4 &#176;C.
		<ol>
			<li>Top off with an additional 150 mL of the medium as an appreciable amount is filtered. It takes 1-2 days to filter 1 L of medium completely.</li>
			<li>Repeat with additional chambers until all the media is filtered. 
			NOTE: Try not to filter more than 350 mL of the medium through the same 0.22 &#181;m filter since mucin will plug the filter over time.</li>
		</ol>
		</li>
		<li>Refrigerate filtered artificial sputum medium at 4 &#176;C until ready for use. Use ASM within one month of preparation for best results.</li>
	</ol>
	</li>
</ol>
2. Oxygen Sparging
<ol>
	<li>Sparging station setup 
	NOTE: This protocol should only need to be done in full once, after which point the setup can be maintained through simple maintenance as necessary. See Figure 1 for a visual schematic of the oxygen sparging system.
	<ol>
		<li>Obtain and properly secure the compressed air and oxygen tanks. 
		CAUTION: High pressure makes the tanks extremely dangerous when mishandled. Ensure that the tanks are completely sealed and secured, there are no leaks when the tank is closed, and that all handling personnel is fully trained in their use.</li>
		<li>Attach an air regulator to the compressed air tank with a wrench. For optimal flow reading on the regulator, attach the regulator as close as possible to an upright position.</li>
		<li>Attach an oxygen regulator to the compressed oxygen tank, attaching as close as possible to an upright position. Depending on the oxygen tank, one may need to invert the direction to tighten.</li>
		<li>Connect the tubing from the regulators to a Y-connector to combine the gas flow from the two tanks.</li>
		<li>Connect the output of the Y-connector to the central T-junction valve.</li>
		<li>Connect one side of the central T-junction valve to a gas pressure gauge.</li>
		<li>Connect the other side of the gas pressure gauge to a 25 mm diameter sterile syringe filter with a 0.22 &#181;m pore size.</li>
		<li>Attach a second 25 mm diameter syringe filter to a syringe without a plunger to be used as a gas release during sparging.</li>
		<li>Connect the final side of the central T-junction valve to a second T-junction valve for the oxygen monitor.</li>
		<li>Connect a 25 mm diameter syringe filter to one side of this second T-junction valve, along with tubing to attach 18 G needles.</li>
		<li>Connect the final side of the second T-junction valve to the oxygen monitoring apparatus.</li>
		<li>Connect a cut-off tube to the other side of the oxygen monitoring apparatus to be used as a gas release during monitoring. 
		CAUTION: When testing/using the oxygen sparging system, take careful note of the position of the T-junctions and ensure it matches the intended path through the system. Failure to do so will result in pressure buildup inside the system and cause components to fail and come apart .</li>
		<li>For the maintenance of the system and to keep it working at optimal performance, the following practices are beneficial.
		<ol>
			<li>Reinforce the connections with liberal amounts of Teflon tape to greatly improve their seal and reduce the chance of components coming apart from the internal pressure.</li>
			<li>Keep combined flow rate under 10 L/min to mitigate maximum pressure and prevent failures.</li>
			<li>If a leak is suspected, use a detergent solution such as commercially available liquid leak detectors to identify its location easily, as it will bubble above any gas leaks. Patch leaks using a Polytetrafluoroethylene tape (e.g., Teflon).</li>
