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Abbreviations: SHIME, Simulator for Human Intestinal Microbiological Ecosystem; TIM, TNO Intestinal Model.

Abbreviations: SHIME, Simulator for Human Intestinal Microbiological Ecosystem; TIM, TNO Intestinal Model.

in 2005 (12), together with the Reading model, are structurally chemostat models having 3-6 sequentially attached fermenting vessels with computer controlled fluid transition systems (Fig. 1) and (Table 1). The Reading model and the EnteroMix® model both simulate only the human colon, and a similar artificial simulator media described by Macfarlane et al. (10) is used in them to simulate the fluid entering the colon from the small intestine. The SHIME model simulates the whole human GIT from stomach to colon using artificial SHIME media, which has much in common with the medium described by Macfarlane and co-workers (10). These three models have three different designs in fluid transition. Fluids are either pumped semi-continuously to the subsequent vessels in three-hour intervals (EnteroMix® model), there is a continuous overflow of fluids between vessels (the Reading model), or the model can be a combination of these two types (SHIME).

Reading Simulator

The Reading simulator (Fig. 1) simulates the gut using a 3 stage continuous culture with three glass vessels (220 ml, 320 ml and 320 ml) and different pH in each vessel (5.8, 6.2, and 6.8); mimicking the human proximal, transverse, and distal colon, respectively.

In the beginning of the simulation, each vessel is inoculated with 100 ml of 20% (wt/vol) of human feces. The system is incubated in a batch overnight, after which a continuous pumping of fresh simulator fluid to the first vessel is started. At the same time a continuous overflow from vessel to vessel begins and the system is run for at least 14 days in order achieve a steady-state condition in the vessels. The excess fluid from the third vessel is collected to a waste container. The total retention time of the system can vary, e.g., between 27 and 67 hours (10). The viability of the microbiota is determined by taking samples at regular intervals from the vessels. After the incubation period, the test substance is added to the system mixed in the fresh simulation fluid and the system is then run to new steady state [e.g., for 22 days (9)]. The last phase is the washout period [e.g., for 50 days (9)] with the original simulation fluid to determine how long the changes induced by the test substance can still be measured in the absence of the substrate itself.

SHIME Model

The current SHIME model is a single six-stage system, where the first three glass vessels simulate stomach and small intestine and the subsequent three glass vessels the large intestine (11a). The original SHIME model (Fig. 2) (11) was a single five-stage system without the stomach compartment. Working volumes in these vessels are 300 ml for stomach and small intestine, 1000 ml for ceacum and ascending colon, 1600 ml for

Figure 2 The original SHIME model. Vessels 1-5 in the figure mimic the different compartments of the human GIT: duodenum + jejunum, ileum, caecum + ascending colon, transverse colon and distal colon, respectively. In the revised version of this system, a vessel representing the stomach has been added before vessel 1. First five pumps work semi-continuously, and pumps between vessels, 3-5 and effluent work continuously. Source: From Ref. 11.

Figure 2 The original SHIME model. Vessels 1-5 in the figure mimic the different compartments of the human GIT: duodenum + jejunum, ileum, caecum + ascending colon, transverse colon and distal colon, respectively. In the revised version of this system, a vessel representing the stomach has been added before vessel 1. First five pumps work semi-continuously, and pumps between vessels, 3-5 and effluent work continuously. Source: From Ref. 11.

transverse colon, and 1200 ml for descending colon. pH is controlled in vessels 2, 3, 4, 5, and 6 in the ranges 5.0-6.5, 6.5-7.0, 5.5-6.0, 6.0-6.5 and 6.5-7.0, respectively.

The system is inoculated by introducing 10 ml supernatant of a human western diet suspension per day to the three first vessels for eight successive days. The remaining three vessels 4-6 representing the different compartments of the colon are inoculated with 50 ml of fecal suspension for 10 successive days. The contents of these three vessels are pumped continuously from vessel to vessel and finally to a discard bottle. The transit time of the whole system is 84 hours.

In the beginning of the simulation, 200 ml of fresh SHIME media (11) is added to vessel 1 (stomach) three times per day. Every 2-3 hours, the acidic (pH 2.0) contents of the first vessel is pumped to vessel 2 (duodenum + jejunum) along with 100 ml of pancreatic juice, supplemented with bile, to neutralize the acidity of the gastric effluent. After four hours the contents of vessel 2 is pumped to vessel 3 (ileum).

After eight days of using SHIME media only, the actual test substrate mixed with the SHIME media is introduced to the system. Feeding of the substrate is continued for 12 days, followed by another SHIME media-only period for 8-10 days. This cycle of three periods is repeated for all the studied substrates and samples are taken after each period.

The EnteroMix® Colon Simulator

The EnteroMix® model (Fig. 3) has four parallel units each comprising four glass vessels, allowing four simulations to be run simultaneously using the same fecal inoculum (12). EnteroMix® model vessels 1, 2, 3, and 4 have the smallest working volumes (6, 8, 10, and 12 ml, respectively) of the three models presented here (Table 1). The pH levels in the vessels (5.5, 6.0, 6.5, and 7.0, respectively) are similar to the other models. Because of the small volumes of vessels, a 40 ml inoculum of 25% wt/vol human feces and only 4 g of test substrate is needed for four parallel 48-hour simulations.

