JP2012034589A - Apparatus for culturing intestine imitation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an apparatus for culturing an intestine imitation maintaining a microbial diversity which is similar to an actual enteric bacterial flora.SOLUTION: The apparatus for culturing the intestine imitation incorporates at least one mechanism enhancing the microbial diversity.

Description

  The present invention relates to an intestinal mimetic culture apparatus. Specifically, the present invention relates to an improved intestinal mimic culture device for increasing the diversity of enteric bacteria.

  In the intestine of humans and animals, there are about 10 trillions of microorganisms called the intestinal flora (flora). In recent years, it has been reported many times that these microorganisms and their metabolism have a great influence on the metabolism and immune mechanism of humans and animals as hosts, as well as the health condition. Along with this, research on intestinal flora and its metabolites has become increasingly popular. In general, human intestinal flora and metabolite profile studies have been conducted using volunteers such as healthy persons and hospital patients, and experimental animals. However, research by human volunteers requires high costs and long-term experiments, and it is necessary to impose burdens such as dietary and drug type restrictions. Also, it is difficult to directly collect intestinal contents or tissues from an ethical and practical viewpoint. Furthermore, the experimental results are susceptible to individual differences such as volunteer age and gender.

  In studies using animal models, some of the above problems are solved. Furthermore, since an immunological test using aseptic animals and various knockout animals is possible, animal experiments using mice / rats are currently widely used. However, in addition to the need for special facilities and equipment for animal breeding, the intestinal environment of humans is completely reduced, such as the concentration of intestinal spoilage in the intestine of mice / rats is significantly lower than that of human manure. It is hard to say that they are simulating. In addition, it is difficult to monitor the local microbial dynamics in the intestine in real time, as in the case of humans.

  As one method for solving this problem, research on intestinal flora cultured by an intestinal mimetic culture apparatus that reproduces the intestinal environment has been recently conducted mainly in Europe. The most commonly used intestinal mimetic culture apparatus is a simple single tank type or a continuous culture system using a multi-stage type culture tank that reproduces the intestinal tract part. To give a typical example, Gibson et al. Investigated the spatial, temporal and nutritional factors in intestinal fermentation, and the heterogeneous factors such as pH, so that the environment of the right and left hemicolons An intestinal mimetic culture device (hereinafter referred to as “3MCS”) provided with a triple stirrer-type culture tank focused on the difference was proposed (Non-patent Document 1). In this model, a medium simulating a dietary degradation mixture from the small intestine outlet is fed continuously or intermittently to the first tank. Accordingly, the first tank is under eutrophic conditions similar to those of the cecum, and the activity of microorganisms can be examined at a high microorganism growth rate and low pH. The second and third tanks have much fewer nutrient sources that microorganisms are likely to ingest, resulting in similar environmental conditions from the transverse colon to the left hemicolon with a low microbial growth rate and near neutral pH.

  Moreover, based on 3MCS, De Boever et al. Have devised a 5-tank system (The Simulator of the Human Intestinal Microbial Ecosystem: SHIME) that includes a non-stirring culture tank that mimics the stomach and small intestine (Non-patent Document 2). .

  On the other hand, TNO in the Netherlands has developed a model called TIM (TNO's in vitro gastrointestinal models, TIM) (Patent Documents 1, 2, and 3, Non-Patent Document 3). The TIM is composed of TIM-1 that simulates peristaltic movement of the stomach and small intestine and TIM-2 that simulates the large intestine. TIM2 is a system that can separate and extract metabolites and microorganisms by culturing intestinal flora in hollow fiber.

  Thus, the intestinal tract culture system has been devised to imitate the shape, movement, pH, etc. of the intestinal tract in various ways. This is because it is difficult to stably maintain and culture a community composed of several hundreds of various microorganisms having different nutrient utilization and optimum pH, such as intestinal flora.

U.S. Pat.No. 5,525,305 European Patent No. 0642382 European Patent No. 0642382

G.R.Gibson, et al .: Appl.Environ.Microbiol. 54: 2750-2755 (1988) P. De Boever, et al .: J Nutr. 130: 2599-2606 (2000) G.T.Macfarlane, et al .: Microbial Ecology. 35: 180-187 (1998)

  By the way, the surface of the human intestine has a complicated structure called a soft net, and it is known that it has a very large specific surface area. However, no studies have been conducted to investigate the maintenance of the diversity of intestinal flora (flora) by enlarging the specific surface area of the intestinal epidermis or imitating the intestinal epidermis in an intestinal mimicking culture apparatus.

  An object of the present invention is to provide an intestinal mimetic culture apparatus that maintains microbial diversity similar to the actual intestinal flora.

  Therefore, the present inventors maintain the diversity of microorganisms similar to the actual intestinal microflora as compared with the conventional apparatus by expanding the specific surface area of the intestinal epidermis or imitating the intestinal epidermis in the intestinal mimicking culture apparatus. I thought that I could do it, and I repeated research. As a result, the present inventors have found that the diversity of microorganisms can be increased by incorporating a structure imitating the intestinal epidermis into the intestinal mimicking culture apparatus.

That is, the present invention includes the following [1] to [8].
[1] An intestinal mimetic culture apparatus characterized in that in the intestinal mimetic culture apparatus, at least one mechanism for increasing microbial diversity is incorporated.
[2] The intestinal mimetic culture apparatus according to [1], wherein the mechanism for increasing microbial diversity is a mechanism for expanding the specific surface area in the culture tank.
[3] The intestinal mimetic culture apparatus according to [2], wherein the mechanism for expanding the specific surface area in the culture tank is a foamable carrier.
[4] The intestinal imitation culture apparatus according to [2], wherein the mechanism for expanding the specific surface area in the culture tank is a non-woven cloth carrier.

[5] The intestinal mimetic culture device according to any one of [1] to [4], wherein the mechanism for increasing microbial diversity is a mechanism for retaining microorganisms in a culture tank.
[6] The intestinal mimetic culture apparatus according to [5], wherein the mechanism for holding microorganisms in the culture tank is a mechanism for releasing a viscous substance secreted from the intestine.

[7] The intestinal mimetic culture device according to [6], wherein the viscous substance secreted from the intestine is mucin.
[8] The intestinal tract according to any one of [1] to [7], wherein the mechanism for increasing microbial diversity is a combination of a mechanism for expanding the specific surface area in the culture tank and a mechanism for increasing microbial diversity. Imitation culture device.

  According to the present invention, an improved intestinal mimetic culture apparatus is provided. Since the intestinal mimetic culture apparatus according to the present invention incorporates a structure imitating the intestinal epidermis area and its structure, the experimenter can easily increase the diversity of microbial species staying in the apparatus. In addition, microorganisms can be stably maintained in the apparatus. Furthermore, the microbial diversity in the intestinal mimetic culture apparatus according to the present invention is similar to the microbial flora staying in the actual intestinal tract.

A schematic diagram of a conventional intestinal mimetic culture apparatus 3MCS is shown.

  The present invention relates to an improved intestinal mimetic culture apparatus. The intestinal mimetic culture apparatus means a continuous culture system provided with one or more culture tanks that reproduce the intestinal environment and imitate the intestinal tract function. The present invention is characterized in that at least one mechanism for increasing microbial diversity is incorporated in the intestinal mimetic culture apparatus.

  The intestinal mimetic culture apparatus used in the present invention can be any apparatus as long as it is a known intestinal mimic culture apparatus. Specifically, it is a device equipped with an environment (culture device) that mimics the intestinal tract function and grows intestinal bacteria. The culture tank volume, temperature, pH, bacteria, enzymes, nutrient conditions, residence time The pressure is controlled. For example, an intestinal imitation culture device (3MCS, Non-Patent Document 1) provided with three agitation-type culture vessels, and a 5-tank system culture device (SHIME, Non-Patent Literature) equipped with an agitation-type culture vessel and an agitation-type culture vessel 2) A culture apparatus (TIM, Non-Patent Document 3) connected with a flow-tube type culture tank can be used. A configuration example (3MCS) of a conventional culture apparatus that can be used is shown in FIG. In FIG. 1, Vessels 1 to 3 are culture vessels, and the medium moves in the direction of the arrow from the medium and each Vessel with a pump (P4) (solid blue line). The timer is connected to the pump P4 as indicated by a long dotted line, and controls the ON / OFF of the pump P4 to adjust the medium flow rate. The short dotted line shows the connection to the pH controller, the pH electrode attached to each culture vessel, and the pumps (P1 to P3) for feeding the pH adjuster to each culture vessel. The pH controller controls ON / OFF of the pumps P1 to P3 so that each culture tank has a set pH based on the measured value at each pH electrode. Examples of pH adjusters that can be used include, but are not limited to, NaOH and KOH.

