US4072565A
Expired / Lapsed
US
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Production of viruses in tissue culture without use of serum
Assignee: Dow Chemical Co
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Filing: 1974-11-04
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Granted: 1978-02-07
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Expires: 1994-11-04
Abstract
Abstract Growth of cells in tissue culture and virus vaccine production in tissue culture in the absence of serum are disclosed. The cells in tissue culture are grown initially in a chemically defined medium containing protamine zinc insulin, and the protamine zinc insulin medium is removed before virus propagation. Supplementation with serum or serum fractions can be eliminated entirely; and improved virus production in a serum free system can be obtained.
Inventors
- 👤 Stefan A. Weiss
- 👤 Harry L. Torney
- 👤 William J. Caldwell
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 17:52
CPC Classifications
A; A61; A61K; A61K39/00; A61K39/12; A61K39/155; A61K39/165; A61K39/20; A61K39/205; C; C12; C12N; C12N5/00; C12N5/0018; C12N5/0037; C12N5/005; C12N7/00; A61K2039/51; A61K2039/525; A61K2039/5254; C12N2500/00; C12N2500/30; C12N2500/34; C12N2501/00; C12N2501/30; C12N2501/33; C12N2760/00; C12N2760/00011; C12N2760/18011; C12N2760/18411; C12N2760/18434; C12N2760/18451; C12N2760/18711; C12N2760/18734; C12N2760/18751; C12N2770/00; C12N2770/00011; C12N2770/32011; C12N2770/32611; C12N2770/32634; C12N2770/32651; C12N2770/36011; C12N2770/36211; C12N2770/36234; C12N2770/36251
Claims Analysis
Total Claims: 19
Claim 1
Independent Claim
108 words
In a method which comprises inoculating a chemically defined tissue culture medium with living animal cells and incubating the cells and medium in tissure culture until significant cell growth is obtained, and inoculating the cells with virus, the improvement wherein the incubation is carried out in the presence of a cell growth enhancing amount of protamine zinc insulin U.S.P., B.P. dispersed in said medium; removing the medium containing protamine zinc insulin after significant cell growth is obtained from the cells; replacing said medium with a tissue culture maintenance medium essentially free of protamine zinc insulin; prior to virus innoculation and continuing incubation of the cells in tissue culture.
Claim 2
Dependent Claim
108 words
In a method which comprises inoculating a chemically defined tissue culture medium with living animal cells and incubating the cells and medium in tissure culture until significant cell growth is obtained, and inoculating the cells with virus, the improvement wherein the incubation is carried out in the presence of a cell growth enhancing amount of protamine zinc insulin U.S.P., B.P. dispersed in said medium; removing the medium containing protamine zinc insulin after significant cell growth is obtained from the cells; replacing said medium with a tissue culture maintenance medium essentially free of protamine zinc insulin; prior to virus innoculation and continuing incubation of the cells in tissue culture.
Claim 3
Dependent Claim
16 words
The method of claim 1 wherein the tissue culture medium is a chemically defined maintenance medium.
Claim 4
Dependent Claim
25 words
The method of claim 2 wherein the tissue culture medium comprises Medium 199 and is essentially free of detectable protein other than protamine zinc insulin.
Claim 5
Dependent Claim
27 words
Method of claim 1 wherein the medium containing protamine zinc insulin is removed and replaced with said maintenance medium within about five days after beginning the incubation.
Claim 6
Dependent Claim
22 words
Method of claim 4 wherein the medium containing protamine zinc insulin is removed and replaced within about 48 hours after beginning incubation.
Claim 7
Dependent Claim
30 words
Method of claim 4 further comprising the step of washing the cells with maintenance medium essentially free of protamine zinc insulin after removal of the medium containing protamine zinc insulin.
Claim 8
Dependent Claim
34 words
Method of claim 4 wherein the medium containing protamine zinc insulin is removed and replaced within about 48 to about 72 hours after beginning incubation, and wherein the cells are primary chick embryo cells.
