WO2017126758A1

WO2017126758A1 - Three-dimensional hepatocyte culture unit, hepatotoxicity assessment system and hepatotoxicity assessment method using same

Info

Publication number: WO2017126758A1
Application number: PCT/KR2016/008549
Authority: WO
Inventors: 윤석주; 오정화; 안재환; 김우근
Original assignee: 한국화학연구원
Priority date: 2016-01-21
Filing date: 2016-08-03
Publication date: 2017-07-27
Also published as: KR20170087756A; KR101864410B1

Abstract

A three-dimensional hepatocyte culture unit is disclosed. The three-dimensional hepatocyte culture unit according to an embodiment of the present invention comprises a first culture structure in which liver-forming cells cultured, and a second culture structure into which the first culture structure can be inserted and which can accommodate therein a fluid comprising at least one of a culture medium for culturing the liver-forming cells and a hepatotoxic material for assessing hepatotoxicity.

Description

3D hepatocyte culture unit, hepatotoxicity evaluation system and hepatotoxicity evaluation method using the same

The present invention relates to a three-dimensional hepatocyte culture unit, hepatotoxicity evaluation system and hepatotoxicity evaluation method using the same.

The liver is a major living organ of an animal, and the main function of the liver is a detoxification function that detoxifies foreign substances. In general, the liver is composed of unit structures called hepatic lobules, and these hepatic lobules may have a substantially hexagonal structure.

On the other hand, since the activity of hepatocytes in the hepatic lobe is greatly influenced by the structure of the hepatic lobe, studies are being conducted to simulate the liver tissue in three dimensions and to implement the function of the liver tissue in an atmosphere similar to the environment of the liver tissue. It is becoming.

For example, hepatocytes extracted from the human body can be cultured for a long time using a microstructure that simulates the boundary between epithelial tissue and blood vessels of the liver and expresses a function of liver tissue such as albumin production to form a structure similar to the bile duct in the human body. Research has been reported to develop liver chips. (See An Artificial Liver Sinusoid With a Microfluidic Endothelial-Like Barrier for Primary Hepatocyte Culture, Lee et al. Biotechnology and Bioengineering, 97, 5, 1312, 2007).

In addition, 2D simulations of hepatocytes (HepG2) and endothelial cells in the shape of hepatic lobular hexagonal structures using photolithography techniques have also been reported. (See Liver-cell patterning Lab Chip: mimicking the morphology of liver lobule tissue, Ho et al, Lab Chip, 2013, 13, 3578)

In addition, studies have been conducted to form hepatic lobular mimetic structures using hydrogels. (See Construction of hepatic lobule-like 3d tissues utilizing cell embedding hydrogel microfibers, Yuya Yajima et al. 18th International Conference on Miniaturized Systems for Chemistry and Life sciences.)

However, the above-mentioned studies are focused only on the local parts of liver tissue and liver function, and can simulate liver tissue in three dimensions, and implement the function of liver tissue in an atmosphere similar to the environment of liver tissue. Is currently in its infancy.

Embodiments of the present invention co-cultivate various cells constituting the liver tissue by simulating the structure and environment of hepatic lobule, which is a unit structure of the liver, and using the 3D hepatocyte culture unit, hepatotoxicity evaluation system, and hepatotoxicity using the same for evaluating hepatotoxicity An evaluation method is provided.

According to an aspect of the present invention, a first culture structure in which liver constituent cells are cultured and provided so that the first culture structure can be inserted therein, a culture medium for culturing the liver constituent cells, and a hepatotoxic substance for hepatotoxicity evaluation The fluid including at least one of the may include a second culture structure that can be accommodated therein.

The second culture structure may include at least one first injection means into which the fluid is injected, at least one first groove portion into which one side of the first culture structure is inserted, and the other side of the first culture structure. It may include at least one second groove portion, and may include a first drain means for draining at least a portion of the fluid injected from the first injection means.

In addition, the first drainage means may be provided to discharge the fluid contained in the second culture structure by lowering from the top to the bottom of the second culture structure.

