WO2014061244A1 - Toxicity screening method - Google Patents

Abstract

A toxicity screening method is a method for determining whether or not a compound of interest is metabolized with a drug-metabolizing enzyme in the liver to cause toxicity. The method comprises the steps of: culturing multiple hepatocytes three-dimensionally (e.g., producing agglomerates); bringing each of the hepatocyte into contact with each of a first solution that does not contain the compound of interest, a second solution that contains the compound of interest, and a third solution that contains both at least one compound capable of inhibiting a drug-metabolizing enzyme reaction and the compound of interest, thereby obtaining multiple hepatocytes that have been contacted respectively with the different solutions; bringing the multiple hepatocytes into contact with a solution containing a fluorescent probe capable of recognizing living cells or a fluorescent probe capable of recognizing dead cells; and obtaining fluorescently stained images using the multiple hepatocytes that are stained, and then determining the presence of toxicity of the compound of interest on the basis of the data of the fluorescently stained images.

Description

Toxicity screening method

The present invention relates to methods of screening compounds, and in particular to analytical techniques.

Hepatocytes have numerous physiological functions and have important functions in the metabolism of drugs, food additives, environmental pollutants and the like. It is said that metabolism involves many enzymes present in hepatocytes, for example, hydrolases such as esters, cytochrome P450 which is a phase I drug metabolizing enzyme that plays an important role in oxidation reactions, and reduction There is an enzyme and a conjugated enzyme that imparts a phase II drug metabolizing enzyme such as sulfuric acid, acetic acid, glutathione or glucuronic acid. Reactive intermediates (reactive metabolites) may be produced by these enzymatic reactions. The chemical structures of reactive metabolites are unstable and short-lived, and most of them are detoxified by endogenous glutathione, but they may also bind to proteins and DNA and exhibit hepatotoxicity. In addition, if a large amount of reactive metabolite is produced, glutathione is depleted and thus binds to surrounding proteins and DNAs, resulting in extremely strong toxicity. As a result, serious liver damage may occur. For this reason, various methods for analyzing metabolite toxicity of hepatocytes have been developed.

For example, Patent Document 1 discloses a method of screening drug candidates for toxic effects on human cells, and a method of measuring the toxicity of idiosyncrasies of test compounds. More specifically, Patent Document 1 discloses a method of screening whether or not a metabolite produced by a test compound being metabolized by a phase I drug metabolizing enzyme exhibits toxicity. A method using cells expressing a phase I drug metabolizing enzyme is disclosed, and it is preferable to use a plurality of cells expressing different cytochrome P450 and to express a phase I drug metabolizing enzyme. It is recommended that the factor II drug metabolizing enzymes be 20 times larger. This eliminates the influence of the phase II drug metabolizing enzyme reaction, since the compound metabolized by the phase II drug metabolizing enzyme may be metabolized by the phase II drug metabolizing enzyme to produce a toxic compound or be detoxified. In order to do this, cells with the above-mentioned characteristics are used. In this way, accurate toxicity can be evaluated by combining methods for intentionally controlling the metabolic function in cells in evaluating the toxicity of metabolites.

In addition, Non-Patent Document 1 discloses culture using human primary hepatocytes by a sandwich method of collagen-matrigel, visualizing the amount of active oxygen, mitochondrial membrane activity, and consumption of glutathione (GSH) which is an antioxidant. Methods have been disclosed to predict the mechanism of toxicity development in the body. However, as shown in Patent Document 1, it is possible that intracellularly metabolized metabolites produced by the phase I drug metabolizing enzyme are metabolized by the phase II drug metabolizing enzyme to produce a substance exhibiting toxicity. In this method, it is not possible to accurately determine which metabolic enzyme has been metabolized or not.

Japanese Patent Publication No. 2005-511093

In Patent Document 1, a plurality of cells in which the phase I drug metabolizing enzyme is significantly higher than the expression amount of the phase II drug metabolizing enzyme are used. For this reason, there is a problem that maintenance and management of cells take time and effort. Although the sandwich method used in Non-Patent Document 1 is excellent in that the function of primary hepatocytes can be maintained for a long time, there is a problem that handling of the gel is not easy and operation becomes complicated. In addition, since matrigel is expensive, there is a problem that the cost is high. On the other hand, although the operation of the general plate culture method is simple and low cost, the cell has a form completely different from that in the living body flatly spread on the culture bottom. For this reason, it is known that cell functions can not be reproduced in vitro. As a result, cell death due to reactive metabolites may not be detectable in plating.
In order to solve this problem, many methods for reproducing tissue structures close to in vivo in vitro, for example, methods for producing aggregate-like masses, have been studied and marketed. For example, three-dimensional culture systems such as AlgiMatrix (registered trademark) are commercially available. In these methods, the operation is complicated and the light transmittance of the carrier is lower than that of the flat plate. Therefore, it is not suitable for the imaging method disclosed in Non-Patent Document 1.
Under these circumstances, in a method of screening a compound, analysis using a three-dimensional cultured cell capable of maintaining a high degree of cell function and identifying a drug metabolizing enzyme that causes toxicity by metabolizing a test compound by an imaging method Technology was required.

The inventors discovered a new screening method for visualizing live cells and dead cells and evaluating whether they are toxic due to metabolites by simultaneously adding a test substance and a metabolic enzyme inhibitor to sterically cultured cells. did.
The toxicity screening method of one embodiment is a method of determining whether a test compound is metabolized by a drug metabolizing enzyme in the liver and exhibits toxicity, and includes the following configuration.
(1) Three-dimensionally culturing a plurality of hepatocytes.
(2) Each of a first solution not containing the test compound, a second solution containing the test compound, and a third solution of one or more inhibitors inhibiting the drug metabolism enzyme reaction and the test compound, Obtaining the plurality of hepatocytes contacted with different solutions by exposing each hepatocyte. Here, each hepatocyte is exposed to any one of the first to third solutions to obtain three types of hepatocytes which are in contact with one of the first to third solutions.
(3) contacting the plurality of hepatocytes with a solution containing a fluorescent probe that recognizes live cells, a fluorescent probe that recognizes dead cells, and a fluorescent probe selected from the group consisting of a combination thereof;
(4) A step of obtaining a fluorescent stained image using the plurality of hepatocytes after staining, and determining the toxicity of the test compound based on the data of the fluorescent stained image.
By simultaneously adding a test substance and a metabolic enzyme inhibitor to sterically cultured cells, it becomes possible to express the same metabolic function as hepatocytes. In addition, by analyzing using a fluorescent probe, it is possible to visualize live cells and dead cells to evaluate whether or not the metabolic enzymes of the cultured hepatocytes are toxic due to the metabolite that metabolized the test compound.

