Cystic Fibrosis Lung³Gel
Culturing microbial samples in a cystic fibrosis airway mucus model, in multi-well plates, and preparing them for sequencing and metabolomics.
Product
Reproducible and high-throughput in vitro models are essential for studying complex microbial communities. This kit is designed for the in vitro modelling of disease-specific human airway mucus.
The protocol describes how to culture microbial samples in Cystic Fibrosis Lung³Gel in a multi-well plate format, and how to prepare the samples for downstream analyses: cell viability, flow cytometry, metabolomics and sequencing.
Before you begin
- The steps below are written for single-strain samples. The same protocol has also been used for co-cultures, microbial consortia and complex microbial communities.
- The gel in this kit can be produced in Mueller Hinton medium and contains porcine stomach type III mucin at 25.0 mg/mL with 16.3 mg/mL NaCl. The composition can be tuned, and custom compositions are available.
- The cross-linking and the oxygen gradient form during production, and change as microbes grow.
- Sterilize all instruments and consumables, and work under aseptic conditions, ideally in a Class II biological safety cabinet. Prepare a sterile 0.9% (w/v) NaCl solution.
Procedure
APreparing the microbial suspension~15 min
- Grow the species of interest, for example Pseudomonas aeruginosa, to logarithmic phase in the appropriate medium.
- Centrifuge to pellet the cells and discard the supernatant.
- Resuspend the pellet in an appropriate culture medium.
- Measure the optical density at 600 nm and adjust the cell concentration as needed.
Critical If the gel was produced in a culture medium, resuspend the pellet in 0.9% (w/v) NaCl rather than in medium.
BCulturing in Cystic Fibrosis Lung³Gel~5 min per plate
- Bring the plates to room temperature. This protocol uses a 24-well plate as the example.
- Add the microbial suspension to each well at a 1:1 volume ratio with the gel: 700 µL per well in a 24-well plate. Other formats are in the table below.
- Cover the plate and incubate at 37 °C, aerobic or anaerobic, for up to 72 hours.
Note To test a molecule such as a biotic or an active principle, let the microbes stabilize in the gel for 24 hours at 37 °C before adding it.
Tip The plates can be sealed with a breathable membrane before incubation, to prevent cross-contamination.
Critical Add the suspension gently on top of the gel. Do not mix. Anaerobic conditions before culturing are not required, but they prevent the loss of obligate anaerobes.
CDissolving the gel~3 min per well
- Aspirate the supernatant gently without disturbing the gel. Wash with 0.9% NaCl, 700 µL per well in a 24-well plate, then aspirate and discard.
- Add Bac³Gel dissolution medium (200 mM sodium citrate) at a 1:1 ratio with the gel volume, and mix until the gel has fully dissolved. Transfer to microcentrifuge tubes.
Note The wash keeps only the microbes that colonized the gel. Dissolution takes longer in larger wells and where exopolysaccharides are present.
| Plate format | Lung³Gel per well |
|---|---|
| 6-well | 3,700 µL |
| 12-well | 1,600 µL |
| 24-well | 700 µL |
| 48-well | 300 µL |
| 96-well | 100 µL |
The microbial suspension and the Bac³Gel dissolution medium are each added at a 1:1 ratio with the gel, so both equal the volume shown.
Downstream analysis
Sequencing
Centrifuge at 14,000 × g for 10 minutes, then use the pellet for DNA extraction.
Metabolomics
Centrifuge at 14,000 × g for 10 minutes, then collect and lyophilize the supernatant.
Other readouts
Also suited to cell viability assessment, flow cytometry, metabolic activity and standard microbiological characterization assays.
Troubleshooting
The gel does not dissolve, or dissolves slowly
Add more 200 mM sodium citrate and mix again. Larger wells and exopolysaccharide-rich cultures take longer.
For research and development use only. Not for diagnostic or clinical use.