KR20210053601A - Apparatus and method for co-culturing anaerobic and aerobic cells using intestinal environment simulation - Google Patents

Abstract

The present invention comprises: a first block including a first porthole and a second porthole penetrating vertically, wherein the first porthole and the second porthole are sealed with a first airtight cover and a second airtight cover, respectively; a second block including first and second holes, which communicate with the first and second portholes, respectively, penetrating vertically and coupled to a lower part of the first block; a third block coupled to a lower part of the second block and including a connecting groove connecting the lower part of the first hole and the lower part of the second hole to each other; and a porous membrane inoculated with animal cells located in the first porthole or the first hole, wherein the upper part of the porous membrane is an anaerobic atmosphere, and the lower part is an aerobic atmosphere. Accordingly, a high-reliability analysis result can be obtained at a low price.

Description

Apparatus and method for co-culturing anaerobic and aerobic cells using intestinal environment simulation}

The present invention relates to an apparatus and method for co-culture of anaerobic-aerobic cells using simulation of intestinal environment, and more particularly, to cultivate host animal cells on the upper surface of a porous membrane, and differentiate between anaerobic bacteria and host immune cells based on this And cultivate, analyze, or simulate and implement the anaerobic or aerobic environment inside and outside the intestine of animals to analyze the growth and physiological activity of each of the intestinal anaerobic and aerobic microorganisms and the physiological interactions of the intestinal microorganisms, or physiological changes in pharmaceuticals It relates to a cell co-culture apparatus and method that can be used for prediction.

Traditionally, in order to analyze the physiological interaction of host-anaerobic intestinal microorganisms, an animal model using a mouse or the like was used.

However, there is a problem in that the use of the animal test model requires high cost and high-intensity labor, and a lot of time must be invested to obtain a desired result.

In addition, since the animal model is genetically different from that of humans, there is a problem that the reliability of the results is deteriorated.

Therefore, there is a need for a method of culturing human intestinal cells in vitro without using such an animal model to analyze physiological interactions of intestinal microorganisms.

As a related prior art, registration patent 10-1618018B (microbial complex culture device, registered on April 27, 2016) describes a device capable of culturing various anaerobic or aerobic microorganisms for livestock and livestock in large quantities. It is not a co-culture of anaerobic or aerobic microorganisms under a simulated environment, and there is no disclosed or implied technology for simultaneously culturing so that the physiological activity and growth characteristics of anaerobic and aerobic microorganisms can be analyzed under such an environment.

As another method, a method of studying the interaction between intestinal microorganisms and intestinal epithelial cells in an aerobic environment using a general transwell type incubator has been proposed, but this method cannot be used for anaerobic co-culture. Except for a small number of aerobic or aerobic anaerobic microorganisms in the intestine, not only cannot grow in the host cell environment, but also cannot simulate the gaseous environment in the intestine, so there is a problem that an analysis result different from the actual human phenotype can be derived.

The technical problem to be solved by the present invention is to provide a co-culture device capable of co-culture of anaerobic and aerobic cells, so that anaerobic bacteria and immune cells of a host can be classified and cultured and analyzed based on intestinal epithelial cells. In addition, it is intended to provide a system capable of predicting and analyzing changes in intestinal physiological activity such as various physiological activity assays and pharmaceuticals by efficiently and easily co-culture at low cost by simulating the intestinal environment of anaerobic intestinal microorganisms.

In addition, another technical problem to be solved by the present invention is to provide a method for co-culture of anaerobic and aerobic cells capable of obtaining accurate analysis results consistent with the actual human phenotype by simulating the gaseous environment in the intestine as it is.

The cell co-cultivation apparatus according to an aspect of the present invention for solving the above-described problems includes a first port hole and a second port hole respectively penetrating vertically, and the first port hole and the second port hole are each first gas tight. A first block sealed with a cover and a second airtight cover, a first hole and a second hole respectively communicating with the first port hole and the second port hole are vertically penetrated, and a second block coupled to a lower portion of the first block , A third block coupled to a lower portion of the second block and including a connection groove connecting a lower portion of the first hole and a lower portion of the second hole to each other, and an animal cell located inside the first port hole or the first hole It comprises a porous membrane inoculated, and the upper portion of the porous membrane is characterized in that the anaerobic atmosphere, the lower portion is an aerobic atmosphere.

In an embodiment of the present invention, the porous membrane may be positioned between the first port hole and the first hole.