According to the present invention, it is possible to provide an analytical technique for identifying metabolic enzymes that cause toxicity by metabolizing a test compound by using a three-dimensional cultured cell capable of maintaining a high degree of cellular function by an imaging method. .

It is a figure which shows the whole culture plate used by one embodiment of this invention. FIG. 2 is a cross-sectional view taken along line II-II of the culture plate shown in FIG. FIG. 2 is another cross-sectional view of the culture plate shown in FIG. 1 along the line II-II. It is a figure which shows the whole culture vessel used by one embodiment of this invention. FIG. 4 is a cross-sectional view of the culture vessel shown in FIG. 3 taken along line IV-IV. It is the schematic showing the state which culture | cultivates an aggregate in culture space. It is a figure which shows the schematic diagram explaining an example of the preferable size of the aggregate cultured by culture space. It is the schematic showing the state which culture | cultivates a spheroid in culture space. It is a figure which shows the schematic diagram explaining an example of the preferable size of the spheroid cultured by culture space. It is a figure which shows the example of another shape of culture space. It is a figure which shows the example of another shape of culture space. It is sectional drawing which shows the example of a shape of the other side of culture space. It is sectional drawing which shows the example of a shape of the further another side of culture space. It is sectional drawing which shows the example of a shape of the further another side of culture space. It is a photograph which shows an example of the culture plate used in the Example. It is a photograph which shows an example of the hepatocyte cultured by the Example. In the examples, it is a photograph of a fluorescent staining image of hepatocytes contacted with solutions (i) to (iii). In a comparative example, it is a photograph of a fluorescence stained picture of hepatocyte which contacted solution (i)-(iii).

Hereinafter, embodiments will be described with reference to the drawings. The following description and drawings are omitted and simplified as appropriate for clarification of the explanation. Components and corresponding parts having the same configuration or function in the drawings are denoted by the same reference numerals, and the description thereof will be omitted.

The following terms are used herein.
The term "aggregate" refers to an embodiment in which a plurality of cells are stacked in two or more layers and have a three-dimensional shape.
The term "cytochrome P450 (CYP)" is an enzyme that plays a role in foreign body (drug) metabolism, which is present in almost all organisms from bacteria to plants to mammals. In animals, they are mainly present in the liver.

The term "well plate" refers to an experimental / inspection instrument consisting of a flat plate with a large number of depressions (holes or wells), wherein each well is used as a test tube or petri dish. The number of wells includes, for example, 6, 24, 96, 384, etc., and the number of wells is more than that. The bottom of the well may be flat or round, or may be a combination of many elongated microtubes (deep well plate).
The term "drug metabolizing enzyme" is a generic term for enzymes involved in reactions to degrade or excrete in vitro substances such as drugs and toxic substances (xenobiotics Xenobiotics, also referred to as foreign substances).
The term "phase I drug metabolizing enzyme" refers to a group of enzymes involved in hydrolysis, oxidation reaction, reduction reaction of ester etc. as a reaction that lowers (degrades) or does not significantly change the molecular weight of the target substance called phase I reaction It is. The oxidation reaction is mainly oxidation by cytochrome P450 (P450).
The term “phase II drug metabolizing enzyme” is a reaction that adds another molecule called a phase II reaction (the molecular weight increases), and as a molecule to be added in a reaction also referred to as conjugation (googau), sulfuric acid, It is a group of enzymes that conjugate acetic acid, glutathione, glucuronic acid and the like. In the present specification, “phase II drug metabolizing enzyme” is also described as “phase II enzyme group”.

In the following description, the term “range from value A to value B” means “value A or more and value B or less” unless otherwise specified.
In addition, “these combinations” described as “A, B,..., C, and combinations thereof” are two or more of A, B,. Means any combination of numbers. In other words, "A, B, ..., C, and a combination thereof" is any one of A, B, ..., C, and any combination of these. It can also be said.

One embodiment provides a new screening system for visualizing live cells and dead cells to evaluate whether or not they are toxic due to a metabolite by simultaneously adding a test substance and a metabolic enzyme inhibitor to cells cultured in three dimensions. In this method, three-dimensional culture can be performed without using a gel as described in Non-Patent Document 1, and furthermore, since it is a culture method with high light permeability, visualization using a microscope is also possible. The toxicity screening method carries out, for example, the procedures of the first to fourth steps. The outline of the first to fourth steps will be described below. The first to fourth steps are obtained by dividing the steps in order to facilitate the description, and the present invention is not limited thereto.
The first step is a culture treatment of culturing a plurality of hepatocytes in a three-dimensional manner.
The second step is a test compound treatment in which a solution containing a test compound, a solution containing a test compound and an inhibitor of a drug metabolizing enzyme, or a solution not containing a test compound is brought into contact with a plurality of hepatocytes cultured sterically.
The third step is fluorescent probe treatment in which a solution containing a fluorescent probe is brought into contact with a plurality of hepatocytes.
The fourth step is a determination process of determining the toxicity of the test compound.
In the following, regarding the evaluation method of one embodiment, the culture vessel used in the culture treatment will be described first, and then the procedure of the toxicity screening method for performing from the culture treatment to the determination treatment will be described in detail.

1. Culture Container The culture container uses a culture container capable of three-dimensionally culturing hepatocytes. In other words, any culture vessel may be used as long as a plurality of cells are stacked to produce hepatocytes having a three-dimensional shape. In particular, it is preferable to produce hepatocytes having an aggregate shape. Hereinafter, a preferred example of a container for forming aggregates will be described as an example of the culture container.

