Method of making a virus using duck embryonic derived stem cell lines

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.

Claims

1. A method of producing a virus, the method 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; 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; k) proliferating the duck cell line obtained in step j) in a serum-free medium; l) infecting the duck cell line of step k) with said virus; m) culturing said infected cells of step l) to allow virus replication; and n) harvesting said virus.

2. The method of claim 1, further comprising adding glutamine and D-glucose to the medium of steps k), l), and m).

3. The method of claim 2, wherein the adding glutamine and D-glucose to the medium of steps k), l), and m) occurs on a daily basis.

4. The method according to claim 2, wherein the glutamine concentration is maintained between 1 mM to 3 mM, preferably around 2 mM.

5. The method according to claim 2, wherein the D-glucose concentration in the culture medium of steps k), l), and m) is maintained between 1 g/l to 10 g/l, preferably around 2 to 3 g/l.

6. The method of claim 1, wherein the cell density in step l) is of at least 1.5 million cells/ml.

7. The method of claim 1, wherein steps k), l), and m) are performed in a cultivation vessel that is a continuous stirred tank bioreactor.

8. The method of claim 1, further comprising adding glutamine and D-glucose to the medium of steps l) and m).

9. The method of claim 1, further comprising adding glutamine and D-glucose to the medium of step l).

10. The method of claim 1, further comprising adding a proteolytic enzyme to the culture medium of step m).

11. The method according to claim 1, wherein steps k), l), and m) are performed at a temperature of 33° C.

12. The method according to claim 1, wherein the withdrawal of IGF-1 and CNTF from the culture medium in step e) is performed simultaneously.

13. The method according to claim 1, wherein said duck is a Pekin duck.

14. The method according to claim 1, wherein said duck is a Muscovy duck.

15. The method according to claim 1, wherein said duck cell line is diploid.

16. The method according to claim 1, wherein the virus is an endogenous avian leucosis virus (ALV-E) or an endogenous avian retrovirus (EAV).

17. The method according to claim 1, wherein said virus is selected from the group comprising poxviruses, orthomyxoviruses, paramyxoviruses, herpes viruses, hepadnaviruses, adenoviruses, parvoviruses, reoviruses, circoviruses, coronaviruses, flaviviruses, togaviruses, birnaviruses, and retroviruses.

18. The method according to claim 17, wherein the poxvirus is selected from the group consisting of Modified Vaccinia Ankara (MVA) virus, Lister-Elstree vaccinia virus, LC16m8 vaccinia virus, CVI78 vaccinia virus, Fowl pox virus, canary pox virus, ALVAC, NYVAC, juncopox virus, mynah pox virus, pigeonpox virus, psittacine pox virus, quail pox virus, sparrow poxvirus, starling pox virus, and Turkey pox virus; the paramyxovirus is selected from the group consisting of measles virus, mumps virus, rubella virus, Sendai virus, Respiratory Syncythial virus (RSV), human para-influenza types I and III, Rinderpest virus, canine distemper virus, Newcastle disease virus, and duck para-influenza virus; the orthomyxovirus is selected from the group consisting of human influenza virus, avian influenza virus, swine influenza virus, equine influenza virus, and feline influenza virus; the togavirus is selected from the group consisting of Sinbis virus, Semliki forest virus, O'nyong'nyong virus, Chikungunya virus, Mayaro virus, Ross river virus, Eastern equine encephalitis virus, Western Equine encephalitis virus, Venezuelan Equine encephalitis virus, and a recombinant togavirus thereof; the retrovirus is selected from the group consisting of reticulo-endotheliosis virus, duck infectious anemia virus, duck spleen necrosis virus, and a recombinant retrovirus thereof; the parvovirus is a duck parvovirus; the adenovirus is selected from the group consisting of fowl adenovirus, goose adenovirus, duck adenovirus, pigeon adenovirus, and a recombinant adenovirus thereof; the birnavirus is Infectious Bursal Disease virus; or the flavivirus is selected from the group consisting of Dengue virus, Japanese encephalitis virus, and West Nile virus.

Description

FIGURES

(1) FIG. 1: Anchorage-Independent chicken EBx cells

(2) FIG. 1A: Anchorage-Independent chicken Valo EBv13 cells In serum free-medium. EBv13 cells were cultured at 37° C. in suspension serum-free medium Excell 65319 (SAFC). EBv13 cells have an homogeneous size and grow in loose clumps into culture. The population doubling time is about 16-18 Hours and the cell density reached in agitated flask vessels were about 4-5 millions cells/ml.

(3) FIG. 1B: Anchorage-independent chicken EB Line 0 cells in serum free-medium

(4) EB Line 0 cells were cultured at 39° C. in suspension serum-free medium Excell 66444 (SAFC). EB Line 0 cells have an homogeneous size and grow in loose clumps.

(5) FIG. 2: Chicken Valo EBv13 cells express high level of Telomerase

(6) EBv13 cells at passage p193 do express high level of telomerase in the same order of magnitude that chicken EB14-O74 cells (see WO03/076601) at passage p164 (Master cell Bank: MCB) or at passage p184 (Workin Cell bank: WCB). Murine embryonic stem cells (mES) were used as a positive control and mouse fibroblast (FED) were used as a negative control.

(7) FIGS. 3A and 3B: Susceptibility of Chicken Valo EBv13 to poxvirus

(8) EBv13 (passage 188) were seeded at 0.4×106 cells/ml in 100 mL F175 flasks either in 40 ml of SFM Excell Medium 65319 or G9916 SFM Medium (SAFC) supplemented with 4 mM Glutamine. The cell growth and infection with MVA-GFP (MOI 10.sup.−2 TCID50/cell) were performed at 37°. One hour post infection, 60 ml of fresh medium were added.

(9) FIG. 3A: cell density kinetics in SFM Excell Medium 65319 or G9916 SFM Medium (SAFC).

(10) FIG. 3B: MVA productivity expressed in TCID50/ml in SFM Excell Medium 65319 or G9916 SFM Medium (SAFC).

(11) FIGS. 4A. 4B and 4C: Transmission Electronic Microscopy analysis of duck EBx cells

(12) FIG. 4A: Micrograph of anchorage-independent duck EBx cells.

(13) FIG. 4B: Transmission Electronic Microscopy analysis of dEBx cells were performed by Dr. A Rivoire (Lyon, France). Duck EBx cells display a typical embryonic stem cells morphology (i.e high nucleo-cytoplasmic ratio) that resemble the phenotype of murine embryonic stem cells and VIVALIS EB14 cells described in WO2006/108846. Duck EBx cells are small round cells with a large nucleus and nucleolus, with short pseudopodia extending from the plasma membrane. They are highly metabolic active with a ribosome and mitochondria rich cytoplasm.

(14) FIG. 4C: Micrograph of duck EBx cells able to grow in suspension, isolated during feeder deprivation, and adapted to grow without additives and serum.

(15) FIGS. 5A and 5B: Telomerase expression In duck EBx cell lines

(16) Telomerase expression during different stages of establishment of duck EBx cells was investigated by using Roche telomerase detection kit (Telomerase OCR ELISA).

(17) FIG. 5A: Telomerase is found to be highly expressed in different adherent duck EBx cell lines just like in chicken EBv13 cells. Duck epithelial cells used as a negative control do not express telomerase.

(18) FIG. 5B: During the process of establishment of suspension duck EBx cells, high level of telomerase expression is maintained. High level of telomerase were investigated in duck EBx cells during feeder deprivation (with or without feeder cells), during the process of adapting duck EB26 cells to suspension and after serum deprivation of dEB24 et dEB26.

(19) Duck EBx cells, such as EB24 and EB26, express high level of telomerase just like chicken EB14 cells. Duck EB66 also express high level of telomerase (Data not shown).

(20) FIGS. 6A and 6B: Duck EBx® cells display no endogenous reverse transcriptase activity

(21) FIG. 6A: Endogenous reverse transcriptase expression was investigated by direct F-PERT analysis (Lovatt et al., 1999, J. Virol. Methods, 82:185-200) in Clean Cells (FRANCE). Duck EBx® cell lines, EB26 and EB5-1, display no endogenous Reverse Transcriptase (RT) activity. High level of RT activity were detected in chicken EB14 and EBv13 cells culture (at different passages) as well as, to a lesser extend, in chicken embryonic libroblast (CEF) derived from Specific Pathogen Free (SPF) chicken strain. CEM cells, which are RTase negative, were used as a negative control to set the detection limit of the assay.

(22) FIG. 6B: Presence of endogenous retroviral particles, either replicative (i.e replication competent) or non-replicative, in the cell culture supernatant of duck and chicken EBx cells were investigated by an ELISA assay detecting the avian leukosis major capsid antigen P27. Duck EBx cell lines, EB26 and EB5-1, as well as chicken EBv13 do not secrete ALV p27 antigen. In the opposite, chicken EB14 cells do express ALV P27 antigen.

(23) FIGS. 7A and 7B: Duck EBx cells do not secrete replicative avian leucosis virus (ALV)

(24) Co-cultivation assay of duck EBx cells with quail QT6 cell line, known to be sensitive to endogenous and exogenous ALVs, were performed in Bioreliance (UK) to detect the presence of endogenous replicative duck viruses.

(25) FIG. 7A: described the principle of QT6 co-culture.

(26) FIG. 7B: The presence of replicative virus is detected by an ELISA assay detecting the avian leukosis major capsid antigen P27.

(27) The assay demonstrates that none of duck EBx® cells tested (dEB26 and dEB51) secrete replicative ALV. RAV-1 virus, which is known to replicate in QT6, were used as a positive control.

(28) FIG. 8: Cell surface expression of receptors SAo2-3 and SAa2-6 in duck EBx and chicken EB14 cell lines

(29) Cells are incubated with digoxygenin labelled lectins: Sambuca nigra agglutinin lectin specifically binds to Sia2-6Gal, while Maackia amurensis agglutinin lectin specifically binds to Sia2-3Gal. Lectins that bind to cells are revealed with anti-digoxygenin antibody FITC-labelled according to well-known techniques by the man skilled in the art. FITC-labelled cells are numbered with a fluorescent cell sorter (FACS). SAa2-3 and SAa2-6 molecules are been described to be the receptors for the avian and human influenza viruses, respectively. Almost all duck EBx cells highly express cell surface receptors SAa2-3 and SAa2-6.

(30) FIGS. 9A and 9B: MVA-GFP virus propagation in infected duck EBx cells

(31) FIG. 9A: Duck EBx® were allowed to form small clumps in T175 stirred tank flasks during cell proliferation in a cell growth SFM medium. Clumps were then infected with 10.sup.−2 TCID.sub.5/cell of MVA-GFP virus and the mixture was diluted in production SFM media. During a 6 days virus propagation period at 37° C., pictures of UV-exposed infected cells were taken daily. The pick of MVA infection was reached at day 4 post-infection (pi). At day 6 μl, the infected cells start to die.

(32) FIG. 9B: MVA-GFP virus titration propagated in duck EBx® cells in a 3 L fed-batch bioreactor. (Left Panel) Duck EBx-derived biomass was allowed to accumulate during cell proliferation phase in Excell growth medium (SAFC). At day 4, cell density reached 4 million cells/ml. Cells were then infected with 10.sup.−1 TCID.sub.50/cell of MVA-GFP virus and the mixture was diluted in 1.5 L Excell medium. During a 6 days virus propagation period at 37° C., samples were collected daily and TCID.sub.50 titration (Right Panel) was performed at the end of the kinetic. A yield of 8.5 log TCID50/ml were reached at 4 p.i. corresponding to a yield of 205 TCID.sub.50/Cell.

