Cell proliferation

Abstract

Cells are grown in a bioreactor and activated with an activator (e.g., a soluble activator complex). The nutrient and gas exchange capabilities of the automated closed cell expansion system allow, for example, seeding at a lower seeding density. Parameters of the cell growth environment are controlled to place cells in specific locations within the bioreactor for efficient nutrient and gas exchange. System parameters are adjusted to shear cell colonies that may form during the expansion phase. Metabolic concentrations are controlled to improve cell growth and viability. Cell retention within the bioreactor is controlled. In embodiments, the cells include T cells. The cells include T cell subpopulations, including, for example, regulatory T cells (Tregs).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 18 / 368,879, filed September 15, 2023, and the benefit of U.S. Provisional Patent Application No. 63 / 407,987, filed September 19, 2022, the entire disclosures of which are incorporated herein by reference.

[0002] The present disclosure relates to cell growth and cell growth systems. [Background technology]

[0003] This section provides background information related to the present disclosure that is not necessarily prior art.

[0004] Cell expansion systems (CES) are used to grow and differentiate cells. Cell expansion systems can be used to grow (e.g., grow) a variety of adherent and suspension cells. Growth of both adherent and non-adherent cell types occurs in the bioreactors of the cell expansion systems. Summary of the Invention [Problem to be solved by the invention]

[0005] This section provides a general overview of the disclosure and is not an exhaustive disclosure of the entire scope or all features. [Means for solving the problem]

[0006] In various aspects, the present disclosure provides a method for expanding cells. The method includes introducing cells into a cell expansion system. The cell expansion system includes a bioreactor and an air removal chamber. The bioreactor includes an inner capillary loop and an outer capillary loop. The flow rate within the bioreactor is less than 0.1 mL / min. The method further includes removing the cells from the air removal chamber, filling the inner capillary loop with a medium containing a protein, and placing the cells in the bioreactor for expansion for a first period of time.

[0007] In at least one exemplary embodiment, the protein comprises a cell signaling molecule.

[0008] In at least one exemplary embodiment, the cell signaling molecule comprises a cytokine.

[0009] In at least one exemplary embodiment, the cytokine comprises recombinant human IL-2 cytokine.

[0010] In at least one exemplary embodiment, the step of disposing the cells in the bioreactor includes disposing the cells in a first location on a first side of the bioreactor.

[0011] In at least one exemplary embodiment, the first side of the bioreactor comprises an outlet side of the bioreactor.

[0012] In at least one exemplary embodiment, the step of disposing the cells in the bioreactor further comprises disposing the cells at a second location, the second location being toward a central location of the bioreactor.

[0013] In at least one exemplary embodiment, the cells migrate toward the second location due to a pressure differential within the bioreactor.

[0014] In at least one exemplary embodiment, the pressure differential is created during operation of the air removal chamber.

[0015] In at least one exemplary embodiment, the method further comprises, after the first period of time, recirculating the cells for a second period of time, disposing the cells for a third period of time, and feeding the cells.

[0016] In at least one exemplary embodiment, the flow rate within the bioreactor is less than 0.02 mL / min.

[0017] In at least one exemplary embodiment, the flow rate in the bioreactor is about 0.01 mL / min.

[0018] In at least one exemplary embodiment, the cells comprise suspension cells.

[0019] In at least one exemplary embodiment, the suspension cells comprise one or more types of T cells.

[0020] In various aspects, the present disclosure provides a cell expansion system comprising: a first pump configured to circulate a first fluid, a second pump configured to circulate a second fluid, a fluid transfer assembly including a bioreactor, the fluid transfer assembly fluidly coupled to the first pump and the second pump, a processor, and a memory in communication with and readable by the processor, the memory containing a set of instructions, the fluid transfer assembly having an air removal chamber, the bioreactor having an inner capillary loop and an outer capillary loop, and a flow rate within the bioreactor being less than 0.1 mL / min, the set of instructions, when executed by the processor, causing the processor to direct loading cells into the fluid transfer assembly, directing removal of the cells from the air removal chamber, directing filling of the inner capillary loop with a medium comprising a protein, and directing placement of the cells in the bioreactor for expansion for a first period of time.

[0021] In at least one exemplary embodiment, the fluid transfer assembly is removably attached to the cell growth system.

[0022] In at least one exemplary embodiment, the fluid transfer assembly comprises a bioreactor.

[0023] In at least one exemplary embodiment, the fluid transfer assembly includes a first fluid transfer assembly that includes a first bioreactor, or the first fluid transfer assembly includes a second fluid transfer assembly that includes a second bioreactor that is smaller than the first bioreactor.

[0024] In at least one exemplary embodiment, the cells comprise suspension cells.

[0025] In at least one exemplary embodiment, the suspension cells comprise one or more types of T cells.

[0026] The drawings herein are for purposes of illustrating selected embodiments only, not all possible implementations, and are not intended to limit the scope of the present disclosure. [Brief explanation of the drawings]

