Terumo BCT, Inc.

A method for functionalizing a hollow fiber membrane for cell expansion, differentiation, harvesting, activation, or any combination thereof of target cells (e.g., natural killer cells) includes contacting a biotinylated molecule to a surface of the hollow fiber membrane including an extracellular matrix component, the biotinylated molecule binding to the extracellular matrix component and having an affinity for the target cells. The biotinylated molecule is selected from the group consisting of: cytokine, epitope, ligand, monoclonal antibody, stains, aptamer, and combinations thereof. The extracellular matrix component selected from the group consisting of: fibronectin, vitronectin, fibrinogen, collagen, laminin, streptavidin, truncations thereof, and combinations thereof.

Embodiments described herein generally provide for expanding cells in a cell expansion system. The cells may be grown in a bioreactor, and the cells may be activated by an activator (e.g., a soluble activator complex). Nutrient and gas exchange capabilities of a closed, automated cell expansion system may allow cells to be seeded at reduced cell seeding densities, for example. Parameters of the cell growth environment may be manipulated to load the cells into a particular position in the bioreactor for the efficient exchange of nutrients and gases. System parameters may be adjusted to shear any cell colonies that may form during the expansion phase. Metabolic concentrations may be controlled to improve cell growth and viability. Cell residence in the bioreactor may be controlled. In embodiments, the cells may include T cells. In further embodiments, the cells may include T cell subpopulations, including regulatory T cells (Tregs), helper, naïve, memory, or effector, for example.

A method of differentiating induced pluripotent stem cells using a hollow fiber bioreactor includes obtaining induced pluripotent stem cells in the hollow fiber bioreactor and causing a differentiating medium to move through the hollow fiber bioreactor. In certain variations, the obtaining of the induced pluripotent stem cells may include expanding the induced pluripotent stem cell using the hollow fiber bioreactor. In other variations, the obtaining of the induced pluripotent stem cells may include introducing the induced pluripotent stem cells into the hollow fiber bioreactor.

Embodiments described herein generally provide for expanding cells in a cell expansion system. The cells may be grown in a bioreactor, and the cells may be activated by an activator (e.g., a soluble activator complex). Nutrient and gas exchange capabilities of a closed, automated cell expansion system may allow cells to be seeded at reduced cell seeding densities, for example. Parameters of the cell growth environment may be manipulated to load the cells into a particular position in the bioreactor for the efficient exchange of nutrients and gases. System parameters may be adjusted to shear any cell colonies that may form during the expansion phase. Metabolic concentrations may be controlled to improve cell growth and viability. Cell residence in the bioreactor may be controlled. In embodiments, the cells may include T cells. In further embodiments, the cells may include T cell subpopulations, including regulatory T cells (Tregs), for example.

A method for using a bioreactor-based system includes circulating a volume including a cell population through a space in a first direction for a first period, the space having a first orientation for the first period; circulating the volume in a second direction for a second period, the space having the first orientation for the second period; circulating the volume in the first direction for a third period, the space having a second orientation for the third period; and circulating the volume in the second direction for a fourth period, the space having the second orientation for the fourth period. The method further includes maintaining a culture medium in the space using bidirectional movement at a first rate, causing the culture medium to move through the space at a second rate that is less than the first rate, and causing a washing fluid to move through the space.

Embodiments described herein generally provide for expanding cells in a cell expansion system. The cells may be grown in a bioreactor, and the cells may be activated by an activator (e.g., a soluble activator complex). Nutrient and gas exchange capabilities of a closed, automated cell expansion system may allow cells to be seeded at reduced cell seeding densities, for example. Parameters of the cell growth environment may be manipulated to load the cells into a particular position in the bioreactor for the efficient exchange of nutrients and gases. System parameters may be adjusted to shear any cell colonies that may form during the expansion phase. Metabolic concentrations may be controlled to improve cell growth and viability. Cell residence in the bioreactor may be controlled. In embodiments, the cells may include T cells. In further embodiments, the cells may include T cell subpopulations, including regulatory T cells (Tregs), helper, na“i”ve, memory, or effector, for example.

A method for preparing chimeric antigen receptor cells using a cell expansion system includes introducing target cells to a bioreactor of the cell expansion system, where the introduction of the target cells includes isolating the target cells from a source. The isolation of the target cells from the source includes contacting the source to one or more identifying component and then causing the source to move through a separating column, where the separating column disposed in the cell expansion system in line with the bioreactor. The method further includes introducing viral vectors to the bioreactor, introducing a transduction reagent to the bioreactor, and removing non-cellular material from the bioreactor by causing material in the bioreactor to flow through a bypass loop, where the bypass loop includes one or more size exclusion filters and the non-cellular material includes unused viral vectors and unused identifying components.

Embodiments described herein generally provide for expanding cells in a cell expansion system. The cells may be grown in a bioreactor, and the cells may be activated by an activator (e.g., a soluble activator complex). Nutrient and gas exchange capabilities of a closed, automated cell expansion system may allow cells to be seeded at reduced cell seeding densities, for example. Parameters of the cell growth environment may be manipulated to load the cells into a particular position in the bioreactor for the efficient exchange of nutrients and gases. System parameters may be adjusted to shear any cell colonies that may form during the expansion phase. Metabolic concentrations may be controlled to improve cell growth and viability. Cell residence in the bioreactor may be controlled. In embodiments, the cells may include T cells. In further embodiments, the cells may include T cell subpopulations, including regulatory T cells (Tregs), helper, naïve, memory, or effector, for example.

A method for producing T-cells using a cell expansion system includes expanding T-cells using a small bioreactor of the cell expansion system, the small bioreactor having a surface area of about 2,000 cm 2 and an intracapillary volume of about 58 milliliters. The method further includes causing T-cells to flow into a small bioreactor of the cell expansion system. A rate at which the T-cells flow into the small bioreactor may be greater than or equal to about 0.007 μL/min/fiber to less than or equal to about 0.0281 μL/min/fiber. The cell expansion system may further include a tubing set that is in fluid communication with the small bioreactor and a volume ratio of the tubing set to the bioreactor may be about 2.96.

Embodiments described herein generally provide for the scheduled feeding of cells in a cell expansion system. A schedule to proactively and automatically increase and/or maintain inlet rates of media to feed cells may be created, in which inlet rates to the intracapillary portion (or extracapillary portion) of a bioreactor may be increased or maintained according to the schedule. Such schedule may be conservative or aggressive or a combination thereof, for example. Multiple schedules may be used. Scheduled media exchanges may also be included. By following a feed schedule, the monitoring of metabolite levels may be optional. Inlet rates may be increased or maintained without manual manipulation. Media usage may also be more predictable. In an embodiment, a custom task(s) may be created to follow a desired feed schedule. In another embodiment, a pre-programmed task(s) may be used for the scheduled feeding of cells.