Cell expansion

Abstract

Embodiments described herein generally provide for expanding cells in a cell expansion system. The cells can be grown in a bioreactor and the cells can be activated by an activating agent (e.g., a soluble activating agent complex). The ability of a closed automated cell expansion system to exchange nutrients and gases, for example, can enable cells to be seeded at reduced cell seeding densities. Parameters of the cell growth environment can be manipulated to load cells to specific locations in the bioreactor for efficient exchange of nutrients and gases. System parameters can be adjusted to shear any cell colonies that can form during the expansion phase. Metabolic concentrations can be controlled to improve cell growth and viability. Cell residence in the bioreactor can be controlled. In some embodiments, the cells can comprise T cells. In yet other embodiments, the cells can comprise a T cell subset, which for example, comprises regulatory T cells (Tregs).

Description

[0001] This application is a divisional application filed on April 2, 2018, with application number 201880030588.1 and invention title "Cell Expansion".

[0002] Cross-references to related applications

[0003] This application claims priority and benefit from the following applications: U.S. Provisional Application Serial No. 62 / 479,721, filed March 31, 2017, entitled "Cell Expansion"; U.S. Provisional Application Serial No. 62 / 479,760, filed March 31, 2017, entitled "Cell Expansion"; U.S. Provisional Application Serial No. 62 / 479,788, filed March 31, 2017, entitled "Cell Expansion in a Bioreactor"; U.S. Provisional Application Serial No. 62 / 549,871, filed August 24, 2017, entitled "Cell Expansion"; and U.S. Provisional Application Serial No. 62 / 647,361, filed March 23, 2018, entitled "Cell Expansion". The disclosures of the foregoing applications are incorporated herein by reference in their entirety, as if all that they teach and serve for all purposes is fully set forth herein. Background Technology

[0004] Cell expansion systems (CES) are used for the expansion and differentiation of cells. CES can be used to expand (e.g., grow) various adherent and suspension cell types. Both adherent and non-adherent cell types can be grown in the bioreactor of a CES. Summary of the Invention

[0005] Embodiments of the present invention generally relate to the amplification of cells in a cell expansion system (CES). According to embodiments, such amplification can occur using a bioreactor or cell growth chamber. In one embodiment, such a bioreactor or cell growth chamber may include a hollow fiber membrane. Such a hollow fiber membrane may include an extracapillary (EC) space and an intracapillary (IC) space. The cell expansion system can amplify a variety of cell types. Embodiments may provide adherent and non-adherent cells for growth or amplification in the cell expansion system. Non-adherent or suspension cells that can be amplified in the system may include, for example, T cells or T lymphocytes. In embodiments, one or more T cell subsets or subsets may be grown. For example, embodiments may provide methods and systems for amplifying regulatory T cells (Tregs) or human regulatory T cells (hTregs).

[0006] In the embodiments, methods and systems for amplifying cells in a closed, automated cell amplification system can be provided. In one embodiment, such a cell amplification system may include a bioreactor or cell growth chamber. In a further embodiment, such a bioreactor or cell growth chamber may include a hollow fiber membrane.

[0007] For example, non-adherent cells (e.g., T cells and / or Treg cells) can be introduced into a hollow fiber bioreactor, wherein the hollow fiber bioreactor comprises multiple hollow fibers. According to one embodiment, the cells can be exposed to a stimulant or activator to stimulate or activate cell proliferation in the hollow fiber bioreactor.

[0008] Closed, automated cell expansion systems containing bioreactors or cell growth chambers may include capabilities such as nutrient and gas exchange. This capability allows cells to be seeded at reduced density compared to cell seeding densities used in culture flasks or other static culture methods. Implementations provide parameters of the cell growth environment to be manipulated to the location within the bioreactor where cells are loaded or introduced to efficiently exchange or deliver nutrients and gases to the growing cells. In implementations, cell-to-cell communication may also be facilitated. For example, in one implementation, the concentration or centralization of cells within the bioreactor can increase cell density to facilitate cell-to-cell communication, such as chemical signaling.

[0009] The implementation scheme may also provide system parameters to be managed to control cell residence in a bioreactor or cell growth chamber. For example, by controlling cell residence in a bioreactor during the cell growth phase, the system can provide efficient gas and nutrient exchange to expanding cells. In implementation schemes, the bioreactor may be designed to provide gas exchange to growing cells, and in some implementation schemes, nutrient exchange is provided. In one exemplary implementation scheme, a bioreactor containing a semi-permeable hollow fiber membrane provides gas and nutrient exchange through the semi-permeable hollow fiber membrane. The semi-permeable hollow fibers of the bioreactor allow essential nutrients (e.g., glucose and / or the culture medium formulated for cell growth) to reach the cells, while metabolic waste (e.g., lactic acid) leaves the system by diffusion across the hollow fiber walls. However, according to the implementation scheme, cells located in the manifolds or other areas outside the hollow fibers of the bioreactor may not receive adequate gas and nutrient exchange, which may lead to cell aggregation and death. Therefore, the implementation scheme relates to providing a method for maintaining a cell population (e.g., a non-adherent cell population) within the hollow fibers of the bioreactor while feeding cells. Retaining cells in a bioreactor can also promote intercellular communication, because as the number of cells in the bioreactor increases, the cell density can increase, and thus improve intercellular communication.

[0010] Additional implementations can provide system features to shear any cell communities, microcommunities, or cell clusters that may form during the expansion phase of cell growth. For example, one implementation provides shearing of cell communities (e.g., communities, microcommunities, or clusters) through a hollow fiber membrane in a bioreactor after a period of expansion. This shearing can reduce the number of cells in a microcommunity, community, or cluster, where a microcommunity, community, or cluster can be a group of one or more attached cells. Implementations can provide a protocol for shearing any community during the expansion phase of growth by circulating the suspended cell culture through, for example, an internal (IC) loop in a hollow fiber capillary (e.g., a hollow fiber with an inner diameter of 215 μm). In implementations, cell communities, microcommunities, or clusters can be sheared to reduce their size. In one implementation, cell communities, microcommunities, or clusters can be sheared to provide a homogeneous suspension of individual cells and improve cell growth / viability. Such capabilities can facilitate continuous perfusion growth of cells (e.g., T cells or Tregs).

[0011] Embodiments of the present invention further relate to using a combination of features for cell expansion to grow cells. For example, in one embodiment, cells can be seeded at a reduced seeding density compared to the cell seeding density used in culture flasks or other static culture methods. For example, in some embodiments, the volume of the fluid used for cell seeding may include about 1 × 10⁻⁶. 4 cells / mL to approximately 1×10 6 cells / mL, for example, about 10 5 Cells / mL. In other embodiments, the volume of the fluid used for cell inoculation may include less than about 1 × 10⁻⁶ cells / mL. 6 Cells / mL. According to some embodiments, cells can be loaded or introduced into a desired location within a bioreactor (e.g., a hollow fiber bioreactor) in a cell expansion system. For example, in one embodiment, cells can be exposed to an activator to activate cell expansion in the hollow fiber bioreactor. For example, according to one embodiment, any cell populations or clusters that may form during cell expansion can be sheared by circulating the cells to generate shear stress and / or shear force. For example, such shear stress and / or shear force can cause one or more cells to divide from the cell population. To provide appropriate gas and nutrient exchange to the growing cell population, further embodiments provide, for example, retaining the cell population (e.g., a suspended cell population) within the hollow fibers of the bioreactor during cell feeding. By concentrating and / or retaining cells within the hollow fibers of the bioreactor during the cell growth phase, cell density can be increased and intercellular communication can be improved. Therefore, a reduced cell seeding density can be used compared to static culture.

[0012] The implementation further provides a cell expansion system for expanding cells, wherein the system may include, for example, a hollow fiber bioreactor including an inlet port and an outlet port; a first fluid flow path having at least opposing ends, wherein a first opposing end of the first fluid flow path is fluidly associated with the inlet port of the bioreactor, and a second end of the first fluid flow path is fluidly associated with the outlet port of the bioreactor, wherein the first fluid flow path is in fluid communication with a capillary portion of the bioreactor; a fluid inlet path fluidly associated with the first fluid flow path; and a first fluid circulation path fluidly associated with the first fluid flow path and the capillary portion of the bioreactor.

[0013] The embodiments further provide a cell expansion system to include a processor for executing instructions to perform the methods described and / or illustrated herein. For example, embodiments of the invention provide for achieving such cell expansion by using one or more protocols or tasks in conjunction with the cell expansion system. Such protocols or tasks may include, for example, pre-programmed protocols or tasks. In embodiments, a user or system operator can select pre-programmed, default, or otherwise previously saved tasks to perform a specific function through the cell expansion system. In other embodiments, such protocols or tasks may include custom or user-defined protocols or tasks. For example, custom or user-defined protocols or tasks can be created via a user interface (UI) and one or more graphical user interface (GUI) elements. A task may include one or more steps.

[0014] As used herein, “at least one,” “one or more,” and “and / or” are open-ended expressions that are both connected and delimited in operation. For example, each of the expressions “at least one of A, B, and C,” “at least one of A, B, or C,” “one or more of A, B, and C,” “one or more of A, B, or C,” and “A, B, and / or C” refers to A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0015] The present invention includes a selection of concepts provided in a simplified form, which are further described in the detailed description below. The present invention is not intended to limit the scope of the claimed subject matter in any way. Features, including equivalents and variations thereof, may be included in addition to those features provided herein. Attached Figure Description

[0016] Embodiments of the invention can be described with reference to the accompanying drawings. In the drawings, the same reference numerals denote the same items. Dashed lines in the drawings may be used to indicate that one or more elements may be optional.

[0017] Figure 1A An implementation scheme for the cell expansion system (CES) is described.

[0018] Figure 1B A front view of an embodiment of the bioreactor is shown, illustrating the circulation path through the bioreactor.

[0019] Figure 1C A shaking device according to an embodiment of the invention is described for rotating or moving a cell growth chamber laterally during operation of a cell expansion system.

[0020] Figure 2 A perspective view of a cell amplification system with a pre-installed fluid delivery device according to an embodiment of the present invention is shown.

[0021] Figure 3 A perspective view of the housing of a cell expansion system according to an embodiment of the present invention is depicted.

[0022] Figure 4 A perspective view of a pre-installed fluid delivery device according to an embodiment of the present invention is shown.

[0023] Figure 5A A schematic diagram of a cell expansion system according to an embodiment of the present invention is depicted, including an operational configuration illustrating fluid motion.

[0024] Figure 5B A schematic diagram of a cell expansion system according to an embodiment of the present invention is depicted, including another operational configuration illustrating fluid motion.

[0025] Figure 5C A schematic diagram of a cell expansion system according to an embodiment of the present invention is depicted, including another operational configuration illustrating fluid motion.

[0026] Figure 6 A schematic diagram of a cell expansion system according to another embodiment of the present invention is shown.

[0027] Figure 7 A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is provided.

[0028] Figure 8A A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is shown.

[0029] Figure 8BA schematic diagram depicting a portion of a cell expansion system according to an embodiment of the present invention is shown.

[0030] Figure 9A A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is provided.

[0031] Figure 9B A graph showing oxygen consumption in a cell expansion system according to an embodiment of the present invention is shown.

[0032] Figure 10A A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is shown.

[0033] Figure 10B A table showing exemplary pump rates that can be used in cell expansion systems according to an embodiment of the present invention is illustrated.

[0034] Figure 11A A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is provided.

[0035] Figure 11B A diagram illustrating the metabolism of expanded cells according to an embodiment of the present invention is shown.

[0036] Figure 11C A diagram illustrating the metabolism of expanded cells according to an embodiment of the present invention is shown.

[0037] Figure 12 A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is shown.

[0038] Figure 13 A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is provided.

[0039] Figure 14 A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is shown.

[0040] Figure 15A A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is shown.

[0041] Figure 15B A graph showing the number of cells versus flow rate during cell expansion according to an embodiment of the present invention is shown.

[0042] Figure 16 A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is provided.

[0043] Figure 17AA flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is provided.

[0044] Figure 17B A view depicting cell expansion in a cell expansion system according to an embodiment of the present invention is shown.

[0045] Figure 17C A diagram illustrating the inner diameter of cell disassociation according to an embodiment of the present invention is shown.

[0046] Figure 18 A graph showing the cell number and flow rate versus culture days during cell expansion according to an embodiment of the present invention is shown.

[0047] Figure 19A A flowchart illustrating the operational characteristics of a process for operating a pump to amplify cells, according to an embodiment of the present invention, is shown.

[0048] Figure 19B A schematic diagram depicting a portion of a cell expansion system according to an embodiment of the present invention is shown.

[0049] Figure 19C A schematic diagram depicting a portion of a cell expansion system according to an embodiment of the present invention is shown.

[0050] Figure 19D A schematic diagram depicting a portion of a cell expansion system according to an embodiment of the present invention is shown.

[0051] Figure 20 A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is shown.

[0052] Figure 21 A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is provided.

[0053] Figure 22 A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is shown.

[0054] Figure 23 A flowchart illustrating the operational features of a process for cell amplification according to an embodiment of the present invention is provided.

[0055] Figure 24 An exemplary processing system for a cell expansion system is described, on which embodiments of the present invention can be implemented.

[0056] Figure 25 A flowchart illustrating an embodiment of the present invention is provided. Detailed Implementation

[0057] The following detailed description, with reference to the accompanying drawings, provides a discussion of exemplary embodiments. The specific embodiments included herein should not be construed as limiting or restricting the invention. Furthermore, although language specific to features, actions, and / or structures may be used to describe the embodiments herein, the claims are not limited to the described features, actions, and / or structures. Those skilled in the art will understand that other embodiments, including modifications, are also within the spirit and scope of the invention. Moreover, any substitutions or additions, including those listed as separate embodiments, may be used, or combined with any other embodiments described herein.

[0058] Embodiments of the present invention generally relate to systems and methods for amplifying cells in a cell expansion system (CES). According to embodiments, such amplification can occur using a bioreactor or cell growth chamber. In one embodiment, such a bioreactor or cell growth chamber may include a hollow fiber membrane. This hollow fiber membrane may include multiple hollow fibers and may include extracapillary (EC) and / or intracapillary (IC) spaces. Embodiments may provide adherent or non-adherent cells for growth or amplification in the cell expansion system. For example, non-adherent or suspension cells (such as T cells or T lymphocytes) may be amplified in the system. In embodiments, one or more T cell subsets or subsets may be grown. For example, embodiments may provide methods and systems for amplifying regulatory T cells (Tregs) and / or human regulatory T cells (hTregs).

[0059] In embodiments, methods and systems for amplifying cells in a closed, automated cell expansion system can be provided. In one embodiment, such a cell expansion system may include a bioreactor or cell growth chamber. In a further embodiment, such a bioreactor or cell growth chamber may include a hollow fiber membrane. The capabilities of such a system (such as nutrient and gas exchange capabilities) allow cells to be seeded at reduced cell seeding densities. Embodiments provide parameters of the cell growth environment to be manipulated to load or introduce cells into a location within the bioreactor to efficiently exchange nutrients and gases into the growing cells. For example, in one embodiment, concentration of cells within the bioreactor can increase cell density.

[0060] In embodiments, non-adherent cell populations (e.g., T cells and / or Treg cells) can be introduced or loaded into a hollow fiber bioreactor, wherein the hollow fiber bioreactor may include multiple hollow fibers. In embodiments, cells can be exposed to an activator to activate cell expansion within the hollow fiber bioreactor. In one embodiment, for example, a "centralized cell loading without circulation" task can be used to introduce multiple cells into a cell expansion system. According to an exemplary embodiment, this task can be performed on days 0 and 4-8. Other days can be used in other embodiments. In embodiments, this cell loading task can cause cells to be centered within the bioreactor to increase cell density. In other embodiments, cells can be located in other parts or regions of the bioreactor to increase cell density. In embodiments, by placing cells in a first location, such as the central region of the bioreactor, cells can receive efficient nutrient and gas exchange.

[0061] In the implementation scheme, a lower cell seeding density can be used compared to the cell seeding density used in static culture methods. In the implementation scheme using a cell expansion system, cells (e.g., Treg or Treg cells) can be increased from 2.54 × 10⁻⁶ to 10⁻⁶ cells. 5 Cells / mL up to 3.69 × 10⁻⁶ 5 Cell seeding density expansion is achieved at cells / mL. In other embodiments, the cell seeding density may be less than about 1 × 10⁻⁶. 6 Cells / mL. Alternatively, it can be obtained from 1.0 × 10⁻⁶ cells / mL. 5 Treg cell seedings are prepared at a cell seeding density of cells / mL. Other methods, such as static Treg cell culture, may use a density of 1.0 × 10⁶ cells / mL. 6 A cell seeding density of Treg cells / mL is used for in vitro expansion. In one embodiment, a lower cell seeding density can be used, for example, due to the overall efficiency of the system in delivering nutrients to the culture environment. In other embodiments, the use of one or more steps in the expansion process, combined with the overall efficiency of the system in delivering nutrients to the culture environment, can make it possible to use a lower initial cell seeding density.

[0062] In one embodiment, automated cell (e.g., Treg) expansion can be performed using a soluble activator complex. In other embodiments, for example, other types of activators, such as beads for stimulating cells, can be used. In other embodiments, cells, such as Treg cells, can be expanded without using bead-based stimulation. In one embodiment, soluble ImmunoCult from stem cell technology can be used. TMHuman CD3 / CD28 / CD2 T cell activators are used for cell expansion to activate and expand Treg cells in the presence of 200 IU / mL cytokine IL-2 using an automated cell expansion system. For example, stimulation using a soluble activator complex can be less expensive than bead-based methods. Other types of activators can be used in other embodiments. Furthermore, other types of cytokines or other growth factors can be used in other embodiments.

[0063] The implementation scheme may also provide system parameters to be adjusted or managed to control cell residence in the bioreactor or cell growth chamber. For example, by controlling cell residence in the hollow fibers of the bioreactor during the cell growth phase, the system can provide efficient gas and nutrient exchange for the expanding cells. In implementation schemes, the bioreactor may be designed to provide gas exchange to growing cells, and in some implementation schemes, to provide nutrient exchange. In one exemplary implementation, a bioreactor including a semi-permeable hollow fiber membrane can provide gas and nutrient exchange through the semi-permeable hollow fiber membrane. In implementation schemes, methods for providing culture medium components (such as various cytokines, proteins, etc.) to growing cells that cannot pass through the membrane may use fluid inflow to the bioreactor side where the cells are growing (e.g., the capillary (IC) side). However, according to the implementation scheme, even low, reduced, or diminished inflow rates, such as minimum (e.g., 0.1 mL / min), may cause cells to aggregate in the bioreactor header. Cells remaining in the bioreactor header may not be able to undergo proper gas and nutrient exchange, which may lead to cell death and aggregation.

[0064] Implementations relate to providing a method for retaining cells (e.g., non-adherent cell populations) in a bioreactor when cells are fed using an ingress (e.g., IC ingress) flow. While embodiments herein may relate to, for example, cells being on the IC side of the membrane during feeding, other embodiments may specify cells being on the EC side of the membrane, wherein, according to embodiments, cells may be contained in a first circulation path and / or a second circulation path. In embodiments, the feeding method may, for example, provide pumping a first volume of fluid (e.g., culture medium or cell growth formulation) into a first port of the bioreactor at a first volumetric flow rate, volumetric flow rate, fluid flow rate, velocity, fluid flow velocity, or volumetric velocity. For example, volumetric flow rate, volumetric flow rate, fluid flow velocity, velocity, fluid flow rate, or volumetric velocity may be used interchangeably. In some embodiments, the flow velocity may be a vector having both velocity and direction. A second volume of fluid may be pumped into a second port of the bioreactor at a second volumetric flow rate, volumetric flow rate, fluid flow rate, velocity, fluid flow velocity, or volumetric velocity. In implementations, for example, such volumetric flow rate, fluid flow rate, velocity, rate of fluid flow, or volumetric velocity can be controlled by one or more pump speeds and / or pump flow rates. Pump speed can generate, cause, or influence the volumetric flow rate or velocity of the fluid to which the pump acts. As used herein, pump speed can be described in implementations as the volumetric flow rate or fluid flow rate generated, caused, or influenced by the pump.

[0065] In one implementation, the second flow velocity of the fluid entering the bioreactor can be opposite in direction to the first flow velocity of the fluid entering the bioreactor. For example, Figure 5B and 5C An exemplary operational configuration according to an embodiment of the invention is shown, illustrating its compatibility with devices such as the CES 500 (e.g., Figure 5B and 5C The flow rate and direction of flow are used in conjunction with the cell expansion system. In an embodiment, a cell expansion system pump (e.g., an IC pump) can be used to control the residence of cells in the bioreactor. In an embodiment, for example, during the expansion phase of growth, cells may be lost from the bioreactor to the IC circulation path or IC loop. In an embodiment, for example, cells closer to the IC inlet port in the bioreactor may receive the freshest growth medium, while cells in the portion of the IC circulation path outside the bioreactor may receive essentially consumed or conditioned medium, which may affect their glycolytic metabolism. Additionally, according to an embodiment, cells in the bioreactor may receive a mixed gas (O2, CO2, N2) input from the gas transport module (GTM) via diffusion from the EC loop circulation, while cells in the portion of the IC circulation path outside the bioreactor may not receive the mixed gas.

[0066] In this embodiment, during the feeding period, cell loss from the hollow fiber membrane (HFM) bioreactor can be reduced by matching, approximately matching, or substantially matching the IC circulation pump rate with the IC inlet pump rate, but in the opposite direction. For example, in this embodiment, an IC inlet pump rate of +0.1 mL / min can be matched, approximately matching, or substantially matching a complementary IC circulation pump rate of -0.1 mL / min to maintain cells in the bioreactor during the growth phase of the cell culture (which could be days 4–7). According to this embodiment, the adjustment of the pump can counteract the forces associated with cell loss from the IC outlet port. In other embodiments, other pump rates can be used. For example, in other embodiments, the pump rates can be different. In this embodiment, other pumps or additional pumps can be used. In one embodiment, fewer pumps can be used. Further, in other embodiments, other time periods can be used.

[0067] In implementations, the metabolic activity of the cell population can influence nutrient parameters. For example, the lactate level of the cell culture can be maintained at or below a predetermined level. In one implementation, the cell culture value can, for example, be maintained at or below about 7 mmol / L. In implementations, by controlling the rate of culture medium addition using a graphical user interface (GUI) of the cell expansion system, lactate metabolic waste products from glycolysis can be maintained at or below a predetermined value during cell (regulatory T cell) expansion. In other implementations, for example, the rate of culture medium addition and / or other settings can be controlled to maintain or attempt to maintain a lactate level ≤ about 5 mmol / L, for example, to improve cell growth and viability. Other concentrations can be used in other implementations.

[0068] Additional implementations can provide system features to shear any cell communities, microcommunities, or cell clusters that may form during the amplification phase. For example, one implementation provides shearing cell communities (e.g., microcommunities) through a hollow fiber membrane in a bioreactor to reduce the number of cells in a microcommunity, community, or cluster, where a community or cluster can be a group of one or more attached cells. In implementations, a Cell Expansion System (CES) bioreactor structure can be used to shear cells (e.g., Treg cells) and microcommunities. In implementations, as cells (e.g., Treg cells) grow, they tend to form microcommunities that can limit the diffusion of nutrients to the center of the colony. This can lead to adverse effects such as necrosis during cell culture. Implementations can provide a protocol for shearing cells during the amplification phase of growth by circulating a suspended cell culture through, for example, an internal (IC) loop in a hollow fiber capillary (e.g., a hollow fiber with an inner diameter of 215 μm). In implementations, cell communities, microcommunities, or clusters can be sheared to reduce the size of cell communities, microcommunities, or clusters. In one implementation, cell communities or clusters can be sheared to provide a single-cell suspension and generate cell growth / viability. In the implementation scheme, this capability can promote continuous perfusion growth of cells (e.g., T cells or Tregs).