In an embodiment of the present invention, the perimeter of the first airtight cover and the second airtight cover, the lower circumference of the first port hole and the second port hole of the first block, and the first hole and the second hole of the second block. It may further include an airtight holding unit positioned around an upper circumference and an upper circumference where the third block is coupled to the second block.

In an embodiment of the present invention, the first block, the second block, and the third block may be made of polycarbonate.

In an embodiment of the present invention, the porous membrane may be nitrocellulose, polycarbonate, or polyester.

In an embodiment of the present invention, the average pore size of the porous membrane may be 0.3 ~ 0.5㎛.

In an embodiment of the present invention, the animal cell may be an intestinal epithelial cell, but is not limited thereto.

In an embodiment of the present invention, anaerobic cells are inoculated in the upper space of the porous membrane, and aerobic cells are inoculated in the lower space, and the upper space and the lower space are further provided with anaerobic cell culture solutions and aerobic cell culture solutions, respectively. Can be.

To this end, as an embodiment, the cell co-culture device may further include a first peristaltic pump outside the anaerobic chamber communicating with the first port hole and a second peristaltic pump outside the anaerobic chamber communicating with the second port hole, The cell co-culture time using the cell co-culture device may be increased by adding an anaerobic cell culture solution through the first peristaltic pump or by adding an aerobic cell culture solution through the second peristaltic pump.

In addition, a cell co-culture method according to another aspect of the present invention includes a first block including a first port hole and a second port hole penetrating vertically, a first hole and a first hole communicating with the first port hole and the second port hole, respectively. A second block having two holes penetrating up and down, coupled to a lower portion of the first block, coupled to a lower portion of the second block, and including a connection groove connecting the lower portion of the first hole and the lower portion of the second hole to each other. A cell co-culture method using a cell co-culture device comprising a third block and a porous membrane positioned inside the first port hole or the first hole, a) the first block, the second block, the third block, And binding each of the porous membranes, b) inoculating and culturing animal cells on the porous membrane, c) injecting an aerobic cell culture solution into the lower space of the porous membrane through the second port hole, and anaerobic cell culture solution. Injecting into the upper space of the porous membrane through the first port hole, d) sealing the second port hole with a second gas tight cover, and when the space above the porous membrane becomes anaerobic in the anaerobic chamber, the first It may include the step of co-culturing the cells after sealing the porthole with the first hermetic cover.

In an embodiment of the present invention, it may further include a step of sterilizing the cell co-culture device, respectively, after steps a) and b).

In particular, after performing step b), the process of sterilizing the cell co-culture device may be washing the inside of the cell co-culture device with a sterilized phosphate buffer solution.

In an embodiment of the present invention, the volume ratio of the aerobic cell culture medium and the anaerobic cell culture medium may be 6 to 7: 1, but is not limited thereto.

In an embodiment of the present invention, in the culturing step b), _{the temperature of the CO 2} incubator is set to 35 to 38°C, and can be cultured for 7 days in a _{4 to 6% CO 2 environment, preferably the CO 2} incubator The temperature of 37 ℃, it can be cultured for 7 days in a _{5% CO 2 environment.}

In an embodiment of the present invention, in the co-culture step of d), the temperature of the anaerobic chamber is set to 35 to 38°C, and incubated for 10 to 13 hours in an environment of _{90% N 2} , 5% CO ₂ , 5% H ₂ It can be, and preferably, the temperature of the anaerobic chamber is 37 ℃, 90% N ₂ , 5% CO ₂ , It can be cultured for 12 hours in an environment of 5% H _2.

In an embodiment of the present invention, the cell co-culture device further comprises a first peristaltic pump outside the anaerobic chamber in communication with the first port hole and a second peristaltic pump outside the anaerobic chamber in communication with the second port hole, the The cell co-culture time using the cell co-culture device may be increased by adding an anaerobic cell culture solution through the first peristaltic pump or by adding an aerobic cell culture solution through the second peristaltic pump.

In an embodiment of the present invention, in step c), immune cells, macrophages, lactic acid bacteria, and pharmaceuticals may be additionally injected into the lower space of the porous membrane.

The present invention provides an apparatus capable of performing co-culture of anaerobic and aerobic cells, and by simulating the gaseous environment in the intestine as it is, there is an effect of increasing the reliability of analysis of physiological interactions of host-anaerobic intestinal microorganisms.

