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WO2014061244A1 - Procédé de dépistage de la toxicité - Google Patents

Procédé de dépistage de la toxicité Download PDF

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Publication number
WO2014061244A1
WO2014061244A1 PCT/JP2013/006068 JP2013006068W WO2014061244A1 WO 2014061244 A1 WO2014061244 A1 WO 2014061244A1 JP 2013006068 W JP2013006068 W JP 2013006068W WO 2014061244 A1 WO2014061244 A1 WO 2014061244A1
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Prior art keywords
hepatocytes
culture
solution
cells
area
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English (en)
Japanese (ja)
Inventor
正剛 佐能
太田　茂
征岳 山頭
洋子 江尻
雅也 細田
賢 綾野
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Kuraray Co Ltd
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Kuraray Co Ltd
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Priority to JP2014541937A priority Critical patent/JP6035343B2/ja
Publication of WO2014061244A1 publication Critical patent/WO2014061244A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/795—Porphyrin- or corrin-ring-containing peptides
    • G01N2333/80—Cytochromes

Definitions

  • 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.
  • 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
  • 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.
  • phase II drug metabolizing enzyme reaction 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.
  • 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.
  • 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.
  • GSH glutathione
  • 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.
  • 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.
  • matrigel is expensive, there is a problem that the cost is high.
  • 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.
  • Non-Patent Document 1 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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
  • It is a figure which shows the schematic diagram explaining an example of the preferable size of the aggregate cultured by culture space.
  • FIG. 1 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.
  • cytochrome P450 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.
  • 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).
  • 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).
  • 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).
  • 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”.
  • the term “range from value A to value B” means “value A or more and value B or less” unless otherwise specified.
  • “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.
  • 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.
  • 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.
  • the culture container uses a culture container capable of three-dimensionally culturing hepatocytes.
  • any culture vessel may be used as long as a plurality of cells are stacked to produce hepatocytes having a three-dimensional shape.
  • a preferred example of a container for forming aggregates will be described as an example of the 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.
  • 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”.
  • FIG. 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 .
  • the bottom part 13 has shown the case where it was produced integrally with the wall 12.
  • the equivalent diameter D refers to the diameter of the inscribed circle inscribed in the culture space 11.
  • 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 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.
  • 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.
  • culture container 10 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).
  • the diameter of the aggregates is between 30 and 200 ⁇ m, 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.
  • 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.
  • 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 ⁇ m.
  • 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.
  • the diameter of the aggregate in order to diffuse or transport the test solution to the center of the aggregate, is preferably at most 200 ⁇ m, preferably 150 ⁇ m or less. In addition, in order to maximize interaction between cells, the diameter of the aggregate is preferably at least 50 ⁇ m, more preferably in the range of 60 ⁇ m to 150 ⁇ m.
  • 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 ⁇ m or more and less than 50 ⁇ m 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 ⁇ m is preferable, and the range of 5 to 10 ⁇ m is more preferable.
  • the angle ⁇ 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.
  • 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.
  • aggregates can be cultured using a well plate, it becomes possible to use an apparatus or the like used in conventional cell culture.
  • 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) .
  • 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.
  • the average diameter of the aggregates is preferably 30 ⁇ m or more and less than 200 ⁇ m, and particularly preferably in the range of 60 ⁇ m to 150 ⁇ m.
  • FIG. 5B shows that the end face of the cut portion of aggregate 9 is formed of five cells 8.
  • the diameter DCL of the cells 8 is 20 ⁇ m and the diameter DSP of the aggregates 9 forms the aggregates 9 of 60 ⁇ m, for example, three cells 8 are formed in a straight line.
  • the diameter DSP of the aggregate 9 forms an aggregate 9 of 150 ⁇ m, 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.
  • 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
  • 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.
  • the aggregate in one test area 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 ⁇ m) 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.
  • 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.
  • 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 ⁇ m or less.
  • 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.
  • the culture space 11 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.
  • a method of making it hydrophilic a method of depositing SiO 2 and a method of performing plasma treatment may be mentioned.
  • aggregates 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.
  • aggregates can be efficiently formed by coating a substance that promotes cell adhesion or a substance that suppresses cell adhesion to control adhesion.
  • poly-L-lysine may be coated to enhance cell adhesion.
  • a culture plate provided with a plurality of wells from the viewpoint of operability.
  • 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.
  • 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.
  • an automatic culture apparatus or an automatic analyzer is used.
  • 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.
  • 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.
  • the hepatocytes are more preferably primary hepatocytes.
  • the equivalent diameter of the hepatocytes to be produced is preferably 30 ⁇ m or more and less than 200 ⁇ m. 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.
  • an apparatus corresponding to the culture method 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • one containing glucose and nutrients such as amino acids and vitamins.
  • DEM Dulbecco's modified Eagle's medium
  • 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 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.
  • the fourth step is a determination process for determining the toxicity of the test compound.
  • 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.
  • the determination condition is satisfied from the fluorescent staining image
  • the test compound is metabolized by a drug-metabolizing enzyme possessed by hepatocytes
  • 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.
  • 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).
  • the second dead area (E) of the fluorescent area that recognizes dead cells of the hepatocytes contacted with the second solution is used.
  • 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).
  • 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
  • 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)
  • 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).
  • 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).
  • 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).
  • 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.
  • the culture step of one embodiment can be three-dimensional culture without using a gel as described in Non-Patent Document 1.
  • 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.
  • 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.
  • FIG. 8 (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 ⁇ m and a height H of 50 ⁇ m in the bottom culture surface 14 (culture bottom). Also, the width W of the wall 12a is 10 ⁇ m.
  • 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.
  • (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 ⁇ g / ml insulin, 1 ⁇ 10 -7 mol / L dexamethasone, 10 mM nicotinamide, 2 mmol / L L-glutamine, 50 ⁇ m ⁇ -mercaptoethanol, 5 mmol / L HEPES, 59 ⁇ g in DMEM / F12 medium ml penicillin, 100 ⁇ g / 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 .
  • medium exchange was performed every one to two days using 0.5 mL of fresh medium of the same composition.
  • FIG. 9 shows cultured hepatocytes. It can be seen that three-dimensional culture is possible.
  • Test compounds Solutions (i) to (iii) shown in Table 1 were used.
  • 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.
  • 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.
  • MCB dichlorobimane
  • 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.
  • 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).
  • 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.

