KR20210036159A - Cell Culture Device and In-vitro Type Apparatus for Multi Testing Inhalation Toxicity of Particles Using The Same - Google Patents

Abstract

The present invention relates to a cell culture device and an in-vitro-type hazardous substance toxicity multi-evaluation device using the same. By exposing harmful substance particles to a first cell tissue in an in vitro method, it is possible to evaluate the direct toxic effect of harmful substance particles on the first cell tissue. By simulating reactions that actually occur in a living body in a method of circulating a cell culture solution of the first cell tissue exposed to the harmful substance particles in a culture well of a second cell tissue, it is possible to simultaneously evaluate an indirect toxic effect of harmful substance particles on the second cell tissue. Accordingly, multi-toxicity evaluation on multiple cells can be performed at the same time.

Description

Cell Culture Device and In-vitro Type Apparatus for Multi Testing Inhalation Toxicity of Particles Using The Same}

The present invention relates to a cell culture device and an in vitro method for evaluating multiple toxicity of harmful substances using the same. More specifically, it is possible to evaluate the direct toxic effect of the toxic substance particles on the first cell tissue by exposing the toxic substance particles to the first cell tissue in an in vitro method, and the cell culture solution of the first cell tissue exposed to the toxic substance particles By simulating the reactions that actually occur in vivo by circulating the cells in the culture well of the second cell tissue, it is possible to simultaneously evaluate the indirect toxic effect of the toxic substance particles on the second cell tissue, thus simultaneously evaluating multiple toxicity to multiple cells. It relates to a cell culture device that enables and an in vitro method of multi-evaluation of toxicity of harmful substances using the same.

If the 20th century was the era of microscopic division, the 21st century can be said to be the nano era. Nanotechnology can be broadly classified into nanomaterials and nanodevices, and environmental and biotechnology-based technologies, depending on the application field.

Such nanotechnology is to artificially manipulate microscopic substances in atomic or molecular units to create materials or devices with new properties and functions. This is today's information technology (IT) and other biotechnology (BT). It is being admired as a state-of-the-art technology for realizing this.

However, although nanotechnology provides many benefits and benefits that are recognized as a new technological revolution throughout the industry, on the other hand, it is also a well-known fact that it has a potential risk. It can be said that it is due to the nature of nanotechnology.

In other words, the smaller the particle, the wider the specific surface area ratio, and the small particle with such a wider specific surface area ratio increases toxicity when reacting with living tissue.As an example, some nanoparticles such as titanium dioxide, carbon powder, diesel particles, etc. It has already been discovered through academic experiments that the smaller the size, the stronger the toxicity, such as causing inflammation. In addition, ultrafine nanoparticles may not be filtered into the airways or mucous membranes and may be lodged deep in the alveoli or migrate to the brain.Moreover, according to recent studies, theories have been reported that when nanoparticles accumulate in the body, they cause disease or central nervous disorder .

Therefore, in recent years, with the development of nanotechnology, the stability evaluation of nanotechnology is also actively progressing. Typically, nanoparticle inhalation toxicity evaluation tests to evaluate the toxicity that occurs when nanoparticles are inhaled and accumulated in the human body are various experiments. It is being studied in animals. The human hazard data obtained through these nanoparticle inhalation toxicity evaluation tests are used as various basic data for nanoparticles throughout industries such as nanofibers, cosmetics, semiconductors, and drug delivery systems.

Recently, inhalation toxicity assessment of these nanoparticles as well as various harmful substances such as mist, dust, gas, vapor, fine particles, ultrafine particles, smog, etc. Toxicity evaluation is in progress, and most of these inhalation toxicity evaluation tests are conducted in the same way. Hereinafter, all of the various harmful substance particles including nanoparticles are collectively referred to as harmful substance particles.

In the inhalation toxicity evaluation test for such harmful substance particles, in general, harmful substance particles are generated in an aerosol state and supplied to an exposure chamber apparatus having an inhalation chamber of a certain size, and a laboratory animal is put into the inhalation chamber of the exposure chamber apparatus. After exposure to inhaled particles of harmful substances through this respiratory tract, various changes in experimental animals are measured.

