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...

<ol>
	<li>Johansson, M., Larsson, J., Hansson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+mucus+layers+of+colon+are+organized+by+the+MUC2+mucin,+whereas+the+outer+layer+is+a+legislator+of+host-microbial+interactions.">The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host-microbial interactions.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (1), 4659-4665 (2011).</li><li>Xu, J., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honor+thy+symbionts.">Honor thy symbionts.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (18), 10452-10459 (2003).</li><li>Jandhyala, S., Talukdar, R., Subramanyam, C., Vuyyuru, H., Sasikala, M., Reddy, D. <a target="_blank" 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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 r...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 0.1mm 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, 30m, 0.25mm ID, 0.25&#181;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 Ecolo...

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 ...

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.

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

Research

JoVE Journal

Bioengineering

13.7K visualizações.  Agricultural Research Service, United States Department of Agriculture. O objetivo deste protocolo é cultivar microbiota intestinal in vitro. Permitirá o estudo da dinâmica da microbiota sem ter que considerar a contribuição de um componente moderno. Usando este sistema in vitro multi estágio, contamos que a comunidade de microbiotas para desenvolver no lumin e na mucosa de várias regiões do cólon pode ser iniciada simultaneamente.Uma demonstração visual deste método é útil devido à complexidade deste sistema in vitro. Comece preenchendo o c...