The authors gratefully acknowledge funding support from the Center for Diabetes, Obesity and Metabolism and the Clinical and Translational Science Center, the Wake Forest School of Medicine, the Department of Defense funding (Grant number: W81XWH-18-1-0118), the Kermit Glenn Phillips II Chair in Cardiovascular Medicine; the National Institutes of Health funded Claude D. Pepper Older Americans Center (funded by P30AG12232); R01AG18915; R01DK114224 and the Clinical and Translational Science Center (Clinical Research Unit, funded by UL1TR001420), is also thankfully acknowledged. We also thank the volunteers for providing fecal samples, and our other lab members for their technical helps during this experiment.

CAUTION: Consult the appropriate Material Safety Data Sheets and follow the instructions and guidelines for appropriate Biosafety Level 2 (BSL-2) training. Follow all the culturing steps as per the standard biosafety rules and use a BSL-2 cabinet using aseptic conditions. Furthermore, fecal samples from different models and human subjects may have potential risk of spreading microbial borne diseases. Immediately seek medical aid in the occurrence of any injury and infection. In addition, the use of human and animal stool samples should be approved through institutional ethical committees and must compliant with protocols to use samples and subject information.
1. Preparation of Culture Media
<ol>
	<li>Preparation of the culture media, prepare nine types of stock solutions
	<ol>
		<li>Solution A (1,000 mL): Dissolve 5.4 g of sodium chloride (NaCl), 2.7 g of potassium dihydrogen phosphate (KH2PO4), 0.16 g of calcium chloride dihydrate (CaCl2&#183;2H2O), 0.12 g of magnesium chloride hexahydrate (MgCl2&#183;6H2O), 0.06 g of manganese chloride tetrahydrate (MnCl2&#183;4H2O), 0.06 g of cobaltous chloride hexahydrate (CoCl2&#183;6H2O), and 5.4 g of ammonium sulfate (NH4)2SO4, in deionized water to make total volume to 1,000 mL.</li>
		<li>Solution B (1,000 mL): Dissolve 2.7 g Potassium Hydrogen Phosphate (K2HPO4) in deionized water to make total volume to 1000 mL.</li>
		<li>Trace mineral solution (1,000 mL): Dissolve 500 mg of disodium ethylenediamine-tetraacetate dihydrate (Na2EDTA), 200 mg of ferrous sulfate heptahydrate (FeSO4&#183;7H2O), 10 mg of zinc sulfate heptahydrate (ZnSO4&#183;7H2O), 3 mg of manganese(II) chloride tetrahydrate (MnCl2&#183;4H2O), 30 mg of phosphoric acid (H3PO4), 20 mg of CoCl2&#183;6H2O, 1 mg of cupric chloride dihydrate (CuCl2&#183;2H2O), 2 mg of nickel(II) chloride hexahydrate (NiCl2&#183;6H2O) and 3 mg of sodium molybdate dihydrate (Na2MoO4&#183;2H2O) in deionized water to make total volume to 1,000 mL. 
		NOTE: This solution is light sensitive, therefore ensure to be stored in dark/black or aluminum wrapped tubes/bottles.</li>
		<li>Water soluble vitamin solution (1,000 mL): Dissolve 100 mg of thiamine hydrochloride (Thiamin-HCl), 100 mg of D-pantothenic acid, 100 mg of Niacin, 100 mg of pyridoxine, 5 mg of P-aminobenzoic acid and 0.25 mg of Vitamin B12 in deionized water to make total volume to 1,000 mL.</li>
		<li>Folate: biotin solution (1,000 mL): Dissolve 10 mg of folic acid, 2 mg of D-biotin and 100 mg of ammonium bicarbonate (NH4HCO3) in deionized water to make total volume to 1,000 mL.</li>
		<li>Riboflavin solution (1,000 mL): Dissolve 10 mg of Riboflavin in 5 mM HEPES (1.19 g/L) solution to make total volume to 1,000 mL.</li>