As such, a test performed in the body of an experimental animal, which is a test subject, is called an in-vivo test, and a test performed by dissecting some tissue cells of the test subject or culturing the cells and adjusting conditions in a separate test tube is performed. It is called an in-vitro test, and recently, in OECD countries, legislation regulating experiments on living organisms has been strengthened, and in terms of protection of animals, in vitro testing is more likely than in vivo testing. Is being demanded.

Currently generally used in vitro toxicity evaluation device includes a cell culture device capable of culturing tissue cells of a test subject, and an exposure chamber device that supplies and exposes harmful substance particles to tissue cells cultured in the cell culture device. And, in a state in which the tissue cells of the test subject are cultured in a cell culture apparatus, the harmful substance particles are exposed for a certain period of time to examine the state of change of the tissue cells.

In this case, since the test is performed only on tissue cells exposed to the harmful material particles for a certain period of time, the toxic effect of the harmful material particles has to be judged only on the tissue cell, and the toxic effect on other tissues related to the tissue cell is not at all. There is a problem that you cannot judge.

In general, since various tissues of living organisms are organically connected and interact with each other, when harmful substance particles are introduced into the lungs through respiration, not only does it have a direct toxic effect on the lung tissues, but also other tissues connected to the lungs, such as the liver, Indirect toxic effects may occur through. That is, when harmful substance particles enter the lungs through breathing, the lungs are primarily affected, and when harmful substance particles spread throughout the body through blood, various tissues throughout the body are also affected, and also harmful substance particles. Pulmonary cells exposed to inflammatory reactions cause an inflammatory reaction, and various inflammatory substances secreted from the lung cells are transmitted to other tissues through blood and so on.

Therefore, in order to accurately evaluate the toxicity of harmful substance particles, it is necessary to comprehensively determine not only the toxic effect on specific tissue cells but also the toxic effect on other surrounding tissue cells. There is a problem in that it is difficult to comprehensively evaluate the toxic effects of harmful substances particles because it is not possible to evaluate the toxic effects on surrounding tissue cells other than the tissue cells exposed to the particles.

Domestic registered patent No. 10-1160307

The present invention was invented to solve the problems of the prior art, and an object of the present invention is to evaluate the direct toxic effect of the toxic substance particles on the first cell tissue by exposing the toxic substance particles to the first cell tissue in an in vitro manner. And, by circulating the cell culture solution of the first cell tissue exposed to the harmful material particles into the culture well of the second cell tissue, by simulating the reactions that actually occur in the body, the indirect effect of the harmful material particles on the second cell tissue. It is to provide a cell culture device capable of simultaneously evaluating the effect of phosphorus toxicity, enabling multiple toxicity evaluation on multiple cells at the same time, and an in vitro method of multiple toxicity evaluation device for harmful substances using the same.

Another object of the present invention is to expose the toxic substance particles only to the first cell tissue, and to keep the toxic substance particles sealed so as not to be exposed to the second cell tissue connected to the cell culture medium of the first cell tissue. A cell culture device that enables a more diverse and accurate toxicity evaluation by controlling the circulation method of the cell culture fluid of the first cell tissue according to the needs of the user and inbiography using the same is possible by removing the influencing variable. It is to provide a device for evaluating multiple toxicity of harmful substances in a tro-type.

The present invention is formed to evaluate the toxicity of harmful substances to a first cell tissue and to evaluate the effect of the first cell tissue on a second cell tissue associated with the first cell tissue. As a cell culture device for multi-evaluation, a first well plate having a first culture well formed therein to store a cell culture medium, and a first well plate formed so as to arrange a first cell tissue on a bottom surface to be inserted and seated in the first culture well And a first cell culture module including a first insert in which at least a portion of the region is formed as a semi-permeable membrane so that the cell culture solution permeates; A second cell culture module formed to cultivate a second cell tissue by storing the cell culture solution therein; And a flow connection line connecting the first cell culture module and the second cell culture module so that the cell culture solution stored in the first cell culture module flows to the second cell culture module. Provides a cell culture device for multi-evaluation of toxicity of harmful substances in a tro-type.