[0027] [Figure 1A] FIG. 1A is a diagram of one embodiment of a cell expansion system (CES), in accordance with at least one exemplary embodiment of the present disclosure. [Figure 1B] FIG. 1B is a front view of an embodiment of a bioreactor showing a circuit path through the bioreactor, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 1C] FIG. 1C is a perspective view of a first bioreactor and a second bioreactor according to at least one exemplary embodiment of the present disclosure. [Figure 1D] FIG. 1D is a diagram of a rocking device for rotationally or laterally moving a cell growth chamber during operation of a cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 2A] FIG. 2A is a front perspective view of a cell growth system in accordance with at least one exemplary embodiment of the present disclosure. [Figure 2B] 2B is an interior perspective view of the cell growth system of FIG. 2A with a pre-mounted fluid transfer device, according to at least one exemplary embodiment of the present disclosure. [Figure 2C] 2C illustrates a rail system and a hook system of a holder of the cell growth system of FIG. 2A, according to at least one exemplary embodiment of the present disclosure. [Figure 3] FIG. 3 is a perspective view of a housing of a cell growth system in accordance with at least one exemplary embodiment of the present disclosure. [Figure 4A] FIG. 4A is a perspective view of a pre-mounted fluid transfer device according to at least one exemplary embodiment of the present disclosure. [Figure 4B] FIG. 4B is a diagram of the pre-mounted fluid transfer assembly of FIG. 4A, according to at least one exemplary embodiment of the present disclosure. [Figure 4C] FIG. 4C is a diagram of the pre-mounted fluid transfer assembly of FIG. 4A, according to at least one exemplary embodiment of the present disclosure. [Figure 4D] 4D is an illustration of a media bag of the pre-mounted fluid transfer assembly of FIG. 4A, according to at least one exemplary embodiment of the present disclosure. [Figure 4E] FIG. 4E is an illustration of a waste bag of the pre-mounted fluid transfer assembly of FIG. 4A, according to at least one exemplary embodiment of the present disclosure. [Figure 4F] 4F is a cross-sectional view of a pressure pod of the pre-mounted fluid transfer assembly of FIG. 4A, according to at least one exemplary embodiment of the present disclosure. [Figure 4G] FIG. 4G is an exploded view of the pressure pod of FIG. 4F, according to at least one exemplary embodiment of the present disclosure. [Figure 4H] FIG. 4H is a diagram of a sampling coil of the pre-mounted fluid transfer assembly of FIG. 4A, according to at least one exemplary embodiment of the present disclosure. [Figure 4I] FIG. 41 is a diagram of an in-line filter of the pre-mounted fluid transfer assembly of FIG. 4A, according to at least one exemplary embodiment of the present disclosure. [Figure 5A] FIG. 5A is a schematic diagram of a cell growth system including an operating configuration illustrating fluid movement, according to at least one exemplary embodiment of the present disclosure. [Figure 5B] FIG. 5B is a schematic illustration of a cell growth system including another operating configuration showing fluid movement, according to at least one exemplary embodiment of the present disclosure. [Figure 5C] FIG. 5C is a schematic illustration of a cell growth system including another operating configuration showing fluid movement, according to at least one exemplary embodiment of the present disclosure. [Figure 6] FIG. 6 is a schematic diagram of a cell growth system in accordance with at least one exemplary embodiment of the present disclosure. [Figure 7A] FIG. 7A is a schematic diagram of a cell growth system, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 7B] FIG. 7B is a schematic diagram of a cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 7C] FIG. 7C is a schematic diagram of a cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 8] FIG. 8 is a flow diagram illustrating operational features of a process for expanding cells, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 9A] FIG. 9A is a flow diagram illustrating operational features of a process for expanding cells, according to at least one exemplary embodiment of the present disclosure. [Figure 9B] FIG. 9B is a schematic diagram of a portion of a cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 10A] FIG. 10A is a flow diagram illustrating operational features of a process for expanding cells, according to at least one exemplary embodiment of the present disclosure. [Figure 10B] FIG. 10B is a graph of oxygen consumption in a cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 11A] FIG. 11A is a flow diagram illustrating operational features of a process for expanding cells, according to at least one exemplary embodiment of the present disclosure. [Figure 11B] FIG. 11B illustrates a table of exemplary pump flow rates for use in a cell growth system, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 12A] FIG. 12A is a flow diagram illustrating operational features of a process for expanding cells, according to at least one exemplary embodiment of the present disclosure. [Figure 12B] FIG. 12B is a graph of cell proliferation metabolism, according to at least one exemplary embodiment of the present disclosure. [Figure 12C] FIG. 12C is a graph of cell proliferation metabolism, according to at least one exemplary embodiment of the present disclosure. [Figure 13] FIG. 13 is a flow diagram illustrating operational features of a process for expanding cells, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 14] FIG. 14 is a flow diagram illustrating operational features of a process for expanding cells, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 15] FIG. 15 is a flow diagram illustrating operational features of a process for expanding cells, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 16A] FIG. 16A is a flow diagram illustrating operational features of a process for expanding cells, according to at least one exemplary embodiment of the present disclosure. [Figure 16B] FIG. 16B shows a graph of cell number versus flow rate during cell growth, according to at least one exemplary embodiment of the present disclosure. [Figure 17] FIG. 17 is a flow diagram illustrating operational features of a process for expanding cells, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 18A] FIG. 18A is a flow diagram illustrating operational features of a process for expanding cells, according to at least one exemplary embodiment of the present disclosure. [Figure 18B] FIG. 18B shows a diagram of cells growing in a cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 18C] FIG. 18C shows a graph illustrating the internal diameter of cell dissociation, according to at least one exemplary embodiment of the present disclosure. [Figure 19] FIG. 19 shows a graph of cell number and flow rate versus days in culture during cell expansion, according to at least one exemplary embodiment of the present disclosure. [Figure 20A] FIG. 20A is a flow diagram illustrating operational features of a process for operating a pump to grow cells, according to at least one exemplary embodiment of the present disclosure. [Figure 20B] FIG. 20B is a schematic diagram of a portion of a cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 20C]FIG. 20C is a schematic illustration of a portion of a cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 20D] FIG. 20D is a schematic illustration of a portion of a cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 21] FIG. 21 is a flow diagram illustrating operational features of a process for expanding cells, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 22] FIG. 22 is a flow diagram illustrating operational features of a process for expanding cells, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 23] FIG. 23 is a flow diagram illustrating operational features of a process for expanding cells, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 24] FIG. 24 is a flow diagram illustrating operational features of a process for expanding cells, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 25] FIG. 25 illustrates an exemplary processing system of a cell growth system in which embodiments of the present disclosure can be implemented, according to at least one exemplary embodiment of the present disclosure. [Figure 26] FIG. 26 is a flow diagram illustrating an exemplary experimental flow, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 27] FIG. 27 is a graph illustrating example results of a coating process, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 28] FIG. 28 is a graph showing example results of cells harvested after a coating process, according to at least one exemplary embodiment of the present disclosure. [Figure 29] FIG. 29 is a graph showing example results of cells harvested after a coating process, according to at least one exemplary embodiment of the present disclosure. [Figure 30] FIG. 30 is a graph showing example results of viral particles measured per day, according to at least one exemplary embodiment of the present disclosure. [Figure 31]FIG. 31 is a graph showing example results of cells harvested after a coating process, according to at least one exemplary embodiment of the present disclosure. [Figure 32] FIG. 32 is a graph showing example results of cells harvested after a coating process, according to at least one exemplary embodiment of the present disclosure. [Figure 33] FIG. 33 is a table illustrating example settings for days 0 to 2, according to at least one exemplary embodiment of the present disclosure. [Figure 34] FIG. 34 is a table illustrating steps for programming a 10 minute coating procedure using CPPT, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 35A] FIG. 35A is a table illustrating virus propagation steps, according to at least one exemplary embodiment of the present disclosure. [Figure 35B] FIG. 35B is a table illustrating virus propagation steps, according to at least one exemplary embodiment of the present disclosure. [Figure 35C] FIG. 35C is a table illustrating virus propagation steps, according to at least one exemplary embodiment of the present disclosure. [Figure 36A] FIG. 36A is a portion of a table showing exemplary task settings (e.g., flow rates, angular velocities, outlets, etc.) for various components (e.g., pumps, rockers, valves, etc.) of a compact cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 36B] FIG. 36B is a portion of a table showing exemplary task settings (e.g., flow rates, angular velocities, outlets, etc.) for various components (e.g., pumps, rockers, valves, etc.) of a compact cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 36C] FIG. 36C is a portion of a table showing exemplary task settings (e.g., flow rates, angular velocities, outlets, etc.) for various components (e.g., pumps, rockers, valves, etc.) of a compact cell growth system according to at least one exemplary embodiment of the present disclosure. [Figure 36D]FIG. 36D is a portion of a table showing exemplary task settings (e.g., flow rates, angular velocities, outlets, etc.) for various components (e.g., pumps, rockers, valves, etc.) of a compact cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 36E] FIG. 36E is a portion of a table showing exemplary task settings (e.g., flow rates, angular velocities, outlets, etc.) for various components (e.g., pumps, rockers, valves, etc.) of a compact cell growth system according to at least one exemplary embodiment of the present disclosure. [Figure 37A] FIG. 37A is a table showing exemplary task settings (e.g., flow rates, angular velocities, outlets, etc.) for various components (e.g., pumps, rockers, valves, etc.) of a standard cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 37B] FIG. 37B is a table showing exemplary task settings (e.g., flow rates, angular velocities, outlets, etc.) for various components (e.g., pumps, rockers, valves, etc.) of a standard cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 37C] FIG. 37C is a table showing exemplary task settings (e.g., flow rates, angular velocities, outlets, etc.) for various components (e.g., pumps, rockers, valves, etc.) of a standard cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 37D] FIG. 37D is a table showing exemplary task settings (e.g., flow rates, angular velocities, outlets, etc.) for various components (e.g., pumps, rockers, valves, etc.) of a standard cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 38A] FIG. 38A is a table showing exemplary task settings (e.g., flow rates, angular velocities, outlets, etc.) for various components (e.g., pumps, rockers, valves, etc.) of a compact cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 38B]FIG. 38B is a table showing exemplary task settings (e.g., flow rates, angular velocities, outlets, etc.) for various components (e.g., pumps, rockers, valves, etc.) of a compact cell growth system, according to at least one exemplary embodiment of the present disclosure. [Figure 38C] FIG. 38C is a table showing exemplary task settings (e.g., flow rates, angular velocities, outlets, etc.) for various components (e.g., pumps, rockers, valves, etc.) of a compact cell growth system according to at least one exemplary embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0028] Exemplary embodiments will be described in more detail with reference to the accompanying drawings.

[0029] The exemplary embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope to those skilled in the art. Numerous specific details are set forth, such as examples of specific components, devices, and methods, to provide a thorough understanding of the embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details are not necessary, that the exemplary embodiments may be embodied in many different forms, and that none of these should be construed as limiting the scope of the present disclosure. In some exemplary embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

[0030] The terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. The terms "comprises," "comprising," "including," and "having" are inclusive and thus specify the presence of stated features, entities, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The method steps, processes, and operations described herein should not be construed as necessarily requiring performance in the particular order described or illustrated, unless specifically identified as such. It should also be understood that additional or alternative steps may be employed.

[0031] When an element or layer is referred to as "on," "engaged to," "connected to," or "coupled to" another element or layer, it may be directly on, directly engaged with, directly connected to, or directly coupled to the other element or layer, or intervening elements or layers may be present. Conversely, when an element is referred to as "directly on," "directly engaged with," "directly connected to," or "directly coupled to" another element or layer, no intervening elements or layers are present. Other terms used to describe relationships between elements should be interpreted similarly (e.g., "between" vs. "directly between," "adjacent" vs. "directly adjacent," etc.). As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0032] Terms such as "first," "second," and "third" may be used herein to describe various elements, components, regions, layers, and / or sections, but these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are used only to distinguish one element, component, region, layer, or section from another region, layer, or section. Terms such as "first," "second," and other numerical terms, when used herein, do not imply a sequence or order unless clearly indicated by context. Thus, a first element, first component, first region, first layer, or first section described below could also be referred to as a second element, second component, second region, second layer, or second section without departing from the teachings of the exemplary embodiments.

[0033] Spatial terms such as "inside," "outside," "beneath," "below," "lower," "upper," and "above" are used herein for ease of description when describing the relationship of one element or feature to another element(s) or feature(s), as shown in the figures. Spatial terms may be intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures were inverted, elements described as being "beneath" or "beneath" other elements or features would change accordingly to be "above" them. Thus, for example, the term "below" can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations), and the spatial descriptions used herein should be interpreted accordingly.

