Patent GB-1070764-A (GB)
Title: Improvements in or relating to a process for culturing tissue cells
IPC: C12N7/00
Filed: 1965-06-21
Abstract: A process for culturing tissue cells for use in preparing vaccines comprises removing cell tissue from a normal animal, growing the cells in consecutive passages in a cell culture medium until a single diploid cell type remains and beyond the time for latent viruses to express themselves in the culture medium, and continuing culture of the surviving cells in consecutive passages in culture medium untill all cells have uniform growth and morphological characteristics, the minimal total number of passages being 75, even 150.  The cells of a DK line from dog kidney cell tissue are epithelial in appearance and diploid, have chromosomal characteristics similar to those of a normal female Canis familiaris, grow uniformly at a fast and reproducible rate, are maintainable in Eagle's medium containing 10% normal bovine serum, can be preserved in 8% glycerine at -60 DEG C. and are capable of supporting growth of such viruses as canine distemper, infectious canine hepatitis, infectious bovine rhinotracheitis, Herpes Simplex, influenza A and B, and rabies (Flury strain of chick-embryo adapted rabies virus or a Wistar strain) viruses. Before such cells are inoculated with a modified virus capable of growing on the cells in a standard culture medium to produce a modified live virus vaccine, the cells of an extended culture continued in consecutive passages for an extended period of time such as approximately two years, are tested to ensure that the cells do not develop oncogenic properties, during which time the master cell seed stock is preserved in the cold.  According to an example, 2.0ml of Snyder Hill distemper virus seed (adapted to grow on the DK cell line by a series of 24 passages at 10-14 day intervals) is added to cell bottles containing DK cells in a growth medium and then incubated for 3-4 days at 35-37 DEG C.  The cells are shaken loose by a solution of trypsin and ethylenediamine tetra-acetic acid, and the infected cells are reincubated using Eagle's medium with 10% normal horse serum. After three days incubation the culture medium is changed to Earle's salt solution; lactalbumin hydrolysate and antibiotics for a further 4 to 7 days. The attenuated live virus vaccine is then harvested and lyophilized. Infectious canine hepatitis vaccine is similarly prepared using modified strains following 14 passages in porcine kidney tissue. Infectious bovine rhinotracheitis virus is first modified by 10 passages in the DK cell line followed by one additional passage in primary bovine embryonic kidney cells. The DK cells can be infected with virus for vaccine production in either monolayer or suspension culture.ALSO:A process for culturing tissue cells for use in preparing vaccines comprises removing cell tissue from a normal animal, growing the cells in consecutive passages in a cell culture medium until a single diploid cell type remains and beyond the time for latent viruses to express themselves in the culture medium, and continuing culture of the surviving cells in consecutive passages in culture medium until all cells have uniform growth and morphological characteristics, the minimal total number of passages being 75, even 150.  The cells of a DK line from dog kidney cell tissue are epithelial in appearance and diploid, have chromosomal characteristics similar to those of a normal female Canis familiaris, grow uniformly at a fast and reproducible rate, are maintainable in Eagle's medium containing 10% normal bovine serum, can be preserved in 8% glycerin at -60 DEG C and are capable of supporting growth of such viruses as canine distemper, infectious canine hepatitis, infectious bovine rhinotracheitis, Herpes Simplex, influenza A and B, and rabies (Flury strain of chick-embryo adapted rabies virus or a Wistar strain) viruses. Before such cells are inoculated with a modified virus capable of growing on the cells in a standard culture medium to produce a modified live virus vaccine, the cells of an extended culture continued in consecutive passages for an extended period of time such as approximately two years are tested to ensure that the cells do not develop oncogenic properties, during which time the master cell seed stock is preserved in the cold.  According to an example, 2.Oml of Snyder Hill distemper virus seed (adapted to grow on the DK cell line by a series of 24 passages at 10-14 day intervals) is added to cell bottles containing DK cells in a growth medium and then incubated for 3-4 days at 35-37 DEG C.  The cells are shaken loose by a solution of trypsin and ethylenediamine tetra-acetic acid, and the infected cells are reincubated using Eagle's medium with 10% normal horse serum. After three days incubation the culture medium is changed to Earle's salt solution, lactalbumin hydrolysate and antibiotics for a further 4 to 7 days. The attenuated live virus vaccine is then harvested and lyophilized. Infectious canine hepatitis vaccine is similarly prepared using modified strains following 14 passages in porcine kidney tissue. Infectious bovine rhinotracheitis virus is first modified by 10 passages in the DK cell line followed by one additional passage in primary bovine embryonic kidney cells.  The DK cells can be infected with virus for vaccine production in either monolayer or suspension culture.