(33) FIGS. 10A and 10B: Influence of Calcium and Magnesium concentration In SFM medium on the size of EBx® cells clumps

(34) FIG. 10A: Chicken EBv13 cells were first cultured in the SFM medium from SAFC Biosciences that comprise a high concentration of calcium (Ca2+) (Approx. 0.79 mM) and magnesium (Mg2+) ions; in this medium, cells produce large aggregates in culture.

(35) Three days alter having changed the cell culture medium with the same SFM medium that comprises a lower concentration of Ca2+(0.03 mM final) and Mg2+(1.6 mM final), the cells form smaller aggregates.

(36) FIG. 10B: Duck EB24, EB26 and EB66 cells were first cultured in the SFM medium from SAFC Biosciences that comprise a high concentration of calcium (Ca2+) (Approx. 0.79 mM) and magnesium (Mg2+) ions; in this medium, cells produce large aggregates in culture.

(37) Three days after having changed the cell culture medium with the same SFM medium that comprises a lower concentration of Ca2+(0.03 mM final) and Mg2+(1.6 mM final), the cells form smaller aggregates.

(38) FIGS. 11A and 11B: Production of Influenza virus strains A In duck EBx cells In 3 L-bioreactors

(39) Duck EBx® biomass was allowed to accumulate at 37° C. during cell proliferation phase in a cell growth medium. Cells were then infected with 10.sup.−4 TCID.sub.50/cell of A/H1N1/Beijing/262/95 or A/H3N2/New York/55/2004 influenza virus, the mixture was diluted in 1.5 L Excell production medium supplemented with 0.75 USP/mL of trypsin and temperature was lowered to 33° C. During a 14 days virus propagation period, samples were collected daily and stored at −80° C.

(40) FIG. 11A: Growth kinetic of duck EBx cells infected with A/H1N1/Beijing/262/95 influenza virus strain

(41) Left panel: Cell density (rhombus, ×10.sup.6 cells.ml.sup.−1) and viral titer in logTCID.sub.50/ml.

(42) Right panel: Total number of cells (square), viability (black circles, %) and haemagglutinin concentration in ug/ml (red circles, %).

(43) The viral yield reached 20 ug of Hemagglutinin per ml of culture supernatant.

(44) FIG. 11B: Growth kinetic of duck EBx cells infected with A/H3N2/New York/55/2004 influenza virus strain

(45) Left panel: Cell density (rhombus, ×10.sup.6 cells.ml.sup.−1)

(46) Right panel: Total number of cells (square), viability (black circles, %) and haemagglutinin concentration in ug/ml (red circles, %).

(47) The viral yield reached 30 ug of Hemagglutinin per ml of culture supernatant.

(48) FIG. 12: Production of influenza virus strain B In duck EBx® cells

(49) Duck EBx® biomass was allowed to accumulate at 37° C. during cell proliferation phase in a cell growth medium. Cells were then infected with 10.sup.−3 TCID.sub.50/cell of B/Jiangsu/10/2003 influenza virus, the mixture was diluted in 1.5 L Excell production medium supplemented with 0.75 USP/mL of trypsin and temperature was lowered to 33° C. During a 14 days virus propagation period, samples were collected daily and stored at −80° C.

(50) Left panel: Cell density (rhombus, ×10.sup.6 cells.ml.sup.−1)

(51) Right panel: Total number of cells (square), viability (black circles, %) and haemagglutinin concentration in ug/ml (red circles, %).

(52) The viral yield reached 25 ug of Hemagglutinin per ml of culture supernatant.

(53) FIG. 13: Analysis of NDV productivity and viral protein expression in suspension duck EB66 cells (MOI 10.sup.3, 0.75 USP/mL trypsin)

(54) Duck and chicken EBx cells are sensitive to and replicate NDV La Sota strain. Titers (in TCID50/ml) of NDV produced in duck EB66 cells increase from day 0 to day 2 μl to reach an average of 10.sup.6.83 .sub.TCID50/mL (FIG. 13 Left Panel).

(55) Western blot analysis (FIG. 13 right Panel) showed NDV viral proteins (HN, Fo/F, NP & M) expression. The viral proteins composition of NDV virus produced in duck EB66 cells are similar to the one obtained with NDV virus produced in chicken EB14 cells. In addition, the kinetic of release for viruses produced in chicken and Duck EBx cells are similar.

(56) FIG. 14: Analysis of recombinant Measles virus replication In suspension duck EB66 cells (MOI 10.sup.−1 or 10.sup.−2) In tissue-culture flasks In serum free medium. Duck EB66 cells are at least as sensitive as VERO cells to infection by Measles Virus. Titers (in TCID50/ml) of recombinant Measles virus expressing Green Fluorescent Protein (GFP) produced in duck EB66 cells reach 10.sup.7 TCID50/mL at day 6 post-infection.

(57) FIGS. 15A and 15B: SSEA-1, EMA-1 & Telomerase expression in duck EB66 cells Telomerase expression at different passages of duck EB66 cultured in roller bottles was investigated by using Roche telomerase detection kit (Telomerase OCR ELISA). SSEA-1 and EMA-1 at different passages of duck EB66 cultured in roller bottles was investigated by FACS analysis.

(58) FIG. 15A: Telomerase is found to be highly expressed in suspension duck EB66 cell line at different passages (138, 144, 147, 150, 154).

(59) FIG. 15B: SSEA-1 and EMA-1 cell surface markers was found to be highly expressed in suspension duck EB66 cell line at different passages (138, 144, 147, 150, 154).

(60) FIG. 16: Karyotype analysis of duck EB66 cells

(61) Duck EB66 cells karyotype was performed by Pr. Franck, ENVL, Lyon. EB66 cells are diploid cells.

EXAMPLES

Example 1

Chicken EBv13 Cell Line from SPF Chicken Strain VALO

(62) 1.1—Raw Material

(63) Eggs

(64) Specific Pathogen Free (SPF) strain called Valo. The valo strain is a white Leghorn strain produced and delivered by Lohmann from Germany. Those SPF chicken eggs, supplied with a certificate of analysis, are tested for: CAV, Avian adenoviruses (group 1, serotypes 1-12 and group 3), EDS, Avian Encephalomyelitis Virus, Avian Leukosis Viruses/RSV (including Serotype ALV-J), Avian Nephritis Virus, Avian Reoviruses, Fowlpox Virus, Infectious Bronchitis Virus, Infectious Bursitis Virus (IBDV), Infectious Laryngo Tracheitis Virus, Influenzavirus Typ A, Marek's Disease Virus, Mycoplasmosis (Mg+Ms), Mycobacterium avium, Newcastle Disease Virus, Reticuloendotheliosis Virus, Salmonella pullorum, Other Salmonella Infections, Avian Rhinotracheitis Virus (ART), Hemophilus paragallinarum. Valo chicken eggs were only submitted to a disinfection with the decontaminant to avoid any risk of contamination linked to the manipulation of eggs during the transport.

(65) Feeder Cells

(66) In the first step of the process of establishment of EBv13, cells from murine origin (STO cells) were used as feeder layer to maintain the pluripotency of chicken stem cells. Those feeder cells are mitotically inactivated by gamma irradiation (45 to 55 Grays) before seeding on plastic. This dose of irradiation is a sub-lethal dose that induces a definitive arrest of the cell cycle but still permits the production of growth factors and extracellular matrix, necessary for the promotion of the cell growth of non differentiated cells.

(67) The STO cell line was derived by A. Bernstein, Ontario Cancer Institute, Toronto, Canada from a continuous line of SIM (Sandos Inbred Mice) mouse embryonic fibroblasts and it was supplied by the American Type Culture Collection (ATCC) (STO Product number: CRL-1503, Batch number 1198713). Fresh feeder layers were prepared twice a week, in general on Monday and Thursday. Exponentially cells were dissociated and counted. A part of cells were seeded for maintenance of viable cultures and another part was irradiated. For irradiation, we prepared a cell suspension at 10×10.sup.6 cells/mL in tubes. Cells were exposed to a 45 to 55 grey dose and were seeded on plastic. After seeding, dishes or plates coated with inactivated feeder cells were used during a maximum of 5 days

(68) Medium

(69) DMEM-HamF12 (Cambrex, Cat no BE04-687) Optipro medium (Invitrogen, Cat no 12309) EX-CELL™ 65195, 60947 and 65319 (SAFC, customized medium)
Additives Glutamine (Cambrex, Cat no BE17-605E) Pencillin/streptomycin (Cambrex, Cat no BE17-602E)) Non essential Amino Acids (Cambrex, Cat no BE13-114E) Sodium pyruvate (Cambrex, Cat no BE13-115) Vitamines (Cambrex, Cat no 13-607C) Beta Mercapto Ethanol (Sigma, Cat no M7522)
Buffer and Fixators PBS 1× (Cambrex, Cat no BE 17-516F) Paraformaidehyde 4% (Sigma, Cat no P6148) KCl 5.6% (Sigma, Cat no P9333) Methanol/Acetic acid (3/1): Methanol (Merck, Cat no K34497209; Acetic acid Sigma Cat no A6283) Colcemid. Karyomax (Gibco, Cat no 15212-046)
Cryoprotective Agent Dimethyl Sulfoxyde (DMSO) (Sigma, Cat no D2650)
Factors
Two different recombinant factors were used: Recombinant Human Ciliary Neurotrophic Factor (CNTF) (Peprotech Inc, Cat no 450-13) Recombinant Human Insulin Like Factor I (IGF1) (Peprotech Inc, Cat no 100-11)
The two factors were produced in E. Coli bacteria.
Fetal Bovine Serum

(70) Non Irradiated Fetal Bovin Serum (FBS) (JRH, Cat No 12103)

(71) The non irradiated serum used in the program was collected and produced in United States. Animals used for collection were USDA inspected and acceptable for slaughter. It was added in the medium during avian stem cells culture. This batch was not submitted to irradiation to avoid the destruction of critical proteins or components identified as essential for the maintenance of stem cells in culture.

(72) Irradiated Serum (JRH, Cat No 12107)

(73) The irradiated batch used in this program was also collected in United States. This irradiated batch was added as supplement in the DMEM medium used for the culture of STO or FED cells (feeder cells). Those cells do not require as stem cells a specific quality of serum for growth and maintenance in culture. To minimize high concentration of serum in the medium we have adapted the STO cells to grow in presence of 4% of FBS only.

(74) Dissociating Agents:

(75) Pronase (Roche, Cat No 165 921)

(76) Pronase is a recombinant protease manufactured by Roche Diagnostics, Germany, used for the dissociation of adherent avian stem cells.

(77) Trypsine EDTA (Cambrex, Cat No BE17-161E)

(78) Trypsine is used for the dissociation of STO or FED cells and at late passages for the dissociation of avian cells adapted to Serum Free Medium. This enzyme of porcine origin is manufactured aseptically according to cGMP referential conditions by a validated sterile filtration method and tested according to current E.P. The raw material, irradiated prior to formulation, is tested for porcine parvovirus in strict compliance with 9/CFR 113.53.

(79) Non Enzymatic Cell Dissociation Solution (Sigma, Cat No C5914)

(80) This agent of dissociation is a ready to use formulation used to gently detach cells from the growing surface of the culture vessel. The formula contains no protein, and allows dislodging of cells without use of enzymes. Cellular proteins are preserved making possible immunochemical studies that are dependent upon the recognition of cell surface proteins. This enzyme was used to detach cell before FACS analysis of biological markers like EMA-1 (Epithelial Membrane Antigen 1) and SSEA1 (Stage Specific Embryonic antigen-1).

