Ms. Audrey Thomas-Gahring is acknowledged for her GC/MS work. We would also like to thank Massimo Marzorati for editing the manuscript.

1. Materials and Preparations
NOTE: The defined medium is purchased as a powder (see Table of Materials). The composition of the defined medium in g/L is the following: Arabinogalactan (1.2), Pectin (2.0), Xylan (0.5), Glucose (0.4), Yeast extract (3.0), Special peptone (1.0), Mucin (2.0), L-cysteine-HCl (0.2).
<ol>
	<li>Prepare the defined medium
	<ol>
		<li>Fill a 4 L flask with 2 L of double distilled, deionized water.</li>
		<li>Add 29.2 g of defined medium powder to the water and mix unit complete homogenized.</li>
		<li>Add a magnetic stirrer and loosely screw on the lid.</li>
		<li>Autoclave at 121 &#176;C for 30 min.</li>
		<li>After cooling, store the prepared defined medium in a refrigerator at 4 &#176;C with constant agitation (magnetic stirrer).</li>
	</ol>
	</li>
	<li>Prepare pancreatic juice
	<ol>
		<li>Autoclave 2L of distilled, deionized water in a 5L vessel with a stirrer bar added, at 121 &#176;C for 30 min.</li>
		<li>After autoclaving, allow the water to cool to room temperature.</li>
		<li>Add 12.5 g NaHCO3, 6 g bile, and 0.9 g pancreatin. Place on magnetic stirrer to mix. 
		NOTE: The pancreatic juice should be prepared fresh every 2-3 days and refrigerated with agitation after it made.</li>
	</ol>
	</li>
	<li>Phosphate buffer
	<ol>
		<li>Place a 1 L flask containing a magnetic stirrer bar on a magnetic stirrer. Add 0.5 L of distilled, deionized water and turn on agitation.</li>
		<li>Next, add 6.8 g KH2PO4, 8.8 g K2HPO4, and 0.1 g sodium thioglycolate. Top off with water to a final volume of 1 L.</li>
		<li>After the components have completely dissolved, adjust the pH to 7.0 using NaOH.</li>
		<li>Pour the buffer into a 2 L vessel with a screw cap lid and autoclave at 121 &#176;C for 30 min. Ensure that the lid is screwed on loosely and not closed tightly.</li>
		<li>After removing from the autoclave, allow the solution to cool to the room temperature. Store the buffer at room temperature for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Prepare Mucin agar carriers
	<ol>
		<li>Autoclave plastic mucin carriers (see Table of Materials), forceps, plastic mesh (tubular shape), and zip ties at 131 &#176;C for 30 min.</li>
		<li>Autoclave 300 mL of double distilled, de-ionized water at 131 &#176;C for 30 min. Store at room temperature until use.</li>
		<li>In a biosafety cabinet with the laminar flow, fill sterile Petri dishes with the plastic mucin carriers using sterile forceps. 
		NOTE: The mucin carriers are hollow plastic circles that are filled with mucin agar. Once filled, the lid can be placed on top and these can be set aside.</li>
		<li>To prepare mucin agar, add 3 g of bacterial agar and 15 g of porcine mucin to the 300 mL of autoclaved water.</li>
		<li>In a fume hood, boil this solution, then remove from the heat source and allow to cool for 1 min. Repeat the boiling and cooling for a total of 3 times.</li>
		<li>Once the glass vessel containing the mucin agar solution is cool enough to touch, move it into the biosafety cabinet with laminar flow.</li>
		<li>In the biosafety cabinet with the laminar flow, pour the liquid mucin agar over the plastic mucin carriers that were placed in the Petri dish. Ensure that all the carriers are filled completely with mucin agar.</li>
		<li>Place the lid back on the Petri dish and allow it to cool under laminar flow.</li>
		<li>To assemble the mucin carriers, take the autoclaved plastic netting and insert mucin carriers containing the solidified mucin agar from the Petri dish using sterile forceps.</li>
		<li>Use zip ties to close the ends of the mesh. In this way the net functions to contain the mucin carriers filled with mucin agar. Store at 4 &#176;C until use.</li>
	</ol>
	</li>
</ol>
2. Set up, Inoculation, and Running of the System
<ol>
	<li>Set up of the culturing system 
	NOTE: The Twin Simulator of the Human Intestinal Microbial Ecosystem was used for this study.
	<ol>
		<li>Place 10 bioreactors containing stirrer bars on magnetic stirrers and turn on agitation. 
		NOTE: Each Unit will consist of 5 bioreactors, so 2 units will require 10 bioreactors.</li>
		<li>Connect 5 bioreactors to each other using silicon tubing in sequence by way of the peristaltic pump (Figure 1). Use the pumps to transverse the fluid contents from one bioreactor to the next.</li>
		<li>Label the bioreactors in the following sequence: Stomach, small intestine, ascending colon, transverse colon, descending colon to represent the order of the gastrointestinal tract. 
		NOTE: The bioreactors can all be identical, or the stomach and small intestine bioreactors can be smaller compared to the colon bioreactors since they will hold less volume. Setting up the bioreactors in this way means that the ascending colon region receives nutrients from the small intestine, the transverse colon region receives nutrients from the ascending colon region, and the descending colon region receives nutrients from the transverse region. In this way, the system is mimicking the sequential movement of nutrients through the gastrointestinal tract.</li>
		<li>Place the defined medium and pancreatic juice in a refrigerator at 4 &#176;C with constant agitation using magnetic stirrers. Using silicon tubing, connect the defined medium to the stomach bioreactor via a peristaltic pump and connect the pancreatic juice to the small intestine via a peristaltic pump.</li>
		<li>Add solidifying agent to a urine drainage bag. Connect the descending colon to the urine drainage bag by way of the peristaltic pump. Dispose of the solidified as biological waste during the experiment.</li>