More specifically, the present invention co-cultures anaerobic microorganisms and host immune cells to contact intestinal epithelial cells while culturing aerobic intestinal epithelial cells. There is an effect of obtaining an analysis result consistent with the phenotype.

In addition, according to the present invention, the anaerobic and aerobic cell co-culture apparatus can perform co-culture of anaerobic and aerobic cells while simplifying the structure, thereby obtaining an analysis result with high reliability at a low price.

1 is a perspective view of a cell co-culture apparatus according to an embodiment of the present invention.
2 is an exploded perspective view of FIG. 1.
3 is a cross-sectional view taken along AA in a combined state of FIG. 1.
4 is a detailed flowchart of a cell co-culture method according to an embodiment of the present invention.
5 is a schematic diagram of intestinal epithelial cells and anaerobic bacteria cultured.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings with respect to the anaerobic-aerobic cell co-culture apparatus and method using the intestinal environment simulation.

The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art, and the embodiments described below may be modified in various other forms, and The scope is not limited to the following embodiments. Rather, these embodiments are provided to make the present invention more faithful and complete, and to completely convey the spirit of the present invention to those skilled in the art.

The terms used in this specification are used to describe specific embodiments, and are not intended to limit the present invention. As used herein, the singular form may include a plural form unless the context clearly indicates a different case. Also, as used herein, “comprise” and/or “comprising” specify the presence of the mentioned shapes, numbers, steps, actions, members, elements and/or groups thereof. And does not exclude the presence or addition of one or more other shapes, numbers, actions, members, elements and/or groups. As used herein, the term “and/or” includes any and all combinations of one or more of the corresponding listed items.

In the present specification, terms such as first and second are used to describe various members, regions, and/or parts, but it is obvious that these members, parts, regions, layers and/or parts are not limited by these terms. . These terms do not imply any particular order, top or bottom, or superiority, and are only used to distinguish one member, region, or region from another member, region, or region. Accordingly, the first member, region, or region to be described below may refer to the second member, region, or region without departing from the teachings of the present invention.

Hereinafter, embodiments of the present invention will be described with reference to the drawings schematically showing embodiments of the present invention. In the drawings, for example, depending on manufacturing techniques and/or tolerances, variations of the illustrated shape can be expected. Accordingly, the embodiments of the present invention should not be construed as being limited to the specific shape of the region shown in the present specification, but should include, for example, a change in shape caused by manufacturing.

1 is a partial exploded perspective view of an anaerobic-aerobic cell co-culture apparatus using a simulation of an intestinal environment according to a preferred embodiment of the present invention, FIG. 2 is an exploded perspective view of FIG. 1, and FIG. 3 is a cross-sectional view of a combined state of FIG. to be.

1 to 3, respectively, the cell co-culture apparatus 1 of the present invention includes three blocks stacked up and down. The first block 10 on the uppermost layer includes a first port hole 11 and a second port hole 12 that are spaced apart from each other and provided vertically.

The first port hole 11 and the second port hole 12 are formed to completely penetrate the first block 10 vertically.

A plurality of fastening holes 70 are provided along the circumference of the first block 10, and the fastening holes 70 correspond to the second block 20 and the third block 30 to be described later. The first block 10, the second block 20, and the third block 30 can be firmly coupled in a stacked state by using a fastening means provided at a position.

The fastening hole 70 may be formed with a tab, and the fastening means at this time may be a bolt.

In the above example, the bolt is illustrated as a fastening means, but any means that can firmly fix the first block 10, the second block 20, and the third block 30 in a downward stacked state in the order of enumeration are irrelevant. Can be used.

It is assumed that an insertion hole into which the first bottom sealing portion 13 and the second bottom sealing portion 14 are inserted is formed in the bottom of the first block 10.

The first bottom sealing part 13 may be an O-shaped ring installed around the bottom surface of the first port hole 11, and the second bottom sealing part 14 is an O-type ring installed around the bottom surface of the second port hole 12. It can be a type ring.

The second block 20 positioned below the first block 10 configured as described above includes a first hole 21 communicating with the first port hole 11 and a first hole 21 communicating with the second port hole 12. Two holes 22 are formed.

Both the first hole 21 and the second hole 22 are holes penetrating the second block 20 vertically.

