Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
226 words
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 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° 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 μM 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° 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 μM insulin, or an equivalent amount of an insulin analogue; 16 nM to 1 μM of a glucocorticoid; and f) incubating said cells at about 37° 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.
Claim 2
Independent Claim
226 words
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 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° 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 μM 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° 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 μM insulin, or an equivalent amount of an insulin analogue; 16 nM to 1 μM of a glucocorticoid; and f) incubating said cells at about 37° 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.
Claim 3
Dependent Claim
22 words
The method of claim 1, wherein said preadipocyte medium, said differentiation medium and said adipocyte medium further comprise 3-10% fetal bovine serum.
Claim 4
Dependent Claim
22 words
The method of claim 1, wherein said differentiation medium and said adipocyte medium further comprise 1-100 μM pantothenate and 1-100 μM biotin.
Claim 5
Dependent Claim
24 words
The method of claim 1, wherein said defined cell culture medium further comprises a biological buffer having a pH of about 7.0 to 7.6.
Claim 6
Dependent Claim
20 words
The method of claim 1, wherein said defined cell culture medium is Dulbeccos Modified Eagle/Hams' F-10 Nutrient Broth (1:1 vol/vol).
Claim 7
Dependent Claim
11 words
The method of claim 1, wherein said thiazolidinedione is BRL 49653.
Claim 8
Dependent Claim
15 words
The method of claim 6, wherein the concentration of said BRL 49653 is 0.5-1.0 μM.
Claim 9
Dependent Claim
10 words
The method of claim 1 wherein said thiazolidinedione is troglitazone.
Claim 10
Dependent Claim
14 words
The method of claim 8, wherein the concentration of said troglitazone is 1-5 μM.
Claim 11
Dependent Claim
13 words
The method of claim 1, wherein said glucocorticoid is dexamethasone, hydrocortisone or cortisol.
Claim 12
Dependent Claim
14 words
The method of claim 1, wherein said cyclic AMP inducer is isobutylmethylxanthine or forskolin.
Claim 13
Dependent Claim
20 words
The medium of claim 11, wherein said isobutylmethylxanthine is present at a final concentration of about 0.2 to 0.5 mM.