Vag³Gel
Culturing cervicovaginal microbiota or single strains in a human cervicovaginal mucus model, in syringes, and preparing them for downstream analysis.
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 cervicovaginal mucus.
The protocol describes how to culture cervicovaginal microbiota and single strains in Vag³Gel in a syringe format, and how to prepare the samples for downstream analyses: cell viability, flow cytometry, metabolomics and sequencing.
Before you begin
- Two tracks are given below. Stages A to C culture a donor cervicovaginal sample. Stage D replaces stage A when you are working with single strains, and is followed by stages B and C unchanged.
- The gel in this kit contains porcine stomach type III mucin at 2.4 mg/mL in the ovulatory version and 3.0 mg/mL in the non-ovulatory and pregnancy versions, with 0.07 mg/mL NaCl or MRS medium. The pregnancy version also contains 5.0 mg/mL albumin. The composition can be tuned, and custom compositions are available.
- Vag³Gel largely favours facultative anaerobes under aerobic incubation. It is especially suited to culturing Lactobacillaceae.
- Sterilize all instruments and consumables, including the menstrual cup, and work under aseptic conditions, ideally in a Class II biological safety cabinet. Prepare a sterile 0.9% (w/v) NaCl solution.
Procedure
ACervicovaginal sample isolation~75 min
- The donor uses a menstrual cup for 60 minutes. Recover it and transfer it into a sterile 50 mL tube.
- Centrifuge the tube with the cup at 8,000 × g for 15 minutes to collect the secretions, then discard the cup.
- Resuspend the recovered sample in up to 100 mL of sterile MRS medium in a sterile flask.
- Homogenize by inverting the flask gently several times and pipetting up and down.
Note Handle the menstrual cup only with gloves, cleaned with a 70% ethanol wipe beforehand.
Critical Use samples as fresh as possible, ideally processed within 30 minutes of collection, and kept on ice. If the gel was produced in MRS, resuspend in 0.9% (w/v) NaCl instead. Unused sample can be aliquoted with glycerol (10% v/v final) and stored at −80 °C.
BCulturing in Vag³Gel~5 min
- Remove the syringe cap and keep it sterile. Discard the first 2 to 3 drops by gently pressing the plunger.
- Using the double syringe mixing method, combine the inoculum and the gel at a 1:1 volume ratio, mixing 6 to 7 times.
- Put the cap back on the syringe holding the inoculum and gel.
- Incubate at 37 °C for up to 72 hours under aerobic conditions, according to the experimental design.
Note To test a molecule such as a biotic or an active principle, let the microbiota stabilize in the gel for 24 hours at 37 °C before adding it with the double syringe method.
Critical Anaerobic conditions before culturing are not required, but they prevent the loss of obligate anaerobes.
CDissolving the gel~5 min per syringe
- Add Bac³Gel dissolution medium (200 mM sodium citrate) at a 1:1 ratio to each sample.
- Mix thoroughly until the gel has fully dissolved. The homogenized samples are ready for downstream assays.
DSingle strains instead of a donor sample~15 min
- Grow the species of interest, for example a Lactobacillus, to logarithmic phase in the appropriate medium.
- Centrifuge to pellet the cells, discard the supernatant and resuspend the pellet in an appropriate culture medium.
- Measure the optical density at 600 nm and adjust the cell concentration as needed.
- Continue with stage B, then stage C.
Critical If the gel was produced in MRS, resuspend the pellet in 0.9% (w/v) NaCl rather than in MRS.
Downstream analysis
Sequencing
Centrifuge at 14,000 × g for 5 minutes, then use the pellet for DNA extraction.
Metabolomics
Centrifuge at 14,000 × g for 10 minutes, then collect and lyophilize the supernatant.
Metabolic activity or optical density
Centrifuge at 14,000 × g for 5 minutes, discard the supernatant and resuspend in 0.9% (w/v) NaCl.
Other readouts
Also suited to cell viability assessment, flow cytometry and standard microbiological characterization assays.
Troubleshooting
For research and development use only. Not for diagnostic or clinical use.