In this case, the second cell culture module includes a second well plate having a second culture well formed therein to store a cell culture solution, and a second cell tissue is disposed on the bottom surface of the second culture well to be cultured. And the flow connection line may be formed to connect the first culture well and the second culture well.

In addition, the second cell culture module includes a second well plate having a second culture well formed therein to store a cell culture solution, and a second cell tissue disposed on a bottom surface thereof. And a second insert in which at least a portion of the region is formed as a semi-permeable membrane so that the cell culture medium is inserted and placed therethrough, and the flow connection line may be formed to connect the first culture well and the second culture well.

In addition, an opening/closing valve may be mounted on the flow connection line to open and close the flow path of the flow connection line.

In addition, a fluid circulation supply pump for supplying and circulating the cell culture solution stored in the first culture well to the second culture well through the flow connection line may be provided.

On the other hand, the present invention, the cell culture device; And an exposure chamber device for supplying a test substance to the first insert of the first cell culture module to expose the first cell tissue to the test substance. .

In this case, the exposure chamber device includes: a case in which an exposure chamber accommodating the first cell culture module and a closed chamber accommodating the second cell culture module are respectively sealed and separated in an inner space; A particle inlet port coupled to one side of the case in communication with the exposure chamber to inject a test material into the exposure chamber; And a suction and discharge port coupled to the other side of the case so as to be in communication with the exposure chamber so as to suck in the exposure chamber space, and the sealed chamber may be sealed so that the test substance injected into the exposure chamber does not flow. have.

In addition, the flow connection line is equipped with an on/off valve capable of opening and closing the flow path of the flow connection line, or a fluid circulation supply pump for supplying and circulating the cell culture solution stored in the first culture module to the second culture module. I can.

According to the present invention, it is possible to evaluate the direct toxic effect of the toxic substance particles on the first cell tissue by exposing the toxic substance particles to the first cell tissue in an in vitro manner, and the cells of the first cell tissue exposed to the toxic substance particles By simulating the reactions that actually occur in vivo by circulating the culture medium into the culture well of the second cell tissue, it is possible to simultaneously evaluate the indirect toxic effect of the toxic substance particles on the second cell tissue. There is an effect that enables evaluation.

In addition, by exposing the toxic substance particles only to the first cell tissue and keeping the second cell tissue connected to the cell culture fluid of the first cell tissue sealed so that the toxic substance particles are not exposed, external influence variables on the second cell tissue are removed. Thus, more accurate evaluation is possible, and the circulation method for the cell culture medium of the first cell tissue is variously adjusted according to the needs of the user, thereby enabling more diverse and accurate toxicity evaluation.

1 is a diagram conceptually showing the configuration of a cell culture device for multi-evaluation of toxicity of harmful substances in an in vitro method according to an embodiment of the present invention;
FIG. 2 is a diagram conceptually showing the configuration of a first cell culture module among the cell culture devices shown in FIG. 1;
3 is a diagram conceptually showing the configuration of a second cell culture module of the cell culture apparatus shown in FIG. 1;
4 is a view conceptually showing another form of a cell culture apparatus for multi-evaluation of toxicity of harmful substances in an in vitro method according to an embodiment of the present invention;
5 is a diagram conceptually showing the configuration of a cell culture apparatus for multi-evaluation of toxicity of harmful substances in an in vitro method according to another embodiment of the present invention;
6 is a diagram conceptually showing the configuration of a multi-evaluation device for toxicity of harmful substances of an in vitro method according to an embodiment of the present invention;
7 is a diagram conceptually showing the configuration of an in vitro method for multi-evaluating toxicity of harmful substances according to another embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. First of all, in adding reference numerals to elements of each drawing, it should be noted that the same elements are assigned the same numerals as possible even if they are indicated on different drawings. In addition, in describing the present invention, when it is determined that a detailed description of a related known configuration or function may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

1 is a diagram conceptually showing the configuration of a cell culture apparatus for multi-evaluation of toxicity of harmful substances in an in vitro method according to an embodiment of the present invention, and FIG. 2 is a first cell culture among the cell culture apparatuses shown in FIG. A diagram conceptually showing a configuration of a module, FIG. 3 is a diagram conceptually illustrating a configuration of a second cell culture module of the cell culture apparatus shown in FIG. 1, and FIG. 4 is a diagram illustrating an embodiment of the present invention. It is a diagram conceptually showing another form of the cell culture device for multi-evaluation of toxicity of harmful substances according to the in vitro method.

