Duck embryonic derived stem cell lines for the production of viral vaccines
AssigneeVivalis
InventorsFabienne Guehenneux, Karine Moreau, Magali Esnault, Majid Mehtali
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Abstract
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.
Claims23
A process for obtaining continuous diploid avian cell lines, named EBx®, derived from avian embryonic stem cells (ES), wherein said avian cell lines do not produce replication-competent endogenous retrovirus particles, said process comprising the steps of:
- a) isolating bird embryo(s) at a developmental stage around oviposition, wherein the genome of said bird does not contain endogenous proviral sequences susceptible to produce replication competent endogenous retroviral particles;
- b) suspending avian embryonic stem (ES) cells obtained by dissociating embryo(s) of step a) in a basal culture medium supplemented with:
- Insulin Growth Factor 1 (IGF-1) and Ciliary Neurotrophic Factor (CNTF);
- animal serum;
- and
- optionally, growth factors selected from the group consisting of Interleukin 6 (IL 6), Interleukin 6 Receptor (IL 6R), Stem Cell Factor (SCF) and Fibroblast Growth Factor (FGF);
- c) seeding the suspension of ES cells obtained in step b) on a layer of feeder cells and further culturing the ES cells for at least one passage;
- d) optionally withdrawing all the growth factors selected from the group consisting of IL-6, IL-6R, SCF, FGF from the culture medium and further culturing the ES cells for at least one passage;
- e) withdrawing IGF-1 and CNTF from the culture medium, and further culturing the cells for at least one passage;
- f) progressively decreasing the concentration of feeder cells in the culture medium so as to obtain a total withdrawal of feeder layer after several passages, and further culturing the cells;
- g) optionally, progressively decreasing the concentration of animal serum in the culture medium so as to obtain a total withdrawal of animal serum after several passages;
- and
- h) obtaining adherent avian EBx® cell lines derived from ES cells capable of proliferating in a basal medium in the absence of growth factors, feeder layer optionally without animal serum, and wherein said continuous diploid avian cell lines do not produce replication-competent endogenous retrovirus particles;
- i) optionally, further adapting adherent avian EBx® cell lines to suspension culture conditions.
Dependencies are inferred from the claim text.