Claim 9
Independent Claim
131 words
A method which comprises inoculating a tissue culture growth medium with a suspension of living animal cells, said growth medium being an intimate mixture of: a. a chemically defined protein-free tissue culture maintenance medium which is adapted to maintain metabolism of said cells but which is insufficient to support growth of said cells into a substantially complete monolayer in tissue culture; and b. an amount of from about 0.05 to about 0.5 units per milliliter of protamine zinc insulin U.S.P., B.P. effective to provide an ultimate growth medium adapted to support such growth medium adapted to support such growth of said cells; and incubating said cells and said growth medium in tissue culture under conditions conducive to growth of a confluent cell monolayer and thereafter inoculating said cells with a virus.
Claim 10
Independent Claim
131 words
A method which comprises inoculating a tissue culture growth medium with a suspension of living animal cells, said growth medium being an intimate mixture of: a. a chemically defined protein-free tissue culture maintenance medium which is adapted to maintain metabolism of said cells but which is insufficient to support growth of said cells into a substantially complete monolayer in tissue culture; and b. an amount of from about 0.05 to about 0.5 units per milliliter of protamine zinc insulin U.S.P., B.P. effective to provide an ultimate growth medium adapted to support such growth medium adapted to support such growth of said cells; and incubating said cells and said growth medium in tissue culture under conditions conducive to growth of a confluent cell monolayer and thereafter inoculating said cells with a virus.
Claim 11
Dependent Claim
59 words
In a method which comprises inoculating a tissue culture medium with a suspension of primary living animal cells and incubating the cells and medium in tissue culture until significant cell growth is obtained, prior to virus inoculation, the improvement wherein the medium is Medium 199 having a cell growth enhancing amount of protamine zinc insulin U.S.P., B.P. dispersed therein.
Claim 12
Dependent Claim
59 words
In a method which comprises inoculating a tissue culture medium with a suspension of primary living animal cells and incubating the cells and medium in tissue culture until significant cell growth is obtained, prior to virus inoculation, the improvement wherein the medium is Medium 199 having a cell growth enhancing amount of protamine zinc insulin U.S.P., B.P. dispersed therein.
Claim 13
Dependent Claim
22 words
A method of claim 9 wherein the cells and medium are incubated under conditions conducive to growth of a confluent cell monolayer.
Claim 14
Dependent Claim
62 words
The method of claim 9 further comprising the step of removing the medium containing protamine zinc insulin after significant cell growth is obtained from the cells and within about five days after beginning incubation, replacing said medium with a tissue culture maintenance medium essentially free of protamine zinc insulin;, and continuing incubation of the cells in tissue culture in said maintenance medium.
Claim 15
Dependent Claim
26 words
The method of claim 11 wherein the medium containing protamine zinc insulin is removed and replaced with said maintenance medium about 48 hours after beginning incubation.
Claim 16
Dependent Claim
24 words
Method of claim 12 wherein the medium containing protamine zinc insulin is removed and replaced about 24 to about 48 hours after beginning incubation.
Claim 17
Dependent Claim
30 words
Method of claim 13 further comprising the step of washing the cells with maintenance medium essentially free of protamine zinc insulin after removal of the medium containing protamine zinc insulin.
Claim 18
Dependent Claim
60 words
A chemcially defined tissue culture growth medium comprising Medium 199 having a cell growth enhancing amount of from about 0.05 to about 0.5 units of protamine zinc insulin U.S.P., B.P. per milliliter dispersed therein said zinc insulin providing essentially the sole source of detectable insulin activity therein, said medium containing from about 1 to about 2 grams glucose per liter.
Claim 19
Dependent Claim
60 words
A chemcially defined tissue culture growth medium comprising Medium 199 having a cell growth enhancing amount of from about 0.05 to about 0.5 units of protamine zinc insulin U.S.P., B.P. per milliliter dispersed therein said zinc insulin providing essentially the sole source of detectable insulin activity therein, said medium containing from about 1 to about 2 grams glucose per liter.
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) | 1981-02-06 | 1981-08-08 | 1982-02-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1985-02-06 | 1985-08-08 | 1986-02-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1989-02-06 | 1989-08-08 | 1990-02-07 | $7700.00 | Window Closed / Lapsed |