			<li>Replace the 25 mm diameter syringe filters in the oxygen sparging system bi-weekly, but this varies with use frequency. Over time, particles caught in the filter reduce the gas flow rate and cause pressure buildups.</li>
			<li>Calibrate the oxygen monitor to 21% oxygen compressed air prior to carrying out measurements.</li>
			<li>Upon completion of the system use, turn off the tanks and bleed the excess gas from the regulators until the flow completely stops.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Serum bottle culture sparging
	<ol>
		<li>Label 500 mL autoclaved serum bottles with sample identifiers, date/time of inoculation, and target oxygen percentage.</li>
		<li>In a biological hood, add 24 mL of the artificial sputum medium to each serum bottle being set up.</li>
		<li>Add 1 mL of the patient sputum homogenized with an 18-G needle (diluted with sterile saline if necessary to obtain sufficient volume of sample for each culture condition) to each serum bottle.</li>
		<li>Using sterile tweezers, place the autoclaved rubber stoppers onto the top of each serum bottle.</li>
		<li>Press down the rubber stoppers, take care not to touch the underside of the stopper with hands.</li>
		<li>Remove the bottles from the hood, apply and crimp the aluminum seals. Remove the center piece from the seals.</li>
		<li>Wipe down the top of bottles with an alcohol wipe and pass them through a Bunsen burner flame.</li>
		<li>Affix a sterile 18-G needle to a plunger-less syringe with a filter. Insert this gas release into the bottle first.</li>
		<li>Affix a sterile 18-G needle to the gas output from the system and insert the gas output needle into the bottle as well.</li>
		<li>Route the T-junctions from the tanks through the oxygen monitor. Verify that the target oxygen concentration is flowing through the system. Target approximately 5 L/min of gas flow.</li>
		<li>Reroute the T-junctions from the tanks through to the gas output. The gas starts to flow through the serum bottle. 
		CAUTION: Pay close attention to the pressure gauge during oxygen sparging. If pressure increases unexpectedly, shut the system off immediately.</li>
		<li>Run oxygen sparge through the serum bottle for 1 min. At 5 L/min, this allows for 10 air exchanges and ensures the internal atmosphere reaches the desired partial pressure.</li>
		<li>Remove the gas release 18-G needle.</li>
		<li>Allow the pressure in the serum bottle to build to +1 atmosphere (2 atmospheres at sea level) and then immediately remove the gas output needle. 
		NOTE: Maintaining pressure aids retention of hyperoxic conditions over time.</li>
		<li>Place the serum bottle into a 37 &#176;C incubator shaker at 150 RPM. Incubate the samples for three 24-h intervals. At each 24-h interval, take an aliquot for downstream analysis, re-sparge the samples and return them to incubation for a total incubation time of 72 h.</li>
	</ol>
	</li>
	<li>Outflow oxygen measurement
	<ol>
		<li>Calibrate the oxygen meter to 21% compressed air, and then turn off the tank.</li>
		<li>Route the serum bottle intake through the oxygen monitor and affix a sterile needle to the end.</li>
		<li>Insert the needle through the rubber stopper into the serum bottle.</li>
		<li>Wait for the outflow reading to stabilize. A low flow rate out of the serum bottles means this may take up to 2 min. Report the peak difference from room air (number furthest from 21%).</li>
		<li>If performing multiple readings, flush the system with compressed air between readings.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