The cell culture apparatus 10 according to an embodiment of the present invention is for evaluating the toxicity of harmful substances in an in vitro method, and evaluating the toxicity of harmful substances to the first cell tissue T1 directly exposed to the particles of the harmful substances, and It is not directly exposed to the material particles and is formed to evaluate the indirect toxic effect on the second cell tissue (T2) associated with the first cell tissue (T1), and the first cell culture module 100 and the second cell culture It is configured to include a module 200 and a flow connection line 300.

The first cell culture module 100 includes a first well plate 110 having a first culture well 111 formed therein to store a cell culture solution S1, and a first cell tissue T1 on the bottom surface thereof. It is formed to be disposed and is configured to include a first insert 120 inserted and seated in the first culture well 111. The first insert 120 is formed with a semi-permeable membrane so that the cell culture medium S1 stored in the first culture well 111 can permeate, and the first cell tissue T1 is the first insert 120. In the cell culture medium (S1) is supplied and cultured.

The second cell culture module 200 is formed to store the cell culture solution S2 therein to cultivate the second cell tissue T2. For example, like the first cell culture module 100, a second well plate 210 having a second culture well 211 formed therein to store a cell culture solution S2, and a second well plate 210 on the bottom surface It is configured to include a second insert 220 that is formed to be able to place the tissue T2 and is inserted and seated in the second culture well 211. In addition, at least a portion of the second insert 220 is formed as a semi-permeable film so that the cell culture solution S2 stored in the second culture well 211 can permeate.

The flow connection line 300 connects the first culture well 111 and the second culture well 211 so that the cell culture solution S1 stored in the first culture well 111 flows into the second culture well 211. Connect. That is, the first culture well 111 and the second culture well 211 are connected to each other through the flow connection line 300 so that the cell culture solution S1 and the second culture well 211 stored in the first culture well 111 are connected to each other. ) Stored in the cell culture solution (S2) may be mixed.

The first cell tissue T1 disposed in the first insert 120 of the first cell culture module 100 is cultured by receiving nutrients from the cell culture solution S1 stored in the first culture well 111, and the second The second cell tissue T2 disposed in the second insert 220 of the cell culture module 200 is cultured by receiving nutrients from the cell culture solution S2 stored in the second culture well 211.

At this time, the first cell culture module 100 is inserted into the exposure chamber 601 of a separate exposure chamber device 20 as shown by a dotted line in FIG. 1 so that the first cell tissue T1 is exposed to the test material. And, the second cell culture module 200 is put into a separate sealed chamber 602 to prevent the second cell tissue (T2) from being exposed to the test material, harmful substances to the first cell tissue (T1). The toxicity evaluation and the evaluation of an indirect effect on the second cell tissue T2 associated with the first cell tissue T1 may be performed simultaneously.

In more detail, when the first cell culture module 100 is put into the exposure chamber 601 and the first cell tissue T1 is exposed to the test material, the first cell tissue T1 is in a state due to the toxicity of the test material. Change will occur. In the first cell tissue T1, a new toxic substance may be generated under the influence of the test substance, and the toxic substance is formed in the first culture well 111 through the semipermeable membrane of the first insert 120 as shown in FIG. 2. ) And flows into the cell culture solution (S1) stored in it. In addition, inflammatory reactions may appear in the first cell tissue T1 due to the influence of the test substance, and such inflammatory reaction substances may be newly generated and introduced into the cell culture solution S1 stored in the first culture well 111. . Of course, the test substance itself absorbed into the first cell tissue T1 may be introduced into the cell culture solution S1. Accordingly, in the cell culture medium S1 of the first culture well 111, various substances such as a test substance or a new toxic substance generated from the first cell tissue T1 are present in a mixed state.

