EP0403139A1
Expired / Lapsed
EP
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Cell culture sytems and media
Assignee: Organogenesis Inc
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Filing: 1990-06-05
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Granted: —
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Expires: 2010-06-05
Abstract
Abstract The present invention provides systems, methods and chemically defined media for the cultivation of cells, particularly epithelial cells. Cells may be cultured with a varied calcium concentration. Furthermore, a calcium concentation in excess of 1.00mM may be used in the practice of the present invention without loss of a proliferative cell population and maintianing high colony forming efficiencies. Population doubling times range from about 16 to about 33 hours. Cells may serially cultivated to achieve from about 20 to about 50 population doublings.
Inventors
- 👤 Nancy Louise Parenteau
- 👤 Eric William Johnson
- 👤 Susan Frances Meunier
- 👤 John Gregory Maresh
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-22 22:32
CPC Classifications
C; C12; C12N; C12N5/00; C12N5/06; C12N5/0602; C12N5/0625; C12N5/0629; A; A61; A61L; A61L27/00; A61L27/50; A61L27/60; C12N5/0068; C12N5/0075; C12N2500/00; C12N2500/05; C12N2500/10; C12N2500/12; C12N2500/14; C12N2500/20; C12N2500/24; C12N2500/25; C12N2500/90; C12N2501/00; C12N2501/01; C12N2501/10; C12N2501/11; C12N2501/30; C12N2501/38; C12N2501/39; C12N2501/392; C12N2501/395; C12N2533/00; C12N2533/50; C12N2533/54
Claims Analysis
Total Claims: 43
Claim 1
Independent Claim
30 words
A cell culture medium comprising, insulin or an insulin-like growth factor; tansferrin or ferrous ion; triiodothyronine or thyroxin; at least one of ethanolamine or o-phosphoryl-ethanolamine; calcium; and a nutrient source.
Claim 2
Dependent Claim
30 words
A cell culture medium comprising, insulin or an insulin-like growth factor; tansferrin or ferrous ion; triiodothyronine or thyroxin; at least one of ethanolamine or o-phosphoryl-ethanolamine; calcium; and a nutrient source.
Claim 3
Dependent Claim
101 words
A cell culture medium in accordance with claim 1, wherein: (a) Insulin is present at from about 0.5 to about 50 ug/ml; (b) Ferrous ion is present at from about 5X10⁻⁸ to about 5X10⁻⁵M; (c) Triiodothyroine is present at from about 2 to about 200 pM; (d) At least one of o-phosphoryl-ethanolamine and ethanolamine is present at from about 10⁻⁶ to about 10⁻²M; (e) Calcium is present at from about 0.005 to about 2:0 mM; and (f) Nutrient source is selected from at least one of DMEM, IDMEM, MEM, M199, RPMI 1640, Ham's F12, Ham's F10, NCTC 109, and NCTC 135.
Claim 4
Dependent Claim
12 words
A cell culture medium in accordance with claim 1, further comprising hydrocortisone.
Claim 5
Dependent Claim
21 words
A cell culture medium in accordance with claim 3, wherein hydrocortisone is present at from about 0.04 to about 4.0 ug/ml.
Claim 6
Dependent Claim
14 words
A cell culture medium in accordance with claim 1, further comprising epidermal growth factor.
Claim 7
Dependent Claim
23 words
A cell culture medium in accordance with claim 5, wherein epidermal growth factor is present at from about 1 to about 50 ng/ml.
Claim 8
Dependent Claim
16 words
A cell culture medium in accordance with claim 1, further comprising a cyclic AMP elevating agent.
Claim 9
Dependent Claim
32 words
A medium according to claim 1, wherein the cAMP elevating agent is selected from at least one of cholera toxin, foreskolin, isoproterenol, methyl isobutyl xanthine, dibutryrl c-AMP, theophylline, caffeine and pertussis toxin.
Claim 10
Dependent Claim
24 words
A cell culture medium in accordance with claim 7, wherein the cyclic AMP elevating agent is present at from about 10⁻⁹ to about 10⁻³M.
Claim 11
Dependent Claim
17 words
A medium according to claim 1, further comprising adenine at from about 0.02 to about 2.0 mM.
Claim 12
Dependent Claim
17 words
A medium according to claim 1, further comprising chelexed serum at from about 0.05% to about 2.00%.
Claim 13
Dependent Claim
16 words
A medium according to claim 1, further comprising progesterone at from about 2X10⁻¹⁰ to about 2X10⁻⁸M.
Claim 14
Dependent Claim
15 words
A medium according to claim 1, further comprising selenium from about 10⁻⁹ to about 10⁻⁷M.
Claim 15
Dependent Claim
46 words
A medium according to claim 1, further comprising at least one of the group consisting of Bovine Hypothalamic Extract from about 5X10⁻⁶ to about 5X10⁻⁴ g/ml, and acidic Fibroblast growth factor, basic Fibroblast growth factor and keratinocyte growth factor at about 10⁻¹⁰ to about 10⁻⁶ g/ml.
Claim 16
Dependent Claim
18 words
A medium in accordance with claim 1, further comprising EGF at from about 1 to about 50 ng/ml.
Claim 17
Dependent Claim
23 words
A medium in accordance with claim 1, wherein the ferrous ion is supplied by ferric sulfate at from about 5X10⁻⁸ to about 5X10⁻⁵M.