The apparatus may further include a receiving case accommodating the second culture structure while closing one end of the first injection means and one end of the first drain means.

In addition, the first culture structure may include a porous matrix that provides a culture space in which the liver constituent cells are cultured.

In addition, the liver constituent cells may include at least one of hepatocytes, kupffer cells and stellate cells. According to another aspect of the present invention, the first culture structure and the at least one first culture structure in which the liver constituent cells are cultured is inserted, the culture medium for culturing the liver constituent cells and hepatotoxic substance for hepatotoxicity evaluation A three-dimensional hepatocyte culture unit having a second culture structure capable of receiving a fluid including at least one therein, a plate on which at least one of the three-dimensional hepatocyte culture units are installed, and located on one side of the plate, A hepatotoxicity evaluation system can be provided that includes a fluid supply member for supplying a fluid to a three-dimensional hepatocyte culture unit.

In addition, the plate is formed on the flow path, the flow path, the fluid is moved, at least one or more second injection means and the second injection means for injecting the fluid from the flow path to the three-dimensional hepatocyte culture unit And second drainage means for draining at least some of the fluid injected therefrom.

In addition, one end of the flow path may be connected to the fluid supply member, and the branch point of the flow path may be a point extending from one end of the flow path connected to the fluid supply member.

The apparatus may further include a fluid discharge member positioned on the other side of the plate to discharge the fluid to the outside.

According to another aspect of the invention, the step of providing a three-dimensional hepatocyte culture unit having a second culture structure is inserted into the first culture structure in which the liver constituent cells are cultured, accommodating the three-dimensional hepatocyte culture unit in a housing case Supplying a fluid including at least one of a culture medium for culturing the liver constituent cells and a hepatotoxic substance for hepatotoxicity evaluation, to the three-dimensional hepatocyte culture unit, and the fluid from the three-dimensional hepatocyte culture unit. Discharging the first culture construct from the second culture construct to measure the degree of necrosis of the liver constituent cells and analyzing cytokines generated in the fluid discharged to the outside. Hepatotoxicity assessment methods may be provided.

According to another aspect of the present invention, there is provided a three-dimensional hepatocyte culture unit having a second culture structure in which a first culture structure in which liver constituent cells are cultured is inserted, wherein the three-dimensional hepatocyte culture unit is at least placed on a plate. Installing at least one, fluid containing at least one of the culture medium for culturing the liver constituent cells in the three-dimensional hepatocyte culture unit using a fluid supply member located on one side of the plate and hepatotoxic substances for hepatotoxicity evaluation Supplying, discharging the fluid from the three-dimensional hepatocyte culture unit to the outside, separating the first culture structure from the second culture structure, measuring the degree of necrosis of the liver constituent cells and discharging to the outside Hepatic venom comprising analyzing cytokines produced in the fluid There are evaluation methods can be provided.

In addition, the providing of the three-dimensional hepatocyte culture unit may include preparing the first culture structure, culturing the liver constituent cells in the first culture structure, and applying the first culture structure to the second culture structure. It may include inserting into the installation.

In addition, the step of preparing the first culture structure, three-dimensional printing (salt leaching), salt leaching process, phase separation (phase separation) process, gas foaming process and electrospinning ) May be performed through at least one process.

In addition, the step of culturing the liver constituent cells in the first culture structure, the step of injecting the liver constituent cells into the first culture structure by stirring with a solution in which the biopolymer dissolved in a liquid medium and the first culture structure Incubating for 24 hours or more may include the step of stabilizing.

The method may further include culturing the liver constituent cells cultured in the first culture structure after inserting the first culture structure into the second culture structure.

In addition, the fluid supplied by the fluid supply member is automatically discharged to the outside according to the flow of the fluid flowing from one side of the plate to the other side of the plate, or the outside by the fluid discharge member located on the other side of the plate Can be discharged.

