CA1220744A - Culture medium and method for the reproduction of viruses in suspension culture - Google Patents
Culture medium and method for the reproduction of viruses in suspension cultureInfo
- Publication number
- CA1220744A CA1220744A CA000427113A CA427113A CA1220744A CA 1220744 A CA1220744 A CA 1220744A CA 000427113 A CA000427113 A CA 000427113A CA 427113 A CA427113 A CA 427113A CA 1220744 A CA1220744 A CA 1220744A
- Authority
- CA
- Canada
- Prior art keywords
- cells
- culture medium
- virus
- culture
- suspension
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
- C12N2740/14061—Methods of inactivation or attenuation
- C12N2740/14064—Methods of inactivation or attenuation by serial passage
Abstract
ABSTRACT OF THE DISCLOSURE
A culture medium and process for the preparation of same are described. The culture medium allows for the reproduction of cells and viruses in suspension. The culture medium includes inorganic salts, amino acids, sugar, anti-biotics, vitamins and at least one substance selected from the group of insulin, transferrin, biotin, peptic peptone and casein peptone. Viruses reproduced in a suspension in such a culture medium yield antigens which can be applied in diagnostic agents or vaccines.
A culture medium and process for the preparation of same are described. The culture medium allows for the reproduction of cells and viruses in suspension. The culture medium includes inorganic salts, amino acids, sugar, anti-biotics, vitamins and at least one substance selected from the group of insulin, transferrin, biotin, peptic peptone and casein peptone. Viruses reproduced in a suspension in such a culture medium yield antigens which can be applied in diagnostic agents or vaccines.
Description
lZZ074~
The invention provides a culture medium and a method for the reproduction of viruses in a suspension of cells.
The viruses reproduced yield antigens which can be applied in diagnos~ic agents and optionally in vaccines.
Often, viruses cannot be reproduced but in monolayer cell cultures, that is, the cells and viruses are repro-duced at the interface of the nutrient medium, which means low space/time yields, much operative expenditure, high cost of medium and increased risk of contamination, because many recipients are required for these operations.
To find cGnditions under which a virus is reproduced ir. a suspension of cells which allows utilization of the total space filled with nutrient solution would bring about a considerable advantage over an interface culture.
Virus monolayer cell cultures are state of the art.
A method for the bovine leukosis virus (BLV) is described in J. Nat. Cancer Inst., vol. 52, 491-497 (1974).
This virus causes enzootic leukosis which is a disease of the hematopoietic, lymphatic system of cattle and brings about heavy losses. In order to recognize animals so infected, an antigen prepared in tissue culture can be applied.
Surprisingly, a medium has been found in which cells and viruses can be reproduced in suspension.
Subject of the invention is therefore a nutrient medium andits use in a process for the reproduction of viruses in a suspension of cells.
The medium contains inorganic salts, amino acids, sugar, antibiotics and vitamins, and one or more substances selected from the group of insulin, transferrin, biotin, peptic peptone and casein peptone.
These substances may be contained in the medium in following amounts:
insulin 1.0 - 10.0 mg/l transferrin 2.0 - 20.0 mg/l b~otin 0.5 - 10.0 mg/l peptic peptone 0.5 - 10.0 g/l casein peptone 0.5 - 10.0 g/l ~Z20~44 Optionally, 0 to 5 volume percent of serum are added to the medium. Suitable cells for the cultures are above all fetal lamb kidney (FLK) cells, the permanent human cell line Hep-2 or baby hamster kidney (BHK) cells (monolayer type).
The cell cultures can b~ established in suspension according to known methods.
Such a suspension culture is suitable for the repro-duction of reoviruses, MKS viruses, the causative organisms of infectious bovine rhinotracheitis (IBR) or of infectious pustular vulvovaginitis, or herpes simplex viruses, especially, however, the bovine leucosis virus (BLV), the para-influenza-3 virus or herpes virus suis.