A general in vitro toxicity test apparatus has no choice but to evaluate its effect only on the first cell tissue T1 exposed to the test substance. The cell culture apparatus 10 according to an embodiment of the present invention includes a first cell tissue ( A second cell culture module 200 capable of culturing T1) is added, and the first culture well 111 of the first cell culture module 100 and the second culture well of the second cell culture module 200 ( By connecting the 211 to each other through the flow connection line 300, the cell culture solution S1 of the first culture well 111 may flow into the second culture well 211.

Accordingly, new toxic substances such as test substances and inflammatory reaction substances contained in the cell culture medium S1 of the first culture well 111 are introduced into the cell culture liquid S2 of the second culture well 211. When these toxic substances are present in the cell culture medium S2 of the second culture well 211, the toxic substances pass through the semi-permeable membrane of the second insert 220 to the second cell tissue T2 as shown in FIG. 3. It is transmitted, and accordingly, the state of the second cell tissue T2 is changed under the influence of such a toxic substance.

According to this structure, the effect of direct exposure to the test substance can be evaluated for the first cell tissue (T1), and the indirect effect of the first cell tissue (T1) affected by exposure to the second cell tissue (T2). Can be evaluated.

In actual animals, when toxic particles are inhaled and exposed to the lungs through breathing, the lungs are primarily affected, and toxic substances particles are distributed throughout the body through the blood, which can affect other tissues. In addition, lung cells exposed to harmful substance particles cause an inflammatory reaction, and various inflammatory reaction substances secreted from lung cells are transmitted to other tissues to affect them.

The cell culture apparatus 10 according to an embodiment of the present invention directly exposes the test substance to the first cell tissue T1, similar to the reaction state inside the body, thereby causing toxicity to the first cell tissue T1. The effect can be evaluated, and by mixing the cell culture solution (S1) of the first cell tissue (T1) with the cell culture solution (S2) of the second cell tissue (T2), toxic substances generated in the first cell tissue (T1) are removed. It can be delivered to the second cell tissue (T2) to evaluate the indirect toxic effect on the second cell tissue (T2).

The first culture well 111 of the first cell culture module 100 and the second culture well 211 of the second cell culture module 200 are connected through a separate flow connection line 300, such a flow connection Even if only the line 300 is formed, the cell culture solution S1 of the first culture well 111 and the cell culture well S2 of the second culture well 211 may be mixed, but both cell culture solutions S1 and S2 For more smooth mixing, a fluid circulation supply pump 500 may be provided as shown in (b) of FIG. 4. In addition, an opening/closing valve 400 for opening and closing the flow path of the flow connection line 300 may be provided to selectively mix the cell culture solutions S1 and S2 according to the user's needs.

At this time, the fluid circulation supply pump 500 may operate so that the cell culture solution S1 of the first culture well 111 flows to the second culture well 211, through which the second cell tissue T2 By preventing the newly generated material from flowing into the first culture well 111, it is possible to evaluate only the exposure effect of the test material on the first cell tissue T1.

In addition, even when the opening/closing valve 400 is mounted, a separate check for allowing only the flow of the cell culture fluid S1 from the first culture well 111 to the second culture well 211 and blocking the opposite flow A valve (not shown) may be additionally mounted.

5 is a diagram conceptually showing the configuration of a cell culture apparatus for multi-evaluation of toxicity of harmful substances in an in vitro method according to another embodiment of the present invention.

1 to 4, a second well plate 210 in which a second culture well 211 is formed for the second cell culture module 200, and a second insert 220 inserted and seated in the second culture well 211 ), but the second cell culture module 200 according to another embodiment of the present invention is a second culture well capable of storing the cell culture solution S2 therein, as shown in FIG. 5. It may include a second well plate 210 on which 211 is formed, and may be formed to be cultured by disposing the second cell tissue T2 on the bottom surface of the second culture well 211. In this case, the second insert 220 is not required for the second cell culture module 200, as shown in FIGS. 1 to 4, and the second cell tissue T2 is not disposed on a separate insert, but is simply a second insert. It may be disposed in the culture well 211 and cultured through the cell culture solution S2.