A first insertion groove 25 and a second insertion groove 26 are formed along the circumference of the first hole 21 and the second hole 22 on the upper surface of the second block 20, and the first insertion groove The first upper surface sealing part 23 and the second upper surface sealing part 24 are inserted and fixed in the 25 and the second insertion groove 26, respectively.

In the combined state of the first block 10 and the second block 20, the first top sealing part 23 comes into contact with the first bottom sealing part 13, and the second top sealing part 24 is It comes into contact with the bottom sealing part 14.

The sealing structure of the present invention as described above is used for the purpose of preventing leakage of the culture medium and the purpose of preventing changes in properties of the culture medium, which will be described later.

That is, the present invention uses a culture solution having aerobic and anaerobic properties, and is configured to block the entrance of oxygen to prevent the properties of the culture solution from being changed.

A porous membrane 60 is installed between the first hole 21 and the first port hole 11. The porous membrane 60 may be made of nitrocellulose.

In particular, by using a porous material, it is possible to cultivate aerobic and anaerobic cells, which will be described in detail later.

Polycarbonate or polyester may be used as the material of the porous membrane 60.

As another requirement of the porous membrane 60, it may be mentioned that the adhesion of cells is strengthened and that an element that interferes with protein analysis does not occur. Considering various conditions, the most suitable material is nitrocellulose.

In addition, the suitability of the porous membrane 60 when applied to the present invention is determined according to the size of the pores. Animal cells such as intestinal epithelial cells as host cells are cultured and located above the porous membrane 60. The anaerobic culture medium is located on the upper side of the porous membrane 60.

At this time, the supply of oxygen may be appropriately adjusted to aerobic cells existing in animal cells cultured on the membrane by the pore size of the porous membrane 60. For this purpose, the average pore size of the porous membrane 60 is preferably 0.2 to 10 μm.

Specifically, when culturing intestinal anaerobic microbial cells in the upper space of host epithelial cells on the upper surface of the porous membrane 60 and co-culturing immune cells such as macrophages in the lower space, the average pore size of the porous membrane 60 The size may be 0.2 to 10 μm, but when co-culturing by inoculating anaerobic and aerobic microorganisms in the upper and lower spaces based on the host epithelial cells, the average pore size of the porous membrane 60 is adjusted to 0.2 to 0.5 μm. I can.

For stable mounting of the porous membrane 60, a seating surface 27, which is a stepped surface lower than the height of the upper surface of the second block 20, is located around the upper side of the first hole 21 of the second block 20. Can be formed.

Although not shown in the drawings, as another embodiment, the porous membrane is provided at a predetermined position inside the first hole 21 or inside the first port hole 11 in addition to between the first hole 21 and the first port hole 11. A predetermined inner circumferential groove may be installed in a predetermined position of and positioned therein.

Depending on the location of the porous membrane, the size of the anaerobic space and the aerobic space in the cell co-culture apparatus may be adjusted.

According to an embodiment of the present invention, the upper space of the porous membrane is an anaerobic space, and the lower space may be composed of an aerobic space, wherein the porous membrane is formed of the first hole 21 and the first port hole 11. The size of the space can be controlled depending on where it is provided.

The standard of the anaerobic atmosphere in the upper anaerobic space and the aerobic atmosphere in the lower aerobic space is the presence or absence of _{oxygen (O 2 ).} That is, there is no oxygen under an anaerobic atmosphere, and there is oxygen under an aerobic atmosphere.

A connection groove 31 is formed on the upper surface of the third block 30 coupled to the lower portion of the second block 20.

As can be seen in FIG. 3, the connection groove 31 connects the lower side of the first hole 21 and the second hole 22 of the second block 20 to each other, and the porous membrane 60 By dividing the first port hole 11 side and the second port hole 12 side of the first block 10 on the basis of, it is possible to supply anaerobic and aerobic culture solutions, respectively.

That is, the second port hole 12, the second hole 22, the connection groove 31, and the first hole 21 are in communication to expose the bottom surface of the porous membrane 60, and the first port hole 11 Through it, the upper surface of the porous membrane 60 may be exposed.

The second port hole 12, the second hole 22, the connection groove 31, and the first hole 21 are an aerobic culture solution, and the first port hole 11 is injected with an anaerobic culture solution.

That is, based on the position of the porous membrane 60, an anaerobic culture solution is injected into the upper space and an aerobic culture solution is injected into the lower space.

A coupling groove 32 to which the airtight holding portion 33 is coupled is formed around the connection groove 31 of the third block 30.

The first airtight cover 40 is attached to the first port hole 11 and the second airtight cover 50 is coupled to the second port hole 12 to provide the air quality (anaerobic or exhaled) of each internal space and contained in each. Make sure that the properties of the culture medium are maintained.

That is, when the entire cell co-culture device is placed in the anaerobic chamber while the anaerobic culture solution is injected into the first port hole 11, oxygen in the first port hole 11 diffuses and disappears, resulting in an anaerobic state. By combining the airtight cover 40, the anaerobic space inside is maintained.

In addition, if the second airtight cover 50 is connected to the second port hole 12 in the state where the aerobic culture solution is injected, the inner space remains in an aerobic state, and the cell co-culture device is then used in the anaerobic chamber. Even if placed in the second port hole 12, the inside of the second port hole 12 may be maintained in an exhaled state.

O-rings 41 and 51 are provided in each of the first airtight cover 40 and the second airtight cover 50 to maintain airtightness.

The first block 10, the second block 20, the third block 30, the first hermetic cover 40 and the second hermetic cover 50 may be made of polycarbonate. Polycarbonate can be autoclaved, has low cost and strong durability.

Hereinafter, a method of co-culturing anaerobic microorganisms and aerobic microorganisms in one device using the cell co-culture device according to a preferred embodiment of the present invention configured as described above will be described in detail.

First, the first block 10, the second block 20, and the third block 30 are stacked and bonded to each other, but the porous membrane 60 is seated on the seating surface 27 of the second block 20 Combine with

As a preferred embodiment of the present invention, the porous membrane 60 is located on the seating surface 27 of the second block 20, but in another embodiment, the inner space of the first block 10 and the second block 20 It can be configured to be located in any one of.

Figure 4 is a flow chart of a cell co-culture method as an embodiment of the present invention.

4, the step of sterilizing the cell co-culture device 1 at high temperature and high pressure (S41), the step of inoculating intestinal epithelial cells on the top of the porous membrane 60 (S42), and the cell co-culture device Injecting (1) into the incubator and culturing the intestinal epithelial cells (S43), washing the cell co-culture device (1) (S44), and adding an aerobic cell medium and an anaerobic bacteria culture medium (S45) ), and co-culturing anaerobic/aerobic cells having different characteristics in the cell co-culture device 1 in the anaerobic chamber (S46), and analyzing physiological interactions between intestinal cells and anaerobic bacteria (S47) ).

In particular, although not specifically shown in FIG. 4, the step of introducing an aerobic cell medium and an anaerobic bacteria culture medium (S45) may include firstly injecting an aerobic cell medium into the lower space of the porous membrane through the second port hole, and The step of sealing the second port hole with a second gas tight cover to prevent oxygen from escaping, the step of moving and mounting the cell co-culture device (1) to the anaerobic chamber, and in the anaerobic chamber, the When the space above the porous membrane becomes anaerobic, injecting an anaerobic cell culture solution into the upper space of the porous membrane through the first port hole, and sealing the first port hole with a first gas tight cover so that oxygen cannot escape. It includes the step of 氣密).

According to another embodiment of FIG. 4, after washing the cell co-incubator (S44), an aerobic cell medium may be injected through each of the first port hole and the second port hole in a clean bench in an aerobic environment. In this case, after the second porthole is sealed, the aerobic cell medium injected on the upper portion of the porous membrane in the anaerobic chamber is removed, and the anaerobic cell culture solution is injected into the upper space of the porous membrane under anaerobic conditions. At this time, the upper space of the porous membrane may be placed at least one day or longer to become anaerobic, but is not particularly limited thereto.

The cell co-culture method of the present invention will be described in more detail below.

First, in step S41, the cell co-culture apparatus 1 of the present invention in which the first block 10, the second block 20, the third block 30, and the porous membrane 60 are combined is sterilized at high temperature and high pressure.

As an example of high-pressure, high-temperature sterilization, it is treated at 121℃ and 15lb psi pressure for about 15 minutes.

Then, intestinal epithelial cells are inoculated on the top of the porous membrane 60 of the cell co-culture device 1, which has been sterilized in step S42. At this time, about 2 million intestinal epithelial cells are adequate.

Then, in step S43, the cell co-culture apparatus 1 including the porous membrane 60 inoculated with the intestinal epithelial cells is introduced into a _{CO 2 incubator to culture the intestinal epithelial cells.}

At this time, _{the temperature of the CO 2} incubator is 37°C and 5% CO ₂ for 7 days, but is not limited thereto.

