Methods for producing nonadherent avian cell lines

Abstract

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.