Derivation and culture of human embryo-derived cells
The present invention concerns methods for deriving and culturing embryonic cells and in particular to methods for maintaining the undifferentiated state of stems cells and cell lines in culture. The invention also concerns cells and cell lines derived by the methods of the invention.
1. A method of derivation of embryo-derived cells or embryo-derived cell lines consisting of the following steps:
a) culturing an embryo under low oxygen tension conditions of about 5% oxygen to the blastocyst stage;
b) separating Inner Cell Mass (ICM) cells of said blastocyst from trophectodermal cells, plating said ICM cells and culturing said ICM cells under low oxygen tension conditions of about 5% oxygen for a time sufficient to produce embryonic cell-like outgrowths;
c) at least once, dissociating said embryonic cell-like outgrowths, replating said dissociated embryonic cell-like outgrowths and culturing said dissociated embryonic cell-like outgrowths under low oxygen tension conditions of about 5% oxygen for a time sufficient to produce further embryonic cell-like outgrowths;
d) analyzing said further embryonic cell-like outgrowths of step c) for embryonic stem cell-like properties;
e) selecting outgrowths with embryonic stem cell-like properties; and
f) isolating and dissociating said outgrowths with embryonic stem cell-like properties selected in step e) and culturing the dissociated cells under low oxygen tension conditions of about 5% oxygen to obtain embryo-derived cells or embryo-derived cell lines.
2. A method of derivation of embryo-derived cells or embryo-derived cell lines consisting of the following steps:
a) incubating a putative zygote in a culture medium under low oxygen tension conditions of about 5% oxygen to derive an embryo;
b) culturing embryo obtained in step a) under low oxygen tension conditions of about 5% oxygen to the blastocyst stage;
c) separating Inner Cell Mass (ICM) cells of said blastocyst from trophectodermal cells, plating said ICM cells and culturing said ICM cells under low oxygen tension conditions of about 5% oxygen for a time sufficient to produce embryonic cell-like outgrowths;
d) at least once, dissociating said embryonic cell-like outgrowths, replating said dissociated embryonic cell-like outgrowths and culturing said dissociated embryonic cell-like outgrowths under low oxygen tension conditions of about 5% oxygen for a time sufficient to produce further embryonic cell-like outgrowths;
e) analyzing said further embryonic cell-like outgrowths of step d) for embryonic stem cell-like properties;
f) selecting outgrowths with embryonic stem cell-like properties;
g) isolating and dissociating said outgrowths with embryonic stem cell-like properties selected in step f) and culturing the dissociated cells under low oxygen tension conditions of about 5% oxygen to obtain embryo-derived cells or embryo-derived cell lines.
3. The method according to claim 1 or 2 , wherein the embryo-derived cell or embryo-derived cell line is a stem cell or stem cell line.