Diploid porcine embryonic cell strains, cultures produced therefrom, and use of said cultures for production of vaccines
AssigneeSyntex (U.S.A.) Inc.
InventorsDale Emil Bordt, Phillip Clay Thomas
Life of this patent
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Abstract
Diploid porcine embryonic cell strains, method of growing same from porcine embryonic tissue, culturing and subculturing said diploid cell strains, and preparing a virus vaccine (exemplary of the virus vaccine thus prepared is a porcine transmissible gastroenteritis virus vaccine) from said diploid cell strain cultures. A novel nutrient growth medium composition for culturing and subculturing by serial subcultivation is also disclosed.
Claims11
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 (.alpha.-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) ______________________________________
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.
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 (.alpha.-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.
A diploid porcine embryonic cell strain having ATCC No. CL. 184 in a suitable culture medium therefor.
Dependencies are inferred from the claim text.