[0034] Various components are referred to herein as "operably associated." As used herein, "operably associated" refers to components that are linked together in an operable manner and includes embodiments in which components are directly coupled as well as embodiments in which other components are disposed between the coupled components. "Operably associated" components can be "fluidically associated." "Fluidly associated" refers to components that are linked together so that fluid can be transported between them. The term "fluidly associated" includes embodiments in which another member is disposed between two fluidly associated members, embodiments in which members are directly connected, and the like. Fluidly associated members may include members that do not contact the fluid but that operate the system by contacting other members (e.g., a peristaltic pump that pumps fluid through a flexible tube by compressing the outside of the tube).

[0035] As used herein, the term "donor" can refer to anyone who provides a fluid, such as whole blood, to an apheresis system. A donor can also be a patient who temporarily provides a fluid to an apheresis system, where the fluid is processed, treated, manipulated, etc., before being returned to the patient.

[0036] As used herein, the term "automatic" and variations thereof refer to any process or operation that occurs without substantial human input when the process or operation is performed. However, a process or operation may be automatic if significant or insignificant human input is used in the execution of the process or operation, but the input is received before the process or operation is performed. Human input is considered significant if it affects how the process or operation is performed. Human input that implies consent to the execution of a process or operation is not considered "critical."

[0037] As used herein, the term "computer-readable medium" refers to any tangible storage and / or transmission medium that participates in providing instructions to a processor for execution. Such media may take many forms, including, but not limited to, nonvolatile media, volatile media, and transmission media. Nonvolatile media include, for example, NVRAM or magnetic or optical disks. Volatile media include dynamic memory such as main memory. Common forms of computer-readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tape, or any other magnetic media, magneto-optical media, CD-ROMs, any other optical media, punch cards, paper tape, any other physical media with patterns of holes, RAM, PROMs, EPROMs, FLASH-EPROMs, solid-state media such as memory cards, any other memory chips or cartridges, carrier waves, or any other medium from which a computer can read. A digital file attachment to an email or other self-contained information archive or set of archives is considered a distribution medium equivalent to a tangible storage medium. When the computer-readable medium is configured as a database, it should be understood that the database may be any type of database, such as relational, hierarchical, object-oriented, and / or the like. Accordingly, the present disclosure is considered to encompass tangible storage or distribution media on which software implementations of the present disclosure are stored, and prior art-recognized equivalents and successor media.

[0038] As used herein, the term "module" refers to any known or later developed hardware, software, firmware, artificial intelligence, fuzzy logic, or combination of hardware and software that is capable of performing the functions associated with that element.

[0039] As used herein, the terms "determining," "calculating," and "computing," as well as variations thereof, are used interchangeably and include any type of methodology, process, mathematical operation, or technique.

[0040] Embodiments of the present disclosure will now be more fully described in connection with apheresis methods and systems with reference to the accompanying drawings. The following embodiments will be described with reference to separating blood components from whole blood. However, this is provided for illustrative purposes only. It should be noted that the embodiments are not limited to the following description. The embodiments are intended for use in products, processes, devices, and systems for separating any complex fluid. Thus, the present disclosure is not limited to separating blood components from whole blood.

[0041] Exemplary embodiments of the present disclosure generally relate to systems and methods for expanding cells in a cell expansion system (CES). According to embodiments, such expansion is achieved through the use of a bioreactor or cell growth chamber. In one embodiment, such a bioreactor or cell growth chamber includes hollow fiber membranes. Such hollow fiber membranes include a plurality of hollow fiber membranes and include an extra-capillary (EC) space and an extra-capillary (IC) space. In embodiments, adherent or non-adherent cells are grown or expanded in the cell expansion system. For example, non-adherent cells or suspension cells (e.g., T cells, T lymphocytes, or CD3+ selected cells) are expanded in the system. In embodiments, one or more subpopulations or subsets of T cells are expanded. For example, in embodiments, methods and systems are provided for expanding regulatory T cells (Tregs) and / or human regulatory T cells (hTregs).

[0042] In some exemplary embodiments, methods and systems are provided for growing cells in a closed, automated cell growth system. In one embodiment, such a cell growth system includes a bioreactor or cell growth chamber. In a further embodiment, such a bioreactor or cell growth chamber includes hollow fiber membranes. The capabilities of such a system (e.g., nutrient supply and gas exchange capabilities) allow cells to be seeded at low cell seeding densities. In embodiments, parameters of the cell growth environment are adjusted to load or introduce cells into a predetermined location within the bioreactor for efficient exchange of nutrients and gases to the growing cells. For example, cell density is increased by concentrating the cells within the bioreactor.

[0043] In an exemplary embodiment, a non-adherent cell population (e.g., T cells) is introduced or loaded into a hollow fiber bioreactor, where the hollow fiber bioreactor includes a plurality of hollow fibers. In an embodiment, the cells are exposed to an activator to activate cell growth within the hollow fiber bioreactor. In one embodiment, a plurality of cells are introduced into a cell growth system, for example, using a "Central Load without Circulation" task. According to an exemplary embodiment, such a task is performed on days 0 and 4-8. In other embodiments, it may be performed on other days. In an embodiment, such a cell loading task results in cell concentration within the bioreactor, increasing cell density. In other embodiments, cells may be placed in other parts or regions of the bioreactor to increase cell density. In an embodiment, placing the cells in a first location (e.g., the central region) of the bioreactor allows the cells to receive efficient nutrient and gas exchange.

[0044] In exemplary embodiments, a lower cell seeding density can be used compared to the cell seeding density used in static culture methods. In embodiments using a cell expansion system, cells (e.g., regulatory T cells (Treg or Treg cells)) can be seeded at a density of 2.54 x 10 5 Cell count / mL to 3.69 x 10 5In another embodiment, the cell seeding density is about 1 x 10 cells / mL. 6 Furthermore, the Treg cell inoculum was less than 1.0 × 10 5 For other methods (e.g., static Treg cell culture), in vitro expansion can be performed at a cell seeding density of 1.0 x 10 cells / mL. 6 A cell seeding density of Treg cells / mL is used. In one embodiment, a lower cell seeding density can be used, for example, due to the efficiency of the overall system in delivering nutrients to the culture environment. In other embodiments, one or more steps used during expansion, combined with the overall efficiency of the system in delivering nutrients to the culture environment, allow the use of a lower initial cell seeding density.

[0045] In an exemplary embodiment, automated cell (e.g., Treg) expansion is performed using a soluble activator complex. In other embodiments, other types of activators can be used, such as beads for cell stimulation or beads for incubation with soluble or surface-immobilized antibodies. In other embodiments, cells (e.g., Treg cells) are expanded without the use of bead-based stimulation. In one embodiment, cell expansion is performed using Stem Cell Technologies soluble ImmunoCult™ human CD3 / CD28 / CD2 T cell activator to activate and expand Treg cells in the presence of 200 IU / mL of the cytokine IL-2 in an automated cell expansion system. For example, the use of a soluble activator complex can reduce the cost of stimulation compared to bead-based protocols. In other embodiments, including co-culture of T cells and antigen-presenting cells for biological antigen stimulation, other types of cell activation activators can be used. Additionally, in other embodiments, other types of cytokines or other growth factors may be used.

[0046] In exemplary embodiments, system parameters are also adjusted or controlled to control cell retention within the bioreactor or cell growth chamber. For example, by controlling cell retention within the bioreactor hollow fibers during the cell growth phase, the system can provide efficient gas and nutrient exchange to the growing cells. In embodiments, the bioreactor may be designed to provide gas exchange, and in some embodiments, nutrient exchange, to the growing cells. In exemplary embodiments, in bioreactors with semipermeable hollow fiber membranes, gas and nutrient exchange occurs through the semipermeable hollow fiber membrane. In embodiments, a method for providing media components (e.g., various cytokines, proteins, etc.) to growing cells that cannot pass through the membrane may use a fluid inlet on the side of the bioreactor where the cells are growing (e.g., the inside capillary (IC) side). However, in certain embodiments, low, reduced, or diminished (e.g., minimal) inlet flow rates can cause cells to collect in the outlet header of the bioreactor. Cells present in the bioreactor header may not receive adequate gas and nutrient exchange, which can lead to cell necrosis and clumping.

[0047] Exemplary embodiments relate to methods for retaining cells (e.g., a non-adherent cell population) within a bioreactor when feeding the cells using a flow inlet (e.g., an IC inlet). In embodiments herein, the cells are on the IC side of the membrane during feeding, but in other embodiments, for example, the cells may be on the EC side of the membrane. According to embodiments, the cells may be contained within a first circuit and / or a second circuit. In embodiments, the feeding method pumps a first volume of fluid (e.g., culture medium or cell growth medium) into a first port of the bioreactor at a first volumetric flow rate, volumetric flow rate, fluid flow rate, flow rate, fluid stream flow rate, or volumetric velocity. For example, the terms volumetric flow rate, fluid flow rate, flow rate, fluid stream flow rate, or volumetric velocity are used interchangeably. In some embodiments, a flow rate is a vector having both speed and direction. A second volume of fluid is pumped into a second port of the bioreactor at a second volumetric flow rate, volumetric flow rate, fluid flow rate, flow rate, fluid stream flow rate, or volumetric velocity. In embodiments, such volumetric flow, fluid flow, flow rate, fluid flow rate, or volumetric rate is controlled by one or more pump speeds and / or pump flow rates. For example, a pump flow rate generates, causes, or influences the volumetric flow rate or rate of a fluid on which the pump acts. As used herein, pump speed or pump flow rate, in embodiments, is described as the volumetric flow rate or fluid flow rate generated, caused, or influenced by a pump.