In this study, an in vitro model was developed to study the effect of hyperoxia on lung microbial communities. This model, based on artificial sputum medium and daily sparging of serum bottles, maintains elevated oxygen concentrations and supports the growth of microbes identified in sputum from pwCF.
There are several critical steps of this approach. First is the choice to use filter-sterilization rather than heat-sterilization of the artificial sputum medium. Filter-sterilization pr...

Cystic fibrosis (CF) is a genetic disease characterized by an inability to clear thick mucus from the lungs, leading to repeated infections and progressive lung function decline that often results in the need for lung transplantation or death. The airway microbiome of people with cystic fibrosis (pwCF) appears to track disease activity1, with a reduction in microbial diversity associated with adverse long-term outcomes2,3. In clinical studies of pwCF, supplemental oxygen therapy has been associated with more advanced disease4,5, though traditionally, the use of oxygen therapy has been viewed as simply a marker for disease severity6. Recent studies from a clinical trial of patients with respiratory failure have shown that higher patient oxygen levels are paradoxically associated with an increase in serious bacterial infections and higher mortality7, suggesting that supplemental oxygen may contribute to disease pathogenesis. The effect of supplemental oxygen on the cystic fibrosis lung microbiome and associated lung and airway microbial communities has not been well studied.
Mechanistic studies often cannot be performed directly on human subjects due to logistical difficulties and potential ethical issues associated with interventions of unknown medical benefit or harm. Translational approaches that integrate human biospecimens into model systems can offer important biological insights in these cases. While the ability to use or tolerate oxygen has traditionally been an important component of microbial classification, little is known about how the therapeutic introduction of supplemental oxygen to the environment might perturb airway microbial communities. To shed light on the unknown effects of supplemental oxygen on the airway microbiomes of pwCF, we needed to address two major challenges; first, the creation of a culture medium that physiologically approximates the composition of CF sputum; second, the creation of a model system that allows the maintenance of elevated oxygen concentrations in culture over extended periods of time.
Artificial sputum media (ASM) are widely used to emulate lung sputum ex vivo8,9,10, but there is no clear consensus on a specific recipe. This protocol describes an artificial sputum medium recipe and preparation strategy carefully designed to physiologically approximate sputum from pwCF. Table 1 outlines the chosen recipe values based on published literature. Basic chemical components and pH were matched to values identified by studies of human CF sputum11,12,13. Low concentration physiological nutrients were added using egg yolk, which was included as 0.25% of the final volume10, as well as vitamin and trace metal mixes14,15. Mucin, the key component of sputum16, was included at 1% w/v14. Although more labor-intensive, filter sterilization was chosen over the more conventional practice of heat sterilization to reduce potential problems from heat-induced denaturation of essential media components10. An additional benefit of filter sterilization is that it generates media that are transparent (heat-sterilization can create turbid media due to precipitation and coagulation of salts and proteins), allowing this artificial sputum media to be used to follow microbial growth based on increases in turbidity.
This model system for the hyperoxic culture is based on anaerobic culturing techniques where oxygen is added rather than removed, creating a model for the effect of supplemental oxygen use for pwCF. Figure 1 and the associated oxygen sparging protocol outlines the components of an oxygen sparging system, which can be obtained at low cost from general laboratory and hospital suppliers. This system enables the mixing of compressed oxygen and air to fixed concentrations ranging from 21%-100% oxygen. The integration of an oxygen sensor allows for the verification of the concentration of the output gas mixture, as well as checking the outflow gas composition of previously sparged serum bottles to verify that the oxygen conditions have been maintained within the desired range.