* Outline of Culture Container FIG. 1 is a view showing the whole culture plate used in one embodiment of the present invention. FIG. 2A is a cross-sectional view taken along line II-II of the culture plate shown in FIG. 1, and FIG. 2B shows a cross-sectional view of another embodiment. The culture plate 1 comprises a plurality of wells 21. The plurality of wells 21 are separated from the adjacent wells 21 by the partition portion 22. A culture vessel 10 is formed in each of the plurality of wells 21.
The structural example of the culture container used by FIG. 3 at embodiment of this invention is shown. FIG. 4 is a cross-sectional view taken along line IV-IV of the culture vessel shown in FIG.
The culture vessel 10 has a culture space 11, a wall 12 and a bottom 13.
The culture space 11 is a region partitioned by the wall 12 and the bottom 13, and serves as a three-dimensional space region (culture region) in which cells are cultured. The culture space 11 is also referred to simply as "space" or "microspace".
The wall 12 is a partition that divides the culture space 11, and can be said to be a convex portion that forms a concavo-convex pattern in the culture container 10. When the culture space 11 is adjacent to the partition 22, the wall 12 may be the same as a part of the wall of the partition 22 as shown in FIG. 2A, or as shown in FIG. 2B. The wall 12 may be disposed adjacent to the wall surface.
The bottom portion 13 functions as a substrate of the culture container 10, and the surface on which the culture space 11 is disposed is a part of the culture region (culture surface). The bottom 13 is the same area as the bottom of each well 21 formed in the culture plate 1, and the bottom of each well 21 is used. The bottom 13 forms the bottom of the culture space 11. The surface of the bottom which is a part of the surface of the bottom 13 which forms the culture space 11 and which is to be a culture region is also referred to as “bottom culture surface 14”.

3 and 4 show the equivalent diameter D, height (depth) H, width (thickness) W of the wall 12 and thickness T of the bottom 13 with respect to the culture space 11 formed in the culture vessel 10 . In FIG.3, 4, the bottom part 13 has shown the case where it was produced integrally with the wall 12. As shown in FIG.
The equivalent diameter D refers to the diameter of the inscribed circle inscribed in the culture space 11. More specifically, the equivalent diameter D is the shape of the surface parallel to the bottom 13 of the culture space 11 (front shape), in other words, the inscribed circle of the shape perpendicular to the direction of the height H of the culture space 11 The diameter of When the shape of the front of the culture space 11 differs depending on the height H, the maximum value of the space area for culturing the hepatocytes is taken as the equivalent diameter.
The height H is a length from the bottom of the culture space 11 (bottom culture surface 14) to the upper surface of the wall 12, and can also be said to be the depth of the culture space 11. When the bottom culture surface 14 is flat, the height H is the same as the height of the wall 12.
The width W of the wall 12 is the thickness of the wall 12 and can also be said to be the distance separating the adjacent culture spaces 11.

In culture container 10 (in other words, in each well 21), a plurality of culture spaces 11 are arranged in an array as shown in FIG. The number or size of culture spaces 11 contained in culture vessel 10 depends on the number of wells 21 (size of wells 21) prepared in culture plate 1 and the sizes of culture spaces 11 and walls 12 . Specifically, as the number of wells 21 increases, the number of culture spaces 11 decreases. In the case of wells 21 of the same size, the number of culture spaces 11 in the wells 21 has a relation of decreasing when the equivalent diameter D is large or the width W is large. FIGS. 1 to 4 are schematic views in which the number of culture spaces 11 is reduced to express the configuration in an easily understandable manner, and the number of culture spaces 11 included in the culture vessel 10 is different from the actual number. In addition, in FIGS. 3 and 4, nine culture spaces 11 are shown. This is shown for the purpose of explanation, and does not correspond to the number of culture spaces 11 included in the actual culture container 10 (each well 21).

We have found that the diameter of the aggregates is between 30 and 200 μm, and in order to make aggregates of this size, the equivalent diameter D is 1 to 5 times the diameter of the desired aggregates, and the height is A culture vessel 10 having a plurality of culture spaces 11 in which H is 0.1 times to 3 times the equivalent diameter D and having a water contact angle of 45 degrees or less on the surface of the culture spaces is used. It has been found that by culturing the cells it is possible to culture aggregates of hepatocytes of the above mentioned diameter.

The size, shape, etc. of the micro-order culture space 11 for forming a desired aggregate and characteristics of the culture surface will be described with reference to FIGS. 1 to 4.
* Size and shape of culture space, etc. After seeding the cells, they will not move over the wall 11 and move to the adjacent culture space 11, that is, the cells may be retained in the culture space 11 until aggregates are formed. It becomes important. Therefore, it is preferable that the height H be 0.1 to 3 times the equivalent diameter D. It is preferable that H be high to retain cells, and high to smoothly supply nutrients. The height H is more preferably 0.2 to 1 times the equivalent diameter D because the height H is preferably lower. Since the equivalent diameter of the aggregate is defined by the culture space 11, the equivalent diameter D of the culture space 11 is preferably in the range of 1 to 5 times the desired diameter of the aggregate and in the range of 1.2 to 4 times More preferable.
For example, in order to form aggregates of hepatocytes having a diameter of 100 μm, the height H of the equivalent diameter is within the range of 1 to 5 times the desired diameter of the aggregate, ie, the equivalent diameter D is 100 to 500 μm. A culture vessel 10 having a bottom 13 in which culture spaces 11 ranging from 0.1 to 3 times are regularly arranged is used.

In one embodiment, in order to diffuse or transport the test solution to the center of the aggregate, the diameter of the aggregate is preferably at most 200 μm, preferably 150 μm or less. In addition, in order to maximize interaction between cells, the diameter of the aggregate is preferably at least 50 μm, more preferably in the range of 60 μm to 150 μm.

The width W of the wall 12 is the thickness of the wall 12 separating the culture space 11 and the culture space 11 adjacent thereto. Therefore, the width W of the wall 12 is 10 μm or more and less than 50 μm in order to prevent migration of cells beyond the upper surface of the wall 12 and to facilitate entry of the cells into the culture space 11. The size of one body or less, that is, the range of 5 to 30 μm is preferable, and the range of 5 to 10 μm is more preferable. Furthermore, from the same viewpoint, the angle θ between the upper surface of the wall 12 and the side surface of the culture space 11 is preferably in the range of 90 to 135 degrees, and more preferably in the range of 90 degrees to 120 degrees.

