Method of culturing eukaryotic cells

Abstract

An apparatus and method to maintain pH within a range conducive for cell growth in a bicarbonate-containing cell culture system without the addition of base. The method relies on the gas transfer characteristics of the bioreactor system to modulate the CO2 transfer to and from the cell culture such that the pH of the cell culture can be maintained within a desired range.

1 . A method for batch culturing eukaryote cells comprising:
 providing cell culture inoculant comprising eukaryotic cells in a bicarbonate-containing culture liquid to a vessel,   said vessel having walls that encapsulate said cell culture and a gas phase head space above said cell culture, and wherein said vessel comprises at least one port that provides an entrance and an egress of gas to and from said head space;   agitating said vessel; and   providing gas to said head space through said port wherein said gas contains an amount of CO 2 , and wherein said amount of CO 2  in said gas is modulated over time to adjust the pH of said cell culture in order to maintain a predetermined pH of said cell culture.   
     
     
         2 . The method of  claim 1  wherein said agitation is by rocking said vessel at a rock rate of 15 to 30 rpm and a rock angle of 5° to 15°. 
     
     
         3 . The method of  claim 2 , wherein the rock rate is between 19 and 25 rpm and the rock angle is between 8° to 12°. 
     
     
         4 . The method of  claim 1  wherein said CO 2  is provided in an amount of between 10% and 0% (v/v) of said gas. 
     
     
         5 . The method of  claim 4  wherein said CO 2  is provided in an amount of between 8% and 2% (v/v) of said gas. 
     
     
         6 . The method of  claim 5  wherein said CO2 is provided in an amount of 8% (v/v) of said gas on day 1, in an amount of 5% (v/v) of said gas on day 2, and in an amount of 2% (v/v) of said gas thereafter. 
     
     
         7 . The method of  claim 1  wherein the flow rate of said gas is between 0 to 1.0 hvm. 
     
     
         8 . The method of  claim 7  wherein the flow rate of said gas is between 0.001 and 0.002 hvm. 
     
     
         9 . The method of  claim 8  wherein the flow rate of said gas is 0.007 hvm. 
     
     
         10 . The method of  claim 1  wherein said eukaryotic cells are vertebrate cells. 
     
     
         11 . The method of  claim 10  wherein said vertebrate cells are selected from the group consisting of frog cells, rabbit cells, rodent cells, sheep cells, goat cells, dog cells, cat cells, cow cells, horse cells, non-human primate cells and human cells. 
     
     
         12 . A method for perfusion culturing eukaryote cells comprising:
 providing cell culture inoculant comprising eukaryotic cells in a bicarbonate-containing culture liquid to a vessel,   said vessel having walls that encapsulate said cell culture and a gas phase head space above said cell culture, and wherein said vessel comprises at least one port that provides an entrance and an egress of gas to and from said head space;   agitating said vessel;   perfusing fresh culture medium into said vessel and removing spent culture medium from said vessel; and   providing gas to said head space through said port to sweep accumulated CO 2  from the headspace of said vessel, thereby modulating the pH of said cell culture in order to maintain a predetermined pH of said cell culture.   
     
     
         13 . The method of  claim 12  wherein said agitation is by rocking said vessel at a rock rate of 15 to 30 rpm and a rock angle of 5° to 15°. 
     
     
         14 . The method of  claim 12 , wherein the rock rate is between 19 and 25 rpm and the rock angle is between 8° to 12°. 
     
     
         15 . The method of  claim 12  wherein said gas contains an amount of O 2  between 0% and 50% (v/v) of said gas. 
     
     
         16 . The method of  claim 15  wherein O 2  is provided in an amount of between 20% and 40% (v/v) of said gas. 
     
     
         17 . The method of  claim 12  wherein O 2  is provided in an amount of 0% (v/v) of said gas on days 1 and 2, in an amount of 30% (v/v) of said gas thereafter. 
     
     
         18 . The method of  claim 12  wherein the flow rate of said gas is between 0 to 1.0 hvm. 
     
     
         19 . The method of  claim 18  wherein the flow rate of said gas is between 0.001 and 0.03 hvm. 
     
     
         20 . The method of  claim 19  wherein the flow rate of said gas is 0.02 hvm. 
     
     
         21 . The method of  claim 12  wherein said eukaryotic cells are vertebrate cells. 
     
     
         22 . The method of  claim 21  wherein said vertebrate cells are selected from the group consisting of frog cells, rabbit cells, rodent cells, sheep cells, goat cells, dog cells, cat cells, cow cells, horse cells, non-human primate cells and human cells. 
     
     
         23 . The method of  claim 1  or  12  wherein said vessel is a rigid container. 
     
     
         24 . The method of  claim 1  or  12  wherein said vessel is a pliable container. 
     
     
         25 . The method of  claim 24  wherein said vessel is a disposable culture bag. 
     
     
         26 . The method of  claim 1  or  12  further comprising a pH monitor for continuously or intermittently monitoring the pH of the cell culture. 
     
     
         27 . The method of  claim 12  wherein said fresh culture medium has a predetermined pH to modulate the pH of said cell culture. 
     
     
         28 . The method of  claim 12  wherein the perfusion of said fresh culture medium is performed through a perfusion device that allows retention of cells in said culture medium. 
     
     
         29 . The method of  claim 1  wherein said gas is provided to said head space such that the cell culture maintains a partial pressure of dissolved CO 2  at a level of about 1 to 200 mmHg. 
     
     
         30 . The method of  claim 29  wherein said gas is provided to said head space such that the cell culture maintains a partial pressure of dissolved CO 2  at a level of about 10 to 150 mmHg. 
     
     
         31 . The method of  claim 30  wherein said gas is provided to said head space such that the cell culture maintains a partial pressure of dissolved CO 2  at a level of about 20 to 120 mmHg. 
     
     
         32 . The method of  claim 1  wherein said gas is provided to said head space such that the cell culture maintains a partial pressure of dissolved CO 2  at a level of about 20 to 80 mmHg. 
     
     
         33 . An apparatus for culturing eukaryote cells comprising:
 a vessel having walls define an interior space of the vessel and separate said interior space from a surrounding environment;   at least one port that provides an entrance and an egress of gas from said vessel;   an agitator reversibly attached to said vessel; and   optionally, a pH monitor having a probe positioned in said interior space.   
     
     
         34 . The apparatus of  claim 33  wherein said vessel is a disposable bioreactor bag. 
     
     
         35 . The apparatus of  claim 33  wherein said vessel comprises a plurality of ports that provides an entrance and an egress of gas from said vessel. 
     
     
         36 . The apparatus of  claim 33  wherein said vessel further comprises a spent medium port to remove spent medium from said vessel and a perfusion port to provide fresh medium to said vessel. 
     
     
         37 . The apparatus of  claim 33  wherein said pH monitor is connected to a computerized automation system wherein said system regulates head space clearance of said vessel to modulate the concentration of CO 2  in said vessel in response to modulation of pH as measured by said pH monitor. 
     
     
         38 . The apparatus of  claim 37  further comprising at least one other monitor to monitor a parameter selected from the group consisting of gas flow, temperature, agitation, perfusion rate of fresh medium, and dissolved CO 2 . 
     
     
         39 . A method for batch culturing eukaryote cells comprising:
 providing cell culture comprising eukaryotic cells in a bicarbonate-containing culture liquid to a vessel,   said vessel having walls that encapsulate said cell culture and a gas phase head space above said cell culture, and wherein said vessel comprises at least one port that provides an entrance and an egress of gas to and from said head space;   agitating said vessel; and   providing gas to said head space through said port wherein said gas contains 8% CO 2  (v/v) of gas during the first day, 5% CO 2  (v/v) of gas the second day and 2% CO 2  (v/v) of gas thereafter.   
     
     
         40 . The method of  claim 39  wherein said vessel is agitated on a rocker at a rock rate of 21-23 rpm. 
     
     
         41 . The method of  claim 39  wherein said vessel is agitated on a rocker at a rock angle of 8-12°. 
     
     
         42 . The method of  claim 41  wherein said vessel is agitated on a rocker at a rock angle of 10°. 
     
     
         43 . A method for perfusion culturing eukaryote cells comprising:
 providing cell culture comprising eukaryotic cells in a bicarbonate-containing culture liquid to a vessel,   said vessel having walls that encapsulate said cell culture and a gas phase head space above said cell culture, and wherein said vessel comprises at least one port that provides an entrance and an egress of gas to and from said head space;   agitating said vessel;   providing gas to said head space through said port wherein said gas is supplemented with at least 30% O 2  (v/v) two days after initiation of perfusion.