[0069] In this implementation, a therapeutic dose of cells (e.g., Tregs) can be expanded and harvested from a cell expansion system. In this implementation, the number of cells harvested can be approximately 1 × 10⁻⁶. 6 Cells to approximately 1×10 10 Cells, such as approximately 1 × 10 9 Cells. In one implementation, the number of cells harvested can be approximately 1 × 10⁻⁶. 8 Up to 1×10 10 One cell, one instance is approximately 7.0 × 10⁶ cells. 8 From approximately 1.4 × 10 9 Individual cells. In some embodiments, the harvested cells may have a viability between about 60% and about 100%. For example, the viability of the harvested cells may be higher than about 65%, higher than about 70%, higher than about 75%, higher than about 80%, higher than about 85%, higher than about 90%, or even higher than about 95%. In some embodiments, the harvested cells may express biomarkers consistent with Tregs. For example, in some embodiments, the cells may express CD4. + CD25 + and / or FoxP3 + Biomarkers. In this embodiment, the harvested cells may contain CD4 at a frequency between approximately 50% and approximately 100%. + CD25 +Phenotype. The harvested cells may contain CD4 at frequencies exceeding approximately 75%, 80%, 90%, or even 95%. + CD25 + Phenotype. In other embodiments, cells may include CD4 at a frequency between about 30% and about 100%. + FoxP3 + Phenotype. In some implementations, the harvested cells may include CD4 at a frequency of more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 55%, more than about 60%, more than about 65%, or even more than about 70%. + FoxP3 + Phenotype.

[0070] The embodiments pertain to the cell expansion system described above. In these embodiments, the cell expansion system is closed, comprising contents that are not directly exposed to the environment. This cell expansion system can be automated. In these embodiments, both adherent and non-adherent, or suspension, cells can be grown in the bioreactor within the cell expansion system. According to these embodiments, the cell expansion system may include a basal culture medium or other types of culture media. A method for replenishing the culture medium for cell growth in the bioreactor of the closed cell expansion system is provided. In these embodiments, the bioreactor used with such a system is a hollow fiber bioreactor. Many types of bioreactors can be used according to embodiments of the invention.

[0071] In some embodiments, the system may include a bioreactor fluidly associated with a first fluid flow path having at least opposite ends, the first opposite end of which is fluidly associated with a first port of a hollow fiber membrane, and a second end of which is fluidly associated with a second port of the hollow fiber membrane. In some embodiments, the hollow fiber membrane comprises a plurality of hollow fibers. The system may further include a fluid inlet path fluidly associated with the first fluid flow path, wherein a plurality of cells may be introduced into the first fluid flow path via the first fluid inlet path. In some embodiments, a pump for transferring intracapsular inlet fluid from an intracapsular culture medium bag to the first fluid flow path and a controller for controlling the operation of the pump are included. In some embodiments, the controller, for example, controls the pump to transfer cells from a cell inlet bag to the first fluid flow path. Another pump for circulating fluid in the first fluid circulation path may also be included, wherein the pump may further include a controller for controlling the operation of the pump. In one embodiment, the controller is, for example, a computing system including a processor. In some embodiments, one or more controllers may be configured to control one or more pumps, such as to circulate fluid at a flow rate within the first fluid circulation path. According to embodiments, multiple controllers can be used, such as a first controller, a second controller, a third controller, a fourth controller, a fifth controller, a sixth controller, etc. Furthermore, according to embodiments of the invention, multiple pumps can be used, such as a first pump, a second pump, a third pump, a fourth pump, a fifth pump, a sixth pump, etc. Additionally, although the invention can relate to culture medium bags, cell entry bags, etc., in embodiments multiple bags can be used, such as a first culture medium bag, a second culture medium bag, a third culture medium bag, a first cell entry bag, a second cell entry bag, a third cell entry bag, etc., and / or other types of containers. In other embodiments, a single culture medium bag, a single cell entry bag, etc., can be used. Furthermore, embodiments may include additional or other fluid paths, such as a second fluid flow path, a second fluid entry path, a second fluid circulation path, etc.

[0072] In one implementation, the system is controlled by, for example, the following: a processor coupled to the cell expansion system; a display device communicating with the processor and operable to display data; and memory communicating with and readable by the processor, containing a series of instructions. In another implementation, for example, when instructions are executed by the processor, the processor receives instructions to start the system. In response to the instructions to start the system, the processor may perform a series of steps to start the system, and then may, for example, receive instructions to perform IC / EC flushing. For example, in response to an instruction to load cells, the processor may perform a series of steps to load cells from a cell feed bag into, for example, a bioreactor.

[0073] According to an embodiment of the present invention, in Figure 1AA schematic diagram of an exemplary cell expansion system (CES) is depicted. CES 10 includes a first fluid circulation path 12 and a second fluid circulation path 14. According to an embodiment, the first fluid flow path 16 has at least opposing ends 18 and 20 associated with the fluid of a hollow fiber cell growth chamber 24 (also referred to herein as a "bioreactor"). Specifically, opposing end 18 may be associated with the first inlet 22 of the cell growth chamber 24, and opposing end 20 may be associated with the first outlet 28 of the cell growth chamber 24. Fluid in the first circulation path 12 flows through a hollow fiber membrane 117 disposed in the cell growth chamber 24 (see [link to original document]). Figure 1B Hollow fiber 116 (see) Figure 1B The interior of the cell growth chamber (and hollow fiber membrane will be described in more detail below). Furthermore, the first fluid flow control device 30 can be operatively connected to the first fluid flow path 16 and can control the flow of fluid in the first circulation path 12.

[0074] The second fluid circulation path 14 includes a second fluid flow path 34, a cell growth chamber 24, and a second fluid flow control device 32. According to an embodiment, the second fluid flow path 34 has at least opposing ends 36 and 38. The opposing ends 36 and 38 of the second fluid flow path 34 can be fluidly associated with the inlet port 40 and outlet port 42 of the cell growth chamber 24, respectively. The fluid flowing through the cell growth chamber 24 can interact with the hollow fiber membrane 117 (see [link to relevant documentation]) within the cell growth chamber 24. Figure 1B The external contact of the hollow fiber membrane includes a plurality of hollow fibers. The second fluid circulation path 14 can be operatively connected to the second fluid flow control device 32.

[0075] Therefore, the first and second fluid circulation paths 12 and 14 can pass through the hollow fiber membrane 117 in the cell growth chamber 24 (see Figure 1B The fluid in the first fluid circulation path 12 flows through the capillary space (“IC”) of the hollow fibers in the cell growth chamber 24. The first circulation path 12 may be referred to as the “IC loop”. The fluid in the second circulation path 14 flows through the capillary space (“EC”) of the cell growth chamber 24. The second fluid circulation path 14 may be referred to as the “EC loop”. According to the embodiment, the fluid in the first fluid circulation path 12 may flow in a co-current or counter-current manner relative to the direction of fluid flow in the second fluid circulation path 14.

[0076] Fluid inlet path 44 may be associated with the fluid in the first fluid circulation path 12. Fluid inlet path 44 allows fluid to enter the first fluid circulation path 12, while fluid outlet path 46 allows fluid to leave CES10. A third fluid flow control device 48 may be operatively associated with fluid inlet path 44. Alternatively, the third fluid flow control device 48 may be associated with the first outlet path 46.

[0077] According to the embodiments, the fluid flow control device used herein may include pumps, valves, clamps, or combinations thereof. Multiple pumps, valves, and clamps may be arranged in any combination. In various embodiments, the fluid flow control device is a peristaltic pump or includes a peristaltic pump. In the embodiments, the fluid circulation path, inlet port, and outlet port may be constructed of piping made of any material.

[0078] Various components are referred to herein as “operably associated.” As used herein, “operably associated” means components that are operably linked together, including embodiments in which components are directly associated, and embodiments in which an additional component is placed between two associated components. “Operably associated” components can be “fluid associated.” “Fluid associated” means components linked together so that fluid can be transferred between them. “Fluid associated” encompasses embodiments in which an additional component is placed between two fluid associated components and directly connected components. Fluid associated components may include components that do not contact the fluid but contact other components to manipulate the system (e.g., a peristaltic pump that pumps fluid through a flexible tube outside a compression tube).

[0079] Generally, any type of fluid (including buffers, protein-containing fluids, and cell-containing fluids) can flow through various circulation, inflow, and outflow pathways. As used herein, “fluid,” “culture medium,” and “fluid culture medium” are used interchangeably.

[0080] Turning Figure 1B An example of a hollow fiber cell growth chamber 100 that can be used with the present invention is shown in a front view. The cell growth chamber 100 has a longitudinal axis LA-LA and includes a cell growth chamber housing 104. In at least one embodiment, the cell growth chamber housing 104 includes four openings or ports: an IC inlet port 108, an IC outlet port 120, an EC inlet port 128, and an EC outlet port 132.

[0081] According to an embodiment of the invention, fluid in the first circulation path enters the cell growth chamber 100 through the IC inlet port 108 at the first longitudinal end 112 of the cell growth chamber 100, enters and passes through the capillary interior side of a plurality of hollow fibers 116 containing a hollow fiber membrane 117 (referred to in various embodiments as the capillary interior (“IC”) side of the hollow fiber membrane or the “IC space”), and exits the cell growth chamber 100 through the IC outlet port 120 located at the second longitudinal end 124 of the cell growth chamber 100. The fluid path between the IC inlet port 108 and the IC outlet port 120 defines the IC portion 126 of the cell growth chamber 100. Fluid in the second circulation path flows into the cell growth chamber 100 through the EC inlet port 128, contacts the capillary exterior or exterior of the hollow fibers 116 (referred to as the “EC side” or “EC space” of the membrane), and exits the cell growth chamber 100 via the EC outlet port 132. The fluid path between the EC inlet port 128 and the EC outlet port 132 includes the EC portion 136 of the cell growth chamber 100. Fluid entering the cell growth chamber 100 via the EC inlet port 128 can come into contact with the outside of the hollow fiber 116. Small molecules (e.g., ions, water, oxygen, lactic acid, etc.) may diffuse through the hollow fiber 116 from the internal space or IC space of the hollow fiber to the outside or EC space, or from the EC space to the IC space. Large molecules (such as growth factors) may typically be too large to pass through the hollow fiber membrane and may remain in the IC space of the hollow fiber 116. In an embodiment, the culture medium can be replaced as needed. The culture medium can also be circulated via an oxygenator or gas transport module to exchange gases as needed. According to an embodiment, cells may be contained within a first circulation path and / or a second circulation path as described below, and may be on either the IC side and / or the EC side of the membrane.

[0082] The material used to manufacture the hollow fiber membrane 117 can be any biocompatible polymer material capable of being made into hollow fibers. According to an embodiment of the invention, one usable material is a synthetic polysulfone-based material.

[0083] In the implementation plan, CES (such as CES 500 (see...) Figure 5A , 5B and 5C) and / or CES 600 (see 5C) and / or CES 600 (see 5C) Figure 6 It may include a device configured to move or “shake” the cell growth chamber, which moves or “shakes” relative to other components of the cell expansion system by attaching the cell growth chamber to a rotating and / or lateral shaking device. Figure 1C An example of such an apparatus is shown, according to one embodiment, wherein the bioreactor 100 is rotatably connected to two rotating rocking components and a lateral rocking component.

[0084] The first rotating agitator 138 causes the bioreactor 100 to rotate about its central axis 142. The rotating agitator 138 can be rotationally associated with the bioreactor 100. In one embodiment, the bioreactor 100 can rotate continuously in a single direction about the central axis 142, either clockwise or counterclockwise. Alternatively, according to another embodiment, the bioreactor 100 can rotate alternately, for example, first clockwise and then counterclockwise about the central axis 142.

[0085] The CES may also include a second rotating oscillating component that rotates the bioreactor 100 about a rotation axis 144. The rotation axis 144 may pass through the center point of the bioreactor 100 and may be perpendicular to the central axis 142. In embodiments, the bioreactor 100 may rotate continuously in a single direction about the rotation axis 144 in a clockwise or counterclockwise direction. Alternatively, the bioreactor 100 may rotate alternately about the rotation axis 144, for example, rotating clockwise first and then counterclockwise about the rotation axis 144. In various embodiments, the bioreactor 100 may also rotate about the rotation axis 144 and be oriented horizontally or vertically relative to gravity.

[0086] In one embodiment, the lateral rocking component 140 may be laterally associated with the bioreactor 100. In another embodiment, the plane of the lateral rocking component 140 moves laterally in the -x and -y directions. According to one embodiment, cell sedimentation in the bioreactor can be reduced by moving the cell-containing culture medium within the hollow fibers.

[0087] The rotation and / or lateral movement of the shaking device can reduce cell sedimentation within the device and decrease the likelihood of cells being trapped within a portion of the bioreactor. According to Stokes' Law, the sedimentation rate of cells in a cell growth chamber is directly proportional to the density difference between the cells and the suspension medium. In some embodiments, repeated 180-degree rotations (rapidly) with pauses (e.g., a total combined time of 30 seconds) as described above keep non-adherent red blood cells, for example, in suspension. In one embodiment, a minimum rotation of about 180 degrees is preferred; however, rotations of up to 360 degrees or greater can be used. Different shaking components can be used individually or in any combination. For example, a shaking component that rotates bioreactor 100 about a central axis 142 can be combined with a shaking component that rotates bioreactor 100 about axis 144. Similarly, clockwise and counterclockwise rotations about different axes can be performed independently in any combination.

[0088] Go to Figure 2 According to some embodiments of the present invention, an embodiment of a cell amplification system 200 having a pre-installed fluid transport assembly is shown. The CES 200 includes a cell amplification unit 202, which includes a hatch or closable door 204 for engaging with a rear portion 206 of the cell amplification unit 202. An internal space 208 within the cell amplification unit 202 includes features adapted to receive and engage a pre-installed fluid transport assembly 210. The pre-installed fluid transport assembly 210 is detachably attached to the cell amplification unit 202 to facilitate relatively rapid replacement of a pre-installed fluid transport assembly 210 used at the same cell amplification unit 202 with a new or unused pre-installed fluid transport assembly 210. The single-cell amplification machine 202 can be operated to grow or amplify a first cell group using a first pre-installed fluid transport assembly 210, and then a second cell group can be grown or amplified using a second pre-installed fluid transport assembly 210 without the need for sterilization between replacements of the first and second pre-installed fluid transport assemblies 210. The pre-installed fluid transport assembly 210 includes a bioreactor 100 and an oxygenator or gas transport module 212 (see also...). Figure 4 According to some implementations, the pipeline guide channel is shown as 214, for receiving various culture media that are piped to the pre-installed fluid transport assembly 210.

[0089] Next, Figure 3 Some embodiments of the invention are shown, in which a pre-installed fluid transport assembly 210 is detachably attached. Figure 2 The rear part 206 of the previous cell expansion machine 202. Figure 3 The closable door 204 is omitted from the text. Figure 2(As shown in the diagram). The rear portion 206 of the cell expansion machine 202 includes several different structures for cooperating with elements of the pre-installed fluid transport assembly 210. More specifically, the rear portion 206 of the cell expansion machine 202 includes multiple peristaltic pumps for cooperating with pump circuits on the pre-installed fluid transport assembly 210, including an IC circulation pump 218, an EC circulation pump 220, an IC inlet pump 222, and an EC inlet pump 224. Furthermore, the rear portion 206 of the cell expansion machine 202 includes multiple valves, including an IC circulation valve 226, a reagent valve 228, an IC culture medium valve 230, an air removal valve 232, a cell inlet valve 234, a washing valve 236, a dispensing valve 238, an EC culture medium valve 240, an IC waste liquid valve or discharge valve 242, an EC waste liquid valve 244, and a harvest valve 246. Several sensors are also associated with the rear 206 of the cell expansion machine 202, including an IC outlet pressure sensor 248, an IC inlet pressure and temperature combination sensor 250, an EC inlet pressure and temperature combination sensor 252, and an EC outlet pressure sensor 254. According to one embodiment, an optical sensor 256 for the air removal chamber is also shown.

[0090] According to some embodiments, a shaft or rocker controller 258 for a rotary bioreactor 100 is shown. A shaft fitting 260 associated with the shaft or rocker controller 258 allows for proper alignment of the shaft through a bore, see, for example, a pipe organizer 424. Figure 4 See, for example, 300 with pre-installed transport component 210. Figure 4 ) or together with the rear 206 of the cell expansion machine 202, 400. The rotation of the shaft or rocker controller 258 gives rotational movement to the shaft fitting 260 and the bioreactor 100. Thus, when the operator or user of the CES 200 attaches a new or unused pre-installed fluid transport assembly 400 ( Figure 4 When the cell expander reaches 202, alignment is relatively simple, as it is properly aligned through orifice 424. Figure 4 The shaft of the pre-installed fluid transport assembly 210 or 400 is equipped with shaft fitting 260.

[0091] Go to Figure 4 This shows a perspective view of a pre-installed fluid transport assembly 400 that is detachably attached. The pre-installed fluid transport assembly 400 can be detachably attached to the cell amplification machine 202. Figure 2 and Figure 3 This facilitates the relatively rapid replacement of pre-installed fluid transport assemblies 400 used at the same cell expansion machine 202 with new or unused pre-installed fluid transport assemblies 400. Figure 4As shown, bioreactor 100 can be attached to a bioreactor coupling including shaft fitting 402. Shaft fitting 402 includes one or more shaft fastening mechanisms, such as shafts for engaging cell expansion machine 202 (e.g., 258). Figure 3 The bias arm or spring member 404 is shown.

[0092] According to some implementation schemes, the pre-installed fluid transport assembly 400 includes pipes 408A, 408B, 408C, 408D, 408E, etc., and various pipe fittings to provide the following... Figure 5A , 5B The fluid paths are shown in 5C and 6. Pump circuits 406A, 406B and 406C are also provided for the pump. In some embodiments, although various culture media can be provided at the location of the cell expansion machine 202, according to some embodiments, the pre-installed fluid transport assembly 400 may include sufficient tubing length to extend outside the cell expansion machine 202 and be weldable to tubing associated with culture medium bags or containers.

[0093] Next, Figure 5A , 5B Figures 5C and 5C illustrate a schematic diagram of an embodiment of the cell expansion system 500, and... Figure 6 A schematic diagram of another embodiment of the cell expansion system 600 is shown. (See diagram below.) Figure 5A , 5B As shown in embodiments 5C and 6, and as described below, cells are grown in the IC space. However, the invention is not limited to these examples, and in other embodiments, cells may be grown in the EC space.

[0094] As pointed out, Figure 5A , 5B The 5C model showcases the CES 500. And... Figure 5A , 5B The 5C depicts structural components that are basically similar to those of CES 500. Figure 5A , 5BFigures 5C and 5C illustrate a possible operational configuration of the structural features of the CES 500 using an embodiment of the invention, showing fluid movement in a first fluid circulation path. As shown, the CES 500 includes a first fluid circulation path 502 (also referred to as a “capillary inner loop” or “IC loop”) and a second fluid circulation path 504 (also referred to as a “capillary outer loop” or “EC loop”). A first fluid flow path 506 may be associated with the fluid in the cell growth chamber 501 to form the first fluid circulation path 502. Fluid flows into the cell growth chamber 501 through an IC inlet port 501A, through hollow fibers in the cell growth chamber 501, and exits via an IC outlet port 501B. A pressure gauge 510 measures the pressure of the culture medium exiting the cell growth chamber 501. The culture medium flows through an IC circulation pump 512, which can be used to control the flow rate of the culture medium. The IC circulation pump 512 may pump fluid in a first direction or a second direction opposite to the first direction. The outlet port 501B may be used as a reverse inlet. For example, in a first configuration, the IC circulation pump can pump fluid in a positive direction, where the fluid enters the IC inlet port 501A. For example, in a second configuration, the IC circulation pump can pump fluid in a negative direction, where the fluid enters the IC outlet port 501B.

[0095] The culture medium entering the IC circuit can be introduced through valve 514. As those skilled in the art will understand, additional valves, pressure / temperature sensors, ports, and / or other devices can be placed at various locations to isolate and / or measure the characteristics of the culture medium at multiple points along the fluid path. Therefore, it should be understood that the schematic diagram shown represents one possible configuration of various components of the CES 500, and modifications to the schematic diagram shown are within the scope of one or more existing embodiments.

[0096] Regarding IC circuit 502, a culture medium sample can be obtained during operation from sample port 516 or sample coil 518. A pressure / thermometer 520 located in the first fluid circulation path 502 allows for the monitoring of culture medium pressure and temperature during operation. The culture medium is then returned to IC inlet port 501A to complete fluid circulation path 502. Cells grown / expanded in cell growth chamber 501 can be flushed from cell growth chamber 501 into harvest bag 599 via valve 598, or redistributed within hollow fibers for further growth.

[0097] The fluid in the second fluid circulation path 504 enters the cell growth chamber 501 through the EC inlet port 501C and exits the cell growth chamber 501 through the EC outlet port 501D. The culture medium in the EC loop 504 can contact the outside of the hollow fibers in the cell growth chamber 501, thereby allowing small molecules to diffuse in and out of the hollow fibers.

[0098] According to one embodiment, a pressure / thermometer 524 disposed in the second fluid circulation path 504 allows for the measurement of the pressure and temperature of the culture medium before it enters the EC space of the cell growth chamber 501. A pressure gauge 526 allows for the measurement of the pressure of the culture medium in the second fluid circulation path 504 after it leaves the cell growth chamber 501. Regarding the EC loop, a sample of the culture medium can be obtained during operation from the sample port 530 or the sample coil.

[0099] In some embodiments, after exiting the EC outlet port 501D of the cell growth chamber 501, the fluid in the second fluid circulation path 504 reaches the oxygenator or gas transfer module 532 via the EC circulation pump 528. The EC circulation pump 528 may also pump fluid in the opposite direction. The second fluid flow path 522 may be fluidly associated with the oxygenator or gas transfer module 532 via the oxygenator inlet port 534 and the oxygenator outlet port 536. In operation, fluid culture medium flows into the oxygenator or gas transfer module 532 via the oxygenator inlet port 534 and exits the oxygenator or gas transfer module 532 via the oxygenator outlet port 536. For example, the oxygenator or gas transfer module 532 adds oxygen to the culture medium in the CES 500 and removes air bubbles therefrom. In various embodiments, the culture medium in the second fluid circulation path 504 may be in equilibrium with the gas entering the oxygenator or gas transfer module 532. The oxygenator or gas transfer module 532 may be any suitably sized oxygenator or gas transfer device. Air or gas flows into the oxygenator or gas transfer module 532 via filter 538 and exits the oxygenator or gas transfer module 532 via filter 540. Filters 538 and 540 reduce or prevent contamination of the oxygenator or gas transfer module 532 and the associated culture medium. During the priming sequence, air or gas purified from the CES 500 may be released into the atmosphere via the oxygenator or gas transfer module 532.

[0100] According to at least one embodiment, cell-containing culture medium (from bag 562) and fluid culture medium from bag 546 can be introduced into first fluid circulation path 502 via first fluid flow path 506. Fluid container 562 (e.g., for introducing cells into bags or saline perfusion solution from an air extraction system) can be fluidly associated with first fluid flow path 506 and first fluid circulation path 502 via valve 564.

[0101] Fluid containers or culture medium bags, 544 (e.g., reagents) and 546 (e.g., IC culture media), may be fluid-associated with a first fluid inlet path 542 via valves 548 and 550, or with a second fluid inlet path 574 via valves 570 and 576, respectively. First and second sterile, sealable inlet perfusion paths 508 and 509 are also provided. An air removal chamber (ARC) 556 may be fluid-associated with a first circulation path 502. The air removal chamber 556 may include one or more ultrasonic sensors at certain measurement locations within the air removal chamber 556, the ultrasonic sensors comprising upper and lower sensors to detect air, fluid absence, and / or gas / fluid interfaces (e.g., air / fluid interfaces). For example, ultrasonic sensors may be used near the bottom and / or top of the air removal chamber 556 to detect air, fluid, and / or air / fluid interfaces at these locations. Embodiments provide for the use of many other types of sensors without departing from the spirit and scope of the invention. For example, optical sensors may be used according to embodiments of the invention. During a priming event or other part of the process, air or gas purified from CES 500 may be discharged to the atmosphere outside air valve 560 via line 558, which may be fluidly associated with air removal chamber 556.

[0102] EC culture medium (e.g., from bag 568) or washing solution (e.g., from bag 566) can be added to the first or second fluid flow path. Fluid container 566 can be fluid-associated with valve 570, which can be fluid-associated with the first fluid circulation path 502 via dispensing valve 572 and the first fluid inlet path 542. Alternatively, by opening valve 570 and closing dispensing valve 572, fluid container 566 can be fluid-associated with the second fluid inlet path 574 and the EC inlet path 584. Similarly, fluid container 568 can be fluid-associated with valve 576, which can be fluid-associated with the first fluid circulation path 502 via the first fluid inlet path 542 and dispensing valve 572. Alternatively, by opening valve 576 and closing dispensing valve 572, fluid container 568 can be fluid-associated with the second fluid inlet path 574.

[0103] An optional heat exchanger 552 can be provided for the introduction of culture medium reagents or rinsing solutions.

[0104] In the IC circuit, fluid can initially enter pump 554 via the IC. In the EC circuit, fluid can initially enter pump 578 via the EC. An air detector 580, such as an ultrasonic sensor, can also be associated with the EC entry path 584.

[0105] In at least one embodiment, first and second fluid circulation paths 502 and 504 are connected to waste line 588. When valve 590 is open, IC culture medium can flow through waste line 588 and into waste bag or discharge bag 586. Similarly, when valve 582 is open, EC culture medium can flow through waste line 588 into waste bag or discharge bag 586.

[0106] In some implementations, cells can be harvested via cell harvesting path 596. Here, cells from cell growth chamber 501 can be harvested by pumping IC culture medium containing cells through cell harvesting path 596 and valve 598 to cell harvesting bag 599.

[0107] Various components of the CES 500 can be contained or housed within the machine or housing (such as the Cell Proliferator 202). Figure 2 and 3 For example, the machine maintains cells and culture medium at a predetermined temperature.

[0108] In one implementation scheme, in response to Figure 5A In the configuration depicted in the CES 500, fluid culture media in the first fluid circulation path 502 and the second fluid circulation path 504 flow in the same direction (co-current configuration) through the cell growth chamber 501. In another embodiment, the CES 500 can also be configured to flow in a counter-current configuration (not shown). Figure 5A In the configuration shown, fluid in the first fluid circulation path 502 enters the bioreactor 501 at the IC inlet port 501A and exits the bioreactor 501 at the IC outlet port 501B. According to the embodiment, in Figure 5B and 5C In the illustrated configuration, the fluid culture medium in the first circulation path 502 can flow from the connector 517 in opposite or opposing directions, allowing fluid to enter the IC inlet port (first port 501A at one end of the bioreactor) and fluid to enter the IC outlet port (second port 501B at the opposite end of the bioreactor) to retain cells within the bioreactor itself. The first fluid flow path can be fluid-associated with the first fluid circulation path via the connector 517. In embodiments, the connector 517 can be, for example, a point or location where fluid flows in opposite directions based on the IC inlet pump and the IC circulation pump. In one embodiment, the connector 517 can be a T-fitting or T-joint. In another embodiment, the connector 517 can be a Y-fitting or Y-joint. The connector 517 can be any type of fitting, connector, fusion piece, passage, pipe, etc., thereby enabling the first fluid flow path to be fluid-associated with the first circulation path. It should be understood that... Figure 5A , Figure 5B and Figure 5C The schematic diagrams and operational configurations shown illustrate possible configurations of various components of the cell expansion system, and modifications to the schematic diagrams and operational configurations shown are within the scope of one or more of these embodiments.

[0109] Go to Figure 6 The diagram illustrates another embodiment of the cell expansion system 600. The CES 600 includes a first fluid circulation path 602 (also referred to as a “capillary inner loop” or “IC loop”) and a second fluid circulation path 604 (also referred to as a “capillary outer loop” or “EC loop”). The first fluid flow path 606 may be associated with the fluid in the cell growth chamber 601 to form the first fluid circulation path 602. Fluid flows into the cell growth chamber 601 through the IC inlet port 601A, through hollow fibers in the cell growth chamber 601, and exits via the IC outlet port 601B. A pressure sensor 610 measures the pressure of the culture medium leaving the cell growth chamber 601. In addition to pressure, in some embodiments, the sensor 610 may also be a temperature sensor that detects the pressure and temperature of the culture medium during operation. The culture medium flows through an IC circulation pump 612, which can be used to control the flow rate of the culture medium. The IC circulation pump 612 may pump fluid in a first direction or a second direction opposite to the first direction. The outlet port 601B may be used as a reverse inlet. Culture medium entering the IC loop may be introduced through a valve 614. As those skilled in the art will understand, additional valves, pressure gauges, pressure / temperature sensors, ports, and / or other devices may be placed at various locations to isolate and / or measure the characteristics of the culture medium at different points along the fluid path. Therefore, it should be understood that the schematic diagrams shown represent one possible configuration of the various components of the CES 600, and modifications to the schematic diagrams shown are within the scope of one or more of these embodiments.

[0110] Regarding IC circulation, a culture medium sample can be obtained from the sample coil 618 during operation. The culture medium is then returned to the IC inlet port 601A to complete the fluid circulation path 602. Cells growing / expanding in the cell growth chamber 601 can be flushed out of the cell growth chamber 601 into the harvest bag 699 via valve 698 and tubing 697. Alternatively, when valve 698 is closed, the cells can be redistributed within the growth chamber 601 for further growth.

[0111] Fluid in the second fluid circulation path 604 enters the cell growth chamber 601 via the EC inlet port 601C and exits the cell growth chamber 601 via the EC outlet port 601D. According to the embodiment, the culture medium in the EC loop can contact the outside of the hollow fibers in the cell growth chamber 601, thereby allowing small molecules to diffuse into and out of the hollow fibers within the chamber 601.

[0112] Pressure / temperature sensor 624, located in the second fluid circulation path 604, allows measurement of the pressure and temperature of the culture medium before it enters the EC space of the cell growth chamber 601. Sensor 626 allows measurement of the pressure and / or temperature of the culture medium in the second fluid circulation path 604 after it leaves the cell growth chamber 601. Regarding the EC loop, a sample of the culture medium can be obtained during operation from the sample port 630 or the sample coil.

[0113] After exiting the EC outlet port 601D of the cell growth chamber 601, the fluid in the second fluid circulation path 604 reaches the oxygenator or gas transfer module 632 via the EC circulation pump 628. According to some embodiments, the EC circulation pump 628 may also pump fluid in the opposite direction. The second fluid flow path 622 may be fluid-dependent on the oxygenator or gas transfer module 632 via inlet port 632A and outlet port 632B. In operation, fluid culture medium flows into the oxygenator or gas transfer module 632 via inlet port 632A and exits via outlet port 632B. For example, the oxygenator or gas transfer module 632 adds oxygen to the culture medium in the CES 600 and removes air bubbles therefrom. In various embodiments, the culture medium in the second fluid circulation path 604 may be balanced with the gas entering the oxygenator or gas transfer module 632. The oxygenator or gas transfer module 632 may be a device of any suitable size for oxygenation or gas transfer. Air or gas flows into the oxygenator or gas transfer module 632 via filter 638 and exits the oxygenator or gas transfer module 632 via filter 640. Filters 638 and 640 reduce or prevent contamination of the oxygenator or gas transfer module 632 and the associated culture medium. During a portion of the perfusion event, air or gas purified from the CES 600 may be released into the atmosphere via the oxygenator or gas transfer module 632.

[0114] In the configuration depicted for the CES 600, fluid culture media in the first fluid circulation path 602 and the second fluid circulation path 604 flow through the cell growth chamber 601 in the same direction (co-current configuration). According to an embodiment, the CES 600 can also be configured to construct flow in a convective manner.

[0115] According to at least one embodiment, culture medium including cells (from a source such as a cell container (e.g., a bag) may be attached to attachment point 662, and fluid culture medium from a culture medium source may be attached to attachment point 646. Cells and culture medium may be introduced into a first fluid circulation path 602 via a first fluid flow path 606. Attachment point 662 may be fluidly associated with the first fluid flow path 606 via valve 664, and attachment point 646 may be fluidly associated with the first fluid flow path 606 via valve 650. A reagent source may be fluidly connected to point 644 and fluidly associated with fluid inlet path 642 via valve 648, or fluidly associated with a second fluid inlet path 674 via valves 648 and 672.

[0116] Air removal chamber (ARC) 656 may be fluid-associated with the first circulation path 602. Air removal chamber 656 may include one or more ultrasonic sensors, including upper and lower sensors, to detect air, fluid deficiency, and / or gas / fluid interface (e.g., air / fluid interface) at certain measurement locations within air removal chamber 656. For example, ultrasonic sensors may be used near the bottom and / or top of air removal chamber 656 to detect air, fluid, and / or air / fluid interface at these locations. Embodiments provide for the use of many other types of sensors without departing from the spirit and scope of the invention. For example, optical sensors may be used according to embodiments of the invention. Air or gas purified from CES 600 during a perfusion event or part of another event may be discharged to the atmosphere outside air valve 660 via line 658, which may be fluid-associated with air removal chamber 656.

[0117] An EC culture medium source can be attached to EC culture medium attachment point 668, and a washing solution source can be attached to washing solution attachment point 666 to add EC culture medium and / or washing solution to a first or second fluid flow path. Attachment point 666 can be fluidly associated with valve 670, which can be fluidly associated with a first fluid circulation path 602 via valve 672 and a first fluid inlet path 642. Alternatively, by opening valve 670 and closing valve 672, attachment point 666 can be fluidly associated with a second fluid circulation path 604 via a second fluid inlet path 674 and a second fluid flow path 684. Similarly, attachment point 668 can be fluidly associated with valve 676, which can be fluidly associated with a first fluid circulation path 602 via a first fluid inlet path 642 and valve 672. Alternatively, by opening valve 676 and closing dispensing valve 672, fluid container 668 can be fluidly associated with a second fluid inlet path 674.

[0118] In the IC circuit, fluid can initially enter pump 654 via the IC. In the EC circuit, fluid can initially enter pump 678 via the EC. An air detector 680, such as an ultrasonic sensor, can also be associated with the EC entry path 684.

[0119] In at least one embodiment, first and second fluid circulation paths 602 and 604 are connected to waste line 688. When valve 690 is open, IC culture medium can flow through waste line 688 to waste bag or discharge bag 686. Similarly, when valve 692 is open, EC culture medium can flow to waste bag or discharge bag 686.

[0120] After the cells have grown in the cell growth chamber 601, they can be harvested via the cell harvesting path 697. Here, with valve 698 open, cells from the cell growth chamber 601 can be harvested by pumping IC medium containing the cells through the cell harvesting path 697 to the cell harvesting bag 699.

[0121] Various components of the CES 600 can be contained or housed within the machine or housing (such as the Cell Amplifier 202). Figure 2 and 3 In this system, for example, the machine maintains cells and culture medium at a predetermined temperature. It should be further noted that in some embodiments, components of the CES 600 and CES 500 can be combined. In other embodiments, the CES may include fewer or additional components than those shown in the CES 500 and CES 600, and still remain within the scope of the invention. Examples of cell expansion systems incorporating features of the present invention are... Cell expansion system, The cell expansion system was manufactured by Terumo BCT in Lakewood, Colorado.

[0122] It should be understood that, Figure 6 The schematic diagrams shown illustrate possible configurations of various components of the cell expansion system, and modifications to the schematic diagrams shown fall within the scope of one or more of these embodiments.

[0123] Examples and further descriptions of cell expansion systems are provided in U.S. Patent No. 8,309,347 (“Cell Expansion System and Method of Use,” published November 13, 2012) and U.S. Patent No. 9,057,045 (“Method of Loading and Distributing Cells in a Bioreactor of a Cell Expansion System,” filed December 15, 2010), which are incorporated herein by reference in their entirety for all their teachings and purposes.

[0124] Although various exemplary embodiments of cell expansion systems and related methods have been described, according to embodiments of the present invention, Figure 7 Exemplary operating steps 700 are shown for a process of amplifying non-adherent or suspension cells in a cell amplification system such as CES 500 or CES 600.

[0125] The process begins at 702, and 700 proceeds to cell preparation 704. In some embodiments, cell preparation 704 can involve many different and optional steps. For example, cells may be collected at 706. Cell collection 706 may involve separating and collecting cells from a donor. In some embodiments, a blood apheresis (e.g., leukocyte apheresis) procedure may be performed to collect a certain amount of lymphocytes from the donor's peripheral blood. The volume of lymphocytes may include the target cell population to be expanded by process 700. In other embodiments, cells may be collected from umbilical cord blood.

[0126] After collection 706, optionally, cells can be separated 708 as part of preparation 704. The cell volume collected in step 706 can include many different cell types, including cells that serve as amplification targets. Optional step 708 can be performed to separate target cells. As an example, target cells can be T cells, such as regulatory T cells. In one embodiment, regulatory T cells can be CD4+CD25+ T cells. Any suitable separation technique can be used to separate cells. For example, cells can be separated using immunomagnetic separation, where antibody-functionalized magnetic beads are contacted with the cells collected in 706. The functionalized magnetic beads can preferentially attach to the target cell population. A magnetic field can then be used to bind the beads to the attached target cell population, while other cells can be removed.

[0127] Cells can optionally be resuspended 710 after separation 708. In one embodiment, cells can be resuspended in a culture medium containing a variety of nutrients and / or reagents that help maintain cell viability. In one embodiment, the culture medium may include at least serum albumin and reagents such as cytokines. In one embodiment, the cytokine may be recombinant human IL-2 cytokine. In one embodiment, the culture medium may contain cytokines at a concentration of 200 IU / ml.

[0128] Following cell preparation 704, process 700 proceeds to cell exposure 712 to activate cells for expansion. Cells may optionally be exposed to a soluble activator 714. An activator, possibly comprising an antibody complex, may be added to the culture medium in which the cells are resuspended. In embodiments, the activator may be a human antibody CD3 / CD28 / CD2 cell activator complex. In some embodiments, the activator may be included in the culture medium used for cell resuspension 710. Optionally, cells may be exposed to beads 716, the surface of which may contain an activator. In embodiments, exposing cells to beads may include adding a predetermined amount of beads to the resuspended cells. Beads may be added in different ratios relative to the number of cells. For example, beads may be added at a ratio of 1 bead: 2 cells. Other embodiments may provide beads added in different ratios, such as 1 bead: 1 cell, 1 bead: 3 cells, etc. These beads may have antibodies on their surface to activate cell expansion. In embodiments, the beads may contain antibodies CD3 / CD28 on their surface.

[0129] Process 700 proceeds to cell expansion 718. As part of cell expansion 718, cells may be loaded into a cell growth chamber for cell expansion, such as a hollow fiber membrane bioreactor. Nutrients may be supplied to the cells 720 to promote their expansion. For example, culture medium may be delivered to the cell growth chamber to provide nutrients for expansion. Cell expansion 718 may also include periodically adding reagents to the cell growth chamber to continue promoting its expansion. For example, in some embodiments, reagents (e.g., cytokines) may be added to the cell growth chamber to promote cell expansion. In one embodiment, the reagent may be an additional IL-2 cytokine, such as recombinant human IL-2 cytokine.

[0130] Also as part of cell expansion 718, the environment within the cell growth chamber can be controlled. For example, gases can be continuously supplied and exchanged to provide a balance of, for example, carbon dioxide and oxygen to the cells expanding in the cell growth chamber. Additionally, the temperature can be controlled within a range optimized for cell expansion. Cell expansion 718 may also include monitoring metabolites 726. For example, lactate and glucose levels can be monitored periodically. Increases or decreases in metabolites can induce changes (e.g., additional nutrient supply, additional reagent addition, additional gas exchange, etc.) to control the environment 724 within the cell growth chamber.

[0131] Next, process 700 proceeds to cell harvesting 728. Further processing or other analysis of the removed cells may optionally be performed in step 730. For example, cells may be characterized to determine cell phenotype. Further processing or other analysis 730 may include, for example, performing flow cytometry to characterize the cell phenotype. Process 700 may then terminate at end operation 732. If further processing / analysis is not desired, process 700 terminates at end operation 732.

[0132] Figure 8A The diagram illustrates operational steps 800 of a process according to an embodiment of the invention, which can be used to place cells or other materials (e.g., proteins, nutrients, growth factors) in a cell growth chamber. In an embodiment, process 800 can be implemented as part of a task of "centrally loading cells without circulation." A start operation 802 is initiated, and process 800 proceeds to step 804, where a first volume of fluid containing cells can be loaded into the cell growth chamber of a cell expansion system. In an embodiment, the cells can comprise non-adherent cells, such as one or more types of T cells. In one embodiment, multiple cells comprise Tregs. It is understood that the cell expansion system described above (such as system CES 500, e.g., Figure 5A ) and CES 600 ( Figure 6 The components are used to load fluid into the first volume of cells. Figure 8B A portion of a cell expansion system is shown, comprising a first inlet pump 840, a first fluid flow path 860, a first fluid circulation pump 848, a first fluid circulation path 852, a cell growth chamber 844, and a second fluid circulation path 854. The first fluid flow path 860 is fluidly associated with the fluid circulation path 852 via a connection 860B. An embodiment may provide that a first volume of fluid 804 containing cells is loaded via the first fluid inlet pump 840 through the first fluid flow path 860 and enters the first fluid circulation path 852. In this embodiment, the first volume of fluid is loaded without activating the first fluid circulation pump 848.

[0133] like Figure 8B As shown, the volume of the first fluid circulation path 852 may include multiple volumes, portions thereof. For example, a first portion of this volume may be the capillary space within the cell growth chamber 844 (when the cell growth chamber is a hollow fiber membrane bioreactor). A second portion of this volume may extend from the connection 860B to the inlet port 844A of the cell growth chamber 844. A third portion may extend from the connection 860B to the outlet port 844A of the cell growth chamber 844.

[0134] Process 800 proceeds to the loading of a second volume of fluid 806. The second volume of fluid may include culture medium and may be introduced into a portion of the first fluid flow path 860. In one embodiment, a predetermined amount of the second volume may be selected to place the first volume 808 into a first portion of the cell growth chamber 844. In one embodiment, the first volume and the second volume of fluid may be the same. In other embodiments, the first volume and the second volume of fluid may be different. In other embodiments, the sum of the first volume and the second volume of fluid may be equal to a percentage of the first fluid circulation path (e.g., path 852). Figure 8B The volume of )).

[0135] In order to accommodate the first volume of fluid, the second volume of fluid must be sufficient to push the first volume into the desired location within the cell growth chamber 844. Therefore, in this embodiment, the second volume of fluid can be approximately the same size as the volume of the first fluid circulation path 852 between the connection 860B and the inlet port 844A. This will, understandably, push the first volume of fluid containing cells into the desired location within the cell growth chamber 844.

[0136] In other embodiments, the first volume of fluid (containing cells) may be placed 808 near the central region 866 of the cell growth chamber 844. In these embodiments, the second volume may be the same as the sum of the volumes of the first fluid circulation path 852 between the connector 860B and the inlet port 844A, and the volume of the first fluid circulation path (e.g., the volume of the space within the capillary) constituted by the cell growth chamber 844 is not occupied by the first fluid circulation path 852 when placed within the cell growth chamber. For example, in one embodiment, the first volume of fluid (containing cells) may be 50 ml. The cell growth chamber may have a volume of, for example, 124 ml. When the first volume is placed around the central region 866, it will occupy 50 ml around the central region 866, leaving 74 ml on either side of the central region 866. Therefore, 50% (or 37 ml) of the 74 ml can be added to the volume between the connector 860B and the inlet port 844A to place 50 ml of the first volume around the central region 866.

[0137] In one implementation, placing the first volume 808 may include adding an additional volume of fluid to place the first volume containing cells into the cell growth chamber. For example, if the desired position of the first volume is not achieved using a second volume, additional fluid may be added to place the first volume 808.

[0138] Process 800 proceeds to inquiry 810 to determine whether the first volume should be repositioned. For example, in one embodiment, the first volume may be placed closer to the inlet port 844A. If it is desired to move the first volume closer to the central region 866 of the cell growth chamber 844, process 800 may cycle back to step 808, where additional fluid may be added to the first fluid circulation path to accommodate the fluid of the first volume.

[0139] If inquiry 810 determines that the first volume does not need to be repositioned, process 800 proceeds to cell feeding 812. In embodiments, cells can be fed using a culture medium comprising a variety of compounds, such as glucose, proteins, growth factors, reagents, or other nutrients. In embodiments, cell feeding 812 may include activating inlet and circulation pumps (e.g., pumps 840 and 848) to deliver a culture medium containing nutrients to cells in cell growth chamber 844. As described below, some embodiments provide for maintaining cells in cell growth chamber 844 during step 812. These embodiments may involve activating pumps, such as inlet and circulation pumps (e.g., pumps 840 and 848 described below), such that the direction of fluid inflow into cell growth chamber 844 can be bidirectional, such as from inlet port 844A and outlet port 844B into cell growth chamber 844.

[0140] Then, process 800 proceeds to cell expansion 814, where cells can be expanded or grown. According to the embodiment, although step 814 is shown after step 812, step 814 may occur before or simultaneously with step 812. Cells can then be removed from the cell growth chamber 816 and collected in a storage container. In the embodiment, step 816 may include multiple sub-steps. For example, cells may be circulated by a circulation pump (e.g., pump 848) before being collected and stored in the container. Process 800 terminates at end operation 830.

[0141] Next, Figure 9A An embodiment of the invention is shown for maintaining cells in a state such as CES 500 (e.g., Figure 5B and 5CExemplary operating step 900 of a process in a bioreactor of a cell expansion system. As described above, cells remaining outside the bioreactor manifold or the bioreactor IC loop may not receive proper gas and nutrient exchange, which can lead to cell aggregation and death. In one embodiment, the bioreactor provides gas and nutrient exchange via a semi-permeable hollow fiber membrane. Importantly, this exchange is efficient because the surface area to volume ratio in a cell expansion system containing a hollow fiber membrane can be much larger than in other cell culture methods (e.g., about 15 times that of a cell culture flask). Such efficiency can be achieved by minimizing the diffusion distance of culture medium components that can pass through the membrane surface where the exchange occurs.

[0142] For example, Figure 9B This demonstrates the effect of cell proliferation (with approximately 3E+09 T cells present in the bioreactor) in cell expansion systems (such as...) Chart 936 shows the oxygen consumption requirements placed on the cell expansion system. Figure 9B The percentage (%) of oxygen (O2) at bioreactor outlet 938 is depicted. For example, according to one embodiment, a sensor for measuring oxygen levels can be placed at the EC outlet of the bioreactor. In another embodiment, a sensor for measuring oxygenation can be placed at the IC outlet of the bioreactor. The percentage of O2 is measured relative to the running time 940 (e.g., in minutes). According to one embodiment, the EC circulation flow rate Q is set to maximize oxygen supply to the cells. EC循环 It can be set to 300 mL / min (942). Figure 9B The changes in oxygenation are shown when the EC circulation rate decreases from 300 mL / min (942) to 50 mL / min (944). For example, cells may consume oxygen (O2) from the culture medium as it flows through the bioreactor. The fluid at 50 mL / min (944) moves more slowly through the bioreactor than at 300 mL / min (942), therefore, at an EC circulation rate of 50 mL / min, cells may have more time or more opportunities to strip oxygen from the medium. Oxygenation then recovers when the EC circulation rate is restored to 300 mL / min (946). Figure 9BThis demonstrates the potential benefits of retaining cells within the fibers themselves where gas transfer occurs, as opposed to being part of the IC circulation path outside the bioreactor (e.g., where cells may lack oxygen). Therefore, it may be advantageous to retain cell populations (e.g., non-adherent cells) within the hollow fibers of the bioreactor during nutrient supply by directing the culture medium flow to both sides of the bioreactor (e.g., the IC inlet and IC outlet ports). In one embodiment, equal distribution of flow rates to the IC inlet and IC outlet ports can be used. In another embodiment, for example, depending on the desired placement of cells within the bioreactor, a larger flow rate can be used to the IC inlet port compared to the IC outlet port, and vice versa.

[0143] return Figure 9A The process begins at 902, and 900 proceeds to loading a disposable kit or pre-installed fluid delivery assembly (e.g., 210 or 400) onto the cell expansion system at 904. The disposable kit can then be perfused at 906, where the device can, for example, be supplied with Lonza without Ca. 2+ / Mg 2+ PBS perfusion 906. When preparing to load and seed cells, the perfusion medium can be replaced using IC / EC flush 908. For example, in one embodiment, the PBS in the system can be replaced with TexMACSGMP basal medium. Next, process 900 proceeds to closing the IC drain valve 910. In an embodiment, the EC drain valve can be opened to allow ultrafiltration of the fluid added to the hollow fibers of the bioreactor, which includes a hollow fiber membrane. The medium can then be conditioned 912. Next, process 900 proceeds to loading cells 914, e.g., suspension cells or non-adherent cells (such as T cells or Tregs). In one embodiment, these cells 914 can be loaded via a “concentrated loading of cells without circulation” task. In another embodiment, these cells 914 can be loaded via a “loading of cells in homogeneous suspension” task. In other embodiments, other loading tasks and / or loading processes can be used.

