KR20170087756A - Three dimensional hepatocyte culturing unit, evaluating system for hepatotoxicity and method using the same - Google Patents
Three dimensional hepatocyte culturing unit, evaluating system for hepatotoxicity and method using the same Download PDFInfo
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- KR20170087756A KR20170087756A KR1020160007723A KR20160007723A KR20170087756A KR 20170087756 A KR20170087756 A KR 20170087756A KR 1020160007723 A KR1020160007723 A KR 1020160007723A KR 20160007723 A KR20160007723 A KR 20160007723A KR 20170087756 A KR20170087756 A KR 20170087756A
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Abstract
A three-dimensional hepatocyte culture unit is disclosed.
The three-dimensional hepatocyte culture unit according to an embodiment of the present invention is provided so that the first culture structure and the first culture structure in which the hepatic constituent cells are cultured are inserted, and the culture medium and hepatic toxicity And a second culture structure in which a fluid containing at least one of toxic substances for evaluation can be received therein.
Description
The present invention relates to a three-dimensional hepatocyte culture unit, a hepatotoxicity evaluation system, and a method for evaluating hepatotoxicity using the same.
The liver is the main living organ of an animal, and the main function of the liver is the detoxification function of detoxifying foreign substances. Generally, the liver is composed of a unitary structure called hepatic lobules, and these liver lobes can have a substantially hexagonal structure.
On the other hand, since the activity of hepatic cells in hepatic lobules is greatly influenced by the structure of hepatic lobules, studies are being carried out to simulate liver tissues three-dimensionally and to realize functions of liver tissues in an atmosphere similar to the environment of liver tissues .
For example, hepatocytes extracted from the human body are cultured for a long time using a microstructure simulating the boundary between the hepatic epithelium and blood vessels, and a liver tissue-like function such as albumin production is expressed to form a structure similar to a bile duct in the human body Researches have been reported on the development of interchip. (An Artificial Liver Sinusoid with a Microfluidic Endothelial-Like Barrier for Primary Hepatocyte Culture, Lee et al., Biotechnology and Bioengineering, 97, 5, 1312, 2007)
It has also been reported that 2D photomicrographs of hepatocytes (HepG2) and endothelial cells in the shape of hepatic hepatocyte structures using photolithography have been reported. (Liver-cell patterning Lab Chip: mimicking the morphology of liver lobule tissue, Ho et al, Lab Chip, 2013, 13, 3578)
In addition, research has been conducted to form an intermediate lobular reshaped structure using a hydrogel. (Construction of hepatic lobule-like 3D tissues using cell embedding hydrogel microfibers, Yuya Yajima et al., 18th International Conference on Miniaturized Systems for Life Science)
However, the above-mentioned studies have focused only on the local part of liver tissue and liver function, and have attempted to simulate liver tissue in three dimensions and to realize the function of liver tissue in an atmosphere similar to liver environment Is currently in its early stages.
Examples of the present invention include a three-dimensional hepatocyte culture unit, hepatotoxicity evaluation system, and hepatotoxicity assay using the hepatocyte culture unit, which co-cultivates various cells constituting liver tissue by simulating the structure and environment of hepatic lobule, Evaluation method.
According to an aspect of the present invention, there is provided a method for culturing hepatocytes, which comprises culturing a first culture construct and a first culture construct in which hepatocytes are cultured, the culture medium for culturing the hepatocytes and the hepatotoxic agent And a second culture structure in which a fluid including at least one of the first culture structure and the second culture structure can be received.
The second culture structure may include at least one or more first injection means into which the fluid is injected, at least one first recessed portion into which one side of the first culture structure is inserted, and at least one second recessed portion into which the other side of the first culture structure is inserted And at least one second recessed portion, and may include first drainage 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 the fluid from the upper portion to the lower portion of the second culture structure.
The method may further include a receiving case for receiving the second culture structure while closing one end of the first injection means and one end of the first drainage means.
In addition, the first culturing structure may include a porous matrix that provides a culture space in which the hepatic cells are cultured.
In addition, the hepatic cells may include at least one of a hepatocyte, a kupffer cell, and a stellate cell.
According to another aspect of the present invention, there is provided a method for culturing hepatocytes, comprising culturing a first culture construct in which liver cells are cultured and at least one first culture construct, A three-dimensional hepatocyte culture unit having a second culture structure in which a fluid containing at least one of them can be received therein; a plate on which at least one three-dimensional hepatocyte culture unit is installed; A liver toxicity evaluation system including a fluid supply member for supplying a fluid to the three-dimensional hepatocyte culture unit may be provided.
The plate may further include at least one flow path through which the fluid flows, at least one second injection means formed on the flow path, for moving the fluid from the flow path and injecting the fluid into the three-dimensional hepatocyte culture unit, And a second drain means for draining at least a portion of the fluid injected from the second conduit.
Further, 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 located on the other side of the plate and discharging the fluid to the outside.
According to another aspect of the present invention, there is provided a three-dimensional hepatocyte culture unit comprising a second culturing structure in which a first culturing structure in which hepatocytes are cultured is inserted, Supplying a fluid containing at least one of a culture medium for culturing the liver-forming cells and a hepatotoxic substance for hepatotoxicity evaluation to the three-dimensional hepatocyte culture unit, supplying the fluid from the three-dimensional hepatocyte culture unit Separating the first cultured structural body from the second cultured structural body to measure the degree of necrosis of the hepatic constituting cells and analyzing the cytokine produced in the fluid discharged to the outside A method for evaluating hepatotoxicity may be provided.
According to another aspect of the present invention, there is provided a three-dimensional hepatocyte culture unit comprising: a three-dimensional hepatocyte culture unit having a second culture structure in which a first culturing structure in which liver cells are cultured is inserted; Comprising the steps of: installing at least one fluid, a fluid containing at least one of a culture medium for culturing the liver cells and a hepatotoxic substance for hepatotoxicity evaluation in the three-dimensional hepatocyte culture unit using a fluid supply member located at one side of the plate Separating the first cultured structural body from the second cultured structural body to measure the degree of necrosis of the hepatic constituting cells, and discharging to the outside And analyzing the cytokine produced in the fluid. There are evaluation methods can be provided.
In addition, the step of providing the three-dimensional hepatocyte culture unit may include the steps of producing the first culture structure, culturing the hepatocytes in the first culture structure, and culturing the first culture structure in the second culture structure As shown in FIG.
In addition, the step of preparing the first culture structure may include a three dimensional printing process, a salt leaching process, a phase separation process, a gas foaming process, and an electrospinning process. ) ≪ / RTI > process.
The step of culturing the hepatocytes in the first culture structure may include the steps of injecting the hepatocytes into the first culture structure by stirring the solution with a solution in which the biopolymer is dissolved in a liquid culture medium, Followed by culturing for 24 hours or longer for stabilization.
In addition, the step of inserting the first culturing structure into the second culturing structure may further include culturing the hepatocytes cultured in the first culturing structure.
The fluid supplied by the fluid supply member may be automatically discharged to the outside in accordance with the flow of the fluid flowing from one side of the plate to the other side of the plate, .
The embodiments according to the present invention are directed to a method for co-culturing liver constituent cells in a three-dimensional hepatocyte culture unit similar to the structure and environment of hepatic lobule, which is a liver unit structure, It is possible to evaluate hepatotoxicity, which reflects the interlinkage between the hepatic constituent cells.
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.
Fig. 3 is a perspective view showing the second cultivation structure of Fig. 1. Fig.
FIG. 4 is an assembled perspective view showing a state in which the three-dimensional hepaticocyte culture unit of FIG. 1 is accommodated in a receiving case.
FIG. 5 is a perspective view showing a hepatotoxicity evaluation system equipped with the three-dimensional hepatocyte culture unit of FIG. 1;
FIG. 6 is a perspective view showing the flow of fluid in the hepatotoxicity evaluation system of FIG. 5; FIG.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, configurations and operations according to embodiments of the present invention will be described in detail with reference to the accompanying drawings. DETAILED DESCRIPTION OF THE INVENTION The following description is one of many aspects of the claimed invention and the following description may form part of the detailed description of the invention.
However, the detailed description of known configurations or functions in describing the present invention may be omitted for clarity.
While the invention is susceptible to various modifications and its various embodiments, it is intended to illustrate the specific embodiments and the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Terms including ordinals such as first, second, etc. may be used to describe various elements, but the elements are not limited by such terms. These terms are used only to distinguish one component from another.
It is to be understood that when an element is referred to as being "connected" or "connected" to another element, it may be directly connected or connected to the other element, .
The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise.
FIG. 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, and FIG. 3 is a perspective view of the second culture structure of FIG. 1 .
1 to 3, the 3D
At least one
Here, the
The
In one embodiment, the
The
At least one or more than one first injection means 210 may be provided at the bottom of the
For example, the first injection means 210 may be provided along three peripheries of the bottom of the
At least one or more of the first recessed
The first drainage means 230 may be provided at a central portion of the
Since the first
In the present embodiment, the
The first drainage 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 move the fluid received in the
The number of the first recessed
As described above, in the three-dimensional
Hereinafter, the case in which the three-dimensional
Referring to Fig. 4, the three-dimensional
The
On the other hand, the three-dimensional
FIG. 5 is a perspective view showing a hepatotoxicity evaluation system equipped with the three-dimensional hepatocyte culture unit of FIG. 1, and FIG. 6 is a perspective view showing a flow of fluid in the hepatotoxicity evaluation system of FIG.
Referring to FIGS. 5 and 6, the
The three-dimensional
At least one three-dimensional
The
The
The second injection means 320 may be formed at each end of the
The second drainage means 330 may be provided so as to be fitted in the first drainage means 230 formed in the
The
The fluid supplied from the
The
Meanwhile, although not shown in FIG. 6, the
The control unit stores data on the amount of fluid that can be received by the three-dimensional
As described above, since the
However, in the present embodiment, it has been described that the hepatotoxicity evaluation is performed by the
Hereinafter, a method for evaluating hepatotoxicity will be described by way of examples.
Example 1: Three-dimensional hepatocyte culture and hepatotoxicity evaluation
(a) Formation of first culture structure
First, a first culture structure with controlled physical properties of pores was prepared using a three-dimensional printing process. Here, the three-dimensional printing process was performed using a nozzle having a diameter of about 0.2 mm, under about 170 ℃ to about 270 ℃ temperature, from about 30mm to about 120 mm s -1 s - was performed with the printing speed of the first.
Polycaprolactone (PCL), polylactic acid (PLA), etc., which are biocompatible and capable of three-dimensional printing, and having high hardness, were used as the material of the first culture structure.
The first culture structure was designed using 3ds Max Design (Autodesk, Inc., San Rafael, CA, USA) as a lattice structure having a diameter of about 0.1 mm to about 0.3 mm.
(b) Liver cell culture
Liver constituent cells were each cultured in the first cultured construct to stabilize the hepatic constituent cells and then inserted into the second cultured construct. Here, the second culture structure used was a cylindrical structure having a diameter of about 4 cm to about 8 cm.
In order to cultivate the liver cells in the first culture structure, the solution obtained by dissolving the biopolymer in the liquid medium and the liver cells were agitated for more than 24 hours to increase the cell attachment rate in the first culture structure .
Human primary hepatoctyes, HepaRG or hepatocarcinoma cell lines (HepG2, Hep3B, Huh7) can be used as the hepatic cells. Cooper cells are available as human kupffer cells or as established cell lines, U937. LX2 cell line, an established cell line, is available for astrocyte mainly.
3: 1 or 4: 1, hepatocyte: stellate cells, 3: 1 or 4: 1) in the second culture structure in a suitable ratio (Hepatocyte: Cooper, After the insertion, the culture medium was filled up to the height of the second culture structure, and the liver-constituting cells were co-cultured.
(c) Assessment of hepatotoxicity
As a positive control hepatotoxic agent, acetoaminophen and Triglitazone were injected into the second culture structure (hereinafter, three-dimensional hepatocyte culture unit) into which the first culture structure was inserted at an appropriate concentration. Then, the three-dimensional hepatocyte culture unit was exposed for a predetermined time, and then the first culture structure was separated from the second culture structure. Then, the first cultured structure exposed to the hepatotoxic substance was analyzed using the cell counting kit-8 (Dojindo Laboratory, Kumanoto, Japan) or the celltiter-glo luminescent cell viability assay (Promega Corp., Madison, Cell viability in the first cultured construct was measured. In addition, the degree of cell necrosis was measured by LDH (lactate dehydrogenase) measurement which is an index of cell damage.
Then, a fluid (for example, a culture medium exposed for a certain period of time after the hepatic toxic substance was injected) was recovered from the three-dimensional hepatocyte culture unit. Interleukin 2, interleukin 6, interleukin 8, and other cytokines were analyzed in the recovered fluid.
As described above, the method for evaluating hepatotoxicity according to an embodiment of the present invention is a method for co-culturing liver-constituting cells in a three-dimensional hepatocyte culture unit similar to the structure and environment of hepatic lobule, which is a liver unit structure, Since the hepatotoxicity assay is continuously performed in the hepatotoxicity assay system employing at least one or more hepatotoxicity assay systems, it is possible to evaluate the hepatotoxicity of hepatocytes by reflecting the correlation between hepatocytes.
While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, You will understand. For example, a person skilled in the art can change the material, size and the like of each constituent element depending on the application field or can combine or substitute the embodiments in a form not clearly disclosed in the embodiments of the present invention, Of the range. Therefore, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive, and that such modified embodiments are included in the technical idea described in the claims of the present invention.
10: Three-dimensional hepatocyte culture unit 15:
100: first culturing structure 200: second culturing structure
210: first injection means 220: first pouring groove
225: second recessed portion 230: first drainage means
300: plate 310:
311: Junction 312: Connection path
320: second injection means 330: second drain means
400: fluid supply member 500: fluid discharge member
1000: Liver toxicity assessment system
Claims (17)
A second culture structure provided so that the first culture structure can be inserted therein and a fluid containing at least one of a culture medium for culturing the hepatic cells and a hepatotoxic substance for hepatotoxicity assessment can be received therein, Wherein the three-dimensional hepatocyte culture unit comprises:
The second culturing structure may comprise:
At least one first injection means through which the fluid is injected;
At least one first recessed portion into which one side of the first culture structure is inserted; And
And a second drainage means for draining at least a part of the fluid injected from the first injection means, wherein the first drainage means has at least one second recessed portion into which the other side of the first culture structure is inserted.
Wherein the first drainage means is provided to discharge the fluid contained in the second culture structure by lowering the fluid from the top of the second culture structure to the bottom.
And a receiving case for accommodating the second culture structure while closing one end of the first injection means and one end of the first drainage means.
The first culture structure may include:
Dimensional hepatocyte culture unit, which is a porous matrix providing a culture space in which the hepatic constituting cells are cultured.
Wherein the hepatic-constituting cells comprise at least one of a hepatocyte, a kupffer cell and a stellate cell.
A plate on which at least one of the three-dimensional hepatocyte culture units is installed; And
And a fluid supply member which is located at one side of the plate and supplies the fluid to the three-dimensional hepatocyte culture unit.
The plate may comprise:
A flow path through which the fluid flows;
At least one second injection means formed on the flow path for moving the fluid from the flow path and injecting the fluid into the three-dimensional hepatocyte culture unit; And
And second drain means for draining at least a part of the fluid injected from said second injection means.
One end of the flow path is connected to the fluid supply member,
Wherein the branch point of the flow path is a point extending from one end of the flow path connected to the fluid supply member.
And a fluid discharge member located on the other side of the plate and discharging the fluid to the outside.
Receiving the three-dimensional hepatocyte culture unit in a receiving case;
Supplying a fluid containing at least one of a culture medium for culturing the liver-forming cells and a hepatotoxic substance for hepatotoxicity evaluation to the three-dimensional hepatocyte culture unit;
Expelling the fluid from the three-dimensional hepatocyte culture unit to the outside;
Separating the first cultured structural body from the second cultured structural body to measure the degree of necrosis of the hepatic constituent cells; And
And analyzing the cytokine produced in the fluid discharged to the outside.
Installing at least one three-dimensional hepatocyte culture unit on a plate;
Supplying a fluid containing at least one of a culture medium for culturing the liver-forming cells and a hepatotoxic substance for hepatotoxicity evaluation to the three-dimensional hepatocyte culture unit using a fluid supply member located at one side of the plate;
Expelling the fluid from the three-dimensional hepatocyte culture unit to the outside;
Separating the first cultured structural body from the second cultured structural body to measure the degree of necrosis of the hepatic constituent cells; And
And analyzing the cytokine produced in the fluid discharged to the outside.
Wherein the providing of the three-dimensional hepatocyte culture unit comprises:
Producing the first culture structure;
Culturing the hepatocytes in the first culture construct; And
And inserting the first culture structure into the second culture structure.
Wherein the step of preparing the first culturing structure comprises:
A liver toxicity evaluation performed through at least one of a three dimensional printing process, a salt leaching process, a phase separation process, a gas foaming process, and an electrospinning process Way.
The step of culturing the liver cells in the first culture structure comprises:
Mixing the hepatic constituent cells with a solution in which the biopolymer is dissolved in a liquid medium, and injecting the mixture into the first culture construct; And
Culturing the first culture construct for at least 24 hours to stabilize the hepatotoxicity.
Further comprising culturing the hepatocyte cultured in the first culture structure after the step of inserting the first culture structure into the second culture structure.
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 A method for evaluating hepatotoxicity.
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WO2019059702A2 (en) * | 2017-09-25 | 2019-03-28 | 아주대학교산학협력단 | Nanofiber-based long-term primary hepatocyte three-dimensional culture system and culturing method |
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WO2015003160A2 (en) * | 2013-07-03 | 2015-01-08 | Colorado State University Research Foundation | Hepatocytes in co-culture and uses thereof |
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