Method for culture of hepatocytes
A method for culturing hepatocytes, wherein hepatocytes embedded in an extracellular matrix is placed on a gas-permeable membrane and the hepatocytes are cultured while being supplied with oxygen from the gas-permeable membrane side. By this, the polarity in the hepatocytes can be induced and a bile canaliculus can be formed in a short period of time. Further, the formed polarity can be maintained for a longer period.
1. A method for producing cultured hepatocytes to form one or more bile canaliculi, comprising disposing hepatocytes embedded in an extracellular matrix on a gas-permeable membrane and culturing said hepatocytes while supplying oxygen from a gas-permeable membrane side, wherein a surface of said gas-permeable membrane is coated with collagen and said hepatocytes embedded in an extracellular matrix are disposed on the collagen-coated side of said gas-permeable membrane, and wherein said extracellular matrix constitutes a groove(s) or hollow, and said hepatocytes are disposed in said groove(s) or hollow, the width of the groove or hollow is not less than 30 μm and not more than 70 μm, and wherein one or more canaliculi are formed by the cultured hepatocytes.
2. A method for evaluating metabolism of a compound, comprising:
(a) disposing hepatocytes embedded in an extracellular matrix on a gas-permeable membrane and culturing said hepatocytes while supplying oxygen from a gas-permeable membrane side, wherein a surface of said gas-permeable membrane is coated with collagen and said hepatocytes embedded in an extracellular matrix are disposed on the collagen-coated side of said gas-permeable membrane, and wherein said extracellular matrix constitutes a groove(s) or hollow, and said hepatocytes are disposed in said groove(s) or hollow, the width of the groove or hollow is not less than 30 μm and not more than 70 μm, and wherein one or more canaliculi are formed;
(b) contacting said cultured hepatocytes with a candidate compound; and,
(c) evaluating metabolism of said compound in said cultured hepatocytes.
3. The method according to claim 1 , wherein said gas-permeable membrane is placed to form a tubular shape.
4. The method according to claim 1 , wherein said gas-permeable membrane is a polydimethylsiloxane membrane.
5. The method according to claim 1 , wherein said gas-permeable membrane is a fluorocarbon membrane.
6. The method according to claim 1 , wherein said collagen is coated by covalent bonding.
7. The method according to claim 1 , wherein said extracellular matrix is a collagen gel or a reconstituted basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma cells.
8. The method according to claim 1 , wherein said extracellular matrix is composed of one or more non-biological components.
9. The method according to claim 1 , wherein the width of the groove or hollow is not less than 30 μm and not more than 50 μm.