[0048] In an exemplary embodiment, the second flow rate of fluid into the bioreactor is opposite to the direction of the first flow rate of fluid into the bioreactor. For example, FIGS. 5B and 5C show exemplary operating configurations illustrating flow rates and flow directions used in a cell expansion system such as CES 500 (e.g., FIGS. 5B and 5C) according to embodiments of the present disclosure. In embodiments, a cell expansion system pump (e.g., an IC pump) can be used to control cell retention within the bioreactor. In embodiments, cells are lost from the bioreactor to the IC circuit or IC loop, e.g., during the expansion phase of growth. In embodiments, cells in the bioreactor located near the IC inlet port, for example, receive the freshest growth medium, while cells in the portion of the IC circuit outside the bioreactor, for example, essentially receive spent or conditioned medium, which influences metabolism. Furthermore, according to embodiments, cells in the bioreactor receive a gas mixture (O, CO, N) input from the gas transfer module (GTM) via diffusion from the EC loop circulation, while cells in the portion of the IC circuit outside the bioreactor do not.

[0049] In an exemplary embodiment, reducing cell loss from a hollow fiber membrane (HFM) bioreactor can be achieved by redirecting half of the inlet flow to flow in the opposite direction of the input flow through the IC loop, effectively introducing cell culture medium to both ends of the bioreactor and retaining cells within the hollow fibers. For example, in one embodiment, during the cell culture growth phase (e.g., days 4-7), to maintain cells in the bioreactor, the IC inlet pump flow rate of +0.2 mL / min is halved to a complementary IC circulation pump flow rate of -0.1 mL / min. This pump adjustment, according to the embodiment, counteracts forces associated with cell loss from the IC outlet port. Other pump flow rates may be used in other embodiments. For example, in other embodiments, the pump flow rate may be different. In embodiments, other pumps or additional pumps may be used. In one embodiment, fewer pumps may be used. Furthermore, in other embodiments, other time periods may be used.

[0050] In exemplary embodiments, the metabolic activity of a cell population can affect feeding parameters. For example, cell culture lactate values ​​are maintained below a predefined level. In one embodiment, cell culture values ​​are maintained, for example, at about 7 mmol / L or below. In embodiments, a graphical user interface (GUI) of a cell expansion system can be used to control the media feed rate to maintain the lactate metabolic waste product from glycolysis below a predetermined value during expansion of cells (e.g., regulatory T cells). In other embodiments, for example, media feed rate and / or other settings are controlled to maintain or attempt to maintain lactate levels, glucose levels, or pH to improve cell growth and viability. In other embodiments, other concentrations may be used.

[0051] In additional embodiments, several system features are utilized to disaggregate cell colonies, microcolonies, or cell clusters that form during the proliferation phase of cell growth. For example, in one embodiment, cell colonies (e.g., microcolonies) passing through a bioreactor hollow fiber membrane can be disaggregated to reduce the number of cells within the microcolony, colony, or cluster, where a microcolony, colony, or cluster is one or more groups of adherent cells. In embodiments, a cell expansion system (CES) bioreactor architecture can be used to disaggregate cell (e.g., Treg cell) microcolonies. In embodiments, as cells (e.g., Treg cells) grow, they tend to form microcolonies. Microcolonies limit the diffusion of nutrients to cells in the center of such colonies, which can lead to adverse effects during cell culture, such as necrosis. In embodiments, a protocol is provided for disaggregating colonies during the proliferation phase of growth, for example, by circulating a suspension cell culture through an inner (IC) loop of a hollow fiber capillary (e.g., having a 215 μm inner diameter hollow fiber). In embodiments, disaggregation of colonies, microcolonies, or clusters of cells can reduce the size of the colonies, microcolonies, or clusters of cells. In one embodiment, disaggregation of colonies or clusters of cells can provide a suspension of single cells, improving cell growth / viability. Such functionality contributes to the continued perfusion growth of cells (e.g., T cells or Tregs).

[0052] In an exemplary embodiment, a therapeutic amount of cells (e.g., Tregs) are expanded in a cell expansion system and harvested from the cell expansion system. In an embodiment, the number of cells at harvest is about 1 x 10 6 cells to approximately 1 x 10 10 cells (e.g., 1 × 10 9 In one embodiment, the number of cells harvested is about 1 x 10 8 From 1×10 10 In one example, approximately 7.0 x 10 8 to approximately 1.4 × 109 In some embodiments, the harvested cells have a viability of about 60% to about 100%. For example, the viability of the harvested cells is greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95%. In some embodiments, the harvested cells express biomarkers consistent with Tregs. For example, in some embodiments, the cells express CD4 + , CD25 + , and / or FoxP3 + In embodiments, the harvested cells express a CD4 biomarker at a frequency of about 50% to about 100%. + CD25 + The harvested cells have a frequency of greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% CD4 + CD25 + In other embodiments, the cells have a frequency of about 30% to about 100% CD4 + FoxP3 + In some embodiments, the harvested cells have a frequency of greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, or even greater than about 70% CD4+ phenotype. + FoxP3 + Other embodiments may be extended to other types of suspension cells, such as T cells, that have different phenotypes, different cell surface markers, and different transcription factor expression.

[0053] Exemplary embodiments relate to cell growth systems, as described above. In embodiments, the cell growth system is closed. Closed cell growth systems include contents that are not directly exposed to the atmosphere. Such cell growth systems may be automated. In embodiments, both adherent and non-adherent (i.e., suspension) types of cells or suspension types can be grown in the bioreactor of the cell growth system. According to embodiments, the cell growth system may include a basal medium or other types of medium. Methods for replenishing medium are provided for cell growth performed in the bioreactor of a closed cell growth system. In embodiments, the bioreactor used with such a system is a hollow fiber bioreactor. Various bioreactors may be used according to embodiments of the invention.

[0054] In some embodiments, the system includes a bioreactor fluidically associated with a first fluid flow path having at least two ends, wherein a first end of the first fluid flow path is fluidically associated with a first port of a hollow fiber membrane, and a second end of the first fluid flow path is fluidically associated with a second port of the hollow fiber membrane. In some embodiments, the hollow fiber membrane includes a plurality of hollow fibers. The system includes a fluid inlet channel fluidically associated with the first fluid flow path, through which a plurality of cells are introduced into the first fluid flow path. In some embodiments, the system further includes a pump that transfers an inner capillary input fluid from an inner capillary medium bag to the first fluid flow path, and a controller that controls operation of the pump. In some embodiments, the controller controls the pump to, for example, transfer cells from a cell input bag to the first fluid flow path. Another pump that circulates fluid in the first fluid circuit is also included. The pump includes a controller that controls operation of the pump. In one embodiment, the controller is, for example, a computing system including a processor. In some embodiments, one or more of the controllers control one or more of the pumps to circulate fluid at a certain flow rate within the first fluid circuit. In some embodiments, multiple controllers (e.g., a first controller, a second controller, a third controller, a fourth controller, a fifth controller, a sixth controller, etc.) may be used. In some embodiments of the present disclosure, multiple pumps (e.g., a first pump, a second pump, a third pump, a fourth pump, a fifth pump, a sixth pump, etc.) may be used. Furthermore, while the present disclosure refers to a medium bag, a cell input bag, etc., multiple bags, e.g., a first medium bag, a second medium bag, a third medium bag, a first cell input bag, a second cell input bag, a third cell input bag, etc., and / or other types of containers, may be used. In other embodiments, a single medium bag, a single cell input bag, etc., may be used. In some embodiments, additional or other flow paths (e.g., a second fluid flow path, a second fluid inlet path, a second fluid circuit, etc.) may be provided.

[0055] In an exemplary embodiment, the system is controlled by, for example, a processor coupled to the cell growth system, a display device in communication with the processor for displaying data, and a memory in communication with the processor and readable by the processor for storing a set of instructions. In an embodiment, when the instructions are executed by the processor, the processor receives, for example, an instruction to prime the system. In response to that instruction, the processor performs a set of steps to prime the system, and then receives, for example, an instruction to perform an IC / EC wash. In response to an instruction to input cells, the processor performs a set of steps to introduce cells into the bioreactor, for example, from a cell input bag.