		<li>Using silicone tubing, connect the water jacket of each vessel in order, and connect each end to the circulating water bath. Fill the circulating water bath with distilled water (this amount will vary depending on the type of circulating water bath used) and set to 37 &#176;C.</li>
		<li>Using silicon tubing, connect the acid and the base to the lid of each vessel by way of peristaltic pumps. The recommended concentrations are 0.5M HCl and 0.5M NaOH.</li>
		<li>Insert the pH probes to each vessel through the designated port in the lid. This port is designed to hold the pH probe and screw into the lid (Figure 1). Set the pH for each region as follows: Stomach, pH = 2; Small intestine, pH = 6.7-6.9; Ascending colon, pH = 5.6-5.9; Transverse colon, pH = 6.15-6.4; Descending colon, pH = 6.6-6.9. 
		NOTE: During the initial phase, the community will differentiate based on the difference in region pH values. However, once the feeding cycles begin, the communities will further mature based on the differences in nutrient input.</li>
		<li>Connect a nitrogen line using silicon tubing to the lid of each vessel using the designated port. The nitrogen line should flow in the following sequence: stomach, small intestine, ascending colon, transverse colon, descending colon. Ensure each unit has its own flush to avoid cross contamination. 
		NOTE: Nitrogen is used to removed oxygen from the system and maintains anaerobiosis during the experiment.</li>
		<li>Insert a metal sample tube through the lid of each bioreactor using the designated port. The metal tube extends down into the bioreactor and is used to collect fluid samples.</li>
		<li>Add a small piece of silicon tubing to the top end of the sample tube and connect a Luer Lock to allow for the use of a syringe during sampling.</li>
	</ol>
	</li>
	<li>Inoculation of the system
	<ol>
		<li>Fill the colon regions with the defined medium using the following volumes: 500 mL in the ascending colon, 800 mL in the transverse colon, and 600 mL in the descending colon.</li>
		<li>Add mucin agar carriers to the colon regions, in an amount proportional to the volume of the reactor. For this experiment, 60 carriers per reactor were used.</li>
		<li>Remove any excess defined medium using the sample tube and a syringe since adding the carriers raises the level of fluid in the bioreactor.</li>
		<li>Turn on the pH probes to adjust the pH in each reactor using the computer.</li>
		<li>Turn on the nitrogen flush for 20 min. 
		NOTE: The fecal homogenate used for inoculation is purchased as 10% feces homogenized in 10% glycerol solution (see Table of Materials).The fecal sample is randomly selected from a pool of donors with the following criteria: An American consuming a typical western diet (not vegan or vegetarian), between 21-45 years of age, antibiotic free for at least 1 year, with an average Body Mass Index (18.5-24.9). For this protocol, only a single donor is used. However, this can be altered due to experimental design.</li>
		<li>Prior to inoculation, thaw the fecal homogenate following the manufacturer&#8217;s guidelines.</li>
		<li>Inoculate each colon reactor by adding the fecal homogenate in an amount equal to 5% of the reactor volume through the sample port, using a 60 mL syringe (25 mL for the ascending colon, 40 mL for the transverse colon, 30 mL for the descending colon).</li>
		<li>Grow the cultures in the bioreactors overnight with pH control.</li>
		<li>After the overnight growth, harvest samples of the luminal fluid from each colon region as detailed below in section 3. After sampling is completed, turn on the computer program to begin feeding.</li>
	</ol>
	</li>
	<li>Daily feeding cycles 
	NOTE: The daily feeding cycles are computer automated.
	<ol>
		<li>Three times a day, pump 140 mL of the defined medium into the stomach bioreactor and allowed to incubate for 1 h.</li>
		<li>After 1 h incubation, pump the contents of the stomach into the small intestine. At the same time, pump 60 mL of pancreatic juice into the small intestine.</li>
		<li>In the small intestine, turn on pH adjustment to pH 6.7-6.9, and allow mixture to incubate for 90 mins. During incubation, turn on the nitrogen flush for each system for a total of 10 min each.</li>
		<li>After the 90 min incubation, turn on the pumps from the small intestine to the ascending colon, the ascending colon to the transverse colon, the transverse colon to the descending colon, and the descending colon to the waste simultaneously.</li>
	</ol>
	</li>
	<li>Experimental timeline 
	NOTE: The in vitro system used here is operated in a continuous fashion for approximately 8 weeks in length. Below is a recommended experimental timeline, however, this can be modified depending on the requirements and objective of the experiment.
	<ol>
		<li>Set up system following steps listed in step 2.1.</li>
		<li>Harvest samples from overnight growth following the protocol below. Begin the daily cycles, feeding three times a day following the protocol above.</li>
		<li>Run the experiment for a total of 2 weeks to allow the bacteria to stabilize. 
		NOTE: Running the system means that the pH is maintained, the daily feeding cycles are followed three times a day, and the mucosal carriers are changed 2-3 times each week.</li>
		<li>After stabilization, run the experiment for 2 weeks as the control period.</li>
		<li>After the control period, use the system to test different components or variables, known as the treatment period.</li>
		<li>After the treatment period, stop adding the components or variables and return the conditions to normal. This is considered the wash-out period and is used to determine whether or not changes to the community are permanent.</li>
