Agent for forming spheroids of hepatocytes and process for culturing hepatocytes for formation of spheroids

Claim 2

The agent according to claim 1 wherein a reducing terminal of said glycosaminoglycan is cleaved.

Claim 3

The agent according to claim 2 wherein a glycosaminoglycan is covalently bound to a lipid through a carboxyl group including lactone, a formyl group or a primary amino group, which is formed at a cleaved reducing terminal of a glycosaminoglycan, or said group of spacer compound introduced in a glycosaminoglycan.

Claim 4

The agent according to claim 2 wherein a lipid is covalently bound to a glycosaminoglycan whose reducing terminal is cleaved through a primary amino group, a carboxyl group or a formyl group of said lipid or said group of a spacer compound introduced in a lipid.

Claim 5

The agent according to claim 1 wherein said covalent bond between a lipid and a glycosaminoglycan is selected from: (1) a CONH bond between a carboxyl group including lactone at a cleaved reducing terminal of a glycosaminoglycan and a primary amino group of a lipid; (2) a CONH bond between a carboxyl group of a uronic acid moiety of a glycosaminoglycan and a primary amino group of a lipid; or (3) a CH₂NH bond between a formyl group at a cleaved reducing terminal of a glycosaminoglycan and a primary amino group of a lipid.

Claim 6

The agent according to claim 1 wherein said glycosaminoglycan to which a lipid is bound via a covalent bond is represented by the following formula: wherein P¹ is a lipid having a primary amino group and GAG is a glycosaminoglycan residue excluding a cleaved reducing terminal and; (1) GAG is located at the 4-position, R³ is located at the 3-position, R¹ is an OH group, R² is a COOH group and R³ is an OH group when GAG is a glycosaminoglycan residue of hyaluronic acid, chondroitin, chondroitin sulfate A, C or E, dermatan sulfate, heparin or heparan sulfate excluding a reducing terminal glucuronic acid moiety or when GAG is a glycosaminoglycan residue of dermatan sulfate excluding a reducing terminal iduronic acid moiety, (2) GAG is located at the 4-position, R³ is located at the 3-position, R¹ is an OSO₃H group, R² is a COOH group and R³ is an OH group when GAG is a glycosaminoglycan residue of chondroitin sulfate D excluding a reducing terminal glucuronic acid moiety or when GAG is a glycosaminoglycan residue of heparin or heparan sulfate excluding a reducing terminal iduronic acid moiety, (3) GAG is located at the 4-position, R³ is located at the 3-position, R¹ is an OH group, R² is a COOH group and R³ is an OSO₃H group when GAG is a glycosaminoglycan residue of chondroitin sulfate K excluding a reducing terminal glucuronic acid moiety, (4) GAG is located at the 4-position, R³ is located at the 3-position, at least one of R¹ and R³ is an OSO₃H group, while the other is an OH group, and R² is a COOH group when GAG is a glycosaminoglycan residue of chondroitin polysulfate excluding a reducing terminal glucuronic acid moiety, (5) GAG is located at the 3-position, R³ is located at the 4-position, each of R¹ and R³ is an OH group and R² is a CH₂OH group when GAG is a glycosaminoglycan residue of keratan sulfate excluding a reducing terminal galactose moiety, (6) GAG is located at the 3-position, R³ is located at the 4-position, each of R¹ and R³ is an OH group and R² is a CH₂OSO₃H group when GAG is a glycosaminoglycan residue of keratan polysulfate excluding a reducing terminal galactose moiety, (7) GAG is located at the 3-position, R³ is located at the 4-position, R¹ is an NHCOCH₃ group, R² is a CH₂OH group and R³ is an OH group when GAG is a glycosaminoglycan residue of hyaluronic acid or chondroitin excluding a reducing terminal hexosamine moiety, (8) GAG is located at the 3-position, R³ is located at the 4-position, R¹ is an NHCOCH₃ group, R² is a CH₂OH group and R³ is an OSO₃H group when GAG is a glycosaminoglycan residue of chondroitin sulfate A or K or dermatan sulfate excluding a reducing terminal hexosamine moiety, (9) GAG is located at the 3-position, R³ is located at the 4-position, R¹ is an NHCOCH₃ group, R² is a CH₂OSO₃H group and R³ is an OH group when GAG is a glycosaminoglycan residue of chondroitin sulfate C or D excluding a reducing terminal hexosamine moiety, (10) GAG is located at the 3-position, R³ is located at the 4-position, R¹ is an NHCOCH₃ group, R² is a CH₂OSO₃H group and R³ is an OSO₃H group when GAG is a glycosaminoglycan residue of chondroitin sulfate E excluding a reducing terminal hexosamine moiety, (11) GAG is located at the 3-position, R³ is located at the 4-position, R¹ is an NHCOCH₃ group, R² is a CH₂OH group and R³ is an OSO₃H group, or R² is a CH₂OSO₃H group and R³ is an OH group or an OSO₃H group, when GAG is a glycosaminoglycan residue of chondroitin polysulfate excluding a reducing terminal hexosamine moiety, (12) GAG is located at the 4-position, R³ is located at the 3-position, R¹ is an NHSO₃H group, R² is a CH₂OSO₃H group and R³ is an OH group when GAG is a glycosaminoglycan residue of heparin excluding a reducing terminal hexosamine moiety, (13) GAG is located at the 4-position, R³ is located at the 3-position, R¹ is an NHCOCH₃ group or an NHSO₃H group, R² is a CH₂OH group when R³ is an OSO₃H group, or R² is a CH₂OSO₃H group when R³ is an OH group or an OSO₃H group, when GAG is a glycosaminoglycan residue of heparan sulfate excluding a reducing terminal hexosamine moiety, (14) GAG is located at the 4-position, R³ is located at the 3-position, R¹ is an NHCOCH₃ group, R² is a CH₂OSO₃H group and R³ is an OH group when GAG is a glycosaminoglycan residue of keratan sulfate or keratan polysulfate excluding a reducing terminal hexosamine moiety.

Claim 7

The agent according to claim 1 wherein said lipid is phosphatidylethanolamine and said glycosaminoglycan is chondroitin sulfate C whose reducing terminal is cleaved.

Claim 8

The agent according to claim 1 wherein said glycosaminoglycan to which a lipid is bound is obtainable by oxidizing a reducing terminal of a glycosaminoglycan to cleave the reducing terminal, lactonizing the cleaved product and reacting the resulting lactone compound with a primary amino group of a lipid.

Claim 9

A process for forming spheroids of hepatocytes which comprises culturing hepatocytes with a lipid-bound glycosaminoglycan as a culture substrate.

Claim 10

The process according to claim 9 wherein the lipid-bound glycosaminoglycan of claim 6 is used as a culture substrate.

Claim 11

The process according to claim 9 wherein the phosphatidylethanolamine-bound chondroitin sulfate C of claim 7 is used as a culture substrate.

Claim 12

Spheroids of hepatocytes obtainable by culturing hepatocytes with a lipid-bound glycosaminoglycan as a culture substrate.

Claim 13

Use of glycosaminoglycan to which a lipid is covalently bound for manufacture of an agent for forming hepatocyte spheroids.