Suspension mode seed train development for adherent cells
AssigneeSarepta Therapeutics, Inc.
InventorMaroof ALAM
Life of this patent
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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.
Claims43
A method of mammalian cell expansion comprising:
- (a) culturing the mammalian cells with a first growth medium comprising serum in a N-2 container;
- (b) removing the mammalian cells from the first growth medium;
- (c) inoculating the 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 mammalian cells in the N-1 container under suspension conditions in the second growth medium;
- (e) passaging the mammalian cells from step (d) at least one time;
- (f) inoculating the mammalian cells with a third growth medium in a bioreactor;
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- (g) culturing the mammalian cells in the bioreactor.
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A method of seed-train expansion of mammalian cells comprising:
- (a) culturing the mammalian cells under adherent conditions in a first growth medium comprising serum, and passaging the mammalian cells at least once;
- (b) removing the mammalian cells from the first growth medium;
- (c) suspending the mammalian cells in a second growth medium comprising no serum or serum at a concentration less than the first growth medium;
- (d) culturing the mammalian cells from step (c) under suspension conditions in the second growth medium;
- (e) passaging the mammalian cells from step (d) at least one time under suspension conditions in the second growth medium;
- (f) inoculating the mammalian cells with a third growth medium in a bioreactor;
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- (g) culturing the mammalian cells in the bioreactor.
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A method of producing a viral particle, comprising:
- (a) culturing mammalian cells under adherent conditions in a first growth medium, and passaging the mammalian cells at least once;
- (b) removing the mammalian cells from the first growth medium;
- (c) suspending the mammalian cells in a second growth medium comprising no serum or serum at a concentration less than the first growth medium;
- (d) culturing the mammalian cells from step (c) under suspension conditions, and passaging the cells at least one time;
- (e) inoculating a third growth medium in a bioreactor with the mammalian cells from step (d);
- (f) transfecting the mammalian cells with a polynucleotide sequence encoding a viral particle, wherein the polynucleotide is a plasmid;
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- (g) culturing the mammalian cells in the bioreactor under conditions in which the viral particle is produced, wherein the viral particle is selected from the group consisting of an AAV, a lentivirus, a herpes virus, a polyoma virus, and a vaccinia virus,
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Dependencies are inferred from the claim text.