	</ol>
	</li>
</ol>
3. Harvesting Samples from the System
NOTE: During the experiment, samples can be harvested from the luminal or mucosal phase of any region at any time, following the below guidelines.
<ol>
	<li>Harvesting luminal samples
	<ol>
		<li>Harvest luminal fluid samples through the sample port that extends into the middle of the culture liquid. Begin by cleaning the Luer Lock on the sample port with either 70% ethanol or the small alcohol pad and allow to dry.</li>
		<li>Ensure that all air is removed from a 30 mL syringe and connect it to the sample port. Draw up and down 3-5 times to ensure proper mixing of the vessel components. After, draw a total of 30 mL of culture.</li>
		<li>Aliquot the luminal samples as needed into sterile falcon tubes and store on ice. After sampling is completed transfer these to a -80 &#176;C freezer.</li>
		<li>For SCFA analysis, 15 mL of the luminal sample is spun at 4 &#176;C, 5,000 x g for 10 min. The supernatant is syringe filtered using a PES 0.2 &#956;M filter. Store the filtered supernatant at -80 &#176;C.
		<ol>
			<li>For DNA analysis, spin 1 mL of luminal fluid at 4 &#176;C, 5,000 x g for 10 min. Discard the supernatant and store the pellet at -80 &#176;C.</li>
			<li>As a backup, store 10 mL of luminal fluid at -80 &#176;C for most experiments.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Harvesting mucosal samples 
	NOTE: Change mucosal carriers every 2-3 days.
	<ol>
		<li>Remove and bring out the prepared mucin carriers from the refrigerator.</li>
		<li>Turn on the nitrogen flush and open the reactor lid quickly. Remove 50% of mucin carriers and add new ones.</li>
		<li>Seal the lid quickly and keep the nitrogen flush on for another 20 min.</li>
		<li>For DNA extraction, aliquot 0.25-0.5 g of agar into a 2 mL tube and stored at -80 &#176;C until needed.</li>
	</ol>
	</li>
</ol>
4. DNA Extraction, Sequencing, and Analysis
NOTE: DNA is extracted using the CTAB DNA extraction method with physical homogenization in a fume hood29. Following extraction, a spectrophotometer is used to quantify the amount of DNA in each sample.
<ol>
	<li>Prepare CTAB buffer
	<ol>
		<li>Dissolve 4.2 g K2HPO4 and 4.09 g NaCl in 200 mL of deionized, distilled water, then autoclave at 121 &#176;C for 30 min.</li>
		<li>Allow the solution to cool to room temperature.</li>
		<li>Add 10 g of Hexadecyltrimethylammonium bromide (CTAB) and heat to 60 &#176;C with agitation.</li>
		<li>After all the CTAB is dissolved, remove the solution from heat and cool to room temperature.</li>
	</ol>
	</li>
	<li>Prepare PEG-6000 solution
	<ol>
		<li>Dissolve 300 g of polyethylene glycol 6,000 and 93.5 g NaCl in 1 L of deionized, distilled water.</li>
		<li>After all particles are dissolved, autoclave the solution at 121 &#176;C for 30 min.</li>
		<li>After autoclaving, cool the solution to room temperature before use.</li>
	</ol>
	</li>
	<li>Perform DNA extraction
	<ol>
		<li>Add 500 &#956;L of CTAB buffer and 500 &#956;L of Phenol-Chloroform-Isoamyl alcohol to the 2 mL sample tube and vortex to homogenize.</li>
		<li>Transfer the entire contents of the sample tube to a 0.1 mm physical homogenizing tube (see Table of Materials).</li>
		<li>Homogenize the tubes for 20 s at the highest setting two times, with a 20 s pause in between, using a physical homogenizer (see Table of Materials).</li>
		<li>Centrifuge the homogenized tubes at 3,000 x g for 5 mins.</li>
		<li>Transfer 300 &#956;L of the supernatant to a clean, 1.7 mL tube and add 500 &#956;L of CTAB buffer to the original tube.</li>
		<li>Homogenize this tube again using the methods in steps 4.3.2-4.3.3 and centrifuge at 3,000 x g for 5 min.</li>
		<li>After centrifugation, transfer another 300 &#956;L of supernatant to the new tube which now contains 600 &#956;L.</li>
		<li>Add a total of 600 &#956;L Chloroform-Isoamyl alcohol to the tube containing 600 &#956;L of the supernatant.</li>
		<li>Invert the tubes to mix, and then centrifuge briefly. Transfer 500 &#956;L of the upper phase to a clean 1.7 mL tube. Ensure only to take the top phase.</li>
		<li>Next, add 1000 &#956;L of Peg-6000 solution, invert the tubes to mix, and then incubate at room temperature for 2 h.</li>
		<li>After incubation, centrifuge the tubes at 18,200 x g, 4 &#176;C, for 10 min.</li>
		<li>Discard the supernatant and clean the pellet with ice cold 70% ethanol.</li>
		<li>Centrifuge the tubes again at 18,200 x g, 4 &#176;C, for 10 min. Discard the supernatant and allow the pellet to dry in the biosafety cabinet under laminar flow.</li>
		<li>After the pellet is dry, add 75 &#956;L of RNAse free, DNAse free, sterile water to each tube.</li>
		<li>Quantify the resuspended DNA by a spectrophotometer and then send it out for 16S rRNA gene sequencing.</li>
	</ol>
	</li>
</ol>
5. Short Chain Fatty Acid (SCFA) Detection and Analysis
<ol>
	<li>Thaw the frozen samples at 40&#176;C for 30 min and extract short chain fatty acids using diethyl ether in a 2:1 (v:v) ratio.</li>
	<li>Transfer the organic phase to a GC/MS (see Table of Materials) equipped with a column, 30 m, 0.25 mm ID, 0.25 &#181;m, (see Table of Materials) where 10 &#181;L is injected.</li>
	<li>Set the injection port temperature and initial column temperature to 260 &#176;C and 125 &#176;C respectively. Hold the initial temperature for 1 min, and then increase this to 250 &#176;C, over 11.5 min with a column flow rate of 1 mL/min. 
	NOTE: The MS temperature for the ion source is 220&#176;C and 250&#176;C for the interface.</li>
	<li>Quantify the amount of each SCFA using standard curves and 2-methylhexanoic acid as the internal standard.</li>
</ol>

