Gradient Gut³Gel Colonic
Culturing human fecal microbiota in a colonic mucus model, in multi-well plates, and preparing the samples 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 human colon mucus layers.
The protocol describes how to culture fecal samples in Gut³Gel using 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 fecal samples. The same protocol has also been used for monocultures, co-cultures and microbial consortia.
- The Gut³Gel in this kit was produced in Brain Heart Infusion (BHI) medium with 50 mg/mL porcine stomach type III mucin. Standard gels are prepared in a salt solution; this protocol used BHI. 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
AFecal sample processing~15 min
- Weigh at least 30 g of fresh fecal sample into a sterilized blender. For each 30 g, add 150 mL of sterile 0.9% NaCl solution. Homogenize until the slurry is uniform.
- Filter the slurry through a sterilized colander into a sterile container to remove large particles. Aliquot into 50 mL tubes and keep on ice until use, for no more than 1 hour.
Tip Filtered slurry can be aliquoted with glycerol (10% v/v final) and stored at −80 °C for later experiments. Do it quickly, to preserve microbial diversity.
Critical Use samples as fresh as possible, ideally processed within 30 minutes of collection. Avoid excessive blending, which damages microbes.
BCulturing in Gut³Gel~5 min per plate
- Bring the Gut³Gel plates to room temperature. This protocol uses a 24-well plate as the example.
- Add fecal slurry 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 microbiota stabilize in Gut³Gel for 24 hours at 37 °C before adding it.
Critical Add the slurry 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 | Gut³Gel per well |
|---|---|
| 6-well | 3,700 µL |
| 12-well | 1,600 µL |
| 24-well | 700 µL |
| 48-well | 300 µL |
| 96-well | 100 µL |
Fecal slurry and Bac³Gel dissolution medium are each added at a 1:1 ratio with the gel, so both equal the volume shown.
Downstream analysis
Sequencing
Centrifuge the dissolved samples 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.