This model is the next step in setting host microdirections for the upper digestive tracts. It is allowing addressing mechanistic research questions and it offers many opportunities for the food, the pharma, and nutraceutical industry and also toxicology. The main advantage of this technique is that it combines a model of the gastrointestinal system with the host micro interface including a complex microbe community.
It is very applicable and it is very flexible as well. This methodology can be adopted using other cell types to assess pathogen viability and subsequent information. Changes can also be used to assist colonization capacity of probiotic mixtures and ultimately the potential effect of bacterias on the per systemic circulation.
To begin, autoclave the medium, and then add five microgram per milliliter of hemen and one microgram per milliliter of menadione. Dispense nine milliliters of the medium into Hungate tubes, and close the tubes with a rubber stopper and an aluminum cap. Then flush the tubes with a mixture of 90%nitrogen and 10%carbon dioxide.
Use the syringe to retrieve one milliliter of anaerobic medium, and transfer it to a 1.5 milliliter microcentrifuge tube. Pick single colonies of each bacterial species and resuspend them in the medium. Then transfer the colonies into the anaerobic modified BHI, and incubate them at 37 degrees celsius for 48 hours.
Following the incubation, use filtered sterilized PBS to dilute the cultures to 10 to the fifth cells per milliliter. Then use flow cytometry with the cyber green propidium iodide stain to measure the cell number. Evaluating the microbiome community composition and viability is essential for avoiding bias in our experiment.
Remove the cell culture medium from the flasks, then use five milliliters of PBS without calcium or magnesium to wash the cells once. Add two milliliters of trypsin EDTA solution to the cells. Distribute the solution by gently moving the flask.
Then remove 1.5 milliliters of the trypsin solution, and incubate the flask at 37 degrees celsius for 10 minutes. After checking under the microscope if the cells have become detached, add five milliliters of supplemented cell culture medium to the flask to inactivate the trypsin, and pipette up and down to obtain a homogenous single cell suspension. Immediately mix a 50 microliter aliquot of the cell suspension with sterile 0.4%trypan blue solution in a 1:1 volume to volume proportion for KCO2 cells for a 1:5 volume to volume for HT29MTX.
Mix by pipetting, and load the suspension into a counting chamber before counting the cells under the microscope. After preparing KCO2 and HT29MTX cell suspensions according to the text protocol, homogenize the suspensions, and add the corresponding volume of KCO2 and HT29MTX cells to a pre-filled sterile container with supplemented cell culture medium. Aspirate the pre-incubated medium from the apical side of the previously prepared chamber.
Gently mix the cell suspension by pipetting, and transfer 1.5 milliliters to the apical compartment of the chamber inserts mixing the cell suspension every one to three wells to ensure constant homogenization. Gently move the plates back and forth and then right to left to evenly spread the cells in the well. Transfer the plates to the incubator, and repeat the movement of the plates.
The sitting of the cells need to be done with constant homogenization of the cell suspension to ensure an even spread in the well. To measure the epithelial barrier integrity before starting the ASA, ensure that tier equipment is fully charged before starting the measurements. Disconnect the tier equipment from the power supply, and cover with a plastic autoclave bag.
Make a hole in the bag to introduce the electrode into the input port on the meter. Then spray the bag with 70%ethanol before transferring the tier equipment into the flow cabinet. To disinfect the electrode, immerse the electrode tips in 70%ethanol for 15 minutes.
Allow the tips to air dry for 15 seconds, then use cell culture medium to rinse the electrode. Allow the cells to come to room temperature inside the flow cabinet. Make sure that the meter is disconnected from the charger, then set the mode switch to Ohms, and turn on the power switch.
Measure the cell resistance by immersing the electrode with the shorter tip in the insert and the longer tip out of the well. Keep the electrode at a 90 degree angle to the plate insert. To simulate oral digestion, transfer three milliliters of the bacterial community in a 50 milliliter sterile tube, and mix with three milliliters of simulated saliva fluid or SSF.
Add 75 units per milliliter of alpha-amylase from human saliva type 9A and two grams per liter of mucin from porcine stomach type 2. Incubate the mixture at 37 degrees celsius and 100 RPM for two minutes. Collect a two milliliter sample for DNA extraction and flow cytometry quantification.
To simulate gastric digestion, add four milliliters of simulated gastric fluid or SGF to three milliliters of the bacteria. Use one molar HCl to adjust the pH to three if necessary. Incubate the sample at 37 degrees celsius and 100 RPM for two minutes.
Then collect a two milliliter sample. For small intestine digestions, add four milliliters simulated intestinal fluid or SIF to the bacteria. Use sodium hydroxide to adjust the pH if necessary.
Then incubate the sample at 37 degrees celsius and 100 RPM for two minutes. And collect a two milliliter sample. After washing and adjusting the cell density of the small intestine digestion according to the text protocol, remove the apical medium from the transwell system, and add 1.5 milliliters of the bacterial suspension.
Co-culture the system under general cell-culture conditions for two hours. Following incubation, measure the tier values to evaluate the integrity of the epithelial barrier as demonstrated earlier in the video. Add 0.5 milliliters of 10 millimolar n-acetylcysteine and HPSS to solubilize the mucus produced by the HT29MTX and to recover the adhered bacteria.
Incubate the plates at 37 degrees celsius for one hour under agitation. Recover the NAC HPSS in a microcentrifuge tube, and use one milliliter of DPBS to wash the cells twice. Add 0.5 milliliters of 0.5%triton-x100 on top of the monolayers, and disrupt the cells by pipetting.
Then quickly recover the liquid in vortex for one minute. Speed is crucial to avoid damaging the bacterial cells with the detergent. Analyze the samples according to the text protocol.
As reported here, the counts of intact cells from individual strains were approximately 10 to the eighth cells per milliliter prior to the creation of the synthetic community while the multi-species microcosm contained above 90%viable cells during the establishment of the community. Based on the live-dead quantification, bacterial viability decreases after each digestion step as the result of acid pH during gastric passage and of the bio-salts contained in the small intestine digestion fluids. The harsh conditions of stomach and small intestine digestion can switch bacteria to viable but not in cultureable cells which are live bacteria that do not either grow or divide.
As demonstrated in this figure, viable cells recovered from the mucosal interface are colored blue in the flow-cytometry plot. However, no growth was observed when these mucus samples were plating. The fraction in which bacteria show higher viability rates is the cellular debris as revealed by plate counting.
The number of colonies may be variable, but the presence of metabolically active bacteria is found in the less diluted samples. After this development, this technique can be used by researches from the fields of nutrigenomics or pharmacomicrobiomics to study the host microcosmically. Following this protocol, other techniques like transcriptome or metabolic analysis can be performed to obtain mechanistic understanding in the successful colonization process accordingly.
After watching this video, you should have a good understanding in how to establish complex in vitro model representative of the digestive processes including the cross talk between the host cells and the rest of the microbiome. Don't forget that working with pathogenic bacteria can be extremely hazardous, and it's important to consider bio-safety precautions.
