Ultra-high density cell banking methods
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 mammalian cell bank directly from a population of cultured cells, the method comprising:
a) culturing the mammalian cells in a perfusion bioreactor to obtain the ultra-high density cell population with a concentration of at least about 1.0×10 8 cells/mL, wherein said perfusion bioreactor is coupled to a cell retention system;
b) harvesting the ultra-high density population directly without performing a concentration step after culturing the mammalian cells in the perfusion bioreactor that is coupled to the cell retention system; and
c) cryopreserving the harvested ultra-high density cell population by adding a cryoprotectant to produce an ultra-high density frozen cell bank,
wherein the ultra-high density frozen cell bank has a concentration of at least about 1.0×10 8 cells/mL and at least 95% post-thaw viability.
2 . The method of claim 1 , wherein the ultra-high density cell population has a concentration selected from the group consisting of about 1.1×10 8 cells/mL, about 1.2×10 8 cells/mL, about 1.3×10 8 cells/mL, about 1.4×10 8 cells/mL, about 1.5×10 8 cells/mL, about 1.6×10 8 cells/mL, about 1.7×10 8 cells/mL, about 1.8×10 8 cells/mL, about 1.9×10 8 cells/mL, and about 2.0×10 8 cells/mL.
3 . The method of claim 1 , wherein the cryopreserving comprises adding dimethyl sulphoxide (DMSO) to the ultra-high density cell population at a final concentration of about 5% to about 10%, vol/vol.
4 . The method of claim 1 , wherein the ultra-high density frozen cell bank is contained in a vial or a cryobag and comprises about 4.5×10 8 cells/vial or about 100×10 8 cells/cryobag.
5 . 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.
6 . The method of claim 1 , wherein the perfusion rate in the perfusion bioreactor is between 0 and 15 reactor volumes per day.
7 . The method claim 1 , wherein the perfusion bioreactor cell culture has a dissolved oxygen concentration (DO) of at least about 30%.
8 . The method of claim 1 , 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-Agl4, and hybridoma cells.
9 . The method of claim 1 , wherein the mammalian cells are transfected cells or the cells express a therapeutic protein.
10 . The method of claim 1 , wherein the bioreactor is rocked at 25 rpm with a rock angle of 12° or at 22 rpm with a rock angle of 10°.
11 . The method of claim 1 , wherein the ultra-high density cell population is cryopreserved by any one of the following:
(i) cooled to and maintained at a temperature of about 4° C. prior to and during the addition of the cryoprotectant and dispensing; or
(ii) maintained at a temperature of about 20° C. to about 26° C. prior to and during the addition of the cryoprotectant; or
(iii) maintained at an uncontrolled cold temperature by using an ice water bath prior to and during the addition of the cryoprotectant.
12 . The method of claim 1 , wherein a total gas flow rate is initially set to 0.2 liters per minute (lpm), followed by increasing the total gas flow rate to 0.4 lpm when the cell concentration is about 3×10 7 cells/mL to about 4×10 7 cells/mL.
13 . The method of claim 1 , wherein the CO2 concentration is reduced to 0% when the cell concentration is about 2×10 7 cells/mL to about 5×10 7 cells/mL.
14 . A method for producing a culture of mammalian cells from an ultra-high density frozen cell bank, the method comprising thawing and culturing the ultra-high density frozen cell bank with a concentration of at least about 1.0×10 8 cells/mL in a perfusion bioreactor to obtain the culture of mammalian cells,
wherein the ultra-high density frozen cell bank was produced by harvesting a cell culture directly from a bioreactor, wherein said bioreactor is coupled to a cell retention system, without performing any concentration steps after culturing the mammalian cells in the perfusion bioreactor and cryopreserving the harvested cell culture after adding a cryoprotectant, and wherein the cultured mammalian cells have at least 95% post-thaw viability.
15 . The method of claim 14 , wherein the ultra-high density frozen cell bank has a concentration selected from the group consisting of about 1.1×10 8 cells/mL, about 1.2×10 8 cells/mL, about 1.3×10 8 cells/mL, about 1.4×10 8 cells/mL, about 1.5×10 8 cells/mL, about 1.6×10 8 cells/mL, about 1.7×10 8 cells/mL, about 1.8×10 8 cells/mL, about 1.9×10 8 cells/mL, and about 2.0×10 8 cells/mL.
16 . The method of claim 14 , 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-Agl4, and hybridoma cells.
17 . The method of claim 14 , wherein the cells are transfected cells or the cells express a therapeutic protein.
18 . The method of claim 14 , wherein the cryoprotectant comprises dimethyl sulphoxide (DMSO) at a final concentration of about 5% to about 10%, vol/vol of the cell culture.
19 . The method of claim 14 , wherein the ultra-high density frozen cell bank is contained in a vial or a cryobag and comprises about 4.5×10 8 cells/vial or about 100×10 8 cells/cryobag.
20 . The method of claim 14 , wherein the cell culture is cryopreserved by any one of the following:
(i) cooled to and maintained at a temperature of about 4° C. prior to and during the addition of the cryoprotectant and dispensing; or
(ii) maintained at a temperature of about 20° C. to about 26° C. prior to and during the addition of the cryoprotectant; or
(iii) maintained at an uncontrolled cold temperature by using an ice water bath prior to and during the addition of the cryoprotectant.
21 . A method for producing an ultra-high density frozen cell bank, the method comprising:
a) harvesting a culture of mammalian cells directly from a bioreactor, wherein the bioreactor is coupled to a non-centrifugal cell retention device, and wherein the harvesting is conducted without performing any concentration step after culturing the mammalian cells in the bioreactor that is coupled to the non-centrifugal cell retention device; and
b) cryopreserving the harvested culture by adding a cryoprotectant to produce the ultra-high density frozen cell bank,
wherein the ultra-high density frozen cell bank has a concentration of at least about 1.0×10 8 cells/mL and at least 95% post-thaw viability in a perfusion culture.