This protocol outlines procedures to create an artificial sputum medium, the construction and use of an oxygen sparging system, and the application of both to culture CF sputum under differential oxygen conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BME Vitamins (100x) Solution</td>
			<td>MilliporeSigma</td>
			<td>B6891</td>
			<td>Concentrated solution of supplemental vitamins.</td>
		</tr>
		<tr>
			<td>Crimper, 30 mm</td>
			<td>DWK Life Sciences</td>
			<td>224307</td>
			<td>Crimper for attaching aluminum seals to serum bottles.</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>MilliporeSigma</td>
			<td>G7021</td>
			<td>Solid glucose powder (dextrorotatory isomer).</td>
		</tr>
		<tr>
			<td>Diaphragm Pump ME 2 NT</td>
			<td>VACUUBRAND</td>
			<td>20730003</td>
			<td>Vacuum pump for vacuum filtration.</td>
		</tr>
		<tr>
			<td>Egg Yolk Emulsion</td>
			<td>HiMedia</td>
			<td>FD045</td>
			<td>Sterile emulsion of 30% egg yolk in saline.</td>
		</tr>
		<tr>
			<td>Ferritin, Cationized from Horse Spleen</td>
			<td>MilliporeSigma</td>
			<td>F7879</td>
			<td>Ferritin (iron-storage protein) solution.</td>
		</tr>
		<tr>
			<td>FIREBOY plus Safety Bunsen Burner</td>
			<td>Integra Biosciences</td>
			<td>144000</td>
			<td>Bunsen burner with user interface and safety features.</td>
		</tr>
		<tr>
			<td>Hydrion pH Paper (1.0&#8211;14.0)</td>
			<td>Micro Essential Laboratory</td>
			<td>94</td>
			<td>pH testing paper for the range of 1.0&#8211;14.0.</td>
		</tr>
		<tr>
			<td>Hydrion pH Paper (4.0&#8211;9.0)</td>
			<td>Micro Essential Laboratory</td>
			<td>55</td>
			<td>pH testing paper for the range of 4.0&#8211;9.0.</td>
		</tr>
		<tr>
			<td>Hydrion pH Paper (6.0&#8211;8.0)</td>
			<td>Micro Essential Laboratory</td>
			<td>345</td>
			<td>pH testing paper for the range of 6.0&#8211;8.0.</td>
		</tr>
		<tr>
			<td>Hypodermic Needle-Pro EDGE Safety Device, 18 G</td>
			<td>Smiths Medical</td>
			<td>401815</td>
			<td>18 G needles with safety caps.</td>
		</tr>
		<tr>
			<td>In-Line Pressure Gauge</td>
			<td>MilliporeSigma</td>
			<td>20469</td>
			<td>Gas pressure gauge for monitoring bottle pressure.</td>
		</tr>
		<tr>
			<td>Innova 42 Incubated Shaker</td>
			<td>Eppendorf</td>
			<td>2231000756</td>
			<td>Combination incubator/orbital shaker.</td>
		</tr>
		<tr>
			<td>Luer-Lok Syringe with Attached Needle</td>
			<td>Becton Dickinson</td>
			<td>309580</td>
			<td>Combination 3 mL syringe and 18 G needle.</td>
		</tr>
		<tr>
			<td>Luer Valve Assortment</td>
			<td>World Precision Instruments</td>
			<td>14011</td>
			<td>Valves for gas flow tubing.</td>
		</tr>
		<tr>
			<td>LSE Orbital Shaker</td>
			<td>ThermoFisher Scientific</td>
			<td>6780-NP</td>
			<td>Orbital shaker to agitate media during filtration.</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate</td>
			<td>MilliporeSigma</td>
			<td>M2773</td>
			<td>Solid epsom salt (magnesium sulfate heptahydrate).</td>
		</tr>
		<tr>
			<td>Medical Air Single Stage Regulator with Flowmeter</td>
			<td>Western Enterprises</td>
			<td>M1-346-15FM</td>
			<td>Air flow rate regulator with 15 L/min meter.</td>
		</tr>
		<tr>
			<td>MEM Amino Acids (50x) Solution</td>
			<td>MilliporeSigma</td>
			<td>M5550</td>
			<td>Concentrated solution of essential amino acids.</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids (100x) Solution</td>
			<td>MilliporeSigma</td>
			<td>M7145</td>
			<td>Concentrated solution of non-essential amino acids.</td>
		</tr>
		<tr>
			<td>Millex-GP Filter, 0.22 &#181;m</td>
			<td>MilliporeSigma</td>
			<td>SLMP25SS</td>
			<td>0.22 &#181;m polyethersulfone membrane sterile syringe filter.</td>
		</tr>
		<tr>
			<td>Milli-Q Academic</td>
			<td>MilliporeSigma</td>
			<td>ZMQS60E01</td>
			<td>Milli-Q sterile water filtration system.</td>
		</tr>
		<tr>
			<td>MiniOX 3000 Oxygen Monitor</td>
			<td>MSA</td>
			<td>814365</td>
			<td>Gas flow oxygen percentage monitor.</td>
