US4673649A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Process and defined medium for growth of human epidermal keratinocyte cells
Assignee: University Patents Inc
•
Filing: 1983-07-15
•
Granted: 1987-06-16
•
Expires: 2003-07-15
Abstract
Abstract Disclosed are novel methods and materials for generating in vitro cultured populations of human epidermal keratinocyte cells having a characteristic colony-forming efficiency of greater than 20%. Novel media preparations and procedures are disclosed that permit isolation, serum-free primary culture and serum-free serial subculture of human epidermal keratinocytes. Also disclosed are procedures and products employing keratinocyte cells grown in serum-free conditions for medical application, i.e., skin grafting.
Inventors
- 👤 Steven T. Boyce
- 👤 Richard G. Ham
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-22 23:22
CPC Classifications
C; C12; C12N; C12N5/00; C12N5/06; C12N5/0602; C12N5/0625; C12N5/0629; C12N2500/00; C12N2500/70; C12N2500/80; C12N2500/84; C12N2500/90; C12N2501/00; C12N2501/10; C12N2501/11; C12N2501/30; C12N2501/33; C12N2501/38; C12N2501/39; C12N2501/392
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
49 words
A transferrin-free medium suitable for stock culture or clonal growth of a population of human keratinocyte cells in a primary culture comprising: complete MCDB153; epidermal growth factor at a concentration range of 1.0 mg/ml to 25.0 ng/ml; and insulin at a concentration range of 0.5 μg/ml to 50.0 μg/ml.
Claim 2
Dependent Claim
49 words
A transferrin-free medium suitable for stock culture or clonal growth of a population of human keratinocyte cells in a primary culture comprising: complete MCDB153; epidermal growth factor at a concentration range of 1.0 mg/ml to 25.0 ng/ml; and insulin at a concentration range of 0.5 μg/ml to 50.0 μg/ml.
Claim 3
Dependent Claim
24 words
The medium according to claim 1 comprising: epidermal growth factor at a concentration of 10 ng/ml; and insulin at a concentration of 5 μg/ml.
Claim 4
Dependent Claim
77 words
The medium according to claim 2 further comprising a member selected from the group consisting of: hydrocortisone at a concentration range of 0.14 μM to 28.0 μM; ethanolamine at a concentration range of 0.01 mM to 1.0 mM; phosphoethanolamine at a concentration range of 0.01 mM to 1.0 mM; calcium chloride at a concentration range of 0.03 mM to 1.0 mM; and whole bovine pituitary extract at a protein concentration range of 7.0 μg/ml to 700.0 μg/ml.
Claim 5
Dependent Claim
56 words
The medium according to claim 3 comprising a member selected from the group consisting of: hydrocortisone at a concentration of 1.4 μM; ethanolamine at a concentration of 0.1 mM; phosphoethanolamine at a concentration of 0.1 mM; calcium chloride at a concentration of 0.1 mM; and whole bovine pituitary extract at a protein concentration of 70 μg/ml.
Claim 6
Dependent Claim
24 words
The medium according to claim 1 comprising: epidermal growth factor at a concentration of 5.0 ng/ml; and insulin at a concentration of 5.0 μg/ml.
Claim 7
Dependent Claim
60 words
The medium according to claim 2 further comprising a member selected from the group consisting of: hydrocortisone at a concentration of 1.4 μM; ethanolamine at a concentration of 0.01 mM; phosphoethanolamine at a concentration of 0.01 mM; calcium chloride at a conccentration in the range of 0.03-1.0 mM; and whole bovine pituitary extract at a protein concentration of 70 μg/ml.
Claim 8
Dependent Claim
78 words
A transferrin-free medium suitable for stock culture of a population of human keratinocyte cells in a primary culture comprising: complete MCDB153; epidermal growth factor at a concentration of 10 ng/ml; insulin at a concentration of 5 μg/ml; hydrocortisone at a concentration of 1.4 μM; ethanolamine at a concentration of 0.1 mM; phosphoethanolamine at a concentration of 0.1 mM; calcium chloride at a concentration of 0.1 mM; and whole bovine pituitary extract at a protein concentration of 70 μg/ml.
Claim 9
Dependent Claim
78 words
A transferrin-free medium suitable for stock culture of a population of human keratinocyte cells in a primary culture comprising: complete MCDB153; epidermal growth factor at a concentration of 10 ng/ml; insulin at a concentration of 5 μg/ml; hydrocortisone at a concentration of 1.4 μM; ethanolamine at a concentration of 0.1 mM; phosphoethanolamine at a concentration of 0.1 mM; calcium chloride at a concentration of 0.1 mM; and whole bovine pituitary extract at a protein concentration of 70 μg/ml.
Claim 10
Dependent Claim
33 words
A transferrin-free medium suitable for supporting a viable population of human keratinocyte cells in frozen storage comprising: complete MCDB153; and epidermal growth factor at a concentration range of 1.0 ng/ml to 25.0 ng/ml.
Claim 11
Dependent Claim
33 words
A transferrin-free medium suitable for supporting a viable population of human keratinocyte cells in frozen storage comprising: complete MCDB153; and epidermal growth factor at a concentration range of 1.0 ng/ml to 25.0 ng/ml.
Claim 12
Dependent Claim
16 words
The medium according to claim 8 comprising: epidermal growth factor at a concentration of 10 ng/ml.
Claim 13
Dependent Claim
79 words
The medium according to claim 8 further comprising a member selected from the group consisting of: hydrocortisone at a concentration range of 0.14 μM to 28.0 μM; calcium chloride at a concentration range of 0.03 mM to 1.0 mM; whole bovine pituitary extract at a protein concentration range of 70 μg/ml to 2800 μg/ml; whole bovine fetal serum at a concentration range of 2.0% to 50.0% v/v; and dimethylsulfoxide at a concentration range of 2.0% v/v to 20.0% v/v.
