Adaption of mammalian cell lines to high cell densities

Abstract

Methods and nutrient media are disclosed useful for adapting mammalian cell lines to culture at increased cell densities.

We claim: 
     
       1. A method for producing an adapted mammalian cell line which grows at increased cell densities, said method comprising: a) initiating a passage by diluting a culture containing mammalian cells with a suitable growth medium, the dilution factor being suitable for the duration of the passage;   b) maintaining pH, dissolved oxygen, and nutrients at non-limiting levels during the passage;   c) repeating steps (a) and (b) at least about 5 times; and   d) obtaining an adapted mammalian cell line with the ability to grow at increased cell densities.   
     
     
       2. The method of claim 1, wherein steps (a) and (b) are repeated about 5 to about 20 times. 
     
     
       3. A method for producing an adapted CHO cell line which grows at increased cell densities, said method comprising: a) initiating a passage approximately 1 to 5 days in duration by diluting a culture containing CHO cells at a density of at least approximately 1×10 6  cells/ml with a suitable growth medium, the dilution factor being suitable for the duration of the passage;   b) maintaining pH, dissolved oxygen, and nutrients in non-limiting levels during the passage;   c) repeating steps (a) and (b) at least about 5 times; and   d) obtaining an adapted CHO cell line with the ability to grow at increased cell densities.   
     
     
       4. The method of claim 3, wherein steps (a) and (b) are repeated about 5 to about 20 times. 
     
     
       5. A method for producing an adapted CHO cell line which grows at increased cell densities, said method comprising: a) initiating a passage approximately 3 to 4 days in duration by diluting a culture containing CHO cells at a density of at least approximately 1×10 6  cells/ml with a suitable growth medium, the dilution factor being suitable to the duration of the passage;   b) maintaining pH, dissolved oxygen, and nutrients at non-limiting levels during the passage;   c) repeating steps (a) and (b) at least about 5 times; and   d) obtaining an adapted CHO cell line with the ability to grow at increased cell densities.   
     
     
       6. The method of claim 5, wherein steps (a) and (b) are repeated about 5 to about 20 times. 
     
     
       7. A method for adapting mammalian cell lines to culture at increased cell densities, said method comprising continuously or periodically diluting a cell culture, containing mammalian cells, with a suitable growth medium, for between approximately 10 to 60 days, while maintaining pH, dissolved oxygen and nutrients at non-limiting levels. 
     
     
       8. A method for adapting CHO cell lines to culture at increased cell densities, said method comprising continuously or periodically diluting a culture containing CHO cells, at a density of at least approximately 1×10 6  cells/ml with a suitable growth medium, at a dilution rate less than approximately 0.029 hr -1 , while maintaining pH, dissolved oxygen and nutrients at non-limiting levels. 
     
     
       9. The method of claim 8, wherein the dilution rate is between approximately 0.018 hr -1  and 0.026 hr -1 .