Suspension mode seed train development for adherent cells
AssigneeSarepta Therapeutics, Inc.
InventorMaroof Alam
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Abstract
The disclosure is directed to a method for seed train expansion of adherent cells comprising culturing cells with a serum-supplemented growth medium in a N-2 vessel; removing the cells from the serum-supplemented medium; inoculating the cells from step into a serum-free growth medium in a N-1 vessel; culturing the cells in the N-1 vessel under suspension conditions; and inoculating a growth medium in a bioreactor with the suspension-cultured cells. In some aspects, the adherent cells are not suspension-adapted. In some aspects, the adherent cells are suspension-adapted. In some aspects, the adherent cells produced by the seed train expansion method are used to produce viral vectors. In some aspects, the viral vectors are AAV vectors.
Claims32
A method of cell expansion of adherent mammalian cells comprising:
- (a) culturing the adherent mammalian cells under adherent conditions with a first growth medium comprising serum in a N-2 container;
- (b) removing the adherent mammalian cells from the first growth medium;
- (c) inoculating the adherent mammalian cells from step (b) into a second growth medium comprising no serum or serum at a concentration less than the first growth medium in a N-1 container;
- (d) culturing the adherent mammalian cells in the N-1 container under suspension conditions in the second growth medium;
- (e) collecting the adherent mammalian cells or the adherent mammalian cells in the second growth medium from step (d) from the N-1 container;
- (f) passaging the adherent mammalian cells from step (d) at least one time under suspension conditions in the second growth medium;
- (g) inoculating the adherent mammalian cells or the adherent mammalian cells in the second growth medium with a third growth medium in a bioreactor;
- and
- (h) culturing the adherent mammalian cells in the bioreactor.
A method of cell expansion of adherent mammalian cells comprising:
- (a) culturing the adherent mammalian cells under adherent conditions with a first growth medium comprising serum in a N-3 container;
- (b) removing the adherent mammalian cells from the first growth medium;
- (c) inoculating the adherent mammalian cells from step (b) into a second growth medium comprising no serum or serum at a concentration less than the first growth medium in a N-2 container;
- (d) culturing the adherent mammalian cells in the N-2 container under suspension conditions in the second growth medium;
- (e) collecting the adherent mammalian cells or the adherent mammalian cells in the second growth medium from step (d) from the N-2 container;
- (f) inoculating the adherent mammalian cells or the adherent mammalian cells in the second growth medium into the second growth medium in a N-1 container;
- (g) culturing the adherent mammalian cells in the N-1 container under suspension conditions in the second growth medium;
- (h) collecting the adherent mammalian cells or the adherent mammalian cells in the second growth medium from step (g) from the N-1 container;
- (i) inoculating the adherent mammalian cells or the adherent mammalian cells in the second growth medium with a third growth medium in a bioreactor;
- and
- (j) culturing the adherent mammalian cells in the bioreactor.
A method of seed-train expansion of adherent mammalian cells comprising:
- (a) culturing the adherent mammalian cells under adherent conditions in a first growth medium comprising serum;
- (b) removing the adherent mammalian cells from the first growth medium;
- (c) suspending the adherent mammalian cells in a second growth medium comprising no serum or serum at a concentration less than the first growth medium;
- (d) culturing the adherent mammalian cells from step (c) under suspension conditions in the second growth medium;
- (e) passaging the adherent mammalian cells from step (d) at least one time under suspension conditions in the second growth medium;
- (f) inoculating the adherent mammalian cells or the adherent mammalian cells in the second growth medium with a third growth medium in a bioreactor;
- and
- (g) culturing the adherent mammalian cells in the bioreactor.
A method of producing a viral particle, comprising:
- (a) culturing adherent mammalian cells under adherent conditions in a first growth medium comprising serum;
- (b) removing the adherent mammalian cells from the first growth medium;
- (c) suspending the adherent mammalian cells in a second growth medium comprising no serum or serum at a concentration less than the first growth medium;
- (d) culturing the adherent mammalian cells from step (c) under suspension conditions in the second growth medium;
- (e) passaging the adherent mammalian cells from step (d) at least one time under suspension conditions in the second growth medium;
- (f) inoculating the adherent mammalian cells or the adherent mammalian cells in the second growth medium with a third growth medium in a bioreactor;
- (g) transfecting the adherent mammalian cells with a polynucleotide sequence encoding a viral capsid protein;
- and
- (h) culturing the adherent mammalian cells in the bioreactor under conditions in which the viral particle is produced.
Dependencies are inferred from the claim text.