[0056] An example of a cell growth system (CES) according to an embodiment of the present invention is shown schematically in FIG. 1A. CES 10 includes a first fluid circuit 12 and a second fluid circuit 14. According to the embodiment, first fluid flow path 16 has at least opposite ends 18, 20 fluidly associated with a hollow fiber cell growth chamber 24 (also referred to as a "bioreactor"). Specifically, end 18 is fluidly associated with a first inlet 22 of cell growth chamber 24, and end 20 is fluidly associated with a first outlet 28 of cell growth chamber 24. In first fluid circuit 12, fluid passes through the interior of hollow fibers 116 (see FIG. 1B) of hollow fiber membranes 117 (see FIG. 1B) disposed in cell growth chamber 24 (the cell growth chamber and hollow fiber membranes are described in more detail below). Additionally, a first flow controller 30 is operably connected to first fluid flow path 16 to control the flow of fluid in first fluid circuit 12.

[0057] Second fluid circuit 14 includes second fluid flow path 34, cell growth chamber 24, and second flow controller 32. According to an embodiment, second fluid flow path 34 has at least opposite ends 36, 38. End 36 of second fluid flow path 34 is fluidly associated with inlet port 40 of cell growth chamber 24, and end 38 is fluidly associated with outlet port 42. Fluid flowing through cell growth chamber 24 contacts the exterior of hollow fiber membrane 117 (see FIG. 1B) of cell growth chamber 24. The hollow fiber membrane comprises a plurality of hollow fibers. Second fluid circuit 14 is operably connected to second flow controller 32.

[0058] Thus, the first fluid circuit 12 and the second fluid circuit 14 are separated in the cell growth chamber 24 by a hollow fiber membrane 117 (see FIG. 1B). The fluid in the first fluid circuit 12 flows through the inner capillary (IC) space of the hollow fibers in the cell growth chamber 24. Therefore, the first fluid circuit 12 is referred to as the "IC loop." The fluid in the second fluid circuit 14 flows through the outer capillary (EC) space of the hollow fibers in the cell growth chamber 24. Therefore, the second fluid circuit 14 is referred to as the "EC loop." Depending on the embodiment, the fluid in the first fluid circuit 12 may flow either cocurrently or countercurrently with respect to the flow of the fluid in the second fluid circuit 14.

[0059] A fluid inlet passage 44 is fluidly associated with the first fluid circuit 12. The fluid inlet passage 44 allows fluid to be introduced into the first fluid circuit 12, and a fluid outlet passage 46 allows fluid to be removed from the CES 10. A third flow control device 48 is operatively associated with the fluid inlet passage 44. Alternatively, the third flow control device 48 may be associated with the fluid outlet passage 46.

[0060] According to embodiments, the flow control device used herein can be a pump, a valve, a clamp, or a combination thereof. Multiple pumps, multiple valves, and multiple clamps may be arranged in any combination. In various embodiments, the flow control device is or includes a peristaltic pump. In embodiments, the fluid circuit, inlet port, and outlet port may be constructed of tubing of any material.

[0061] Generally, any type of fluid can flow through the circulation, inlet, and outlet channels, such as buffers, protein-containing fluids, cell-containing fluids, etc. As used herein, the terms "fluid," "media," and "fluid medium" are used interchangeably.

[0062] 1B is a front view of an example hollow fiber cell growth chamber 100, or bioreactor 100, for use with the present disclosure. Cell growth chamber 100 has a longitudinal axis LA-LA and includes a cell growth chamber housing 104. In at least one embodiment, cell growth chamber housing 104 has four openings or ports: IC inlet port 108, IC outlet port 120, EC inlet port 128, and EC outlet port 132.

[0063] According to an embodiment of the present invention, fluid in a first circuit enters cell growth chamber 100 through IC inlet port 108 at first longitudinal end 112 of cell growth chamber 100, passes through the capillary interior of a plurality of hollow fibers 116 comprising hollow fiber membrane 117 (referred to in various embodiments as the intra-capillary ("IC") side of the hollow fiber membrane or the "IC space"), and exits cell growth chamber 100 through IC outlet port 120 located at second longitudinal end 124 of cell growth chamber 100. The flow path between IC inlet port 108 and IC outlet port 120 constitutes IC portion 126 of cell growth chamber 100. Fluid in a second circuit enters cell growth chamber 100 through EC inlet port 128, contacts the extra- or extra-capillary exterior of hollow fibers 116 (referred to as the "EC side" or "EC space" of the membrane), and exits cell growth chamber 100 through EC outlet port 132. The flow path between the EC inlet port 128 and the EC outlet port 132 constitutes the EC portion 136 of the cell growth chamber 100. Fluid entering the cell growth chamber 100 through the EC inlet port 128 contacts the outside of the hollow fibers 116. Small molecules (e.g., ions, water, oxygen, lactate, metabolites, nutrients, gases, etc.) can diffuse through the hollow fibers 116 from the interior, or IC space, of the hollow fibers to the exterior, or EC space, or from the EC space to the IC space (e.g., continuous perfusion). For example, the hollow fibers 116 have a diffusion distance of 200 microns, allowing for more efficient transfer of gases and nutrients compared to flask-based systems. High molecular weight molecules, such as growth factors, are typically too large to pass through the hollow fiber membrane and remain within the IC space of the hollow fibers 116. The hollow fibers 116 experience lower shear stress compared to stirred bioreactors and wave-shaking bioreactors. In embodiments, the medium may be changed as needed. If necessary, the medium may also be circulated through an oxygenator or gas transfer module to exchange gases. In embodiments, as described below, cells may be contained within the first and / or second circuits and may reside on the IC and / or EC sides of the membrane.

[0064] The material used to fabricate hollow fiber membrane 117 can be any biocompatible polymeric material that can be formed into hollow fibers. According to one embodiment of the present invention, one material used is a synthetic polysulfone-based material.

[0065] According to some embodiments, as shown in FIG. 1C , two or more different sizes of cell growth chambers 100 may be provided separately or included in a single disposable kit. For example, a pair of cell growth chambers 100A and 100B may be provided separately or included in a single disposable kit. In at least one exemplary embodiment, cell growth chamber 100A is smaller than cell growth chamber 100B and accommodates fewer cells for growth. For example, cell growth chamber 100A is one-tenth, or 0.1 times, the size of cell growth chamber 100B. In at least one exemplary embodiment, cell growth chamber 100A (i.e., small cell growth chamber 100A) is advantageous for experiments, pediatrics, and other events (e.g., skin grafts) using small batches of starter cells. Because small cell growth chamber 100A can grow small cell batches, use of small cell growth chamber 100A is advantageous when the initial cell source is too small for cell growth chamber 100B. Small cell batches may be transferred to cell growth chamber 100B for further growth. Thus, small cell growth chamber 100A provides additional opportunities for using CES 10 when cell growth chamber 100B is not optimal. In some exemplary embodiments, CES 10 automatically recognizes whether cell growth chamber 100A or cell growth chamber 100B is installed and identifies appropriate process parameters, as described further below. By including two or more sizes of cell growth chamber 100, CES 10 increases its versatility. For example, bone marrow-derived mesenchymal stem cells (MSCs), adipose tissue-derived MSCs, umbilical cord MSCs, fibroblasts, keratinocytes, HEK293T cells, human embryonic stem cells (ESCs), periosteum-derived cells, induced pluripotent stem cell-derived MSCs (IPS-MSCs), neural stem cells (NSCs), osteochondroprogenitor cells, endothelial cells, dendritic cells, induced pluripotent stem cells (iPSCs), T cells, viral vectors, exosome production, and expansion of adherent and suspension cell types in autologous and allogeneic doses are available.For example, multiple cell growth chambers 100 for the CES 10 provide the ability to expand anywhere from 300 million up to 1 billion MSCs or up to 25 billion T cells per run.

[0066] In at least one exemplary embodiment, a CES (e.g., CES500 (FIGS. 5A, 5B, and 5C) and / or CES600 (FIG. 6)) is provided with an apparatus configured to move, or "rock," the cell growth chamber relative to other components of the cell growth system by attaching it to a rotational and / or lateral rocking device. FIG. 1D illustrates one such exemplary apparatus. In one embodiment, in the apparatus, the cell growth chamber 100 is rotationally connected to two rotational rocking components and one lateral rocking component.

[0067] The first rotational oscillating component 138 rotates the cell growth chamber 100 about a central axis 142 of the cell growth chamber 100. The first rotational oscillating component 138 is rotationally associated with the cell growth chamber 100. In some embodiments, the cell growth chamber 100 can continuously rotate in a single direction (clockwise or counterclockwise) about the central axis 142. Alternatively, the cell growth chamber 100 can be rotated alternately, for example, first clockwise and then counterclockwise about the central axis 142.

[0068] The CES also includes a second rotational rocking component that rotates the cell growth chamber 100 about a rotation axis 144. The rotation axis 144 passes through a center point of the cell growth chamber 100 and is perpendicular to the central axis 142. In embodiments, the cell growth chamber 100 can be continuously rotated in a single direction, either clockwise or counterclockwise, about the rotation axis 144. Alternatively, the cell growth chamber 100 can be rotated alternately about the rotation axis 144, for example, first clockwise and then counterclockwise. In various embodiments, the cell growth chamber 100 can be rotated about the rotation axis 144 and positioned in either a horizontal or vertical orientation relative to gravity.