		<li>Hemin solution (10 mL): Dissolve 5,000 mg of Hemin in 10 mM sodium hydroxide (NaOH) (0.4 g/L) solution to make total volume to 10 mL.</li>
		<li>Short-chain fatty acid mix (10 mL): Combine 2.5 mL of N-valerate, 2.5 mL of isovalerate, 2.5 mL of isobutyrate and 2.5 mL: DL-&#945;-methylbutyrate. 
		NOTE: This solution is recommended to use in fume hood to avoid smell and fumes.</li>
		<li>Resazurin (1,000 mL): Dissolve 1 g of resazurin in deionized water and make total volume to 1,000 mL.</li>
	</ol>
	</li>
	<li>Medium used for in vitro anaerobic fermentation
	<ol>
		<li>To prepare this media, mix 330 mL of Solution A, 330 mL of Solution B, 10 mL of Trace mineral solution, 20 mL of Water soluble vitamin solution, 5 mL of Folate:biotin solution, 5 mL of Riboflavin solution; 2.5 mL of Hemin solution, 0.4 mL of Short chain fatty acid mix, 1 mL of Resazurin, 0.5 g of yeast extract, 4 g of sodium carbonate (Na2CO3), 0.5 g of Cysteine HCl-H2O, and 0.5 g of Trypticase, and add 296.1 mL of distilled water.</li>
		<li>Check the pH and ensure it is around 7.0, if not, adjust the pH with 1 N HCl or 1 N NaOH. Sterilize by vacuum filtering the media using a bottle filter under the aseptic workstation.</li>
		<li>Alternatively, mix all the components (except vitamin and hemin solutions) and autoclave at 121 &#176;C for 20 min and let it cool down to room temperature. Simultaneously, filter-sterilize vitamin and hemin solutions using 0.22 &#181;m membrane filters and add these to the autoclaved and cooled media before dispensing.</li>
	</ol>
	</li>
</ol>
2. Preparation of Anaerobic Chamber and Required Material
<ol>
	<li>Keep all the materials, solutions and tools needed for the fermentation experiment inside the anaerobic chamber at least 48 h before the start of the experiment, to ensure that any residual oxygen associated with tools and soluble oxygen in buffers/solutions is removed and all the materials are acclimatized to the set anaerobic conditions. 
	NOTE: Materials needed inside the anaerobic chamber (48 h earlier of experiment to start): (i) fermentation media; (ii) anaerobic solution (prepared according to section 4.1), (iii) vortexer, (iv) weighing balance, (v) muslin cheese cloths, (vi) scissors, (vii) funnel, (viii) 1.5, 2.0, 15, 50 mL tubes, (ix) pipettor (2, 20, 200 and 1,000 &#181;L) and compatible pipet tips, (x) gun pipet and pipets (5 and 10 mL), (xi) paper tissues, (xii) markers, (xiii) tube stands for different tubes, (xiv) waste box, (xv) O2 indicator and (xvi) 70% ethanol spray bottle (disinfectant).</li>
</ol>
3. Preparation of Tubes and Fibers
<ol>
	<li>Weight 300 mg of inulin and transfer to a 50 mL tube followed by the aseptically addition of 26 mL of fermentation media already prepared and stored in the anaerobic chamber. Prepare one blank tube for each fecal sample type and experimental tube(s) (according to the number of compounds being tested), in triplicate.</li>
	<li>Leave these tubes inside the anaerobic chamber for around 24 h to allow hydration of samples before starting the fermentation experiment. Ensure the tube temperature is 37 &#176;C at the time of inoculation, therefore bring the tubes in the incubator enclosed within the anaerobic chamber.</li>
</ol>
4. Preparation of Inoculum
<ol>
	<li>Anaerobic dilution solution (at least 48 h before fermentation experiment): Dissolve 5 g of NaCl, 2 g of glucose and 0.3 g of Cysteine-HCl in deionized water and make total volume to 1,000 mL. Autoclave and store it inside the anaerobic chamber at least 48 h before use.</li>