Claim 18
Dependent Claim
23 words
A medium in accordance with claim 1, wherein the ferrous ion is supplied by transferrin at from about 0.05 to about 50 ug/ml.
Claim 19
Dependent Claim
13 words
A medium in accordance with claim 1, wherein the cells are epithelial cells.
Claim 20
Dependent Claim
15 words
A medium in accordance with claim 1, wherein the cells are epidermal cells or keratinocytes.
Claim 21
Dependent Claim
97 words
A cell culture system comprising, a cell culture medium in accordance with claim 1 and a substrate for the cells comprising plastic, glass, collagen, fibronectin, laminin, heparan sulfate proteoglycan, microcarriers coated with collagen, fibronectin, laminin or heparan sulfate proteo glycan, or a tissue equivalent produced by a method comprising: a. combining a collagen solution with a contractile agent under conditions to form a gel mixture having the contractile agent dispersed within the gel mixture; and b. maintaining the gel mixture prepared in step (a) under conditions which permit contraction of the gel mixture to form a tissue-equivalent.
Claim 22
Dependent Claim
17 words
The system of claim 20, wherein the collagen is native Type I collagen derived from bovine tendon.
Claim 23
Dependent Claim
32 words
A method of culturing epithelial cells comprising, inoculating a growth substrate in accordance with claim 14 with the epithelial cells, and maintaining the cell culture system under conditions to promote cell growth.
Claim 24
Dependent Claim
11 words
Epithelial cells produced in accordance with the method of claim 22.
Claim 25
Dependent Claim
23 words
The method of claim 22, wherein cell growth is characterized by a population doubling time of from about 16 to about 33 hours.
Claim 26
Dependent Claim
21 words
The method of claim 22, wherein the cells are serially cultured to achieve from about 20 to about 50 population doublings.
Claim 27
Dependent Claim
41 words
The method of claim 22, wherein the method further comprises adding calcium at a concentration greater than about 1.0 mM to enable stratification and differentiation of the cells while maintaining a colony forming efficiency of from about 20 to about 60%.
Claim 28
Dependent Claim
20 words
The method of claim 24 or 25, wherein the innoculum of cells contains from about 30 to about 1000 cells/cm².
Claim 29
Dependent Claim
31 words
A method of culturing epithelial cells comprising, inoculating a medium in accordance with claim 1 with the epithelial cells, and maintaining the cells and medium under conditions to promote cell growth.
Claim 30
Dependent Claim
11 words
Epithelial cells produced in accordance with the method of claim 28.
Claim 31
Dependent Claim
23 words
The method of claim 28, wherein cell growth is characterized by a population doubling time of from about 16 to about 33 hours.
Claim 32
Dependent Claim
21 words
The method of claim 28, wherein the cells are serially cultured to achieve from about 20 to about 50 population doublings.
Claim 33
Dependent Claim
20 words
The method of claim 30 or 31, wherein the innoculation of cells contains from about 30 to about 1000 cells.
Claim 34
Dependent Claim
37 words
The method of claim 22, wherein cell growth comprises the rapid expansion of primary cells with a colony forming efficiency of from about 20 to about 100%, and a proliferative capacity in excess of 50 population doublings.
Claim 35
Dependent Claim
35 words
The method of claim 33 further comprising adding calcium in excess of 1.0 mM to enable stratification and differentiation of the cells while maintaining a colony forming efficiency of from about 20 to about 60%.
Claim 36
Dependent Claim
52 words
A method of culturing epithelial cells comprising plating epithelial cells onto collagen-coated microcarriers in a cell culture system in accordance with claim 14, wherein the cells are plated at a plating efficiency of from about 20 to about 40%, and maintaining the microcarriers and plated cells under conditions to promote cell growth.
Claim 37
Dependent Claim
25 words
The method of claim 35, wherein a colony forming efficiency of from about 20 to about 100% is maintained when the cell growth reaches confluence.
Claim 38
Dependent Claim
34 words
The method of claim 35, wherein calcium concentration is increased from 0.08 to about 1.8mM during the rapid proliferative phase when cell growth is approaching confluence to increase adherence of the cells to microcarriers.
Claim 39
Dependent Claim
32 words
A method of producing keratinocytes comprising the steps of: a. innoculating a growth substrate in accordance with claim 20 with keratinocytes; and b. maintaining the system under conditions to promote cell growth.
Claim 40
Dependent Claim
21 words
A method of producing a sheet of keratinocytes comprising enzymatically detaching a sheet of keratinocytes prepared in accordance with claim 38.
Claim 41
Dependent Claim
12 words
Keratinocytes produced in accordance with the method of claim 38 or 39.
Claim 42
Dependent Claim
54 words
A method of epidermalizing a dermis comprising: a. innoculating a tissue equivalent in accordance with claim 20 with epidermal cells; b. maintaining the cell culture system under conditions to promote cell growth; and c. adding calcium to physiological concentration and maintaining the system under conditions to enable development of a fully keratinized epidermal layer.
Claim 43
Dependent Claim
19 words
A medium according to claim 2 wherein the nutrient source is 75% calcium free DMEM and 25% Ham's F-12.
References Cited (Backward Citations)
FR2532948A1
Cited
US4673649A
Cited
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