Embodiments according to the present invention co-culture the liver constituent cells in a three-dimensional hepatocyte culture unit similar to the structure and environment of hepatic lobule, a liver unit structure, and hepatotoxicity evaluation in a hepatotoxicity evaluation system employing at least one three-dimensional hepatocyte culture unit Since the continually proceeds, hepatotoxicity evaluation reflecting the mutual linkage between the liver constituent cells is possible.

1 is a perspective view showing a three-dimensional hepatocyte culture unit according to an embodiment of the present invention.

FIG. 2 is a perspective view showing the first culture structure of FIG. 1. FIG.

3 is a perspective view illustrating the second culture structure of FIG. 1.

4 is a perspective view showing a combined state of the three-dimensional hepatocyte culture unit of Figure 1 accommodated in the housing case.

5 is a perspective view showing a hepatotoxicity evaluation system in which the three-dimensional hepatocyte culture unit of FIG. 1 is installed.

6 is a perspective view showing the flow of the fluid in the hepatotoxicity evaluation system of FIG.

Hereinafter, with reference to the accompanying drawings will be described in detail the configuration and operation according to the embodiment of the present invention. The following description is one of several aspects of the invention that can be claimed, and the following description may form part of the detailed description of the invention.

However, in describing the present invention, a detailed description of known configurations or functions may be omitted to clarify the present invention.

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all changes, equivalents, and substitutes included in the spirit and scope of the present invention.

Terms including ordinal numbers such as first and second may be used to describe various components, but the components are not limited by the terms. These terms are only used to distinguish one component from another.

When a component is said to be 'connected' or 'connected' to another component, it may be directly connected to or connected to that other component, but it may be understood that another component may exist in between Should be.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise.

1 is a perspective view showing a three-dimensional hepatocyte culture unit according to an embodiment of the present invention, Figure 2 is a perspective view showing a first culture structure of Figure 1, Figure 3 is a perspective view showing a second culture structure of Figure 1 .

1 to 3, the 3D hepatocyte culture unit 10 may include a first culture structure 100 and a second culture structure 200.

At least one first culture structure 100 may be provided and may be inserted into the second culture structure 200. In this case, the number of the first culture structures 100 inserted into the second culture structure 200 may vary depending on the purpose and / or conditions of the user.

Here, the first culture structure 100 may have a porous structure, for example, the first culture structure 100 may be a lattice structure. In this case, the lattice may have the shape of a groove or the shape of a hole. In addition, the lattice may be freely applied as the shape of the lattice if the liver constituent cells have a predetermined space to be cultured. In addition to the lattice structure, the first culture structure 100 may be implemented in various forms such as a sponge structure, a hydrogel, a solution in which a biopolymer is dissolved in a liquid medium.

The first culture structure 100 may be composed of, for example, polycaprolactone (PCL), polylactic acid (polylactic acid, PLA) and the like. However, this is only an example, and if the material is biocompatible and has a hardness of a certain level or more, it may be applied as a material constituting the first culture structure 100. In the first culture structure 100, liver constituent cells including at least one of hepatocytes, cooper cells, and stellate cells may be cultured.

In one embodiment, the first culture structure 100 may be inserted into the second culture structure 200 in a state that the liver constituent cells are cultured for each lattice. However, this is an example, after the first culture structure 100 is manufactured, the liver is inserted into the first culture structure 100 while the first culture structure 100 is inserted into the second culture structure 200. The constituent cells can also be cultured.

The second culture structure 200 may be inserted into the first culture structure 100, and may have a receiving space for accommodating a fluid such as culture medium, hepatotoxic substances, etc., the receiving space is an example of a cylindrical shape Can be formed. To this end, the second culture structure 200 may include a first injection means 210, a first recess 220, and a first drain means 230.

At least one first injection means 210 may be provided at the bottom of the second culture structure 200. In addition, the first injection means 210 may be spaced apart at predetermined intervals along the circumference of the bottom of the second culture structure 200.

For example, a total of three first injection means 210 may be provided along the circumference of the bottom of the second culture structure 200, and three first injection means 210 may be provided with a second drainage means 230. It may be formed at equal intervals on the circumference of the imaginary circle with the center. Here, the first injection means 210 may be formed in the form of a hole. In addition, fluid such as culture medium, hepatotoxic substances, etc. may be injected into the second culture structure 200 through the first injection means 210.