Of course, culture conditions such as temperature, internal gas, and period of the incubator may vary according to the characteristics of the animal cells inoculated on the porous membrane 60.

Then, as in step S44, the cell co-culture device 1 in which the intestinal epithelial cells are cultured _{is taken out from the CO 2} incubator, and the inside is washed with a sterilized phosphate buffer saline. At this time, it is preferable to wash twice.

Then, as in step S45, the aerobic cell medium and the anaerobic culture medium are added.

The aerobic cell medium is injected into the second port hole 12 and moves to the second hole 22, the connection groove 31, and the first hole 21, which are mutually communicated with the second port hole 12. Then, the aerobic cell culture solution is filled in the lower space based on the porous membrane 60.

According to an embodiment, the aerobic cell medium may be injected into the first port hole 11 of the first block 10 and filled in the upper space of the porous membrane 60.

The second port hole 12 is sealed so that oxygen cannot escape, and then it is moved to the anaerobic chamber to mount the space above the porous membrane of the cell co-culture device under anaerobic conditions. Then, when the anaerobic state is reached, the anaerobic bacteria culture solution is injected into the first port hole 11 of the first block 10 and filled in the upper space of the porous membrane 60. In the embodiment in which the aerobic cell medium is also filled in the upper space of the porous membrane 60, the anaerobic culture medium injected at this time replaces the aerobic cell medium.

That is, an anaerobic culture solution is added to the upper side of the porous membrane 60 in which the intestinal epithelial cells, which are the first port holes 11, are cultured, and an aerobic cell medium is introduced through the second port hole 12 to form the porous membrane 60. The bottom of the aerobic cell medium is in contact.

After injecting the aerobic cell medium through the second port hole 12, the second port hole 12 is covered with a second hermetic cover 50. This is to maintain the internal gas of the second port hole 12, the second hole 22, the connection groove 31, and the first hole 21 communicated in an exhaled atmosphere with oxygen.

On the other hand, when a predetermined time has elapsed by placing the cell co-culture device 1 in the anaerobic chamber, oxygen diffuses into the anaerobic chamber from the inner space of the first port hole 11, that is, the upper space of the porous membrane 60, and is anaerobic without oxygen. It becomes a condition. At this time, the first port hole 11 is covered with the first gas tight cover 40 to maintain the inner space of the first port hole 11 in an anaerobic atmosphere.

In one embodiment of the present invention, the first port hole 11 of the first block 10 is an anaerobic atmosphere, and the second port hole 12, the second hole 22, the connection groove 31, and the first One hole 21 is maintained in an aerobic atmosphere, and aerobic and anaerobic cells can be co-cultured. However, it is not necessarily limited to this embodiment, and various embodiments of the space arrangement of an anaerobic atmosphere and an aerobic atmosphere are possible according to the position of the porous membrane 60.

At this time, 5 ml of the anaerobic culture medium and 33 ml of the aerobic cell medium may be injected, but the present invention is not limited thereto. Preferably, the volume ratio of the anaerobic bacteria culture medium and the aerobic bacteria culture medium may be injected in the range of 1:6 to 1:7.

In such a state, the cell co-culture apparatus 1 of the present invention is cultured in an anaerobic chamber as in step S46.

Conditions of the anaerobic chamber _{are preferably 90% N 2} , 5% CO ₂ , and 5% H ₂ environments at 37° C., but are not necessarily limited thereto.

It may be cultured for 12 hours under the conditions of the anaerobic chamber, but the culture conditions may be set differently depending on the type of microorganism to be cultured and the property characteristics thereof.

The time to be cultured in the anaerobic chamber is the time during which the anaerobic and aerobic spaces are maintained in the cell co-culture device (1).

If oxygen is insufficient in the lower space of the porous membrane 60, an environment in which aerobic bacteria can no longer grow, and various methods of oxygen injection for maintaining an aerobic atmosphere can be used depending on the characteristics of the aerobic bacteria.

Although not shown in the drawings, according to an embodiment, the culture time may be increased or adjusted using at least one peristaltic pump that communicates with the outside of the anaerobic chamber during culture.