		</tr>
		<tr>
			<td>MOPS Buffer (1 M, pH 9.0)</td>
			<td>Boston BioProducts</td>
			<td>BBM-90</td>
			<td>MOPS buffer for adjusting media pH.</td>
		</tr>
		<tr>
			<td>Mucin from Porcine Stomach</td>
			<td>MilliporeSigma</td>
			<td>M2378</td>
			<td>Mucin (glycosylated gel-forming protein) powder.</td>
		</tr>
		<tr>
			<td>Natural Polypropylene Barbed Fitting Kit</td>
			<td>Harvard Apparatus</td>
			<td>72-1413</td>
			<td>Connectors for gas flow tubing.</td>
		</tr>
		<tr>
			<td>Nextera XT DNA Library Preparation Kit</td>
			<td>Illumina</td>
			<td>FC-131-1096</td>
			<td>Library preparation for identification during sequencing.</td>
		</tr>
		<tr>
			<td>NovaSeq 6000 Sequencing System</td>
			<td>Illumina</td>
			<td>770-2016-025-N</td>
			<td>Shotgun sequencing platform for generating sample reads.</td>
		</tr>
		<tr>
			<td>Oxygen Single Stage Regulator with Flowmeter</td>
			<td>Western Enterprises</td>
			<td>M1-540-15FM</td>
			<td>Oxygen flow rate regulator with 15 L/min meter.</td>
		</tr>
		<tr>
			<td>Oxygen Tubing with 2 Standard Connectors</td>
			<td>SunMed</td>
			<td>2001-01</td>
			<td>Tubing for connecting gas system components.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 10x, pH 7.4</td>
			<td>Molecular Biologicals International</td>
			<td>MRGF-6235</td>
			<td>Concentrated phosphate-buffered saline solution.</td>
		</tr>
		<tr>
			<td>PC 420 Hot Plate/Stirrer</td>
			<td>Marshall Scientific</td>
			<td>CO-PC420</td>
			<td>Combination hot plate/stirrer.</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>MilliporeSigma</td>
			<td>P9541</td>
			<td>Solid potassium chloride salt.</td>
		</tr>
		<tr>
			<td>PTFE Disposable Stir Bars</td>
			<td>ThermoFisher Scientific</td>
			<td>14-513-95</td>
			<td>Disposable magnetic stir bars.</td>
		</tr>
		<tr>
			<td>PTFE Thread Seal Teflon Tape</td>
			<td>VWR</td>
			<td>470042-938</td>
			<td>Teflon tape for reinforcing gas system connections.</td>
		</tr>
		<tr>
			<td>Q-Gard 2 Purification Cartridge</td>
			<td>MilliporeSigma</td>
			<td>QGARD00D2</td>
			<td>Purification cartridge for Milli-Q system.</td>
		</tr>
		<tr>
			<td>Reusable Media Storage Bottles</td>
			<td>ThermoFisher Scientific</td>
			<td>06-423A</td>
			<td>Bottles for mixing and storing culture media.</td>
		</tr>
		<tr>
			<td>Rubber Stopper, 30 mm, Gray Bromobutyl</td>
			<td>DWK Life Sciences</td>
			<td>224100-331</td>
			<td>Rubber stoppers for serum bottles.</td>
		</tr>
		<tr>
			<td>Serum Bottle with Molded Graduations, 500 mL</td>
			<td>DWK Life Sciences</td>
			<td>223952</td>
			<td>Glass serum bottles for sealed culturing.</td>
		</tr>
		<tr>
			<td>Small Bore Extension Set</td>
			<td>Braun Medical</td>
			<td>471960</td>
			<td>Tubing extension with luer lock connectors.</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>MilliporeSigma</td>
			<td>S3014</td>
			<td>Solid sodium chloride salt.</td>
		</tr>
		<tr>
			<td>Spike-in Control I (High Microbial Load)</td>
			<td>ZymoBIOMICS</td>
			<td>D6320</td>
			<td>Spike-in microbes (I. halotolerans and A. halotolerans) for absolute microbial load calculations</td>
		</tr>
		<tr>
			<td>Stericup Quick Release Sterile Vacuum Filtration System</td>
			<td>MilliporeSigma</td>
			<td>S2GPU02RE</td>
			<td>250 mL 0.22 &#181;m vacuum filtration chamber.</td>
		</tr>
		<tr>
			<td>Super Sani-Cloth Germicidal Disposable Wipes</td>
			<td>Professional Disposables International</td>
			<td>H04082</td>
			<td>Disposable germicidal wipes for sterilization.</td>
		</tr>
		<tr>
			<td>Trace Metals Mixture, 1000x</td>
			<td>ThermoFisher Scientific</td>
			<td>NC0112668</td>
			<td>Concentrated solution of physiological trace metals.</td>
		</tr>
		<tr>
			<td>Unlined Aluminum Seal, 30 mm</td>
			<td>DWK Life Sciences</td>
			<td>224187-01</td>
			<td>Aluminum seals crimped over top of rubber stoppers.</td>
		</tr>
		<tr>
			<td>USP Medical Grade Air Tank</td>
			<td>Airgas</td>
			<td>AI USP200</td>
			<td>Compressed air tank for input to sparging system.</td>
		</tr>
		<tr>
			<td>USP Medical Grade Oxygen Tank</td>
			<td>Airgas</td>
			<td>OX USP200</td>
			<td>Compressed oxygen tank for input to sparging system.</td>
		</tr>
	</tbody>
</table>

