Cell culture medium for human liver epithelial cell line

Claim 2

A cell culturing medium suitable for primary culture of normal human hepatocytes comprising the following ingredients: an effective amount of ornithine to allow proliferation of human hepatocytes; an effective cell growth promoting amount of growth factor inactivated serum; retinoic acid at concentration of 1 to 300 nM; an effective amount of calcium ions, which is represented by a concentration from 0.2 to 1 mM, high enough to promote human hepatocyte cell growth but low enough to avoid the inducement of terminal cellular differentiation; an effective cell growth promoting amount of glucose; an effective amount of insulin to aid cells in glucose uptake; an effective cell growth promoting amount of hydrocortisone; an effective amount of epidermal growth factor to bind epidermal growth factor receptor on cells; an effective amount of transferrin to increase DNA synthesis in cells; an effective amount of cholera toxin to increase DNA synthesis in cells; an effective amount of triiodothyronine to increase DNA synthesis in cells; an effective growth promoting amount of mammalian hormones; and an effective cell growth promoting amount of lipoprotein, cholesterol, phospholipids and fatty acids, wherein said medium contains no arginine and has a low endotoxin concentration, thereby providing a culture medium which supports the growth of normal human hepatocyte cells and does not support the growth of fibroblast cells.

Claim 3

A method for selectively culturing normal adult human liver epithelial cells, which comprises: providing a primary tissue culture from the liver of an adult human; and culturing said primary tissue culture in the cell culturing medium of claim 1.

Claim 4

A cell culturing medium for primary culture of normal human hepatocytes which comprises: an effective amount of ornithine to allow proliferation of human hepatocytes; an effective cell growth promoting amount of growth factor inactivated serum; an effective cell growth promoting amount of the essential amino acids except arginine; an effective growth promoting amount of water soluble vitamins; an effective cell growth promoting amount of coenzymes; an effective cell growth promoting amount of sodium ions; an effective cell growth promoting amount of calcium ions; an effective cell growth promoting amount of glucose; an effective amount of insulin to aid cells in glucose uptake; an effective cell growth promoting amount of hydrocortisone; an effective amount of epidermal growth factor to bind epidermal growth factor receptor on cells; an effective amount of transferrin to increase DNA synthesis in cells; an effective amount of cholera toxin to increase DNA synthesis in cells; an effective amount of triiodothyronine to increase DNA synthesis in cells; an effective amount of retinoic acid to increase DNA synthesis in cells, said amount resulting in a concentration of 1 to 300 nM; an effective growth promoting amount of mammalian hormones; and an effective cell growth promoting amount of lipoprotein, cholesterol, phospholipids and fatty acids, wherein said medium contains no arginine and has a low concentration of endotoxin, thereby providing a medium which supports the growth of normal human hepatocyte cells and does not support the growth of fibroblast cells.

Claim 5

A cell culturing medium for primary culture of normal human hepatocytes which comprises: an effective amount of ornithine to allow proliferation of human hepatocytes; an effective cell growth promoting amount of growth factor inactivated serum; an effective cell growth promoting amount of the essential amino acids except arginine; an effective growth promoting amount of water soluble vitamins; an effective cell growth promoting amount of coenzymes; an effective cell growth promoting amount of sodium ions; an effective cell growth promoting amount of calcium ions; an effective cell growth promoting amount of glucose; an effective amount of insulin to aid cells in glucose uptake; an effective cell growth promoting amount of hydrocortisone; an effective amount of epidermal growth factor to bind epidermal growth factor receptor on cells; an effective amount of transferrin to increase DNA synthesis in cells; an effective amount of cholera toxin to increase DNA synthesis in cells; an effective amount of triiodothyronine to increase DNA synthesis in cells; an effective amount of retinoic acid to increase DNA synthesis in cells, said amount resulting in a concentration of 1 to 300 nM; an effective growth promoting amount of mammalian hormones; and an effective cell growth promoting amount of lipoprotein, cholesterol, phospholipids and fatty acids, wherein said medium contains no arginine and has a low concentration of endotoxin, thereby providing a medium which supports the growth of normal human hepatocyte cells and does not support the growth of fibroblast cells.