Preferably, the anaerobic cell culture solution can be added through the first peristaltic pump outside the anaerobic chamber communicating with the first port hole 11 of the cell co-culture device 1, and the second port hole of the cell co-culture device 1 (12) The aerobic cell culture solution may be added through a second peristaltic pump outside the anaerobic chamber which is communicated with.

In this case, the anaerobic cell culture solution is continuously filled in the upper space of the porous membrane 60 through the first peristaltic pump to supply nutrients for the activity of anaerobic bacteria.

In addition, the aerobic cell culture medium is continuously filled in the lower space of the porous membrane 60 through the second peristaltic pump to exchange the aerobic cell medium, thereby increasing the culture time of the aerobic cells.

A schematic diagram of a state in which anaerobic bacteria of intestinal epithelial cells are cultured is shown in FIG. 5.

Referring to FIG. 5, intestinal epithelial cells 2 are cultured on the upper surface of the porous membrane 60, and anaerobic bacteria culture solution capable of culturing anaerobic bacteria in intestinal epithelial cells ( 3) is filled.

In addition, the aerobic cell culture solution 4 is filled in the lower space of the porous membrane 60.

Although not shown in Figure 5, during cultivation by sealing each porthole of the cell co-culture apparatus of the present invention with an airtight cover, some oxygen through the porous membrane 60 to the bottom contacting bottom of the intestinal epithelial cells 2 Under the supplied conditions, aerobic bacteria of intestinal epithelial cells can be cultured, and anaerobic bacteria are cultured because oxygen is not supplied to the anaerobic culture medium (3) in the upper space of the intestinal epithelial cells (2).

The porous membrane 60 not only serves as a support for cell growth, but also can separate the anaerobic-aerobic culture medium.

Then, the analysis is performed as in step S47. The method of analysis is to analyze the culture medium itself, or wash the cell co-culture device (1) with a phosphate buffer solution, and then extract protein bodies or metabolites of intestinal epithelial cells and microbial cells to analyze the physiological interaction of the host and anaerobic microorganisms I can.

That is, each of the anaerobic bacteria and aerobic bacteria cultured in step S46 grows in the upper and lower spaces based on the intestinal epithelial cells (2) to generate metabolic products such as various proteins. In particular, aerobic bacteria and metabolites of host immune cells can escape to the lower space through the porous membrane 6, and thus samples can be collected and analyzed through the second port hole 12.

Metabolites of anaerobic bacteria can be analyzed by collecting a sample through the first port hole 11.

In another embodiment of the present invention, host immune cells such as macrophages may be additionally injected into the aerobic cell medium 4 to be cultured and analyzed. Since the environment is simulated as it is, the interaction between anaerobic intestinal microbes-intestinal epithelial cells-immune cells can also be analyzed, centering on intestinal epithelial cells. It is preferable to use human-derived intestinal epithelial cells.

In various embodiments of the present invention, immune cells and macrophages are injected into the lower space of the intestinal epithelial cells 2, or lactic acid bacteria or pharmaceuticals are additionally injected into the upper space of the intestinal epithelial cells 2 to analyze physiological activity You can do it.

Specifically, the present invention simulates the human body, and the interaction can be observed by treating probiotics such as Bifidobacteirum species or Lactobacillus species.

In addition, it can be used for predicting research results in animal experimental models using a co-culture device.

In addition, an experiment to observe physiological changes caused by pathogens by co-culturing Clostridium difficile with intestinal epithelial cells and macrophages. We can observe the immune system processes of intestinal epithelial cells and macrophages against cytotoxin produced by Clostridium difficile.

In addition, by culturing intestinal microbes in an anaerobic microbial culture space and treating prebiotics such as fructooligosaccharide or inulin, it is possible to confirm changes in the microbial community in the intestine. The efficacy of prebiotics can be studied through analysis of changes in the activity of epithelial cells.

It is obvious to those of ordinary skill in the art that the present invention is not limited to the above embodiments and can be variously modified and modified within the scope not departing from the technical gist of the present invention. will be.