design and development of a model to study the effect of supplemental oxygen on the cystic fibrosis airway microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

These protocols were applied to 50 expectorated sputum samples from pwCF presenting for routine care to an outpatient cystic fibrosis clinic at Massachusetts General Hospital in Boston, Massachusetts. Each patient&#39;s sputum was cultured under 21%, 50%, and 100% oxygen conditions using the artificial sputum medium, with 0.5 mL aliquots taken from each culture at 24 h, 48 h, and 72 h of culture time for testing. Cultures were photographed when extractions were made to track visual changes. In addition, a 0.5 mL aliquot ...

Watch this Scientific Journal Video about Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome at JoVE.com

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

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2.2K Views.  Massachusetts General Hospital. The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Video: Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson&#39;s Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can jeopardize their spontaneous breathing, topical anesthesia of the airway is a popular technique. The spray-as-you-go technique represents one of the simplest opportunities to anesthetize the airway mucosa. The application of local anesthetic through the working channel of the flexible endoscope is a widespread practice for anesthetists as well as pulmonologists. There is neither need for additional devices nor special training as a pre-requisite to perform this technique. However, a known clinical problem is the coughing and gagging reflex that may occur when the liquid anesthetic strikes the airway mucosa and other sensitive structures like the vocal cords. This can be avoided by the use of oxygen applied through the working channel with the aim of fogging the local anesthetic into finer particles. Furthermore, the oxygen flow provides a higher oxygen supply and contributes to a better view, dispersing mucus secretions and blood away from the lens. Using an atomizer with a high oxygen flow of 10 L/min we maximized these benefits, caused less coughing and had more satisfied and therefore cooperative patients. Possible, but very rare complications of using oxygen flow including gastric insufflation, organ rupture or barotrauma did not arise. We attribute the complication-free use of high oxygen flow to the design of the set, which permits flow and pressure release.

The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Rectal Organoid Morphology Analysis (ROMA): A Diagnostic Assay in Cystic Fibrosis

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two disease-causing CFTR mutations can be identified. Physiological CFTR assays (nasal potential difference, intestinal current measurement) have been included in the diagnostic algorithm but are not always readily available or feasible (e.g., in infants). Rectal organoids are 3D structures that grow from stem cells isolated from crypts of a rectal biopsy when cultured under specific conditions. Organoids from non-CF subjects have a round shape and a fluid-filled lumen, as CFTR-mediated chloride transport drives water into the lumen. Organoids with defective CFTR function do not swell, retaining an irregular shape and having no visible lumen. Differences in morphology between CF and non-CF organoids are quantified in the &#39;Rectal Organoid Morphology Analysis&#39; (ROMA) as a novel CFTR physiological assay. For the ROMA assay, organoids are plated in 96-well plates, stained with calcein, and imaged in a confocal microscope. Morphological differences are quantified using two indexes: The circularity index (CI) quantifies the roundness of organoids, and the intensity ratio (IR) is a measure of the presence of a central lumen. Non-CF organoids have a high CI and low IR compared to CF organoids. ROMA indexes perfectly discriminated 167 subjects with CF from 22 subjects without CF, making ROMA an appealing physiological CFTR assay to aid in CF diagnosis. Rectal biopsies can be routinely performed at all ages in most hospitals and tissue can be sent to a central lab for organoid culture and ROMA. In the future, ROMA might also be applied to test the efficacy of CFTR modulators in vitro. The aim of the present report is to fully explain the methods used for ROMA, to allow replication in other labs.

Rectal Organoid Morphology Analysis ROMA: A Diagnostic Assay in Cystic Fibrosis

This protocol describes rectal organoid morphology analysis (ROMA), a novel diagnostic assay for cystic fibrosis (CF). Morphological characteristics, namely the roundness (circularity index, CI) and the presence of a lumen (intensity ratio, IR), are a measure of CFTR function. Analysis of 189 subjects showed perfect discrimination between CF and non-CF.