Claim 6

A method for selectively culturing normal adult human liver epithelial cells, which comprises: providing a primary tissue culture from the liver of an adult human; and culturing said primary tissue culture in the cell culturing medium of claim 3.

Claim 7

An aqueous cell culturing medium suitable for primary culture of normal human hepatocytes which comprises: water; an effective amount of ornithine to allow proliferation of human hepatocytes; an effective cell growth promoting amount of growth factor inactivated serum; an effective cell growth promoting amount of all of the essential amino acids, except arginine; an effective cell growth promoting amount of water soluble vitamins; an effective cell growth promoting amount of coenzymes; an effective cell growth promoting amount of sodium ions; an effective amount of calcium ions, which is represented by a concentration from 0.2 to 1 mM, high enough to promote human hepatocyte growth but low enough to avoid the inducement of cellular differentiation; an effective cell growth promoting amount of glucose; insulin in an amount of 1 to 10 μg/ml; hydrocortisone in an amount of 0.05 to 1 μM; epidermal growth factor in an amount of 1 to 25 ng/ml; transferrin in an amount of 1 to 10 μg/ml; cholera toxin in an amount of 5 to 50 ng/ml; triiodothyronine in an amount of O1 to 100 nM; retinoic acid in an amount of 1 to 30 nM; an effective amount of mammalian pituitary extract to provide hormones and mitogenic factors necessary for culturing human hepatocytes; an effective cell growth promoting amount of lipoprotein, cholesterol, phospholipids and fatty acids; an effective cell growth promoting amount of conditioned medium derived from HepG2 hepatoblastoma cells or a mutant of said HepG2 cells; and an effective amount of a buffer to maintain the pH of the medium between 6.7 and 7.6, wherein said medium contains no arginine and has a low concentration of endotoxin, thereby providing a medium which supports the growth of normal human hepatocyte cells and does not support the growth of fibroblast cells.

Claim 8

An aqueous cell culturing medium suitable for primary culture of normal human hepatocytes which comprises: water; an effective amount of ornithine to allow proliferation of human hepatocytes; an effective cell growth promoting amount of growth factor inactivated serum; an effective cell growth promoting amount of all of the essential amino acids, except arginine; an effective cell growth promoting amount of water soluble vitamins; an effective cell growth promoting amount of coenzymes; an effective cell growth promoting amount of sodium ions; an effective amount of calcium ions, which is represented by a concentration from 0.2 to 1 mM, high enough to promote human hepatocyte growth but low enough to avoid the inducement of cellular differentiation; an effective cell growth promoting amount of glucose; insulin in an amount of 1 to 10 μg/ml; hydrocortisone in an amount of 0.05 to 1 μM; epidermal growth factor in an amount of 1 to 25 ng/ml; transferrin in an amount of 1 to 10 μg/ml; cholera toxin in an amount of 5 to 50 ng/ml; triiodothyronine in an amount of O1 to 100 nM; retinoic acid in an amount of 1 to 30 nM; an effective amount of mammalian pituitary extract to provide hormones and mitogenic factors necessary for culturing human hepatocytes; an effective cell growth promoting amount of lipoprotein, cholesterol, phospholipids and fatty acids; an effective cell growth promoting amount of conditioned medium derived from HepG2 hepatoblastoma cells or a mutant of said HepG2 cells; and an effective amount of a buffer to maintain the pH of the medium between 6.7 and 7.6, wherein said medium contains no arginine and has a low concentration of endotoxin, thereby providing a medium which supports the growth of normal human hepatocyte cells and does not support the growth of fibroblast cells.

Claim 9

The medium of claim 5, wherein said pituitary extract is bovine pituitary extract.

Claim 10

The medium of claim 5, which contains 100 to 700 μg/ml of a mixture of lipoprotein, cholesterol, phospholipids, and fatty acids with low endotoxin.

Claim 11

A method for selectively culturing normal adult human liver epithelial cells, which comprises: providing a primary tissue culture from the liver of an adult human; and culturing said primary tissue culture in the cell culturing medium of claim 5.