At this time, the flow connection line 300 is similarly formed to connect the first culture well 111 and the second culture well 211, and the flow connection line 300 includes the cell culture solution S1 of the first culture well 111 As described above, the opening/closing valve 400 or the fluid circulation supply pump 500 may be mounted so that) can flow smoothly into the second culture well 211. In this case, since the method in which the cell culture solution S1 of the first culture well 111 flows into the second culture well 211 is the same as described above, a detailed description thereof will be omitted.

In this way, only the second well plate 210 in which the second culture well 211 is formed without the second insert 220 is provided for the second cell culture module 200, and the cell culture solution is placed in the second culture well 211. By storing (S2) and culturing the second cell tissue (T2), and allowing the cell culture solution (S1) of the first culture well 111 to flow into the second culture well 211, the first exposed to harmful substance particles The influence of the cell tissue T1 on the second cell tissue T2 can be made more similar to the actual environment, so that more accurate test results can be obtained.

This is because, when harmful substances are exposed to the lungs and transferred to other tissues through blood or lymphatic vessels, they are exposed directly to the apical surface of the cells without passing through the basement membrane of other tissue cells.

That is, when harmful substance particles are exposed to the lungs, the exposure route of the harmful substance particles is exposure to the epithelial cells of the lung cells, the epithelial cells pass through the basement membrane, the vascular endothelial cells pass the basement membrane, and the vascular endothelial cells pass through the apical part , Blood vessel entry, and exposure to the apical part of other organ cells. At this time, it does not pass through the basement membrane of other organ cells.

The cell culture apparatus according to another embodiment of the present invention is configured as described above for the second cell culture module 200, so that the harmful substance particles in the same manner as the exposure route of the harmful substance particles actually generated in the living body. An exposure path is formed. That is, when the harmful substance particles are exposed to the first cell tissue, for example, lung cells, the harmful substance particles are exposed to the epithelial cells, pass through the basement membrane of the epithelial cells, pass through the basement membrane of the vascular endothelial cells, and the vascular endothelium. Passes through the apical part of the cell, moves to the second culture well 211 in which the second tissue cells are cultured through the flow connection line 300, and passes through the cell basement membrane in the second culture well 211 It is exposed to the apical part of the second cell tissue without doing so. Here, in the first cell culture module 100, the semipermeable membrane of the first insert 120 performs the function of the cell basement membrane, but the second insert 220 is not provided in the second cell culture module 200, so the cell basement membrane Function is not performed.

Therefore, the cell culture apparatus according to another embodiment of the present invention is directly in the second culture well 211 in a form in which a separate second insert 200 is not provided in the second cell culture module 200. Tissue cells (T2) are arranged and configured to be cultured, and through this, a path to exposure to harmful substance particles is formed in a form very similar to the exposure route of the particles to be actually generated in the body, so that more accurate test results can be obtained.

6 is a diagram conceptually showing the configuration of an in vitro method for multi-evaluating toxicity of harmful substances according to an embodiment of the present invention, and FIG. 7 is a diagram illustrating an in vitro method of harmful substances according to another embodiment of the present invention. It is a diagram conceptually showing the configuration of the multiple toxicity evaluation device.

In the apparatus for multi-evaluating toxicity of harmful substances of an in vitro method according to an embodiment of the present invention, the cell culture apparatus 10 and the first cell culture module 100 are accommodated in the exposure chamber 601 and the first insert It is configured to include an exposure chamber device 20 for supplying the test material to the inner space of 120 to expose the first cell tissue T1 to the test material.

The exposure chamber device 20 is separated in an inner space such that an exposure chamber 601 accommodating the first cell culture module 100 and a closed chamber 602 accommodating the second cell culture module 200 are sealed, respectively. Particle inlet port 700 coupled to one side of the case 600 in communication with the exposure chamber 601 so that the test material P can be injected into the formed case 600 and the exposure chamber 601, and exposure It is configured to include a suction and discharge port 800 coupled to the other side of the case 600 in communication with the exposure chamber 601 so as to suck the inner space of the chamber 601.

