Methods for preparing cells and viruses
View Patent ↗Methods for preparing cells and viruses such as poxviruses are provided herein.
1. A method for preparing cells competent for poxvirus replication from a biological tissue, the method comprising:
(a) providing a biological tissue comprising avian primary cells susceptible to infection with a poxvirus that replicates in the cells;
(b) exposing the biological tissue to a solution comprising a protease, and agitating the biological tissue in an orbital shaker running at a speed of at least 95 revolutions per minute (rpm), thereby causing cells of the biological tissue to be dissociated and be released into the solution;
(c) inactivating the protease in the solution;
(d) separating the released cells from the non-dissociated tissue components; and
(e) infecting the released cells with the poxvirus, wherein the released cells are not centrifuged at any point prior to infection with the poxvirus.
2. The method of claim 1 , wherein the biological tissue provided in (a) comprises avian embryo tissue.
3. The method of claim 2 , wherein the avian embryo tissue is chicken embryo tissue.
4. The method of claim 1 , wherein the protease is trypsin.
5. The method of claim 4 , wherein trypsin is present in the solution of (b) at a concentration of 0.25% g/L or less.
6. The method of claim 1 , wherein the protease is inactivated by addition of a solution comprising serum.
7. The method of claim 6 , wherein sufficient serum is added to the solution in (c) to produce a concentration of less than 10% serum in the solution.
8. The method of claim 4 , wherein the trypsin is inactivated in (c) by addition of a trypsin inhibitor to the solution.
9. The method of claim 7 , wherein the trypsin inhibitor is soybean trypsin inhibitor.
10. The method of claim 1 , wherein (d) comprises passing the solution containing the released cells through a filter.
11. The method of claim 2 , wherein the avian embryo tissue is obtained from one or more embryos.
12. The method of claim 4 , wherein the trypsin is recombinant.
13. The method of claim 12 , wherein the recombinant trypsin is produced in a non-mammalian cell.
14. The method of claim 13 , wherein the recombinant trypsin is produced in a bacterial cell.
15. The method of claim 1 , wherein the poxvirus is a vaccinia virus or an avipox virus.
16. The method of claim 15 , wherein the avipox virus is a fowlpox virus or canarypox virus.
17. The method of claim 1 , wherein the cells in (e) are infected in a medium in which the concentration of serum is 0.1% or less.
18. The method of claim 1 , further comprising
(f) incubating the infected cells in a culture medium under conditions in which poxvirus replication occurs in the cells.
19. The method of claim 18 , further comprising
(g) isolating poxvirus from the incubated infected cells.
20. The method of claim 19 , wherein the poxvirus is isolated from the cells in (g) without the use of centrifugation.
21. The method of claim 19 , further comprising
(h) isolating poxvirus from the medium.
22. The method of claim 21 , wherein the poxvirus is isolated using filtration.
23. The method of claim 19 , wherein the poxvirus is isolated from the cells and the medium using filtration.
24. The method of claim 1 , wherein the released cells are infected with poxvirus in (e) and added to a container for incubation at the same time.
25. The method of claim 1 , wherein the agitation runs at a speed of 130-150 revolutions per minute (rpm), and wherein the protease is inactivated by addition of a solution comprising serum.
26. The method of claim 25 , wherein the poxvirus is vaccinia or fowlpox virus.
27. A method for producing poxvirus in vitro, the method comprising:
(a) providing a biological tissue comprising avian primary cells susceptible to infection with a poxvirus that replicates in the cells;
(b) exposing the biological tissue to a solution comprising a protease, and agitating the biological tissue in an orbital shaker running at a speed of at least 95 revolutions per minute (rpm), thereby causing cells of the biological tissue to be dissociated and be released into the solution;
(c) inactivating the protease in the solution;
(d) separating the released cells from the non-dissociated tissue components;
(e) infecting the released cells with the poxvirus, wherein the released cells are not centrifuged at any point prior to infection with the poxvirus;
(f) incubating the cells in medium under condition in which the poxvirus replicates in the cells; and
(g) isolating poxvirus from the cells and medium without the use of centrifugation.
28. The method of claim 27 , wherein the cells are avian embryonic cells.
29. The method of claim 28 , wherein the cells are avian embryonic dermal cells.
30. The method of claim 27 , wherein the poxvirus is a vaccinia virus or an avipox virus.
31. The method of claim 30 , wherein the avipox virus is a fowlpox or a canarypox.
32. The method of claim 27 , wherein the cells are prepared from avian embryo tissue.