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The authors have no competing financial interests. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

In vitro culturing systems have been developed to study the gut microbiota of the large intestine. They use apparatuses designed to simulate the physiological conditions of the gastro intestinal tract, promoting the growth of a mature gut microbial community for each region of the colon33. While the concept is logical and comprehensible, the actual running of in vitro culturing systems to study the gut microbiota requires precision and an understanding of what is required and expected to produce reliable results.
The required elements for an in vitro gut microbiota experiment are that the community must reach stability after inoculation and that it must maintain the microbial diversity of the fecal inoculum. If the experiment is run properly, these two required elements are achieved readily and predictably. Critical steps in the protocol that will affect system stability and diversity are the following: The preparation of defined medium and pancreatic juice must be accurate, and delivery of these substrates must be consistent. Anaerobic conditions must be maintained during the experiment. The pH of each intestinal region must be accurately maintained over time. Changes in these three parameters during the experiment may result in population fluctuations and variable metabolite production.
Using this type of multi-stage in vitro system has several advantages. Notably, they simulate the different regions of the colon. They can also be designed to simulate in vitro digestion of food in the upper GIT, including the enzymatic components from saliva, and the addition of pancreatin and bile. A mucosal phase can be incorporated into each colon reactor, adding further complexity8. This results in the development of the region and the phase specific microbial communities that can be individually analyzed and compared. These types of systems can be readily manipulated, depending on experimental design through physical reconfiguration, alterations of the physiological parameters such as pH, temperature, or transit time, or inoculation with specific fecal samples26. Importantly, research has illustrated that the communities developed in these in vitro systems represent the gut microbial community11,26,34.
While the use of multi-stage in vitro systems can be used to mechanistically study the gut microbiota, they do have limitations. First, while the physiological conditions are programmable, they are fixed using specific average values, thus representing the average person. This is a simplification since there is considerable inter-individual variatiability12,23. Second, although the lack of mammalian components is one reason to use an in vitro system, it also must be considered a limitation. The lack of certain mammalian components, like the immune system, can result in differences between the in vitro model and the corresponding in vivo microenvironment12,23. However, these limitations do not detract from the value and insight that can be gained from using in vitro systems to mechanistically study the gut microbiota. Finally, the cultures are grown in glass bioreactors with no absorption of water or metabolites. This is an important difference, and the effect can be seen in the measurement of SCFAs where the total amounts of SCFAs are increasing from the ascending to transverse to descending colon regions (Figure 7). This is opposite of what is observed in vivo, where the highest concentrations of SCFAs are found proximally, in the ascending region, and the lowest concentrations found distally36. However, when the net production of SCFAs is considered for each region, then it can be determined that the most SCFA production is occurring in the ascending colon. It should also be mentioned here that bacterial load was not measured in this experiment. Differences in bacterial load between the regions in this system may have an effect on the total amounts of SCFAs produced.
In conclusion, it is well known that the gut microbiota plays a role in both human health and disease, and it is considered to function as a mediator between diet and metabolic health37,38. Here, the application of an in vitro system to study the gut microbiota was discussed, and the results from an experiment demonstrating the development of a stable community were presented. An in vitro system has many advantages and promotes the development of a complex and dynamic community, which is illustrated in the described experiment. This type of system is best used to study the interactions and changes within the gut microbiota community in response to external factors, such as food components and medicines. The results of these in vitro studies can then be supplemented with in vivo studies, to gain a deep understanding of the function and contribution of the gut microbiota to both health and disease.

