Ultra-high density cell banking methods

Abstract

Provided are methods for the creation of ultra-high density cryopreserved cell banks. In certain embodiments, these methods employ altered perfusion culture techniques that allow for production of ultra-high density cell cultures that can be cryopreserved at unexpectedly high cell densities without the need for any cell concentration steps, while retaining excellent cell viability and quality.

1 . A method for producing an ultra-high density frozen cell bank directly from a population of cultured cells, the method comprising:
 a) culturing cells in a perfusion bioreactor to obtain an ultra-high density cell population with a concentration of at least about 1.0×10̂8 cells/mL, wherein the perfusion bioreactor is coupled to a cell retention system; and   b) adding cryoprotectant to the ultra-high density cell population to produce an ultra-high density frozen cell bank, wherein the ultra-high density frozen cell bank has a concentration of at least about 1.0×10̂8 cells/mL, and   
       wherein no additional concentrating step is performed between culturing the cells and adding cryoprotectant to the ultra-high density cell population. 
     
     
         2 . The method of  claim 1 , wherein the cell retention system comprises an alternating tangential flow filtration system comprising a filter. 
     
     
         3 . The method of  claim 2 , wherein the filter has a surface area of at least about 0.08 m 2 . 
     
     
         4 . The method of  claim 3 , wherein the filter has a surface area of about 0.3 m 2  to about 0.5 m 2 , about 0.5 m 2  to about 1.0 m 2 , about 0.7 m 2  to about 0.8 m 2 , about 1.0 m 2  to about 2.0 m 2 , about 2.0 m 2  to about 3.0 m 2 , about 3.0 m 2  to about 4.0 m 2 , or about 4.0 m 2  to about 5.5 m 2 . 
     
     
         5 . The method of  claim 2 , wherein the filter has a pore size selected from the group consisting of 0.7 μm, 1.2 μm, and 7 μm. 
     
     
         6 . The method of  claim 1 , wherein the ultra-high density cell population has a cell density selected from the group consisting of about 1.0×10̂8 cells/mL, about 1.1×10̂8 cells/mL, about 1.2×10̂8 cells/mL, about 1.3×10̂8 cells/mL, about 1.4×10̂8 cells/mL, about 1.5×10̂8 cells/mL, about 1.6×10̂8 cells/mL, about 1.7×10̂8 cells/mL, about 1.8×10̂8 cells/mL, about 1.9×10̂8 cells/mL, and about 2.0×10̂8 cells/mL. 
     
     
         7 . The method of  claim 1 , wherein adding cryoprotectant comprises adding dimethyl sulphoxide (DMSO) to the ultra-high density cell population at a final concentration of about 5% to about 10%, vol/vol. 
     
     
         8 . The method of  claim 1 , wherein the production of a high density cell bank comprises freezing at least a portion of the ultra-high density cell population in a container appropriate for storage under cryopreservation conditions. 
     
     
         9 . The method of  claim 8 , wherein the container is a vial. 
     
     
         10 . The method of  claim 9 , wherein the ultra-high density frozen cell bank comprises about 4.5×10̂8 cells/vial. 
     
     
         11 . The method of  claim 8 , wherein the container is a cryobag. 
     
     
         12 . The method of  claim 11 , wherein the ultra-high density frozen cell bank comprises about 100×10̂8 cells/cryobag. 
     
     
         13 . The method of  claim 1 , wherein the ultra-high density frozen cell bank has a cell density of at least about 1.1 10̂8 cells/mL. 
     
     
         14 . The method of  claim 1 , wherein the perfusion rate in the perfusion bioreactor is between about 0.02 nL/cell/day to about 0.5 nL/cell/day. 
     
     
         15 . The method of  claim 1 , wherein the perfusion rate in the perfusion bioreactor is between 0 and 15 reactor volumes per day. 
     
     
         16 . The method of  claim 1 , wherein the perfusion bioreactor cell culture has a pH of between about 6.8 to about 7.2. 
     
     
         17 . The method of  claim 1 , wherein the perfusion bioreactor cell culture has a dissolved oxygen concentration (DO) of at least about 30%. 
     
     
         18 . The method of  claim 1 , wherein the bioreactor is a flexible bag bioreactor. 
     
     
         19 . The method of  claim 1 , wherein the ultra-high density frozen cell bank has a post-thaw cell viability of at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. 
     
     
         20 . The method of  claim 1 , wherein the cells are mammalian cells. 
     
     
         21 . The method of  claim 20 , wherein the mammalian cells are selected from the group consisting of: CHO, CHO-DBX11, CHO-DG44, CHO-S, CHO-K1, Vero, BHK, HeLa, COS, MDCK, HEK-293, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0, CRL7030, HsS78Bst cells, PER.C6, SP2/0-Ag14, and hybridoma cells. 
     
     
         22 . The method of  claim 1 , wherein the cells are transfected cells. 
     
     
         23 . The method of  claim 1 , wherein the cells express a therapeutic protein. 
     
     
         24 . The method of  claim 1 , wherein:
 a) the perfusion bioreactor comprises a flexible bag bioreactor;   b) the filter has a filter surface area of at least 0.08 m 2  and a molecular mass cut off (MWCO) size of at least 50 kDa;   c) the cryoprotectant added to the ultra-high density cell population is DMSO; and   d) the ultra-high density frozen cell bank comprises about 5% to about 10%, vol/vol DMSO.   
     
     
         25 . The method of  claim 1 , wherein the pH and DO of the culture are controlled by automated methods. 
     
     
         26 . The method of  claim 1 , wherein the pH and DO of the culture are controlled by non-automated methods. 
     
     
         27 . The method of  claim 26 , wherein the pH and DO are controlled through any one or more of the following: adjustment of the mixture of gases that are introduced to the culture, adjustment of the rock rate of the bioreactor, or adjustment of the rock angle of the bioreactor. 
     
     
         28 . The method of  claim 27 , wherein the bioreactor is rocked at 25 rpm with a rock angle of 12°. 
     
     
         29 . The method of  claim 27 , wherein the bioreactor is rocked at 22 rpm with a rock angle of 10°. 
     
     
         30 . The method of  claim 1 , wherein the ultra-high density cell population is cooled to and maintained at a temperature of about 4° C. prior to and during the addition of the cryoprotectant and dispensing. 
     
     
         31 . The method of  claim 1 , wherein the ultra-high density cell population is maintained at a temperature of about 20° C. to about 26° C. prior and during the addition of the cryoprotectant and dispensing. 
     
     
         32 . The method of  claim 1 , wherein the ultra-high density cell population is maintained at an uncontrolled cold temperature by using an ice water bath prior to and during the addition of the cryoprotectant and dispensing. 
     
     
         33 . The method of  claim 1 , wherein the bioreactor comprises a built-in filter.