At least one first recess 220 may be provided on a circumferential wall of the second culture structure 200, and one side of the first culture structure 100 may be inserted into the first recess 220. have.

The first drainage means 230 may be provided in the central portion of the second culture structure 200, the other side of the first culture structure 100 is inserted, and substantially the number of the first recesses 220. The second recess 225 having the same number may be provided. Here, the first drain means 230 may be formed in a cylindrical shape as an example.

As such, since the first culture structure 100 is fitted into the first groove portion 220 and the second groove portion 225, the first culture structure 100 can be selectively inserted into the second culture structure 200. Since the number of the first culture structures 100 to be inserted into the two culture structures 200 can be freely changed, hepatocyte culture efficiency and / or hepatotoxicity evaluation efficiency can be increased.

However, in the present embodiment, the first culture structure 100 is described as being fitted by the first groove portion 220 and the second groove portion 225, the first culture structure 100 and the second culture structure. The connection relationship of the 200 is not limited thereto. For example, the first culture structure 100 may be fixed by controlling magnetism, pressure, and the like between the second culture structures 200.

The first drain means 230 may drain at least a portion of the fluid injected through the first injection means 210. Specifically, the first drainage means 230 may be provided to allow the fluid contained in the second culture structure 200 to be discharged by moving from the top to the bottom of the second culture structure 200. In addition, the height of the first drainage means 230 may be formed to be relatively lower than the height of the second culture structure (200). Accordingly, even when the fluid is continuously injected into the second culture structure 200 through the first injection means 210, the fluid may be drained in advance by the first drain means 230 having a relatively low height. Therefore, the fluid can be prevented from overflowing from the second culture structure 200 to the outside. However, this is only an example, and in some cases, the height of the first drainage means 230 may be the same as the height of the second culture structure 200.

In the present exemplary embodiment, the first recess 220 and the second recess 225 are each provided with twelve, but the number of the first recess 220 and the second recess 225 is limited thereto. The number of the first recesses 220 and the second recesses 225 may vary depending on the number of the first culture structures 100 inserted into the second culture structures 200.

As described above, the three-dimensional hepatocyte culture unit 10 according to an embodiment of the present invention is a liver constituent cell in the second cell culture structure 200 which is a structure that simulates the structure and environment of hepatic lobule which is the basic structure of the liver. The cultured first culture structure 100 may be selectively inserted and used for evaluating hepatotoxicity. In some cases, the hepatotoxicity evaluation may be performed using liver constituent cells cultured in the first culture construct 100, or the first culture construct 100 in which the liver constituent cells are cultured may be stored in the second culture construct 200. Insertion may be performed using additionally cultured liver constituent cells.

Hereinafter, a case where the three-dimensional hepatocyte culture unit 10 is used alone will be described with reference to FIG. 4. 4 is a perspective view showing a combined state of the three-dimensional hepatocyte culture unit of Figure 1 accommodated in the housing case.

Referring to FIG. 4, the 3D hepatocyte culture unit 10 may be accommodated in the accommodation case 15.

The accommodating case 15 may have a hexagonal shape and may further include an accommodating space having a cylindrical shape therein to accommodate the three-dimensional hepatocyte culture unit 10. When the second culture structure 200 in which the first culture structure 100 is inserted is accommodated in the housing case 15, the first injection means 210 of the second culture structure 200 is accommodated by the storage case 15. One end of) and one end of the first drainage means 230 may be closed. Accordingly, the loss of fluid that may occur due to the first injection means 210 and the first drain means 230 can be minimized. As such, the housing case 15 may be a member required when the 3D hepatocyte culture unit 10 is to be used alone.