FIG. 5A is a schematic view showing a state in which the aggregate is cultured in the culture space 11. In FIG. 5A, the aggregate 9 is indicated by a circle using the cross-sectional view shown in FIG. Aggregates 9 are cultured in each of a plurality of culture spaces 11.
In the case of culturing on the culture plate 1 shown in FIG. Therefore, in order to form a plurality of culture spaces 11 in each well 21, it becomes possible to culture a plurality of aggregates under the same conditions in each well 21. In addition, since aggregates can be cultured using a well plate, it becomes possible to use an apparatus or the like used in conventional cell culture.
Assuming that the diameter DSP of the aggregate 9 is a value dsp (dsp is a positive numerical value), the equivalent diameter D of the culture space 11 is preferably in the range from the value dsp to five times the value dsp (dsp ≦ D ≦ 5 dsp) . Further, the height H of the culture space 11 is preferably in the range of 0.3 times the value dsp to 25 times (5 × 5) the value dsp (0.3 dsp ≦ H ≦ 25 dsp).

FIG. 5B is a schematic view illustrating an example of a preferable size of the aggregate cultured in the culture space. FIG. 5B is a view schematically showing an end face of a cut portion cut along the equivalent diameter D of the aggregate 9. As described above, the average diameter of the aggregates is preferably 30 μm or more and less than 200 μm, and particularly preferably in the range of 60 μm to 150 μm. FIG. 5B shows that the end face of the cut portion of aggregate 9 is formed of five cells 8. For example, in the case where the diameter DCL of the cells 8 is 20 μm and the diameter DSP of the aggregates 9 forms the aggregates 9 of 60 μm, for example, three cells 8 are formed in a straight line. Similarly, when the diameter DSP of the aggregate 9 forms an aggregate 9 of 150 μm, it is formed, for example, by arranging five cells 8 on a straight line. FIG. 5B schematically shows the cells 8 aligned on a straight line for ease of explanation, and the cells 8 are not necessarily aligned on a straight line.
In addition, the steric cells to be cultured may be in the case of culturing spheroids, which is a subordinate concept of aggregate 9. FIG. 5C is a schematic view showing a state in which the spheroid 9a is cultured in the culture space 11, and FIG. 5D is a schematic view illustrating an example of a preferable size of the spheroid 9a cultured in the culture space. The spheroid 9 a is a cell mass which is formed more spherically than the aggregate 9.

In addition, it is preferable that the aggregate in one test area (one well, one petri dish) contains 70% or more of the whole whose diameter is within the half width range. In other words, it is preferable that the diameters of the aggregates have the same size. The reason is as follows. First, since it is known that the value of the metabolic activity varies depending on the size of the aggregate, high accuracy results can not be obtained when aggregates of various diameters are mixed. In addition, it is known that the metabolic function of small aggregates (less than 50 μm) is extremely low. That is, small aggregates may have a small amount of metabolite and cell death may not be observed. When determining the toxicity, there is a possibility that the presence or absence of the toxicity can not be accurately determined because a part where cell death is occurring and a part where it does not exist are mixed in one well.

The shape of the culture space 11 (the shape of the front) or the shape of the plane parallel to the bottom 13 is not limited to the shape shown in FIG. 3 and may be, for example, a shape as shown in FIGS. Also, it may be another shape (such as an ellipse or a rhombus). In order to form an aggregate having a higher density and uniform diameter, it is preferable that the structure is symmetrical.
The shape of the side surface of the culture space 11 is not limited to the shape shown in FIG. 4, and may be, for example, the shape shown in FIGS. 7A to 7C.

As materials constituting the culture vessel 10, acrylic resin, polylactic acid, polyglycolic acid, styrene resin, acrylic / styrene copolymer resin, polycarbonate resin, polyester resin, polyvinyl alcohol resin, ethylene / vinyl alcohol It is selected from the group consisting of a base copolymer resin, a thermoplastic elastomer, a vinyl chloride resin, a silicone resin, and a combination thereof.

The thickness T of the bottom portion 13 of the culture vessel 10 is preferably 1 mm or less from the viewpoint of observability. However, 1 mm or more may be sufficient as long as it does not affect observation with a microscope, and the thickness T of the bottom portion 13 is not limited. By securing the observability of the bottom 13 of the culture vessel, it is possible to observe the cultured aggregate using the culture plate as it is. By securing the observability of the culture vessel, it becomes possible to apply an imaging technique such as fluorescence staining observation by an immunohistological method by using the culture vessel as it is.
In addition, the total light transmittance of the polymer constituting the bottom 13 of the culture vessel 10 is preferably 85% or more and less than 99%. The total luminous transmittance is measured in accordance with Japanese Industrial Standard (JIS K7375). By increasing the total light transmittance, the observability of the aggregate cultured on the bottom 13 can be secured. Furthermore, the thickness of the bottom of the culture vessel 10 (well) is preferably 300 μm or less.

* Characteristics of Culture Surface Next, the characteristics of the culture surface on which cells are cultured, that is, the wall 12 surrounding the culture space 11 and the bottom culture surface 14, in particular, the hydrophilization treatment will be described. The culture surface can not cover the surface because the culture medium contains the culture medium in each culture space 11, and when the coating solution is used, the solution does not enter the culture space 11. For this reason, it is preferable to make a water contact angle 45 degrees or less. More preferably, it is in the range of 0 degrees to 20 degrees. Further, the value of the water contact angle is assumed to be a value obtained by preparing and measuring a flat plate on which the concavo-convex pattern of the culture space 11 and the wall 12 is not formed, under the same conditions as the culture vessel 10.

When the culture space 11 is arranged in an array, if the surface is highly hydrophobic and the water contact angle exceeds 45 degrees, that is, if the wettability is low, air bubbles will form in the space when the culture medium or the coating solution is added. It may be easy to enter, resulting in spaces where cells can not be cultured. Therefore, it is necessary to perform hydrophilization so that the water contact angle is 45 degrees or less. As a method of making it hydrophilic, a method of depositing SiO ₂ and a method of performing plasma treatment may be mentioned.