[0069] In some embodiments, lateral rocking component 140 is associated with cell growth chamber 100 for lateral movement. In embodiments, the plane of lateral rocking component 140 moves in the x and y directions, thereby reducing cell settling in the cell growth chamber due to movement of cell-containing media within the hollow fibers.

[0070] The rotational and / or lateral movement of the rocking device reduces cell settling within the device and also reduces the likelihood of cells becoming trapped in certain areas of the cell growth chamber. The settling rate of cells in the cell growth chamber is proportional to the density difference between the cells and the suspension medium, according to Stokes' law. In certain embodiments, as described above, alternating 180-degree rotations (fast) and pauses (e.g., for a total time of 30 seconds) maintains the suspension of, for example, non-adherent red blood cells. Exemplary embodiments can rotate the cell growth chamber 100 from a minimum rotation of about 180 degrees to a maximum rotation of about 290 to about 300 degrees, although rotations up to 360 degrees or more can also be used. Different rocking components can be used separately or in combination. For example, a rocking component that rotates the cell growth chamber 100 about central axis 142 can be combined with a rocking component that rotates the cell growth chamber 100 about axis 144. Similarly, clockwise and counterclockwise rotations about different axes can be independently combined.

[0071] 2A and 2B, an embodiment of a cell growth system 200 having a pre-mounted fluid transfer assembly according to an embodiment of the present invention is shown. The CES 200 includes a cell growth apparatus 202. The cell growth apparatus 202 has a hatch or closable door 204 that engages with a rear portion 206 of the cell growth apparatus 202. An interior space 208 within the cell growth apparatus 202 has functional features to receive and securely engage the pre-mounted fluid transfer assembly 210. The pre-mounted fluid transfer assembly 210 is removably attached to the cell growth apparatus 202, allowing for relatively quick replacement of a used fluid transfer assembly 210 with a new or unused pre-mounted fluid transfer assembly 210 in the cell growth apparatus 202. Operation of a single cell growth apparatus 202 allows for the growth or propagation of a first set of cells using a first pre-mounted fluid transfer assembly 210, followed by the growth or propagation of a second set of cells using a second pre-mounted fluid transfer assembly 210 without sterilizing the cell growth apparatus 202 when the first pre-mounted fluid transfer assembly 210 is replaced with the second pre-mounted fluid transfer assembly 210. The pre-mounted fluid transfer assembly 210 may include a cell growth chamber 100 and an oxygenator or gas transfer module 212 (see FIG. 4A). An embodiment of a tubing guide slot is shown as 214 for receiving tubing for various media connected to the pre-mounted fluid transfer assembly 210.

[0072] The front face 262 of the CES 200 includes a user interface 264. The user interface 264 includes a display 268, such as a touch screen, to allow a user to, for example, enter data, search for data, enter test protocols, switch displays, view data, view alarms, etc. Additionally or alternatively, the user interface 264 may include one or more buttons or switches for entering information, controlling a display, or performing other functions. Input from the user interface 264 is transmitted to a control system, as described below. The front face 262 of the CES 200 may also include one or more lights or other visual signals to indicate alarms.

[0073] A holder 270, such as a bag holder or disposable holder, extends from a top surface 272 of the CES 200. The holder 270 includes a vertical leg 274 and a horizontal leg 276, arranged, for example, as an L-shaped support 278. A line support or clamp 280 is attached to the vertical leg 274 of the L-shaped support 278 to support a portion of the disposable assembly, as described below. For example, the line support 280 supports a tube of the disposable assembly relative to the vertical leg 274. A plurality of racks 282 are supported by the horizontal leg 276 of the L-shaped support 278. Each of the plurality of racks 282 supports one or more bags of a disposable set, as described below. For example, as shown in FIG. 2C , each of the plurality of racks includes a rail system 284, a hook system 286, or both the rail system 284 and the hook system 286. Rail system 284 includes sidewalls 288 extending parallel to one another and defining a channel 290 therebetween. Sidewalls 288 are formed of metal, plastic, composite, or other suitable material. In the exemplary embodiment, sidewalls 288 each have a second sidewall or bumper on its inner surface, defining a neck 292, i.e., narrow portion, of channel 290. For example, second sidewall 293 is formed of a resilient or flexible material such as rubber. Alternatively, second sidewall 293 may be formed of a non-resilient material such as metal, plastic, composite, or other suitable material. For example, neck 292 is the narrow portion of channel 290 that retains the top of a bag or disposable within channel 290. Rail system 284 functions to distribute the load of the bag being supported and reduce rupture, stretching, or tearing of the bag material. Rail system 284 can accommodate a variety of different bag types and sizes. The bags suspended by rail system 284 include rods that are received within channel 290 to retain the bags therein, as described below. The rods within the bags have a diameter greater than the width of neck 292 defined by second sidewall 293.

[0074] A slot 294 is formed in the bottom surface of the second side wall 293. The slot 294 is positioned on a side of the second side wall 293 adjacent to the side wall 288. The slot 294 is positioned to receive one or more hooks 296 of the hook system 286 therein. For example, the slot 294 in one second side wall 293 of each rail system 284 may receive a hook 296 of the hook system 286, while the slot 294 in the other second side wall 293 of the rail system 284 may be left empty. The hooks 296 may be U-shaped or J-shaped and are configured to secure the bag at the opening of the bag.

[0075] The cell growth system 200 and / or cell growth apparatus 202 include a barcode scanner configured to scan barcodes on disposables, media, formulations, etc. used with the cell growth system 200 and / or cell growth apparatus 202. The barcode scanner is configured to collect data from the scanned barcodes and transfer the data to a computing system 2400 as described herein. The barcode scanner may be hardwired or wirelessly connected. The barcode scanner may be a handheld device or may be configured within a user interface or fixed on the housing of the cell growth apparatus 202. The barcode scanner may be compatible to read customer barcodes, generic barcodes, supplier barcodes, or any barcode, commercially available or not.

[0076] Figure 3 shows the rear portion 206 of the cell growth device 202 prior to the removably attachment of a pre-mounted fluid transfer assembly 210 (see Figure 2B) according to an embodiment of the present disclosure. The closable door 204 (see Figures 2A and 2B) is omitted from Figure 3. The rear portion 206 of the cell growth device 202 includes several different components that operate in conjunction with the components of the pre-mounted fluid transfer assembly 210. Specifically, the rear portion 206 of the cell growth device 202 includes several peristaltic pumps (IC circulation pump 218, EC circulation pump 220, IC inlet pump 222, EC inlet pump 224) that cooperate with the pump loops in the pre-mounted fluid transfer assembly 210. The rear portion 206 of the cell growth apparatus 202 also includes a number of valves (IC circulation valve 226, reagent valve 228, IC media valve 230, air removal valve 232, cell inlet valve 234, wash valve 236, distribution valve 238, EC media valve 240, IC waste or outlet valve 242, EC waste valve 244, and harvest valve 246). Additionally, a number of sensors (IC outlet pressure sensor 248, combined IC inlet pressure / temperature sensor 250, combined EC inlet pressure / temperature sensor 252, and EC outlet pressure sensor 254) are associated with the rear portion 206 of the cell growth apparatus 202. Additionally shown is an optical sensor 256 for the air removal chamber, according to one embodiment.

[0077] According to an embodiment, an axis or swing control 258 is shown for rotating the cell growth chamber 100. An axis fitting 260 associated with the axis or swing control 258 ensures proper alignment of the axis access opening (e.g., opening 424 (FIG. 4A) in the tubing housing 300 (FIG. 4A)) of the pre-mounted fluid transfer assembly 210 or 400 relative to the rear portion 206 of the cell growth device 202. Rotation of the axis or swing control 258 imparts rotational motion to the axis fitting 260 and the cell growth chamber 100. Thus, when an operator or user of the CES 200 installs a new or unused pre-mounted fluid transfer assembly 400 (FIG. 4A) on the cell growth device 202, alignment is a relatively simple matter of properly orienting the axis access opening (e.g., opening 424 in FIG. 4A) of the pre-mounted fluid transfer assembly 210, 400 relative to the axis fitting 260.

[0078] Figure 4A is a perspective view of a removable pre-mounted fluid transfer assembly 400. The pre-mounted fluid transfer assembly 400 is removably attached to the cell growth apparatus 202 (Figures 2B and 3), allowing for relatively quick replacement of a used pre-mounted fluid transfer assembly 400 with a new or unused pre-mounted fluid transfer assembly 400 on the cell growth apparatus 202. As shown in Figure 4A, the cell growth chamber 100 is attached to a bioreactor coupling that includes a shaft fitting 402. The shaft fitting 402 has one or more shaft fastening mechanisms (such as biased arms or spring members 404) for engaging the shaft (258 in Figure 3) of the cell growth apparatus 202.