The viruses can be worked up in known manner, in order to obtain antigens suitable for diagnostic application or vaccines.
The following examples illustrate the invention~
Example 1 From a 20 liter fermenter containing a stock of FLK
cells infected with BLV, 6 liters of cell suspension were removed, introduced into a 50 liter fermentor and diluted with 19 liters of novel KPL-2 medium + 5 ~ of calf serum.
Composition of KPL-2-medium Basic_medium: NaCl 7.2 g/l K Cl 0.4 g/l g 4 2 0.2 g/l Fe(NO3)3 x 9H2O 0.0001 g/l Na H2 P4 x 2H2O 0 07g/l K H2 P4 0 03 g/l Na2 H PO4 x 2H2O 0.037 g/l Ca Cl2 x 2H2 0.225 g/l neomycin (pure base)0.1 g/l phenol red 0.02 g/l glucose 3.5 g/l Na HCO 1.55 g/l ~22074~
Vitarins: 1 oo-fola according to Eagles 1959 double -Amino acid~:L-cystine 1.6 g/l L-tryptophane 0.8 g/l L-glutamine 30.0 g/l 5 Additives: insulin 2.5 mg/l transferrin 5.0 mg/l biotin 2.0 mg/l peptic peptone 1.25 g/l (Oxoid, L49J
casein peptone 1.25 g/l (Serva,C/TLG).
The suspension culture was incubated at 37C with stirring. From the 24th hour after preparation on, the culture was gassed with 1 liter of air per liter of culture and hour. 5 Days after preparation, the culture was harvested, the cells were separated by centrifugation, and the supernatant was worked up in known manner to give the BLV
antigen (van Maaten and Miller, Bibl. Haematol. (1976) 43, 350-362). By concentration, 250 ml of a leukosis antigen solution having a titer of 1:32 in the agar gel precipitation (AGP) were obtained from these Z5 liters of culture liquid.
Example 2 From a 20 liter fermentor containing FLK cells infected with BLV, 5 liters of cell suspension were removed, introduced into a 50 liter fermentor, and diluted with 30 liters of novel KPL-2 medi~m + 2.5 % of calf serum. With stirring, the suspension culture was incubated at 36C. 30 Hours after preparation, the culture was gassed with 0.5 liter of air per liter of culture and hour.
5 Days after preparation, the culture was harvested, the cells were separated by centrifugation, and the supernatent was worked up in known manner to yield BLV antigen.
By concentration, 350 ml of a solution of leukosis antigen having a titer of 1:32 in the AGP were obtained from the 35 liters of culture supernatant.
Example 3 From a Roux dish, HEP-2 ce~ls were separated in known manner by means of trypsin, introduced into 1 liter of .:
- 5 - HOE 82/B 00~
KPL-2 medium, transferred to a 2 liter fermentor, and maintained in suspension at 37C. After 4 days, the cells had increased from 200,000 cells/ml to 2,000,000 cells/ml.
This cell suspension was then di-luted in a ratio of 1:3 with novel KPL-2 medium, and infected with parainfluenza-3 virus. After 48 hours, the culture was harvested. The KBR
titer of the virus harvest was 1:16.
Exam~le 4 From a Roux dish, HEP-2 cells were separated in known manner by means of trypsin, introduced into 1 liter of RPMI
1640 medium containing 2.5 mg of insulin, 5 mg of transferrin and 2 m~ of biotin, transferred to a 2 liter fermentcr, and maintained in suspension at 37C. After 4 days, the cells had increased from 170,000 cells/ml to
The invention provides a culture medium and a method for the reproduction of viruses in a suspension of cells.
The viruses reproduced yield antigens which can be applied in diagnos~ic agents and optionally in vaccines.
Often, viruses cannot be reproduced but in monolayer cell cultures, that is, the cells and viruses are repro-duced at the interface of the nutrient medium, which means low space/time yields, much operative expenditure, high cost of medium and increased risk of contamination, because many recipients are required for these operations.
