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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

The gut microbiota is a community of micro-organisms that reside in the human gastrointestinal tract (GIT).  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 milieu^1,2. The composition and functionality of the gut microbiota change spatially along the GIT, forming region specific communities, with the most diversity found distally^2,3,4,5. 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 source^1,5,8. 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 pathogens^1,3,9. 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 status^3,9,10,11,12. 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 autism^3,9,10,11,12.  Metabolites produced from the gut microbiota have a global effect, reaching locations far from the gut^12,13. 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 pigs^15-19. 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 humans^20,21. 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 factors^2,4,22.  There are also restrictions on what can be tested in humans, and which type of samples can be harvested at what times⁴. 

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.  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 communities^5,6,7,8.

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 studies^11,22.

Depending on the hypothesis in question and the desired results, in vitro studies can be performed in numerous ways.  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.

Protokół

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).

1. Prepare the defined medium
   1. Fill a 4 L flask with 2 L of double distilled, deionized water.
   2. Add 29.2 g of defined medium powder to the water and mix unit complete homogenized.
   3. Add a magnetic stirrer and loosely screw on the lid.
   4. Autoclave at 121 °C for 30 min.
   5. After cooling, store the prepared defined medium in a refrigerator at 4 °C with constant agitation (magnetic stirrer).
2. Prepare pancreatic juice
   1. Autoclave 2L of distilled, deionized water in a 5L vessel with a stirrer bar added, at 121 °C for 30 min.
   2. After autoclaving, allow the water to cool to room temperature.
   3. 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.
3. Phosphate buffer
   1. 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.
   2. 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.
   3. After the components have completely dissolved, adjust the pH to 7.0 using NaOH.
   4. Pour the buffer into a 2 L vessel with a screw cap lid and autoclave at 121 °C for 30 min. Ensure that the lid is screwed on loosely and not closed tightly.
   5. 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.
4. Prepare Mucin agar carriers
   1. Autoclave plastic mucin carriers (see Table of Materials), forceps, plastic mesh (tubular shape), and zip ties at 131 °C for 30 min.
   2. Autoclave 300 mL of double distilled, de-ionized water at 131 °C for 30 min. Store at room temperature until use.
   3. 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.
   4. To prepare mucin agar, add 3 g of bacterial agar and 15 g of porcine mucin to the 300 mL of autoclaved water.
   5. 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.
   6. Once the glass vessel containing the mucin agar solution is cool enough to touch, move it into the biosafety cabinet with laminar flow.
   7. 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.
   8. Place the lid back on the Petri dish and allow it to cool under laminar flow.
   9. 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.
   10. 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 °C until use.

2. Set up, Inoculation, and Running of the System

1. Set up of the culturing system
   NOTE: The Twin Simulator of the Human Intestinal Microbial Ecosystem was used for this study.
   1. 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.
   2. 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.
   3. 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.
   4. Place the defined medium and pancreatic juice in a refrigerator at 4 °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.
   5. 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.
   6. 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 °C.
   7. 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.
   8. 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.
   9. 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.
   10. 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.
   11. 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.
2. Inoculation of the system
   1. 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.
   2. 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.
   3. Remove any excess defined medium using the sample tube and a syringe since adding the carriers raises the level of fluid in the bioreactor.
   4. Turn on the pH probes to adjust the pH in each reactor using the computer.
   5. 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.
   6. Prior to inoculation, thaw the fecal homogenate following the manufacturer’s guidelines.
   7. 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).
   8. Grow the cultures in the bioreactors overnight with pH control.
   9. 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.
3. Daily feeding cycles
   NOTE: The daily feeding cycles are computer automated.
   1. Three times a day, pump 140 mL of the defined medium into the stomach bioreactor and allowed to incubate for 1 h.
   2. 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.
   3. 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.
   4. 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.
4. 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.
   1. Set up system following steps listed in step 2.1.
   2. Harvest samples from overnight growth following the protocol below. Begin the daily cycles, feeding three times a day following the protocol above.
   3. 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.
   4. After stabilization, run the experiment for 2 weeks as the control period.
   5. After the control period, use the system to test different components or variables, known as the treatment period.
   6. 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.

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.