The gut microbiota is a community of micro-organisms that reside in the human gastrointestinal tract (GIT).&#160; This community reaches maximum concentration in the colon, which is estimated to hold 1013-1014 bacteria, from 500-1,000 species, that live in symbiosis with the colon milieu1,2. The composition and functionality of the gut microbiota change spatially along the GIT, forming region specific communities, with the most diversity found distally2,3,4,5. For each anatomical region, separate microbial communities reside in the lumen and on the mucosal lining6. The lumen community has more direct access to nutrients as substrates move through the luminal compartment7. Despite this, some bacteria reside preferentially in the mucus layer, utilizing mucin produced by the colon cells as an energy source1,5,8. The difference in microenvironments between the luminal and mucosal phases results in divergence and the development of phase specific communities. Together, these communities provide metabolic functions, such as nutrient metabolism and the production of vitamins, and immunological functions, such as preventing the colonization of human pathogens1,3,9. The gut microbiota also works functionally in conjugation with the human colon cells3.
As an important part of the human GIT, it is not surprising that the gut microbiota is known to contribute to both host health and disease status3,9,10,11,12. A shift in the gut microbial population has been associated with multiple human diseases, including GIT disorders like intestinal bowel disease (IBD) and intestinal bowel syndrome (IBS), but also other diseases, such as obesity, circulatory disease, and autism3,9,10,11,12.&#160; Metabolites produced from the gut microbiota have a global effect, reaching locations far from the gut12,13. For example, the gut-brain axis is associated with mental disorders like anxiety and depression14. Therefore, studying the gut microbiota is important to multiple fields of research, and is applicable to many diseases, even those not often associated with the GIT.
While it is widely acknowledged that studying the gut microbiota is important, it is a complicated endeavor. Multiple animal models are available, from small animals like zebrafish, rats, and mice, to larger ones like monkeys and pigs15-19. However, the application of these animals in terms of the human gut microbiota is not straightforward, since these animals have a unique bacterial community that has evolved based on environment and diet, and they are anatomically distinct from humans20,21. The use of human subjects removes the question of relevance yet introduces another set of challenges. Human studies are expensive, time consuming, and are ethically constrained11. Moreover, confounding factors influence the gut microbiota in human studies, including age or developmental stage, environment, diet, medication, and genetic factors2,4,22.&#160; There are also restrictions on what can be tested in humans, and which type of samples can be harvested at what times4.&#160;
One critical disadvantage of using an in vivo system to study the gut microbiota is the presence of mammalian components. The gut microbiota and human cells interact with one another, and in an in vivo setting, it is impossible to distinguish the two. The metabolites produced by the gut microbiota are taken in by the colon cells, so measurements cannot be calculated with precision. Therefore, any mechanistic study must be limited to end-point measurements11. Another major disadvantage for in vivo studies is the inability to harvest samples from the different regions of the GIT longitudinally23. This does not allow for the assessment of changes that may occur in the microenvironments of the colon over time12.&#160; Many in vivo studies, including human studies, rely on analysis of fecal samples to detect changes to the gut microbiota12. While this is informative, it does not provide data on the gut microbiota across the GIT and does not differentiate between the luminal and mucosal communities5,6,7,8.
For the gut microbiota, the application of an in vitro method is required to study the dynamics of the bacterial community, without interference from the mammalian components. Using an in vitro method allows for the tight control of environmental conditions10, testing of multiple parameters simultaneously, and the ability to sample longitudinally, and in large volumes11. Since an in vitro method utilizes a mechanical device and not a host, no considerations are needed for age, environment, diet, or genetic background. These systems can be used to test either the entire gut microbiota community, only selected organisms, or even single strains. Importantly, in vitro results are reproducible, yet retain a level of diversity comparable to in vivo studies11,22.
Depending on the hypothesis in question and the desired results, in vitro studies can be performed in numerous ways.&#160; They can utilize single-vessel systems and simple methods, such as incubating samples with fecal homogenate24 or performing single batch cultures over the course of 24-48 h25. They can also be accomplished using single-vessel systems and more complex methods, such as using a chemostat system to produce a stable gut microbial community11. However, the use of a single reactor can over-simplify the microbiota12 since it only represents one section of the colon, even though the colon is composed of the ascending, transverse, and descending regions.
In order to study the gut microbiota community that develops in the different regions of the colon (the ascending, transverse, and descending regions), a complex, multi-stage system can be employed. In these systems, multiple vessels are set up to mimic the different regions of the colon, so the gut microbiota of the ascending, transverse, and descending regions are cultivated independently. These vessels are connected, using pumps to move substrates in sequence, from the ascending to the transverse to the descending colon regions, mimicking the flow of nutrients through the GIT.
The objective of this study was to demonstrate how a 5-stage in vitro culture system (see Table of Materials) can be used to cultivate the gut microbiota community, and to demonstrate community dynamics in terms of stability and composition. In this system, one vessel represents the stomach and one represent the small intestine. The colon is divided into three regions (ascending, transverse, descending), with one vessel representing each region26. In this experimental setup, two complete systems were run in parallel, with Unit 1 containing mucin carriers to represents the mucosal surface and Unit 2 containing no mucin carriers. The communities that developed in the luminal and mucosal phases of each region were compared to each other, and to the fecal inoculum over time using 16S rRNA gene sequencing and SCFA analysis. The results presented demonstrate the type of community, both in terms of composition and functionality, which can be produced from this type of in vitro system.

