Serum-free mammalian cell culture medium, and uses thereof
View Patent ↗The present invention provides a cell culture medium formulation that supports the in vitro cultivation, particularly in suspension, of mammalian cells, particularly epithelial cells and fibroblast cells, and methods for cultivating mammalian cells in suspension in vitro using these media. The media comprise a basal medium and a polyanionic or polyanionic compound, preferably a polysulfonated or polysulfated compound, and more preferably dextran sulfate. The present invention also provides chemically defined, protein-free eukaryotic cell culture media comprising an iron chelate and zinc, which is capable of supporting the growth (and particularly the high-density growth of mammalian cells) in suspension culture, increasing the level of expression of recombinant protein in cultured cells, and/or increasing virus production in cultured cells.
1. A method for producing a recombinant protein in vitro, comprising culturing a recombinant CHO cell in suspension, in a serum-free and protein-free culture medium comprising zinc at a final concentration of about 0.00007 to 0.00073 g/L, and iron at a final concentration of about 0.00028 to 0.011 g/L, under conditions suitable for supporting the cultivation of the CHO cell to a density of about 5×10 6 cells/ml, in said suspension and expression of said recombinant protein, the conditions comprising culturing the CHO cell at about 37 degrees Celsius in a humidified atmosphere containing about 3-20% carbon dioxide in air, and wherein said serum-free and protein-free culture medium supports increased levels of cell growth as compared to culture in FMX-8 medium.
2. The method for producing a recombinant protein in vitro according to claim 1 wherein said recombinant protein is isolated.
3. The method for producing a recombinant protein in vitro according to claim 1 wherein the serum-free and protein-free culture medium comprises at least one polyanionic or polycationic compound.
4. The method for producing a recombinant protein in vitro according to claim 3 wherein said polyanionic compound is a polysulfonated or polysulfated compound.
5. The method for producing a recombinant protein in vitro according to claim 4 wherein said polysulfonated or polysulfated compound is selected from the group consisting of dextran sulfate, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, pentosan sulfate and a sulfated proteoglycan.
6. The method for producing a recombinant protein in vitro according to claim 4 wherein said polysulfonated or polysulfated compound is dextran sulfate.
7. The method for producing a recombinant protein in vitro according to claim 6 wherein said dextran sulfate has an average molecular weight of about 5,000 to about 25,000 daltons.
8. The method for producing a recombinant protein in vitro according to claim 6 wherein said dextran sulfate has an average molecular weight of about 5,000 to about 10,000 daltons.
9. The method for producing a recombinant protein in vitro according to claim 1 wherein said recombinant CHO cell is cultured on a microcarrier.
10. The method for producing a recombinant protein in vitro according to claim 1 wherein said iron is a transferrin substitute and the zinc is an insulin substitute.
11. The method for producing a recombinant protein in vitro according to claim 1 , wherein the serum-free and protein-free medium is sufficient for the growth of the recombinant CHO cell.
12. The method for producing a recombinant protein in vitro according to claim 1 , wherein the expression of said recombinant protein is not required for the serum-free and protein-free cultivation of the cell.
13. The method for producing a recombinant protein in vitro according to claim 1 , wherein the serum-free and protein-free growth of the cell does not require the co-production of an exogenous protein within the CHO cell.