(81) 1.2—Process of Establishment of EBv13 Cell Line

(82) Eggs are opened, the yolk were separated from the albumen during the opening. The embryos were removed from the yolk either directly with the aid of a Pasteur pipette, or with the aid of a small absorbent filter paper (Whatmann 3M paper), cut out beforehand in the form of a perforated ring with the aid of a punch. The diameter of the perforation were about 5 mm. These small rings were sterilized using dry heat for about 30 minutes in an oven. This small paper ring is deposited on the surface of the yolk and centered on the embryo which is thus surrounded by the paper ring. The latter is then cut out with the aid of small pairs of scissors and the whole removed is placed in a Petri dish, filled with PBS or with a physiological saline. The embryo thus carried away by the ring were cleaned of the excess yolk in the medium and the embryonic disk, thus free of the excess vitellin, is collected with a Pasteur pipette.

(83) The chicken Valo embryos were placed in a tube containing physiological medium (1×PBS, Tris Glucose, medium, and the like). The Valo embryos were then mechanically dissociated and inoculated on a layer of feeder STO cells into complete culture medium at 39° C. The feeder cells were seeded in flask at around 2.7×10.sup.4 cell/cm.sup.2. The complete culture medium is composed of basal commercial medium DMEM-Ham F12 supplemented with 10% fetal call serum, with IGF1 and CNTF at a final concentration of 1 ng/ml, and with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 1 mM, with beta-mercapto-ethanol at a final concentration of 0.2 mM, glutamine at a final concentration of 2.9 mM, with an initial mixture of antibiotics containing penicillin at a final concentration of 100 U/ml and streptomycin at a final concentration of 100 μg/ml. Rapidly after the first passages of the cells, the mixture of antibiotics is no longer added to the medium. The expression rapidly is understood to mean after the first 3 to 5 passages in general.

(84) When the avian ES cells from chicken Valo embryos is passaged from a culture dish to another, the seeding of culture dishes was performed with around between 7×10.sup.4/cm.sup.2 to 8×10.sup.4/cm.sup.2 of avian ES cells in the complete culture medium. Preferably, the seeding is made with around 7.3×10.sup.4/cm.sup.2 (4×10.sup.6 cells/55 cm.sup.2 or 4×10.sup.6 cells/100 mm dish). The avian cells, preferably the avian embryonic cells of step a) are cultured during several passages in the complete medium. At passage 15, the complete medium was depleted in growth factors IGF1 and CNTF. The depletion is made directly in one step, from one passage to another. The embryonic stem cells, preferably the avian embryonic cells are cultured during several passages in the complete medium without IGF1 and CNTF growth factors.

(85) Then depletion of feeder cells were performed after the depletion of growth factors IGF1 and CNTF by a progressive decreasing of feeder cells concentration over several passages. Practically, the same concentration of the feeder cells were used for 2 to 4 passages, then a lower concentration of the feeder cells were used for an additional 2 to 4 passages, and so on. The flask were originally seeded with around 2.7×10.sup.4 feeder cells/cm.sup.2, then around 2.2×10.sup.4 feeder cells/cm.sup.2, then around 1.8×10.sup.4 feeder cells/cm.sup.2, then around 1.4×10.sup.4 feeder cells/cm.sup.2, then around 1.1×10.sup.4 feeder cells/cm.sup.2, then around 0.9×10.sup.4 feeder cells/cm.sup.2, then around 0.5×10.sup.4 feeder cells/cm.sup.2. Then the flask were seeded with 6.5×10.sup.4 avian cells/cm.sup.2 to 7.5×10.sup.4 avian cells/cm.sup.2 and without feeder cells. The depletion of feeder cells started at around passage 21 and ended at around passage 65. During the depletion of feeder cells, the chicken Valo ES cells were seeded in culture flask at a lower concentration than in step a), about around 4×10.sup.4 cell/cm.sup.2 to 5×10.sup.4 cell/cm.sup.2. In the hypothesis that Valo ES cells were not in good shape following a decrease of feeder cells concentration in the flask, then the avian cells are cultured for additional passages with the same feeder cells concentration before to pursue the feeder cells depletion.

(86) The serum depletion were performed after the growth factor and the feeder cells depletion. At the beginning of serum depletion, the culture medium were composed of basal commercial medium DMEM-HamF12 supplemented with 10% fetal calf serum and with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 1 mM, with beta-mercaptoethanol at a final concentration of 0.2 mM, glutamine at a final concentration of 2.9 mM. The chicken Valo cells were adapted to the growth in a serum free medium culture in a two steps process: first, the chicken Valo cells were rapidly adapted to a culture medium composed of commercial serum free medium (SFM), preferably ExCell 60947 (SAFC Biosciences) supplemented with 10% fetal calf serum and with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 1 mM, with beta-mercaptoethanol at a final concentration of 0.2 mM, glutamine at a final concentration of 2.9 mM. Once this rapid adaptation to a new medium (DMEM-HamF12 to Excell 60947) was performed, a second step is performed consisting of a slow adaptation to decreasing concentration of animal serum in the SFM medium were initiated. Serum depletion was performed by a progressive decreasing starting from 10% serum, then 7.5%, then 5%, then 2.5%, then 1.25%, then 0.75% of serum concentration in SFM cell culture medium to finally reach 0% serum in SFM cell culture medium. Serum depletion started at passage 103 and ended at passage 135.

(87) At the end of the process of deprivation of serum when the remaining concentration of serum in SFM medium was either 0.75% or 0%, the adaptation of anchorage-dependent EBv13 cells to suspension culture started. Among the several attempts performed to isolate anchorage-independent EBv13 isolates, 62.5% of the attempts were successful and allow to get different isolates of suspension EBv13 cells. One isolate of EBv13 cells were selected according to the population doubling time (around 18 h), the optimal cell concentration into flask culture (around 4 million cell/ml), the cell viability, the cell culture homogeneity (presence and size of cells clumps) and the easiness to manipulate the cells (FIG. 1).

(88) At the end of serum depletion, anchorage dependent chicken Valo cells, named EBv13 were able to grow in absence of grow factors, in absence of feeder cells, in serum free medium. EBv13 Cells were then adapted to growth at 37° C., by progressively decreasing cell culture temperature of 0.5° C./day.

Example 2

Chicken EB Line 0 Cell Line from SPF Chicken Strain ELL-0

(89) 2.1—Raw Material

(90) Eggs:

(91) Chicken Specific Pathogen Free (SPF) strain called ELL-0 (East Lansing Line 0) was provided by the Avian Disease and Oncology Laboratory (USDA-ARS-MWA, USA). Those SPF chicken eggs, are produced from a flock tested intensively to various poultry pathogens. Disease tested include: Salmonella pullorum, Salmonella gallinarum, mycoplasma gallisepticum, mycoplasma synoviae, Avian Leukosis virus A-D and J, Marek's disease virus, Reticuloendotheliosis virus. Avian adenovirus. Infectious bronchitis, Infectious bursal disease, Avian Influenza, Newcastle disease, Avian encephalomyelitis and Avian Reovirus. Line 0 chicken eggs were only submitted to a desinfection with the decontaminant to avoid any risk of contamination linked to the manipulation of eggs during transportation.

(92) Feeder Cells

(93) In the first step of the process of establishment of EB Line 0, cells from murine origin (STO cells) were used as feeder layer to maintain the pluripotency of chicken stem cells. Those feeder cells are mitotically inactivated by gamma irradiation (45 to 55 Grays) before seeding on plastic. This dose of irradiation is a sub-lethal dose that induces a definitive arrest of the cell cycle but still permits the production of growth factors and extracellular matrix, necessary for the promotion of the cell growth of non differentiated cells.

(94) The STO cell line was derived by A. Bernstein, Ontario Cancer Institute, Toronto, Canada from a continuous line of SIM (Sandos Inbred Mice) mouse embryonic fibroblasts and it was supplied by the American Type Culture Collection (ATCC) (STO Product number: CRL-1503, Batch number 1198713). Fresh feeder layers were prepared twice a week. Exponentially cells were dissociated and counted. A part of cells were seeded for maintenance of viable cultures and another part was irradiated. For irradiation, we prepared a cell suspension at 10×10.sup.6 cells/mL in tubes. Cells were exposed to a 45 to 55 grey dose and were seeded on plastic. After seeding, dishes or plates coated with inactivated feeder cells were used during a maximum of 5 days.

(95) Media

(96) DMEM-HamF12 (Cambrex, Cat no BE04-687) Medium GTM-3 (Sigma, Cat no G9916) Medium EX-CELL™ 66522, 65788 and 66444 (SAFC, customized medium)
Additives Glutamine (Cambrex, Cat no BE17-605E) Pencillin/streptomycin (Cambrex, Cat no BE17-602E)) Non essential Amino Acids (Cambrex, Cat no BE13-114E) Sodium pyruvate (Cambrex, Cat no BE13-115) Vitamines (Cambrex, Cat no 13-607C) Beta Mercapto Ethanol (Sigma, Cat no M7522) Yeastolate (SAFC, Cat no 58902C)
Buffer and Fixators PBS 1× (Cambrex, Cat no BE17-516F)
Cryoprotective Agent Dimethyl Sulfoxyde (DMSO) (Sigma, Cat no D2650))
Factors
Six different recombinant factors were used: Recombinant Human Ciliary Neurotrophic Factor (CNTF) (Peprotech Inc, Cat no 450-13) Recombinant Human Insulin Like Factor I (IGF1) (Peprotech Inc, Cat no 100-11) Recombinant Human Interleukin 6 (IL6) (Peprotech Inc, Cat no 200-06) Recombinant Human soluble Interleukin 6 receptor (slL6r) (Peprotech Inc, Cat no 200-06 R) Recombinant Human Stem Cell Factor (SCF) (Peprotech Inc, Cat no 300-07) Recombinant Human basic Fibroblast Growth Factor (bFGF) (Peprotech Inc, Cat no 100-18B)

(97) All those factors, excepted IL6r, are produced in E. Coli bacteria. Soluble IL6r is expressed in transfected HEK293 cells.

(98) Fetal Bovine Serum

(99) Non Irradiated Fetal Bovin Serum (FBS) (SAFC, Cat No 12003)

(100) The non irradiated serum used in the program was collected and produced in Australia. Animals used for collection were USDA inspected and acceptable for slaughter. It was added in the medium during avian stem cells culture. This batch was not submitted to irradiation to avoid the destruction of critical proteins or components identified as essential for the maintenance of stem cells in culture.

(101) Irradiated Serum (JRH, Cat No 12007)

(102) The irradiated batch used in this program was collected in Australia. This irradiated batch was added as supplement in the DMEM medium used for the culture of STO or FED cells (feeder cells). Those cells do not require as stem cells a specific quality of serum for growth and maintenance in culture. To minimize high concentration of serum in the medium we have adapted the STO cells to grow in presence of 4% of FBS only.

(103) Dissociating Agents:

(104) Trypzean ((Sigma, Cat No 73449)

(105) 2.2—Process of Establishment of the Line 0 Cell Line

(106) Embryos from 13 eggs from Line 0 chicken were collected according to the process described in Example 1.2. Then, the Line 0 embryos were placed in a tube containing PBS 1×. Embryos were then mechanically dissociated and inoculated on a layer of feeder STO cells into complete culture medium at 39° C. The feeder cells were seeded in dishes at around 2.7×10.sup.4 cell/cm.sup.2. The complete culture medium is composed of basal commercial medium DMEM-Ham F12 supplemented with 10% fetal calf serum, with IGF1, CNTF, bFGF, IL6, IL6r and SCF at a final concentration of 1 ng/ml, and with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 1 mM, with beta-mercapto-ethanol at a final concentration of 0.2 mM, glutamine at a final concentration of 2.9 mM, with yeastolate 1× and with an initial mixture of antibiotics containing penicillin at a final concentration of 100 U/ml and streptomycin at a final concentration of 100 μg/ml. After 7 passages, the mixture of antibiotics is no longer added to the medium.