Claim 12

An aqueous cell culturing medium suitable for primary culture of normal human hepatocytes which comprises: water; an effective cell growth promoting amount of all of the essential amino acids, except arginine; an effective cell growth promoting amount of growth factor in activated serum; an effective growth promoting amount of water soluble vitamins; an effective cell growth promoting amount of coenzymes; an effective cell growth promoting amount of sodium ions; calcium ions in a concentration of 0.2 to 1 mM; an effective cell growth promoting amount of glucose; insulin in an amount of 1 to 10 μg/ml; hydrocortisone in an amount of 0.05 to 1 μM; epidermal growth factor in an amount of 1 to 25 ng/ml; transferrin in an amount of 1 to 10 μg/ml; cholera toxin in an amount of 5 to 50 ng/ml; triiodothyronine in an amount of 1 to 100 nM; retinoic acid in an amount of 1 to 300 nM; an effective amount of mammalian pituitary extract to provide hormones and mitogenic factors necessary for culturing normal adult human liver epithelial cells; an effective cell growth promoting amount of conditioned medium derived from HepG2 hepatoblastoma cells or a mutant of said HepG2 cells; and 10- 70μ g/ml of a mixture of lipoprotein, cholesterol, phospholipids, and fatty acids with low endotoxin; and an effective amount of a buffer to maintain the pH of the medium between 6.7 and 7.6, wherein said medium contains no arginine, thereby providing a medium which supports the growth of normal human hepatocyte cells but does not support the growth of fibroblast cells.

Claim 13

An aqueous cell culturing medium suitable for primary culture of normal human hepatocytes which comprises: water; an effective cell growth promoting amount of all of the essential amino acids, except arginine; an effective cell growth promoting amount of growth factor in activated serum; an effective growth promoting amount of water soluble vitamins; an effective cell growth promoting amount of coenzymes; an effective cell growth promoting amount of sodium ions; calcium ions in a concentration of 0.2 to 1 mM; an effective cell growth promoting amount of glucose; insulin in an amount of 1 to 10 μg/ml; hydrocortisone in an amount of 0.05 to 1 μM; epidermal growth factor in an amount of 1 to 25 ng/ml; transferrin in an amount of 1 to 10 μg/ml; cholera toxin in an amount of 5 to 50 ng/ml; triiodothyronine in an amount of 1 to 100 nM; retinoic acid in an amount of 1 to 300 nM; an effective amount of mammalian pituitary extract to provide hormones and mitogenic factors necessary for culturing normal adult human liver epithelial cells; an effective cell growth promoting amount of conditioned medium derived from HepG2 hepatoblastoma cells or a mutant of said HepG2 cells; and 10- 70μ g/ml of a mixture of lipoprotein, cholesterol, phospholipids, and fatty acids with low endotoxin; and an effective amount of a buffer to maintain the pH of the medium between 6.7 and 7.6, wherein said medium contains no arginine, thereby providing a medium which supports the growth of normal human hepatocyte cells but does not support the growth of fibroblast cells.

Claim 14

A method for culturing normal adult human liver epithelial cells, which comprises: culturing normal adult human liver epithelial cells in the cell culturing medium of claim 9.

Claim 15

The method of claim 10, wherein said cells are cultured for at least 10 rounds of DNA synthesis.

Claim 16

A mammalian cell culturing medium, consisting essentially of: an effective amount of ornithine to allow proliferation of human hepatocytes; an effective amount of growth factor inactivated serum; retinoic acid in an amount of 1 to 300 nm; and calcium ions in an amount of 0.2 to 1 mM, wherein said medium contains essentially no arginine.

Claim 17

A mammalian cell culturing medium, consisting essentially of: an effective amount of ornithine to allow proliferation of human hepatocytes; an effective amount of growth factor inactivated serum; retinoic acid in an amount of 1 to 300 nm; and calcium ions in an amount of 0.2 to 1 mM, wherein said medium contains essentially no arginine.

Claim 18

The cell culturing medium of claim 12, wherein said growth factor inactivated serum is chemically denatured serum.

Claim 19

A cell culturing medium suitable for primary culture of normal human hepatocytes comprising the following ingredients: an effective amount of ornithine to allow proliferation of human hepatocytes; an effective cell growth promoting amount of growth factor inactivated serum; retinoic acid at a concentration of 1 to 300 nM; an effective amount of calcium ions, which is represented by a concentration from 0.2 to 1 mM, high enough to promote human hepatocyte growth but low enough to avoid the inducement of cellular differentiation; an effective cell growth promoting amount of glucose; an effective amount of insulin to aid cells in glucose uptake; an effective cell growth promoting amount of hydrocortisone; an effective amount of epidermal growth factor to bind epidermal growth factor receptor on cells; an effective amount of transferrin to increase DNA synthesis in cells; an effective amount of cholera toxin to increase DNA synthesis in cells; an effective amount of triiodothyronine to increase DNA synthesis in cells; an effective amount of triiodothyronine to increase DNA synthesis in cells; an effective amount of mammalian hormones; and wherein said medium contains no arginine, thereby providing a medium which supports the growth of normal human hepatocyte cells but does not support the growth of fibroblast cells.

Claim 20

A cell culturing medium suitable for primary culture of normal human hepatocytes comprising the following ingredients: an effective amount of ornithine to allow proliferation of human hepatocytes; an effective cell growth promoting amount of growth factor inactivated serum; retinoic acid at a concentration of 1 to 300 nM; an effective amount of calcium ions, which is represented by a concentration from 0.2 to 1 mM, high enough to promote human hepatocyte growth but low enough to avoid the inducement of cellular differentiation; an effective cell growth promoting amount of glucose; an effective amount of insulin to aid cells in glucose uptake; an effective cell growth promoting amount of hydrocortisone; an effective amount of epidermal growth factor to bind epidermal growth factor receptor on cells; an effective amount of transferrin to increase DNA synthesis in cells; an effective amount of cholera toxin to increase DNA synthesis in cells; an effective amount of triiodothyronine to increase DNA synthesis in cells; an effective amount of triiodothyronine to increase DNA synthesis in cells; an effective amount of mammalian hormones; and wherein said medium contains no arginine, thereby providing a medium which supports the growth of normal human hepatocyte cells but does not support the growth of fibroblast cells.

Claim 21

A cell culturing medium suitable for primary culture of normal human hepatocytes which comprises: an effective amount of ornithine to allow proliferation of human hepatocytes; an effective cell growth promoting amount of growth factor inactivated serum; an effective cell growth promoting amount of the essential amino acids, except arginine; an effective growth promoting amount of water soluble vitamins; an effective cell growth promoting amount of coenzymes; an effective cell growth promoting amount of sodium ions; an effective cell growth promoting amount of calcium ions; an effective cell growth promoting amount of glucose; an effective amount of insulin to aid cells in glucose uptake; an effective cell growth promoting amount of hydrocortisone; an effective amount of epidermal growth factor to bind epidermal growth factor receptor on cells; an effective amount of transferrin to increase DNA synthesis in cells; an effective amount of cholera toxin to increase DNA synthesis in cells; an effective amount of triiodothyronine to increase DNA synthesis in cells; an effective amount of retinoic acid to increase DNA synthesis in cells, said amount resulting in a concentration of 1 to 300 nM; an effective growth promoting amount of mammalian hormones; wherein said medium contains no arginine, thereby providing a medium which supports the growth of normal human hepatocyte cells but does not support the growth of fibroblast cells.

Claim 22

A cell culturing medium suitable for primary culture of normal human hepatocytes which comprises: an effective amount of ornithine to allow proliferation of human hepatocytes; an effective cell growth promoting amount of growth factor inactivated serum; an effective cell growth promoting amount of the essential amino acids, except arginine; an effective growth promoting amount of water soluble vitamins; an effective cell growth promoting amount of coenzymes; an effective cell growth promoting amount of sodium ions; an effective cell growth promoting amount of calcium ions; an effective cell growth promoting amount of glucose; an effective amount of insulin to aid cells in glucose uptake; an effective cell growth promoting amount of hydrocortisone; an effective amount of epidermal growth factor to bind epidermal growth factor receptor on cells; an effective amount of transferrin to increase DNA synthesis in cells; an effective amount of cholera toxin to increase DNA synthesis in cells; an effective amount of triiodothyronine to increase DNA synthesis in cells; an effective amount of retinoic acid to increase DNA synthesis in cells, said amount resulting in a concentration of 1 to 300 nM; an effective growth promoting amount of mammalian hormones; wherein said medium contains no arginine, thereby providing a medium which supports the growth of normal human hepatocyte cells but does not support the growth of fibroblast cells.