  In addition, the bacteria (microorganisms) to be used are not particularly limited as long as they are conventional intestinal bacteria. , Coprococcus, Clostridium (eg, C. coccoides subgroup, C. lituseburense subgroup, C. leptum subgroup, C. botulinum subgroup), Faecalibacterium, Sarsina Genus (Sarcina), Staphylococcus, Enterococcus, Streptococcus, Atopobium, Eubacterium, Lactobacillus, Lactobacillus, Collinsella ), Eggerthella, Slacki a), Peptostreptococcus, Anaerococcus, Finegoldia, Parvimonas, Peptoniphilus, Fibrobacter, Acholeplasma (Acholeplasma) ), Anaeroplasma, Fusobacterium, Bifidobacterium, Ruminococcus, Lactococcus, Megasphaera (eg M. elsdenii), Bacteria belonging to the genus Mycoplasma are known as bacteria that inhabit the digestive tract. In the present invention, any bacteria can be used as long as it is the above-mentioned enteric bacteria. For example, enteric bacteria can be derived from feces. Moreover, the detection method, isolation method, systematic classification, etc. of these enteric bacteria are also well-known.

  In the present invention, at least one mechanism for increasing microbial diversity is incorporated into the culture tank in the intestinal mimicking culture apparatus. Such a mechanism for increasing microbial diversity means a mechanism that enables the growth of a wide variety of microorganisms in the intestinal mimetic culture apparatus, specifically, a mechanism that increases the specific surface area in the culture tank, and A mechanism for holding the microorganisms in the culture vessel is included.

  The “mechanism for expanding the specific surface area in the culture tank” is a mechanism that mimics the surface area of the intestinal tract, specifically a porous structure, but a structure that can increase the specific surface area. Any may be used. Such a mechanism (porous structure) can be, for example, a foamable carrier (sponge), a non-woven fabric carrier, a porous gel, a hollow fiber membrane, etc., and such a structure should be obtained as a commercial product. Is possible. Examples of the foamable carrier include micro breath (manufactured by Aion Co., Ltd.), biotube (manufactured by JFE Engineering Co., Ltd.), APG (manufactured by Nisshinbo Chemical Co., Ltd.), and porous cellulose carrier (manufactured by EYELA). Etc. Examples of the non-woven fabric carrier include porous nonwoven fabrics made of materials such as cotton, cellulose, nylon, polyester, polypropylene, and glass wool. Examples of the hollow fiber membrane include porous hollow fiber membranes made of materials such as polyethylene, polyacrylonitrile, polysulfone, and polyvinylidene fluoride.

  The “mechanism for enlarging the specific surface area in the culture tank” may exist in the culture tank of the intestinal mimetic culture apparatus, or may be fixed. For example, the intestinal mimetic culture apparatus of the present invention can be constructed by adding a porous structure in each Vessel of the conventional intestinal mimic culture apparatus shown in FIG.

  In addition, the “mechanism for holding microorganisms in the culture tank” is a mechanism for releasing or holding a substance that promotes the growth and maintenance of intestinal microorganisms and imitating the structure of the intestinal epidermis. It is a mechanism that releases or retains viscous substances secreted from the body. Such a viscous substance is not particularly limited, and can be at least one selected from the group consisting of mucin polysaccharides (mucins 1 to 7), intestinal cell epidermal proteins, and antibodies against microbial bacteria. . Mucin glycoproteins preferred as viscous substances are known in the art and can be obtained as commercial products.