(107) When the avian ES cells from chicken Line 0 embryos are transferred from a culture dish to another, the seeding of culture dishes was performed with around between 7×10.sup.4/cm.sup.2 to 8×10.sup.4/cm.sup.2 of avian ES cells in the complete culture medium. Preferably, the seeding is made with around 7.3×10.sup.4/cm.sup.2 (4×10.sup.6 cells/55 cm.sup.2 or 4×10.sup.6 cells/100 mm dish). The avian cells, preferably the avian embryonic cells of step a) are cultured during several passages in the complete medium supplemented with 10 or 15% of FBS. At passage 7, the complete medium was depleted in growth factors bFGF, IL6, IL6r and SCF. The depletion was made directly in one step, from one passage to another. The embryonic stem cells, preferably the avian embryonic cells, were cultured during several passages in the complete medium without those 4 growth factors. At passage 12, the 2 last factors IGF1 and CNTF were removed from the medium and cells were amplified without factor.

(108) To promote cell growth 3 base medium were used successively: DMEM Ham F12 from passage 1 to passage 18, Exell GTM-3 from passage 18 to passage 26 and a mixture of Excell 66788 and Excell 66522 after passage 26.

(109) After passage 30, depletion of feeder cells was performed by a progressive decreasing of feeder cells concentration over several passages following the step by step process previously described. During this phase of feeder deprivation, some cells able to grow in suspension were isolated using Excell 66444 as growth medium and serum deprivation was initiated (FIG. 1B).

Example 3

Duck EBx Cell Line EB66

(110) 3.1—Raw Material

(111) Duck Eggs

(112) Duck eggs from Peking strains GL30 were obtained from GRIMAUD FRERES SELECTION (La Corbiére, Roussay France). The parent ducks were vaccinated against Escherichia Coli (Autogenous vaccine Coli 01 & 02), Pasteurella multocida (Landavax), Duck viral hepatitis (Hepatovax), Erysipelothrix rhusiopathiae (Ruvax), Avian metapneumovirus (Nemovac), Salmonella typhimurium & Enteridis (Autogenous vaccine), Riemerella antipestifer (Autovaccine Riemerella), Avian metapneumovirus (Nobilis RTV inactive) and Erysipelothrix rhusiopathiae (Ruvax). After receipt, fertilized Peking duck eggs were submitted to a disinfection in an hypochloryde bath followed by a decontamination with Fermacidal (Thermo) to avoid any risk of contamination linked to dusts attached on the shell.

(113) Feeder Cells

(114) In the first step of the process, cells from murine origin (STO cells) were used as feeder layer to maintain the pluripotency of duck stem cells. Those feeder cells are mitotically inactivated by gamma irradiation (45 to 55 Grays) before seeding on plastic. This dose of irradiation is a sub-lethal dose that induces a definitive arrest of the cell cycle but still permits the production of growth factors and extracellular matrix, necessary for the promotion of the cell growth of non differentiated cells. The STO cell line was derived by A. Bernstein, Ontario Cancer Institute, Toronto, Canada from a continuous line of SIM (Sandos Inbred Mice) mouse embryonic libroblasts and it was supplied by the American Type Culture Collection (ATCC) (STO Product number: CRL-1503, Batch number 1198713). Fresh feeder layers were prepared twice a week. Exponentially cells were dissociated and counted. A part of cells were seeded for maintenance of viable cultures and another part was irradiated. For irradiation, we prepared a cell suspension at 10×10.sup.6 cells/mL in tubes. Cells were exposed to a 45 to 55 grey dose and were seeded on plastic. After seeding, dishes or plates coated with inactivated feeder cells were used during a maximum of 5 days.

(115) Medium

(116) Medium EX-CELL™ 65788, 65319, 63066 and 66444 (SAFC, customized medium) Medium GTM-3 (Sigma, Cat no G9916) DMEM-HamF12 (Cambrex, Cat no BE04-687) DMEM (Cambrex, Cat no BE 12-614F)
Additives Glutamine (Cambrex, Cat no BE17-605E) Pencillin/streptomycin (Cambrex, Cat no BE17-602E)) Non essential Amino Acids (Cambrex, Cat no BE13-114E) Sodium pyruvate (Cambrex, Cat no BE13-115) Vitamines (Cambrex, Cat no 13-607C) Beta Mercapto Ethanol (Sigma, Cat no M7522) Yeastolate (SAFC, Cat no 58902C)
Buffer and Fixators PBS 1× (Cambrex, Cat no BE 17-516F)
Cryoprotective Agent Dimethyl Sulfoxyde (DMSO) (Sigma, Cat no D2650)
Factors

(117) Two different recombinant factors were used: Recombinant Human Ciliary Neurotrophic Factor (CNTF) (Peprotech Inc, Cat no 450-13) Recombinant Human Insulin Like Factor I (IGF1) (Peprotech Inc. Cat no 100-11)

(118) Those 2 factors are produced in E. Coli bacteria.

(119) Fetal Bovine Serum

(120) Non Irradiated Fetal Bovin Serum (FBS) (JRH, Cat No 12003)

(121) The non irradiated serum used in the program was collected and produced in Australia. Animals used for collection were USDA inspected and acceptable for slaughter. It was added in the medium during avian stem cells culture. This batch was not submitted to irradiation to avoid the destruction of critical proteins or components identified as essential for the maintenance of stem cells in culture.

(122) Irradiated Serum (JRH, Cat No 12107)

(123) The irradiated batch used in this program was collected in United States. This irradiated batch was added as supplement in the DMEM medium used for the culture of STO cells (feeder cells). Those cells do not require as stem cells a specific quality of serum for growth and maintenance in culture. To minimize high concentration of serum in the medium we have adapted the STO cells to grow in presence of 4% of FBS only.

(124) Dissociating Agents:

(125) Pronase (Roche, Cat No 165 921)

(126) Pronase is a recombinant protease manufactured by Roche Diagnostics, Germany, used for the dissociation of adherent avian stem cells.

(127) Trypsine EDTA (Cambrex, Cat No BE17-161E)

(128) Trypsine is used for the dissociation of STO cells and at late passages for the dissociation of avian cells adapted to Serum Free Medium. This enzyme of porcine origin is manufactured aseptically according to cGMP referential conditions by a validated sterile filtration method and tested according to current E.P. The raw material, irradiated prior to formulation, is tested for porcine parvovirus in strict compliance with 9/CFR 113.53.

(129) Trypzean (Sigma, Cat No T3449)

(130) Trypzean solution is formulated with a recombinant bovine trypsin, expressed in corn and manufactured by Sigma Aldrich utilizing ProdiGene's proprietary transgenic plant protein expression system. This product is optimized for cell dissociation in both serum free and serum-supplemented adherent cell cultures.

(131) Non Enzymatic Cell Dissociation Solution (Sigma, Cat No C5914)

(132) This agent of dissociation is a ready to use formulation used to gently detach cells from the growing surface of the culture vessel. The formula contains no protein, and allows dislodging of cells without use of enzymes. Cellular proteins are preserved making possible immunochemical studies that are dependent upon the recognition of cell surface proteins. This enzyme was used to detach cell before FACS analysis of biological markers like EMA-1 (Epithelial Membrane Antigen 1) and SSEA1 (Stage Specific Embryonic antigen-1).

(133) 3.2—Process of Establishment of Duck EBx Cell Line EB66

(134) Around 360 Fertilized duck eggs were opened, the yolk were separated from the albumen during the opening. The embryos were removed from the yolk with the aid of a small absorbent filter paper (Whatmann 3M paper), cut out beforehand in the form of a perforated ring with the aid of a punch. The diameter of the perforation is about 5 mm. These small rings were sterilized using dry heat for about 30 minutes in an oven. In practice, during the step of embryo collection, a small paper ring is deposited on the surface of the yolk and centered on the embryo which is thus surrounded by the paper ring. The latter is then cut out with the aid of small pairs of scissors and the whole removed is placed in a Petri dish, filled with PBS. The embryo thus carried away by the ring were cleaned of the excess yolk in the medium and the embryonic disk, thus free of the excess vitellin, were collected with a Pasteur pipette.

(135) The duck embryos were placed in 50 mL tubes containing PBS 1×. The duck embryos were then mechanically dissociated, washed with PBS, and seeded on an inactivated layer of feeder STO cells into complete culture medium at 39° C., 7.5% CO.sub.2. The feeder cells were seeded in 6 well plates or dishes at around 2.7×10.sup.4 cell/cm.sup.2. The complete culture medium is composed of serum free medium DMEM-Ham F12 supplemented with 10% fetal bovine serum, with IGF1, CNTF, at a final concentration of 1 ng/ml, and with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 0.1 mM, with beta-mercapto-ethanol at a final concentration of 0.5 mM, glutamine at a final concentration of 2.1 mM, penicillin at a final concentration of 100 U/ml, streptomycin at a final concentration of 100 μg/ml and yeastolate 1×. Rapidly at the passage 4, the mixture of antibiotics is no longer added to the medium.

(136) The duck ES cells were cultured in the DMEM-Ham F12 medium up to passage 4. After passage 4, the base medium is modified and DMEM-Ham F12 complete medium is replaced by the SFM GTM-3 medium supplemented with 10% fetal bovine serum, with IGF1, CNTF, at a final concentration of 1 ng/ml, with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 0.1 mM, with beta-mercapto-ethanol at a final concentration of 0.5 mM, glutamine at a final concentration of 2.1 mM and yeastolate 1×. The duck ES cells were further cultured during 14 passages in this new medium of culture, then growth factors deprivation was performed at passage 18. IGF1 and CNTF were simultaneously removed from the medium, thus from passage 19 to passage 24, the medium of culture was GTM-3 medium supplemented with 10% FBS, with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 0.1 mM, with beta-mercapto-ethanol at a final concentration of 0.5 mM, glutamine at a final concentration of 2.1 mM and yeastolate 1×.

(137) When the duck ES cells from Peking duck embryos are passaged from a culture dish to another, the seeding of culture dish was performed with around between 7×10.sup.4/cm.sup.2 to 12×10.sup.4/cm.sup.2 of duck ES cells in the complete culture medium.

(138) Then, after passage 24, depletion of feeder cells were performed by a progressive decrease of feeder cells concentration over several passages. The dishes were originally seeded with around 2.7×10.sup.4 feeder cells/cm.sup.2, then around 1.8×10.sup.4 feeder cells/cm.sup.2 between passage 25 and 31, then around 1.4×10.sup.4 cells/cm.sup.2 between passage 32 and 35, then around 1×10.sup.4 feeder cells/cm.sup.2 between passage 36 and 41, then around 0.7×10.sup.4 feeder cells/cm.sup.2 between passage 42 and 44, and finally from passage 45 dishes were seeded only with avian cells and without feeder cells. At the end of the feeder depletion, the dishes are seeded with 9×10.sup.4 avian cells/cm.sup.2 to 12.7×10.sup.4 avian cells/cm.sup.2. The depletion of feeder cells started at passage 25 and ended at passage 45. During the depletion of feeder cells, the duck ES cells are seeded in culture dishes at a higher concentration than in step a), about around 9×10.sup.4 cell/cm.sup.2 to 12.7×10.sup.4 cell/cm.sup.2 After several passages without feeder cells, growth parameters (Population Doubling Time (PDT) and Density) are studied to confirm cell stability and robustness and to initiate the deprivation of amino acids, vitamins, beta mercaptoethanol, sodium pyruvate and yeastolate. Cells are considered as enough robust to be submitted to such deprivation if, PDT is lower than around 40 hours and cell density higher than around 26×10.sup.4 cells/cm.sup.2.

(139) In the case of the present duck EBx® cells development, named EB66, deprivation of vitamins, sodium pyruvate, non essential amino acids and beta mercaptoethanol were initiated at passage 52. All those additives were removed simultaneously from the medium. Thus, between passage 52 and passage 59, the medium of culture is SFM GTM-3 supplemented with glutamine, yeastolate and FBS.