<table><tbody><tr><td>TWINSHIME</td><td>Prodigest</td><td>NA</td><td></td></tr><tr><td>Defined medium (Adult M-SHIME growth medium with starch)</td><td>Prodigest</td><td>NA</td><td></td></tr><tr><td>Masterflex tubing</td><td>cole Parmer</td><td>NA</td><td></td></tr><tr><td>Urine Drainage bag</td><td>Bard</td><td>NA</td><td></td></tr><tr><td>Labsorb</td><td>Sigma-Aldrich</td><td>NA</td><td></td></tr><tr><td>Fecal sample</td><td>Openbiome</td><td>NA</td><td></td></tr><tr><td>Syringes</td><td>Becton Dickson</td><td>NA</td><td></td></tr><tr><td>Defined medium</td><td>Prodigest</td><td>NA</td><td></td></tr><tr><td>Oxgall Bile</td><td>Becton Dickson</td><td>NA</td><td></td></tr><tr><td>Pancreatin</td><td>Sigma-Aldrich</td><td>NA</td><td></td></tr><tr><td>Glass ware</td><td>Ace Glass</td><td>NA</td><td></td></tr><tr><td>Porcine mucin</td><td>Sigma-Aldrich</td><td>NA</td><td></td></tr><tr><td>Bacteriological agar</td><td>Sigma-Aldrich</td><td>NA</td><td></td></tr><tr><td>Sterilization pouches</td><td>VWR</td><td>NA</td><td></td></tr><tr><td>BeadBug</td><td>Benchmark Scientific</td><td>NA</td><td></td></tr><tr><td>Triple-Pure High Impact Zirconium 0.1 mm Bead beater tube</td><td>Benchmark Scientific</td><td>NA</td><td></td></tr><tr><td>RNAse free, DNAse free, sterile water</td><td>Roche</td><td>NA</td><td></td></tr><tr><td>Shimadzu QP2010 Ultra GC/MS</td><td>Shimadzu</td><td>NA</td><td></td></tr><tr><td>Stabilwax-DA column, 30 m, 0.25 mm ID, 0.25 &amp;micro;m</td><td>Restek</td><td>NA</td><td></td></tr><tr><td>plastic mucin carriers</td><td>Prodigest</td><td>NA</td><td></td></tr></tbody></table>

applying advanced in vitro culturing technology to study the human gut microbiota