  The “mechanism for holding microorganisms in the culture tank” may be present as it is in the culture tank of the intestinal mimetic culture apparatus, or may be fixed, or the “specific surface area in the culture tank is determined. It may be present in combination with the “enlarging mechanism”. For example, an intestinal secretory substance as a “mechanism for holding microorganisms in a culture tank” can be held on an effervescent carrier or a nonwoven fabric carrier as a “mechanism for expanding the specific surface area in the culture tank”. As an example, as described in Example 2, a suitable mechanism can be obtained by mixing a viscous substance in low-concentration agar and applying it to a porous structure.

  Since the intestinal mimetic culture apparatus according to the present invention incorporates a structure imitating the intestinal epidermis area and its structure, the experimenter can easily increase the diversity of microbial species staying in the apparatus, Moreover, microorganisms can be stably maintained in the apparatus. Therefore, it is useful for applications such as a method for determining the behavior of substances and drugs in the intestine and a method for determining the effects of substances and drugs on intestinal bacteria. Specifically, the test substance is cultured in the intestinal mimetic culture apparatus according to the present invention, and the growth or proliferation of intestinal bacteria in the intestinal mimic culture apparatus is determined, thereby affecting the growth or proliferation of intestinal bacteria. Substances can be screened. Moreover, it is possible to measure the release profile of the test drug by culturing the test drug in the intestinal mimetic culture apparatus according to the present invention and measuring the release of the test drug in the intestinal mimic culture apparatus. In addition, the use of the intestinal mimicking culture apparatus of the present invention is not particularly limited as long as the conventional apparatus is used.

  Hereinafter, the present embodiment will be described in more detail with reference to examples. The present invention is not limited to these.

[Reference Example 1]
The conventional intestinal mimetic culture device 3MCS shown in Fig. 1 that mimics the basic environmental conditions in the large intestine such as pH, temperature, and residence time, but does not have any structure that mimics the epidermis, using pig feces as a microbial source. The culture test used was performed. The culture tanks were the same for all three tanks, and they were made by remodeling a glass Duran bottle so that a pH electrode, a medium supply / discharge tube, and a pH adjusting agent charging tube could be attached to the side. The number of detected microbial species was 11, 14 and 14 types in Vessel 1, Vessel 2 and Vessel 3, respectively.

  In addition, bacterial flora detected from swine feces (Non-patent Document 4: Michael A. Cotta, Terence R. Whitehead and Rhonda L. Zeltwanger, "Isolation, characterization and comparison of bacteria from swine faecesand manure storage pits," Environmental Microbiology 5 (9): 737-745 (2003)) and the vessel Vessel 3 that imitates the sigmoid colon / rectum, as shown in Table 1, 3 out of 9 groups matched. Although the culture apparatus of Fig. 1 succeeded in culturing many anaerobic bacteria that are usually observed in the intestinal flora, only about 1/3 of the flora can be reproduced compared to the swine dung flora. As a result.

[Example 1]
In order to show the effect of the intestinal mimetic culture apparatus in which the structure was introduced, a culture apparatus in which a porous carrier was added to the conventional apparatus shown in FIG. 1 was constructed. The carrier used was micro-brace (high density microorganism fixed carrier, manufactured by Aion Co., Ltd.), and the amount according to the surface area of the large intestine 0.20 m ² (Vessel 1: 0.18g, Vessel 2: 0.26g, Vessel 3: 0.26g) ) Was added.

After autoclave (121 ° C, 20 min) with medium and stirrer in Vessel 1-3, the device is inoculated with pig feces in a clean bench and pre-sterilized with UV (20 UV in clean bench). Min irradiation) pH electrodes were placed in Vessels 1-3. After that, N ₂ gas was passed through the filter for 20 min to replace the inside with N ₂ gas, which was anaerobic.

  The residence time of the apparatus was set as shown in Table 2 according to the residence time at each part of the large intestine. Here, the retention time (Retention time) in each Vessel was set to the general fecal residence time of an adult.

  The swine dung was filtered with 10 g of physiological saline that had been sterilized by autoclaving (121 ° C., 20 min) to remove solid matter. 100 mL of each of the filtered pig feces was inoculated into Vessels 1-3. The final amount of pig feces in each Vessel was 3.3 g. After inoculation, in order to stabilize the microflora in swine feces, the vessel was pre-cultured for 24 hours while stirring in the Vessel, and then the culture apparatus was started to perform sampling every day.