(140) Following a short period of adaptation to the new conditions of culture, temperature decreasing was initiated. This decrease was performed progressively between passage 60 and passage 67. After passage 67 cells were able to grow at 37° C. After passage 67, the base medium GTM-3 was replaced by a new SFM base medium called Excell 65788. So, after passage 67 the culture medium was Excell 65788 supplemented with 10% FBS, 2.5 mM glutamine and 1× yeastolate. At passage 80, 4×10.sup.6 cells were transferred in a Ultra Low Attachment (ULA) dish maintained under constant agitation to initiate anchorage-independent cells growth. To promote the growth as suspension, the base medium was modified and percentage of serum was decreased from 10% to 5% for the seeding in the ULA dish. Thus from passage 80 to passage 85 the medium of culture was SFM GTM-3 supplemented with 5% FBS, 2.5 mM glutamine and 1× yeastolate. Slow decrease of FBS was initiated on EB66 cell suspension after passage 85. Serum depletion was performed by a progressive decreasing starting from 2.5% serum, then 1.5% of serum concentration in SFM cell culture medium to finally reach 0% serum in SFM cell culture medium. Serum depletion started at passage 86 and ended at passage 94. At the end of serum depletion, anchorage independent dEB66 cells were able to grow at 37° C. in absence of grow factors, in absence of feeder cells, in serum free medium.

(141) Alter the obtaining of EB66 duck cells that are able to grow at 37° C. in the SFM GTM-3 supplemented by 2.5 mM glutamine, some further adaptation to SFM media were made by dilution or progressive adaptation in new SFM formulations as Excell 63066, Excell 66444, Excell CHO ACF for example. The subcloning of suspension duck EB66 cell could also realized in presence or absence of yeastolate

Example 4

Duck EBx Cell Line EB26

(142) 4.1—Raw Material

(143) Duck Eggs, Feeder Cells, Additives, Buffers and Fixators, Cryopreservative Agents, Fetal Calf Serum & Dissociating Agents (Idem as Example 3).

(144) Duck eggs from Peking strains GL30 were used.

(145) Medium

(146) Medium EX-CELL 65319, 63066 and 66444 (SAFC, customized medium) Medium GTM-3 (Sigma, Cat no G9916) DMEM (Cambrex, Cat no BE 12-614F)
Factors

(147) Six different recombinant factors were used: Recombinant Human Ciliary Neurotrophic Factor (CNTF) (Peprotech Inc, Cat no 450-13) Recombinant Human Insulin Like Factor I (IGF1) (Peprotech Inc, Cat no 100-11) Recombinant Human Interleukin 6 (IL6) (Peprotech Inc, Cat no 200-06) Recombinant Human soluble Interleukin 6 receptor (slL6r) (Peprotech Inc, Cat no 200-06 R) Recombinant Human Stem Cell Factor (SCF) (Peprotech Inc, Cat no 300-07) Recombinant Human basic Fibroblast Growth Factor (bFGF) (Peprotech Inc, Cat no 100-18B)

(148) All those factors, excepted IL6r, are produced in E. Coli bacteria. Soluble IL6r is expressed in transfected HEK293 cells.

(149) 4.2—Process of Establishment of Duck EBx Cell Line EB26

(150) The duck embryos were collected as previously described with EB66. The duck embryos were placed in 50 mL tubes containing PBS 1×. The duck embryos were then mechanically dissociated, washed in PBS, and seeded on an inactivated layer of feeder STO cells into complete culture medium at 39° C., 7.5% CO.sub.2. The feeder cells were seeded in 6 well plates or dishes at around 2.7×10.sup.4 cell/cm.sup.2. The complete culture medium is composed of serum free medium GTM-3 supplemented with 5% fetal bovine serum, with IGF1, CNTF, 11-6, 11-6R, SCF and FGF at a final concentration of 1 ng/ml, and with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 0.1 mM, with beta-mercapto-ethanol at a final concentration of 0.5 mM, glutamine at a final concentration of 2.1 mM, penicillin at a final concentration of 100 U/ml, streptomycin at a final concentration of 100 μg/ml and yeastolate 1×. Rapidly after the first passages of the cells, the mixture of antibiotics is no longer added to the medium. The expression rapidly is understood to mean alter the first 3 to 9 passages in general. The duck ES cells were cultured in the complete medium up to passage 9. After passage 9, the complete medium is partially depleted in factors. Thus, between passage 10 and 13, SCF, IL6, IL6r and bFGF were removed for the medium and only recombinant IGF1 and CNTF were maintained at a concentration of 1 ng/mL. A simultaneous decease of concentration of IGF1 and CNTF is secondly performed between passage 13 and 16 to finally obtain cells able to grow without recombinant factors at passage 17. The factor depletion were made by a progressive adaptation to lower concentrations of factors. When the duck ES cells from Pekin duck embryos were passaged from a culture dish to another, the seeding of culture dish was performed with around between 7×10.sup.4/cm.sup.2 to 12×10.sup.4/cm.sup.2 of duck ES cells in the complete culture medium. Preferably, the seeding is made with around 7.3×10.sup.4/cm.sup.2 (4×10.sup.6 cells/55 cm.sup.2 or 4×10.sup.6 cells/100 mm dish). After depletion of recombinant factors, a decrease of yeastolate were performed at passage 23 reaching the final concentration at 0.5×. Then, after passage 31, depletion of feeder cells were performed by a progressive decrease of feeder cells concentration over several passages. The dishes were originally seeded with around 2.7×10.sup.4 feeder cells/cm.sup.2, then around 1.8×10.sup.4 feeder cells/cm.sup.2 between passage 32 and 38, then around 1.4×10.sup.4 cells/cm.sup.2 between passage 39 and 44, then around 1×10.sup.4 feeder cells/cm.sup.2 between passage 45 and 47, then around 0.7×10.sup.4 feeder cells/cm.sup.2 between passage 48 and 50, and finally from passage 51 dishes were seeded only with avian cells and without feeder cells. At the end of the feeder depletion, the dishes are seeded with 9×10.sup.4 avian cells/cm.sup.2 to 12.7×10.sup.4 avian cells/cm.sup.2. The depletion of feeder cells started at passage 32 and ended at passage 51. During the depletion of feeder cells, the duck ES cells are seeded in culture dishes at a higher concentration than in step a), about around 9×10.sup.4 cell/cm.sup.2 to 12.7×10.sup.4 cell/cm.sup.2. After several passages without feeder cells, growth parameters (Population Doubling Time (PDT) and Density) were studied to confirm cell stability and robustness and to initiate the cell growth as suspension. Cells are considered as enough robust to be submitted to a culture in suspension if, PDT is lower than around 40 hours and cell density higher than around 26×10.sup.4 cells/cm.sup.2. Moreover, cells morphology should be: round, refringent, very small and the cells shall not attached to the plastic dish too much.

(151) In the case of the EB26 cell development, culture in suspension were initiated at passage 53. 7×10.sup.6 cells were transferred in a Ultra Low attachment dish and maintained under constant agitation at around 50 to 70 rpm. For the next passages, cells were seeded in T175 flasks (Sarsted, ref 831812502) at a concentration comprise between 0.4 to 0.5×10.sup.6 cells/mL. Following a short period of adaptation to the new conditions of culture, cells PDT decreased from around 160 H to 40 hours. Regarding this good evolution, at passage 59, a new set of deprivation was performed. Thus vitamins, sodium pyruvate, beta-mercaptoethanol and non essential amino acids were removed. Thus after passage 59, the culture medium was supplemented with 5% FBS, 0.5× yeastolate and 2.5 mM glutamine only. The serum depletion is performed on cell suspensions already depleted in growth factor, feeder cells, vitamins, non essential amino acids, sodium pyruvate and beta-mercaptoethanol. Serum depletion was performed by a progressive decreasing starting from 5% serum, then 2.5%, then 1.5%, of serum concentration in SFM cell culture medium to finally reach 0% serum in SFM cell culture medium. Serum depletion started at passage 61 and ended at passage 79. At the end of serum depletion, anchorage independent duck EB26 cells were able to grow at 39° C. in absence of grow factors, in absence of feeder cells, in serum free medium. EB26 cells were then adapted to growth in absence of 0.5× yeastolate at 37° C. by decreasing cell culture temperature at passage 80.

(152) After the obtaining of EB26 cells that are able to grow at 370 in the SFM GTM-3 supplemented by 2.5 mM glutamine, some further adaptation were made by dilution or progressive adaptation on new SFM formulations as Excell 63066, Excell 66444, Excell CHO ACF. The subcloning of suspension duck EB26 cell could also realized in presence or absence of yeastolate.

Example 5

Duck EBx Cell Line EB24

(153) 5.1—Raw Material

(154) Duck Eggs, Feeder Cells, Additives, Buffers and Fixators, Cryopreservative Agents, Fetal Calf Serum & Dissociating Agents (Idem as Example 3).

(155) Duck eggs from Peking strains GL30 were used.

(156) Medium

(157) Medium EX-CELL™ 65319, 63066 and 66444 (SAFC, customized medium) Medium GTM-3 (Sigma, Cat no G9916) DMEM F12 (Cambrex, Cat no BE04-687) DMEM (Cambrex, Cat no BE 12-614F)
Factors

(158) Six different recombinant factors were used: Recombinant Human Ciliary Neurotrophic Factor (CNTF) (Peprotech Inc, Cat no 450-13) Recombinant Human Insulin Like Factor I (IGF1) (Peprotech Inc, Cat no 100-11) Recombinant Human Interleukin 6 (IL6) (Peprotech Inc, Cat no 200-06) Recombinant Human soluble Interleukin 6 receptor (sIL6r) (Peprotech Inc, Cat no 200-06 R) Recombinant Human Stem Cell Factor (SCF) (Peprotech Inc, Cat no 300-07) Recombinant Human basic Fibroblast Growth Factor (bFGF) (Peprotech Inc, Cat no 100-18B)

(159) All those factors, excepted IL6r, are produced in E. Coli bacteria. Soluble IL6r is expressed in transfected HEK293 cells.

(160) 5.2—Process of Establishment of Duck EBx® Cell Line EB24

(161) The duck embryos were collected as previously described with EB66. The duck embryos were placed in 50 mL tubes containing PBS 1×. The duck embryos are then mechanically dissociated and seeded on an inactivated layer of feeder STO cells into complete culture medium at 39° C., 7.5% CO.sub.2. The feeder cells were seeded in 6 well plates or dishes at around 2.7×10.sup.4 cell/cm.sup.2. The complete culture medium is composed of serum free medium DMEM-Ham F12 supplemented with 10% fetal bovine serum, with IGF1, CNTF, 11-6, II-6R, SCF and FGF at a final concentration of Ing/ml, and with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 0.1 mM, with beta-mercapto-ethanol at a final concentration of 0.5 mM, glutamine at a final concentration of 2.1 mM, penicillin at a final concentration of 100 U/ml, streptomycin at a final concentration of 100 μg/ml and 1× yeastolate. Rapidly after the first passages of the cells, the mixture of antibiotics is no longer added to the medium. The expression rapidly is understood to mean after the first 3 to 9 passages in general.