In addition, in order to form aggregates efficiently in the culture vessel 10, it is preferable to increase cell adhesion in primary hepatocytes and to suppress cell adhesion in the case of established hepatocytes.
After performing the above-described hydrophilization treatment, aggregates can be efficiently formed by coating a substance that promotes cell adhesion or a substance that suppresses cell adhesion to control adhesion. For example, after plasma treatment to reduce the water contact angle to 45 degrees or less, poly-L-lysine may be coated to enhance cell adhesion.

2. Procedure from culture treatment to judgment treatment In the toxicity screening method of one embodiment, it is preferable to use a culture plate provided with a plurality of wells from the viewpoint of operability. In particular, it is more preferable to perform culture treatment (first step) to fluorescence probe treatment (third step) in the same well 21 (in other words, in the same cell container 10). In particular, it is preferable that a culture vessel having a plurality of culture spaces formed by the concavo-convex pattern be formed in each well of the culture plate. Therefore, in one embodiment, the case of performing the steps from the culture treatment to the fluorescent probe treatment in one well 21 of the plurality of wells 21 using the plurality of wells 21 of the culture plate 1 shown in FIG. 1 will be described Do. In other words, culture treatment, test compound treatment, and fluorescent probe treatment are performed in one well 21 and cells are not moved to another well 21 in each treatment.
For example, when screening a large number of compounds simultaneously, an automatic culture apparatus or an automatic analyzer is used. In such a case, in order to reduce the risk of contamination, it is desirable to perform the test compound treatment and the fluorescent probe treatment without transferring the sterically cultured hepatocytes formed by the culture treatment to another container. .
In addition, it is preferable to select any of 6, 24, 48, 96, and 384-well culture plates according to the number of specimens using a culture plate (well plate) shape having a plurality of wells.

* Culture treatment In the culture treatment, a plurality of culture spaces 11 formed by the culture vessel 10 described above are used. A plurality of culture spaces 11 are formed in each well 21 in the well plate. The hepatocytes are three-dimensionally cultured in each culture space 11 to form a plurality of hepatocytes in a plurality of culture spaces 11. In other words, cells are three-dimensionally cultured in a well plate to form a plurality of hepatocytes of a desired size.
The source of hepatocytes to be cultured is preferably selected from any of human, rodent, rat, dog and monkey. In addition, the hepatocytes are more preferably primary hepatocytes.

The equivalent diameter of the hepatocytes to be produced is preferably 30 μm or more and less than 200 μm.
It is preferable to carry out the addition treatment after confirming whether the formed aggregate expresses a metabolic enzyme that metabolizes the test compound.

The method for obtaining the hepatocytes in the aggregate form is not particularly limited, such as roller bottle culture, spinner flask culture, hanging drop culture and the like. However, in these methods, since an apparatus corresponding to the culture method is used, it is necessary to perform aggregate formation treatment and contact treatment in separate containers. The inventors discovered that the aggregate formation treatment and the contact treatment can be performed in the same container by using the well 21 in which the culture container 10 described above is formed. This simplifies the operation and makes it possible to add the first to third media to the cells without moving the formed aggregates. In particular, it becomes possible to use for an automatic culture apparatus in the screening of medicines which evaluate many compounds at once. In addition, it becomes possible to prevent cell damage and contamination.

* Test compound treatment In the test compound treatment, any of the first to third solutions containing the test compound or not containing the test compound is exposed to sterically cultured hepatocytes. The first solution is a control solution containing no test substance. The second solution is a test solution containing a test compound. The third solution is a mixed solution in which one or more compounds (inhibitors of drug metabolizing enzyme) that inhibit the drug metabolizing enzyme reaction are mixed with a test compound. By exposing any of the first to third solutions to each hepatocyte, a plurality of hepatocytes contacted with different solutions are obtained. In other words, a plurality of hepatocytes exposed to the first solution, a plurality of hepatocytes exposed to the second solution, and a plurality of hepatocytes exposed to the third solution are obtained.
As a solvent used for the first to third solutions to be brought into contact with each hepatocyte, it is preferable to use a medium containing no phenol red in order to reduce background when obtaining a fluorescent staining image. In addition, it is preferable not to contain serum in order to eliminate the influence on cells by cytokines contained in the serum. On the other hand, when each test solution is brought into contact with cells for 48 hours or more, serum in the range of 0.1 to 1% may be added to maintain the physiological function of the cells.

The solvent of each of the first to third solutions may have an osmotic pressure of 200 to 315 mOsm / kg · H ₂ O, and may have a buffer action in the pH range of 6.8 to 8.4. In addition, in order to keep the physiological function of the cells constant, it is preferable to use one containing glucose and nutrients such as amino acids and vitamins. For example, by using a mixed medium of Dulbecco's modified Eagle's medium (DEM: Dulbecco's Eagle medium) and Nutrient Mixture F-12, the physiological function can be kept constant.
The concentrations of the test substance and the drug metabolizing enzyme inhibitor are prepared by bringing a solution of any drug concentration into contact with hepatocytes in the range of 1 hour to 96 hours, and adopting a concentration with a survival rate of over 80%. If the concentration is too low, it is assumed that no toxicity due to reactive metabolites is observed, so it is preferable to use a solution of a test substance with a concentration as high as possible without exceeding a viability of 80% as the maximum concentration. It is more preferable to use multiple concentrations of the test substance in the range of 1/2 to 1/100 of the maximum concentration.

* Fluorescent Probe Treatment Fluorescent probe treatment brings the solution containing the fluorescent probe into contact with a plurality of hepatocytes. The fluorescent probe is selected from the group consisting of a fluorescent probe that recognizes living cells, a fluorescent probe that recognizes dead cells, and a combination thereof.
A confocal laser microscope or a fluorescence microscope is used as an apparatus for obtaining a fluorescent stained image.