To find cGnditions under which a virus is reproduced ir. a suspension of cells which allows utilization of the total space filled with nutrient solution would bring about a considerable advantage over an interface culture.
Virus monolayer cell cultures are state of the art.
A method for the bovine leukosis virus (BLV) is described in J. Nat. Cancer Inst., vol. 52, 491-497 (1974).
This virus causes enzootic leukosis which is a disease of the hematopoietic, lymphatic system of cattle and brings about heavy losses. In order to recognize animals so infected, an antigen prepared in tissue culture can be applied.
Surprisingly, a medium has been found in which cells and viruses can be reproduced in suspension.
Subject of the invention is therefore a nutrient medium andits use in a process for the reproduction of viruses in a suspension of cells.
The medium contains inorganic salts, amino acids, sugar, antibiotics and vitamins, and one or more substances selected from the group of insulin, transferrin, biotin, peptic peptone and casein peptone.
These substances may be contained in the medium in following amounts:
insulin 1.0 - 10.0 mg/l transferrin 2.0 - 20.0 mg/l b~otin 0.5 - 10.0 mg/l peptic peptone 0.5 - 10.0 g/l casein peptone 0.5 - 10.0 g/l ~Z20~44 Optionally, 0 to 5 volume percent of serum are added to the medium. Suitable cells for the cultures are above all fetal lamb kidney (FLK) cells, the permanent human cell line Hep-2 or baby hamster kidney (BHK) cells (monolayer type).
The cell cultures can b~ established in suspension according to known methods.
Such a suspension culture is suitable for the repro-duction of reoviruses, MKS viruses, the causative organisms of infectious bovine rhinotracheitis (IBR) or of infectious pustular vulvovaginitis, or herpes simplex viruses, especially, however, the bovine leucosis virus (BLV), the para-influenza-3 virus or herpes virus suis.
The viruses can be worked up in known manner, in order to obtain antigens suitable for diagnostic application or vaccines.
The following examples illustrate the invention~
Example 1 From a 20 liter fermenter containing a stock of FLK
cells infected with BLV, 6 liters of cell suspension were removed, introduced into a 50 liter fermentor and diluted with 19 liters of novel KPL-2 medium + 5 ~ of calf serum.
Composition of KPL-2-medium Basic_medium: NaCl 7.2 g/l K Cl 0.4 g/l g 4 2 0.2 g/l Fe(NO3)3 x 9H2O 0.0001 g/l Na H2 P4 x 2H2O 0 07g/l K H2 P4 0 03 g/l Na2 H PO4 x 2H2O 0.037 g/l Ca Cl2 x 2H2 0.225 g/l neomycin (pure base)0.1 g/l phenol red 0.02 g/l glucose 3.5 g/l Na HCO 1.55 g/l ~22074~
Vitarins: 1 oo-fola according to Eagles 1959 double -Amino acid~:L-cystine 1.6 g/l L-tryptophane 0.8 g/l L-glutamine 30.0 g/l 5 Additives: insulin 2.5 mg/l transferrin 5.0 mg/l biotin 2.0 mg/l peptic peptone 1.25 g/l (Oxoid, L49J
casein peptone 1.25 g/l (Serva,C/TLG).
The suspension culture was incubated at 37C with stirring. From the 24th hour after preparation on, the culture was gassed with 1 liter of air per liter of culture and hour. 5 Days after preparation, the culture was harvested, the cells were separated by centrifugation, and the supernatant was worked up in known manner to give the BLV
antigen (van Maaten and Miller, Bibl. Haematol. (1976) 43, 350-362). By concentration, 250 ml of a leukosis antigen solution having a titer of 1:32 in the agar gel precipitation (AGP) were obtained from these Z5 liters of culture liquid.