(162) The duck ES cells are cultured in the DMEM-Ham F12 complete medium up to passage 7. After passage 7, the base medium is modified and DMEM-Ham F12 complete medium is replaced by the GTM-3 complete medium supplemented with 10% fetal bovine serum, with IGF1, CNTF, 11-6, II-6R, SCF and FGF at a final concentration of ing/ml, with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 0.1 mM, with beta-mercapto-ethanol at a final concentration of 0.5 mM, glutamine at a final concentration of 2.1 mM, penicillin at a final concentration of 100 U/ml, streptomycin at a final concentration of 100 μg/ml and yeastolate 1×. Thus, at passage 11, the serum concentration is decreased at 5% and SCF, IL6, IL6r and bFGF are removed for the medium. So, from passage 11, the medium is composed of 5% FBS, with IGF1 and CNTF at a final concentration of 1 ng/mL with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 0.1 mM, with beta-mercapto-ethanol at a final concentration of 0.5 mM, glutamine at a final concentration of 2.1 mM, penicillin at a final concentration of 100 U/ml, streptomycin at a final concentration of 100 μg/ml and yeastolate 1×. A simultaneous withdrawal of IGF1 and CNTF is performed at passage 22. No recombinant factors are present in the GTM-3 culture medium after passage 22. Duck cells were maintained in a such medium between passage 23 and passage 28.

(163) When the duck ES cells from Pekin duck embryos are passaged from a culture dish to another, the seeding of culture dish was performed with around between 7×10.sup.4/cm.sup.2 to 12×10.sup.4/cm.sup.2 of duck ES cells in the complete culture medium. Preferably, the seeding is made with around 7.3×10.sup.4/cm.sup.2 (4×10.sup.6 cells/55 cm.sup.2 or 4×10.sup.6 cells/100 mm dish). Then, after passage 28, depletion of feeder cells is performed by a progressive decrease of feeder cells concentration over several passages. The dishes were originally seeded with around 2.7×10.sup.4 feeder cells/cm.sup.2, then around 1.8×10.sup.4 feeder cells/cm.sup.2 between passage 29 and 33, then around 1.4×10.sup.4 cells/cm.sup.2 between passage 34 and 37, then around 1×10.sup.4 feeder cells/cm.sup.2 between passage 38 and 42, then around 0.7×10.sup.4 feeder cells/cm.sup.2 between passage 43 and 46, and finally from passage 47 dishes were seeded only with avian cells and without feeder cells. At the end of the feeder depletion, the dishes are seeded with 9×10.sup.4 avian cells/cm.sup.2 to 12.7×10.sup.4 avian cells/cm.sup.2. The depletion of feeder cells started at passage 29 and ended at passage 47. During the depletion of feeder cells, the duck ES cells are seeded in culture dishes at a higher concentration than in step a), about around 9×10.sup.4 cell/cm.sup.2 to 12.7×10.sup.4 cell/cm.sup.2. After several passages without feeder cells, growth parameters (Population Doubling Time (PDT) and Density) were studied to confirm cell stability and robustness and to initiate the cell growth as suspension. Cells are considered as enough robust to be submitted to a culture in suspension if, PDT is lower than around 40 hours and cell density higher than around 26×10.sup.4 cells/cm.sup.2. Moreover, cells morphology should be: round, refringent, very small and the cells shall not attached to the plastic dish too much. In the case of the EB24 cell development, culture in suspension is initiated at passage 48. 8×10.sup.6 cells were transferred in a Ultra Low attachment dish and maintained under constant agitation at around 50 to 70 rpm. For the next passages, cells were seeded in T175 flasks (Sarsted, ref 831812502) at a concentration comprise between 0.4 to 0.5×10.sup.6 cells/mL. Following a short period of adaptation to the new conditions of culture, cell PDT decreased from around 248 H to 128 hours and the next step of deprivation is then performed. Thus at passage 52, vitamines, non essential amino acids, sodium pyruvate and beta mercaptethanol are removed. Regarding the good evolution of the PDT reaching 44 hours, at passage 56, from passage 57, the serum deprivation was initiated. Thus from passage 57, the culture medium GTM-3 was supplemented with 5% FBS, 1× yeastolate and 2.5 mM glutamine only. The serum depletion is performed on cell suspensions already depleted in growth factors, feeder cells, vitamins, non essential amino acids, sodium pyruvate and beta-mercaptoethanol. Serum depletion was performed by a progressive decreasing starting from 5% serum, then 2.5%, then 2%, then 1.5% of serum concentration in SFM cell culture medium to finally reach 0% serum in SFM cell culture medium. Serum depletion started at passage 57 and ended at passage 77. During this serum depletion, adaptation to growth at 37° C. was also performed. Thus at passage 65, cells growing in the culture medium supplemented with 2.5% FBS were transferred at 37° C. avoiding a progressive temperature shift. At the end of serum depletion, anchorage independent duck EB24 cells were able to grow at 37° C. in absence of grow factors, in absence of feeder cells, in serum free medium.

(164) After the obtaining of duck EB24 cells able to grow at 37° C. in the SFM GTM-3 supplemented by 2.5 mM glutamine, some further adaptation were made by dilution or progressive adaptation in new SFM formulations as Excell 63066, Excell 66444, Excell CHO ACF. The subcloning of suspension duck EB24 were performed, an duck EB24-12 subclone were selected because of its good performance to efficiently replicate viruses.

Example 6

SPF Duck Muscovy EBx Cell Line

(165) 6.1—Raw Material

(166) Duck Eggs:

(167) Duck SPF eggs from Muscovy strains were obtained from Le Couvoir de Cerveloup (France). Those SPF duck eggs, are produced from a flock tested intensively to various poultry pathogens. Disease tested include: Salmonella gallinarum-pullorum, Mycoplasma synoviae, Mycoplasma meleagridis, Mycoplasma galliepticum, Marek's disease virus, Avian Influenza, Type 2 Paramyxovirus, Type 3 Paramyxovirus, Newcastle disease, Type 3 Adenovirus (EDS), Gumboro disease, Avian reovirus, Reticuloendotheliosis virus, Avian encephalomyelitis, infectious rhinotracheitis virus and Chlamydiosis. Muscovy duck eggs were only submitted to a disinfection with the decontaminant to avoid any risk of contamination linked to the manipulation of eggs during the transport.

(168) Feeder Cells (See Previous Examples)

(169) Media

(170) Medium EX-CELL™ 66444 (SAFC, customized medium) Medium GTM-3 (Sigma, Cat no G9916) DMEM-HamF12 (Cambrex, Cat no BE04-687)
Additives Glutamine (Cambrex, Cat no BE17-605E) Pencillin/streptomycin (Cambrex, Cat no BE17-602E)) Non essential Amino Acids (Cambrex, Cat no BE13-114E) Sodium pyruvate (Cambrex, Cat no BE13-115) Vitamines (Cambrex, Cat no 13-607C) Beta Mercapto Ethanol (Sigma, Cat no M7522) Yeastolate (SAFC, Cat no 58902C)
Buffer and Fixators: PBS 1× (Cambrex, Cat no BE 17-516F)
Cryoprotective Agent Dimethyl Sulfoxyde (DMSO) (Sigma, Cat no D2650)
Factors

(171) Two different recombinant factors were used: Recombinant Human Ciliary Neurotrophic Factor (CNTF) (Peprotech Inc, Cat no 450-13) Recombinant Human Insulin Like Factor I (IGF1) (Peprotech Inc, Cat no 100-11)

(172) Those 2 factors are produced in E. Coli bacteria.

(173) Fetal Bovine Serum

(174) Non Irradiated Fetal Bovin Serum (FBS) (JRH, Cat No 12003)

(175) The non irradiated serum used in the program was collected and produced in Australia. Animals used for collection were USDA inspected and acceptable for slaughter. It was added in the medium during avian stem cells culture. This batch was not submitted to irradiation to avoid the destruction of critical proteins or components identified as essential for the maintenance of stem cells in culture.

(176) Irradiated Serum (JRH, Cat No 12007)

(177) The irradiated batch used in this program was collected in Australia. This irradiated batch was added as supplement in the DMEM medium used for the culture of STO cells (feeder cells). Those cells do not require as stem cells a specific quality of serum for growth and maintenance in culture. To minimize high concentration of serum in the medium we have adapted the STO cells to grow in presence of 4% of FBS only.

(178) Dissociating Agents:

(179) Pronase (Roche, Cat no 165 921) Trypzean (Sigma, cat no T3449)
6.2—Process of Establishment of Muscovy Duck EBx Cell Line

(180) Embryos from 20 fertilized SPF eggs from Muscovy ducks were collected according to the process described in Example 3. The duck embryos were placed in 50 mL tubes containing PBS 1×. The duck embryos were then mechanically dissociated, washed with PBS, and seeded in a well of a 12 well plate coated with an inactivated layer of feeder STO cells. Duck Embryonic cells were seeded into complete culture medium and transferred at 39° C., 7.5%5% CO.sub.2. The feeder cells were seeded at around 2.7×10.sup.4 cell/cm.sup.2. The complete culture medium used is composed of DMEM-Ham F12 supplemented with 10% fetal bovine serum, with IGF1, CNTF, at a final concentration of 1 ng/ml, and with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 0.1 mM, with beta-mercapto-ethanol at a final concentration of 0.5 mM, glutamine at a final concentration of 2.1 mM, penicillin at a final concentration of 100 U/ml, streptomycin at a final concentration of 100 μg/ml and yeastolate 1×. At passage 2, the DMEM-HamF12 base medium is replaced by GTM-3 base medium. The mixture of antibiotics is no longer added to the medium after passage 4.

(181) The duck ES cells were cultured in the complete GTM-3 medium up to passage 8. After passage 8, concentration of IGF1 and CNTF are reduced to 0.5 ng/mL. The duck ES cells were further cultured during 2 passages in this new medium of culture, then growth factor deprivation was performed at passage 10. IGF1 and CNTF were simultaneously removed from the medium.

(182) Thus from passage 10 to passage 37, the medium of culture was GTM-3 medium supplemented with 10% FBS, with 1% non-essential amino acids, with 1% of mixture of vitamins of commercial origin, with sodium pyruvate at a final concentration of 0.1 mM, with beta-mercapto-ethanol at a final concentration of 0.5 mM, glutamine at a final concentration of 2.1 mM and yeastolate 1×.

(183) When the duck ES cells isolated from Muscovy duck embryos are passaged from a culture dish to another, the seeding was performed with around 12×10.sup.4/cm.sup.2 of duck ES cells in the culture medium. Some conditioned medium can be occasionally used for cell seeding to improve cell recovery post dissociation.

(184) Then, after passage 37, depletion of feeder cells was performed by a progressive decreasing of feeder cells concentration over several passages following the step by step process previously described.

(185) During this phase of feeder deprivation, some cells able to grow in suspension were isolated and adapted to grow without additives and serum (FIG. 4C). Anchorage-independent Muscovy duck EBx cells express ES cells markers, such as telomerase, SSEA-1 and EMEA-1 (data not shown).

Example 7

EBx Cell Lines Characterization

(186) 7.1—Chicken Valo EBv13 Cells Characterization

(187) 7.1.1—Telomerase Activity

(188) Telomerase detection is achieved by using the Telo TAGGG Telomerase PCR ELISA developed by Roche Applied Science (Telomeric Repeat Amplification Protocol (TRAP)—Cat. No. 11 854 666 910) according to the supplier protocol. The Telo TAGGG Telomerase PCR ELISA allows amplification of Telomerase-mediated elongation products combined with non radioactive detection following an ELISA protocol. The assay is valid if absorbance value of the negative control is less than or equal to 0.25 A.sub.450 nm-A.sub.690 nm and if absorbance value of the positive control is higher than or equal to 1.5 A.sub.450 nm-A.sub.690 nm when using 1×10.sup.3 cell equivalents in the assay. Samples are regarded as telomerase positive if the difference in absorbance is higher than 0.2 A.sub.450 nm-A.sub.690 nm units Two controls were used: the negative control is murine fibroblasts (FED cells) and the positive controls are FGB8 cells (Embryonic Stem cells established by Vivalis from 129 SV mouse embryos) and chicken EB14-O74 cells previously established in WO 03/076601.

(189) Results obtained are summarized on the figure No 2. EBv13 cells do express high level of telomerase. At passage p193 and 195, the telomerase activity is equivalent to the one of chicken EB14-O74 cells.