* Determination Process The fourth step is a determination process for determining the toxicity of the test compound. In the determination process, a fluorescent stained image is obtained using a plurality of stained hepatocytes, and the toxicity of the test compound is determined based on the data of the fluorescent stained image. When it is detected that the determination condition is satisfied from the fluorescent staining image, it is determined that the test compound (reactive metabolite) metabolized by the hepatocytes is a factor of cytotoxicity. In other words, when the test compound is metabolized by a drug-metabolizing enzyme possessed by hepatocytes, it can be determined that a reactive metabolite is produced and becomes a factor of cytotoxicity.
The determination of the fluorescent staining image uses a fluorescent region or a fluorescent intensity. Three types of determination conditions are shown below. It can be determined using any one or more of these determination conditions.

When using a fluorescent region, there are the following two types of determination conditions.
The fluorescent region is defined as follows.
A fluorescent region that recognizes living cells of hepatocytes contacted with the first solution is referred to as a first live region (A).
A fluorescent region that recognizes living cells of hepatocytes contacted with the second solution is referred to as a second live region (B).
A fluorescent region that recognizes living cells of hepatocytes contacted with the third solution is referred to as a third live region (C).
Let it be the first dead area (D) of the fluorescent area that recognizes dead cells of the hepatocytes contacted with the first solution.
The second dead area (E) of the fluorescent area that recognizes dead cells of the hepatocytes contacted with the second solution is used.
Let it be the third dead area (F) of the fluorescent area that recognizes dead cells of the hepatocytes contacted with the third solution.

The first determination condition is that at least one of the following (1) and (2) determines that the test compound that has been metabolized by hepatocytes is a factor of cytotoxicity.
(1) The first live area is larger than the second live area, and the second live area is smaller than the third live area (A> B and B <C).
(2) The first dead area is smaller than the second dead area, and the second dead area is larger than the third dead area (D <E and E> F)
The second judgment condition is
The value obtained by dividing the first dead area by the first live area is smaller than the value obtained by dividing the second dead area by the second live area, and the value obtained by dividing the second dead area by the second live area is the third When it is larger than the value obtained by dividing the dead area by the third live area, it is determined that the test compound that has been metabolized by hepatocytes is a factor of cytotoxicity. It will be the following relational expression when it expresses with a symbol.
(D / A) <(E / B) and (E / B)> (F / C)

In the case of using the fluorescence intensity, the following determination conditions are satisfied.
The fluorescence intensity is defined as follows.
The fluorescence intensity for recognizing living cells of the hepatocytes brought into contact with the first solution is taken as a first life intensity (G).
The fluorescence intensity for recognizing live cells of the hepatocytes brought into contact with the second solution is taken as the second life intensity (H).
The fluorescence intensity for recognizing live cells of the hepatocytes brought into contact with the third solution is taken as the third life intensity (I).
Let the first death intensity (J) of the fluorescence intensity that recognizes dead cells of the hepatocytes brought into contact with the first solution.
The second death intensity (K) of the fluorescence intensity for recognizing dead cells of the hepatocytes contacted with the second solution is used.
The third death intensity (L) of the fluorescence intensity for recognizing dead cells of the hepatocytes contacted with the third solution is used.
The determination condition is determined that the test compound that has been metabolized by hepatocytes is a factor of cytotoxicity when at least one of the following (3) and (4).
(3) The first green strength is larger than the second green strength, and the second green strength is smaller than the third green strength (G> H and H <I).
(4) The first dead strength is smaller than the second dead strength, and the second dead strength is larger than the third dead strength (J <K and K> L).

The region to be subjected to the fluorescence observation is determined by a method of comparing the fluorescence intensity or the area (region) of the hepatocytes brought into contact with the first to third solutions visually or quantitatively. For this reason, the state of the cells prior to the test compound treatment operation needs to be the same in all observation areas. Even in the case of culturing under the same conditions (cell seeding density, medium, etc.), it is assumed that the adhesion area of cells on the bottom of the culture differs depending on the field of view. Therefore, before performing the test compound treatment operation, a microscope is used in advance to select several places having the same area (area) in which the hepatocytes brought into contact with the first to third solutions are present, and select several steps. Then, it is preferable to shoot a predetermined place.
When this method can not be used, it is preferable to calculate and determine the ratio of the area of dead cells to the area of living cells (area of dead cells / area of living cells).

As described above, according to one embodiment, whether the test substance is toxic due to a metabolite metabolized by a metabolic enzyme by simultaneously adding the test substance and the metabolic enzyme inhibitor to sterically cultured cells It can provide a new screening system to visualize and evaluate live and dead cells.
In addition, since the culture treatment of one embodiment can evaluate which metabolic enzyme is caused when a single cell expresses toxicity as compared to the culture method of Patent Document 1, multiple cells are maintained. It is excellent at the point which can save time and effort. In addition, the culture step of one embodiment can be three-dimensional culture without using a gel as described in Non-Patent Document 1. Furthermore, in the screening method of one embodiment, the process from the culture treatment to the fluorescent probe treatment uses a culture plate with high light permeability and uses the same culture plate in a series of treatments. Therefore, it is excellent in that visualization with a microscope is also possible.

Although the example of the toxicity screening method of one embodiment is described, one embodiment is not limited to these examples.

[Example]
1. Cell preparation (culture treatment)
(1-1) Preparation of Hepatocytes Primary rat hepatocytes used for culture were prepared as follows. A surflow indwelling needle was inserted into the portal vein of a 6-week-old SD rat, and the blood containing an EDTA solution was flushed to perform blood removal, and then the collagenase solution was refluxed. Thereafter, the collagenase solution-treated liver was added to the culture solution, and hepatocytes were dispersed by pipetting with a female pipette. The hepatocyte suspension was washed three times to remove cells other than hepatocytes, and the isolated hepatocytes were used for culture.

(1-2) Culture container A culture plate (24 well culture plate) 1a having 24 wells 21a shown in FIG. 8 was used. A plurality of culture vessels 10a are formed on the bottom culture surface of each well 21a by a concavo-convex pattern (fine pattern). Each culture container 10a has a culture space 11a formed with a corresponding diameter D of 200 μm and a height H of 50 μm in the bottom culture surface 14 (culture bottom). Also, the width W of the wall 12a is 10 μm.
The above-mentioned concavo-convex pattern was produced by photolithography and Ni electrolytic plating was performed to obtain a mold having a corresponding concavo-convex shape. Using the mold, pattern transfer was performed on polystyrene by hot embossing, and a resin base of the above dimensions was produced. A silicon dioxide film was formed to 100 nm on the surface of the resin base material by vacuum deposition, and gamma ray sterilization was performed to obtain a plurality of cell containers 10 formed by the concavo-convex pattern. The hepatocytes were cultured in a plurality of culture spaces 11 formed by a plurality of culture vessels 10.

