Patent ID: 8148132

Claim:
A process of infecting avian embryonic derived stem cells with a poxvirus and replicating said poxvirus in said avian embryonic derived stem cells, wherein said avian embryonic derived stem cells are obtained by a method comprising the following steps: A) culturing avian embryonic stem cells in a complete culture medium complemented in serum containing: i) exogenous growth factors comprising the trophic factors stem cell factor (“SCF”), Insulin Growth Factor (“IGF-1”), and basic Fibroblast Growth Factor (“bFGF”), and cytokines whose action is through a receptor which is associated with the gp130 protein, said cytokines being selected from the group consisting of leukemia inhibitory factor (“LIF”), interleukin 11 (“IL-11”), interleukin 6 (“IL-6”), interleukin 6 receptor (“IL-6R”), ciliary neurotrophic factor (“CNTF”), oncostatin and cardiotrophin; and ii) an inactivated feeder layer; B) passaging by modifying the culture medium so as to obtain the withdrawal of said exogenous growth factors, of the serum and/or of the feeder layer; and C) establishing an adherent or non-adherent cell culture capable of proliferating in a basal medium in the absence of exogenous growth factors, serum and/or inactivated feeder layer; wherein said process of infecting and replicating a poxvirus in said avian embryonic derived stem cells comprises the steps of: a) proliferating said avian embryonic derived stem cells in a cultivation vessel, in suspension as clumped cells, in a first serum-free medium, allowing said cells to aggregate to each other to form cell clumps, wherein the transfer of the cell culture from the culture vessel to another is performed by dilution without disrupting the cell clumps; b) infecting said clumped cells with the poxvirus when the cell density is of at least 1.5 million cells/ml; c) shortly before infection, simultaneously to infection, or shortly after infection adding to the cell culture a second serum-free medium; d) further culturing said infected cells in order to allow virus replication and propagation; and e) harvesting said virus; wherein said cell clumps are maintained during steps, a, b, c, and d.