[0144] In one embodiment, for example, the loaded cells 914 may be suspended in a solution containing a culture medium to feed the cells, for example, during and after the loading. In another embodiment, such a solution may contain both a culture medium for feeding the cells and a soluble activator complex that stimulates the cells (e.g., T cells). For example, this loading of 914 may occur on day 0.

[0145] After loading cells 914, cells 916 can be further fed. During this feeding 916, it may be necessary to control the cell residence time within the bioreactor itself. By adjusting the flow control parameter 918, during the expansion phase of growth, cells can be retained within the bioreactor itself, rather than being lost, for example, in a portion of the IC loop from the bioreactor to the outside of the bioreactor. According to an embodiment, by retaining cells within the bioreactor, cells in the bioreactor can be closer to the IC inlet port, where such cells can receive the freshest growth medium. On the other hand, cells in the IC loop may be receiving a consumable or conditioned medium that could affect, for example, their glycolytic metabolism. Additionally, according to one embodiment, cells in the bioreactor may receive a mixed gas (e.g., oxygen, carbon dioxide, and nitrogen) input from the gas transfer module (GTM) via diffusion from the EC loop cycle, while cells in other parts of the IC loop may not receive this mixed gas. It should be noted that while embodiments may provide for retaining cells within the bioreactor itself, other embodiments may provide for holding cells in any location to improve nutrient delivery and / or gas exchange. Therefore, the implementation provides for using other locations to retain or control cell dwell time without departing from the spirit and scope of the invention.

[0146] return Figure 9A In process 900, according to an embodiment, cell loss in the hollow fiber membrane bioreactor can be reduced by matching, approximately matching, or substantially matching, the IC circulation pump rate with the IC inlet pump rate, but in the opposite direction. The IC inlet pump 920 can be adjusted to produce a first flow rate or volumetric flow rate, and the IC circulation pump 922 can be adjusted to produce a second countercurrent flow rate or a second counter-volume flow rate, wherein the volumetric flow rate or fluid flow rate or fluid velocity can be considered as the volume of fluid passing through per unit time (which may be represented by the symbol "Q"). In an embodiment, for example, an IC inlet pump rate of 0.1 mL / min can be matched, approximately matched, or substantially matched with a complementary IC circulation pump rate of -0.1 mL / min to maintain cells in the bioreactor during the growth phase of cell culture (e.g., possibly 4-7 days). For example, such pump adjustment 918 can make it possible to counteract any forces associated with cell loss from the IC outlet port of the bioreactor.

[0147] Next, the cells can be allowed to grow or expand 924. The cells are not limited to growth or expansion in step 924; instead, they can also expand in, for example, steps 914, 916, 918, 920, and 922. Process 900 can then proceed to harvesting operation 926, in which the cells can be transferred to a harvest bag or container. The disposable kit 932 can then be removed from the cell expansion system, and process 900 terminates at ending operation 934.

[0148] Alternatively, starting from harvesting operation 926, process 900 may optionally proceed to enable further processing / analysis 928. This further processing 928 may include, for example, characterization of the phenotype of the harvested cells. Starting from the optional further processing / analysis step 928, process 900 may proceed to optionally reload any remaining cells 930. Process 900 may then proceed to unload the disposable kit 932, and process 900 may terminate at end operation 934. Alternatively, process 900 may proceed from further processing / analysis step 928 to unload the disposable kit 932. Process 900 may then terminate at END operation 934.

[0149] Next, Figure 10A Exemplary operational steps 1000 for a process for culturing cells according to an embodiment of the present invention are shown, the process being compatible with, for example, a CES 500 (e.g., Figure 5B and 5C This is used in conjunction with a cell expansion system. For example, the process 1002 is initiated, and process 1000 proceeds to loading a disposable kit onto the cell expansion system, perfusing the kit, performing IC / EC rinsing, conditioning the culture medium, and loading cells, such as suspension or non-adherent cells. Next, process 1000 proceeds to feeding cells during a first time period 1004. In an embodiment, a first inflow rate and a first circulation rate can be used. As an example, a first IC inflow rate and a first IC circulation rate can be used, wherein the first IC inflow rate can be controlled by an IC inflow pump (e.g., a first pump), and the first IC circulation rate can be controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, an IC inlet pump (554) can deliver a volumetric flow rate of 0.1 mL / min into the IC inlet port (501A) of the bioreactor (501), and an IC circulation pump (512) can deliver a complementary IC circulation volumetric flow rate or fluid flow rate of -0.1 mL / min into the IC outlet port (501B) of the bioreactor (501), wherein the negative sign ("-") used in -0.1 mL / min indicates the direction of the IC circulation pump (512) to induce or generate a countercurrent rate during the growth phase of the cell culture to hold the cells in the bioreactor.

[0150] During cell culture and the use of an IC pump to control cell residence in the bioreactor through flow and countercurrent characteristics, cells continue to grow and expand. As a result, cells may require additional culture media, such as glucose and / or cell growth formulations, to support the continuously expanding population. Efforts can also be made to control the lactate level of the expanding cell population. In embodiments, the lactate level of the cell culture can be maintained at or below about 20 mmol / L, at or below about 15 mmol / L, at or below about 10 mmol / L, or even at or below about 7 mmol / L. In other embodiments, for example, the rate of culture medium addition and / or other settings can be controlled to attempt to maintain, for example, a lactate level ≤ about 5 mmol / L to improve cell growth and viability. Other concentrations may be used in other embodiments.

[0151] In an exemplary embodiment, efforts can be made to control the lactate level to ≤ about 7 mmol / L by simultaneously increasing both the IC inlet (+) pump rate and the IC circulation (-) pump rate within the lumen of the hollow fiber membrane from ±0.1 to ±0.4 mL / min over multiple time periods, depending on the embodiment, such as several days (days 4-8). For example, Figure 10BTable 1018 provides exemplary IC pumping rates for use in “feeding cells” tasks, such as feeding cells via a cell expansion system (e.g., CES500). Table 1018 provides example time periods 1020 (e.g., days) compared to example IC pumping rates 1022 to generate volumetric flow rates to both sides of the bioreactor to maintain cells in the bioreactor. For example, on days 0-4 (1024), an IC inlet or outlet pump rate of 0.1 mL / min (1026) and an IC circulation pump rate of -0.1 mL / min (1028) can be used; on day 5 (1030), an IC inlet pump rate of 0.2 mL / min (1032) and an IC circulation pump rate of -0.2 mL / min (1034) can be used; on day 6 (1036), an IC inlet pump rate of 0.3 mL / min (1038) and an IC circulation pump rate of -0.3 mL / min (1040) can be used; and on day 7 (1042), an IC inlet pump rate of 0.4 mL / min (1044) and an IC circulation pump rate of -0.4 mL / min (1046) can be used. Although exemplary pumping rates of ±0.1 to ±0.4 mL / min for nutrient supply to cells while holding them in the bioreactor during the growth phase of cell cultures are provided in Table 1018 of Figure 10, other pumping rates and resulting flow rates may be used according to embodiments without departing from the spirit and scope of the invention. For example, although an increment of ±0.1 mL / min is shown in this example for increasing the nutrient supply flow rate, other increments (e.g., ±0.005 mL / min, ±0.05 mL / min, etc.) may also be used to increase the nutrient supply flow rate in the embodiments. Figure 10B The time periods (e.g., days) and pump rates in Table 1018 are for illustrative purposes only and are not intended to be limiting.

[0152] return Figure 10A Process 1000 proceeds from feeding cells during a first time period 1004 to increasing the first inflow rate by a first amount to achieve a second inflow rate 1006. For example, as described above. Figure 10BIn the embodiment described, the IC inlet pump rate (+) can be increased from 0.1 mL / min to 0.2 mL / min to produce an IC inlet flow rate of 0.2 mL / min. Furthermore, the IC circulation pump rate (-) can simultaneously be increased from -0.1 mL / min to -0.2 mL / min to produce an IC circulation flow rate of -0.2 mL / min. Therefore, the first circulation flow rate is increased by a first amount to achieve a second circulation flow rate 1008. For example, cells can then be fed at the second inlet flow rate and the second circulation flow rate 1010 for a second time period to maintain cells in the bioreactor and on the outside of the manifold and on the outside of a portion of the IC circulation path in the bioreactor. After the feeding 1010 for the second time period, for example, if it is not desired to continue feeding and / or expanding cells, process 1000 can be terminated at an end operation 1016. Alternatively, process 1000 can optionally continue to increase or otherwise change the feeding flow rate 1012. Any number of feeding time periods can exist, as indicated by ellipsis 1014. After the required number of feeding periods 1014, process 1000 then terminates at the end operation 1016. Although Figure 10A and 10B Process 1000 illustrates an "increase" in flow rate, but other adjustments can be made. For example, the flow rate can be decreased or remained essentially the same from one feeding period to the next. Considerations such as metabolic activity can determine how the flow rate is regulated. Figure 10A and Figure 10B The "increase" in flow rate is for illustrative purposes only and is not intended to impose any restrictions.

[0153] Turning Figure 11A According to embodiments of the present invention, it is provided that can be used with, for example, CES 500 (e.g., Figure 5B and 5CExample operating steps 1100 for a cell supply process used in conjunction with a cell expansion system. Start-up operation 1102 is initiated, and process 1100 proceeds to loading a disposable kit onto the cell expansion system, perfusing the device, performing IC / EC flushing, conditioning the culture medium, and loading cells, such as suspension or non-adherent cells. Next, process 1100 proceeds to supply cells during a first time period 1104. In embodiments, a first inflow rate or flow rate and a first circulation rate or flow rate may be used. As an example, a first IC inflow flow rate and a first IC circulation flow rate may be used, wherein the first IC inflow flow rate is controlled by an IC inflow pump (e.g., a first pump), and the first IC circulation flow rate may be controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, an IC inlet pump (554) can deliver a volumetric flow rate of 0.1 mL / min into the IC inlet port (501A) of the bioreactor (501), and an IC circulation pump (512) can deliver a complementary IC circulation volumetric flow rate or fluid flow rate of -0.1 mL / min into the IC outlet port (501B) of the bioreactor (501), wherein the negative sign ("-") used in -0.1 mL / min indicates the direction of the IC circulation pump (512) to induce or generate a countercurrent rate during the growth phase of the cell culture to hold the cells in the bioreactor.

[0154] During cell feeding and while the residence time of cells in the bioreactor is controlled using the flow and countercurrent characteristics of an IC pump, cells continue to grow and expand. As a result, cells may require additional culture media, such as glucose and / or cell growth formulations, to support the continuously expanding population. Efforts can also be made to control the lactate level of the expanding cell population. In an exemplary embodiment, efforts can be made to maintain the lactate level at ≤approximately 7 mmol / L by simultaneously increasing both the IC inlet (+) pump rate and the IC circulation (-) pump rate within the lumen of the hollow fiber membrane from ±0.1 to ±0.4 mL / min over multiple time periods (depending on the embodiment, e.g., several days (days 4-8)). Figure 10B Table 1018 and as discussed above. Although in Figure 10B Table 1018 provides exemplary pumping rates of ±0.1 to ±0.4 mL / min for feeding cells while maintaining them in the bioreactor during the growth phase of cell cultures. Other pumping rates and resulting flow rates may be used according to the embodiments without departing from the spirit and scope of the invention. Figure 10B The time periods (e.g., days) and pump rates in Table 1018 are for illustrative purposes only and are not intended to be limiting.

[0155] return Figure 11AProcess 1100 proceeds from the donation of cells during a first time period 1104 to increasing the first entry rate or flow rate by a first amount to achieve a second entry rate or flow rate 1106. As described above. Figure 10B In the depicted embodiment, the IC inlet pump rate (+) can be increased from 0.1 mL / min to 0.2 mL / min to produce an IC inlet flow rate of 0.2 mL / min. Furthermore, the IC circulation pump rate (-) can simultaneously be increased from -0.1 mL / min to -0.2 mL / min to produce an IC circulation flow rate of -0.2 mL / min. Therefore, the first circulation rate or flow rate is increased by a first amount to achieve a second circulation rate or flow rate 1108. Cells can then be fed at the second inlet rate or flow rate and at the second circulation rate or flow rate 1110 during a second time period to maintain the cells, for example, outside the bioreactor and outside a portion of the IC circulation path outside the manifold or bioreactor. After the second stage of feeding 1110, for example, if continued feeding and / or cell expansion is not required, the process 1100 can be terminated at the end operation 1116.

[0156] Alternatively, process 1100 may optionally determine whether to regulate the nutrient supply rate or flow rate based on metabolic activity, wherein process 1100 proceeds to optional query 1112 to determine whether to regulate the nutrient supply based on metabolic levels. Monitoring glucose and / or lactate levels can facilitate the regulation of the flow rate of cell expansion system media, such as IC medium, to support the expansion of cells, such as Tregs, in a bioreactor, such as a hollow fiber bioreactor.

[0157] like Figure 11B and 11C As shown, the implementation provides lactate levels for, for example, controlling cell expansion runs or procedures involving hTreg amplification. Graphs 1118 and 1132 indicate that measurements of glucose and lactate levels can be used to adjust the flow rate of the cell expansion system medium, such as IC50 medium, to support, for example, the amplification of Tregs. Figure 11B Graph 1118 is provided, which shows the metabolism of expanded hTregs, where such cell expansion can occur in events such as In a cell expansion system, glucose concentrations (mg / dL) 1120 and lactate concentrations (mmol / L) 1122 for various cell expansion runs 1124 and 1125 across time periods (e.g., day 1126) are shown. In these Treg runs 1124 and 1125, considerable effort may be required, for example, by simultaneously adjusting the IC inlet (+) pump rate and IC circulation pump rate within the hollow fiber membrane lumen from ±0.1 liters to ±0.4 mL / min to control the lactate level of the expanded cell population to ≤ about 7 mmol / L. In other embodiments, different pump rates may be used. As shown, according to the illustrated embodiment, the lowest glucose level during Treg cell expansion can be 264 mg / dL at concentration 1128 on day 7 (Q1584) to 279 mg / dL at concentration 1130 on day 8 (Q1558). As depicted, the basal glucose concentration in the cell growth formulation medium running at 1124 can, for example, be in the range of 325 mg / dL to 335 mg / dL. In other embodiments, it may be necessary to maintain lactate levels ≤ about 5 mmol / L to improve cell growth and viability. In embodiments, a graphical user interface (GUI) element can be used to control the rate of medium addition and to maintain lactate metabolic waste products from glycolysis below defined levels during cell expansion.

[0158] Turning Figure 11C Figure 1132 shows the metabolism of expanded hTregs, where such cell expansion can occur in events such as... In a cell expansion system, glucose consumption (mmol / day) 1134 and lactate production (mmol / day) 1136 are shown for various cell expansion runs 1138 and 1139 spanning time periods (e.g., day 1140). To control lactate levels to ≤ about 7 mmol / L, for example, in one embodiment, the IC inlet (+) pump rate and IC circulation (-) pump rate can be simultaneously increased from ±0.1 to ±0.4 mL / min. For example, graph 1132 shows IC circulation and IC supply rates of ±0.1 mL / min (1142); ±0.2 mL / min (1144); ±0.3 mL / min (1146); and ±0.4 mL / min (1148). Other embodiments may use other flow rates. Provided in Figure 11B and 11C The flow rates used and shown are for illustrative purposes only and are not restrictive. Note that although the direction of IC entering the pump may be shown as positive (+) and the direction of IC circulating in the pump may be shown as negative (-), these directions are provided for illustrative purposes only and depend on the configuration of the pump used.

[0159] return Figure 11A And with optional inquiry 1112, if it is not desired to measure metabolic activity and / or regulate nutrient levels based on such measurements, process 1100 proceeds to "No" to end operation 1116, and process 1100 terminates. Alternatively, if it is desired to regulate nutrient levels based on metabolic activity, process 1100 proceeds to "Yes" to optional step 1114 to continue increasing the rate of culture medium addition and nutrient supply to the expanding cell population. Although step 1114 is shown as a step, this step may involve many adjustments to the rate of culture medium addition, such as increasing the IC inflow rate and increasing the IC circulation rate. Step 1114 is shown as a step for illustrative purposes only and is not intended to be limiting. After any adjustments to the rate of culture medium addition, process 1100 proceeds to optional inquiry 1112 to determine whether to continue measuring metabolic levels and / or regulating nutrient supply. If it is not desired to continue measuring metabolic activity and / or regulating nutrient levels based on metabolic activity, process 1100 proceeds to "No" to end operation 1116, and process 1100 terminates. Although Figure 11A Process 1100 illustrates an "increase" in rate or flow rate, but other adjustments can be made to the flow rate. For example, the rate or flow rate may decrease or remain substantially the same from one feeding period to the next. The types of adjustments that may be made may depend on an assessment of the metabolic activity of the growing cell population. For example, providing... Figure 11A The "increase" in flow rate is for illustrative purposes only and is not intended to restrict it.

[0160] Next, Figure 12 The invention illustrates an embodiment of a cell expansion system using, for example, the CES 500. Figure 5B and 5CExemplary operational steps 1200 of the process of holding cells in a certain position during culture. Start-up operation 1202 is initiated, and process 1200 proceeds to loading a disposable kit onto the cell expansion system, perfusion device, performing IC / EC flushing, conditioning the culture medium, and loading cells, e.g., suspension or non-adherent cells. Next, process 1200 proceeds to culture cells during a first time period 1204. In embodiments, a first inflow rate and a first circulation rate can be used. As an example, a first IC inflow rate and a first IC circulation rate can be used, wherein the first IC inflow rate can be generated and controlled by an IC inflow pump (e.g., a first pump) and the first IC circulation rate can be generated and controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, an IC inlet pump (554) may allow a volumetric flow rate of 0.1 mL / min to enter the IC inlet port (501A) of the bioreactor (501), and an IC circulation pump (512) may allow a complementary IC circulation volumetric flow rate or fluid flow rate of -0.1 mL / min to enter the IC outlet port (501B) of the bioreactor (501), wherein the negative sign ("-") used in -0.1 mL / min indicates the direction of the IC circulation pump (512) to induce or generate a backflow rate during the growth phase of the cell culture to hold the cells in the bioreactor. In another embodiment, the first IC circulation flow rate may be a percentage of the first IC inlet flow rate. For example, according to an embodiment, the first IC circulation flow rate may be about fifty percent (50%), or about half (1 / 2), or another percentage or portion of the first IC inlet flow rate.

[0161] For example, during cell nutrient supply (and expansion) and the use of an IC pump to control cell residence in the bioreactor through flow and countercurrent properties, cells continue to grow and expand. As a result, cells may require additional culture medium, such as glucose and / or cell growth formulations, to support the continuously expanding population. In embodiments, efforts can be made to increase the rate of culture medium addition to nutrient supply to the expanding cell population. In an exemplary embodiment, an increase in the IC inlet pump rate (+) can cause a first increase in the IC inlet flow rate to achieve a second IC inlet flow rate of 1206. For example, according to one embodiment, the IC inlet flow rate can be increased by a first amount of 0.1 mL / min to achieve a second IC inlet flow rate of 0.2 mL / min. According to other embodiments, other adjustments can be made to the IC inlet flow rate.

[0162] Next, according to the implementation scheme, the second IC circulation flow rate can be set / adjusted or configured to be equal to a percentage / part or fraction of the second IC inflow flow rate 1208. For example, according to one implementation scheme, the second IC circulation flow rate can be set, adjusted or configured to be equal to about fifty percent (50%) or about half (1 / 2) of the IC inflow flow rate, or according to the implementation scheme, another percentage or part of the IC inflow flow rate. According to one implementation scheme, depending on the value of the first IC circulation flow rate, adjusting the IC circulation pump rate (-) can cause an increase in the second IC circulation flow rate. In another implementation scheme, the IC circulation pump rate can be adjusted to produce or cause a decrease in the second IC circulation flow rate, such that the IC circulation flow rate can be substantially equal to a predetermined percentage or predetermined fraction of the second IC inflow flow rate. In yet another implementation scheme, the IC circulation pump rate may not be adjusted. For example, if the first IC inflow flow rate is equal to 0.1 mL / min and the first IC circulation flow rate is equal to -0.1 mL / min, if the second IC inflow flow rate is increased to 0.2 mL / min, the second IC circulation flow rate can be set to (-1 / 2). * (Second Q) IC进入 (where Q) IC进入 (Is the IC inflow rate) or (-1 / 2) * (0.2 mL / min), which provides a second Q of -0.1 mL / min IC循环 (where Q) IC循环 (for IC circulation flow rate), and without adjusting the IC circulation pump speed to achieve this second Q. IC循环 .

[0163] Turn Figure 5B and 5C According to an embodiment of the invention, these figures depict the operational configuration of the cell expansion system 500, showing the movement of fluid in a first circulation path. Figure 5B and 5CThe configuration illustrates, for example, the division of flow rates to the IC inlet port (e.g., the first port) and the IC outlet port (e.g., the second port) to hold cells in a cell growth chamber or bioreactor. As discussed above with respect to CES 500, in the IC loop or first circulation path 502, fluid can initially be propelled by the IC inlet pump 554. This fluid can be propelled, for example, in a first direction, such as the positive direction. Fluid can flow into the cell growth chamber or bioreactor 501 through the hollow fibers in the cell growth chamber or bioreactor 501 via the IC inlet port 501A, and can flow out through the IC outlet port 501B. Culture medium can flow through the IC circulation pump 512, which can be used to control the flow rate of the culture medium. The IC circulation pump can pump fluid in the first direction or in a second direction opposite to the first direction. The outlet port 501B can, for example, be used as an inlet in the opposite direction. In one embodiment, for example, the IC circulation pump 512 can pump fluid in a direction opposite to the direction of the IC inlet pump. As an example, the direction of IC entering the pump can be positive (+), while the direction of IC circulation pump can be negative (-) to cause backflow, thereby allowing fluid to enter both sides of the bioreactor to maintain the cells in the bioreactor.

[0164] In one embodiment, a first portion of the fluid may branch at connection 517 to flow into the IC inlet port (501A) of bioreactor 501. In one embodiment, the IC circulation pump 512 may operate at a pump rate that matches, approximately matches, or substantially matches the rate of the IC inlet pump 554, but in the opposite direction, so that a second portion of the fluid may branch at connection 517 to flow into the IC outlet port (501B) of bioreactor 501. For example, an IC inlet pump rate of +0.1 mL / min may be matched, approximately matches, or substantially matches a complementary IC circulation pump rate of -0.1 mL / min to maintain cells in the bioreactor during the growth phase of cell culture. This pump regulation strategy during nutrient supply can counteract the forces associated with cell loss from the IC outlet. According to one embodiment, this type of nutrient supply using pump regulation to induce flow and countercurrent into the IC inlet port (501A) and IC outlet port (501B) of the bioreactor, respectively, can be referred to as an improved nutrient supply method. In another exemplary embodiment, the IC circulation pump rate can be adjusted so that the flow rate into the IC outlet port 501B can be equal to approximately fifty percent (50%) or approximately half (1 / 2) of the IC inflow rate, or in other embodiments, another percentage of the IC inflow rate, but in the opposite direction. For example, with an IC inflow pump rate of 0.4 mL / min, the IC circulation pump rate can be set or configured to -0.2 mL / min. Other percentages or portions can be used in other embodiments.

[0165] According to the implementation plan, Figure 5Band Figure 5C The operational configuration of fluid movement in the CES 500 is illustrated, showing the flow rates and counter-flow rates for holding cells in a cell growth chamber or bioreactor 501. According to the embodiment, for example, this flow rate is... Figure 5B In the diagram, "X" represents a flow velocity of 503; "(-1 / 2)X" represents a flow velocity of 505 (where the negative sign "-" indicates direction, and the direction of flow velocity 505 is determined by...). Figure 5B (Indicated by the directional arrows in the figure); and (1 / 2)X” flow rate 507 indicates that approximately 1 / 2 or the first portion of the flow rate branches at the connection 517 to enter the IC inlet port (501A) of the bioreactor 501, and approximately 1 / 2 or the second portion of the flow rate branches at the connection 517 to enter the IC outlet port (501B) of the bioreactor 501. As shown, the sum of the first and second portions can be substantially equal to the total flow rate 503, which is pumped by the IC inlet pump 554. Depending on the flow rate and counterflow rate used to hold cells in the bioreactor or cell growth chamber 501, other types of IC inlet pump rates can be used to set, configure, or adjust the IC circulation pump. Thus, according to the embodiment, Figure 5C This shows flow rates such as "X" flow rate 511; "-(y%)*X" flow rate 513 (where the negative sign ("-") indicates direction, and the direction of flow rate 513 is determined by...). Figure 5C The direction arrows in the text indicate the direction; and "(100%-y%)*X" flow rate 515, where "y" equals a numerical percentage. In the implementation, the sum of flow rate 513 and flow rate 515 is substantially equal to flow rate 511.

