CA1064395A - Cell strain and virus culture - Google Patents

Abstract

CELL STRAIN AND VIRUS CULTURE

Abstract A cell culture system capable of supporting viral growth comprises a canine lung cell strain in combination with a nutrient medium. A typical embodiment of the cell strain are those designated A.T.C.C. No. CL-175. Viruses cultivated in the cell culture system incorporating cells of the canine lung cell strain provide antigenic material for the preparation of vaccines.

Description

1 06 43 ~ 5 CELL STRAIN AND VIRUS CULTURE

This invention relates to the preparation of a new cell strain, to the cultivation of viruses therein, and to the production of vaccines.
Although primary animal cells are frequently used as culture media for growing viruses, it is common knowledge that : such cells are generally undesirable since repeated production of cultures from fresh tissues is necessary. This is ~mpracti-cal from an economic standpoint when large quantities are re-quired for commercial production of vaccines. Such primary cells can also carry bacterial and viral contaminants. Efforts are constantly being made ~herefore to discover stable, viable cell strains or cell lines which are economically suitable for the propagation of viruses in the production of human and veterinarian vaccines. A number of cell cultures have been described but very few have, for one or more reasons, proven acceptable. For example~ some become unstable over a period of time and do not maintain uniform growth rates. Other3 acquire a potential for carcinogenesis. In so~e, multiplica-tion rates and split ratios from one passage to the next areunsatisfactory.
A new cell strain has been discovered which can be cultivated and ~ub-cultivated for up to about 35 pa~sages with-out showing any changes in its characteristics. It is suit-` :

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able for growing a number of viruses for vaccine production.
The new cell strain of the present invention is a diploid cell strain derived from embryonic canine lung tissue (CLDLu~.
The cells are derived from the minced lung in a growth medium, preferably Eagle's MEM supplemented with fetal calf serum and containing antibiotics and a fungicide. Master cell stock (MCS~ from the fifteenth subculture is frozen and from this MCS, cell cultures from an additional twenty passages (MCS~20) are obtained and preserved for characterization studies.
Doubling of cell population in each passage occurs in from

2 to 4 days at 35-37 C.
The following characteristics, obtained from studies on cells from the M~S and MCS~20 passages, define the new cell strain of this invention:
1. The cells are fibroblast-like.
2. The cells are diploid as demonstrated by their karyology.

3. The cells are free of mycoplasma, bacteria, fungi and adventitious viral a8ents.

4. The cells are non-tumorigenic and non-oncogenic.

5. The cells are confirmed as being canine by modal diploid chromosome number of 78 and by ~mmunofluores-cence tests.
The present invention thus includes a cell culture --system comprising the fetal canine lung diploid cell strain in a nutrie~t medium which ls suitable for the growth of a number of viruses. Preferably such a cell culture system in-.~

