METHODS FOR PRODUCING NONADHERENT AVIAN CELL LINES
The present invention relates to a method for producing avian cell lines, comprising gradual or complete withdrawal of growth factors, serum and/or feeder layer so that the established lines are adherent or nonadherent cells capable of proliferating indefinitely in a basic culture medium. The invention also relates to the cells derived from such lines which are particularly useful for the production of substances of interest.
1 . An avian cell line obtained from embryonic stem cells of fertilized eggs characterized by a status of proliferation and non differentiation wherein said cell line is capable of proliferating in a basal culture medium free of exogenous growth factors and cytokines, serum, and/or feeder layer for at least 50 days and wherein said cell line has at least one of the following features:
a very visible nucleola and very small nucleoplasm,
an endogenous alkaline phosphatase activity,
an endogenous telomerase activity, and
reactivity with specific antibodies selected from the group of antibodies SSEA-1 (TEC01), SSEA-3 and EMA-1.
2 . The avian cell line according to claim 1 , wherein said embryonic stem cells originate from blastodermal disks of fertilized eggs.
3 . The avian cell line according to claim 1 , wherein said embryonic stem cells are chicken embryonic stem cells.
4 . The avian cell line according to claim 1 , wherein said cell line has been obtained by a method comprising the following steps:
a) culturing embryonic stem cells obtained from blastodermal disks of fertilized eggs in a culture medium comprising:
at least a growth factor selected from the group of SCF, IGF-1 and bFGF and at least one cytokine selected from LIF, IL-11, IL-6, IL-6R, CNTF, oncostatin and cardiotrophin;
an inactivated feeder layer of STO cells; and
fetal calf serum at a concentration of 12 to 8%,
b) after about twenty passages, modifying the culture medium:
by gradual withdrawal of said growth factors and cytokines; or
by gradual withdrawal of the feeder layer; or
by decreasing the fetal calf serum concentration to reach a low concentration of 2%, tending toward 0% (serum-free medium), and
c) establishing adherent or non-adherent cell lines capable of proliferating in a basic medium in the absence of exogenous growth factors and cytokines, serum or the inactivated feeder layer.
5 . The avian cell line according to claim 4 , wherein the cell culture medium of step a) contains at least one cytokine, bFGF, and SCF.
6 . The avian cell line according to claim 4 , wherein the cell culture medium of step a) contains at least the growth factors and cytokines consisting of SCF, IGF-1, CNTF, IL-6, soluble IL-6R, and bFGF.
7 . The avian cell line according to claim 6 , wherein that the cell culture medium of step a) further contains IL-11.
8 . The avian cell line according to claim 4 , wherein the culture medium modification of step b) comprises gradual withdrawal of said growth factors and cytokines followed by progressive deprivation of said feeder layer of STO cells and/or the decreasing of the concentration of fetal calf serum to reach a low concentration of 2%, tending toward 0% (serum-free medium).
9 . The avian cell line according to claim 1 , wherein said cell line is an adherent cell line.
10 . The avian cell line according to claim 1 , wherein said cell line is a non-adherent cell line.
11 . The avian cell line according to claim 10 , wherein said non-adherent cell line has been obtained by inoculating the cells obtained in step c) at high-density into a bacteriological dish followed by several passages in mere dilution.
12 . The avian cell line according to claim 11 , wherein the initial inoculation density is of at least 1×10 6 cells/mL.
13 . The avian cell line according to claim 1 , characterized by a status of proliferation and non differentiation and wherein said cell line has the following features:
a very visible nucleola and very small nucleoplasm,
an endogenous alkaline phosphatase activity,
an endogenous telomerase activity, and
reactivity with specific antibodies selected from the group of antibodies SSEA-1 (TEC01), SSEA-3 and EMA-1.
14 . The avian cell line according to claim 13 , wherein said cell line presents reactivity with an antibody selected from the group consisting of antibodies SSEA-1 (TEC01) and EMA-1.
15 . The avian cell line according to claim 1 , wherein said cell line is capable of proliferating for at least 100 days.
16 . The avian cell line according to claim 1 , wherein said cell line is capable of proliferating for at least 150 days.
17 . The avian cell line according to claim 1 , wherein said cell line is capable of proliferating for at least 300 days.
18 . The avian cell line according to claim 1 , wherein said cell line is capable of proliferating for at least 600 days.
19 . The avian cell line according to claim 1 , wherein said basal culture medium is selected from the group of medium consisting of DMEM, GMEM, HamF12, and McCoy's 5A supplemented with additives.
20 . The avian cell line according to claim 19 , wherein said additives are chosen from non-essential amino acids, vitamins, and sodium pyruvate.