Preparation of Artificial Sputum Medium to Emulate Cystic Fibrosis Lung Sputum Ex Vivo

Abstract

Source: Vieira, J., et al. Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome. J. Vis. Exp. (2021).

This video demonstrates the preparation of an artificial sputum medium that mimics the viscosity, nutrient composition, and pH of cystic fibrosis lung sputum. The resulting medium provides a physiologically relevant platform for ex vivo studies.

Protocol

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.

1. 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.
   1. Mix the constituent chemical stock solutions.
      1. Prepare 1 M NaCl stock: Add 58.44 g of NaCl per liter of sterile water.
      2. Prepare 1 M KCl stock: Add 74.55 g of KCl per liter of sterile water.
      3. Prepare 1 M MgSO₄ stock: Add 246.47 g of MgSO₄·7H₂O per liter of sterile water, or 120.37 g of anhydrous MgSO₄ per liter of sterile water.
      4. Prepare 1 M glucose stock: Add 180.16 g of glucose per liter of sterile water.
   2. 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 °C and 15 PSI for 30 min.
   3. Add 80.59 mL of sterile water to the empty 250 mL bottle.
   4. Add 152.30 mL of 1 M NaCl stock to the mix.
   5. Add 15.8 mL of 1 M KCl stock to the mix.
   6. Add 610 µL of 1 M MgSO₄ stock to the mix.
   7. Add 700 µL of 1 M glucose stock to the mix.
2. 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.
   1. Add 450 mL of sterile water to an empty 1 L bottle.
   2. Add 50 mL of 10x Phosphate-Buffered Saline (PBS) to the bottle.
   3. Add a disposable magnetic stir bar to the bottle.
   4. Autoclave the bottle containing PBS and the stir bar.
   5. Measure out 10 g of mucin powder and add it to the PBS.
   6. Shake the bottle vigorously for preliminary mixing.
   7. Place the bottle onto a hot plate with a magnetic stirrer. Set the heat to medium-high, targeting 50 °C and stirring speed to 1100 rpm. Ramp up the speed gradually so that the bar does not fly off the magnet.
      1. Allow to heat and stir for 15 min.
      2. Pick up the bottle with heat-resistant gloves. Observe to check if the mucin powder settles out of the solution.
      3. If mucin powder is not fully dissolved, return the bottle to the heat/stirrer for 5-minute intervals until it is completely dissolved.
   8. Allow the mucin mix to cool to room temperature.
3. Artificial sputum biological mix (ASBM)​
   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.
   1. Thaw the 100x vitamin stock in a 4 °C fridge or on ice.​
      NOTE: Pre-portion the vitamin stock into 10 mL aliquots to minimize the number of freeze/thaw cycles.
   2. Add 124.24 mL of sterile water to the empty autoclaved 250 mL bottle.
   3. Add 25.76 mL of 50x essential amino acid stock to the mix.
   4. Add 80.14 mL of 100x non-essential amino acid stock to the mix.
   5. Add 10 mL of (thawed) 100x vitamin stock to the mix.
   6. Add 1 mL of 1000x trace metals stock to the mix.
   7. Add 8.33 mL of 30% egg yolk emulsion to the mix.
   8. Add 400 µL of 10 g/L ferritin stock to the mix.
   9. Mix the solution well via manual shaking.
4. Artificial sputum medium (ASM)
   1. Add 250 mL of ASCM to the 1 L bottle containing ASMM.
   2. Add 250 mL of ASBM to the medium bottle.
   3. 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.
   4. Refrigerate the resulting artificial sputum medium at 4 °C until it is ready for filtration.
   5. To start the filtration process, transfer 200 mL of unfiltered artificial sputum medium to a vacuum filtration system with a 0.22 µm pore size filter.
   6. 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 °C.
      1. 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.
      2. 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 µm filter since mucin will plug the filter over time.
   7. Refrigerate filtered artificial sputum medium at 4 °C until ready for use. Use ASM within one month of preparation for best results.

Table 1: Artificial sputum medium recipe derived from review of literature. (Column 1) Reagents and key values in the formulation of artificial sputum medium. (Columns 2-7) Comparison of recipes from extant literature. (Columns 8-9).

Column 1		Column 2	Column 3	Column 4	Column 5	Column 6	Column 7	Column 8	Column 9
Value		Comstock	Kirchner	Sriramulu	Palmer	Flynn	Gallagher	Lai	Source
Mucin		2% w/v	0.5% w/v	0.5% w/v	-	1% w/v	2% w/v	1% w/v	Flynn
Sodium Chloride		85.5 mM	85.5 mM	85.5 mM	66.6 mM	89.8 mM	85.5 mM	152.3 mM	Lapierre
Potassium Chloride		29.5 mM	29.5 mM	29.5 mM	15.8 mM	-	2.95 mM	15.8 mM	Palmer
Magnesium Sulfate		-	-	-	0.6 mM	1 mM	1 mM	0.61 mM	Palmer
Iron Sulfate		-	-	-	0.0036 mM	-	-	-	-
Ammonium Chloride		-	-	-	2.3 mM	60 mM	-	-	-
Monopotassium Phosphate		-	-	-	2.5 mM	60 mM	-	-	-
Glucose		-	-	-	3.2 mM	13 mM	40 mM	0.7 mM	Sambeek
Lactate		-	-	-	9 mM	-	-	-	-
Essential Amino Acids		14.45x	0.25 g/L	0.25 g/L	Per Acid	0.5x	0.375x	1.29x	Palmer
Non-Essential Amino Acids		28.9x	0.25 g/L	0.25 g/L	Per Acid	0.25x	0.5x	8.01x	Palmer
Vitamins		-	-	-	-	-	1x	1x	Gallagher
Trace Metals		-	-	-	-	1x	1x	1x	Flynn
Egg Yolk		0.25%	0.25%	0.25%	-	-	0.25%	0.25%	Kirchner
Ferritin		0.0003 g/L	-	-	-	-	0.0004 g/L	0.0004 g/L	Gallagher
Salmon Sperm DNA		1.4 g/L	4 g/L	4 g/L	-	-	1.4 g/L	-	-
DPTA		-	-	0.0059 g/L	-	-	-	-	-
pH		-	6.9	-	6.8	-	-	6.3	Lapierre
Storage		0	4°	-	-	4°	4°	4°	Kirchner
Sterilization		Autoclave	Filter	Autoclave	-	Autoclave	Autoclave	Filter	Kirchner