	<li>Fecal inoculum preparation (at the day of fermentation experiment): Weigh 5 g of fresh fecal sample in a 50 mL conical tube, add anaerobic dilution solution for a final volume of 50 mL (1:10 w/v) and vortex for 15 min or until completely homogenized. Filter the homogenized mixture through 4 layers of sterile cheesecloth (autoclaved) and use it immediately for inoculation in the tubes containing media. 
	NOTE: The fecal samples from a group of subjects can be pooled if the experimental objective is to compare the effect of a given compound/ingredient on overall healthy versus diseased fecal microbiota in general.</li>
</ol>
5. Fermentation and Sampling
<ol>
	<li>Prepare tubes according to the section 4.2 and inoculate blank/control and experimental tubes with 4 mL of diluted and filtered fecal inoculum. Incubate the inoculated tubes at 37 &#176;C inside the anaerobic chamber. Shake the tubes once every hour by inverting gently to re-suspend the fibers and inoculum.</li>
	<li>Collect samples as frequently as needed, for example, hourly to 0, 3, 6, 9 and 24 h during fermentation by taking a 2 mL aliquot sample out into a 2 mL tube from respective fermenting tubes.</li>
	<li>Measure the pH of the aliquots using a laboratory pH meter (by directly inserting the pH electrode in the sample); centrifuge the remaining sample at 14,000 x g for 10 min at 4 &#176;C. Immediately freeze the supernatant and pellet in liquid nitrogen, and after snap freezing, store the supernatant for SCFAs analysis and pellet for microbiome analysis at -80 &#176;C.</li>
</ol>
6. Short Chain Fatty Acids (SCFAs) and Lactate Quantification
NOTE: The SCFAs and lactate in the supernatant of microbiome culture can be measured exactly according to the methods detailed elsewhere13,14,15,16.
<ol>
	<li>Briefly, thaw the snap frozen supernatants obtained in section 5 from control and treatment samples on ice and carry out all further processing steps on ice. Filter the supernatant through a 0.45&#8201;&#181;m membrane filter and use the cell-free samples to measure the concentrations of SCFAs and lactate using HPLC system with DAD detector at 210&#8201;nm, equipped with an HPX-87H column. Use inject volume of 10 &#956;L for each sample and use H2SO4 (0.005&#8201;N) to elute the column at a flow rate of 0.6&#8201;mL/min at 35&#8201;&#176;C.</li>
</ol>
7. Fecal Microbiome Analysis
NOTE: Perform microbiome analysis following the methods and pipeline detailed elsewhere7,13,14,17.
<ol>
	<li>Briefly, extract genomic DNA from approximately 200&#8201;mg of fecal slurry pellets by using a fecal DNA extraction kit13.</li>
	<li>Amplify the hypervariable region of the bacterial 16S rRNA gene by using the barcoded primers as per the method described elsewhere13, using the primer sequences as described in the Earth Microbiome Project protocol18.</li>
	<li>Purify the resulting amplicons with magnetic purification beads and quantify by Picogreen or an equivalent method. Pool the purified PCR products in equal molar concentration and sequence on a sequencer13.</li>
	<li>Process the resulting sequences for de-multiplexing, quality filtering and clustering, taxonomic assignment and rarefaction, and downstream analyses by using the Quantitative Insights into Microbial Ecology (QIIME) software as per the methods described by Caporaso et al.19.</li>
</ol>

