Method for evaluating effect of cytokine on metabolic activity of cytochrome p450, and drug screening method

Abstract

Provided is a technique that uses an established hepatocyte cell line in a method for evaluating an effect of a cytokine on a metabolic activity of a cytochrome P450 and in a method for evaluating a drug which interacts with a cytokine. The method for evaluating an effect of a cytokine on a metabolic activity of a cytochrome P450 includes: culturing an established hepatocyte cell line by using a culture chamber ( 10 ) including culture rooms ( 11 ), to thereby form spheroids ( 9 ); and evaluating the presence or absence of induction or attenuation of the cytochrome P450 after bringing a spheroid-shaped established hepatocyte cell line into contact with a test solution containing the cytokine in the culture chamber for one hour or more and less than 96 hours.

1 . A method for evaluating an effect of a cytokine on a metabolic activity of a cytochrome P450, the method comprising:
 evaluating the presence or absence of induction or attenuation of a metabolic function of the cytochrome P450 after bringing a spheroid-shaped established hepatocyte cell line into contact with a test solution containing a cytokine in a culture chamber for one hour or more and less than 96 hours.   
     
     
         2 . The method according to  claim 1 , wherein an average value of diameters of spheroids formed from the established hepatocyte cell line is equal to or more than 50 μm and less than 200 μm, and spheroids having a diameter within a half-width range account for 70% or more of all spheroids. 
     
     
         3 . The method according to  claim 1 , wherein the test solution is a serum-free culture medium. 
     
     
         4 . The method according to  claim 1 , wherein at least three types of test solutions are used, the at least three types of test solutions containing the cytokine at a concentration equal to a reference concentration, at a concentration that is 10 times higher than the reference concentration, and at a concentration that is 100 times higher than the reference concentration, respectively, the reference concentration being one of cytokine concentrations of the test solutions which are in a range from 0.1 times to 50 times higher than an average value of blood levels of cytokines in a healthy human. 
     
     
         5 . The method according to  claim 1 , wherein at least three types of test solutions are used, the at least three types of test solutions containing the cytokine at a concentration equal to a reference concentration, at a concentration that is 10 times higher than the reference concentration, and at a concentration that is 100 times higher than the reference concentration, respectively, the reference concentration being one of cytokine concentrations of the test solutions which are in a range from 0.1 times to 50 times higher than an average value of blood levels of the cytokine secreted from a patient with a disease. 
     
     
         6 . The method according to  claim 1 , wherein a step of culturing the established hepatocyte cell line into a spheroid shape is carried out in a well and a step of bringing the spheroid-shaped established hepatocyte cell line into contact with the test solution containing the cytokine is carried out in the same well. 
     
     
         7 . The method according to  claim 1 , wherein one well of a culture plate having a plurality of wells is used as the culture chamber, and the method comprises:
 a step of forming, in the one well, a spheroid of the established hepatocyte cell line by using a culture medium containing 10% serum;   a step of removing the culture medium from the one well;   a step of adding the test solution containing the cytokine to the one well; and   a step of bringing the test solution containing the cytokine into contact with the spheroid in the one well for one hour or more and less than 96 hours.   
     
     
         8 . The method according to  claim 1 , wherein
 the culture chamber is formed so as to include a plurality of culture rooms in each well of a culture plate including a plurality of wells,   each of the culture rooms is a space having a height and a surface with a length corresponding to an equivalent diameter, and a value obtained by dividing the height by the equivalent diameter is in a range from 0.3 to 2, and   at least two culture rooms having the equivalent diameter in a range from 100 μm to 1000 μm are disposed at a bottom of the culture rooms.   
     
     
         9 . The method according to  claim 8 , wherein adjacent culture rooms among the plurality of culture rooms are partitioned by a wall having a thickness in a range from 2 μm to 50 μm. 
     
     
         10 . The method according to  claim 8 , wherein an angle formed between an upper surface and a side surface of a wall partitioning the culture rooms is in a range from 90 degrees to 135 degrees. 
     
     
         11 . The method according to  claim 8 , wherein a bottom surface of each of the plurality of culture rooms is processed by glass processing so that the bottom surface has a water contact angle of 45 degrees or less. 
     
     
         12 . The method according to  claim 8 , wherein a bottom surface of each of the plurality of culture rooms is processed in such a manner that a functional group is formed on the bottom surface by a plasma treatment and the bottom surface has a water contact angle of 45 degrees or less. 
     
     
         13 . The method according to  claim 1 , wherein a polymer selected from the group consisting of a polymer having hydrophilic and hydrophobic properties that vary depending on a polymer temperature or light, a hydrophilic polymer chain that inhibits cell adhesion, phospholipid, a phospholipid-polymer complex, poly(2-hydroxyethyl methacrylate) (PHEMA), polyvinyl alcohol, agarose, chitosan, polyethyleneglycol, and albumin, or a combination thereof, is immobilized on a surface of the culture chamber. 
     
     
         14 . The method according to  claim 1 , wherein a mixture of a polymer selected from the group consisting of a hydrophilic polymer chain that inhibits cell adhesion, phospholipid, a phospholipid-polymer complex, poly(2-hydroxyethyl methacrylate) (PHEMA), polyvinyl alcohol, agarose, chitosan, polyethyleneglycol, and albumin, or a combination thereof, and a polymer selected from the group consisting of poly-L-lysine, poly-L-lysine, collagen, laminin, and fibronectin, each of which is a polymer that promotes cell adhesion properties, or a combination thereof is immobilized on a surface of the culture chamber. 
     
     
         15 . The method according to  claim 1 , wherein the culture chamber is a resin molded product that is made of one selected from the group consisting of acrylic resin, polylactic acid, polyglycolic acid, styrene resin, acrylic-styrene copolymer resin, polycarbonate resin, polyester resin, polyvinyl alcohol resin, ethylene-vinylalcohol copolymer resin, thermoplastic elastomer, vinyl chloride resin, and silicon resin, or a combination thereof. 
     
     
         16 . The method according to  claim 1 , wherein a total light transmittance of polymers that form a bottom portion of the culture chamber is equal to or more than 85% and less than 99%. 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled)