Patent ID: 6153432
Filing Date: 2000-11-28
Classification: A61L,C12N

Abstract:
A method for differentiating human preadipocytes into adipocytes, comprising:a) plating isolated human preadipocytes at a density of about 25,000 to 30,000 cells/cm.sup.2 in a preadipocyte medium comprising a chemically defined cell culture medium having or supplemented with 1.0-4.5 g/liter glucose;b) incubating said cells at about 37.degree. C. for about 4-48 hours until said cells are about 95-100% confluent;c) replacing said preadipocyte medium with a differentiation medium comprising a defined cell culture medium having or supplemented with 1.0-4.5 g/liter glucose, 0.2 to 0.5 mM isobutylmethylxanthine; 100 nM to 1 .mu.M of a glucocorticoid, and a concentration of Peroxisome Proliferator Activated Receptor gamma agonist which is a thiazolidinedione, effective to stimulate differentiation of human preadipocytes;d) incubating said cells at about 37.degree. C. for about 2-4 days;e) replacing said differentiation medium with an adipocyte medium comprising a defined cell culture medium having or supplemented with 1.0-4.5 g/liter glucose; 100 nM to 1 .mu.M insulin, or an equivalent amount of an insulin analogue; 16 nM to 1 .mu.M of a glucocorticoid; andf) incubating said cells at about 37.degree. C. for about 1-2 weeks and refeeding said cells with said adipocyte medium at least every 3-4 days; wherein said preadipocyte medium, said differentiation medium and said adipocyte medium are maintained at a pH of about 7.0 to 7.6 when in contact with said cells.