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The authors have nothing to disclose.

The in vitro fecal slurry fermentation model presented here is a simple single-batch model to approximate the effects of different substrates and microbial strains (e.g., prebiotics and probiotics) on the composition of human fecal microbiota as well as its metabolic activities in terms of fecal pH and SCFAs levels. The results presented herein demonstrate that the inoculation of inulin decreases the fecal pH and significantly increases the levels of SCFAs and lactate in inulin-treated fecal specimen as compared to non-treated fecal microbiota culture (Figure 1). In addition, the gut microbiota signature also appears to be different between inulin-treated and untreated samples (Figure 2 and Figure 3). These data exemplify how this system can reflect the effects of inulin on fecal microbiome diversity and composition as well as its metabolic activities. In addition, depending on specific experimental objectives and hypotheses, a variety of other factors can also be measured using this system. Furthermore, in addition to 16S rRNA gene sequencing, other analyses such as whole microbial metagenome sequencing (using Whole genome sequencer) or qPCR and culture methods targeted at specific single or multiple genera, species and strains (e.g., bifidobacteria, lactobacilli, Akkermansia, Enterobacteriacaea, clostridia, etc.) can also be executed. In addition, different chromatographic procedures for the analysis of SCFAs, such as LC-MS, GC, GC-MS, GC-FID, and HPLC can also be exploited based on experimental requirement and availability. Nevertheless, it should be noted that sample preparation for these procedures may vary according to the instruments and conditions required for operation20.
Although the use of fresh fecal specimen would yield best reproducible results; however, snap frozen fecal sample (as used in the experiment presented herein) can also be used efficaciously as most of the bacteria can be revived from it once resuscitated to body temperature and no more decomposition happens after the freezing21. Using frozen sample can be particularly advantageous when it is impossible to obtain fresh fecal samples from specific donor(s) on planned day of the experiment, or from the same donor when an experiment needs to be replicated. It should be noted, however, that the fecal samples should be snap frozen using liquid nitrogen and immediately stored at -80 &#176;C until further use. In addition, to avoid the exposure of the frozen fecal samples to air (oxygen), the samples should be transferred to the anaerobic chamber as soon as possible/immediately after taking out from the freezer and used right away (repeated thawing should be avoided). All subsequent experiments including sample thawing, inoculum preparation and processing should be done inside the anaerobic chamber.
The intestinal organic environment and pH during nutrient fermentation in large bowel are very important particularly in view of the fact that an abnormally reduced pH indicates increased acidity due to the substrate utilization. Hence, more rapid pH reduction can correspond to more rapid substrate utilization22. Although, in this model, the pH has not been controlled, however, inclusion of identical non-treated control is highly recommended to make direct comparisons. The SCFAs produced in the colon as a result of the fermentation of indigestible polysaccharides by the gut microbiota can further influence diverse mechanisms related to the maintenance of the host health. These metabolites contribute in gluconeogenesis and lipid biosynthesis, act as an energy source for colonocytes, and can also have health benefits including immune modulation, controlled/improved gut barrier functions, and neuromodulation23,24,25,26. In addition, SCFAs are also known to influence several biological pathways including hormones, endocannabinoid system, cell proliferation and death, bone health, mineral absorption, gut motility, intestinal pH, and invert effects on the gut microbiome and microbial metabolic function. Therefore, knowledge of the profile and concentrations of intestinal/ fecal SCFAs can be an important component while evaluating the efficacy of specific microbiota modulators6. Of course, besides SCFAs, the gut microbiome also produces many other important metabolites (e.g., ammonium, vitamins, histamine). Hence, the supernatant specimens collected during such in vitro fermentation experiments can also be evaluated for global metabolomics analyses to discover novel gut microbiome-derived metabolites that can be influenced by specific microbiome modulators.
The system described here has many advantages, such as the ease, simplicity, cost effectiveness, and the general adoptability of the experimental set-up. However, there are few limitations as well. For example, the system does not pertain to the interaction of prebiotics (or other interventions used) in the upper digestive system (e.g., saliva, stomach, small intestine) before being exposed to the microbiota of the large intestine. However, such steps can be adopted and incorporated according to specific experimental requirements. Also, an acidic and/or enzymatic hydrolysis process may need to be performed before fermentation if using specific substrates including whole food or digestible food because only the indigestible part of such foods reaches to the colon to be fermented by the lower gut microbiota. In addition, the composition of gut microbiota may shift due to specific culture conditions during fermentation. For example, it was observed that up to 9 h of incubation, microbiota changes are much closer to normal than at 24 h. However, after 24 h of incubation, although pH and SCFAs levels increased substantially, gut microbiota composition showed that Proteobacteria counts increased while the microbial diversity reduced. However, it remains unknown how precisely and closely the increased accumulation of microbial metabolites accompanied with the lowered fecal pH is associated with specific microbiota signatures.
In summary, a simple protocol to simulate the ex vivo microbiota ecosystem is described herein. The system enables researchers to test different interventions for modulation of the microbiota diversity, composition and metabolic function that can influence diverse features of the host intestinal and overall health.

The human microbiota is a complex community consisting of bacteria, archaea, viruses and eukaryotic microbes1, that inhabit the human body internally and externally. Recent evidences have established the fundamental role of the gut microbiota and the gut microbiome (the entire collection of microbes and their genes found in the human gastrointestinal tract) in various human diseases including obesity, diabetes, cardiovascular diseases, and cancer1,2,3. Additionally, the microorganisms living in our gut produce a wide spectrum of metabolites which significantly affect our health and can also contribute to the pathophysiology of several diseases as well as a variety of metabolic functions4,5. Abnormal changes (perturbations) in the composition and function of this gut microbial population are generally termed as &#34;gut dysbiosis&#34;. Dysbiosis is usually associated with an unhealthy state of the host and hence can be differentiated from the normal (homeostatic) microbial community associated with a healthy control state of the host. Specific patterns of gut microbiome dysbiosis are often found in various different diseases1,2,3,6,7.
The fermentation of undigested food, particularly the fermentable carbohydrates/fibers, by the gut microbiota not only yields energy but also produces divergent metabolites including short-chain fatty acids (SCFAs), lactate, formate, carbon dioxide, methane, hydrogen, and ethanol6. In addition, the gut microbiota also produces a number of other bioactive substances such as folate, biotin, trimethylamine-N-oxide, serotonin, tryptophan, gamma-aminobutyric acid, dopamine, norepinephrine, acetylcholine, histamine, deoxycholic acid, and 4-ethylphenyl sulfate. This occurs primarily through the utilization of intrinsic metabolic fluxes within the host-microbe niche, which contributes in several body processes, metabolic functions and epigenetic changes1,8,9,10. However, the effects of various interventions on such microbial products remain unkown or unclear due to the lack of easy, efficient and reproducible protocols. The human gut microbiota composition is an extremely complex and diverse ecosystem, and hence, many questions about its role in human health and disease pathology still remain unanswered. The effects of many common gut microbiome modulators (e.g., probiotics, prebiotics, antibiotics, fecal transplantation and infections) on the composition and metabolic functions of the intestinal microbiota remain largely elusive. In addition, the examination and validation of these effects in vivo is difficult, particularly because most of the nutrients and metabolites produced by the gut microbiota are absorbed or disposed of simultaneously and rapidly in the gut; therefore, measuring the production, amount and processing of these metabolites (e.g., SCFAs) in vivo still remains a practical challenge. Indeed, physiological models such as animals and human subjects are critical for determining the role of gut microbiome and its modulation on host health, but these may not be suitable for large-scale screening of different types of microbiome modulators due to ethical, monetary or time constraints. To this end, in vitro and/or ex vivo models, such as culturing of gut microbiota in vitro and then intervening with different microbiota modulators, can offer time- and money-saving opportunities and hence can allow for preliminary or large-scale screening of various components (such as probiotics, prebiotics, and other interventional compounds) to examine/predict their effects on the fecal microbiota diversity, composition and metabolic profiles. Studies using such in vitro and ex vivo systems of the gut microbiome may facilitate further understanding of the host-microbiome interactions that contribute to host health and disease, and could also lead to finding novel therapies that target the microbiome to ameliorate host health and prevent and treat various diseases1.
Although the in vitro gut microbiota culture systems cannot truly replicate the actual intestinal conditions, several laboratories have endeavored to develop such models, some of which have been found practicable to some extent and have been successfully used for different purposes. One of the recent gut models is the Simulator of the Human Intestinal Microbial Ecosystem, which mimics the entire human gastrointestinal tract, including the stomach, small intestine, and different regions of the colon. However, such technically complex models may not be accessible to other research facilities worldwide. Therefore, there is still a critical need for the development of new alternative models that are relatively simple, affordable and practical for laboratories studying the microbiome modulators and their effects on gut microbiota and host health. Hence, the use of an in vitro (or ex vivo) fecal microbiota culture system would be useful for studying the effects of such interventions11,12. Specifically, the effect of different prebiotics on the microbiota fermentation capacity in terms of periodic changes in the gut microbiota diversity and composition, the fecal pH, and the levels of microbial metabolites including SCFAs and lactate can be studied13. Herein, using inulin (one of the most widely studied prebiotic components) as an example of the microbiome modulator, a step-by-step protocol of this simple ex vivo microbiota batch-culture system is described to demonstrate its use to estimate the changes in the fecal microbiota and microbial metabolites following intervention with the microbiome modulators.