The case 600 includes a case body 610 in the form of a container whose upper surface is opened so that the first cell culture module 100 and the second cell culture module 200 are seated and accommodated, and an open upper surface of the case body 610 It is configured to include a case cover 620 coupled to. A separation partition wall 621 is formed on the lower surface of the center of the case cover 620 to separate the inner space of the case 600 into the exposure chamber 601 and the sealed chamber 602. The sealing chamber 602 is sealed by the separation partition wall 621 so that the tasting material P injected into the exposure chamber 601 does not flow.

The flow connection line 300 connecting the first cell culture module 100 and the second cell culture module 200 is disposed to pass through the separation partition wall 621, and the flow connection line 300 connects the separation partition wall 621 A sealing member 630 may be inserted and interposed in a portion penetrating the airtight chamber 602 to seal the sealed chamber 602.

Particle inlet port 700 is coupled to penetrate the case cover 620 up and down, and the lower end of the case cover 620 is coupled to the case body 610, the first cell tissue of the first insert 120 ( It is located in the inner space of the first insert 120 so as to be adjacent to T1), and is formed in the shape of a fallopian tube in which the inner flow path increases toward the bottom. The test substance P supplied toward the first cell tissue T1 of the first insert 120 through the particle inlet port 700 is discharged to the outside through the suction and discharge port 800.

On the other hand, on the lower surface of the case cover 620, the case cover 620 is in close contact along the upper circumference of the first insert 120 and the second insert 220 in a state coupled to the case body 610, and the first insert A sealing member 640 capable of sealing the inner space of the 120 and the second insert 220 may be mounted.

According to this structure, the first cell tissue T1 disposed in the first insert 120 of the first cell culture module 100 is a test substance P supplied through the particle inlet port 700 in the exposure chamber 601. ), the second cell tissue T2 disposed in the second insert 220 of the second cell culture module 200 is sealed in the closed chamber 602 without exposure to the test substance P. maintain. As described above, in a state in which the first cell tissue T1 and the second cell tissue T2 are separated and located in separate spaces, the cell culture solution S1 of the first culture well 111 is removed through the flow connection line 300. 2 It flows into the culture well 211. Accordingly, for the first cell tissue T1, direct toxicity assessment for the test material may be performed according to the exposure of the test material P, and for the second cell tissue T2, the first cell tissue T1 may be evaluated. Toxic substances generated according to the toxic effect are introduced into the cell culture solution S2 of the second culture well 211 from the cell culture solution S1 of the first culture well 111 and transferred to the second cell tissue T2, Accordingly, it is possible to perform an indirect toxicity evaluation on the second cell tissue (T2).

In the above, the second cell culture module 200 of the cell culture device is inserted into the second well plate 210 in which the second culture well 211 is formed and the second culture well 211 as shown in FIG. 6. Although described in the form in which the seated second insert 220 is provided, as described in FIG. 5, the cell culture apparatus according to an embodiment of the present invention includes a second cell culture module 200 as shown in FIG. 7. Includes a second well plate 210 in which a second culture well 211 capable of storing the cell culture solution S2 is formed, and a second cell tissue T2 is disposed on the bottom surface of the second culture well 211 It can be formed so that it can be cultured. In this case, as described above, the second insert 220 is not required in the second cell culture module 200, and the second cell tissue T2 is not disposed in a separate insert, but simply in the second culture well 211. It can be arranged and cultured through the cell culture solution (S2).

Since the configuration of the second cell culture module 200 has been described in detail in FIG. 5, detailed descriptions are omitted here.

However, with respect to the sealing member 640 mounted on the lower surface of the case cover 620 described above, one sealing member 640 is the first insert while the case cover 620 is coupled to the case body 610. Formed to seal the inner space of the first insert 120 by intimate contact along the upper circumference of 120, and the other sealing member 640 is a state in which the case cover 620 is coupled to the case body 610 In close contact along the upper circumference of the second culture well 211 and may be formed to seal the inner space of the second culture well 211.