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

The above protocol describes set up, inoculation, and running of a 5-stage in vitro system to study the gut microbiota of the colon. To generate the data presented below, following DNA extraction, 16S rRNA marker gene DNA sequencing of the V1V2 region was performed using the high throughput sequencing (e.g., MiSeq Illumina platform) by the Microbiome Center at the Children&#8217;s Hospital of Philadelphia27. QIIME (Quantitative Insight into Microbial Ecology) version 1.928 was used to process the sequencing data and statistical analysis was performed on the R environment for statistical computing29. Taxonomic assignments were generated using the Greengenes 16S reference database30,31. OTU relative abundance values were calculated by dividing the OTU read count by the total number of reads in the sample. SCFA levels were quantified using the protocol described.
The in vitro communities achieve a steady state equilibrium
The ability to study the gut microbiota and short chain fatty acid (SCFA) production in vitro with a multi-stage system requires that the microbial communities reach a steady state. This occurs after inoculation when the taxa have become established in their niches, and the composition of the community and their metabolites are no longer fluctuating. In a steady state, the community and its products remain the same over time. For in vitro gut microbiota studies, stability is a key requirement; without stability, it is impossible to determine whether or not observed changes are occurring due to the experimental conditions, or if they are due to variation. According to what has been proposed in the literature, a community can be considered stable when there is 80% similarity between time points32.
Using the results of 16S rRNA marker gene sequencing, Principal Coordinates Analysis (PCoA) plots based on unweighted and weighted UniFrac distances were generated for the communities that developed in each region of the system over time (Figure 2). An unweighted analysis compares the communities in terms of the presence/absence of species. The weighted analysis compares them based not only on the presence/absence of species but also considers their abundance. For Unit 1, this included both the luminal and mucosal phases, and for Unit 2 only the luminal phase. Based on this analysis, it was observed that the community changed appreciably between days 1 and 7. However, from day 11 post inoculation until the end of the experiment, the samples occupied a small region of the PCoA space. This was true for sample-sample distance scores based on OTU presence-absence (Figure 2A) or on OTU abundance (Figure 2B). Thus, it was observed that, after 11 days, the abundance and the types of bacteria were stable from one sample point to the next. This pattern was observed for both the luminal and mucosal phases of all three colon regions of Unit 1 and the luminal phase of Unit 2. These results illustrated that both the luminal and mucosal phases reach stability and that this occurred at the same time.
System stability was also probed by analyzing the production of short chain fatty acids (SCFAs) over time. The three most prominent SCFAs (propionic acid, butanoic acid, and acetic acid)38 were measured in each sample over the course of the experiment using gas chromatography GC/MS. These measurements revealed that propionic acid, butanoic acid, and acetic acid fluctuated from the start of the experiment until day 15 post inoculation (Figure 3A-C). After day 15, the amounts of these SCFAs produced in each colon region remained constant, with only minimal changes occurring until the end of the experiment (Figure 3A-C). The difference between time points was an average of 6.8% for propionic acid, 7.2% for acetic acid, and 8.02% for butanoic acid. This suggests that similar to the community composition, the metabolic properties of the community entered a steady state, as indicated by the production of stable amounts of SCFAs over time. Both Unit 1 and Unit 2 produced similar amounts of SCFAs, with no significant differences between the two (p &#62; 0.05). This indicated that the production of SCFAs was not affected by the presence or absence of the mucosal community. It should be noted that the point of stability determined in this experiment is similar to that reported previously, in which it was stated that community stabilization in a 5-stage in vitro system occurred approximately 2 weeks post inoculation33,34,35.
The communities developed in the in vitro system are similar to the inoculum
The second required element for an in vitro gut microbiota experiment is that the community developed in the model preserves the microbial diversity of the fecal inoculum. Since the system used in this experiment provides for three distinct colon regions it cannot be expected that any one region be exactly the same as the fecal inoculum. However, it is expected that the members of each community are derived from the fecal inoculum, and all together maintain a similar level of diversity as the inoculum.
Based on the results of 16S rRNA marker gene sequencing, the average, stable community for the luminal and mucosal phase of each colon region was determined (Days 15-28 post inoculation) and compared to the community of the fecal inoculum (Figure 4A). These results demonstrate that the communities which developed in the individual colon regions of the in vitro system were similar to the fecal inoculum in composition. The two most prominent orders in the reactor, Clostridiales and Bacteroidales, matched those observed in the inoculum. However, several low-abundance bacterial orders in the colon regions were different from the inoculum, with the most prominent differences between orders Burkholderiales and Synergistales (Figure 4A).
The alpha diversity for in vitro system was compared to the inoculum, as another measure of community structure after stabilization. The Shannon index, calculated for each region over time, reached a similar level of diversity as the inoculum for all regions, except for luminal samples from the ascending region (Figure. 4B). Taken together, these results demonstrate that the in vitro culture system was able to produce a community comparable to the fecal inoculum, both in terms of composition and diversity (Figure 4A-B).
Using an in vitro system allows for the development of the region and the phase specific communities
The three colon regions represented in this system are maintained at different pH values and receive different nutrient supplies. (This was mentioned in the protocol section). Based on this, it is expected that the communities in these regions will differentiate 33. The stable (Days 15-28) luminal communities in both Unit 1 and Unit 2 were determined at the family level and plotted according to relative abundance (Figure 5). The divergence between the three colon regions in terms of the abundance of specific taxa, demonstrates that each region develops a unique community. This is also supported by the results of Figure 2 and Figure 4A. In Figure 2, the mature communities that developed in each region cluster at different locations in the PCoA chart. In Figure 4A, the communities in each colon region differed in the percentages of the dominant order members.
For this experiment, Unit 1 was provided with mucosal carriers, while Unit 2 had no mucin carriers. Based on this experimental design, the contribution of the mucosal surface could be examined. For comparison purposes, the stable (Days 15-28) luminal and mucosal communities for Unit 1, at the family level, were plotted together according to relative abundance (Figure 6). It is clear that the composition of the luminal and mucosal communities for all three colons differ in the abundance of some taxa, the most prominent being Lachnospiraceae and Bacteroidaceae. These results also demonstrate that the mucosal communities in the three regions are different from each other. For example, Clostridiaceae is enriched in the mucosal communities of the descending and transverse regions, and Veillonellaceae is higher in the mucosal phase of the ascending colon, but not in the transverse or descending colon regions. Taken together, the results presented in these figures show that there is a clear difference between the communities in each phase and each region for some taxa, illustrating that the composition between the regions is similar and that there is a difference in abundance.
Although analysis of DNA sequencing reveals the development of a region-specific community, there is no apparent difference in the production of SCFAs between regions. The average ratio of Acetic Acid: Propionic Acid: Butanoic Acid for the stable community (Day 15-28) was calculated for each region (Figure 7A). While the ratios of the different SCFA remained fairly similar, there is an increase in the total amounts of SCFAs produced between the ascending, transverse and descending regions, with the highest levels found in the descending region (Figure 7B). This was true for both Unit 1 and Unit 2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59054/59054fig1.jpg" /> 
Figure 1: Illustration of the 5-stage, in vitro experimental design. The complete system consists of the following components: A circulating water bath, nitrogen flow, a set of glass bioreactors, a set of magnetic stirrer bars and magnetic stirrers, pH probes, a computer-controlled console containing 40 peristaltic pumps, a computer monitor, and a refrigerator. The main system is composed of a set of bioreactors mimicking the stomach, small intestine, and the ascending, transverse, and descending colon regions. Two complete units are set up to run in parallel, providing for an experimental and a control group. This means that 10 bioreactors, 10 pH probes, and 10 magnetic stirrers are required for this experiment. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59054/59054fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59054/59054fig2.jpg" /> 
Figure 2: The in vitro community stabilizes by day 11 post inoculation. PCoA analysis based on (A) Unweighted and (B) Weighted Unifrac distances for the luminal and mucosal phase of each region over time for Unit 1 and for the luminal phase of Unit 2. The plot is faceted into the components after calculating the PCoA axes. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59054/59054fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59054/59054fig3.jpg" /> 
Figure 3: The production of SCFAs by the in vitro system stabilize by day 15 post inoculation. Measurements of Propionic Acid, Butanoic Acid, and Acetic Acid over time for the (A) Ascending colon (B) Transverse colon and (C) Descending colon. The experiment was performed in triplicate. The results represent an average of three independent measurements, with error bars depicting the standard deviation. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59054/59054fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59054/59054fig4.jpg" /> 
Figure 4: Comparison of the stable communities from the in vitro system to the fecal inoculum. (A) The stable communities (D15-28), at the order level were averaged and formatted in pie charts for both the mucosal and luminal phase of each colon region, and for the fecal inoculum. (B) The Shannon diversity for the mucosal and luminal phase of each colon region compared to the fecal inoculum (black dotted line). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59054/59054fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59054/59054fig5.jpg" /> 
Figure 5: The luminal and mucosal phase in each colon region promote the growth of distinct communities. The average relative abundance, at the family level, for the stable communities (D15-28) for the luminal and mucosal phase, and the inoculum, were calculated and plotted together for each colon region. Error bars represent the standard deviation between timepoints. AC1 = Ascending colon Unit 1; TC1 = Transverse colon Unit 1; DC1 = Descending colon Unit 1; AC2 = Ascending colon Unit 2; TC2 = Transverse colon Unit 2; DC2 = Descending colon Unit 2. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59054/59054fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59054/59054fig6.jpg" /> 
Figure 6: Each colon region of the in vitro system develops a unique community. The average relative abundance, at the family level, for the stable communities (D15-28) in each colon region, and the fecal inoculum, were calculated and plotted together. The experiment was performed in triplicate. The results represent an average of three independent measurements, with error bars depicting the standard deviation. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59054/59054fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59054/59054fig7.jpg" /> 
Figure 7: The ratio of Acetic Acid: Propionic Acid: Butanoic Acid is similar for each colon region. (A) The amounts of acetic acid, propionic acid, and butanoic acid for the stable communities (D15-28) of each colon region were calculated and converted to a ratio. (B) The ratios were plotted as percentages for each colon region. Error bars represent the standard deviation between timepoints. AC1= Ascending colon Unit 1; TC1 = Transverse colon Unit 1; DC1 = Descending colon Unit 1; AC2 = Ascending colon Unit 2; TC2 = Transverse colon Unit 2; DC2 = Descending colon Unit 2. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59054/59054fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota at JoVE.com