<table><tbody><tr><td>Ammonium Bicarbonate (NH&lt;sub&gt;4&lt;/sub&gt;HCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>217255</td><td></td></tr><tr><td>Ammonium Sulfate (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4 &lt;/sub&gt;</td><td>TGI</td><td>C2388</td><td>Toxic</td></tr><tr><td>Calcium Chloride Dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>C3306</td><td>Irritating</td></tr><tr><td>Cobaltous Chloride Hexahydrate (CoCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>255599</td><td></td></tr><tr><td>Cupric Chloride Dihydrate (CuCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Acros organics</td><td>2063450000</td><td>Toxic, Irritating</td></tr><tr><td>Cysteine-HCl</td><td>Sigma-Aldrich</td><td>C121800</td><td></td></tr><tr><td>D-biotin</td><td>Sigma-Aldrich</td><td>B4501</td><td></td></tr><tr><td>D-Pantothenic acid</td><td>Alfa Aesar</td><td>A16609</td><td></td></tr><tr><td>Disodium Ethylenediaminetetraacetate Dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;EDTA)</td><td>Biorad</td><td>1610729</td><td></td></tr><tr><td>DL-&amp;alpha;-methylbutyrate</td><td>Sigma-Aldrich</td><td>W271918</td><td></td></tr><tr><td>Ferrous Sulfate Heptahydrate (FeSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>F8263</td><td>Toxic</td></tr><tr><td>Folic acid</td><td>Alfa Aesar</td><td>J62937</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Hemin</td><td>Sigma-Aldrich</td><td>H9039</td><td></td></tr><tr><td>Hepes</td><td>Alfa Aesar</td><td>A14777</td><td></td></tr><tr><td>Isobutyrate</td><td>Alfa Aesar</td><td>L04038</td><td></td></tr><tr><td>Isovalerate</td><td>Alfa Aesar</td><td>A18642</td><td></td></tr><tr><td>Magnesium Chloride Hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Manganese Chloride Tetrahydrate (MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>221279</td><td></td></tr><tr><td>Niacin (Nicotinic acid)</td><td>Sigma-Aldrich</td><td>N4126</td><td></td></tr><tr><td>Nickel(Ii) Chloride Hexahydrate (NiCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Alfa Aesar</td><td>A14366</td><td>Toxic</td></tr><tr><td>N-valerate</td><td>Sigma-Aldrich</td><td>240370</td><td></td></tr><tr><td>P-aminobenzoic acid</td><td>MP China</td><td>102569</td><td>Toxic, Irritating</td></tr><tr><td>Phosphoric Acid (H&lt;sub&gt;3&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P5811</td><td></td></tr><tr><td>Potassium Dihydrogen Phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P5504</td><td></td></tr><tr><td>Potassium Hydrogen Phosphate (K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>1551128</td><td></td></tr><tr><td>Pyridoxine</td><td>Alfa Aesar</td><td>A12041</td><td></td></tr><tr><td>Resazurin</td><td>Sigma-Aldrich</td><td>R7017</td><td></td></tr><tr><td>Riboflavin</td><td>Alfa Aesar</td><td>A11764</td><td></td></tr><tr><td>Sodium carbonate (Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>1613757</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher BioReagents</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Fisher Chemicals</td><td>S320</td><td></td></tr><tr><td>Sodium Molybdate Dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Acros organics</td><td>206375000</td><td></td></tr><tr><td>Thiamine Hydrochloride (Thiamin-HCl)</td><td>Acros organics</td><td>148991000</td><td></td></tr><tr><td>Trypticase</td><td>BD Biosciences</td><td>211921</td><td></td></tr><tr><td>Vitamin B12</td><td>Sigma-Aldrich</td><td>V2876</td><td></td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>70161</td><td></td></tr><tr><td>Zinc Sulfate Heptahydrate (ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>Z0251</td><td></td></tr><tr><td>0.22 &amp;micro;m membrane filter</td><td></td><td></td><td></td></tr><tr><td>AMPure magnetic purification beads</td><td>Agencourt</td><td></td><td></td></tr><tr><td>Anaerobic chamber with incubatore</td><td>Forma anaerobic system, Thermo Scientific, USA</td><td></td><td></td></tr><tr><td>Bottle filter</td><td>Corning</td><td></td><td></td></tr><tr><td>Cheesecloth</td><td></td><td></td><td></td></tr><tr><td>Illumina MiSeq sequencer</td><td>Miseq reagent kit v3</td><td></td><td></td></tr><tr><td>pH meter</td><td></td><td></td><td></td></tr><tr><td>Qiagen PowerFecal kit</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Quantitative Insights into Microbial Ecology (QIIME) software</td><td></td><td></td><td></td></tr><tr><td>Qubit-3 fluorimeter</td><td>InVitrogen</td><td></td><td></td></tr><tr><td>Vortex</td><td>Thermoscientific</td><td></td><td></td></tr><tr><td>Waters-2695 Alliance HPLC system</td><td>Waters Corporation</td><td></td><td></td></tr></tbody></table>

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.