[0079] According to embodiments, the pre-mounted fluid transfer assembly 400 includes tubing 408A, 408B, 408C, 408D, 408E, etc. and fittings, which provide the fluid flow paths shown in FIGS. 5A, 5B, 5C, and 6, as described below. Pump loops 406A, 406B, and 406C are also provided for the pumps. While various media may be provided where the cell growth device 202 is located, according to embodiments, the pre-mounted fluid transfer assembly 400 includes tubing of sufficient length to extend outside the cell growth device 202, allowing tubing associated with a media bag or media container to be welded.

[0080] 4B-4H show various views of the assemblies and components of a pre-mount fluid transfer assembly 400. In an exemplary embodiment, the pre-mount fluid transfer assembly 400 includes a media bag 410 (FIG. 4D), a waste bag 412 (FIG. 4E), a pressure pod 414 (FIGS. 4F-4G), a sampling coil 416 (FIG. 4H), a cell input bag, an in-line filter 420 (FIG. 4I), tubing, a filter, or a combination thereof. For example, the pre-mount fluid transfer assembly 400A of FIG. 4B includes a cell growth chamber 100B. The pre-mount fluid transfer assembly 400B of FIG. 4C includes a cell growth chamber 100A. Except for the differences in size of the cell growth chambers 100A, 100B, the other components of the pre-mount fluid transfer assemblies 400A, 400B are the same. This allows for efficient manufacturing of the pre-mount fluid transfer assembly 400.

[0081] As shown in FIGS. 4D and 4E , the premounted fluid transfer assembly 400 includes one or more media bags 410 and one or more waste bags 412. In some embodiments, the media bag 410 is the same as the waste bag 412. Identical media bag 410 and waste bag 412 allow for efficient and low-cost manufacturing. As shown in FIGS. 4D and 4E , the media bag 410 and waste bag 412 are formed from a single sheet of material and include a container portion 422 and a hanger portion 425. For example, the sheet of material, media bag 410, and waste bag 412 are formed from a triple-layer EVA (ethylene vinyl acetate), which is less breathable than PVC to maintain a more stable pH. This makes the material "friendly" to the media, and such non-breathable materials extend the shelf life of the media. For example, the sheet of material is welded to form the container portion 422 and the hanger portion 425. The container portion 422 may be a pocket within the media bag 410 or waste bag 412. Reservoir portion 422 is sized to hold a particular amount of fluid (e.g., culture medium). For example, reservoir portion 422 may be sized to hold 5 L of fluid. Alternatively, reservoir portion 422 may be sized to hold an amount of fluid appropriate for a desired function.

[0082] The hanger portion 425 includes an opening for receiving the hook 296, as described with reference to FIG. 2C . The hanger portion 425 includes a rod 426 secured within a longitudinal opening or channel 428 in the medium bag 410 or waste bag 412. The rod 426 may be a cylindrical rod or an extruded polygonal rod that engages with the rail system 284, as described with reference to FIG. 2C . For example, the rod 426 rests on top of the second side wall 293 when the bag is inserted into the channel 290 between the side walls 288. The rod 426 has a larger diameter or width than the neck 292, so that the hanger portion 425 does not slide through the neck 292 and the medium bag 410 or waste bag 412 is suspended within the rail system 284. Additionally or alternatively, the medium bag 410 may be a different material, size, or configuration than the waste bag 412.

[0083] The pair of ports 430 are located on opposite sides of the container portion 422 from the hanger portion 425. The pair of ports 430 includes an inlet port 430A and an outlet port 430B. The inlet port 430A is engaged with tubing or fluid flow into the container portion 422. The outlet port 430B is engaged with tubing or fluid flow out of the container portion 422. The pair of ports 430, i.e., the dual port design of the medium bag 410 or waste bag 412, allows multiple medium bags 410 or multiple waste bags 412 to be connected together or daisy-chained, as described below.

[0084] 4F and 4G, a pressure pod 414 is disposed in the housing of the pre-mounted fluid transfer assembly 400. The pressure pod 414 may be configured to detect fluid pressure through a fluid line, tubing, or along a fluid flow path. The pressure pod 414 includes a single outlet housing 433, a diaphragm 432, and a retaining ring 434. The single outlet housing 433 includes a tubular inlet port 436 and a closure port 438. The inlet port 436 is a tubular port configured to receive fluid into the single outlet housing 433. The closure port 438 may be similar to the tubular inlet port 436, but is blocked by a plug 440 to prevent fluid flow therethrough. The plug 440 may be formed of plastic or elastomer. The single outlet housing 433 includes a body 442 integrally formed with the inlet port 436 and the closure port 438. The body 442 defines an interior space 444 therein, e.g., a cylindrical interior space 444. The diaphragm 432 may be a circular, planar diaphragm housed within the interior space 444 of the body 442. For example, the diaphragm 432 is a resilient material that deflects under fluid pressure. The retaining ring 434 is disposed at the end of the body 442 of the single outlet housing 433 opposite the inlet port 436 and the occlusion port 438 to secure the diaphragm 432 within the body 442. For example, the retaining ring 434 may be press-fit or threaded onto the end of the body 442 opposite the inlet port 436 and the occlusion port 438.

[0085] 4H and 41 illustrate exemplary tubing included in the pre-mounted fluid transfer assembly 400. In at least one exemplary embodiment, the sampling coil 416 shown in FIG. 4H is a tube configured to take a sample of fluid in one of the fluid flow paths. In at least one exemplary embodiment, the in-line filter 420 shown in FIG. 41 includes a filter positioned within the tube to filter substances from the fluid traveling through the fluid flow path.

[0086] Next, Figures 5A, 5B, and 5C are schematic diagrams of an embodiment of a cell growth system 500. Figure 6 is a schematic diagram of another embodiment of a cell growth system 600. Figures 7A-7C are schematic diagrams of yet another embodiment of a cell growth system. In the embodiments shown in Figures 5A, 5B, 5C, and 6, cells are grown in the IC space, as described below. However, the present disclosure is not limited to such examples. In other embodiments, cells may be grown in the EC space.

[0087] As previously mentioned, Figures 5A, 5B, and 5C illustrate CES 500. While Figures 5A, 5B, and 5C illustrate substantially similar components of CES 500, Figures 5A, 5B, and 5C illustrate possible operations for fluid movement in a first fluid circuit using structural features of CES 500, according to embodiments of the present disclosure. As shown, CES 500 includes first fluid circuit 502 (also referred to as the "capillary inner loop" or "IC loop") and second fluid circuit 504 (also referred to as the "capillary outer loop" or "EC loop"). First fluid flow path 506 is fluidly associated with cell growth chamber 501 to form first fluid circuit 502. Fluid enters cell growth chamber 501 through IC inlet port 501A, passes through hollow fibers within cell growth chamber 501, and exits through IC outlet port 501B. Pressure measuring device 510 measures the pressure of the medium leaving cell growth chamber or bioreactor 501. The medium flows through IC circulation pump 512, which is used to control the medium flow rate. IC circulation pump 512 can pump fluid in a first direction or a second direction opposite the first direction. IC outlet port 501B can be used as an inlet in the reverse direction. For example, in a first configuration, the IC circulation pump can pump fluid in the forward direction, where fluid enters IC inlet port 501A. In a second configuration, the IC circulation pump can pump fluid in the reverse direction, where fluid enters IC outlet port 501B.

[0088] Media entering the IC loop may enter through valve 514. As will be appreciated by those skilled in the art, additional valves, pressure gauges, pressure / temperature sensors, ports, and / or other devices may be placed in various locations to isolate and / or measure media properties in certain portions of the fluid path. Thus, it should be understood that the illustrated schematic is one possible configuration for elements of CES 500 and that variations are possible within the scope of one or more embodiments.

[0089] For IC loop 502, samples of medium are obtained during operation from sample port 516 or sample coil 518. Pressure / temperature measurement device 520 located in first fluid circuit 502 allows the pressure and temperature of the medium to be measured during operation. The medium then returns to IC inlet port 501A, completing fluid circuit 502. Cells grown / expanded in cell growth chamber 501 are flushed out of cell growth chamber 501 and passed through valve 598 into cell harvest bag 599 or redistributed into the hollow fibers for further growth.

[0090] In second fluid circuit 504, fluid enters cell growth chamber 501 through EC inlet port 501C and leaves cell growth chamber 501 through EC outlet port 501D. In EC loop 504, medium contacts the outside of the hollow fibers of cell growth chamber 501, thereby allowing diffusion of small molecules into and out of the hollow fibers.

[0091] In embodiments, the pressure and temperature of the medium can be measured by pressure / temperature measuring device 524 located in second fluid circuit 504 before the medium enters the EC space of cell growth chamber 501. After the medium leaves cell growth chamber 501, the pressure of the medium in second fluid circuit 504 can be measured by pressure measuring device 526. For EC loops, samples of the medium are obtained from sample port 530 or from a sample coil during operation.

