Methods of genetically modifying animal cells

Abstract

This invention relates to improved methods of genetically modifying animal cells by decreasing the distance between cells and genetic modification agents in order to increase the efficiency of genetic modification and/or reduce use of gene modification agents.

1 . A method of culturing and transducing animal cells comprising:
 adding media and animal cells into a device that includes a gas permeable, liquid impermeable growth surface,   wherein at least a portion of said animal cells are T cells that gravitate to said growth surface and said growth surface is in a horizontal position, and   said media is at a first height and said T cells are at a first concentration of T cells per milliliter of media, and   allowing a period of time during which said growth surface is in contact with ambient gas and said T cells increase in quantity thereby creating a second concentration of T cells per milliliter of media, and   reducing the height of the media to a second height in the device to creating a third concentration, of T cells per milliliter of media, and   adding a quantity of genetic modification agents, and   allowing a period of time whereby said genetic modification agents act to transduce at least a portion of said T cells.   
     
     
         2 . The method of  claim 1  wherein, at least a portion of said animal cells that are added into the device are peripheral blood mononuclear cells. 
     
     
         3 . The method of  claim 1  wherein after a portion of said T cells are transduced, media is added to the device, thereby elevating media to a third height, and allowing a period of time during which the transduced T cells increase in quantity. 
     
     
         4 . The method of  claim 1  wherein said animal cells are at a first concentration of 4 million to 20 million cells per milliliter of media. 
     
     
         5 . The method of  claim 1  wherein said animal cells are at a first concentration of 5 million to 20 million cells per milliliter of media. 
     
     
         6 . The method of  claim 1  wherein said animal cells are at a first concentration of 6 million to 20 million cells per milliliter of media. 
     
     
         7 . The method of  claim 1  wherein the media in the device is not in a static state during at least a portion of the period of time whereby said genetic modification agents act to transducer at least a portion of said animal cells. 
     
     
         8 . The method of  claim 1  wherein said genetic modification agent is comprised of lentivirus. 
     
     
         9 . The method of  claim 1  wherein said gas permeable, liquid impermeable material is in contact with a gas permeable material support. 
     
     
         10 . The method of  claim 1  wherein the ratio of the number of genetic modification agents to the quantity of animal cells added to the device is not greater than 2.