The goal of this protocol is to culture gut microbiota in vitro. It will allow for the study of microbiota dynamics without having to consider the contribution of a modern component. Using this multi-stage in vitro system, we count the microbiota community to develop in the lumin and on the mucosal of multi-regions of the colon can be started simultaneously.
A visual demonstration of this method is helpful due to the complexity of this in vitro system. Begin by filling the ascending colon with 500 milliliters of defined medium, the transverse colon with 800 milliliters of defined medium, and the descending colon with 600 milliliters of defined medium. Add mucin agricarriers to the colon regions in an amount proportional to the volume of the reactor, and use the sample tube and syringe to remove any excess defined medium from each reactor.
Use the pH probes to adjust the pH in each reactor. And turn on the nitrogen flush for 20 minutes. Next, thaw the fecal homogenate according to the manufacturer's guidelines before loading a 60 milliliter syringe with the fecal homogenate sample.
Then, inoculate each colon reactor through the sample port with an amount of homogenate equal to 5%of each reactor volume for overnight culture with pH control. The next morning, clean the luer lock on the sample port of each bioreactor with alcohol. When the ports have dried, connect the 30 milliliter syringe, cleared of all air, to one of the sample ports.
Withdraw the plunger three to five times to ensure a thorough mixing of the vessel components before aspirating 15 milliliters of the culture's supernate from the bioreactor. Then transfer the luminal sample into a sterile Falcon tube and place on ice. For DNA analysis, collect one to three milliliters of luminal fluid using a five milliliter syringe.
Collect the supernate form the other bioreactors in the same manner. To set aside sample material for short chain fatty acid analysis, centrifuge 15 milliliters of the luminal sample of interest, and syringe filter the supernate through a 0.2 micrometer pore filter for minus 80 degree Celsius storage. To set aside sample material for DNA analysis, centrifuge one milliliter of luminal fluid and store the pellet at minus 80 degrees Celsius.
For mucosal sample harvest, remove the prepared mucin carriers from four degrees Celsius storage and turn on the nitrogen flush. Quickly open the reactor lid and replace 50%of the carriers in each reactor with new ones. When all of the carriers have been replaced, quickly reseal the lid and maintain the nitrogen flush for another 20 minutes.
Then aliquot 0.25 to 0.5 grams of mucin carrier agar into individual two milliliter tubes for minus 80 degree Celsius storage until DNA extraction. Three times a day the system automatically cycles. This begins with 140 milliliters of defined medium transferred into the stomach bioreactor followed by a one hour incubation with pH control.
At the end of the incubation, the contents of the stomach are transferred into the small intestine along with 60 milliliters of pancreatic juice. In the small intestine bioreactor, the pH is adjusted automatically to 6.7 to 6.9, and the contents incubate for 90 minutes. During this incubation, the nitrogen flush for each system is turned on for a total of 10 minutes.
At the end of this incubation, the contents from the small intestine are transferred into the ascending colon, from the ascending colon to the transverse colon, from the transverse colon to the descending colon, and from the descending colon to the waste. In this representative principal coordinates analysis, the community changed depreciably between days one and seven. However, from day 11 post inoculation until the end of the experiment, the samples occupied a small region of the principal coordinates analysis space for both sample to sample distance scores based on operational taxonomic unit, presence to absence, and abundance.
The measurement of three prominent short-chained fatty acids reveals that Propionic acid, Butanoic acid, and Acetic acid fluctuate from the start of the experiment until day 15 post inoculation. After day 15, the amounts of these acids produced in each colon region remained constant with only minimal changes until the end of the experiment. Analysis of the stable community for the luminal and mucosal phase of each colon region demonstrates that the communities that develop in the individual colon regions of the in vitro system are similar to the fecal inoculum composition.
Comparison of the alpha diversity of the in vitro system reveals a similar level of diversity as that of the inoculum for all of the regions except the luminal samples from the ascending region. Demonstrating that the in vitro culture system is able to produce a community comparable to the fecal inoculum both in terms of composition and diversity. To produce a stable gut microbiota community using this protocol, it is critical to ensure that solutions are prepared accurately and that anerobic conditions and pH parameters are maintained throughout the experiment.
Following this procedure, samples can be analyzed for community composition using next gen DNA sequencing followed by bioinformatic analysis and for functionality using metabolomics.