10f~;4395 corporates fetal calf serum. A preferred composition in the cell culture system comprises an inoculum of about 4 x 106 of the fetal canine lung cells in a solution which is about 80-90~ with respect to Eagle's MEM, about 0.15% with respect to Earle's BSS with bicarbonate and from about 10 to abou~
20Z fetal calf serum, preferably about 20~. Other tissue cul-ture media formulations may be used for supporting the fetal canine lung cells.
The cell culture system according to the present invention can be used for the cultivation of a varie~y of viruses including rhabdoviruses, for example PV-12 and HEP
Flury strains of rabies virus; paramyxoviruses, for example parainfluenza virus type 3 and canine distemper virus (Cabasso strain); orbiviruses, for example bluetongue virus, and herpesviruses, for example infectious bovine rhinotracheitis virus. The cell strain of the present invention in combina-tion with the nutrient medium has demonstrated marked superi-ority over a number of other cell strains or cell lines in its ability to support growth of a number of viruses, in parti-cular rabies virus, which results in very high titers of the virus. The cell culture system can be used for producing rabies virus for vaccines suitable for both veterinary and human application.
The production of vaccines also falls within the scope of the present invention. Viruses cultivated from the cell culture system of this invention are further processed to obtain vaccines by methods known in the art. Thus the end 10~;4395 product of a virus cultivation may be used as such or may be attenuated or inactivated by any known procedures to give non-disease producing antigenic material which can be incor-porated into an administrable fonm and dosage as a vaccine for st~mulating the production of antibodies by the host for active immunization. The dosage form includes all prepara-tions which are suitable for intranasal, oral, or parenteral administration and can include physiologically acceptable ad-~uvants or other delayed release means, as well as salt~ and/or buffering agents.
; The fetal canine lung cell strain of the present invention has been deposited in a recognized culture collection, namely the American Type Culture Collection, Rockville, Mary-land 20852 U.S.A., A.T.C.C. No. CL-175.
The in~e~tion will be better understood by the follow-ing examples.
. . .
Example 1 Production of th~ Cell Strain Lung tissue obtained from a female fetus asepti-cally removed from a bltch in about her 50th day of gestation was minced? trypsinized for 5 minutes at 37~ C. u~ing 0.25 percent trypsin solution, and allowed to grow in a 75 cm.
bottle in a medium composed of 89.85 percent Eagle's MEM in 0.15 percent Earle's BSS with sodium bicarbonate (1.43 g./L.) supplemented with 10 percent fetal bovine serum. Antibiotics (penicillin, 100 units/ml. and streptomycin, 100 mcg/ml.) and fungicide (amphotericin B, 2.5 mcg./ml.) were included. Cell population approximately doubled at 35-37C. between two and three days. Following trypsinization and cultivation in the same medium for two additional passages, improved cell growth was obtained by increasing the fetal bovine serum to 20 percent in the fourth to the fifteenth passage. In the fifteenth æub-cultivation, the medium was further modified by the inclusion of 0.01 M HEPES buffer. The cells obtained in the fifteen~h sub-culture were frozen to provide a master cell seed stock (MCS) which was used in the same modified medium and sub-cultured for twenty additional passages (MCS~20). Cells from the fifteenth passage (MCS) and cells fram the thirty-fifth pa~sage (MCS~20) were then characterized.
Cells from MCS and MCS~20 both were fibroblast-like and were free of mycoplasma, yeast, bacteria and fungi [deter-mined for example in accordance with Standard Requirement P-72 of ~he United States Department of Agriculture (USDA) Animal ~-Plant Health Inspection Service, APHIS, Veterinary Services (formerly Veterinary Biologics Services, VBS)].
Results of the chromosome snaly8is of the cells are shown in Table I below and show the cells to be canine with retention of the diploid karyotype.