(1-3) Culture Method Primary rat hepatocytes were seeded in a medium at 1 × 10 ⁵ cells / cm ² and cultured for 5 days.
The culture solution used for culture was prepared as follows. 10% fetal bovine serum, 1 μg / ml insulin, 1 × 10 ^-7 mol / L dexamethasone, 10 mM nicotinamide, 2 mmol / L L-glutamine, 50 μm β-mercaptoethanol, 5 mmol / L HEPES, 59 μg in DMEM / F12 medium ml penicillin, 100 μg / ml streptomycin, 20 ng / ml EGF were added. Hepatocytes were seeded at a concentration of 1.0 × 10 ⁵ cells / cm ² on the concavo-convex patterned substrate, and cultured at 37 ° C. for 5 minutes with ⁵ vol% CO ₂ . In addition, medium exchange was performed every one to two days using 0.5 mL of fresh medium of the same composition.

(1-4) Cultured Cells FIG. 9 shows cultured hepatocytes. It can be seen that three-dimensional culture is possible.

2. Test conditions and procedures (2-1) Test compounds Solutions (i) to (iii) shown in Table 1 were used. As a test substance, acetaminophen and 1-aminobenzotriazole (ABT) were used as an inhibitor of cytochrome P450.
Acetaminophen is known to be metabolized by CYP2E1 belonging to the cytochrome P450 species and to exhibit toxicity.

(2-1) Test procedure Item 1. Hepatocytes are sterically cultured according to the procedure described in cell preparation.
Next, after the medium is sucked and washed with phosphate buffer, any one of solutions (i) to (iii) of Table 1 is added to each well to obtain a plurality of wells to which different solutions are added. In each well, hepatocytes and the added solution were allowed to react for 24 hours.
After the reaction, two fluorescent reagents, Calcein-AM and MCB, were used to stain live cells. Calcein-AM is cell permeable and is hydrolyzed by intracellular esterases to form Calcein and exhibits green fluorescence. In addition, since cell death is known to occur by depletion of glutathione, cells were stained using MCB (monochlorobimane) that reacts with glutathione and emits blue fluorescence.

3. Test result (visual observation result)
FIG. 10 shows photographs of fluorescent stained images of hepatocytes contacted with solutions (i) to (iii).
The column of Calcein is a fluorescent stained image stained with Calcein-AM. The row of MCB is a fluorescent stained image stained with MCB. The Merge column is an image obtained by combining the fluorescent stained image of Calcein-AM and the fluorescent stained image of MCB.
The difference in the fluorescence intensity is observed by the solutions (i) to (iii). It can be judged that Calcein which recognizes living cells and MCB show toxicity as A> B and B <C, and acetaminophen is metabolized by cytochrome P450.

[Comparative example]
1. Cell preparation (culture treatment)
The culture plate used was a commercially available culture bottom plate (Becton Dickinson, Falcon (registered trademark) γ-ray-sterilized flat 24-well culture plate).
Rat primary hepatocytes were seeded at 1 × 10 ⁵ cells / cm ² and cultured for 7 days.
The conditions were the same as in the example except for the above.
2. Test conditions and procedures (2-1) Test compounds Solutions (i) to (iii) shown in Table 2 were used.

(2-1) Test procedure Item 1. The hepatocytes are cultured using planar culture plates according to the procedure described in cell preparation.
Next, after the medium is sucked and washed with phosphate buffer, any one of solutions (i) to (iii) of Table 2 is added to each well to obtain a plurality of wells to which different solutions are added. In each well, hepatocytes and the added solution were allowed to react for 24 hours.
After the reaction, two fluorescent reagents, Calcein-AM and MCB, were used to stain live cells.
3. Test result (visual observation result)
FIG. 11 shows photographs of fluorescent stained images of hepatocytes contacted with solutions (i) to (iii). FIG. 11 shows the results for each staining reagent stained in each row, as in FIG.
No toxicity could be detected at the same level of staining intensity for all solutions (i) to (iii).

It goes without saying that the test substance, the metabolic enzyme, and the inhibitor thereof used in the above-described examples are examples, and the evaluation method of one embodiment can be applied to other inhibitors.

The present invention is not limited to the embodiments described above. Within the scope of the present invention, each element of the above embodiments can be changed, added or converted into contents that can be easily considered by those skilled in the art.

This application claims priority based on Japanese Patent Application No. 2012-231226, filed Oct. 18, 2012, the entire disclosure of which is incorporated herein.

DESCRIPTION OF SYMBOLS 1, 1a culture plate 8 cell 9 aggregate 9a spheroid 10, 10a culture container 11 and 11a culture space 12 wall 13 bottom part 14 bottom part culture surface 21, 21a well 22 partition part

Claims (20)

