Generation of duck cell lines
View Patent ↗The present invention relates to the development and manufacturing of viral vaccines. In particular, the invention relates to the field of industrial production of viral vectors and vaccines, more in particular to the use of avian embryonic stem cells, preferably the EBx® cell line derived from duck embryonic stem cells, for the production of viral vectors and viruses. The invention is particularly useful for the industrial production of viral vaccines to prevent viral infection of humans and animals.
1. A method of making a duck cell line comprising:
a) isolating a duck embryo around oviposition;
b) dissociating the embryo of step a) into cells;
c) seeding the dissociated cells of step b) in basal medium comprising insulin like growth factor 1 (IGF-1), ciliary neurotrophic factor (CNTF), animal serum, and a layer of feeder cells;
d) culturing the cells of step c) for at least one passage;
e) withdrawing the IGF-1 and CNTF from the culture of step d);
f) culturing the cells of step e) for at least one passage;
g) progressively withdrawing the feeder cells from the culture of step f);
h) culturing the cells of step g) for at least one passage;
i) progressively withdrawing the animal serum from the culture medium of step h) over several passages; and
j) adapting the cells of step i) to suspension culture conditions such that a duck cell line capable of proliferating in basal medium in the absence of exogenous growth factors, a feeder layer, and animal serum is obtained, wherein the duck cell line does not produce replication-competent endogenous retrovirus particles.
2. The process according to claim 1 , wherein the withdrawal of the growth factors IGF-1 and CNTF from the culture medium in step e) is performed simultaneously.
3. The process according to claim 1 , wherein said duck is a Pekin duck.
4. The process according to claim 1 , wherein said duck is a Muscovy duck.
5. The process according to claim 1 wherein said duck cell line has at least one of the following characteristics:
an endogenous telomerase activity,
a diameter of around 10 μm;
a population doubling time of around 30 hours or less at 37° C.; and
expresses one or more additional markers selected from the group consisting of alkaline phosphatase, SSEA-1, EMA-1, and ENS-1.
6. The process according to claim 1 , wherein said duck cell line is diploid.
7. The process according to claim 1 , wherein said duck cell line does not produce replication-competent endogenous avian leucosis virus (ALV-E) and/or endogenous avian virus (EAV) retrovirus particles.
8. The process according to claim 1 wherein the genome of said duck cell line does not contain proviral sequence of avian leucosis virus (ALV-E) and/or endogenous avian virus (EAV) susceptible to produce replication competent endogenous retroviral particles.
9. The method according to claim 1 , wherein the concentration of IGF-1 and CNTF in step c) each is 1 ng/ml.
10. The method according to claim 1 , wherein the concentration of animal serum in step c) is of 5% to 10%.
11. The method according to claim 1 , wherein the feeder cells in step c) are mitotically inactivated mouse fibroblast cells.