(190) 7.1.2—ES Cells Biological Markers

(191) Embryonic stem cells are characterized by the expression of biological markers expressed on the cell membrane. The expression of EMA-1 (Epithelial Membrane Antigen-1) and SSEA-1 (Stage Specific Embryonic Antigen-1) on EBv13 cells were evaluated by FACS analysis. After 10 minutes of fixation with PFA 4% (Para-formaldehyde), cell samples and controls are rinsed and pre-incubated with monoclonal antibodies specific of EMA-1 or SSEA-1. A second antibody conjugated to FITC is used for detection of cells expressing the 2 biological markers selected. Samples were analyzed by flow cytometry using a FACS (Flow Activated Cell Sorter) from Coulter.

(192) FACS analysis was done on mouse fibroblasts cells (FED cells) as a negative control, murine ES FGB8 cells as a positive control, chicken EB14-O74 cells as a positive control EBx cells and EBv13 cells. As expected FED cells do not express biological markers whereas FGB8 and EB14-O74 cells present an important staining, respectively of, 60.13% and 78.7 for EMA-1 and 94.45% and 95% for SSEA-1 (data not shown). Chicken valo EBv13 cells population do not present any staining for EMA1 (2%) and a very light one for SSEA-1 (22%).

(193) 7.1.3—Karyotype

(194) Karyotype analysis was performed to check the cell diploidy and the avian origin of EBv13 cells. Cells in the exponential phase of growth were harvested and treated 2 hours by colcemid (0.02 μg/mL). After washing and centrifugation, an hypotonic choc is performed on cells with KCl (0.56%) during 20 minutes. Subsequently, EBv13 cells were fixed in methanol/acetic acid (3/1) and stored overnight at −20° C. The day after, metaphasis were spotted on glass, stained by a wright/giemsa solution and observed under microscope. Several series of metaphases were observed confirming the chicken origin of EBv13 cells. No evidence of polyploidy is observed.

(195) 7.1.4—Influence of Cell Culture Medium Composition on the Clumps Size of EBv13 Cells

(196) The inventors have found that the concentration of Calcium and Magnesium in the serum-free medium used for the EBx cells culture and infection have an impact on the clumps size.

(197) FIG. 10 shows the decrease in clumps size when EBv13 cells are passed from a medium with a high to a low Ca2+ and Mg2+ concentration.

(198) 7.2—Duck EBx Cell Lines Characterization

(199) 7.2.1—Duck EBx Cells Morphology

(200) Transmission Electronic Microscopy analysis of dEBx® cells were performed by Dr. A Rivoire (Lyon, France). Duck EBx® cells display a typical embryonic stem cells morphology (i.e high nucleo-cytoplasmic ratio) that resemble the phenotype of murine embryonic stem cells and VIVALIS EB14 cells described in WO2006/108846. Duck EBx® cells are small round cells (diameter˜10 pun) with a large nucleus and nucleolus, with short pseudopodia extending from the plasma membrane (FIG. 4A. FIG. 4B and FIG. 4C). They are highly metabolic active with a ribosome and mitochondria rich cytoplasm. They contain numerous intracellular vacuoles, a very developed Golgi system and a granulous reticulum endoplasmic.

(201) 7.2.2—Telomerase Expression of Duck EBx® Cells

(202) Telomerase expression during different stages of establishment of in duck EBx® cells was investigated by using Roche telomerase detection kit (Telomerase OCR ELISA). Telomerase is found to be highly expressed in adherent duck EBx® cells, as well as during feeder deprivation, during the process of adapting duck EBx® cells to suspension and during feeder deprivation.

(203) FIG. 5 shows that duck EB24 and EB26 express high level of telomerase, just like chicken EB14 cells. Duck EB66 also express high level of telomerase all along cell passages. This high telomerase activity is stable in EB66 cells after adaptation in different SFM (FIG. 15).

(204) 7.2.3—Duck EBx® Cells Display No Endogenous Reverse Transcriptase Activity

(205) Endogenous reverse transcriptase expression was investigated by direct F-PERT analysis (Lovatt et al., 1999, J. Virol. Methods, 82:185-200) in Clean Cells (FRANCE). Duck EBx® cell lines, EB24 (data not shown), EB66 (data not shown), EB26 and EB51, display no endogenous Reverse Transcriptase (RT) activity (FIG. 6A). RT activity were detected in chicken EB14 cells culture as well as, to a lesser extend, in chicken Embryonic fibroblast derived from Specific Pathogen Free (SPF) chicken strain.

(206) The presence of endogenous retroviral particles, either replicative or non-replicative, in the cell culture supernatant of duck and chicken EBx® cells were investigated by an ELISA assay detecting the avian leukosis major capsid antigen P27 (FIG. 6B). All Duck EBx® cell lines (EB26, EB51, EB24, EB66 . . . ), as well as chicken EBv13 do not secrete ALV p27 antigen. In the opposite, chicken EB14 cells do express ALV P27 antigen.

(207) 7.2.4—Duck EBx Cells do not Secrete Replicative Avian Leucosis Virus (ALV)

(208) Co-cultivation assay of duck EBx cells with quail QT6 cell line, known to be sensitive to endogenous and exogenous ALVs, were performed to detect the presence of endogenous replicative duck viruses. FIG. 7A described the principle of QT6 co-culture. The presence of replicative virus is detected by an ELISA assay detecting the avian leucosis major capsid antigen P27. The assay demonstrates that none of duck EBx cells tested secrete replicative (i.e replication competent) ALV (FIG. 7B).

(209) 7.2.5—Duck EBx Cells Express Avian and Human Influenza Virus Receptors

(210) The detection of receptors to avian (Siaα2-3Gal) and human (Siaα2-6Gal) influenza viruses on duck EBx cells were performed by fluorescent cell sorter analysis by using digoxygenin labelled lectins (Boehringer): Sambuca nigra (SNA) agglutinin lectin specifically binds to Siaα2-6Gal; Maackia amurensis (MAA) agglutinin lectin specifically binds to Siaα2-3Gal.

(211) Chicken EB14 and duck EBx cells were washed in 10 mM HEPES, 150 mM NaCl pH7.5 and resuspended in the same buffer at a 5.10.sup.6 final concentration. Cells were incubated 30 min on ice, then for an additional 15 to 30 minutes in presence of SNA or MAA. Lectin treated cells were washed in 10 mM HEPES, 150 mM NaCl pH7.5, prior to incubation on ice during 15 to 30 minutes with FITC-labelled anti-digoxygenin antibody. Then cells are washed in NaCl 0.9% and FACS analyzed. Chicken EB14 and duck EBx cells express cell surface receptors comprising oligosaccharides with Siaα2-6Gal and Siaα2-3Gal residues (FIG. 8).

(212) 7.2.6—Karyotype

(213) Karyotype analysis was performed to check the cell diploidy and the avian origin of duck EB24 and EB66 cells. Cells in the exponential phase of growth were harvested and treated 3 to 6 hours by colcemid (0.6 mg/mL). After washing and centrifugation, an hypotonic choc is performed on cells with KCl (0.56%) during 20 minutes. Subsequently, duck EB24 and EB66 cells were fixed in methanollacetic acid (3/1) and stored overnight at −20° C. The day alter, metaphasis were spotted on glass, stained by a wright/giemsa solution and observed under microscope.

(214) Several series of metaphases were observed confirming the duck origin of EBx cells. No evidence of polyploidy were observed. FIG. 16 shows diploid karyotype of duck EBx66 cells (FIG. 16).

Example 6

Poxvirus Replication in Chicken EBv13 Cell Line

(215) Susceptibility of EBv13 cells to infection with poxvirus was investigated using a recombinant Modified Vaccinia Ankara (MVA) encoding a GFP gene (Green Fluorescent Protein).

(216) The following protocol were used: Three days before infection. 0.4×10.sup.6 EBv13 cells (passage 188)/mL are seeded in T175 flasks under 40 mL of SFM Excell 65319 (SAFC) supplemented with 4 mM Glutamine. The infection is performed at a multiplicity of infection of 10.sup.2 TCID50/cell (MVA-GFP stock is at 10e9.7 TCID/ml). One hour post infection, 60 ml of fresh medium is added to the flask. The culture and the infection were performed at 37° C., 7.5% CO.sub.2 and agitated at 60 rpm. Each day post infection an aliquot of the cell suspension is collected and frozen. At the end of the kinetic, an evaluation of the productivity is performed following the TCID50 method. Briefly, the titration of infectious MVA-GFP viruses was performed on DF-1 cells. Cells were seeded in 96 flat-bottom well plates at a density of 15×10.sup.3 cells/well in DMEM medium (Biowhittaker) supplemented with 5% foetal calf serum (FCS) (SAFC) and 2 mM L-glutamine (Biowhittaker). Twenty-four hours later, cells were infected with ten fold serially diluted samples in DMEM and incubated for one week at 37° C., 5% CO.sub.2 in a humidified atmosphere. Virus infectivity was measured through microscopic observation of global cytopathic effect (CPE) and UV-exposed infected cells. Then, TCID50 titers were calculated according the Reed and Muench method (1938, A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493-97). All along the experiment cell proliferation and viability are monitored. Chicken Valo EBv13 cells appear to be highly sensitive to MVA-GFP infection (FIGS. 3A-3B).

Example 8

Poxvirus Replication in Duck EBx Cell Lines

(217) Susceptibility of duck EBx cells to infection with poxvirus was investigated using a recombinant Modified Vaccinia Ankara encoding a GFP. The virus titration was performed as previously described for chicken EBv13 cells.

(218) 8.1—Cell Culture Method

(219) Duck EBx cells were stored in cryovials in liquid nitrogen at −196° C. (20×10.sup.6 cells/vial). The cryovial is directly thawed into a +37° C. pre-warmed water bath. The cell suspension is put into a 50 ml sterile tube with 30 ml pre-warmed culture medium. Alter centrifugation (5 min at 300±20 g, at room temperature), 15 ml of fresh culture medium is added on the pellet and gently homogenised. The sample is numbered using Trypan blue. Numeration has to be ≧20×10.sup.6 cells and viability has to be >70% to guarantee a good culture.

(220) The cell suspension is plated into a T75 cm.sup.2 flask and incubate at +37° C. under an 7.5% CO.sub.2 atmosphere on an orbital shaker at 50 rpm. Fresh medium is then added daily. The cells are then passaged to increase cells biomass to seed a 3 L-bioreactor. 320.10.sup.6 cells are needed to inoculate a 3 L-bioreactor. A sample is taken after gently mixing to perform a numeration using trypan blue to determine cell density. A 150 mL cell mix is prepared in order to obtain a cell concentration of 0.4×10.sup.6 cells.ml.sup.−1 into the 800 ml final culture volume in the bioreactor. Prior to seed cells, the pH is set in the vessel to 7.2 (because pH will be decrease by CO.sub.2 surface injection). The pO.sub.2 is set to 50% O.sub.2 saturation (the mass flow controller is adjusted to 100% which correspond to a maximum sparger flow rate to 50 ml.min.sup.−1). At the beginning of the process, the pH is maintained by CO.sub.2 surface injection, later, it is controlled by addition of 7.5% NaHCO.sub.3. The surface aeration is started with air at a flow rate of 0.3 ml.min.sup.−1. Cell numeration is performed on a routine basis.