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...

applying-advanced-vitro-culturing-technology-to-study-human-gut

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

13.9K Views.  Agricultural Research Service, United States Department of Agriculture. Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

Video: Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Immunology and Infection

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and inflammatory mediators, which dynamically alter community composition and microbial metabolic output. Understanding how physiological culture conditions affect microbial communities and diversity and their metabolic capacity will contribute important knowledge of their impact on health and diseases. Here, we present a protocol adapted from the template published by Auchtung et al. to create mini-bioreactor arrays that can cultivate complex fecal communities, define bacterial consortiums or single strains under precise conditions, including nutrient availability, temperature, flow rate, pH, and oxygen content. We describe the process for building the mini-bioreactor system, including adaptations to improve limitations in the published protocol. We also discuss the setup of the mini-bioreactor system under anaerobic conditions with the use of MEGA media (adapted from Han et al.), which is a rich media supporting the growth of diverse bacteria. We describe the inoculation of gut humanized mouse fecal samples into the mini-bioreactor system, followed by the establishment of complex community cultures within the mini-bioreactor system, which can be grown under continuous flow with aseptic sampling to monitor community composition. This system is adaptable to dietary changes and other cultural modifications. The techniques described here allow for the characterization of diverse, fastidious fecal communities under dynamic conditions or in response to perturbation in isolation from host-derived factors.

This protocol details the assembly of mini-bioreactor arrays to be utilized for continuous flow culture of complex fecal communities under anaerobic conditions. We also discuss the methods for assembly, inoculation, and sampling of the reactors for further analysis.

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Biology

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.
Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.
Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

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Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing