Diploid porcine embryonic cell strains, cultures produced therefrom, and use of said cultures for production of vaccines

Claim 2

A nutrient growth medium for culturing and subculturing a diploid porcine embryonic cell strain which comprises: ______________________________________ L-glutamine 146 to 584 mg/liter Lactalbumin hydrolysate 2,500 to 10,000 mg/liter NaHCO.sub.3 213 to 4,400 mg/liter Vitamin E (α-tocopherol acetate) 0.05 to 0.2 mg/liter Polysorbate 2.5 to 10 mg/liter (Solubilized lipid source) Cholesterol 0.1 to 0.4 mg/liter Bovine, Equine, to provide a final concentration Ovine, Caprine of from about 0.1% to about 20% or Porcine Serum Eagle's Minimum Essential q.s. to 1 liter Medium containing Earle's Salts pH (adjusted with 6.0 to 8.0 acid or base) ______________________________________

Claim 3

A nutrient growth medium according to claim 1 wherein the following amounts of ingredients are present: ______________________________________ L-glutamine 250 to 350 mg/liter Lactalbumin hydrolysate 4,000 to 6,000 mg/liter NaHCO.sub.3 300 to 2,500 mg/liter Vitamin E (α-tocopherol 0.08 to 0.12 mg/liter acetate) Tween 80 (polyoxyethylene 3 to 7 mg/liter sorbitan monooleate) Cholesterol 0.15 to 0.3 mg/liter Bovine serum to provide a final concentration of from about 5% to about 15% Eagle's Minimum Essential Medium containing Earle's Salts q.s. to 1 liter pH (adjusted with hydrochloric 7.1 to 7.3 acid) ______________________________________

Claim 4

A diploid porcine embryonic cell strain characterized by: A. freedom from i. specified viral contaminants as measured by cytopathology, hemadsorption, inclusion body staining, and fluorescent antibody techniques, ii. specified bacterial contaminants as measured by sterility testing, iii. mycoplasma contamination as measured by brothagar subculturing; B. nontumorigenicity in immunologically depressed hamsters; C. possessing a substantially constant degree of viral susceptibility; D. capable of maintaining substantial diploidy and not becoming senescent after at least 36 subculturings, while remaining free from morphological transformation and chromosomal anomalies; and E. retention of marker chromosomes; and a suitable culture medium therefor.

Claim 5

A diploid porcine embryonic cell strain characterized by: A. freedom from i. specified viral contaminants as measured by cytopathology, hemadsorption, inclusion body staining, and fluorescent antibody techniques, ii. specified bacterial contaminants as measured by sterility testing, iii. mycoplasma contamination as measured by brothagar subculturing; B. nontumorigenicity in immunologically depressed hamsters; C. possessing a substantially constant degree of viral susceptibility; D. capable of maintaining substantial diploidy and not becoming senescent after at least 36 subculturings, while remaining free from morphological transformation and chromosomal anomalies; and E. retention of marker chromosomes; and a suitable culture medium therefor.

Claim 6

The diploid porcine embryonic cell strain of claim 3 which has been subcultured six times with eleven cell doublings and having American Type Culture Collection Accession No. CL 184 (ATCC No. CL 184).

Claim 7

A diploid porcine embryonic cell strain of claim 3 capable of being infected by and supportive of the growth of viruses selected from the group consisting of: ______________________________________ transmissible gastroenteritis (TGE) virus, porcine parvovirus (PPV) virus, parainfluenza.sub.3 (PI.sub.3) virus, rabies (R) virus, enteric cytopathic porcine orphan (ECPO) virus, bovine virus diarrhea (BVD) virus, reovirus (RV) virus, bovine enterovirus (BEV) virus, bovine adenovirus (BAV) virus, and bovine parvovirus (BPV) virus. ______________________________________

Claim 8

A diploid porcine embryonic cell strain of claim 4 capable of being infected by and supportive of the growth of a living, attenuated TGE virus.

Claim 9

Process for the preparation of a diploid porcine embryonic cell strain characterized by: A. freedom from i. specified viral contaminants as measured by cytopathology, hemadsorption, inclusion body staining, and fluorescent antibody techniques, ii. specified bacterial contaminants as measured by sterility testing, iii. mycoplasma contamination as measured by brothagar subculturing; B. nontumorigenicity by inoculation of immunologically depressed hamsters; C. possessing a substantially constant degree of viral susceptibility; D. capable of maintaining substantial diploidy and not becoming senescent after at least 36 subculturings, while remaining free from morphological transformation and chromosomal anomalies; and E. retention of marker chromosomes, which comprises; 1. disassociating the cells from a porcine embryo; 2. culturing the said disassociated cells in a nutrient growth medium comprising: ______________________________________ L-glutamine 146 to 584 mg/liter Lactalbumin hydrolysate 2,500 to 10,000 mg/liter Serial No. 700,251 NaHCO.sub.3 213 to 4,400 mg/liter Vitamine E 0.05 to 0.2 mg/liter (α-tocopherol acetate) Polysorbate 2.5 to 10 mg/liter (Solubilized lipid source) Cholesterol 0.1 to 0.4 mg/liter Bovine, Equine, Ovine, to provide a final concentration Caprine or Porcine Serum of from about 0.1% to about 20% Eagle's Minimum Essential q.s. to 1 liter Medium containing Earle's Salts pH (adjusted with acid or base) 6.0 to 8.0 ______________________________________ 3. removing the cultured diploid porcine embryonic strain cells from the nutrient medium by mechanical means, or by exposure to an enzyme, or by exposure to a chelating agent, or by exposure to a mixture of enzymes and chelating agents; and 4. serially subculturing according to step (2) followed by removal of the cultured diploid porcine embryonic strain cells according to step (3), said serial subcultures and removals being carried out at least 42 times.

Claim 10

Process for the preparation of a diploid porcine embryonic cell strain characterized by: A. freedom from i. specified viral contaminants as measured by cytopathology, hemadsorption, inclusion body staining, and fluorescent antibody techniques, ii. specified bacterial contaminants as measured by sterility testing, iii. mycoplasma contamination as measured by brothagar subculturing; B. nontumorigenicity by inoculation of immunologically depressed hamsters; C. possessing a substantially constant degree of viral susceptibility; D. capable of maintaining substantial diploidy and not becoming senescent after at least 36 subculturings, while remaining free from morphological transformation and chromosomal anomalies; and E. retention of marker chromosomes, which comprises; 1. disassociating the cells from a porcine embryo; 2. culturing the said disassociated cells in a nutrient growth medium comprising: ______________________________________ L-glutamine 146 to 584 mg/liter Lactalbumin hydrolysate 2,500 to 10,000 mg/liter Serial No. 700,251 NaHCO.sub.3 213 to 4,400 mg/liter Vitamine E 0.05 to 0.2 mg/liter (α-tocopherol acetate) Polysorbate 2.5 to 10 mg/liter (Solubilized lipid source) Cholesterol 0.1 to 0.4 mg/liter Bovine, Equine, Ovine, to provide a final concentration Caprine or Porcine Serum of from about 0.1% to about 20% Eagle's Minimum Essential q.s. to 1 liter Medium containing Earle's Salts pH (adjusted with acid or base) 6.0 to 8.0 ______________________________________ 3. removing the cultured diploid porcine embryonic strain cells from the nutrient medium by mechanical means, or by exposure to an enzyme, or by exposure to a chelating agent, or by exposure to a mixture of enzymes and chelating agents; and 4. serially subculturing according to step (2) followed by removal of the cultured diploid porcine embryonic strain cells according to step (3), said serial subcultures and removals being carried out at least 42 times.

Claim 11

The process according to claim 7 wherein in step (2) the nutrient growth medium comprises: ______________________________________ L-glutamine 250 to 350 mg/liter Lactalbumin hydrolysate 4,000 to 6,000 mg/liter NaHCO.sub.3 300 to 2,500 mg/liter Vitamin E (α-tocopherol 0.08 to 0.12 mg/liter acetate) Tween 80 (polyoxyethylene 3 to 7 mg/liter sorbitan monooleate) Cholesterol 0.15 to 0.3 mg/liter Bovine serum to provide a final concentration of from about 5% to about 15% Eagle's Minimum Essential Medium containing Earle's Salts q.s. to 1 liter pH (adjusted with hydrochloric 7.1 to 7.3 acid) ______________________________________ and in step (3) the means used for removal of the cultured diploid porcine embryonic strain cells are mechanical or with an enzyme in conjunction with a chelating agent and the said serial subcultures and removals are carried out at least 42 times.

Claim 12

The process of claim 7 wherein a virus selected from the group consisting of: ______________________________________ transmissible gastroenteritis (TGE) virus, porcine parvovirus (PPV) virus, parainfluenza.sub.3 (PI.sub.3) virus, rabies (R) virus, enteric cytopathic porcine (ECPO) virus, orphan bovine virus diarrhea (BVD) virus, reovirus (RV) virus, bovine enterovirus (BEV) virus, Bovine adenovirus (BAV) virus, and bovine parvovirus (BPV) virus. ______________________________________ is grown on the diploid porcine embryonic strain cells thus produced and a vaccine is produced therefrom.

Claim 13

The process of claim 8 wherein a living, attenuated TGE virus is grown on the diploid porcine embryonic cell strain ATCC No. CL 184 thus produced and a vaccine is produced therefrom.

Claim 14

A diploid porcine embryonic cell strain having ATCC No. CL. 184 in a suitable culture medium therefor.

Claim 15

A diploid porcine embryonic cell strain having ATCC No. CL. 184 in a suitable culture medium therefor.