Coating a bioreactor in a cell expansion system
Inventors
FRANK, Nathan D. • NANKERVIS, Brian J. • Hlavinka, Dennis J. • DILORENZO, Thomas G.
Assignees
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Publication Number
US-12077739-B2
Publication Date
2024-09-03
Expiration Date
Abstract
Embodiments described herein generally provide for the expansion of cells in a cell expansion system using an active promotion of a coating agent(s) to a cell growth surface in some embodiments. A coating agent may be applied to a surface, such as the cell growth surface of a hollow fiber in a bioreactor, by controlling the movement of a fluid in which a coating agent is suspended, by changing flow rates, by changing flow directions, by rotation of the bioreactor, and/or combinations thereof.
Core Innovation
The invention relates to a method of growing cells in a bioreactor of a cell expansion system by actively controlling fluid movement and timing to coat the bioreactor and promote cell attachment and expansion. A first fluid comprising a reagent is circulated through the bioreactor at a first flow rate for a first period of time to allow at least a first portion of the reagent to coat the bioreactor, and then at a second flow rate slower than the first flow rate for a second period of time to allow a second portion of the reagent to coat the bioreactor.
After the reagent-coating periods, a second fluid is circulated through the bioreactor to remove a third portion of the reagent from the bioreactor. Next, a third fluid comprising a plurality of cells is circulated through the bioreactor, circulation of the third fluid is reduced from the third flow rate, and the bioreactor is maintained in a first orientation for a third predetermined period of time to allow at least a portion of the plurality of cells to settle.
The bioreactor is then rotated to a second orientation about 180 degrees from the first orientation and maintained in the second orientation for a fourth period of time to allow at least a second portion of the plurality of cells to settle. Finally, at least the first and the second portion of cells are expanded in the bioreactor. Dependent refinements specify additional settling-stage flow-rate slowdown, multiple stepwise periods, constraints on period durations, and a bioreactor that includes a hollow fiber membrane.
Claims Coverage
The independent claim includes 4 inventive features. It covers staged reagent coating at different flow rates, reagent removal, staged cell introduction with circulation reduction, and controlled settling using defined bioreactor orientations including about 180-degree rotation before expanding the settled cell portions.
Staged reagent coating via different flow rates
Circulating a first fluid comprising a reagent through a bioreactor at a first flow rate for a first period of time to allow at least a first portion of the reagent to coat the bioreactor, and then at a second flow rate slower than the first flow rate for a second period of time to allow a second portion of the reagent to coat the bioreactor.
Reagent removal after coating
Circulating a second fluid through the bioreactor to remove a third portion of the reagent from the bioreactor.
Cell loading with reduced circulation
Circulating a third fluid through the bioreactor, wherein the third fluid comprises a plurality of cells, reducing circulation of the third fluid from the third flow rate, and maintaining the bioreactor in a first orientation for a third predetermined period of time to allow at least a portion of the plurality of cells to settle.
Orientation-controlled settling with about 180-degree rotation
After the third predetermined period of time, rotating the bioreactor to a second orientation that is about 180 degrees from the first orientation, and after the rotating, maintaining the bioreactor in the second orientation for a fourth period of time to allow at least a second portion of the plurality of cells to settle; and expanding at least the first and the second portion of cells in the bioreactor.
Overall, the claim coverage centers on sequentially coating the bioreactor with a reagent using different flow rates, removing excess reagent, introducing cells with reduced circulation, and using controlled bioreactor orientations including an about 180-degree rotation to promote cell settling before expanding the settled cell portions.
Stated Advantages
Improved yields/harvest yields and faster/greater expansion reported in the described comparisons.
Improved uniformity of coating/loading and enhanced cell distribution/settling supported by the described rationale and outcomes.
Comparative improvements in viability and doubling time reported in the described comparisons.
Documented Applications
Cell expansion system (CES) workflows for growing cells in a bioreactor using hollow-fiber systems with reagent coating/loading and subsequent cell settling before expansion.
Gravity-influenced attachment and expansion studies described as showing increased seeded/harvested cells and faster/greater expansion.
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