Meanwhile, the three-dimensional hepatocyte culture unit 10 may be accommodated in the accommodating case 15 and used alone, as described above. As described below, the three-dimensional hepatocyte culture unit 10 includes at least one three-dimensional hepatocyte culture unit 10. It is also possible to be used as the hepatotoxicity evaluation system 1000. Hereinafter, the detailed configuration of the hepatotoxicity evaluation system 1000 will be described with reference to FIGS. 5 and 6.

FIG. 5 is a perspective view showing a hepatotoxicity evaluation system in which the three-dimensional hepatocyte culture unit of FIG. 1 is installed, and FIG. 6 is a perspective view showing the flow of fluid in the hepatotoxicity evaluation system of FIG. 5.

5 and 6, the hepatotoxicity evaluation system 1000 may include at least one three-dimensional hepatocyte culture unit 10. The hepatotoxicity evaluation system 1000 may include a three-dimensional hepatocyte culture unit 10, a plate 300, and a fluid supply member 400. In some embodiments, the hepatotoxicity evaluation system 1000 may further include a fluid discharge member 500.

The three-dimensional hepatocyte culture unit 10 includes a first culture structure 100, a second culture structure 200 including a first injection means 210, a first recess 220, and a first drainage means 230. It may include. However, since the three-dimensional hepatocyte culture unit 10 has been described with reference to FIGS. 1 to 4, the overlapping description of the previously described portions will be omitted.

At least one three-dimensional hepatocyte culture unit 10 may be installed on the plate 300, and may include a flow path 310, a second injection means 320, and a second drain means 330.

The flow path 310 may be formed inside the plate 300, and as shown by a large arrow in FIG. 6, the constituent cells are cultured while the fluid including the culture medium and the hepatotoxic substance flows along the flow path 310. Or hepatotoxicity assessment may be made. However, this is only an example, and the flow path 310 may be formed on the bottom surface of the plate 300. Here, the bottom surface of the plate 300 means a surface in which the three-dimensional hepatocyte culture unit 10 is in contact with the plate 300.

In addition, the flow path 310 may be formed by segmenting the number of three-dimensional hepatocyte culture unit 10 installed in the hepatotoxicity evaluation system 1000, the second drainage means 330 on the upstream side and the downstream side adjacent thereto. It may include a connection passage 312 for connecting the second injection means 320 of the. In addition, a branch point 311 may be formed at the end of the second injection means 320 side of the connection path 312 included in the flow path 310, and the flow path 310 is at least two branches by the branch point 311. Can be cracked. As the flow path 310 is branched by the branch point 311 as described above, the flow path 310 is formed at the periphery of the three-dimensional hepatocyte culture unit 10 when viewed from the center of one three-dimensional hepatocyte culture unit 10. It may extend along at least a portion.

Meanwhile, second injection means 320 may be formed at each end of the branch point 311 and the flow path 310 branching from the branch point 311. The second injection means 320 may be formed to protrude vertically from the plate 300, the first injection of the three-dimensional hepatocyte culture unit 10 when the three-dimensional hepatocyte culture unit 10 is installed in the plate 300 It may be formed at a position that can penetrate the means 210. Accordingly, the fluid flowing through the flow passage 310 rises to the top of the three-dimensional hepatocyte culture unit 10 by the second injection means 320, and the risen fluid is from the top of the three-dimensional hepatocyte culture unit 10. It can spread inside. To this end, the second injection means 320 may be provided in the form of microtubules, for example, and may be configured to allow the fluid to rise by capillary action.

The second drainage means 330 may be provided to be fitted to the first drainage means 230 formed in the second culture structure 200 of the three-dimensional hepatocyte culture unit 10. Accordingly, the diameter of the second drain means 330 may be formed to be substantially smaller than the diameter of the first drain means 230. This second drainage means 330 is an amount exceeded if the amount of the fluid injected from the second injection means 320 exceeds the amount of fluid that the three-dimensional hepatocyte culture unit 10 can accommodate. The fluid may be lowered from the top to the bottom of the three-dimensional hepatocyte culture unit 10 to drain the fluid.

The fluid supply member 400 may be located at one side of the plate 300 and may be, for example, a fluid supply motor capable of continuously supplying fluid to the three-dimensional hepatocyte culture unit 10 installed at one end of the plate 300. Can be. In addition, the fluid supply member 400 may be connected to one end of the flow path 310 to continuously inject the fluid into the three-dimensional hepatocyte culture unit 10.

On the other hand, the fluid supplied from the fluid supply member 400 may be automatically discharged to the outside according to the flow of the fluid flowing from one side of the plate 300 to the other side of the plate 300, the fluid discharged to the outside Can be used to assess hepatotoxicity. However, the method of discharging the fluid to the outside is not limited thereto. If necessary, the fluid may be discharged to the outside by the fluid discharge member 500. Specifically, the fluid discharge member 500 may include a plate. It may be located on the other side of the 300, for example, may be a fluid discharge motor capable of discharging the fluid from the three-dimensional hepatocyte culture unit 10 installed on the other end of the plate (300). In addition, the fluid discharge member 500 may be connected to the other end of the flow path 310 to continuously discharge the fluid from the three-dimensional hepatocyte culture unit 10.

Although not shown in FIG. 6, the hepatotoxicity evaluation system 1000 may further include a sensor and a controller. In this case, the sensor may be provided in the vicinity of the second drainage means 330 of the three-dimensional hepatocyte culture unit 10 to sense the amount of the fluid continuously injected into each three-dimensional hepatocyte culture unit 10. For example, a flow sensor, a level sensor, or the like may be used. However, the installation position and type of the sensor are not limited thereto.

In addition, the control unit pre-stores data about the amount of fluid that can be accommodated in the three-dimensional hepatocyte culture unit 10, and compares the amount of the fluid sensed by the sensor with the amount of the pre-stored fluid, On the basis of this, the fluid injection speed of the fluid supply member 400 and the fluid discharge speed of the fluid discharge member 500 may be controlled. Accordingly, it is possible to prevent the inundation of the fluid which may occur in the process of continuously supplying the fluid to the three-dimensional hepatocyte culture unit 10, and to control the amount of the fluid more accurately, the hepatotoxicity evaluation system 1000 ) Can be operated.

As described above, since the hepatotoxicity evaluation system 1000 according to an embodiment of the present invention continuously injects and exchanges a culture medium into a plurality of three-dimensional hepatocyte culture units 10 connected to each other, an oxygen permeation amount is increased. It can improve the function of liver constituent cells. Hepatotoxicity can then be injected into the cultured liver constituent cells to continue the hepatotoxicity assessment.

However, in the present embodiment, it has been described that the hepatotoxicity evaluation is performed by the hepatotoxicity evaluation system 1000 including the plurality of three-dimensional hepatocyte culture units 10, but this is only an example. In some cases, the three-dimensional hepatocyte culture unit 10 may be accommodated in the accommodating case 15, and then used alone to evaluate hepatotoxicity.

Hereinafter, the liver toxicity evaluation method will be described with reference to Examples.

Example 1: 3D Hepatocyte culture and Hepatotoxicity evaluation

(a) forming a first culture construct

First, using a three-dimensional printing process, a first culture structure was prepared in which the physical properties of the pores were controlled. Here, the three-dimensional printing process used a nozzle having a diameter of about 0.2 mm, about 170 Under a temperature of ^o C to about 270 ^o C, a printing speed of about 30 mm s ⁻¹ to 120 mm s ⁻ ¹ was performed.

As the material of the first culture structure, biocompatible, three-dimensional printing is possible, and strong polycaprolactone (PCL), polylactic acid (polylactic acid, PLA), and the like were used.

The first culture construct was designed using 3ds Max Design (Autodesk, Inc., San Rafael, Calif., USA) with a lattice structure of about 0.1 mm to about 0.3 mm in diameter.

(b) liver constitutive cell culture

Each of the liver constituent cells was cultured in the first culture construct to stabilize the liver constituent cells, and then inserted into the second culture construct. Here, the second culture structure was a cylindrical structure having a diameter of about 4cm to about 8cm.

In order to incubate the liver constituent cells in the first culture construct, the solution was dissolved in a liquid medium and the liver constituent cells were agitated for 24 hours or more, thereby increasing the cell settling rate in the first culture construct. .

As the liver constituent cells, human primary hepatoctyes, HepaRG or liver cancer cell lines (HepG2, Hep3B, Huh7) and the like can be utilized. Cooper cells can be utilized as a human kupffer cell or established cell line U937. As an astrocyte cell line, LX2 cell line which is an established cell line can be utilized.

The first culture construct in which the liver constituent cells are primaryly cultured is applied to the second culture construct in an appropriate ratio (hepatocytes: Cooper cells, 3: 1 or 4: 1, hepatocytes: astrocytes, 3: 1 or 4: 1). After insertion, the culture medium was co-cultured by filling the height of the second culture construct.

(c) hepatotoxicity assessment

As a positive control hepatotoxic substance, acetoaminophen and triglitazone were injected into the second culture structure (hereinafter, 3-dimensional hepatocyte culture unit) into which the first culture structure was inserted at an appropriate concentration. Subsequently, after exposing the three-dimensional hepatocyte culture unit for a predetermined time, the first culture construct was separated from the second culture construct. Subsequently, the first culture construct exposed to the hepatotoxic substance was subjected to the cell counting kit-8 (Dojindo Laboratory, Kumanoto, Japan) or The celltiter-glo® luminescent cell viability assay (Promega Corp, Madison, USA). Cell viability in the first culture construct was measured. In addition, the degree of cell necrosis was measured by measuring lactate dehydrogenase (LDH), a cell damage index.

Subsequently, fluid (eg, culture medium exposed for a period of time after injection of hepatotoxic material) was recovered from the three-dimensional hepatocyte culture unit. Cytokines (cytokines) such as interleukin2, interlekin6, and interleukin8 generated in the recovered fluid were analyzed.

As described above, the method for evaluating hepatotoxicity according to an embodiment of the present invention comprises co-culturing liver constituent cells in a three-dimensional hepatocyte culture unit similar to the structure and environment of hepatic lobule, which is a unit structure of the liver, and Since hepatotoxicity evaluation is continuously performed in the hepatotoxicity evaluation system employing at least one or more, hepatotoxicity evaluation reflecting the mutual link between the liver constituent cells is possible.

Although embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may implement the present invention in other specific forms without changing the technical spirit or essential features thereof. I can understand that. For example, those skilled in the art can change the material, size, etc. of each component according to the application field, or combine or replace the embodiments in a form that is not clearly disclosed in the embodiments of the present invention, this is also the present invention It will not go beyond the scope of the. Therefore, the above-described embodiments are to be considered in all respects as illustrative and not restrictive, and such modified embodiments should be included in the technical spirit described in the claims of the present invention.

[Description of the code]

10: three-dimensional hepatocyte culture unit 15: receiving case

100: first culture structure 200: second culture structure

210: first injection means 220: first groove portion

225: second groove portion 230: first drain means

300: plate 310: euro

311: junction 312: connection

320: second injection means 330: second drain means

400: fluid supply member 500: fluid discharge member

1000: Hepatotoxicity Assessment System

Claims

A first culture construct in which liver constituent cells are cultured; And

The second culture structure is provided so that the first culture structure can be inserted, the fluid containing at least one of the culture medium for culturing the liver constituent cells and hepatotoxic substances for hepatotoxicity evaluation Three-dimensional hepatocyte culture unit comprising a.
The method of claim 1,

The second culture structure,

At least one first injection means into which the fluid is injected;

At least one first recess into which one side of the first culture structure is inserted; And

And a first drainage means for draining at least a portion of the fluid injected from the first injection means, and having at least one second recess portion into which the other side of the first culture structure is inserted.
The method of claim 2,

And the first drainage means is provided to lower and discharge the fluid contained in the second culture structure from the top to the bottom of the second culture structure.
The method of claim 2,

And a receiving case accommodating the second culture structure while closing one end of the first injection means and one end of the first drain means.
The method of claim 1,

The first culture structure,

A three-dimensional hepatocyte culture unit is a porous matrix that provides a culture space in which the liver constituent cells are cultured.
The method of claim 1,

Wherein said liver constituent cells comprise at least one of hepatocytes, kupffer cells and stellate cells.
A first culture structure in which the liver constituent cells are cultured and the first culture structure are inserted and installed, and a fluid containing at least one of a culture medium for culturing the liver constituent cells and a hepatotoxic substance for hepatotoxicity evaluation is contained therein. A three-dimensional hepatocyte culture unit having a second culture structure;

A plate on which at least one three-dimensional stem cell culture unit is installed; And

Located on one side of the plate, hepatotoxicity evaluation system comprising a fluid supply member for supplying a fluid to the three-dimensional hepatocyte culture unit.
The method of claim 7, wherein

The plate,

A flow path through which the fluid is moved;

At least one second injection means formed on the flow path and configured to move the fluid from the flow path and inject the fluid into the three-dimensional hepatocyte culture unit; And

And a second drain means for draining at least a portion of the fluid injected from said second injection means.
The method of claim 8,

One end of the flow path is connected to the fluid supply member,

And said branch point of said flow path is a point extending from one end of said flow path connected with said fluid supply member.
The method of claim 7, wherein

Located on the other side of the plate, the liver toxicity evaluation system further comprises a fluid discharge member for discharging the fluid to the outside.
Providing a three-dimensional hepatocyte culture unit having a second culture structure in which a first culture structure in which liver constituent cells are cultured is inserted;

Accommodating the three-dimensional hepatocyte culture unit in a housing case;

Supplying a fluid comprising at least one of a culture medium for culturing the liver constituent cells and a hepatotoxic substance for hepatotoxicity evaluation to the three-dimensional hepatocyte culture unit; discharging the fluid from the three-dimensional hepatocyte culture unit to the outside Making a step;

Separating the first culture construct from the second culture construct and measuring the degree of necrosis of the liver constituent cells; And

Hepatotoxicity evaluation method comprising the step of analyzing the cytokine generated in the fluid discharged to the outside.
Providing a three-dimensional hepatocyte culture unit having a second culture structure in which a first culture structure in which liver constituent cells are cultured is inserted;

Installing at least one three-dimensional hepatocyte culture unit on a plate;

Supplying a fluid comprising at least one of a culture medium for culturing the liver constituent cells and a hepatotoxic substance for hepatotoxicity evaluation to the three-dimensional hepatocyte culture unit using a fluid supply member located on one side of the plate;

Discharging the fluid from the three-dimensional hepatocyte culture unit to the outside;

Separating the first culture construct from the second culture construct and measuring the degree of necrosis of the liver constituent cells; And

Hepatotoxicity evaluation method comprising the step of analyzing the cytokine generated in the fluid discharged to the outside.
The method of claim 12,

Providing the three-dimensional hepatocyte culture unit,

Preparing the first culture construct;

Culturing the liver constituent cells in the first culture construct; And

Hepatotoxicity evaluation method comprising the step of inserting the first culture structure into the second culture structure.
The method of claim 13,

Preparing the first culture structure,

Hepatotoxicity assessment performed by at least one of three dimensional printing process, salt leaching process, phase separation process, gas foaming process and electrospinning process Way.
The method of claim 13,

Culturing the liver constituent cells in the first culture construct,

Injecting the liver constituent cells into the first culture structure by stirring the solution of the biopolymer in a liquid medium; And

Hepatotoxicity evaluation method comprising the step of stabilizing the first culture structure for 24 hours or more.
The method of claim 13,

And inserting the first culture construct into the second culture construct, and further culturing the liver constituent cells cultured in the first culture construct.
The method of claim 12,

The fluid supplied by the fluid supply member is automatically discharged to the outside according to the flow of the fluid flowing from one side of the plate to the other side of the plate, or discharged to the outside by the fluid discharge member located on the other side of the plate Methods of evaluating hepatotoxicity.