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Karyotypic analysis for the MCS and MCS~20 cells also con-firmed the morphological similarity between the 20 passage range.
Tests conducted in accord with VBS Standard Require-ment P-72 showed the cells to be free of adventitious viral agents.
Tumorigenicity and oncogenicity tests were performed in cortisone-stressed hamsters in accord with VBS Standard Re-quirement P-72, Attachment B, as published by the Veterinary Biologics Division of the USDA, July 1, 1970. There was no evidence of tumor formation and no abnormalities were detected - in cheek pouches or organs in the body cavities of the hamsters.
The test for non-canine cells by i~munofluorescence technique in accord with VBS Standard Requirement P-72, Attach-ment D, published July 1, 1970, confirmed canine identity of the cell strain and demonstrated freedom from contamination with heterologous species of cells in concurrent use in our labora-tories.
Exam~le 2 General Method for the Cul~ivation of ~ 1 Strain CLDLu cells are infected either in suspension or following formation of a confluent monolayer in culture vessels such as tubes or 32 oz. bottles. Appropriately diluted virus, such as 0.2 ml. of a 10 2-0 to 10 3-5 dilution of virus, re-presenting a multiplicity of infection of 0.0001 to 1.0, is added to the culture vessels containing the cells. If the virus , - . -. . . . ~ ................ : : .
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is added to suspended cells an adsorption period of about one hour at 34-37 C. is required. Diluted virus which is added to cell monolayers remains in con~act with the cells for a period of time such as 0.5 to 1 hour at 34-37C. to permit adsorption to take place. For cells infected in sus-pension, growth medium consisting of about 80-85 percent Eagle's MEM in about 0.15 percent Earle's balanced salt solu-tion, supplemented with 15 to 20Z fetal calf serum provides the necessary nutrients to permit monolayer formation to take place. Exclusion of microbial contaminants is fostered by the presence of combinations of antibiotic formulations such as 100 units/ml. of penicillin and 100 mcg./ml. of streptomycin or 30 units/ml. neomycin and 30 units/ml. of polymyxin, plus : 2.5 mcg./ml. of amphotericin B. The pH of the growth and maintenance medium is controlled by the addition of 0.01 M HEPES
buffer and O.lg% sodium bicarbonate.
; Following incubation at 34-37C. for a period suf- -ficient to permit viral replication to take place, infected fluids are harvested when, in the case of cytolytic viruses, 70-90% of the infected cells exhibit a typical cytopathogenic effect. For non-cytolytic viruses, the infected fluids are harvested when the peak of viral growth has taken place as shown by previous growth curve titrations, typically 2-7 days after infection. Multiple cycles of viral harvests of in-fected fluids are also possible in the case of non-cytolytic viruses, by removal of spent medium, the addition of fresh medium, and continued incubation of the infected cells until .
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a new peak of viral growth is achieved in 1-3 days.
Example 3 Cultivation of Rhabdovirus in the Cell Strain .
To adapt rhabdoviruses to the cell culture system, PV-12 or HEP Flury strains of rabies virus are grown in CLDLu cells for 5-9 passages to prepare seed virus. Three or four days after the cells are infected in suspension, when fluores-cent antibody tests show 10-50Z fluorescing cells, the in-fected cells are detached from the glass wall of the culture vessel with trypsin-EDTA solution and resuspended in growth medium. Cells from each culture vessel used in the first cycle of growth are added to each of two fresh culture vessels for the second cycle of growth. Three to four days following the 1:2 cell split, fluorescent antibody tests of the infected ;~ cells demonstrate 100% rabies antigen positive cells. The in-fected fluids are harvested, pooled and stored at -70C. in aliquot~. The titers o~ these virus harvests range from 105-5 to 107 5 mouse LD50~s/ml ExamPle 4 Cultlvation of Paramyxoviru5 in the Cell Strain Parainfluenza virus type 3 is prepared from viru8 infected ATCC PK-15 cells, this material representing 3-4 ., tissue culture transers of the virus. Four to six days after inoculation of the CLDLu cell cultures, the cells show marked degeneration at which time they are harvested. Cells and fluids are pooled and stored at -70C. in aliquots. The titers : _ g _ ., :
:' of such virus harvests range from 106 to 10 TCID50, Similarly, canine dis~emper virus (Cabasso strain) is prepared from virus infected chick embryo suspensions, this material representing 40-45 subculture transfers of the virus. Three to four days after inoculation of the CLDLu cell suspension, the cells show some degeneration at which time they are harvested. Cells and fluids are pooled and stored at -70C. in aliquots. The titers of these virus harvests range from 103- to 104- TCID50.S/ml Example 5 Cultivation of HerPesvirus in the Cell Strain Infectious bovine rhinotracheitis virus is prepared from virus infected pr~mary bovine kidney cells, ~his material representing 3-4 tissue culture transfers of the virus. Four to six days after inoculation of the CLDLu cultures, about 75X
of the cells are affected, at which time they are harvested.
Cells and fluids are pooled and stored at -70C. in aliquots.
Titration of virus harvests typically give a range from 105-5 to 10 TCID50's/ml Example 6 Cultivation of Orbivirus in the Cell Strain Bluetongue virus is prepared from virus infected chick embryo suspension, this material representing 45-50 subculture transfers of the virus. Four to seven days after inoculation of the CLDLu cultures, about 75X of the cells :

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are affected, at which time they are harvested. Cells and fluids are pooled and stored at -70C. in aliquots. Titra-tion of virus harvests typically give a range from 105- to 10 TCID50 ' s/

Claims (23)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In the process for the preparation of a viral vaccine, the steps which comprise (a) inoculating a culture.
system comprising cells of a fetal canine lung cell strain of the diploid karyotype in combination with a nutrient culture medium with a virus to which said cells are susceptible, (b) cultivating the growth of said virus in said medium and (c) harvesting the resultant viral replication from said medium.

2. The process as defined in claim 1 wherein said fetal canine lung cells correspond to those designated ATCC No. CL-175.

3. The process as defined in claim 1 or claim 2 wherein said virus is rabies virus, infectious bovine rhinotracheitis virus, canine distemper virus, parainfluenza virus type 3, or bluetongue virus.

4. A viral vaccine whenever prepared according to claim 1.

5. In the process for the preparation of a rabies vaccine, the steps which comprise (a) inoculating a culture system comprising cells of a fetal canine lung cell strain of the diploid karyotype in combination with a nutrient culture medium with rabies virus, (b) cultivating the growth of said virus in said medium and (c) harvesting the resultant viral replication from said medium.

6. The process as defined in claim 5 wherein said fetal canine lung cell strain corresponds to that designated ATCC No. CL-175.

7. A rabies vaccine whenever prepared according to the process as defined in claim 5 or claim 6 or an obvious biochemical equivalent thereof.

8. A process for the preparation of a vaccine which comprises:
(a) maintaining a viable culture of a diploid fetal canine lung cell strain in a nutrient culture medium, said cells being fibroblast-like and having a chromosome number of 78;
(b) inoculating a first portion of the cell culture with a rabies virus;
(c) serially passaging the virus for a number of passages sufficient to effect adaptation of the virus to the cells;
(d) inoculating a second portion of the cell culture with the adapted rabies virus;
(e) cultivating the adapted rabies virus; and (f) harvesting the cultivation of the adapted rabies virus.

9. The process as defined in claim 8 wherein the diploid fetal canine lung cell strain corresponds to that designated ATCC No. CL-175.

10. The process as defined in claim 8 wherein the nutrient culture medium comprises fetal bovine serum.

11. A rabies vaccine whenever produced accord-ing to the process as defined in claim 8 or claim 9.

12. In the process for the preparation of an infectious bovine rhinotracheitis vaccine, the steps which comprise (a) inoculating a culture system comprising cells of a fetal canine lung cell strain of the diploid karyotype in combination with a nutrient culture medium with infectious bovine rhinotracheitis virus, (b) cultivating the growth of said virus in said medium and (c) harvesting the resultant viral replication from said medium.

13. The process as defined in claim 12 wherein said fetal canine lung cells correspond to those designated ATCC No. CL-175.

14. An infecious bovine rhinotracheitis vaccine whenever prepared according to the process as defined in claim 12 or claim 13 or an obvious biochemical equivalent thereof.

15. In the process for the preparation of a parainfluenza type 3 vaccine, the steps which comprise (a) innoculating a culture system comprising cells of a fetal canine lung cell strain of the diploid karyotype in com-bination with a nutrient culture medium with a parainfluenza type 3 virus, (b) cultivating the growth of said virus in said medium and (c) harvesting the resultant viral replica-tion from said medium.

16. The process as defined in claim 15 wherein said fetal canine lung cells correspond to those designated ATCC No. CL-175.

17. A parainfluenza virus type 3 vaccine whenever prepared according to the process as defined in claim 15 or elaim 16 or an obvious biochemical equivalent thereof.

18. In the process for the preparation of a canine distemper vaccine, the steps which comprise (a) innoculating a culture system comprising cells of a fetal canine lung cell strain of the diploid karyotype in combination with a nutrient culture medium with a canine distemper virus, (b) cultivating the growth of said virus in said medium and (c) harvesting the resultant viral replication from said medium.

19. The process as defined in claim 18 wherein said fetal canine lung cells correspond to those designated ATCC No. CL-175.

20. A canine distemper vaccine whenever prepared according to the process as defined in claim 18 or claim 19 or an obvious biochemical equivalent thereof.

21. In the process for the preparation of a blue-tongue vaccine, the steps which comprise (a) innoculating a culture system comprising cells of a fetal canine lung cell strain of the diploid karyotype in combination with a nutrient culture medium with a bluetongue virus, (b) cultivating the growth of said virus in said medium and (c) harvesting the resultant viral replication from said medium.

22. The process as defined in claim 21 wherein said fetal canine lung cells correspond to those designated ATCC No. CL-175.

23. A bluetongue vaccine whenever prepared according to the process as defined in claim 21 or claim 22 or an obvious biochemical equivalent thereof.