The above description is merely illustrative of the technical idea of the present invention, and those of ordinary skill in the art to which the present invention pertains will be able to make various modifications and variations without departing from the essential characteristics of the present invention. Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but to explain the technical idea, and the scope of the technical idea of the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

10: cell culture device
20: exposure chamber device
100: first cell culture module
110: first well plate
111: first culture well
120: first insert
200: second cell culture module
210: second well plate
211: second culture well
220: second insert
300: flow connection line
400: on-off valve
500: fluid circulation supply pump
600: case
601: exposure chamber
602: sealed chamber
610: case body
620: case cover
630, 640: sealing member
700: particle inlet port
800: suction and discharge port

Claims (8)

Cells for multi-evaluation of toxicity of harmful substances of an in vitro method formed to evaluate the toxicity of harmful substances to the first cell tissue and to evaluate the effect of the first cell tissue on the second cell tissue associated with the first cell tissue As a culture device,
A first well plate in which a first culture well capable of storing a cell culture solution is formed, and a first well plate is formed so as to arrange a first cell tissue on the bottom surface to be inserted and seated in the first culture well, and at least a part of it so that the cell culture solution is permeable. A first cell culture module including a first insert whose region is formed of a semi-permeable film;
A second cell culture module formed to cultivate a second cell tissue by storing the cell culture solution therein; And
A flow connection line connecting the first cell culture module and the second cell culture module so that the cell culture solution stored in the first cell culture module flows to the second cell culture module
Cell culture apparatus for multi-evaluation of toxicity of harmful substances of an in vitro method, comprising: a.
The method of claim 1,
The second cell culture module
It includes a second well plate in which a second culture well capable of storing a cell culture solution is formed therein, and is formed to be cultured by arranging a second cell tissue on the bottom surface of the second culture well,
The flow connection line is a cell culture apparatus for multi-evaluation of toxicity of harmful substances of an in vitro method, characterized in that formed to connect the first culture well and the second culture well.
The method of claim 1,
The second cell culture module
A second well plate in which a second culture well capable of storing a cell culture solution is formed, and a second well plate is formed so as to arrange a second cell tissue on the bottom surface, and is inserted and seated in the second culture well, at least partially so that the cell culture solution is permeable. The region comprises a second insert formed of a semi-permeable film,
The flow connection line is a cell culture apparatus for multi-evaluation of toxicity of harmful substances of an in vitro method, characterized in that formed to connect the first culture well and the second culture well.
The method according to any one of claims 1 to 3,
The cell culture apparatus for multi-evaluation of toxicity of harmful substances of an in vitro method, characterized in that the flow connection line is equipped with an on/off valve to open and close the flow path of the flow connection line.
The method according to any one of claims 1 to 3,
A cell culture apparatus for multi-evaluation of toxicity of harmful substances of an in vitro method, characterized in that a fluid circulation supply pump for supplying and circulating the cell culture solution stored in the first culture module to the second culture module through the flow connection line is provided.
The cell culture device according to any one of claims 1 to 3; And
Exposure chamber apparatus for supplying a test material to the first insert of the first cell culture module to expose the first cell tissue to the test material
In vitro method of toxicity multi-evaluation device, characterized in that it comprises a.
The method of claim 6,
The exposure chamber device
A case in which an exposure chamber accommodating the first cell culture module and a closed chamber accommodating the second cell culture module are respectively sealed and separated in an inner space;
A particle inlet port coupled to one side of the case in communication with the exposure chamber to inject a test material into the exposure chamber; And
A suction and discharge port coupled to the other side of the case in communication with the exposure chamber so as to suck in the exposure chamber space
Including, wherein the closed chamber is a hazardous substance toxicity multi-evaluation apparatus of the in vitro method, characterized in that the sealed form so that the test material injected into the exposure chamber does not flow.
The method of claim 7,
The flow connection line is equipped with an on/off valve capable of opening and closing the flow path of the flow connection line, or a fluid circulation supply pump for supplying and circulating the cell culture solution stored in the first culture module to the second culture module. In vitro method of hazardous substance toxicity multi-evaluation device.

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