The simulation begins by filling the vessels of each of the four units with 0.9 mM anaerobic NaCl (3, 5, 7, and 9 ml to vessels 1, 2, 3, and 4, respectively) and inoculating the

Figure 3 The EnteroMix® model. The figure represents the initial volumes of the system before fresh medium is added to begin the simulation. The vessels V1 to V4 are mimicking different sections of the human colon: caecum C ascending, transverse, descending, and distal colon, respectively. pH controlling and semi-continuous fluid transitions are operated via opening and closing of computer controlled valves (S).

Figure 3 The EnteroMix® model. The figure represents the initial volumes of the system before fresh medium is added to begin the simulation. The vessels V1 to V4 are mimicking different sections of the human colon: caecum C ascending, transverse, descending, and distal colon, respectively. pH controlling and semi-continuous fluid transitions are operated via opening and closing of computer controlled valves (S).

first vessel with 10 ml of fecal inoculum. The inoculum is mixed in the vessel with NaCl and 10 ml of the mixed culture is pumped to the next vessel. This procedure continues through the vessels and finally the excess inoculum is pumped to waste container from the fourth vessel. After three hours of the incubation, 3 ml of fresh simulator media with (three test channels) or without (one control channel) test substance is pumped to the first vessel. The media is fermented in the first vessel for three hours, after which 3 ml of the fermented media is transferred to the second vessel, and 3 ml of fresh media is pumped to the first vessel. This procedure of transferring liquid to the next vessel continues through all the vessels, so that finally after 15 hours, when 3 ml of fermented fluid has been transferred from vessel four to the waste container for the first time, vessels 1, 2, 3, and 4 have respective volumes of 6, 8, 10, and 12 ml of fermenting fluid. The fermentation and three-hourly fluid transfers continue for 48 hours, after which the system is stopped and samples are collected from each vessel.

Other Simulators

In addition to simulate different parts of the GIT, chemostat-type simulators have also been used to simulate the oral cavity, in particular to investigate plaque formation (13); and to simulate the urinary bladder to investigate antibiotic sensitivity of urinary tract infection-causing pathogens (14). These simulators usually consist of a single chemostat.

Non-Chemostat Models

The third type of model is actually comprised of two complementary parts, the TIM (TNO Intestinal Model) systems 1 and 2 introduced by Minekus et al. in 1995 (15) and 1999 (16). The TIM 1 system (Fig. 4) comprises eight sequentially attached glass modules and mimics the stomach and small intestine, while the TIM 2-system consists of four glass modules in a loop mimicking the proximal colon of monogastric animals (Fig. 5). These

Figure 4 TIM 1 model. The model is mimicking the different sections of the human small intestine: the gastric compartment (1), duodenum (2), jejunum (3) and ileum (4). Gastric (5) and intestinal secretions (6), peristaltic valve pumps (7) and dialysis devices (8) are also included in this simulator. Source: From Ref. 17.

Figure 4 TIM 1 model. The model is mimicking the different sections of the human small intestine: the gastric compartment (1), duodenum (2), jejunum (3) and ileum (4). Gastric (5) and intestinal secretions (6), peristaltic valve pumps (7) and dialysis devices (8) are also included in this simulator. Source: From Ref. 17.

dynamic models differ from the three previously presented models in two main aspects: fluid transportation from vessel to vessel is executed via peristaltic valve-pumps and there is a constant absorption of water and fermentation products through dialysis membranes. In both systems the peristaltic movement of the intestinal fluid flowing in a flexible tube in the middle of the modules is achieved by changing the pressure of the 37°C heated water circulating between the module walls and the flexible tube. The peristaltic pressure around the flexible tube is controlled via computer-controlled valves to mimic the gastric emptying times. For the simulation of intestinal absorption TIM 1 has two integrated 5 kDa dialysis membranes, after jejunal and ileal modules, and TIM 2 has one, a hollow-fiber membrane (molecular mass cut-off value 50 kDa) in the lumen of the system. The TIM 1 dialysis membranes allow real-time collection of absorbable metabolites and water that would be absorbable in the human jejunum and ileum. In the tube membrane of TIM 2 circulates dialysis fluid allowing absorption of e.g., water, and short-chain fatty acids. The pH-values are monitored in each compartment.

In a TIM 1 simulation, a homogenized human meal is introduced into the gastric compartment in pre-set times. From the stomach, the fluid is pumped through the following six compartments. During the simulation, the secretion of enzymes, bile, and pancreatic juice and the pH-controlling of the stomach (a pH gradient from 5.0 to 1.8 in 80 minutes from the beginning) and duodenum (constant pH 6.5) is regulated via computer.

In a TIM 2 simulation the model is first inoculated with 200 ml of fecal inoculum. Microbiota is allowed to adapt to the conditions for 16 hours, after which the actual simulation is started by adding ileal medium semi-continuously with or without the tested substrate to the system. The pH is constantly maintained constant at 5.8 representing the pH-level in the proximal colon. Samples can be taken both from the lumen of the simulator and from the dialysis liquid during the simulation.

Figure 5 TIM 2 model: The model represents the human proximal colon in one loop-shaped system: peristaltic mixing with flexible walls inside (a), pH electrode (b), alkaline pump (c), dialysis system (d), fluid level sensor (e), nitrogen inlet (f), peristaltic valves (g), sample port (h), gas sampling (i) and ileal medium reservoir. Source: From Ref. 18.

Figure 5 TIM 2 model: The model represents the human proximal colon in one loop-shaped system: peristaltic mixing with flexible walls inside (a), pH electrode (b), alkaline pump (c), dialysis system (d), fluid level sensor (e), nitrogen inlet (f), peristaltic valves (g), sample port (h), gas sampling (i) and ileal medium reservoir. Source: From Ref. 18.

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