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BME Vitamins (100x) Solution	MilliporeSigma	B6891	Concentrated solution of supplemental vitamins.
Crimper, 30 mm	DWK Life Sciences	224307	Crimper for attaching aluminum seals to serum bottles.
D-(+)-Glucose	MilliporeSigma	G7021	Solid glucose powder (dextrorotatory isomer).
Diaphragm Pump ME 2 NT	VACUUBRAND	20730003	Vacuum pump for vacuum filtration.
Egg Yolk Emulsion	HiMedia	FD045	Sterile emulsion of 30% egg yolk in saline.
Ferritin, Cationized from Horse Spleen	MilliporeSigma	F7879	Ferritin (iron-storage protein) solution.
FIREBOY plus Safety Bunsen Burner	Integra Biosciences	144000	Bunsen burner with user interface and safety features.
Hydrion pH Paper (1.0–14.0)	Micro Essential Laboratory	94	pH testing paper for the range of 1.0–14.0.
Hydrion pH Paper (4.0–9.0)	Micro Essential Laboratory	55	pH testing paper for the range of 4.0–9.0.
Hydrion pH Paper (6.0–8.0)	Micro Essential Laboratory	345	pH testing paper for the range of 6.0–8.0.
Hypodermic Needle-Pro EDGE Safety Device, 18 G	Smiths Medical	401815	18 G needles with safety caps.
In-Line Pressure Gauge	MilliporeSigma	20469	Gas pressure gauge for monitoring bottle pressure.
Innova 42 Incubated Shaker	Eppendorf	2231000756	Combination incubator/orbital shaker.
Luer-Lok Syringe with Attached Needle	Becton Dickinson	309580	Combination 3 mL syringe and 18 G needle.
Luer Valve Assortment	World Precision Instruments	14011	Valves for gas flow tubing.
LSE Orbital Shaker	ThermoFisher Scientific	6780-NP	Orbital shaker to agitate media during filtration.
Magnesium Sulfate Heptahydrate	MilliporeSigma	M2773	Solid epsom salt (magnesium sulfate heptahydrate).
Medical Air Single Stage Regulator with Flowmeter	Western Enterprises	M1-346-15FM	Air flow rate regulator with 15 L/min meter.
MEM Amino Acids (50x) Solution	MilliporeSigma	M5550	Concentrated solution of essential amino acids.
MEM Non-Essential Amino Acids (100x) Solution	MilliporeSigma	M7145	Concentrated solution of non-essential amino acids.
Millex-GP Filter, 0.22 µm	MilliporeSigma	SLMP25SS	0.22 µm polyethersulfone membrane sterile syringe filter.
Milli-Q Academic	MilliporeSigma	ZMQS60E01	Milli-Q sterile water filtration system.
MiniOX 3000 Oxygen Monitor	MSA	814365	Gas flow oxygen percentage monitor.
MOPS Buffer (1 M, pH 9.0)	Boston BioProducts	BBM-90	MOPS buffer for adjusting media pH.
Mucin from Porcine Stomach	MilliporeSigma	M2378	Mucin (glycosylated gel-forming protein) powder.
Potassium Chloride	MilliporeSigma	P9541	Solid potassium chloride salt.
PTFE Disposable Stir Bars	ThermoFisher Scientific	14-513-95	Disposable magnetic stir bars.
PTFE Thread Seal Teflon Tape	VWR	470042-938	Teflon tape for reinforcing gas system connections.
Q-Gard 2 Purification Cartridge	MilliporeSigma	QGARD00D2	Purification cartridge for Milli-Q system.
Reusable Media Storage Bottles	ThermoFisher Scientific	06-423A	Bottles for mixing and storing culture media.
Rubber Stopper, 30 mm, Gray Bromobutyl	DWK Life Sciences	224100-331	Rubber stoppers for serum bottles.
Serum Bottle with Molded Graduations, 500 mL	DWK Life Sciences	223952	Glass serum bottles for sealed culturing.
Small Bore Extension Set	Braun Medical	471960	Tubing extension with luer lock connectors.
Sodium Chloride	MilliporeSigma	S3014	Solid sodium chloride salt.
Trace Metals Mixture, 1000x	ThermoFisher Scientific	NC0112668	Concentrated solution of physiological trace metals.
Unlined Aluminum Seal, 30 mm	DWK Life Sciences	224187-01	Aluminum seals crimped over top of rubber stoppers.