1. Harvesting luminal samples
   1. 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.
   2. 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.
   3. Aliquot the luminal samples as needed into sterile falcon tubes and store on ice. After sampling is completed transfer these to a -80 °C freezer.
   4. For SCFA analysis, 15 mL of the luminal sample is spun at 4 °C, 5,000 x g for 10 min. The supernatant is syringe filtered using a PES 0.2 μM filter. Store the filtered supernatant at -80 °C.
      1. For DNA analysis, spin 1 mL of luminal fluid at 4 °C, 5,000 x g for 10 min. Discard the supernatant and store the pellet at -80 °C.
      2. As a backup, store 10 mL of luminal fluid at -80 °C for most experiments.
2. Harvesting mucosal samples
   NOTE: Change mucosal carriers every 2-3 days.
   1. Remove and bring out the prepared mucin carriers from the refrigerator.
   2. Turn on the nitrogen flush and open the reactor lid quickly. Remove 50% of mucin carriers and add new ones.
   3. Seal the lid quickly and keep the nitrogen flush on for another 20 min.
   4. For DNA extraction, aliquot 0.25-0.5 g of agar into a 2 mL tube and stored at -80 °C until needed.

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.

1. Prepare CTAB buffer
   1. Dissolve 4.2 g K₂HPO₄ and 4.09 g NaCl in 200 mL of deionized, distilled water, then autoclave at 121 °C for 30 min.
   2. Allow the solution to cool to room temperature.
   3. Add 10 g of Hexadecyltrimethylammonium bromide (CTAB) and heat to 60 °C with agitation.
   4. After all the CTAB is dissolved, remove the solution from heat and cool to room temperature.
2. Prepare PEG-6000 solution
   1. Dissolve 300 g of polyethylene glycol 6,000 and 93.5 g NaCl in 1 L of deionized, distilled water.
   2. After all particles are dissolved, autoclave the solution at 121 °C for 30 min.
   3. After autoclaving, cool the solution to room temperature before use.
3. Perform DNA extraction
   1. Add 500 μL of CTAB buffer and 500 μL of Phenol-Chloroform-Isoamyl alcohol to the 2 mL sample tube and vortex to homogenize.
   2. Transfer the entire contents of the sample tube to a 0.1 mm physical homogenizing tube (see Table of Materials).
   3. 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).
   4. Centrifuge the homogenized tubes at 3,000 x g for 5 mins.
   5. Transfer 300 μL of the supernatant to a clean, 1.7 mL tube and add 500 μL of CTAB buffer to the original tube.
   6. 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.
   7. After centrifugation, transfer another 300 μL of supernatant to the new tube which now contains 600 μL.
   8. Add a total of 600 μL Chloroform-Isoamyl alcohol to the tube containing 600 μL of the supernatant.
   9. Invert the tubes to mix, and then centrifuge briefly. Transfer 500 μL of the upper phase to a clean 1.7 mL tube. Ensure only to take the top phase.
   10. Next, add 1000 μL of Peg-6000 solution, invert the tubes to mix, and then incubate at room temperature for 2 h.
   11. After incubation, centrifuge the tubes at 18,200 x g, 4 °C, for 10 min.
   12. Discard the supernatant and clean the pellet with ice cold 70% ethanol.
   13. Centrifuge the tubes again at 18,200 x g, 4 °C, for 10 min. Discard the supernatant and allow the pellet to dry in the biosafety cabinet under laminar flow.
   14. After the pellet is dry, add 75 μL of RNAse free, DNAse free, sterile water to each tube.
   15. Quantify the resuspended DNA by a spectrophotometer and then send it out for 16S rRNA gene sequencing.

5. Short Chain Fatty Acid (SCFA) Detection and Analysis

1. Thaw the frozen samples at 40°C for 30 min and extract short chain fatty acids using diethyl ether in a 2:1 (v:v) ratio.
2. Transfer the organic phase to a GC/MS (see Table of Materials) equipped with a column, 30 m, 0.25 mm ID, 0.25 µm, (see Table of Materials) where 10 µL is injected.
3. Set the injection port temperature and initial column temperature to 260 °C and 125 °C respectively. Hold the initial temperature for 1 min, and then increase this to 250 °C, over 11.5 min with a column flow rate of 1 mL/min.
   NOTE: The MS temperature for the ion source is 220°C and 250°C for the interface.
4. Quantify the amount of each SCFA using standard curves and 2-methylhexanoic acid as the internal standard.

Wyniki

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’s Hospital of Philadelphia²⁷. QIIME (Quantitative Insight into Microbial Ecolo...

Dyskusje

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...

Materiały

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