1. A toxicity screening method for determining whether a test compound is metabolized by a drug metabolizing enzyme in the liver and exhibits toxicity,
   Three-dimensionally culturing a plurality of hepatocytes;
   Each of the first solution not containing the test compound, the second solution containing the test compound, and the third solution of one or more inhibitors inhibiting the drug-metabolizing enzyme reaction and the test compound Obtaining the plurality of hepatocytes contacted with different solutions by exposing to
   Contacting the plurality of hepatocytes with a solution containing a fluorescent probe selected from the group consisting of a fluorescent probe that recognizes living cells, a fluorescent probe that recognizes dead cells, and a combination thereof;
   Obtaining a fluorescent stained image using the plurality of hepatocytes after staining, and determining the toxicity of the test compound based on the data of the fluorescent stained image;
   Toxicity screening methods including:
2. In the step of determining the toxicity of the test compound, a fluorescent region recognizing live cells of hepatocytes contacted with the first solution is recognized as a first live region, and live cells of hepatocytes contacted with the second solution are recognized. Second fluorescent region, the fluorescent region that recognizes living cells of hepatocytes in contact with the third solution, the third biological region, fluorescence that recognizes dead cells of the liver cells in contact with the first solution A first dead area of the area, a second dead area of a fluorescent area that recognizes dead cells of the hepatocytes contacted with the second solution, a fluorescent area that recognizes dead cells of the hepatocytes contacted with the third solution In the case of the third death zone,
   First live area> second live area, and second live area <third live area, and
   First dead area <second dead area, and second dead area> third dead area,
   2. The toxicity screening method according to claim 1, wherein the test compound that has been metabolized by each hepatocyte is determined to be a factor of cytotoxicity when it is at least one of the above.
3. In the step of determining the toxicity of the test compound, a fluorescent region recognizing live cells of hepatocytes contacted with the first solution is recognized as a first live region, and live cells of hepatocytes contacted with the second solution are recognized. Second fluorescent region, the fluorescent region that recognizes living cells of hepatocytes in contact with the third solution, the third biological region, fluorescence that recognizes dead cells of the liver cells in contact with the first solution A first dead area of the area, a second dead area of a fluorescent area that recognizes dead cells of the hepatocytes contacted with the second solution, a fluorescent area that recognizes dead cells of the hepatocytes contacted with the third solution In the case of the third death zone,
   (First dead area / first live area) <(second dead area / second live area) and (second dead area / second live area)> (third dead area / third live area) 2. The toxicity screening method according to claim 1, wherein the test compound that has been metabolized by each hepatocyte is determined to be a factor of cytotoxicity at one time.
4. In the step of determining the toxicity of the test compound, the fluorescence intensity that recognizes living cells of the hepatocytes contacted with the first solution has a first life intensity, and the living cells of the hepatocytes contacted with the second solution are recognized The second fluorescence intensity is the second fluorescence intensity, the fluorescence intensity of the living cells of the hepatocytes contacted with the third solution is recognized. The third fluorescence intensity is the fluorescence intensity of dead cells of the hepatocytes contacted with the first solution. Strong first death intensity, second death intensity of fluorescence intensity to recognize dead cells of hepatocytes contacted with the second solution, fluorescence intensity of dead cells of hepatocytes contacted to the third solution If the third death intensity,
   First green strength> second green strength, and second green strength <third green strength,
   1st death intensity <2nd death intensity and 2nd death intensity> 3rd death intensity,
   2. The toxicity screening method according to claim 1, wherein the test compound that has been metabolized by each hepatocyte is determined to be a factor of cytotoxicity when it is at least one of the above.
5. The plurality of hepatocytes are cultured using a culture plate having a plurality of wells,
   The plurality of hepatocytes are cultured in each well, and any one of the first to third solutions is added to each well and reacted without transferring the plurality of cultured hepatocytes to another container. The toxicity screening method according to any one of claims 1 to 4, wherein the fluorescent probe is subsequently added to each of the wells.
6. The toxicity screening method according to any one of claims 1 to 5, wherein each hepatocyte forms an aggregate in which cells are aggregated.
7. The toxicity screening method according to claim 6, wherein the equivalent diameter of the aggregate is 30 μm or more and less than 200 μm.
8. The compound according to any one of claims 1 to 7, wherein the one or more compounds that inhibit the drug metabolizing enzyme reaction are selected from the group consisting of a cytochrome P450 enzyme group, a phase II drug metabolizing enzyme, and a combination thereof. The toxicity screening method according to any one of the above.
9. The one or more compounds that inhibit the drug metabolizing enzyme reaction are selected from the group consisting of cytochrome P450 enzymes, which are selected from the group consisting of CYP1A1, CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2C19, CYP2C11, CYP2D6, and CYP2E1. The toxicity screening method according to any one of claims 1 to 7, which is characterized.
10. The toxicity screening method according to any one of claims 1 to 9, wherein the plurality of hepatocytes are selected from any of human, rodent, rat, dog, and monkey. .
11. The toxicity screening method according to any one of claims 1 to 10, wherein the plurality of hepatocytes are primary hepatocytes.
12. The plurality of hepatocytes are cultured using a culture plate having a plurality of wells,
    Each well is formed with a culture vessel having a plurality of culture spaces,
    The plurality of culture spaces are formed such that the height from the bottom of each culture vessel is surrounded by a wall in the range of 25 to 500 μm, and spaces in the range of 50 to 1000 μm in equivalent diameter are regularly arranged.
    The step of culturing the plurality of hepatocytes in a three-dimensional manner comprises producing aggregates of the plurality of hepatocytes using the plurality of culture spaces, according to any one of claims 1 to 4. Toxicity screening method.
13. The toxicity screening method according to claim 12, wherein the thickness of the wall is 10 μm or more and less than 50 μm.
14. The toxicity screening method according to claim 12 or 13, wherein the total light transmittance of the polymer constituting the bottom of the culture vessel is 85% or more and less than 99%.
15. The culture vessel is made of acrylic resin, polylactic acid, polyglycolic acid, styrene resin, acrylic / styrene copolymer resin, polycarbonate resin, polyester resin, polyvinyl alcohol resin, ethylene / vinyl alcohol copolymer resin, The toxicity screening method according to any one of claims 12 to 14, which is a resin molded product selected from the group consisting of thermoplastic elastomers, vinyl chloride resins, silicone resins, and combinations thereof.
16. The thickness of the bottom part of the said culture container is 300 micrometers or less, The toxicity screening method as described in any one of the Claims 12 thru | or 15 characterized by the above-mentioned.
17. The toxicity screening method according to any one of claims 12 to 16, wherein the bottom culture surface of the culture vessel is hydrophilized.
18. The toxicity screening method according to any one of claims 12 to 17, wherein the bottom culture surface of the culture vessel is made of glass and treated to have a water contact angle of 45 degrees or less.
19. The bottom culture surface of the said culture container was made to form a functional group by plasma processing, and was processed so that a water contact angle might be 45 degrees or less. Toxicity screening method.
20. The hydrophilized bottom culture surface is further selected from the group consisting of poly-L-lysine, poly-D-lysine, collagen, laminin, fibronectin, and combinations thereof, which are polymers promoting cell adhesion. The toxicity screening method according to claim 17, characterized in that the polymer to be immobilized is immobilized.

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