Example 2 From a 20 liter fermentor containing FLK cells infected with BLV, 5 liters of cell suspension were removed, introduced into a 50 liter fermentor, and diluted with 30 liters of novel KPL-2 medi~m + 2.5 % of calf serum. With stirring, the suspension culture was incubated at 36C. 30 Hours after preparation, the culture was gassed with 0.5 liter of air per liter of culture and hour.
5 Days after preparation, the culture was harvested, the cells were separated by centrifugation, and the supernatent was worked up in known manner to yield BLV antigen.
By concentration, 350 ml of a solution of leukosis antigen having a titer of 1:32 in the AGP were obtained from the 35 liters of culture supernatant.
Example 3 From a Roux dish, HEP-2 ce~ls were separated in known manner by means of trypsin, introduced into 1 liter of .:
- 5 - HOE 82/B 00~
KPL-2 medium, transferred to a 2 liter fermentor, and maintained in suspension at 37C. After 4 days, the cells had increased from 200,000 cells/ml to 2,000,000 cells/ml.
This cell suspension was then di-luted in a ratio of 1:3 with novel KPL-2 medium, and infected with parainfluenza-3 virus. After 48 hours, the culture was harvested. The KBR
titer of the virus harvest was 1:16.
Exam~le 4 From a Roux dish, HEP-2 cells were separated in known manner by means of trypsin, introduced into 1 liter of RPMI
1640 medium containing 2.5 mg of insulin, 5 mg of transferrin and 2 m~ of biotin, transferred to a 2 liter fermentcr, and maintained in suspension at 37C. After 4 days, the cells had increased from 170,000 cells/ml to
2,000,000 cells/ml. This cell suspension was then diluted in a ratio of 1:3 with novel RPMI 1640 + insulin, transferrin and biotin, and infected with PI3 virus. After 48 hours, the culture was harvested. The KBR titer was 1:20.
Example 5 1 Liter of cell suspension H~P-2 cells of the 'th suspension passage containing 2,200,000 cells/ml was diluted with 9 liters of RPMI 1640 + 2.5 mg of insulin, 5 mg of transferri~ and 2 mg of biotin, and introduced into a 2 liter ferme~tor. The culture was maintained in suspension at 37C. After 4 days, the cells had increased anew to 2,000,000 cells/ml. They were diluted in a ratio of 1:3 with fresh RPMI 1640 medium + insulin, transferrin and biotin, and infected with PI3 virus. 48 Hours after the infection, the virus harvest had a KBR titer of 1:20.
Example 6 From a Roux dish, BHK monolayer cells were separated by means of trypsin, introduced into 1 liter of KPL-2 medium, transferred to a 2 liter fermentor, and maintained in suspension at 37C. After 48 hours, the cells had increased from 180,000 cells/ml to 2,400,000 cells/ml. The culture was diluted in a ratio of 1:3 with fresh KPL-2 medium, and infected with herpes virus suis. 24 Hours there-after the culture was harvested. In the solid phase immuno-adsorbent test, a titer of 1:30 was found.
.
Example 5 1 Liter of cell suspension H~P-2 cells of the 'th suspension passage containing 2,200,000 cells/ml was diluted with 9 liters of RPMI 1640 + 2.5 mg of insulin, 5 mg of transferri~ and 2 mg of biotin, and introduced into a 2 liter ferme~tor. The culture was maintained in suspension at 37C. After 4 days, the cells had increased anew to 2,000,000 cells/ml. They were diluted in a ratio of 1:3 with fresh RPMI 1640 medium + insulin, transferrin and biotin, and infected with PI3 virus. 48 Hours after the infection, the virus harvest had a KBR titer of 1:20.
Example 6 From a Roux dish, BHK monolayer cells were separated by means of trypsin, introduced into 1 liter of KPL-2 medium, transferred to a 2 liter fermentor, and maintained in suspension at 37C. After 48 hours, the cells had increased from 180,000 cells/ml to 2,400,000 cells/ml. The culture was diluted in a ratio of 1:3 with fresh KPL-2 medium, and infected with herpes virus suis. 24 Hours there-after the culture was harvested. In the solid phase immuno-adsorbent test, a titer of 1:30 was found.
