Suspension mode seed train development for adherent cells
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Publication Number
US-12577540-B2
Publication Date
2026-03-17
Expiration Date
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.
Core Innovation
The invention relates to a method of cell expansion of adherent mammalian cells using a hybrid seed-train. The method includes culturing adherent mammalian cells under adherent conditions in a first growth medium comprising serum, removing the adherent mammalian cells from the first growth medium, and inoculating the cells into a second growth medium comprising no serum or serum at a concentration less than the first growth medium in a container. The cells are then cultured under suspension conditions in the second growth medium, collected, and passaged at least one time under suspension conditions.
The hybrid seed-train continues by inoculating the adherent mammalian cells or the cells in the second growth medium with a third growth medium in a bioreactor and culturing the cells in the bioreactor. In some embodiments, the expansion is performed using additional staging between the serum-containing first growth medium and the suspension culture, including a progression with collection steps at each suspension stage. The method includes cultivating the adherent mammalian cells under suspension conditions in multiple containers before transfer to the bioreactor.
The invention also covers seed-train expansion for adherent mammalian cells and production of viral particles. A method of producing a viral particle further includes transfecting the adherent mammalian cells with a polynucleotide sequence encoding a viral capsid protein and culturing under conditions in which the viral particle is produced.
Claims Coverage
The independent claims are clm-00001, clm-00014, clm-00017, and clm-00020. Across these claims, the core inventive workflow is expanded for cell expansion with container staging and a bioreactor transfer, seed-train expansion, and viral particle production including a polynucleotide encoding a viral capsid protein.
Hybrid seed-train with serum-containing adherent culture followed by serum-reduced suspension culture
Culturing adherent mammalian cells under adherent conditions in a first growth medium comprising serum; removing the adherent mammalian cells from the first growth medium; inoculating the adherent mammalian cells into a second growth medium comprising no serum or serum at a concentration less than the first growth medium in a container; culturing the adherent mammalian cells under suspension conditions in the second growth medium; collecting the cells; and passaging the adherent mammalian cells at least one time under suspension conditions in the second growth medium.
Bioreactor inoculation using a third growth medium after suspension passaging
Inoculating the adherent mammalian cells or the adherent mammalian cells in the second growth medium with a third growth medium in a bioreactor; and culturing the adherent mammalian cells in the bioreactor.
Multi-stage container suspension expansion prior to bioreactor inoculation
Culturing the adherent mammalian cells under adherent conditions with a first growth medium comprising serum in a first container; inoculating into a second growth medium comprising no serum or serum at a concentration less than the first growth medium in a second container; culturing under suspension conditions in the second container; collecting the cells; inoculating into the second growth medium in a third container; culturing under suspension conditions in the third container; collecting; inoculating with a third growth medium in a bioreactor; and culturing the adherent mammalian cells in the bioreactor.
Seed-train expansion method with suspension passaging at least once before bioreactor transfer
Culturing the adherent mammalian cells under adherent conditions in a first growth medium comprising serum; removing the adherent mammalian cells from the first growth medium; suspending the adherent mammalian cells in a second growth medium comprising no serum or serum at a concentration less than the first growth medium; culturing the adherent mammalian cells under suspension conditions in the second growth medium; passaging the adherent mammalian cells at least one time under suspension conditions in the second growth medium; inoculating the adherent mammalian cells with a third growth medium in a bioreactor; and culturing the adherent mammalian cells in the bioreactor.
Viral particle production by suspension-mode seed-train transfer plus polynucleotide encoding a viral capsid protein
Culturing adherent mammalian cells under adherent conditions in a first growth medium comprising serum; removing the adherent mammalian cells from the first growth medium; suspending the adherent mammalian cells in a second growth medium comprising no serum or serum at a concentration less than the first growth medium; culturing under suspension conditions in the second growth medium; passaging at least one time under suspension conditions in the second growth medium; inoculating with a third growth medium in a bioreactor; transfecting the adherent mammalian cells with a polynucleotide sequence encoding a viral capsid protein; and culturing the adherent mammalian cells in the bioreactor under conditions in which the viral particle is produced.
Across clm-00001, clm-00014, clm-00017, and clm-00020, the claim set covers a staged transition from adherent, serum-containing growth to serum-reduced suspension conditions with at least one suspension passaging, followed by inoculation into a bioreactor using a third growth medium for further culturing. In the viral-particle claim, this workflow is combined with transfection using a polynucleotide sequence encoding a viral capsid protein and culturing under conditions in which the viral particle is produced.
Stated Advantages
Reduced contamination risk by avoiding trypsin during passaging.
Documented Applications
Producing viral vectors/viral particles, particularly AAV vectors.
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