Avian cell lines useful for the production of substrates of interest

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 - 53 . (canceled) 
   
   
       54 . A method for producing a nonadherent avian cell line capable of proliferating in a basal medium in the absence of exogenous trophic factors and cytokines, said method comprising culturing embryonic stem cells obtained from blastodermal disks of fertilized eggs in a culture medium, wherein:
 (a) in at least the first passage, said embryonic stem cells are cultured in a primary medium comprising:
 (i) the trophic factor insulin-like growth factor 1 (IGF-1) and the cytokine ciliary neurotrophic factor (CNTF); 
 (ii) an inactivated feeder comprising mouse fibroblast STO cells; and 
 (iii) a basal medium, wherein the basal medium is supplemented with fetal calf serum at an initial concentration of from 8 to 12%; 
   (b) the cells obtained in step (a) are further cultured in a primary medium that has been modified by progressively depriving the primary medium of said trophic factors and said cytokines defined in (i) of step (a), wherein the progressive withdrawal of each trophic factor and cytokine is carried out by successive passage of the cells in a culture medium having a lower concentration of at least one of said trophic factors or said cytokines as compared to the culture medium of the prior passage, until the medium is free of all exogenous trophic factors and cytokines; and   (c) the cells obtained in step (b) are inoculated at a high density into a bacteriological dish to produce a nonadherent avian cell line capable of proliferating in a basal medium in the absence of exogenous trophic factors and cytokines.   
   
   
       55 . The method of  claim 54 , wherein the nonadherent avian cell line obtained in step (c) is capable of proliferating in a basal medium in the absence of exogenous trophic factors and cytokines for at least 50 days. 
   
   
       56 . The method of  claim 54 , further comprising the following step (d):
 (d) proliferating in suspension the nonadherent cells derived from the lines obtained in step (c) in a medium free of exogenous trophic factors and cytokines.   
   
   
       57 . The method of  claim 54 , wherein the cells derived from the lines obtained in step (c) have a high nucleocytoplasmic ratio, an endogenous alkaline phosphatase activity, an endogenous telomerase activity, and a reactivity with specific antibodies selected from the group of antibodies SSEA-1, (TEC01) and EMA-1. 
   
   
       58 . The method of  claim 54 , wherein the cells used in step (a) are obtained by suspending cells from blastodermal disks of fertilized eggs in a culture medium comprising the tropic factors and cytokines as defined in (i) of step (a), wherein said cells are inoculated into a feeder comprising mouse fibroblast STO cells, incubated, and then collected. 
   
   
       59 . The method of  claim 54 , further comprising the following step (d):
 (d) proliferating cells derived from the lines obtained in step (c) in a basic medium selected from the group consisting of DMEM, GMEM, HamF12 and McCoy, wherein the medium is supplemented with various additives selected from the group consisting of nonessential amino acids, vitamins and sodium pyruvate.   
   
   
       60 . The method of  claim 54 , wherein the primary medium of step (a) further comprises any one or more of the following trophic factors and cytokines: stem cell factor (SCF), basic fibroblast growth factor (bFGF), interleukin 6 (IL-6), soluble IL-6 receptor (sIL-6r) and interleukin 11 (IL-11). 
   
   
       61 . The method of  claim 60 , wherein the primary medium of step (a) further comprises the following trophic factors and cytokines: stem cell factor (SCF), basic fibroblast growth factor (bFGF), interleukin 6 (IL-6), and soluble IL-6 receptor (sIL-6r). 
   
   
       62 . The method of  claim 60 , wherein the primary medium of step (a) further comprises the following trophic factors and cytokines: stem cell factor (SCF) and interleukin 11 (IL-11). 
   
   
       63 . The method of  claim 60 , wherein the primary medium of step (a) further comprises the following trophic factors and cytokines: stem cell factor (SCF), basic fibroblast growth factor (bFGF), interleukin 6 (IL-6), soluble IL-6 receptor (sIL-6r) and interleukin 11 (IL-11). 
   
   
       64 . The method of  claim 54 , wherein said embryonic stem cells obtained from blastodermal disks of fertilized eggs are chicken embryonic stem cells. 
   
   
       65 . The method of  claim 54 , wherein the basal medium in step a) (iii) is a basal medium selected from the group consisting of DMEM, GMEM, HamF12 and MacCoy medium.