[0166] In one embodiment, for example, all or substantially all of the flow from the first fluid flow path 506 may flow from the connection 517 to the IC inlet port (501A) of the bioreactor 501. In another embodiment, all or substantially all of the flow from the first fluid flow path 506 may flow from the connection 517 to the IC outlet port (501B) of the bioreactor 501. In yet another embodiment, a first portion of the flow from the first fluid flow path 506 may flow from the connection 517 to the IC inlet port (501A), and a second portion of the flow from the first fluid flow path 506 may flow from the connection 517 to the IC outlet port (501B). In embodiments, the percentage of the IC inlet flow rate relative to the IC circulation flow rate can be set in the range of about 0% to about 100%. In other embodiments, this percentage may be between about 25% and about 75%. In other embodiments, this percentage may be between about 40% and about 60%. In other embodiments, this percentage may be between about 45% and about 55%. In embodiments, this percentage may be about 50%. It should be understood that... Figure 5Band Figure 5C The operational configurations shown represent possible configurations for various operations of the cell expansion system, and modifications to the shown configurations are within the scope of one or more of these embodiments.

[0167] return Figure 12 Cells can be fed (and expanded) at a second inflow rate and a second circulation rate 1210 during a second time period to maintain the cells, for example, in a bioreactor and outside a portion of the bioreactor manifold or IC circulation path. After the second stage of feeding 1210, for example, if continued feeding and / or cell expansion is not required, process 1200 can terminate at end operation 1216. Alternatively, process 1200 can optionally continue to increase or otherwise change the feeding flow rate 1212. Any number of feeding time periods can exist, as indicated by ellipsis 1214. After the desired number of feeding time periods 1214, process 1200 can then terminate at end operation 1216. Although Figure 12 Process 1200 illustrates an "increase" in flow rate, but other adjustments can be made to the flow rate. For example, the flow rate can be decreased or remain essentially the same from one feeding period to the next. Considerations such as metabolic activity can determine how the flow rate is regulated. Figure 12 The "increased" traffic in the text is provided for illustrative purposes only and is not intended to limit.

[0168] Turn Figure 13 According to embodiments of the invention, exemplary operational steps 1300 are provided for a process of feeding cells while holding them in a first location (e.g., in a bioreactor), which can be used with, for example, a CES 500 (e.g., Figure 5B and 5CThis is used in conjunction with a cell expansion system. The process begins at start operation 1302, and proceeds to loading a disposable kit onto the cell expansion system, perfusion device, IC / EC flushing, conditioning the culture medium, and loading cells, e.g., suspension or non-adherent cells. Next, process 1300 proceeds to supplying cells during a first time period 1304. In this embodiment, a first inflow rate and a first circulation rate can be used. As an example, a first IC inflow rate and a first IC circulation rate can be used, wherein the first IC inflow rate can be generated and controlled by an IC inflow pump (e.g., a first pump) and the first IC circulation rate can be generated and controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, IC inlet pump 554 may allow a volumetric flow rate of 0.1 mL / min to enter the IC inlet port 501A of bioreactor 501, and IC circulation pump 512 may allow a complementary IC circulation volumetric flow rate or fluid flow rate of -0.1 mL / min to enter the IC outlet (501B) of bioreactor 501, wherein the negative sign ("-") used in -0.1 mL / min indicates the direction of the IC circulation pump (512) to induce or generate a backflow rate during the growth phase of the cell culture to hold the cells in the bioreactor. In another embodiment, the first IC circulation flow rate may be a percentage of the first IC inlet flow rate. For example, according to an embodiment, the first IC circulation flow rate may be about fifty percent (50%), or about half (1 / 2), or another percentage or portion of the first IC inlet flow rate.

[0169] During cell nutrient supply (and expansion) and while cell residence in the bioreactor is controlled using the flow and countercurrent characteristics of an IC pump, cells continue to grow and expand. As a result, cells may require additional culture medium, such as glucose and / or cell growth formulations, to support the continuously expanding population. In embodiments, efforts can be made to increase the rate of culture medium addition to nutrient supply to the expanding cell population. In an exemplary embodiment, increasing the IC inlet pump rate (+) can increase the IC inlet flow rate by a first amount to achieve a second IC inlet flow rate of 1306. For example, according to one embodiment, a first amount of IC inlet flow rate of 0.1 mL / min can be increased to achieve a second IC inlet flow rate of 0.2 mL / min. Other adjustments to the IC inlet flow rate can be made according to other embodiments.

[0170] Next, according to the embodiments, the second IC circulation flow rate can be set, configured, or adjusted to be equal to a percentage, part, or fraction of the second IC inflow flow rate 1308. According to one embodiment, for example, the second IC circulation flow rate can be set, configured, or adjusted to be equal to approximately fifty percent (50%) or approximately half (1 / 2) of the IC inflow flow rate, or another percentage or part according to the embodiments. In one embodiment, for example, all or substantially all of the flow from the first fluid flow path 506 can flow from the connection 517 to the IC inlet port (501A) of the bioreactor 501. In another embodiment, all or substantially all of the flow from the first fluid flow path 506 can flow from the connection 517 to the IC outlet port (501B) of the bioreactor 501. In yet another embodiment, a first portion of the flow from the first fluid flow path 506 can flow from the connection 517 to the IC inlet port (501A), and a second portion of the flow from the first fluid flow path 506 can flow from the connection 517 to the IC outlet port (501B). In one embodiment, the percentage of the IC inflow rate relative to the IC circulation flow rate can be set to range from about 0% to about 100%. In other embodiments, this percentage can be between about 25% and about 75%. In other embodiments, this percentage can be between about 40% and about 60%. In other embodiments, this percentage can be between about 45% and about 55%. In one embodiment, this percentage can be about 50%.

[0171] According to one embodiment, depending on the value of the first IC circulation flow rate, adjusting the IC circulation pump rate (-) can result in an increase in the second IC circulation flow rate. In another embodiment, the IC circulation pump rate can be adjusted to produce or cause a decrease in the second IC circulation flow rate, such that the IC circulation flow rate can be substantially equal to a predetermined percentage or fraction of the second IC inflow rate. In yet another embodiment, the IC circulation pump rate may not be adjusted. For example, according to one embodiment, if the first IC inflow rate is equal to 0.1 mL / min and the first IC circulation flow rate is equal to -0.1 mL / min, and the second IC inflow rate is increased to 0.2 mL / min, then the second IC circulation flow rate can be set to (-1 / 2). * (Second Q) IC进入 (where Q) IC进入 (Is the IC inflow rate) or (-1 / 2) * (0.2 mL / min), which provides a second Q of -0.1 mL / min IC循环 (where Q) IC循环 (for IC circulation flow rate), and without adjusting the IC circulation pump speed to achieve this second Q. IC循环 .

[0172] For example, cells can then be fed (and expanded) at a second IC inlet flow rate and a second IC circulation flow rate 1310 during a second time period to maintain the cells in the bioreactor and outside a portion of the bioreactor manifold or IC circulation path. For example, after the feeding 1310 during the second time period, if continued feeding and / or cell expansion is not required, process 1300 can be terminated at end operation 1316.

[0173] In the implementation scheme, the first time period, the second time period, the third time period, the fourth time period, the fifth time period, etc., may each include one or more days (and / or hours and / or minutes). For example, according to the implementation scheme, the time period may be one (1) day to fourteen (14) days. However, in other implementation schemes, for example, the time period may be less than one (1) day or greater than fourteen (14) days. In one implementation scheme, the first time period for providing care may include day 0, day 1, day 2, day 3, and day 4; the second time period for providing care may include day 5; the third time period for providing care may include day 6; and the fourth time period for providing care may include day 7. In another embodiment, the first time period may include day 0, day 1, and day 2 (e.g., a duration of approximately 3 days); the second time period may include day 3, day 4, and day 5 (e.g., a duration of approximately 3 days); the third time period may include day 6, day 7, and day 8 (e.g., a duration of approximately 3 days); the fourth time period may include day 9 and day 10 (e.g., a duration of approximately 2 days); and the fifth time period may include day 11, day 12, and day 13 (e.g., a duration of approximately 3 days). Depending on the embodiment, the time periods may be of different durations. Each time period may be measured in days, hours, minutes, and / or portions thereof.

[0174] return Figure 13 Process 1300 may optionally continue to optional query 1312 to determine whether to adjust the nutrient supply rate or flow rate based on metabolic activity or metabolic level. For example, for the expansion of Tregs, it may be necessary to control the lactate level of the expanded cell population to ≤ about 7 mmol / L. For example, when it is desirable to maintain the lactate level in the cell culture at or below about 7 mmol / L, the rate of culture medium addition can be controlled during cell (e.g., regulatory T cells) expansion. In other embodiments, it may be necessary to maintain the lactate level ≤ about 5 mmol / L to improve cell growth and viability. In embodiments, a graphical user interface (GUI) element may be used to control the rate of culture medium addition and maintain lactate metabolic waste products from glycolysis below a predetermined level during cell expansion.

[0175] At optional inquiry 1312, if it is not desired to measure metabolic activity and / or regulate nutrient levels based on such measurements, process 1300 proceeds to "No" to end operation 1316, and process 1300 terminates. Alternatively, if it is desired to regulate nutrient levels based on metabolic activity, process 1300 proceeds to "Yes" to optional step 1314 to continue increasing the rate of culture medium addition and nutrient supply to the expanding cell population. Although step 1314 is shown as a single step, this step may involve numerous adjustments to the rate of culture medium addition, such as increasing the IC inflow rate and increasing the IC circulation rate. Step 1314 is shown as a single step for illustrative purposes only and is not intended to be limiting. After any adjustments to the rate of culture medium addition, process 1300 proceeds to optional inquiry 1312 to determine whether to continue measuring metabolic levels and / or regulating nutrient supply. If it is not desired to continue measuring metabolic activity and / or regulating nutrient levels based on metabolic activity, process 1300 proceeds to "No" to end operation 1316, and process 1300 terminates. Although Figure 13 Process 1300 illustrates an "increase" in rate or flow rate, but other adjustments can be made to the flow rate. For example, the rate or flow rate may decrease or remain substantially the same from one feeding period to the next. The types of adjustments that may be made may depend on the assessment of the metabolic activity of the growing cell population. For example, providing... Figure 13 The "increase" in flow rate is for illustrative purposes only and is not intended to restrict it.

[0176] Next, Figure 14 An exemplary operational step 1400 for maintaining cells during cell feeding, according to an embodiment of the invention, is provided, which can be used with, for example, a CES 500 (e.g., Figure 5B and 5C This is used in conjunction with a cell expansion system. Operation 1402 is initiated, and process 1400 proceeds to loading a disposable tubing kit or pre-installed fluid delivery assembly (e.g., 210 or 400) 1404 onto the cell expansion system. Next, the system can be perfused 1406. In one embodiment, for example, a user or operator can provide instructions to the system to perfuse by, for example, selecting a task for perfusion. In one embodiment, such a task for perfusion may be a pre-programmed task. Next, an IC / EC flushing task 1408 can be performed, where, for example, fluid can be replaced in the IC and EC circulation loops. Depending on the embodiment, the replacement volume can be determined by the number of IC and EC volumes exchanged.

[0177] Next, to maintain an appropriate or desired gas concentration across the filaments in the bioreactor membrane, a conditioning task 1410 can be performed so that the medium is balanced with the provided gas supply before cells are loaded into the bioreactor. For example, contact between the medium and the gas supply provided by the gas delivery module (GTM) or oxygenator can be provided by adjusting the EC circulation rate. The system can then be maintained in the appropriate or desired state until, for example, a user or operator is ready to load cells into the bioreactor. In one embodiment, the system can be conditioned, for example, with a medium such as a complete medium. A complete medium can be any medium source used for cell growth. In one embodiment, the system can be conditioned, for example, with a serum-free medium. In one embodiment, the system can be conditioned with a basal medium. Any type of medium understood by those skilled in the art can be used.

[0178] For example, next, process 1400 proceeds to loading cells 1412 from the cell inlet bag into the bioreactor. In one embodiment, for example, the cells in the cell inlet bag may be in solution with a culture medium to feed cells 1414. In another embodiment, the cells in the cell inlet bag may be in solution with a culture medium to feed cells 1414 and also contain a soluble activator complex that can stimulate cells (e.g., T cells or Tregs). In one embodiment, cells (and the nutrient solution in the embodiment) may be loaded from the cell inlet bag into the bioreactor until the bag is empty. Cells (and the nutrient solution in the embodiment) may be removed from the air removal chamber into the bioreactor. In one embodiment, a "loading cells in homogeneous suspension" task may be performed to load cells (and the nutrient solution in the embodiment). In another embodiment, a "centralized loading cells without circulation" task may be performed to load cells (and the nutrient solution in the embodiment) into a specific, for example, central region of the bioreactor. Other loading methods and / or loading tasks may be used depending on the embodiment.

[0179] Next, process 1400 proceeds to inquiry 1416 to determine whether to retain cells (e.g., non-adherent cells or suspension cells, such as T cells or Tregs) in the bioreactor (e.g., a hollow fiber bioreactor) using an improved feeding method. For example, it may be necessary to place the cells within the bioreactor itself, outside the rest of the bioreactor's manifold or IC loop. If it is not desired to retain the cells within the bioreactor itself using an improved feeding method, process 1400 proceeds to "No" to expand the cells 1426, where, for example, the cells may continue to grow / expand using the culture medium initially fed in step 1414.

[0180] On the other hand, if it is desired to retain the cells within the bioreactor itself, process 1400 proceeds to an improved feeding 1418, where cells can be fed using flow rates from the IC inlet port (501A) and the IC outlet port (501B) of the bioreactor 501 to retain the cells within the bioreactor. To do this, an inlet volumetric flow rate or inlet velocity can be introduced into the first fluid path (506) 1420. For example, an IC inlet velocity can be introduced into the fluid path (506) 1420. An inlet pump (554), such as a first peristaltic pump (in one embodiment), can operate at a predetermined revolutions per minute (RPM) to induce a predetermined IC inlet volumetric flow rate or IC inlet velocity in the fluid within the first fluid flow path (506) 1420. According to one embodiment, a processor and / or controller can direct or control, for example, the first and / or second pumps to operate at a predetermined amount of RPM. Depending on the speed and direction of the IC circulation pump (512), a first flow rate or a first portion of the modified IC inlet flow rate may enter the IC inlet port 501A or the first port 1422 of the bioreactor (501). The pump speed may depend on the diameter of the pump (e.g., a peristaltic pump) or the pump configuration. Other types of pumps may also be used, where the pump speed may depend on the configuration of the pump used. The IC circulation pump (512), such as a second peristaltic pump (in one embodiment), may operate at a predetermined amount of RPM and in the opposite direction to the first pump to induce or generate a second flow rate or a second portion of the predetermined IC circulation flow rate or IC inlet flow rate to enter the IC outlet port 501B or the second port 1424 of the bioreactor (501). For example, the implementation may provide approximately half (approximately 1 / 2) of the IC inlet flow rate, or a first portion branching at connection 517 to enter the IC inlet port (501A) of bioreactor 501, and approximately half (or 1 / 2) of the IC inlet flow rate, or a second portion branching at connection 517 to enter the IC outlet port (501B) of bioreactor 501. As shown, the sum of the first and second portions may be substantially equal to the IC inlet flow rate 503 (e.g., as...). Figure 5B The IC inlet flow rate 503 can be pumped by the IC inlet pump 554. According to the embodiment, in order to maintain cells in the bioreactor or cell growth chamber 501, other types of fractional or percentage IC inlet pump rates can be used to set, or the IC circulation pump can be configured or adjusted.

[0181] After providing the cells with this flow and countercurrent properties to hold them in the bioreactor, process 1400 proceeds to cell growth / expansion 1426. Although cell expansion is shown in step 1426, the cells may also grow / expand in one or more other steps (such as 1412, 1414, 1416, 1418, 1420, 1422, 1424). From expansion step 1426, process 1400 proceeds to cell harvesting or removal 1430. Process 1400 may then terminate at end operation 1432. According to the embodiment, if any additional steps are required before harvesting, such as continued application of a second improved feeding method or other types of feeding methods, process 1400 proceeds to optional “other” step 1428. From optional step 1428, process 1400 is performed to harvest or remove cells from bioreactor 1430, and process 1400 may then terminate at end operation 1432.

[0182] Turn Figure 15A According to embodiments of the invention, exemplary operational steps 1500 are provided for a process of feeding cells to hold them in a first location, such as the bioreactor itself, which can be used with, for example, a CES 500 (e.g., Figure 5B and 5C This is used in conjunction with a cell expansion system. Operation is initiated at 1502, and process 1500 proceeds to loading a disposable tubing kit or pre-installed fluid delivery assembly (e.g., 210 or 400) 1504 onto the cell expansion system. Next, the system can be perfused 1506. In one embodiment, for example, a user or operator can provide instructions to the system to perfuse by, for example, selecting a task for perfusion. In one embodiment, such a task for perfusion may be a pre-programmed task. Next, an IC / EC flushing task 1508 can be performed, where, for example, fluid can be replaced in the IC and EC circulation loops. Depending on the embodiment, the replacement volume can be determined by the number of IC and EC volumes exchanged.

[0183] Next, to maintain an appropriate or desired gas concentration across the filaments in the bioreactor membrane, a conditioning task 1510 can be performed so that the medium is balanced with the provided gas supply before cells are loaded into the bioreactor. For example, contact between the medium and the gas supply provided by the gas delivery module (GTM) or oxygenator can be provided by adjusting the EC circulation rate. The system can then be maintained in the appropriate or desired state until, for example, a user or operator is ready to load cells into the bioreactor. In one embodiment, the system can be conditioned, for example, with a medium such as a complete medium. A complete medium can be any medium source used for cell growth. In one embodiment, the system can be conditioned, for example, with a serum-free medium. In one embodiment, the system can be conditioned with a basal medium. Any type of medium understood by those skilled in the art can be used.

[0184] For example, next, process 1500 proceeds to the operation of loading cells 1512 from the cell inlet bag into the bioreactor. In one embodiment, for example, the cells in the cell inlet bag may be in solution with a culture medium to nourish the cells. In another embodiment, the cells in the cell inlet bag may be in solution with a culture medium to nourish the cells and also contain a soluble activator complex that can stimulate cells (e.g., T cells or Tregs). In one embodiment, cells may be loaded from the cell inlet bag into the bioreactor until the bag is empty. Cells may be removed from the air removal chamber into the bioreactor. In one embodiment, a "cell loading under uniform suspension" task may be performed to load the cells. In another embodiment, a "concentrated cell loading without circulation" task may be performed to load the cells into a specific, for example, central region of the bioreactor. Depending on the embodiment, other loading methods and / or loading tasks may be used.

[0185] Next, process 1500 proceeds to feed cells for each first process within the first time period 1514. In one embodiment, cells can be fed at a minimum or low feed rate, for example, a minimum or low feed rate that can meet the feed requirements of such a cell population when it begins to grow / expand. For example, an IC inlet pump rate of +0.1 mL / min can be used during this first time period to induce or generate a first fluid flow rate of 0.1 mL / min. According to the embodiment, although this example provides a low or minimum feed rate of 0.1 mL / min, the low or minimum feed rate can be greater than or equal to about 0.01 mL / min and less than or equal to about 0.1 mL / min. In the embodiment, the low or minimum feed rate can be greater than 0.1 mL / min. If it is desired to reduce cell loss in the hollow fiber membrane bioreactor during this first time period, the +0.1 mL / min IC inlet pump rate can be matched or approximately or substantially matched with a complementary -0.1 mL / min IC circulation pump rate during the cell culture growth phase to maintain cells in the bioreactor. Other pump speeds and resulting fluid flow rates can be used in other implementations.

[0186] Next, according to the implementation plan, process 1500 proceeds to inquiry 1516 to determine whether to adjust the nutrient supply rate to retain cells within the bioreactor itself, while also considering cell population growth and increased nutrient demand. For example, Figure 15B This shows an increased nutrient supply rate in response to an increased cell population. For example... Figure 15B As shown, graph 1528 illustrates the effects of cell expansion systems, such as... The cell number in a cell expansion system is relative to the IC flow rate. In one embodiment, the IC flow rate includes the culture medium used to feed the cells, and therefore may also be referred to as the IC culture medium flow rate. The cell number 1530 is shown relative to the IC flow rate (mL / min) 1532. As shown, according to one embodiment, the IC flow rate increases from 0.1 mL / min to 0.2 mL / min to 0.3 mL / min as the number of cells increases and the nutrient requirements for the growth of the expanded cell population increase. In the illustrated embodiment, as shown by line 1534, a substantially linear relationship may exist between the cell number and the IC flow rate.

[0187] return Figure 15AUpon inquiry 1516, if no adjustment of the feed rate is desired, process 1500 proceeds to "No" to expand cells 1520, where, for example, the cells can continue to grow / expand using the culture medium supplied during the first time period 1514. Although cell expansion is shown in step 1520, cells may also grow / expand during, for example, one or more other steps (such as 1512, 1514, 1516, 1518). On the other hand, if it is desired to adjust the feed rate while holding the cells in the bioreactor, process 1500 proceeds to "Yes" to feed the cells in each of the second processes during the second time period 1518. In one embodiment, such a second process, for example, "feeding," may involve feeding the cells at a feed rate substantially the same as that used during the first time period. In another embodiment, the second process may include feeding the cells at a different feed rate compared to the feed rate used during the first time period. In one embodiment, the IC inflow rate may be increased, and the IC circulation flow rate may be set, configured, or adjusted to be equal to a percentage, portion, or fraction of the IC inflow rate. For example, according to one embodiment, the IC circulation flow rate can be set to approximately fifty percent (50%) or approximately half (1 / 2) of the IC inflow flow rate value, or another percentage or portion according to the embodiment. According to one embodiment, it can be determined whether to set, configure, or adjust the IC circulation flow rate to a percentage, fraction, or portion of the IC inflow flow rate based on the value of the IC inflow flow rate. For example, one embodiment provides a method for retaining cells in a bioreactor (where Q) when feeding cells using an IC inflow flow rate. IC循环 =IC circulation rate (mL / min); Q IC进入 =IC inflow rate (mL / min):

[0188] When Q IC进入 When Q is ≥0.2 mL / min IC循环 = (-)1 / 2*Q IC进入

[0189] as well as

[0190] When Q IC进入 When Q = 0.1 mL / min, IC循环 =(-)Q IC进入 .

[0191] Although the above equations provide different calculations of the IC circulation flow rate based on IC inflow rate values ​​(e.g., 0.2 mL / min or 0.1 mL / min), other IC inflow rate values ​​used for this determination may be used according to other embodiments. Furthermore, while approximately fifty percent (50%) or approximately half (1 / 2) is used in this example, other percentages, proportions, fractions, and / or portions may be used according to embodiments. Returning to process 1500, after feeding the cells with this flow and countercurrent characteristic as part of a second process 1518 to hold the cells in the bioreactor, process 1500 continues to grow / expand the cells 1520. Although cell expansion is shown in step 1520, the cells may also be grown / expanded in one or more other steps (such as 1512, 1514, 1516, 1518). Starting from expansion step 1520, process 1500 proceeds to harvest or remove cells 1524. Process 1500 may then terminate at end operation 1526. If any additional steps are required before harvesting cells, process 1500 proceeds to optional “other” step 1522, depending on the implementation scheme. From optional step 1522, process 1500 continues to harvest or remove cells from bioreactor 1524, and process 1500 may then terminate at end operation 1526.

[0192] Next, Figure 16 Exemplary operational steps 1600 of a process for culturing cells according to an embodiment of the present invention are shown, which can be used with, for example, a CES 500 (e.g., Figure 5B and 5CThis is used in conjunction with a cell expansion system. For example, in start-up operation 1602, a disposable kit is loaded onto the cell expansion system, the system is perfused, IC / EC rinsed, the culture medium is conditioned, and cells are loaded. Next, process 1600 proceeds to feed cells during a first time period 1604. In one embodiment, cells can be fed at a minimum or low feed rate, for example, when a cell population begins to grow / expand, a minimum or low feed rate can meet the feeding needs of such a population. For example, an IC inlet pump rate of +0.1 mL / min can be used during this first time period. According to the embodiment, although this example provides a low or minimum feed rate of 0.1 mL / min, the low or minimum feed rate can be greater than or equal to about 0.01 mL / min and less than or equal to about 0.1 mL / min. In the embodiment, the low or minimum feed rate can be greater than 0.1 mL / min. If it is desired to reduce cell loss in the hollow fiber membrane bioreactor during this first time period, the IC inlet pump rate of +0.1 mL / min can be matched, approximately matched, or substantially matched with the complementary IC circulation pump rate of -0.1 mL / min during the cell culture growth phase to maintain cells in the bioreactor during the cell culture growth phase.