Claims (18)

A first block including a first port hole and a second port hole respectively penetrating vertically, wherein the first port hole and the second port hole are sealed with a first gas tight cover and a second gas tight cover, respectively;
A second block through which a first hole and a second hole respectively communicating with the first port hole and the second port hole vertically penetrate and are coupled to a lower portion of the first block;
A third block coupled to a lower portion of the second block and including a connection groove connecting a lower portion of the first hole and a lower portion of the second hole to each other; And
Including a porous membrane inoculated with animal cells positioned inside the first port hole or the first hole,
Cell co-culture apparatus, characterized in that the upper portion of the porous membrane is an anaerobic atmosphere, and the lower portion is an aerobic atmosphere.
The method of claim 1,
The porous membrane is a cell co-culture apparatus, characterized in that located between the first port hole and the first hole.
The method of claim 1,
A circumference where the first gas tight cover and the second gas tight cover are sealed,
A lower circumference of the first port hole and the second port hole of the first block,
The upper circumference of the first hole and the second hole of the second block, and
The cell co-cultivation apparatus further comprises an airtight holding portion positioned around the upper circumference of the third block coupled to the second block.
The method of claim 1,
The first block, the second block and the third block,
Cell co-culture device, characterized in that the polycarbonate material.
The method of claim 1,
The porous membrane,
Cell co-culture device, characterized in that the nitrocellulose (Nitrocellulose), polycarbonate or polyester.
The method of claim 1,
Cell co-culture apparatus, characterized in that the average pore size of the porous membrane is 0.2 ~ 10㎛.
The method of claim 1,
The cell co-culture device, characterized in that the animal cells are intestinal epithelial cells.
The method of claim 1,
Cell co-culture apparatus, characterized in that the anaerobic microorganisms are cultured in the upper space of the porous membrane, and immune cells are cultured in the lower space.
The method of claim 8,
Cell co-culture apparatus, characterized in that the anaerobic microorganism culture medium and the immune cell culture medium are further provided in each of the upper space and the lower space.
The method of claim 1,
Anaerobic cells are inoculated in the upper space of the porous membrane, and aerobic cells are inoculated in the lower space,
The cell co-culture apparatus, wherein the upper space and the lower space are each further provided with an anaerobic cell culture solution and an aerobic cell culture solution.
A first block including a first port hole and a second port hole vertically penetrating;
A second block through which a first hole and a second hole respectively communicating with the first port hole and the second port hole vertically penetrate and are coupled to a lower portion of the first block;
A third block coupled to a lower portion of the second block and including a connection groove connecting a lower portion of the first hole and a lower portion of the second hole to each other; And
A cell co-culture method using a cell co-culture device including the first port hole or a porous membrane positioned inside the first hole,
a) combining the first block, the second block, the third block, and the porous membrane, respectively;
b) inoculating and culturing animal cells on the porous membrane;
c) injecting an aerobic cell culture solution into the lower space of the porous membrane through the second port hole, sealing the second port hole with a second hermetic cover, and placing it in the anaerobic chamber;
d) When the space above the porous membrane becomes anaerobic in the anaerobic chamber, an anaerobic cell culture solution is injected into the upper space of the porous membrane through the first port hole, and the first port hole is sealed with a first gas tight cover. Cell co-culture method comprising the step of co-culture the cells.
The method of claim 11,
Cell co-culture method further comprising the step of sterilizing the cell co-culture device after the step a).
The method of claim 11,
The cell co-culture method further comprising the step of washing the inside of the cell co-culture device with a sterilized phosphate buffer solution after step b).
The method of claim 11,
Cell co-culture method, characterized in that the volume ratio of the aerobic cell culture medium and the anaerobic cell culture medium is 6 to 7: 1.
The method of claim 11,
The culturing step of b),
A cell co-culture method, characterized in that the temperature of the CO ₂ incubator is set to 35 to 38° C. and _{cultured for 7 days in a 4 to 6% CO 2 environment.}
The method of claim 11,
The co-culture step of d),
Cell co-culture method, characterized in that the temperature of the anaerobic chamber is 35 to 38 ℃, 90% N ₂ , 5% CO ₂ , and cultured for 10 to 13 hours in an environment of 5% H _2.
The method of claim 11,
The cell co-culture device further includes a first peristaltic pump outside the anaerobic chamber in communication with the first port hole and a second peristaltic pump outside the anaerobic chamber in communication with the second port hole,
Cell co-culture time using the cell co-culture device,
Cell co-culture method, characterized in that it can be increased by adding an anaerobic cell culture solution through the first peristaltic pump or by adding an aerobic cell culture solution through the second peristaltic pump.
The method of claim 11,
A cell ball, characterized in that in step c), immune cells or macrophages are additionally injected into the lower space of the porous membrane, or lactic acid bacteria or pharmaceuticals are additionally injected into the upper space of the porous membrane in step d). Culture method.

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