  As a result of the flora analysis, the flora changed greatly during the first day from the start of operation of the device, and then the flora was stabilized after the eighth day of culture. Bacteroides, Clostridium, Lactobacillus, Eubacterium, Listeria, Streptococcus, Enterococcus, and other groups were detected as the bacterial flora after the 8th day of stable culture. The total number of microorganisms was Vessel 1 (V1), Vessel 2 (V2 ) And Vessel 3 (V3) were 20, 15 and 16 types, respectively, and compared to the results in the initial bioreactor shown in Reference Example 1, the diversity of the flora increased.

  In addition, when comparing the flora results with the flora detected in swine feces (Non-patent Document 4) and the flora in Vessel 3 that mimics the sigmoid colon / rectum, T-RF was detected. Except for the values, 6 out of 9 groups were detected, resulting in increased flora diversity over the initial bioreactor shown in Reference Example 1 (Table 3).

[Example 2]
Example 1 shows that specific surface area is an important factor in the construction of an intestinal mimetic culture apparatus, but in addition, goblet cells covering the luminal surface of epithelial cells in the human large intestine. ) A "mucin-retaining culture device" that reproduces a thick mucus layer mainly composed of the mucous substance (glycoprotein) secreted from the mucous substance was constructed.

  The mucin layer not only protects the epithelium from mechanical damage, but also contains factors that promote the repair of epithelial cells, and also plays an important role in the intestinal environment, such as preventing the outflow of microorganisms. It is considered.

  The structure to be introduced into this “mucin-retaining culture apparatus” was prepared by impregnating the carrier used in Example 1 with a highly viscous liquid in which agar and mucin were mixed. The amount of structure added is the same as in Example 1.

  Mucin is impregnated on a carrier. First, 0.8 g of agar is dissolved in 100 mL of milli Q water, autoclaved (121 ° C, 15 min), then UV sterilized (UV irradiation for 20 minutes in a clean bench) (pig) 2 g of stomach-derived mucin type III (manufactured by Sigma) was added and dissolved. The micro-breath carrier was put before the solution was solidified, and the carrier was degassed and impregnated with mucin gel for 15 minutes using a suction pump.

  As a result of the microbial flora analysis, the numbers of microorganisms detected were 10, 11 and 11 in Vessel 1 (V1), Vessel 2 (V2) and Vessel 3 (V3), respectively. Compared to the culture results of introducing the control agar gel carrier, the diversity of the flora increased (Table 4).

  Furthermore, when the bacterial flora in the carrier was compared with a culture device in which a control agar gel carrier was introduced, it was confirmed that more types of microorganisms were growing in the “mucin-retaining type culture device” (Table 5). .

  From the above results, in the “mucin-retaining culture apparatus” impregnated with mucin, the diversity of the flora increased compared to the case of agar alone. Thus, it has been found that introduction of mucin into the culture apparatus (immobilization) increases the diversity of microorganisms in the culture tank.

  According to the present invention, an improved intestinal mimetic culture apparatus is provided. Since the intestinal mimetic culture apparatus according to the present invention incorporates a structure imitating the intestinal epidermis area and its structure, the experimenter can easily increase the diversity of microbial species staying in the apparatus. In addition, microorganisms can be stably maintained in the apparatus. Furthermore, the microbial diversity in the intestinal mimetic culture apparatus according to the present invention is similar to the microbial flora staying in the actual intestinal tract.

Claims (4)

  An intestinal mimetic culture apparatus, wherein at least one mechanism for increasing microbial diversity is incorporated in the intestinal mimetic culture apparatus.   The intestinal mimetic culture apparatus according to claim 1, wherein the mechanism for increasing microbial diversity is a mechanism for expanding the specific surface area in the culture tank.   The intestinal mimetic culture apparatus according to claim 1 or 2, wherein the mechanism for increasing microbial diversity is a mechanism for holding microorganisms in a culture tank.   The intestinal mimetic culture apparatus according to any one of claims 1 to 3, wherein the mechanism for increasing microbial diversity is a combination of a mechanism for expanding a specific surface area in a culture tank and a mechanism for increasing microbial diversity. .

Images