[0193] Next, process 1600 proceeds to inquiry 1606 to determine whether the feeding rate can be adjusted to address the increasing cell number and / or to continue efforts to hold the cells in the bioreactor. If it is desired to adjust the feeding rate while holding the cells in the bioreactor, process 1600 proceeds to "yes" to feed the cells according to the second process within the second time period 1608. In one embodiment, such a second process, for example, "feeding," may involve feeding the cells at a feeding rate substantially the same as that used during the first time period. In another embodiment, the second process may include feeding the cells at a different feeding rate compared to the feeding rate used during the first time period. In one embodiment, the IC inflow rate may be increased, and the IC circulation rate may be set, configured, or adjusted to be equal to a percentage, part, or fraction of the IC inflow rate. For example, according to one embodiment, the IC circulation rate may be set to be equal to about fifty percent (50%) or about half (1 / 2) of the IC inflow rate value, or another percentage or part according to the embodiment. According to one embodiment, it may be determined whether the IC circulation rate is set, configured, or adjusted to a percentage, fraction, or part of the IC inflow rate based on the value of the IC inflow rate. For example, one implementation provides a method for retaining cells in a bioreactor when cells are fed with an IC flow rate (where Q...). IC循环 =IC circulation rate (mL / min); Q IC进入 =IC inflow rate (mL / min):

[0194] When QIC进入 ≥0.2mL / min, Q IC循环 = (-)1 / 2*Q IC进入

[0195] as well as

[0196] When Q IC进入 When Q = 0.1 mL / min, IC循环 =(-)Q IC进入 .

[0197] Although the above equations provide different calculations of the IC circulation flow rate based on IC inflow rate values ​​(e.g., 0.2 mL / min or 0.1 mL / min), other IC inflow rate values ​​used for this determination may be used according to other embodiments. Furthermore, while approximately fifty percent (50%) or approximately half (1 / 2) is used in this example, other percentages, proportions, fractions, and / or portions may be used according to embodiments. Returning to process 1600, after feeding the cells with this flow and countercurrent characteristic as part of a second process 1608 to hold the cells in the bioreactor, process 1600 proceeds to inquiry 1610 to determine whether to monitor or measure the metabolic activity (e.g., glucose consumption and / or lactate production) of the growing cell population. If monitoring of metabolic activity is not desired, process 1600 proceeds to "No" to expand the cells 1616, in which the cells may continue to grow / expand using the same culture medium supplied, for example, in the first time period 1604 and / or the second time period 1608. Although cell expansion is shown in step 1616, cells can also be grown / expanded in, for example, one or more other steps (such as 1604, 1606, 1608, 1610, 1612, 1614).

[0198] Returning to query 1610, if it is desired to monitor or measure the metabolic activity of the growing cell population, process 1600 clicks "Yes" to continue feeding the cells based on their metabolic activity and / or measurements, or to adjust the feeding rate accordingly. In one embodiment, monitoring glucose and / or lactate levels can facilitate the regulation of the culture medium flow rate (e.g., IC flow rate) to support the expansion of cells (e.g., T cells or Tregs) in a bioreactor (e.g., a hollow fiber bioreactor). In one embodiment, for example, the cell culture lactate value can be maintained below about 7 mmol / L. In another embodiment, during the expansion of cells (e.g., regulatory T cells), the lactate metabolic waste products from glycolysis can be maintained, for example, below about 7 mmol / L, by controlling the rate of culture medium addition, for example, through the use of a graphical user interface (GUI) of a cell expansion system. In other embodiments, for example, the rate of culture medium addition and / or other settings can be controlled to attempt to maintain lactate levels ≤ about 5 mmol / L, for example, to improve cell growth and viability. Other concentrations may be used in other embodiments.

[0199] For example, depending on the metabolic measurements and the desired lactate level, process 1600 proceeds to continue feeding cells according to the second process 1612 or adjust the feeding rate 1614. For example, according to one embodiment, cell feeding can continue according to the second process 1612, wherein the measurement of the second metabolic activity shows, for example, a lactate level ≤ about 5 mmol / L. In another embodiment, cell feeding can continue according to the second process 1612, wherein the measurement of the second metabolic activity shows, for example, a lactate level ≤ about 7 mmol / L. From the second step 1612 to continue feeding cells, process 1600 returns to query 1610 to continue monitoring the metabolic activity of the growing cell population.

[0200] Depending on the metabolic measurements and their desired levels, process 1600 proceeds to adjusting the feed rate 1614, where cells can be fed according to additional processes over additional time periods. According to embodiments, such additional processes and additional time periods may include, for example, a third process over a third time period, a fourth process over a fourth time period, a fifth process over a fifth time period, and so on. In one embodiment, for example, such an additional process may involve feeding cells at a feed rate substantially the same as during the first and / or second time periods. In another embodiment, the additional process may involve feeding cells at a different feed rate compared to the feed rate used during the first and / or second time periods. For example, in one embodiment, the IC inflow rate may be increased, and the IC circulation rate may be matched, closely matched, or substantially matched to the IC inflow rate, but in the opposite direction. In another embodiment, the IC inflow rate may be increased, and the IC circulation rate may be set, configured, or adjusted to be equal to a percentage, fraction, or portion of the IC inflow rate, and in the opposite direction. While adjusting the feed rate 1614 in step 1614 illustrates “additional” processes and “additional” time periods, any number of processes and time periods can be used to adjust the feed rate based on metabolic activity.

[0201] Process 1600 returns to query 1610 from adjusting the feed rate 1614. If no adjustment or further adjustment of the feed rate is desired, process 1600 proceeds to "No" to amplify the cells 1616, where the cells can continue to grow / amplify using the culture medium supplied during the first time period 1604, the second time period 1608, and / or the additional time period 1614. Although cell amplification is shown in step 1616, the cells may also grow / amplify in, for example, one or more other steps (such as steps 1604, 1606, 1608, 1610, 1612, 1614). Starting from amplification step 1616, process 1600 proceeds to harvest or remove cells 1618 from the bioreactor and into, for example, a harvest bag or container. Process 1600 may then terminate at end operation 1622. Alternatively, starting from harvest operation 1618, process 1600 may optionally proceed to enable further processing / analysis 1620. This optional further processing / analysis 1620 may include, for example, characterizing the phenotype of the harvested cells (e.g., T cells or Tregs). Then, from the optional further processing / analysis step 1620, process 1600 may terminate at end operation 1622.

[0202] Figure 17AThe diagram illustrates operational steps 1700 of a cell expansion process that can be used with a cell expansion system according to an embodiment of the present invention. As described below, according to an embodiment of the invention, process 1700 may include the step of shearing cells already expanded in a cell growth chamber. In embodiments, these steps may be implemented as part of an "improved cycle" task. Start-up operation 1702 is initiated, and process 1700 proceeds to loading a fluid 1704 containing cells into the cell growth chamber of the cell expansion system. In embodiments, the cells may comprise non-adherent cells, such as one or more types of T cells. In one embodiment, the cells comprise Tregs.

[0203] Process 1700 proceeds to exposing the cells to an activator 1706. An activator, which may include an antibody complex, may be added to the fluid loaded in step 1704. In this embodiment, the activator may be a soluble human antibody CD3 / CD28 / CD2 cell activator complex. Process 1700 proceeds to a first time period for cell amplification 1708. Step 1708 may include feeding 1710 cells. Nutrients may be provided to the cells to promote their amplification. For example, a culture medium containing glucose, protein, and reagents may be delivered to a cell growth chamber to provide nutrients for cell amplification.

[0204] The initial timeframe 1708 for cell amplification can be based on the time required for the formation of cell communities, microcommunities, or clusters. A cell community, microcommunity, or cluster can be a group of one or more attached cells. In implementations, cells (e.g., Tregs) may benefit from cell contact. Cell contact can stimulate signaling that promotes amplification and growth. However, after a period of amplification, cells may attach to each other, forming cell communities, microcommunities, or clusters. Without being bound by theory, it is believed that after the initial timeframe 1708 of cell amplification, cells can form relatively large, continuing-growing cell communities, microcommunities, or clusters. Cell communities, microcommunities, or clusters may create necrotic centers where nutrients (e.g., glucose), gases (e.g., oxygen), and reagents (e.g., activators) cannot reach the cells at the center of the cell community, microcommunity, or cluster. As a result, cell amplification conditions at the center of these cell communities, microcommunities, or clusters may cause the amplification rate to slow down (e.g., increase doubling time), or these conditions may lead to cell necrosis.

[0205] In implementations, to enable cell proliferation 1708, the first time period can be between about 5 hours and about 48 hours. In some implementations, the first time period can be greater than about 6 hours, greater than about 12 hours, greater than about 24 hours, or even greater than about 48 hours. In other implementations, the first time period can be less than about 72 hours, less than about 60 hours, less than about 48 hours, less than about 36 hours, less than about 24 hours, or even less than about 12 hours. After the first time period, process 1700 proceeds to cycle 1712 to shear cell communities, microcommunities, or clusters during a second time period. Step 1712 can be performed to reduce the size of cell communities, microcommunities, or clusters. In implementations, the second time period can be less than about 120 minutes, such as between about 60 minutes and about 0.5 minutes. In other implementations, the second time period can be based on the volume of fluid introduced into the first circulation path.

[0206] Figure 17B Multiple views 1750, 1760, and 1770 show cells in a volume of fluid (1752) that can be expanded in a cell growth chamber as part of process 1700. For example, in some embodiments, the cell growth chamber may be a hollow fiber bioreactor. In these embodiments, for example, views 1750, 1760, and 1770 may show cells in the fibers of a hollow fiber bioreactor. 1750A, 1760A, and 1770A are magnified in portions of views 1750, 1760, and 1770, respectively. Referring to view 1750, cells can be shown after they have been loaded 1704, exposed to activator 1706, and expanded 1710 within a time period, for example, a first time period. As shown in view 1750, multiple cell communities 1754A-E have been formed.

[0207] To reduce the number and size of cells in cell communities, microcommunities, or clusters 1754A-E, step 1712 may circulate the fluid and cells through a first fluid circulation path. Without being bound by theory, it is believed that circulation can generate forces acting on the cell community, including shear stress, as indicated by arrow 1756 in the enlarged portion 1760A. Shear stress 1756 can provide sufficient force to separate cells within the cell community. As shown in view 1760, as circulation continues, the cell community may begin to break down into smaller sizes. View 1770 illustrates the cells after circulation in a second time period. As shown in view 1770, the size of the cell community has decreased, with some communities completely separated into single cells. In some embodiments, the cyclic shearing step 1712 may be performed until the cells and fluid comprise a single-cell suspension.

[0208] In other embodiments, cell communities, microcommunities, or clusters may be preserved after the shearing cycle 1712. For example, colony 1754F in the enlarged portion 1770A illustrates that some reduced-size communities may be preserved after step 1712. In embodiments, the remaining cell communities, microcommunities, or clusters (e.g., 1754F) may be between about 25 micrometers and about 300 micrometers. In other embodiments, the shearing cycle 1812 may reduce the size of the cell communities, microcommunities, or clusters (e.g., 1754F), so that the cell communities, microcommunities, or clusters may be between about 50 micrometers and about 250 micrometers. In other embodiments, step 1712 may reduce the size of the cell communities, microcommunities, or clusters to between about 75 micrometers and about 200 micrometers. In some embodiments, after step 1712, the size of the cell communities, microcommunities, or clusters may be less than about 200 micrometers, for example, about 100 micrometers.

[0209] In implementations, the size of the remaining cell communities, microcommunities, or clusters may be a function of certain structural features of the cell growth chamber. As mentioned above, in some implementations, the cell growth chamber may be a hollow fiber bioreactor with hollow fibers. It is understood that the cell communities, microcommunities, or clusters are subjected to shear stress each time they come into contact with the sidewalls of the hollow fibers during their circulation. This contact can more effectively reduce the size of the cell communities. When the inner diameter is large, contact with the sidewalls may not occur frequently, such as in conventional methods that may utilize pipettes to induce shear stress to reduce community size. Figure 17C The difference in inner diameter between one embodiment of a hollow fiber (e.g., 215 micrometers) 1772 and a pipette tip 1774 (762 micrometers) is shown, which can be used to dissociate attached cells from cell communities, microcommunities, or clusters. In this embodiment, the smaller inner diameter of the hollow fiber is considered to more effectively reduce the size of the cell community during the cycle to the shearing step 1712.

[0210] In some implementations, the second time interval for cyclically cutting 1712 can be less than about 120 minutes, less than about 90 minutes, less than about 60 minutes, less than about 30 minutes, or even less than about 15 minutes. In some implementations, the second time interval can be between about 15 minutes and about 1 minute, such as about 4 minutes.

[0211] Following the second time period, process 1700 proceeds to the third time period, where cells are moved into the cell growth chamber 1714. In step 1714, due to the recirculation to shear step 1712, cells not placed in the cell growth chamber are moved back into the cell growth chamber during the third time period. In embodiments, this may involve activating one or more pumps to introduce fluid into a fluid circulation path. For example, fluid may be introduced from the inlet and outlet ports of the cell growth chamber into a first fluid flow path and then into the cell growth chamber. The movement of fluid into the cell growth chamber from the inlet and outlet ports can move cells back into the cell growth chamber.

[0212] In some embodiments, the fluid used in the step of transferring cells back to cell growth chamber 1714 may include reagents that promote cell growth. For example, in embodiments, the fluid may be a culture medium comprising glucose, protein, or other reagents. In one embodiment, the fluid may include one or more supplements. In one embodiment, the fluid is a complete culture medium and contains cytokines (e.g., a human IL-2 cytokine supplement). The addition of the fluid may be referred to as bolus addition. In embodiments, the combination of steps 1712 and 1714 may be referred to as cyclic and bolus addition.

[0213] In other embodiments, the third time period may be based on the volume of fluid introduced into the cell growth chamber during the cyclic shearing step 1712. For example, in one embodiment, step 1712 may be performed until approximately 300 ml, approximately 250 ml, approximately 200 ml, or approximately 150 ml have been introduced into the fluid circulation path.

[0214] Following the third time period, process 1700 proceeds to amplification 1716 during the fourth time period. Similar to step 1708, step 1716 may include cell feeding 1718. Nutrients may be supplied to the cells to promote their amplification. For example, a culture medium containing glucose, protein, and reagents may be delivered to the cell growth chamber to provide nutrients for cell amplification.

[0215] Similar to the first time period, the fourth time period can be based on the time taken for cell community formation. In some embodiments, the fourth time period can be from about 5 hours to about 48 hours. In some embodiments, the fourth time period can be greater than about 6 hours, greater than about 12 hours, greater than about 24 hours, or even greater than about 48 hours. In other embodiments, the fourth time period can be less than about 72 hours, less than about 60 hours, less than about 48 hours, less than about 36 hours, less than about 24 hours, or even less than about 12 hours. Because more cells may be present in the cell growth chamber, in some embodiments, the fourth time period can be shorter than the first time period.

[0216] Following the fourth time interval, process 1700 proceeds to step 1720 to cyclically shear the fifth time interval to reduce the second cell community. In one embodiment, step 1720 may use a first circulation rate. However, in other embodiments, the circulation rate used at step 1720 may be greater than or less than the first circulation rate.

[0217] After the fifth time period, process 1700 proceeds to step 1722, in which cells not placed in the cell growth chamber can be moved back into the cell growth chamber during a sixth time period. Fluid can be introduced from the fluid inlet path into the first fluid flow path and into the cell growth chamber from the inlet and outlet ports. The movement of fluid into the cell growth chamber from the inlet and outlet ports moves the cells back into the cell growth chamber. In some embodiments, the fluid used to move the cells back into the cell growth chamber may include reagents that promote cell growth. For example, in one embodiment, the fluid may be a culture medium comprising glucose, protein, or other reagents. In one embodiment, the fluid may include one or more supplements. In one embodiment, the fluid is a complete culture medium and includes cytokines (e.g., human IL-2 cytokine).

[0218] As shown in optional steps 1724 and ellipsis 1726, process 1700 may optionally include additional cycles of cell amplification, cycling, and migration. The amplification, cycling, and migration steps may be performed sequentially for a period of time. For example, in some embodiments, the steps may be performed every four days, every three days, every two days, once a day, twice a day, or three times a day, for a period of approximately two days to approximately twenty days (e.g., approximately 10 days). In some embodiments, the steps may be performed at different time intervals. For example, in one embodiment, the steps may be performed after three days, and then every other day. As another example, the steps may be performed after two days, and then twice a day. These are merely examples, and other embodiments may utilize other time intervals.

[0219] For example, Figure 18 Curve 1800 shows the effects of cycling and high-dose addition at different times during cell expansion. Curve 1808 shows the effects of cell expansion systems (e.g., [missing information]). In a cell expansion system, the relationship between cell number 1802 and cell culture days 1804 is shown. Curve 1806 illustrates the relationship between cell number 1802 and cell culture days 1804 in a cell expansion system (e.g., [missing information]). The relationship between IC50 flow rate (1818) and cell culture days (1804) on the cell expansion system. As shown in curve 1806, the IC50 flow rate was maintained at 0.1 mL / min for the first three days. On day six, the flow rate increased to 0.2 mL / min, and after day seven, it increased to 0.3 mL / min. The increase in flow rate with increasing culture days (1804) corresponds to an increase in cell number. Similarly, as... Figure 18 The steps shown involve several cycles and high-dose additions (e.g., 1810, 1812, 1814, and 1816). Cycles with high-dose addition of 1810 were performed after 3.5 days of cell culture. Another cycle with high-dose addition of 1812 was performed after 4.5 days of cell culture. Another cycle with high-dose addition of 1814 was performed after 6 days of cell culture, and another cycle with bolus addition of 1816 was performed after 6.5 days of cell culture. Observations of curves 1806 and 1808 suggest that multiple cycles and high-dose additions (1810–1816), combined with increased IC50 flow rates, may have a positive impact on cell expansion rates (e.g., cell count).

[0220] Return to reference Figure 17A Process 1700 proceeds to cell removal 1728 from the cell growth chamber. In an embodiment, this may involve harvesting cells. Step 1728 may include additional steps, such as cyclical steps (e.g., 1712 and 1720) before or during cell removal from the cell growth chamber. Process 1700 terminates at end operation 1730.

[0221] Figure 19A Operational steps 1900 of a process for operating a pump that can be used in a cell expansion system according to an embodiment of the invention are shown. As described below, according to an embodiment of the invention, process 1900 may include the step of activating the pump during the reduction of cells in a cell cluster already expanded in a cell growth chamber. In embodiments, these steps may be implemented as part of an "improved cycle" task. In embodiments, the steps of process 1900 may be performed by a computer processor. A start-up operation 1902 is initiated, and process 1900 proceeds to step 1904, whereby a first pump is activated at a first flow rate to introduce a first volume of fluid comprising cells into the capillary portion of the bioreactor of the cell expansion system. In embodiments, the first pump may be an inlet pump.

[0222] After activating the first pump at a first flow rate, process 1900 proceeds to activating the first pump 1906 at a second flow rate to introduce a nutrient-rich culture medium into the capillary portion of the bioreactor within a first time period. Nutrients may include, for example, proteins, glucose, and other compounds for nourishing and promoting cell proliferation. For example, see reference... Figure 19BThe first pump 1960 can be activated at a second flow rate to introduce culture medium into the inlet port 1962A of the bioreactor 1962.

[0223] In some implementations, the first time period can be based on the time taken for cell community formation. In other implementations, the first time period can be from about 5 hours to about 48 hours. In some implementations, the first time period can be greater than about 6 hours, greater than about 12 hours, greater than about 24 hours, or even greater than about 48 hours. In other implementations, the first time period can be less than about 72 hours, less than about 60 hours, less than about 48 hours, less than about 36 hours, less than about 24 hours, or even less than about 12 hours.

[0224] Then, process 1900 continues by starting the second pump at a third flow rate to direct fluid into bioreactor 1908. An optional step 1908 may be performed to start pump 1964 to direct a portion of the fluid introduced in step 1906 to, for example, the outlet port 1962B of bioreactor 1962 to feed cells.

[0225] Then, process 1900 continues by activating the second pump at a fourth flow rate 1910 to circulate the cells during the second time period and reduce the number of cells in the cell clusters within the bioreactor. In this embodiment, the cells circulate in the first fluid circulation path. (Reference) Figure 19C The second pump 1964 may be activated in step 1910 to circulate fluid through the first fluid circulation path 1966, as shown by arrows 1968A-D.

[0226] Unbound by theory, it is believed that after a period of time, the expanded cells can form cell communities, microcommunities, or clusters. Cell communities may create necrotic centers where nutrients and proteins (e.g., activators) do not reach. As a result, cell expansion conditions at the centers of these cell communities, microcommunities, or clusters may slow down the expansion rate (e.g., increase doubling time) and may lead to cell necrosis. Step 1910 can be performed to reduce the size of the cell communities, microcommunities, or clusters.

[0227] In some embodiments, the fourth flow rate can be high enough to induce shear. For example, in some embodiments, the fourth flow rate can be as high as about 1000 ml / min. In other embodiments, the fourth flow rate can be from about 100 ml / min to about 600 ml / min, for example, 300 ml / min.

[0228] Following the second time period, process 1900 proceeds to step 1912, where the first pump is activated at a fifth flow rate to introduce fluid through the first fluid flow path during the third time period. In an embodiment, the fluid introduced into the first portion of the first fluid flow path can transfer the first cell (potentially in the first fluid flow path due to step 1910) back into the cell growth chamber through inlet port 1962A. Reference Figure 19D Pump 1960 can be activated to introduce fluid into the first fluid flow path 1970. As indicated by arrows 1968A and 1968B, fluid flows from the first fluid flow path 1970 into inlet port 1962A. This moves the first cells outside bioreactor 1962 back into bioreactor 1962. It is believed that moving cells from the first fluid flow path back into the cell growth chamber improves overall cell expansion because cell growth conditions are optimized within the cell growth chamber.

[0229] In some embodiments, the fluid introduced into the first fluid flow path in step 1912 may include one or more materials that promote cell expansion (e.g., reagents). For example, in one embodiment, the fluid may be a culture medium containing glucose or other nutrients for nutrient supply to the cells. In one embodiment, the fluid may include reagents, which may include additional activators for further activating cell expansion. The use of a fluid containing specific reagents or other materials to transfer cells back to the cell growth chamber and expose cells to other agents that can promote expansion (e.g., growth factors, proteins, etc.) may improve cell expansion. In one embodiment, the additional fluid used in step 1912 may be referred to as a high-dose addition.

[0230] Furthermore, after the third time period, in step 1914, a second pump can be activated at a sixth flow rate to move the second portion of fluid introduced through the first fluid flow path and the second cell into the cell growth chamber through outlet port 1962B. In an embodiment, the second portion of the fluid can be used to move the second cell (which may be in the first fluid flow path due to step 1910) back into the cell growth chamber through the outlet port. Reference Figure 19D As shown, pump 1964 can be activated to move the fluid introduced into the first fluid flow path 1970 to the outlet port 1962B. As indicated by arrow 1968C, pump 1964 moves the fluid and cells in the first fluid circulation path through outlet port 1962B into bioreactor 1962. Figure 19D As shown, the sixth flow velocity can be in the opposite direction to the fourth flow velocity. Figure 19C This fluid movement moves cells outside bioreactor 1962 back into bioreactor 1962.

[0231] In the implementation scheme, the sixth flow rate can be less than the fifth flow rate, as described above, which moves the fluid into the first fluid flow path. It is understood that the fifth flow rate can cause a certain volume of fluid to be introduced into a portion of the circulation path 1966 based on the fifth flow rate. The sixth rate can be set to cause a portion of this volume to move towards the outlet port 1962B.

[0232] In some implementations, the sixth flow rate can be set as a percentage of the fifth flow rate. For example, the sixth flow rate can be less than or equal to about 90% of the fifth flow rate. In some implementations, the sixth flow rate can be set to be less than or equal to about 80% of the fifth flow rate. In other implementations, the sixth flow rate can be less than or equal to about 70% of the fifth flow rate. In other implementations, the sixth flow rate can be less than or equal to about 60% of the fifth flow rate. In some implementations, the sixth flow rate can be less than or equal to about 50% of the fifth flow rate.