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Abstract

Un procédé de dépistage de la toxicité consiste en un procédé permettant de déterminer si un composé d'intérêt est métabolisé ou pas dans le foie, par une enzyme impliquée dans le métabolisme des médicaments, pour être ainsi à l'origine d'une toxicité. Ledit procédé comprend les étapes consistant à cultiver de multiples hépatocytes de façon tridimensionnelle (par exemple sous la forme d'agglomérats) ; à amener chacun desdits hépatocytes en contact avec une première solution qui ne contient pas le composé d'intérêt, avec une deuxième solution qui contient le composé d'intérêt et avec une troisième solution qui contient à la fois au moins un composé capable d'inhiber une réaction faisant intervenir une enzyme impliquée dans le métabolisme des médicaments et le composé d'intérêt, ce qui permet d'obtenir de multiples hépatocytes ayant été respectivement mis en contact avec les différentes solutions ; à mettre les multiples hépatocytes en contact avec une solution contenant une sonde fluorescente capable de reconnaître des cellules vivantes ou avec une sonde fluorescente capable de reconnaître des cellules mortes ; et à obtenir des images de fluorescence des multiples hépatocytes colorés, puis à déterminer la toxicité ou pas du composé d'intérêt sur la base des données fournies par les images de fluorescence.
PCT/JP2013/006068 2012-10-18 2013-10-10 Procédé de dépistage de la toxicité Ceased WO2014061244A1 (fr)

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WO2018064675A1 (fr) * 2016-09-30 2018-04-05 Novira Therapeutics, Inc. Restriction métabolique dans des dosages basé sur des cellules
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US10377555B2 (en) 2015-04-15 2019-08-13 Dow Global Technologies Llc Flexible container with a spray valve
JP2017035081A (ja) * 2015-08-13 2017-02-16 サントリーホールディングス株式会社 動物細胞毒性又は抗酸化能の試験方法
JP2023093648A (ja) * 2015-11-25 2023-07-04 ディスカバリー ライフ サイエンシズ エルエルシー タンパク質を発現するためのシステム及び方法
JP2022046639A (ja) * 2015-11-25 2022-03-23 コーニング インコーポレイテッド タンパク質を発現するためのシステム及び方法
EP3519814B1 (fr) * 2016-09-30 2021-06-02 Novira Therapeutics, Inc. Restriction métabolique dans des dosages basé sur des cellules
WO2018064675A1 (fr) * 2016-09-30 2018-04-05 Novira Therapeutics, Inc. Restriction métabolique dans des dosages basé sur des cellules
JP2018108038A (ja) * 2016-12-28 2018-07-12 クアーズテック株式会社 細胞培養担体
RU2715388C1 (ru) * 2018-09-17 2020-02-27 Федеральное государственное бюджетное учреждение "Государственный научный центр Российской Федерации имени А.И. Бурназяна" (ФГБУ ГНЦ ФМБЦ им. А.И. Бурназяна ФМБА России) Способ подготовки и цитомный анализ гепатоцитов лабораторных животных и человека для оценки цитогенетического и цитотоксического действия факторов разной природы
WO2020149246A1 (fr) * 2019-01-18 2020-07-23 東洋製罐グループホールディングス株式会社 Récipient de culture, procédé de culture et dispositif de culture
JP2020202816A (ja) * 2019-06-19 2020-12-24 リプロサポートメディカルリサーチセンター株式会社 細胞を凍結する前段階で使用される器具
WO2020255444A1 (fr) * 2019-06-19 2020-12-24 リプロサポートメディカルリサーチセンター株式会社 Outil pour manipuler des cellules
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JP7785276B2 (ja) 2021-06-17 2025-12-15 三井化学株式会社 肝毒性の評価方法

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