The protocol is used to demonstrate the effect of a specific prebiotic (i.e., inulin on the microbiota composition and metabolic activities in terms of changes in the fecal pH and the concentration of lactate and SCFAs in the feces of healthy human subjects over different time-points following treatment with inulin). The fecal pH, the fecal levels of lactate and SCFAs (Figure 1), and the microbiota composition (Figure 2 and Figure 3) are measured at 0 (baseline), 9 and 24 h of incubation with or without inulin. The results demonstrate how fecal microbiota composition and its metabolic activities are modulated during in vitro fermentation with or without inulin treatment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59524/59524fig1r.jpg" /> 
Figure 1: Changes in fecal pH (a), lactate (b) and short-chain fatty acids viz. acetate (c), propionate (d) and butyrate (e) in human feces at 0 (baseline), 9 and 24 h of anaerobic fermentation with or without inulin. Values presented here are Mean &#177; SEM of triplicate samples. *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.001, vs. baseline. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59524/59524fig1large.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/59524/59524fig2.jpg" /> 
Figure 2: Changes in the microbiota diversity and composition in human feces at 0 (baseline), 9 and 24 h of anaerobic fermentation with or without inulin. (a) Weighted and (b) unweighted Unifrac measures of beta-diversity visualized using Principle Coordinate Analysis (PCoA). (c-f) Indices of alpha-diversity viz. phylogenetic diversity (PD whole tree (c); species richness (Chao1; d); observed number of operational taxonomic units (OTUs, e); and species evenness (Shannon index, f)). Relative abundance of major phyla (g) and genera (h). Values presented here are Mean &#177; SEM of triplicate samples. *P &#60; 0.05, **P &#60; 0.01, vs. baseline. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59524/59524fig2large.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/59524/59524fig3.jpg" /> 
Figure 3: Linear discriminant analysis (LDA) effect size (LEfSe) analysis of gut microbiota changes following 9 h and 24 h incubation with (INU) or without (CTL) inulin. Taxonomic cladogram derived from LEfSe analysis of 16S sequences (relative abundance &#8805; 0.5%) representing the differentially abundant taxa between different group of samples. The brightness of each dot is proportional to its effect size (i.e., the taxon abundance). Only taxa passing LDA threshold value of &#62;2.4 are shown here. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59524/59524fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota at JoVE.com

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

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 ...

an-vitro-batch-culture-model-to-estimate-effects-interventional

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.5K Views.  Wake Forest School of Medicine. Gezien de opkomende rol van darmmicrobioom in verschillende menselijke ziekten, verschillende darmmicrobioom modulators zoals probiotica, prebiotica, diëten en drugs kunnen helpen bij het verbeteren van darmmicrobioom-geassocieerde gezondheidsaandoeningen. Er is echter geen snel en betaalbaar screeningsysteem beschikbaar om de effecten van dergelijke darmmicrobioommodulatoren te voorspellen, maar dit systeem is eenvoudig genoeg om te voorspellen hoe bepaalde interventies van invloed kunnen z...

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

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Cancer Research

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 ...

Bioengineering

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.

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 ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

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 ...

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field&#39;s understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

Medicine

Fecal Microbiota Transplantation and Detection of Prevalence of IgA-Coated Bacteria in the Gut

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the biological function of intestinal bacteria as a whole or at the species level. Several different FMT methods have been published. Here, we present an FMT protocol that successfully depletes gut microbiota in a matter of days, followed by transplantation of fecal microbiota from fresh or frozen donor intestinal contents to conventional mice. Real time-PCR is applied to test the efficacy of bacterial depletion. Sequencing of the 16S ribosomal RNA (rRNA) is then applied to test the relative abundance and identity of gut microbiota in recipient mice. We also present a flow cytometry-based detection method of immunoglobulin A (IgA)-coated bacteria in the gut.

We established an efficient way to deplete intestinal bacteria in three days, and subsequently transplant fecal microbiota via gavage of fecal fluid prepared from fresh or frozen intestinal contents in mice. We also present an optimized method to detect the frequency of IgA-coated bacteria in the gut.

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the ...

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

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