[0233] In the implementation scheme, the sixth flow rate is based at least in part on the difference between the first volume between the second pump and the inlet port and the second volume between the second pump and the outlet port. (Reference) Figure 19D As shown, portions of the first circulation path can have different volumes. For example, in one embodiment, the first volume between the second pump 1964 and the inlet port 1962A can have a first volume, and the second volume between the second pump 1964 and the outlet port 1962B can have a second volume that is different from (e.g., larger) than the first volume. In these embodiments, in order to move cells back from the circulation path into the bioreactor, such that the cells typically arrive at the bioreactor at approximately the same time, the sixth flow rate can be set at least in part based on the differences in these volumes.

[0234] It is understood that when fluid enters fluid circulation path 1966 from the first fluid flow path 1970, the fluid flows towards inlet port 1962A at a flow rate set at the first pump 1960. When pump 1964 is activated, it redirects at least a portion of the fluid to outlet port 1962B. In an embodiment, the second volume (the volume between 1964 and outlet port 1962B) can be larger than the first volume. Therefore, in order to move more fluid into the second volume, the second pump 1964 can be set to a percentage of the rate of pump 1960.

[0235] In one embodiment, in step 1912, pump 1960 can be set to 100 ml / min. In this embodiment, the second volume (from pump 1964 to outlet port 1962B) can be larger than the first volume (from pump 1964 to inlet port 1962A). To account for the additional volume, in step 1914, pump 1964 can be set to 70 ml / min. This embodiment allows cells to arrive at bioreactor 1962 approximately simultaneously within a third time period.

[0236] In the implementation, as indicated by ellipses 1916 and optional step 1918, process 1900 may optionally perform steps 1906-1914 multiple times. The following initiation steps: first pump (at a second rate) 1906, second pump (at a fourth rate) 1910, first pump (at a fifth rate) 1912, and second pump (at a sixth rate) 1914 can be performed continuously for a period of time. As described above, the following steps can be performed to feed cells, circulate cells to disrupt cell communities, microcommunities, or clusters, and transfer cells back to the cell growth chamber. For example, in some implementations, the steps may be performed every three days, every two days, daily, twice daily, or three times daily for a period of approximately two days to approximately twenty days (such as 10 days). In some implementations, the steps may be performed at different time intervals. For example, in one implementation, the steps may be performed after three days and then every other day. As another example, the steps may be performed after two days and then twice daily. This is only one example, and other implementations may utilize other time intervals. Process 1900 terminates at end operation 1920.

[0237] Note that in some embodiments, process 1900 may include additional steps. For example, a shaking device may be attached to the bioreactor, and after the first time period (and during the second time period), when the first pump is started in step 1910, the shaking device may be activated to rotate the bioreactor as part of the circulating cells to reduce the number of cells in the cell cluster. This is merely one example, and other embodiments of process 1900 are not limited thereto.

[0238] Figure 20 An embodiment according to the present invention is shown, which can be used with, for example, CES 500 (e.g., Figure 5A ) or CES 600 ( Figure 6Exemplary operating steps 2000 for a cell expansion process used with a cell expansion system. Start-up operation 2002 is initiated, and process 2000 proceeds to loading a disposable tube kit 2004 onto the cell expansion system. Next, the system can be perfused 2006. In one embodiment, for example, a user or operator can instruct the system to perform perfusion by selecting a task for perfusion. In one embodiment, such a task for perfusion may be a pre-programmed task. Process 2000 then proceeds to an IC / EC flushing task 2008, in which fluids in the IC and EC circulation loops are replaced. The replacement volume depends on the amount of IC and EC volumes exchanged.

[0239] Next, to maintain an appropriate or desired gas concentration on the fibers throughout the bioreactor membrane, a conditioning task 2010 can be performed to allow the culture medium to reach equilibrium with the supplied gas supply before cells are loaded into the bioreactor. For example, rapid contact between the culture medium and the gas supply provided by the gas delivery module or oxygenator can be achieved by using a high EC circulation rate. The system can then be maintained in the appropriate or desired state until, for example, the user or operator is ready to load cells into the bioreactor. In one embodiment, the system can be conditioned using a complete culture medium, for example. The complete culture medium can be any culture medium source used for cell growth. In one embodiment, for example, the complete culture medium may contain α-MEM (α-MEM) and fetal bovine serum (FBS). Any type of culture medium understood by those skilled in the art can be used.

[0240] Next, process 2000 is performed, for example, to centrally load cells without circulation 2012 from the cell entry bag into the bioreactor. In an embodiment, the task of "centrally loading cells without circulation" can be used, where a first volume of fluid comprising multiple cells can be loaded at a first flow rate into a cell expansion system, wherein the cell expansion system includes a cell growth chamber. A second volume of fluid comprising culture medium can then be loaded at a second flow rate into a portion of the first fluid circulation path, for example, to place the first volume of fluid in a first portion of the cell growth chamber. In one embodiment, the cell growth chamber or the first portion of the bioreactor may comprise approximately the central region of the bioreactor. In one embodiment, the first volume is the same as the second volume. In one embodiment, the first flow rate is the same as the second flow rate. In another embodiment, the first volume is different from the second volume. In another embodiment, the first flow rate is different from the second flow rate. In one embodiment, for example, the sum of the first and second volumes may be equal to a percentage or proportion of the volume of the first fluid circulation path, such as the total volume. For example, the sum of the first and second volumes may be, for example, about 50% of the volume of the first fluid circulation path (e.g., the total volume). In one embodiment, fluid in a first fluid circulation path flows through the capillary in-cell (IC) space of a bioreactor or cell growth chamber. In one embodiment, fluid in a second fluid circulation path flows through, for example, the capillary out-of-cell (EC) space of a cell growth chamber or bioreactor. In one embodiment, for example, the sum of the first and second volumes may be about 50% of the volume of the capillary in-cell (IC) loop, or another percentage or proportion according to the embodiment. In one embodiment, the sum of the first and second volumes may be about 50% of the volume of another applicable fluid path, loop, etc., or another percentage or proportion according to the embodiment. According to the embodiment, other percentages or proportions may be used, including, for example, any percentage from about 1% to about 100%.

[0241] Following cell loading 2012, the next process 2000 proceeds to donor cell 2014. According to the embodiment, cells can grow / expand 2016. Although step 2016 is shown after step 2014, step 2016 can occur before or concurrently with step 2014. Next, process 2000 proceeds to query 2018 to determine whether any cell communities, microcommunities, or clusters have formed. A cell community, microcommunity, or cluster can be a group of one or more attached cells. If a cell community, microcommunity, or cluster has formed, process 2000 proceeds to "Yes" to shear 2020 any cell community, microcommunity, or cluster. For example, after expanding multiple cells in a first time period, cells can be circulated at a first circulation rate in a second time period to reduce the number of cells in the cell community, microcommunity, or cluster. In the embodiment, circulating cells at the first circulation rate can cause shear stress in the cell community, where one or more cells in the cell community may separate from the cell community. In one embodiment, for example, reducing the number of cells in a cell community, microcommunity, or cluster can provide a single-cell suspension. In one embodiment, circulating cells to cleave any community, microcommunity, or cluster 2020 can be used, for example, every two (2) days during cell culture to maintain uniform cell density and nutrient diffusion. Other time periods may also be used according to the embodiment. In one embodiment, for example, cleavage of any microcommunity, community, or cluster can begin on or after day 4. Other dates or time periods may be used to begin this cleavage according to the embodiment. After cleavage 2020, process 2000 can then return to the donor cells 2014.

[0242] If it is determined at query 2018 that no cell communities or clusters are to be cut, or if no cell communities or clusters are present, process 2000 proceeds to "No" to resuspend cells 2022. In an embodiment, circulating cells may be used to uniformly resuspend cells that may have loosely adhered during culture. In an embodiment, step 2022 may include circulating cells to uniformly resuspend those cells that may have loosely adhered before the start of a harvest task or other task to remove cells from the bioreactor. After cell resuspension in 2022, process 2000 proceeds to harvest cells 2024. Optionally, further processing or other analysis of the removed cells may be performed in step 2026, and then process 2000 may terminate at end operation 2028. If further processing / analysis is desired, process 2000 terminates at end operation 2030.

[0243] According to the implementation plan, transfer to Figure 21In process 2100, the start operation 2102 is initiated, and process 2100 proceeds to loading the disposable kit 1204 onto the cell expansion system. The disposable kit can then be perfused 2106, and an IC / EC washing step 2108 can occur. Next, the culture medium 2110 can be adjusted. Process 2100 then proceeds to loading cells 2112, such as suspension cells or non-adherent cells, such as T cells or Tregs. In one embodiment, such cells 2112 can be loaded via a "concentrated loading of cells without circulation" task. In another embodiment, such cells 2112 can be loaded via a "loading of cells in uniform suspension" task.

[0244] Next, according to the implementation scheme, process 2100 begins with the donation of cells 2114, which may start on day 0. The cells can grow and expand 2116, and, for example, by day 3, it may be necessary to add a large dose of fluid to the IC circuit and redistribute the cells 2118. In one implementation scheme, this large dose of fluid may include reagents such as cytokines or other growth factors. In another implementation scheme, this large dose of fluid may, for example, include reagents and basal culture medium.

[0245] Following this large-dose addition and redistribution of cells, process 2100 proceeds to cell refeeding 2120, which, for example, may occur on day 3. Using this refeeding 2120, system parameters 2122 (such as controlling the flow rate of one or more pumps) can be controlled to achieve complementary flow and countercurrent settings to allow fluid to enter the bioreactor from both the IC inlet and IC outlet ports. For example, the IC inlet pump 2124 can be adjusted or directed to generate flow, while the IC circulation pump 2126 can be adjusted or directed to generate countercurrent. For example, an IC inlet pump rate of 0.1 mL / min can be matched, approximately matched, or substantially matched with a complementary IC circulation pump rate of -0.1 mL / min to maintain cells in the bioreactor during the growth phase of cell culture (e.g., in an embodiment, possibly 4-7 days). This control of setting 2122 makes it possible to counteract any forces associated with cell loss from the IC outlet port of the bioreactor.

[0246] Process 2100 then proceeds to inquiry 2128, in which, for example, it is determined whether to continue adding reagents on other dates or at other time intervals, or to perform another large dose addition. If it is expected that additional reagents or other large doses will be added and cells will be redistributed, process 2100 branches to "Yes" to add reagents and redistribute cells 2118. Depending on the implementation, for example, this large dose addition and cell redistribution may occur on subsequent days 6 and 9.

[0247] If it is not desired, or not desired at all, to continue adding large doses (e.g., reagents) and redistributing cells, process 2100 proceeds to "No" to harvest cells 2130, where cells can be transferred to a harvest bag or container. Process 2100 then terminates at end operation 2136.

[0248] Alternatively, starting from harvesting operation 2130, process 2100 may optionally proceed to enable further processing / analysis 2132. This further processing 2132 may include characterizing, for example, the phenotype of the harvested cells (e.g., T cells or Tregs). From the optional further processing / analysis step 2132, process 2100 may continue to optionally reload any remaining cells 2134. Process 2100 may then terminate at ending operation 2136.

[0249] Process 2200 illustrates the operational steps of a process for amplifying cells in a cell expansion system according to an embodiment of the present invention. In some embodiments, process 2200 can be used to amplify T cells. As shown, various steps can be performed during a 14-day protocol to amplify cells. Startup operation 2202 is initiated, and process 2200 proceeds to day 0, where a disposable kit is loaded onto the cell expansion system 2206. The disposable kit can then be perfused 2208, where the 2208 kit can be perfused with PBS (e.g., Lonza without Ca2+ / Mg2+). When ready to load cells, the perfusion medium can be exchanged using IC / EC wash buffer 2210. For example, according to one embodiment, the PBS in the system can be replaced with TexMACS GMP basal medium. The medium 2212 can then be conditioned. Conditioning of medium 2212 can be performed to bring the medium into equilibrium with the provided gas supply before loading cells into the bioreactor.

[0250] Next, on day 0, process 2200 proceeds to loading cells 2214, such as suspension cells or non-adherent cells, such as T cells or Tregs. In one embodiment, such cells 2214 can be loaded via a "concentrated loading of cells without circulation" task. In another embodiment, such cells 2214 can be loaded via a "loading of cells with uniform suspension" task.

[0251] On day 3, 2216, a large dose of cytokines may be added during cell redistribution, 2218. In one embodiment, cell redistribution may be combined with the large dose addition to mix the cells and expose them more thoroughly to the cytokines (e.g., IL-2) that may be present in the large dose addition. In another embodiment, redistribution may also disrupt any cell communities or clusters that may have already formed. In one embodiment, redistribution may first occur by circulating the cells in a fluid circulation path. The large dose addition may then be added during the process of pushing the cells back into the bioreactor, such as by introducing fluid into the fluid circulation path to push the cells back into the bioreactor. After redistribution and the large dose addition, 2218, process 2000 proceeds to donor cells, 2220.

[0252] On day 6 (2222), cells can be redistributed again with another large dose (2224). Redistribution disrupts cell communities or clusters that may have formed during days 3-5. Large doses expose cells to other agents that promote expansion. Process 2000 continues until day 6 when cells are fed (2226). On day 9 (2228), cells can be redistributed again with a large dose (2230). Redistribution may disrupt cell communities or clusters that formed during days 6-8. Large doses expose cells to other supplements that promote expansion. Process 2000 continues until day 9 when cells are fed (2232).

[0253] On days 11-13 2234, cells 2236 can be redistributed using a large dose addition. This redistribution may disrupt the cell communities or clusters formed during days 9-10. The large dose addition can expose the cells to other agents that promote expansion. Process 2000 then proceeds to cell 2238 for feeding. In this embodiment, steps 2236 and 2238 can be performed on each of days 11, 12, and 13. This is likely due to the result of cell expansion during days 0-10 and the presence of a large number of cells in the bioreactor. Performing cell redistribution and large dose addition can promote cell expansion by more frequently disrupting cell communities and clusters and mixing them with the agents in the large dose addition. Process 2200 terminates at end operation 2240.

[0254] Process 2300 illustrates the operational steps of a process for expanding cells (e.g., suspension cells or non-adherent cells) in a cell expansion system according to embodiments of the present invention. In some embodiments, process 2300 can be used to expand T cells, such as Tregs. The combination of steps in process 2300 can enable the expansion of cells to a useful clinical quantity using an initial low seeding density.

[0255] The process 2302 begins, and process 2300 proceeds to loading the disposable kit 2304 onto the cell expansion system. The disposable kit can then be perfused 2206, whereby the kit can be perfused with, for example, PBS (e.g., Lonza without Ca2+ / Mg2+). When ready to load cells, the perfusion medium can be exchanged using IC / EC flush 2308. For example, according to one embodiment, the PBS in the system can be replaced with TexMACS GMP basal medium. The medium 2310 can then be conditioned. Conditioning of medium 2310 can be performed to equilibrate the medium with the supplied gas before loading cells into the bioreactor.

[0256] Process 2300 proceeds to 2312, loading an inlet volume containing fluid with cells. In an embodiment, the cells may comprise non-adherent cells, such as one or more types of T cells, for example, Tregs. In one embodiment, the cells comprise Tregs. An embodiment may provide loading of the inlet volume containing fluid with cells into an IC circulation path via an IC inlet pump. In an embodiment, loading the inlet volume 2312 occurs without activating the IC circulation pump.

[0257] Process 2300 proceeds to placing the inlet volume into the first portion of bioreactor 2314. In one embodiment, placement can be achieved by introducing a second volume of fluid, which may include culture medium and can be introduced into a portion of the IC circulation path to push the cell inlet volume into the first position of the bioreactor. In one embodiment, the inlet volume and the second volume of fluid may be the same. In other embodiments, the inlet volume and the second volume of fluid may be different. In other embodiments, the sum of the inlet volume and the second volume of fluid may be equal to a percentage of the volume of the IC circulation path.

[0258] After placement into inlet volume 2314, process 2300 proceeds to expose cells to activator 2316 to activate cell expansion. In some embodiments, cells may be exposed to a soluble activator 2316. In some embodiments, an activator comprising an antibody complex may be added to the culture medium and may be included in the inlet volume or added subsequently, such as with a second volume. In embodiments, the activator may be, for example, a human antibody CD3 / CD28 / CD2 cell activator complex.

[0259] Process 2300 proceeds to the first time period 2318, during which cells are fed in each first process. In an embodiment, cells can be fed at a minimum or low feeding rate, for example, at the first time period 2320 when the cell population begins to grow / expand, and the minimum or low feeding rate is sufficient to meet the needs of the population. For example, an IC inlet pump rate of +0.1 mL / min can be used during this first time period. If it is desired to reduce cell loss in the hollow fiber membrane bioreactor during this first time period, the +0.1 mL / min IC inlet pump rate can be matched, approximately matched, or substantially matched with a complementary -0.1 mL / min IC circulation pump rate to maintain cells in the bioreactor during the growth phase of the cell culture.

[0260] Starting at 2320, process 2300 involves cell expansion in the second time period 2322. Cell expansion in the second time period 2322 may also involve feeding cells according to a second process in the second time period 2324. In one embodiment, this second process may involve feeding cells at, for example, a feeding rate substantially the same as that used in the first time period. In another embodiment, the second process may involve feeding cells at a different feeding rate compared to the feeding rate used during the first time period. For example, the feeding rate may be increased due to cell expansion in the first time period.

[0261] During the second time period 2322, while cells are being expanded, cells may also be circulated to shear cell communities or clusters 2326. Step 2326 may include circulating cells in the IC circulation pathway to shear cell communities or clusters that may have formed in the first time period. The shearing of communities or clusters 2326 step may reduce the number of cells in the cell community or cluster. In an embodiment, circulating to shear 2326 may cause shear stress in the cell community, causing one or more cells in the cell cluster to separate from the cell community.

[0262] The next step, 2300, proceeds to harvesting operation 2328, where cells can be transferred to harvest bags or containers. In this embodiment, a therapeutic dose of cells can be harvested. In this embodiment, the cells harvested at operation 2328 can be in quantities of 1 × 10⁻⁶ cells. 9 On the order of magnitude of cells. In this implementation, the harvested cells may have a viability of approximately 75% to approximately 95%.

[0263] Process 2300 may then optionally proceed to enable further processing / analysis 2330. This further processing may include characterizing, for example, the phenotype of the harvested cells (e.g., T cells or Tregs). In one embodiment, the harvested cells may express biomarkers consistent with those of Tregs. For example, the cells may express CD4+, CD25+, and / or FoxP3+ biomarkers. In one embodiment, the harvested cells may include the CD4+CD25+ phenotype at a frequency greater than about 80%. In other embodiments, the cells may include the CD4+FoxP3+ phenotype at a frequency greater than about 55%. Process 2300 may then terminate at an end operation 2332.

[0264] According to embodiments of the present invention, the operational steps depicted in the above figures are provided for illustrative purposes and can be rearranged, combined into other steps, used in parallel with other steps, etc. Fewer or additional steps may be used in embodiments without departing from the spirit and scope of the invention. Furthermore, in some embodiments, steps (and any sub-steps) such as perfusion, conditioning of the culture medium, and loading of cells can be performed automatically, such as by a processor carrying pre-programmed tasks stored in memory; these steps are provided for illustrative purposes only. Further, Figure 10B An example pump speed setting, for example for nutrient cells, is depicted for illustrative purposes. Other pump speeds, flow rates, directions, etc., can be used according to embodiments of the invention.

[0265] Examples and further descriptions of tasks and schemes, including custom and pre-programmed tasks, for use with cell expansion systems are provided in U.S. Patent Application Serial No. 13 / 269,323 (“Configurable Method and System for Growing and Harvesting Cells in a Hollow Fiber Bioreactor System,” filed October 7, 2011) and U.S. Patent Application Serial No. 13 / 269,351 (“Customizable Method and System for Growing and Harvesting Cells in a Hollow Fiber Bioreactor System,” filed October 7, 2011), the entire contents of which are incorporated herein by reference.

[0266] Next, Figure 24 Exemplary components of a computing system 2400 on which embodiments of the present invention can be implemented are shown. For example, the computing system 2400 can be used in embodiments where a cell expansion system uses a processor to perform tasks (such as custom tasks or pre-programmed tasks) as part of processes such as those shown and / or described herein. In embodiments, for example, pre-programmed tasks may include following “IC / EC flushing” and / or “donor cells”.

[0267] The computing system 2400 may include a user interface 2402, a processing system 2404, and / or a memory 2406. As those skilled in the art will understand, the user interface 2402 may include an output device 2408 and / or an input device 2410. The output device 2408 may include one or more touchscreens, wherein the touchscreen may include a display area for providing one or more application windows. The touchscreen may also be an input device 2410, which may, for example, receive and / or capture physical touch events from a user or operator. As those skilled in the art will understand, the touchscreen may include a liquid crystal display (LCD) with a capacitive structure that enables the processing system 2404 to infer the location of the touch event. The processing system 2404 may then map the location of the touch event to a UI element presented at a predetermined location in the application window. According to embodiments, the touchscreen may also receive touch events via one or more other electronic structures. As those skilled in the art will understand, other output devices 2408 may include a printer, a speaker, etc. Other input devices 2410 may include a keyboard, other touch input devices, a mouse, a voice input device, etc.

[0268] According to embodiments of the present invention, the processing system 2404 may include a processing unit 2412 and / or memory 2414. The processing unit 2412 may be a general-purpose processor operable to execute instructions stored in the memory 2414. According to embodiments, the processing unit 2412 may include a single processor or multiple processors. Further, in embodiments, each processor may be a multi-core processor having one or more cores to read and execute individual instructions. As will be understood by those skilled in the art, processors may include general-purpose processors, application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), other integrated circuits, etc.

[0269] According to the implementation scheme, memory 2414 may include any short-term or long-term memory for data and / or processor-executable instructions. As those skilled in the art will understand, memory 2414 may include, for example, random access memory (RAM), read-only memory (ROM), or electrically erasable programmable read-only memory (EEPROM). As those skilled in the art will understand, other storage media may include, for example, CD-ROM, magnetic tape, digital versatile disc (DVD) or other optical storage, magnetic tape, disk storage, magnetic tape, other magnetic storage devices, etc.

[0270] Memory 2406 can be any long-term data storage device or component. According to embodiments, memory 2406 may include one or more systems described in conjunction with memory 2414. Memory 2406 may be permanent or removable. In embodiments, memory 2406 stores data generated or provided by processing system 2404.

[0271] Example

[0272] The following description includes compatibility with, for example, CES 500 (e.g., Figure 5A , 5B 5C) and / or CES 600 Figure 6 Examples of schemes / methods / processes used in conjunction with cell expansion systems. For example, they implement aspects of the implementation scheme. Although specific features may be described in the examples, such examples are provided for illustrative and descriptive purposes only. For example, although the examples may provide expansion of T cells and / or Treg cells, other and / or additional cell types and / or combinations thereof may be used in other embodiments. Although specific parameters, characteristics, and / or values ​​are described according to some embodiments, such as using CES, for example... Cell expansion systems are described, but these parameters, characteristics, and / or values ​​are for illustrative purposes only. The invention is not limited to the examples and / or specific details provided herein.

[0273] Furthermore, the examples provided herein are not intended to limit other implementations, which may include different or additional steps, parameters, or other features. In some implementations, exemplary methods or schemes including steps (and any sub-steps) can be performed automatically, such as by a processor performing pre-programmed tasks stored in memory. In other implementations, steps (and any sub-steps) can be performed by a combination of automatic and manual operation. In further implementations, steps (and any sub-steps) can be performed by an operator or user or by other manual means.

[0274] While exemplary data may be provided in such instances, such exemplary data is provided for illustrative purposes only and is not intended to limit other implementations, which may include different steps, parameters, values, materials or other characteristics.

[0275] Example 1

[0276] method

[0277] Conventional Treg cell culture

[0278] CD4 separated by immunomagnetic assay + CD25 + Tregs can be obtained from peripheral blood of healthy adult donors via leukoablation (HemaCare Corporation, Van Nuys, CA), and can then be sterile filtered TexMACS in Terumo BCT. TM 1.0 × 10⁻⁶ in GMP medium 5Cells were expanded to a concentration of 200 IU / mL using three T25 flasks (7 mL / flask) supplemented with a mixture of recombinant human IL-2 IS-1 super cytokine (Miltenyi Biotec GmbH, Bergisch Gladbach) and Gibco PSN 100X antibiotics (Thermo Fisher Scientific, Waltham, MA). The actively growing Treg cell suspension was subsequently used as inoculum in each of the three (3) run of the Quantum Cell Expansion System experiments. In the absence of microbeads, soluble tetramer Immunocult was used. TM Human antibody CD3 / CD28 / CD2 cell activator complex (Stem Cell Technologies, Vancouver, BC) was used to co-stimulate inoculum and Tregs amplified using the Quantum system at a concentration of 25 μL / mL. For the Treg inoculum, co-stimulation could be performed on days 0 and 9, while for Quantum system Treg amplification, co-stimulation could be performed on day 0. The Quantum system HFM bioreactor is characterized by a capillary inner ring volume of 177.1 mL and a surface area of ​​21000 cm². 2 .

[0279] Quantum system Treg amplification

[0280] According to the implementation plan, two (2L) bags of sterile filtered culture medium can be prepared using a Quantum 4L Culture Bag Set (Cat. 21021) for the scale-up of Tregs in the Quantum system. A 2L complete culture bag containing TexMACS GMP, IL-2, and PSN antibiotics can be used to supply the IC compartment, while a 2L basal culture bag containing TexMACS GMP and PSN antibiotics can be used to supply the EC inlet compartment of the bioreactor. After perfusing the Quantum system (Lonza Cat. 17-516Q, Walkersville, MD) with PBS, the culture bags can be connected to the appropriate IC and EC inlet lines using a TSCD-Q Terumo sterile welder. The complete culture medium can be protected from light exposure.

[0281] Aseptic techniques can be used to achieve a total cell loading of 4.5-6.5 × 10⁻⁶ cells per run. 7Tregs were resuspended in 50 mL of complete culture medium, which was then introduced into the Quantum system bioreactor using a Quantum cell entry bag (Cat. 21020). Other disposable bags, such as a 4 L Quantum CES medium bag (Cat. 21021) and a 4 L waste bag (Cat. 21023), can also be used during Treg scale-up operations.

[0282] After completing the task of "concentrated loading of cells without circulation", cells can be loaded into 177 mL of complete culture medium at a rate of 2.5–3.7 × 10⁻⁶. 5 The concentration was 2.1–3.1 × 10³ cells / mL, or an average of 2.1–3.1 × 10³ cells / cm² within the lumen of a hollow fiber membrane bioreactor or capillary (IC). 2 Tregs were inoculated on the Quantum system running system (n=3).

[0283] Days 0-4

[0284] Example Quantum Custom Task

[0285] Donor cells, improved IC / EC exchange and regulatory medium for regulatory T cells, examples

[0286] Using a Terumo BCT TSCD-Q aseptic welding machine, attach TexMACS GMP complete medium with IL-2 supplement (200 IU / mL) to the Quantum system's IC medium line. Connect the TexMACS basal medium to the EC medium line. Perform IC / EC flushing and conditioning tasks separately. The complete medium can be used for IC exchange or flushing, and the basal medium can be used for EC exchange or flushing to conserve the amount of IL-2 and activator complex.

[0287] Before introducing cells, the system was placed on modified "donor cells". On days 5, 6 and 7, the IC ingress rate (Q1) and IC circulation rate (Q2) were increased to match the rates to 0.2, 0.3 and 0.4 mL / min, respectively, but on days 4, 5 and 6 the opposite was true or it was necessary to maintain lactate levels between 5 and 8 mmol / L.

[0288] Table 1: Donor Cells, Improved Methods, Examples

[0289]

[0290] Example Quantum custom task:

[0291] Cells are loaded centrally without circulation, as in the example.

[0292] Objective: This task enables the concentrated distribution of suspended cells within the bioreactor membrane, while simultaneously allowing them to flow in the extracapillary (EC) circulation loop. The pump flow rate to the IC loop can be set to zero.

[0293] Before loading cells into the Quantum system using a centralized loading method without loops, input the modifications to the task.

[0294] Table 2: Centralized cell loading without circulation, modified, example.

[0295]

[0296] Table 3: Examples of centralized cell loading without circulation

[0297]

[0298] Return to the default cell feeding task as needed and continue using the cell feeding task expansion protocol.

[0299] Table 4: Donor cells, improvements, examples.

[0300]

[0301]

[0302] Day 4 or later:

[0303] Resuspend Treg cells during cell culture or before harvest, as in the example.

[0304] The purpose of the improved cycle task is to uniformly suspend loosely adhered cells during the culture process or before the start of the "harvest task".

[0305] In addition, this task can be used to cleave the Treg cell population every two (2) days during cell culture to maintain uniform cell density and nutrient diffusion from day 4 onwards. If this task is used to cleave the population during culture, the Quantum system may revert to the modified “donor cell” task.

[0306] Table 5: Cell circulation and resuspension, returning cells to the bioreactor and providing nutrients, examples.

[0307]

[0308] With improved Quantum harvest task, example

[0309] Table 6: Results, Improvements, and Examples

[0310]

[0311]

[0312] Harvested cells can be removed from the Quantum system via RF welding for further evaluation and analysis.

[0313] Post-harvest analysis

[0314] Harvested cells can be counted in the 5-50 μm range using the Vi-CELL XR 2.04 Cell Viability Analyzer (Beckman Coulter), and membrane integrity can be quantified using the trypan blue dye exclusion method.

[0315] metabolism

[0316] T cell metabolism can be monitored via the daily Quantum EC sample port using the i-STAT handheld analyzer (Abbott Point of Care, NJ, Princeton, NJ). The i-STAT G box (Cat.03P83-25) and i-STAT CG4+ box (Cat.03P85-50) were used for glucose and lactate concentrations, respectively.

[0317] Cell surface biomarker expression

[0318] Human regulatory T cells (natural and induced) constitute a small fraction (2-10%) of all T cells in human cord blood and peripheral blood. Functionally, Tregs are responsible for maintaining immune homeostasis, including the regulation of immune tolerance in both innate and adaptive responses. Furthermore, the transcriptional regulation of the forkhead box P3 (FoxP3) gene product is known to be related to CD4+. + CD25 + FoxP3 + CD127 lo / - Treg phenotype and antigen-presenting cells (APCs) and effector T cells (T cells) eff IL-2 is associated with immunosuppression. The binding of IL-2 to the CD25 / IL-2 receptor (Rα) and the activation of the STAT5 transcription factor are involved in Foxp3 induction. Foxp3 inhibition upregulates the activity of several genes (such as CTLA-4, TNFRSF18, and IL2RA) and downregulates IL-2 by binding to histone acetyltransferase KAT5 and histone deacetylase HDAC7.

[0319] The frequency of the Treg phenotype of the harvested cell surface biomarkers can be quantified by flow cytometry. For this purpose, cells can be stained using the following antibody-drug conjugates, with gating applied to unstained live cells: FixableViability. 780 (eBioscience 65-0865), mouse anti-human CD4-PE (BD Pharmingen 561844), anti-CD4-Alexa Fluor 647 (BD Pharmingen 557707), anti-CD4-FITC (BD Pharmingen 561842), anti-CD4-FITC (BD Pharmingen 555346), anti-CD25-PE (BD Pharmingen 555432), anti-CD127-PE (BD Pharmingen 557938), anti-CD45RO-PE (BD Pharmingen 347967), and anti-FoxP3-Alexa Fluor 647 (BD Pharmingen 560045). Sample data were acquired on a bead-compensated BD Canto II flow cytometer equipped with FACSDiva v6.1.3 software, using 1×10⁸ samples per sample. 6 Cells and a total of 20,000 events.

[0320] Experimental flowchart, examples

[0321] Experimental flowchart as follows Figure 25 As shown.

[0322] Possible results

[0323] Preliminary studies of Tregs in static cultures may indicate that these cells tend to form microcolonies with a diameter of approximately 100 μm. Isolating these cells every two days during culture medium exchange may help limit cell necrosis and restore the cells to a high-density single-cell suspension using 1000 μL pipette tips with an ID of 762 μm. Alternatively, the process of maintaining a single-cell suspension could be accomplished more efficiently in an automated HFM bioreactor (e.g., in the Quantum system) with a pre-programmed daily cycle task. Furthermore, such automated feeding tasks can reduce the possibility of contamination while maintaining a continuous flow of nutrients to the Treg cultures, as they can be performed in a functionally closed system.

[0324] Possible Treg cell density and viability

[0325] Table 7: Potential Harvest from Bottle T25

[0326] <![CDATA[8.71×10 5 Cells / cm2 vitality 81.90％ Stimulation cycle 2 Doubling 4.9

[0327] Table 8: Potential Gains from Quantum

[0328]

[0329]

[0330] Preliminary cell seeding density experiment

[0331] When preparing immunomagnetically selected cells from donors for expansion in an automated bioreactor, a series of static growth experiments can be performed to determine whether growth can be achieved at a rate less than 1.0 × 10⁻⁶. 6 Culture stimulated Tregs at a seeding density of 1.0 × 10⁶ cells / mL. This can be achieved by seeding 1.0 × 10⁶ cells / mL in 18 wells of a 24-well tissue culture plate. 5 Cells / mL or in wells of TexMACS GMP medium supplemented with IL-2 (200 IU / mL) and PSN antibiotics, this part of the study was performed. These cells were also co-stimulated with 25 μL / mL of a soluble anti-CD3 / CD28 / CD2 mAb complex on days 0 and 9. Cells could be collected manually and counted by Vi-CELL XR on day 14.

[0332] Table 9: Summary of possible static plate tests for Treg cell seeding.

[0333] Abbreviations: Doubling (DS), Doubling Time (DT).

[0334]

[0335] After harvesting, cell samples can be pooled for Treg biomarker analysis by flow cytometry. Possible results may indicate that, in static culture, CD4... + C25 + CD4 + CD127 - and CD4 + FoxP3 + The frequencies of the phenotypes were 90.9%, 79.7%, and 31.6%, respectively. CD4 + C25 + Phenotypic (>70%) is usually the most reliable determinant for Treg biomarker identification because FoxP3+ detection is highly dependent on permeability methods and cell viability.

[0336] Data from this static plate test, when cultured in the presence of a soluble co-stimulatory anti-CD3 / CD28 / CD2mAb complex and serum-free medium, may indicate that human Tregs can thrive at cell seeding densities of approximately 10⁻⁶ cells / year. 5 It may expand when the number of cells / mL is 1.

[0337] Treg metabolism

[0338] Regulatory T cells depend on mitochondrial metabolism and have the ability to oxidize multiple carbon sources, namely lipids or glucose. It is well known that Tregs, upon entering a highly proliferative state, shift their metabolism from fatty acid oxidation (FAO) to glycolysis due to mTOR's regulation of glycolysis and fatty acid metabolism. Furthermore, it has been shown that glycolysis may be essential for the generation and repression of human inducible Tregs, as investigated by regulating the expression of FoxP3 variants via the IL-2 / STAT5 / enolase-1 promoter signaling pathway and 2-Deoxy-D-glucose inhibition. Therefore, monitoring glucose and lactate levels could facilitate the regulation of Quantum system medium flow rates to support Treg expansion in hollow fiber bioreactors. Initially, it was thought that Tregs temporarily reduced their metabolic rate before entering the cell cycle and proliferating. This may be supported by the transient decrease in glycolysis and mTOR activity in freshly isolated human Tregs prior to TCR stimulation. Specifically, mTORC1 may be thought to increase the expression of glucose transporters, such as Glut-1-mediated glucose transport, due to the upregulation of the mTOR pathway.

[0339] Possible results from three (3) amplifications from three separate Treg cell aliquots may suggest that glucose consumption and lactate production appear to be correlated in each Quantum system run. All three in vitro amplification experiments may indicate that glucose consumption in Tregs may increase above baseline levels by day 1, while two of the three experiments may indicate that lactate production levels may increase above baseline levels by day 2. Decreased cell viability upon thawing may result in a lag in lactate production rates, which may be reflected in cell harvest. In the most actively growing Treg cultures, the maximum glucose consumption rates in two of the three experiments were likely 1.618 and 2.342 mmol / day on days 8 and 7, respectively. At the same time points, the maximum lactate production rates were likely 2.406 and 3.156 mmol / day.

[0340] Throughout the Treg expansion process, lactate levels could be controlled to ≤7 mmol / L by simultaneously increasing the IC input (+) and IC circulation (-) pump rates within the hollow fiber membrane lumen from ±0.1 to ±0.4 on days 4–8. The lowest glucose levels during Treg cell expansion were likely between 264 mg / dL on day 7 (Q1584) and 279 mg / dL on day 8 (Q1558). A basal glucose concentration of 325–335 mg / dL in the cell growth formulations used for these feasible expansions was found to be supportive when used in conjunction with Quantum system flow rate regulation.

[0341] Regulatory T cell biomarker expression

[0342] In this feasibility study, the assessment of Treg cell harvesting by flow cytometry may focus on CD4. + CD25 + FoxP3 + On T cell subsets: In T lymphocytes, the human CD4 gene on chromosome 12 encodes a membrane glycoprotein that interacts with the major histocompatibility complex (MHC) class II and plays an early role in initiating T cell activation. In regulatory T cells, the human CD25 (IL2R) gene on chromosome 10 encodes the IL-2 receptor and functions by isolating the cytokine IL-2. In regulatory T cells, the human fork / winged box P3 gene on chromosome X encodes the FoxP3 transcription factor, which is likely essential for Treg repressor function. The FoxP3 gene product binds to the promoter regions of the CD25, CTLA-4, and IL-2, IL7R, and IFN-γ genes, thereby upregulating CD25 and CTLA-4 and repressing the transcription of IL-2, IL7R, and IFN-γ genes. The CD127 gene encodes the IL-7 receptor, and Tregs are typically characterized by low CD127 (IL-7R) expression compared to conventional T cells. However, certain Treg subsets are known to express high levels of CD127 during activation in vitro and in vivo, which may be related to higher Treg survival rates when cells are incubated with IL-7. The CD45RO gene product is expressed on Tregs derived from immature thymus glands, which lose CD45RA and express CD45RO upon activation.

[0343] Table 10: Percentage of parental population expressing possible regulatory T cell biomarkers. *For FoxP3+ frequency, low cell viability at Q1567 thaw / harvest, and incomplete permeability.

[0344]

[0345]

[0346] According to one implementation scheme, CD4 cells harvested from the Quantum system + CD25 + The mean expression frequency of the Treg phenotype is likely 85.5%, which is consistent with published CD4... + CD25 + Release criteria >70% are better compared to other methods. In Q1567 Treg amplification, CD4 + CD127 低The elevated population frequency (74.2%) likely reflects low cell viability in this particular thawed cell sample, as these cells can only be cultured with IL-2 as a cytokine supplement. In cells operated via two Quantum systems with both inoculation and harvest viability exceeding 80%, CD4... + FoxP3 + The expression frequency is likely 61.6%. This finding may be related to FoxP3. + The release version specifications are ≥60% consistent. Furthermore, the results of this two billion cell expansion are comparable to CD4 in the original donor Treg cell samples that may have been obtained from HemaCare BioResearch Products. + CD25 + (87.30%), CD25 + (47.76%) and FoxP3 + (59.64%) better expression of biomarkers.

[0347] Additional flow cytometry analyses can be performed on cryopreserved Treg cells in Q1584 amplification buffer run by a third-party laboratory using fluorescence Minus One (FMO) gating, different staining agents, and different instruments. The FMO control is a gated control used to interpret cell populations by subtracting the diffusion of fluorescent dyes used to quantify the frequency of a specific marker from the distribution of all fluorescent dyes in the data plot. For example, flow results from a third-party laboratory might indicate that, starting from the Q1584 run, CD4+... + CD25 + The frequency of the Treg cell population was likely 95.4%, which is favorable compared to the 90.5% found in the Terumo BCT CES lab. Incomplete staining with an alternative anti-FoxP3-PE clonal staining agent may limit the quantification of this internal biomarker by third-party laboratories, but the punctuation pattern may indicate high FoxP3 expression in the Q1584 specimen. + A subset of Treg cells, but possibly not observed in control Treg cell reference samples. While interesting, further studies may be needed to confirm these observations.

[0348] Harvest yield

[0349] During the Q1558, Q1567, and Q1584 runs, which were defined within the 5–50 μm range for 50 samples per run, the average diameter of surviving Treg cells (excluding trypan blue) in the Quantum system was approximately 10.89, 11.04, and 11.06 μm, respectively. This can be compared to the average cell diameters of 11.91, 12.40, and 7.83 μm in the flasks at the time of bioreactor inoculation.

[0350] These possible cell diameter data may indicate that the cell diameter harvested from the Quantum system may be more uniform than that of cells expanded in inoculation bottles.

[0351] The possible harvest data for the Treg Quantum system are summarized in Table 11. Furthermore, when comparing the results of the Q1554 / 1584 harvest with those of the Q1567 harvest, CD4... + CD25 + The effect of cell viability can be significant when inoculating bioreactors. The viability of the bioreactor inoculum from the Q1554 / 1584 amplification experiments may be 32-41% higher than that of the bioreactor inoculum from the Q1567 run. This could be due to variations in original cell isolation, cryopreservation techniques, or storage time, as the cell aliquots (HemaCare PB425C-2; Lot14034019) that may have been used in this study may have originated from the same donor on February 11, 2014.

[0352] Table 11: Possible expansion of Treg cells from inoculation bottles to Quantum system harvest. Abbreviations: DS - population doubling, DT - population doubling time (hours). A. Harvest data may be based on Vi-Cell XR counts with trypan blue to ensure membrane integrity. B. Note: Treg cell inoculation bottles may receive two (2) rounds of co-stimulation on days 0 and 9; while Tregs harvested from the Quantum system may receive one (1) round of co-stimulation on day 0.

[0353]

[0354] The purpose of this feasibility study may be to determine whether the Quantum system can use commercially available supplements to support Tregs at 7.0 × 10⁻⁶. 7 -1.4×10 9 Cellular-scale expansion. Using a soluble anti-CD3 / CD28 / CD2 mAb co-stimulatory complex, at a seeding density of less than 1.0 × 10⁻⁶. 6 Cells / mL not exceeding eight (8) days, two of the three bioreactors obtained from Q1554 and Q1584 could produce an average of 1.56 × 10⁻⁶ cells / mL. 9 Treg. During Q1554 / 1584 operation, the average harvested cell density in the IC loop of the Quantum system bioreactor was 8.81 × 10⁻⁶. 6 Cells / mL or 7.43 × 10⁻⁶ 4 cells / cm 2 .

[0355] Possible conclusions

[0356] The results of this feasibility study are likely to be exploratory in nature and not necessarily designed to cover all technical options. For example, reducing Treg co-stimulation from two (2) activation events to one (1) activation event could be considered. This would allow for improvements to the methods used in the automated Quantum system expansion of immunomagnetically isolated regulatory T cells. Our attempt here may be to define certain technical aspects of the culture process that could contribute to further investigation of Treg expansion within the Quantum system platform. In this case, possible findings could suggest that these possible conclusions or observations are reasonable and could contribute to the production of regulatory T cells for research, development, or manufacturing purposes.

[0357] When the cytokine IL-2 was supplemented in the Quantum automated hollow fiber bioreactor, it was cultured and identified as FoxP3. + / CD25 + Human Treg cells can be expanded using a soluble co-stimulatory anti-CD3 / CD28 / CD2 monoclonal antibody (mAb) T cell complex in the absence of co-stimulatory mAb-coated magnetic beads.

[0358] From less than 1×10 6 Cells / mL or less than 6.6 × 10⁻⁶ 4 cells / cm 2 The cell seeding density was optimal for the efficient amplification of human Tregs in the Quantum system. Therefore, cells ranging from 7.0 × 10⁻⁶ cells could be collected in less than 14 days. 8 Up to 1.4×10 9 The goal of Tregs between cells can achieve an average (n=3) of 1.09 × 10⁻⁶. 9 Total cells. On average, 85.5% of cells expressed Treg CD4. + CD25 + Phenotypically, an average of 42.9% of cells expressed CD4. + FoxP3 + Phenotype (n=3). In Quantum system expansion with two (2) billion cells, an average of 61.6% of the total cells expressed CD4. + FoxP3 + Phenotype. Due to cell number limitations, one of the three Quantum Treg cell expansion runs using CD4 cells could be validated via human IMSR by a third-party laboratory. + CD25 + Express.

[0359] Human Tregs can be successfully cultured and expanded in the Quantum system by concentrated seeding of cells within the lumen (IC loop) of an automated hollow fiber bioreactor.

[0360] The IC input (+0.1 to +0.4 mL / min) and IC circulation (-0.0 to -0.4 mL / min) of the culture medium can be adjusted in parallel to support the Treg cell expansion process, maintain lactate levels ≤7 mmol / L, and maintain single-cell suspensions of Treg cultures by shuffling cell microcommunities through the Quantum system HFM bioreactor cavity at an IC circulation rate of 300 mL / min under functionally closed conditions.

[0361] Example 2

[0362] The table below provides an exemplary protocol for T-cell expansion over several days, with exemplary task settings (e.g., flow rate, rotation angle, outlet, etc.) for different components of the cell expansion system (e.g., pump, rocker, valve, etc.). This protocol can be followed in the following order:

[0363] Day 0: Loading kits, perfusion, adding culture medium, loading cells and starting nutrient supply.

[0364] Day 3: While redistributing cells, add a large dose of cytokines to the IC loop. Restart feeding.

[0365] Day 6: While redistributing cells, add a large dose of cytokines to the IC loop. Restart feeding.

[0366] Day 9: While redistributing cells, add a large dose of cytokines to the IC loop. Restart feeding.

[0367] Days 11-13: Harvest; reload the remaining cells. Harvest (Day 14)

[0368] Settings table: Changes made compared to the exemplary factory settings are highlighted in bold and underline.

[0369] Table 12: IL-2 concentrations and numbers in complete culture medium, examples

[0370]

[0371] Table 13: Volume and Amount of IL-2 Added in Large Dose, Examples

[0372]

[0373] Table 14: Day 0-2 Setup, Example

[0374]

[0375]

[0376] Table 15: Days 3-5 Setup, Example

[0377]

[0378]

[0379] Table 16: Settings for Days 6-8, Example

[0380]

[0381] Table 17: Day 9-10 Setup, Example

[0382]

[0383]

[0384] Table 18: Day 11 Setup, Example

[0385]

[0386]

[0387] Table 19: Day 12 Setup, Example

[0388]

[0389] Table 20: Day 13 Setup, Example

[0390]

[0391]

[0392] Table 21: Day 14 Setup, Example

[0393]

[0394] It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and structures of this invention without departing from the scope of the invention. Therefore, it should be understood that the invention is not limited to the specific examples given. Rather, the invention is intended to cover modifications and variations within the scope of the appended claims and their equivalents.

Claims

1. A method for amplifying cells in a cell expansion system, the method comprising: A first fluid containing a first volume of multiple cells is loaded into the cell expansion system, wherein the cell expansion system includes a cell growth chamber; Based on the first volume of the first fluid, a second volume of the second fluid is determined to be loaded into a portion of the first fluid circulation path to place the first volume of the first fluid in a first portion of the cell growth chamber, wherein the second fluid comprises a culture medium, and wherein the first portion of the cell growth chamber comprises a central portion of the cell growth chamber; Based on the determined second volume of the second fluid, the second volume of the second fluid is loaded into the cell amplification system to place the first volume of the first fluid in the central portion of the cell growth chamber; A third volume of a third fluid is loaded to supply the plurality of cells; and Expand the plurality of cells.

2. The method of claim 1, wherein, The cell expansion system includes a second fluid circulation pathway.

3. The method of claim 2, wherein, The fluid in the first fluid circulation path flows through the capillary space of the cell growth chamber.

4. The method of claim 3, wherein, The fluid in the second fluid circulation path flows through the capillary space outside the cell growth chamber.

5. The method of claim 1, wherein, The first volume of fluid containing the plurality of cells is loaded without activating the capillary circulation pump.

6. The method of claim 1, wherein, The first volume of the fluid is the same as the second volume of the fluid.

7. The method of claim 1, wherein, The first volume of the fluid is different from the second volume of the fluid.

8. The method of claim 1, wherein, The plurality of cells includes one or more subsets of T cells.

9. The method of claim 8, wherein, One or more subsets of T cells include regulatory T cells (Tregs).

10. The method of claim 1, wherein, The cell growth chamber includes a hollow fiber membrane.

11. The method of claim 1, further comprising: The cells are stimulated with an activator to activate cell proliferation.

Images