.
Claims (7)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A culture medium comprising inorganic salts, amino acids, sugar, antibiotics, vitamins and at least one substance selected from the group of insulin, transferrin, biotin, peptic peptone and casein peptone.
2. A culture medium as claimed in claim 1, which comprises from 1 to 10 mg/1 of insulin, from 2 to 20 mg/1 of transferrin, from 0.5 to 10 mg/1 of biotin, from 0.5 to 10 g/1 of peptic peptone and/or 0.5 to 10 g/1 of casein peptone.
3. A process for the reproduction of a virus in a sus-pension of cells in which the virus is reproduced in a medium as claimed in claim 1.
4. A process for the reproduction of a virus in a sus-pension of cells in which the virus is reproduced in a medium as claimed in claim 2.
5. A diagnostic agent containing a virus reproduced as claimed in claim 3 or claim 4.
6. A vaccine containing a virus reproduced as claimed in claim 3 or claim 4.
7. A culture medium as claimed in claim 1, which includes insulin, transferrin and biotin.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19823216163 DE3216163A1 (en) | 1982-04-30 | 1982-04-30 | CULTURAL MEDIUM AND METHOD FOR VIRUS REPRODUCTION IN SUSPENSION CULTURE |
| DEP3216163.8 | 1982-04-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1220744A true CA1220744A (en) | 1987-04-21 |
Family
ID=6162375
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000427113A Expired CA1220744A (en) | 1982-04-30 | 1983-04-29 | Culture medium and method for the reproduction of viruses in suspension culture |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0093375B1 (en) |
| JP (1) | JPS58201984A (en) |
| AR (1) | AR230999A1 (en) |
| CA (1) | CA1220744A (en) |
| DE (2) | DE3216163A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4554251A (en) * | 1983-11-07 | 1985-11-19 | The Ohio State University Research Foundation | Process and medium for culturing ant venom gland cells |
| US5024947A (en) * | 1987-07-24 | 1991-06-18 | Cetus Corporation | Serum free media for the growth on insect cells and expression of products thereby |
| KR100507794B1 (en) * | 2003-02-11 | 2005-08-17 | 한미약품 주식회사 | Method for preparing concentrated retroviral particle dispersion |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2553827A1 (en) * | 1975-11-29 | 1977-06-08 | Dow Chemical Co | Protamine zinc insulin additives - to tissue culture media for prepn of viral vaccines esp mumps and german measles |
| GB1480690A (en) * | 1975-12-18 | 1977-07-20 | Dow Chemical Co | Tissue culture media |
| US4205126A (en) * | 1978-01-01 | 1980-05-27 | Cartaya Oscar A | Serum-free cell culture media |
| USRE30753E (en) * | 1980-01-07 | 1981-09-29 | The Regents Of The University Of Minnesota | Process for the large scale production of human growth hormone by serial secondary suspension culture |
-
1982
- 1982-04-30 DE DE19823216163 patent/DE3216163A1/en not_active Withdrawn
-
1983
- 1983-04-26 JP JP58072326A patent/JPS58201984A/en active Granted
- 1983-04-26 EP EP83104059A patent/EP0093375B1/en not_active Expired
- 1983-04-26 DE DE8383104059T patent/DE3363494D1/en not_active Expired
- 1983-04-28 AR AR292843A patent/AR230999A1/en active
- 1983-04-29 CA CA000427113A patent/CA1220744A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| EP0093375A1 (en) | 1983-11-09 |
| JPH0569505B2 (en) | 1993-10-01 |
| DE3216163A1 (en) | 1983-11-03 |
| AR230999A1 (en) | 1984-08-31 |
| DE3363494D1 (en) | 1986-06-19 |
| EP0093375B1 (en) | 1986-05-14 |
| JPS58201984A (en) | 1983-11-25 |
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|---|---|---|---|
| MKEX | Expiry |