Claim 14
Dependent Claim
59 words
The medium according to claim 10 comprising a member selected from the group consisting of: hydrocortisone at a concentration of 1.4 μM; calcium chloride at a concentration of 0.1 mM; whole bovine pituitary extract at a protein concentration of 700 μg/ml; whole bovine fetal serum at a concentration of 20.0% v/v; and dimethylsulfoxide at a concentration of 10% v/v.
Claim 15
Dependent Claim
62 words
A transferrin-free medium suitable for supporting a viable population of human keratinocyte cells in frozen storage comprising: complete MCDB153; epidermal growth factor at a concentration of 10 ng/ml; hydrocortisone at a concentration of 1.4 μM; calcium chloride at a concentration of 1.0 mM; whole bovine pituitary extract at a protein concentration of 70 μg/ml; and dimethylsulfoxide at a concentration of 10% v/v.
Claim 16
Dependent Claim
62 words
A transferrin-free medium suitable for supporting a viable population of human keratinocyte cells in frozen storage comprising: complete MCDB153; epidermal growth factor at a concentration of 10 ng/ml; hydrocortisone at a concentration of 1.4 μM; calcium chloride at a concentration of 1.0 mM; whole bovine pituitary extract at a protein concentration of 70 μg/ml; and dimethylsulfoxide at a concentration of 10% v/v.
Claim 17
Dependent Claim
90 words
A population of human epidermal keratinocyte cells in a frozen state in the medium of claim 12 and having a colony forming efficiency greater than 20% in a transferrin-free medium comprising: complete MCDB153; epidermal growth factor at a concentration of 10 ng/ml; insulin at a concentration of 5 μg/ml; hydrocortisone at a concentration of 1.4 μM; ethanolamine at a concentration of 0.1 mM; phosphoethanolamine at a concentration of 0.1 mM; calcium chloride at a concentration of 0.1 mM; and whole bovine pituitary extract at a protein concentration of 70 μg/ml.
Claim 18
Dependent Claim
83 words
The population of cells according to claim 13 wherein said cells form a monolayer in a culture containing a transferrin-free medium comprising: complete MCDB153; epidermal growth factor at a concentration of 5.0 ng/ml; insulin at a concentration of 5.0 μg/ml; hydrocortisone at a concentration of 1.4 μM; ethanolamine at a concentration of 0.1 mM; phosphoethanolamine at a concentration of 1.0 mM; calcium chloride at a concentration in the range of 0.3-1.0 mM; and whole bovine pituitary extract at a concentration of 70 μg/ml.
Claim 19
Dependent Claim
61 words
A transferrin-free medium suitable for supporting a viable population of human keratinocyte cells in frozen storage comprising: complete MCDB153; epidermal growth factor at a concentration of 10 ng/ml; hydrocortisone at a concentration of 1.4 μM; calcium chloride at a concentration of 1.0 mM; whole bovine fetal serum at a concentration of 20.0% v/v; and dimethylsulfoxide at a concentration of 10% v/v.
Claim 20
Dependent Claim
61 words
A transferrin-free medium suitable for supporting a viable population of human keratinocyte cells in frozen storage comprising: complete MCDB153; epidermal growth factor at a concentration of 10 ng/ml; hydrocortisone at a concentration of 1.4 μM; calcium chloride at a concentration of 1.0 mM; whole bovine fetal serum at a concentration of 20.0% v/v; and dimethylsulfoxide at a concentration of 10% v/v.
Claim 21
Dependent Claim
77 words
A transferrin-free medium suitable for clonal growth of a population of human epidermal keratinocyte cells comprising: complete MCDB153; epidermal growth factor at a concentration of 5.0 ng/ml; insulin at a concentration of 5.0 μg/ml; hydrocortisone at a concentration of 1.4 μM; ethanolamine at a concentration of 0.1 mM; phosphoethanolamine at a concentration of 1.0 mM; calcium chloride at a concentration in the range of 0.3-1.0 mM; and whole bovine pituitary extract at a concentration of 70 μg/ml.
Claim 22
Dependent Claim
77 words
A transferrin-free medium suitable for clonal growth of a population of human epidermal keratinocyte cells comprising: complete MCDB153; epidermal growth factor at a concentration of 5.0 ng/ml; insulin at a concentration of 5.0 μg/ml; hydrocortisone at a concentration of 1.4 μM; ethanolamine at a concentration of 0.1 mM; phosphoethanolamine at a concentration of 1.0 mM; calcium chloride at a concentration in the range of 0.3-1.0 mM; and whole bovine pituitary extract at a concentration of 70 μg/ml.
Claim 23
Dependent Claim
60 words
A method for establishing a population of human epidermal keratinocyte cells in vitro which comprises separating the epidermal tissue layer from the dermal tissue layer of human foreskin tissue, dispersing cells from said epidermal tissue layer to form a suspension of single cells, and growing an effective number of said cells in the medium of claim 2 under cell-culturing conditions.
Claim 24
Dependent Claim
23 words
A method for storing a viable population of human epidermal keratinocyte cells which comprises freezing said cells in the medium of claim 8.
Claim 25
Dependent Claim
26 words
A method for obtaining a population of human epidermal keratinocyte cells which comprises clonally growing said cells in the medium of claim 1 under cell-culturing conditions.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1990-06-15 | 1990-12-15 | 1991-06-16 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-06-15 | 1994-12-15 | 1995-06-16 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-06-15 | 1998-12-15 | 1999-06-16 | $7700.00 | Window Closed / Lapsed |