(221) Alter 3 days of culture, cell density should be higher than 4-5×10.sup.6 cells.ml.sup.−1. If the expected cell density is reached, the virus infection is performed at a MOI of 10.sup.−4. The vessel temperature is set to 33° C. The virus strain is thawed on ice. The infection mix is prepared in 10 ml of production medium. After inoculation of the infection mix into the bioreactor, viral adsorption is performed during 1 hour. The final production medium is prepared: in 1.5 L of production medium, trypsin is added in order to obtain a final concentration in the vessel of 0.3 U.ml.sup.−1 (2.3 L on the whole). The pre-warmed final production medium is then added. Every day a sample of approximately 15 ml is collected from the bioreactor to perform cell numeration, cell morphology analysis and to observe CPE. The metabolites such as glutamate, glutamine, lactate and glucose are analyzed all along the culture with the BioProfile Basic software. Concentration of the metabolites is adjusted if necessary. For example, glutamine concentration is adjusted to 2 mM if necessary. The glucose concentration is adjusted to 2 g.Math.L.sup.−1 if necessary.

(222) Virus titration is carried-out at the end of the experiment using all collected samples.

(223) 8.2—Results

(224) 8.2.1—Cell Growth Kinetics of Duck EBx® Cells in a 3 L Fedbatch Bioreactor

(225) Duck EBx® cells are routinely cultured in stirred-tank bioreactor. Duck EBx® derived biomass is allowed to accumulate at 37° C. in a cell growth medium until a cell density of 5-6.10.sup.6 cells/mL was reached. Then the mixture is diluted from around 3 to 10 fold, and cell growth kinetic is followed-up over a 10 days period. In such conditions, cell density of 12 to 20 million cells/ml is routinely reached around day 5 to 8. Thus Duck EBx® cells display a range of splitting ratio that goes at least up to 10 to 15 fold.

(226) 8.2.2—Influence of Cell Culture Medium Composition on the Clumps Size During MVA-GFP Virus Infection of Duck EBx Cells

(227) The inventors have found that the concentration of Calcium and Magnesium in the serum-free medium used for the EBx cells culture and infection may have an impact on the clumps size. The presence of small clumps of duck EBx cells improves virus infection and propagation, leading to high MVA virus titers (FIG. 9A).

(228) 8.2.3—MVA Virus Production in 3 L-Bioreactor

(229) Duck EBx®-derived biomass was allowed to accumulate during cell proliferation phase in Excell 66444 growth medium. Cells were then infected with 10.sup.−2 TCID.sub.50/cell of MVA-GFP virus and the mixture was diluted in Excell 66444 production medium. Following addition of fresh Excell medium, cell density dropped down on day 2, and at day 4, the cell density of infected cells increased and reached 12 million cell/ml. In such conditions, the MVA-GFP productivity is high. Since at day 4 post-infection, the MVA-GFP titer is around 10.sup.8 TCID50/ml (FIG. 9B). A MVA-GFP yield of 205 TCID50/cell was obtained in duck EBx® cells.

Example 9

Production of Influenza Virus in Duck EBx Cell Lines

(230) 9.1—Materials & Methods

(231) 9.1.1—Influenza Virus Infectivity Assay (TCID50)

(232) Titration of infectious influenza viruses was performed on MDCK cells. In brief, cells were seeded in 96 flat-bottom well plates at a density of 3×10.sup.3 cells/well in UltraMDCK medium supplemented with 2.5 mM L-glutamin. Twenty-four hours later, cells were infected with ten fold serially diluted samples in UltraMDCK containing 6 μg.Math.mL.sup.−1 trypsin-EDTA and incubated for one week at 33° C., 5% CO.sub.2 in a humidified atmosphere. Virus replication was then tested in an HA assay using chicken red blood cells and TCID50 titers were calculated according the Reed and Muench method (1938)*._*Reed L, Muench H, 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493-97.

(233) 9.1.2—Single Radial Immuno-Diffusion Assay (SRID)

(234) The concentration of haemagglutinin in samples derived from influenza virus infected-EB14 cells, was determined as described by Wood and colleagues*. Briefly, glass plates were coated with an agarose gel containing anti-Influenza serum (recommended concentration provided by NIBSC). After the gel has set, 10 μL of appropriate dilutions of the reference and the samples were loaded in 3 mm Øpunched wells. Following a 18-24 h incubation in a moist chamber at room temperature, plates were soaked in 0.9% NaCl and washed in distilled water. The gel was then pressed and dried. The plates were stained on Coomassie Brillant Blue solution for 15 min and destained twice in a mixture of methanol and acetic acid until clearly defined stained zones became visible. After drying the plates, the diameter of the stained zones surrounding antigen wells were measured in two directions at right angles. Dose-response curves of antigen dilutions against the surface were constructed and the results were calculated according to standard slope-ratio assay methods. *Wood J M. Et al. “An improved single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen: application for potency determinations of inactivated whole virus and subunit vaccines” (J. Biol. Stand., 1977, 5(3):237-47).

(235) 9.1.3—Western Blot Analysis of Influenza Hemagglutinin Protein

(236) SDS-PAGE was performed as described by Laemmli UK (1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 259:680-685) in 10% polyacrylamide gel. Denaturated proteins (1% SDS, 70 mM β-mercaptoethanol) were transferred to polyvinylidene dilluoride membrane (hybond P, Amersham) by a semidry blotting procedure (Kyhse-Andersen J (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose (J Biochem Biophys Methods 10:203-209). Blots were blocked for 1 h at room temperature with a mixture composed of 5% fat dry milkpowder in TBST suplemented with 1% FCS (SAFC). Then, the blots were incubated overnight in blocking solution supplemented with specific polyclonal anti-HA sheep serum (1:500 (NIBSC). The blots were washed 6 times with TBST and incubated for 1 h at room temperature with a hrp-conjugated rabbit anti-sheep IgG polyclonal antibody (1:5000 (Rockland) in blocking solution. After 6 washes with TBST, the protein-conjugate complex was finally revealed using chemiluminescence (ECL kit, Amersham) and films (Hyperfilm, Amersham).

(237) 9.2—Influenza Virus Infection of Duck EBx® Cells in 3 L-Bioreactor

(238) 9.2.1—Materials and Equipment

(239) Cell Thawing Material T75 cm.sup.2 flasks (Sarstedt, Cat#831813502) Culture medium (serum free medium) L-Glutamine 200 mM (Biowhittaker, Cat#BE17-605E) Orbital agitator IKA KS260 (Fisher Bioblock, Cat#F35044)
Cell Amplification Material T175 cm.sup.2 flasks (Sarstedt, Cat#831812502) Culture medium (serum free medium): Excell 65319 (JRH, Cat#65319-1000M1687) added with 2.5 mM glutamine L-Glutamin 200 mM (Biowhittaker, Cat#BE17-605E) D (+) Glucose (45%) (Sigma, Cat#G8769)
Production Material Production medium (serum free medium): Excell 65629 (JRH, Cat#65629) supplemented with 2.5 mM glutamine L-Glutamin 200 mM (Biowhittaker, Cat#BE17-605E) D (+) Glucose (45%) (Sigma, Cat#G8769) Trypzean 1× (Sigma, Cat#T3449) 7.5% bicarbonate sodium solution (Sigma, Cat#205-633-8) Influenza virus strain (frozen at −80° C.)
9.2.2—Cell Culture Method
(Idem as for MVA Replication—Example 7.1)

(240) Virus titration, haemmaglutinin assays (HAU) and HA antigen quantifications (western blot. SRID) are carry out at the end of the experiment using all collected samples.

(241) 9.3—Results

(242) The inventors demonstrate that duck EBx cells are a reliable and efficient cell substrate for the replication of various strains A and B of influenza virus. Influenza virus production can be performed in various vessels, such as flasks and spinner (data not shown) and bioreactors. Reproducible and efficient fedbatch process of production of influenza virus in 3 L and 30 L stirred tank bioreactors were obtained by the inventors. Viral yield above 15 mg/l and up to 50 mg/l of haemagglutinin are routinely obtained in flasks and in bioreactors with strains A and B of influenza virus (FIGS. 11 and 12).

Example 10

Newcastle Disease Virus Replication in Duck EBx Cell Lines

(243) Susceptibility of duck EBx cells to infection with Newcastle Disease virus was investigated using a NDV La Sota strain.

(244) 10.1—Methods

(245) Duck EBx® cells were grown in Excell medium (SFAC) in T175 flasks at 37° C. under 7.5% CO.sub.2 atmosphere on an orbital shaker at 60 rpm. At day 0, cells are seeded at 0.4×10.sup.6 cells/mL in 40 ml fresh medium. Cell culture was incubated at 37° C., 7.5% CO.sub.2 under shaking (60 rpm). Cell growth kinetics were followed until cell density has reached a concentration between 4×10.sup.6 to 6×10.sup.6 cells/ml (usually at day 3 post seeding). At that point, cells are inoculated with NDV La Sota strain at two different MOI (10.sup.−3 and 10.sup.−4 TCID.sub.50/cells) and incubated for one additional hour at 37° C., 7.5% CO.sub.2 under shaking (60 RPM). Then the cell culture was diluted with the addition of 60 mL fresh viral production medium and the incubation pursued at 37° C. and 7.5% CO.sub.2 under shaking (60 rpm). The cell growth and virus production kinetics were performed over 7 days. As a source of protease, recombinant trypsin (SAFC) was added every day in the culture medium; two concentration of trypsin (0.4 and 0.75 USP/mL) were tested. Daily aliquots were removed for cell numeration, virus titration and Western blotting analysis.

(246) The samples were separated using 10% SDS-PAGE and blotted onto PDVF membrane (Amersham) by the semi-dry technique. Immunodetection was performed using chicken polyclonal antiserum against NDV (1:2000, CHARLES RIVER laboratories), followed by Alkaline phosphatase-conjugated rabbit anti-chicken (1:5000, SIGMA). Bound secondary antibody was detected using the ECL-Chemiluminescence detection system kit (ROCHE).

(247) 10.2—Results

(248) Duck and chicken EBx cells are sensitive to and replicate NDV La Sota strain. Titers (in TCID50/ml) of NDV produced in duck EBx® cells increase from day 0 to day 2 μl to reach an average of 10.sup.6.83 .sub.TCID50/mL (FIG. 13 left panel).

(249) Western blot analysis (FIG. 13 right Panel) showed NDV viral proteins (HN, Fo/F, NP & M) expression. The viral proteins composition of NDV virus produced in duck EBx® cells are similar to the one obtained with NDV virus produced in chicken EBx® cells. In addition, the kinetic of release for viruses produced in chicken and Duck EBx cells are similar.

Example 11

Measles Virus Replication in Duck EB66 Cells

(250) Susceptibility of duck EB66 cells to infection with measles virus was investigated using a recombinant measles virus expressing green fluorescent protein.

(251) 11.1—Methods

(252) EB66 cells were grown in Excell medium in T175 flasks at 37 CC under 7.5% CO.sub.2 atmosphere on an orbital shaker at 60 rpm. At day 0, cells are seeded at 0.4×10.sup.6 cells/mL in 40 ml fresh medium. Cell culture was incubated at 37° C., 7.5% CO.sub.2 under shaking (60 rpm). Cell growth kinetics were followed until cell density has reached a concentration between 4×10.sup.6 to 6×10.sup.6 cells/ml (usually at day 3 post seeding). At that point, cells are inoculated with recombinant measles virus at two different MOI (10.sup.−1 and 10.sup.−2 TCID.sub.50/cells) and incubated for one additional hour at 37° C., 7.5% CO.sub.2 under shaking (60 RPM). Then the cell culture was diluted with the addition of 60 mL fresh viral production medium and the incubation pursued at 37° C. and 7.5% CO.sub.2 under shaking (60 rpm). The cell growth and virus production kinetics were performed over 7 days. Daily aliquots were removed for cell numeration and virus titration.

(253) 11.2—Results

(254) EB66 cells are sensitive to and replicate measles virus. In non optimised conditions, titers (in TCID50/ml) of measles produced in EB66 cells reach an average of 10.sup.7 TCID50/mL (FIG. 14).