[0092] In embodiments, after leaving the EC outlet port 501D of the cell growth chamber 501, the fluid in the second fluid circuit 504 passes through an EC circulation pump 528 to an oxygenator or gas transfer module 532. The EC circulation pump 528 is capable of pumping fluid in both directions. The second fluid flow path 522 is fluidly associated with the oxygenator or gas transfer module 532 via an oxygenator inlet port 534 and an oxygenator outlet port 536. During operation, the fluid medium enters 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. The oxygenator or gas transfer module 532 adds oxygen and removes air bubbles from the medium in the CES 500, for example. In various embodiments, the medium in the second fluid circuit 504 is in equilibrium with the gas entering the oxygenator or gas transfer module 532. Oxygenator or gas transfer module 532 can be any oxygenator or gas transfer device of appropriate size. Air or gas enters oxygenator or gas transfer module 532 through filter 538 and exits oxygenator or gas transfer module 532 through filter 540. Filters 538, 540 reduce or prevent contamination of oxygenator or gas transfer module 532 and associated media. Air or gas purged from CES 500 during part of the priming process can be vented to atmosphere through oxygenator or gas transfer module 532.

[0093] In at least one embodiment, the cell-containing medium (from bag 562) and fluid medium from bag 546 are introduced into first fluid circuit 502 via first fluid flow path 506. A fluid container 562 (e.g., a cell inlet bag or saline priming fluid for priming air out of the system) is fluidly associated with first fluid flow path 506 and first fluid circuit 502 via valve 564.

[0094] A fluid container, i.e., media bag 544 (e.g., a reagent) is fluidly associated with first fluid inlet line 542 via valve 548 or with second fluid inlet line 574 via valve 576, and a fluid container 546 (e.g., IC media) is fluidly associated with first fluid inlet line 542 via valve 550 or with second fluid inlet line 574 via valve 570. Sterile and sealable first and second input priming lines 508, 509 are also provided. An air elimination chamber (ARC) 556 is fluidly associated with first circuit 502. Air elimination chamber 556 may have one or more ultrasonic sensors, including upper and lower sensors, for detecting air, lack of fluid, and / or a gas / fluid boundary (e.g., an air / fluid boundary) at specific measurement points within air elimination chamber 556. 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 boundaries at those locations. In embodiments, other types of sensors may be used without departing from the scope of the present disclosure. For example, optical sensors may be used in accordance with embodiments of the present disclosure. Air or gas purged from the CES 500 during part of a priming process or other protocol may be vented to the atmosphere through a line 558 fluidly associated with the air removal chamber 556 and through an air valve 560 during the air removal operation.

[0095] EC medium (e.g., from bag 568) or wash solution (e.g., from bag 566) is added to the first or second fluid flow path. Fluid container 566 is fluidly associated with valve 570. Valve 570 is fluidly associated with first fluid circuit 502 via distribution valve 572 and first fluid inlet path 542. By opening valve 570 and closing distribution valve 572, fluid container 566 can be fluidly associated with second fluid circuit 504 via second fluid inlet path 574 and EC inlet path 584. Similarly, fluid container 568 is fluidly associated with valve 576. Valve 576 is fluidly associated with first fluid circuit 502 via first fluid inlet path 542 and distribution valve 572. By opening valve 576 and closing distribution valve 572, fluid container 568 can be fluidly associated with second fluid inlet path 574.

[0096] An optional heat exchanger 552 may be provided for the introduction of media reagents or for the introduction of wash fluids.

[0097] In the IC loop, fluid is first pumped by IC inlet pump 554. In the EC loop, fluid is first pumped by EC inlet pump 578. An air detector 580, such as an ultrasonic sensor, may be associated with EC inlet line 584.

[0098] In at least one embodiment, first and second fluid circuits 502, 504 are connected to a waste line 588. When valve 590 is opened, IC medium flows through waste line 588 to waste or outlet bag 586. Similarly, when valve 582 is opened, EC medium flows through waste line 588 to waste or outlet bag 586.

[0099] In an embodiment, cells are harvested through cell harvest channel 596. Here, cells from cell growth chamber 501 are harvested by pumping the IC medium containing the cells through cell harvest channel 596 and valve 598 into cell harvest bag 599.

[0100] The various components of the CES 500 are housed within a device or housing, such as a cell growth device 202 (see FIGS. 2B and 3), which maintains the cells and culture medium, for example, at a predetermined temperature.

[0101] In the configuration shown for CES 500 in FIG. 5A , the fluid medium in first fluid circuit 502 and second fluid circuit 504, in one embodiment, flows through cell growth chamber 501 in the same direction (co-current configuration). In other embodiments, CES 500 may be configured to flow in a counter-current configuration (not shown). In the configuration shown in FIG. 5A , the fluid in first fluid circuit 502 enters bioreactor 501 at IC inlet port 501A and exits bioreactor 501 at IC outlet port 501B. In the configuration shown in FIGS. 5B and 5C , in embodiments, the fluid medium in first fluid circuit 502 flows in opposite directions from connection 517, entering an IC inlet port at one end of the bioreactor, i.e., first port 501A, and entering an IC outlet port at the other end of the bioreactor, i.e., second port 501B, to retain cells within the bioreactor itself. The first fluid flow path may be fluidly associated with the first fluid circuit via connection 517. In embodiments, connection 517 may be a point or location where fluid flows in opposite directions, for example, based on the direction of the IC inlet pump and the direction of the IC circulation pump. In one embodiment, connection 517 may be a T-piece or T-junction. In other embodiments, connection 517 may be a Y-piece or Y-junction. Connection 517 may be any type of fitting, coupling, weld, passage, tubing, etc. that allows the first fluid flow path to be fluidly associated with the first circulation path. It should be understood that the schematics and operational configurations shown in FIGS. 5A, 5B, and 5C represent possible configurations of the various elements of the cell growth system, and that modifications to the illustrated schematics and operational configurations are within the scope of one or more of the present embodiments.

[0102] FIG. 6 is a schematic diagram of another embodiment of a cell growth system 600. The CES 600 includes a first fluid circuit 602 (also referred to as an "inner capillary loop" or "IC loop") and a second fluid circuit 604 (also referred to as an "outer capillary loop" or "EC loop"). A first fluid flow path 606 is fluidly associated with a cell growth chamber 601 to form the first fluid circuit 602. Fluid enters the cell growth chamber 601 through an IC inlet port 601A, passes through hollow fibers within the cell growth chamber 601, and exits through an IC outlet port 601B. A pressure sensor 610 measures the pressure of the medium leaving the cell growth chamber 601. In addition to pressure, the sensor 610 may be a temperature sensor that senses medium pressure and medium temperature during operation. The medium flows through an IC circulation pump 612, which is used to control the medium flow rate. The IC circulation pump 612 can pump fluid in a first direction or a second direction opposite the first direction. Outlet port 601B can be used as an inlet in the reverse direction. Media entering the IC loop can enter through valve 614. As will be appreciated by those skilled in the art, additional valves, pressure gauges, pressure / temperature sensors, ports, and / or other devices can be placed in various locations to isolate media and / or measure media properties in certain portions of the fluid path. Therefore, it should be understood that the illustrated schematic is one possible configuration for elements of CES 600 and that variations are possible within the scope of one or more embodiments.

[0103] For the IC loop, samples of media are taken from sample coil 618 during operation, and the media returns to IC inlet port 601A, completing fluid circuit 602. Cells grown / expanded in cell growth chamber 601 are flushed out of cell growth chamber 601 and into harvest bag 699 via valve 698 and cell harvest channel 697. Alternatively, if valve 698 is closed, the cells can be redistributed back into chamber 601 for further growth.

[0104] In second fluid circuit 604, fluid enters cell growth chamber 601 through EC inlet port 601C and leaves cell growth chamber 601 through EC outlet port 601D. In the EC loop, medium contacts the outside of the hollow fibers of cell growth chamber 601, thereby allowing diffusion of small molecules into and out of the hollow fibers within chamber 601.

[0105] Before the medium enters the EC space of cell growth chamber 601, the pressure and temperature of the medium can be measured by pressure / temperature sensor 624 located in second fluid circuit 604. After the medium leaves cell growth chamber 601, the pressure and / or temperature of the medium in second fluid circuit 604 can be measured by sensor 626. For the EC loop, a sample of the medium is obtained from sample port 630 or from a sample coil during operation.

[0106] After leaving the EC outlet port 601D of the cell growth chamber 601, the fluid in the second fluid circuit 604 passes through an EC circulation pump 628 to an oxygenator or gas transfer module 632. In embodiments, the EC circulation pump 628 is also capable of pumping fluid in both directions. The second fluid flow path 622 is fluidly associated with the oxygenator or gas transfer module 632 via an inlet port 632A and an outlet port 632B of the oxygenator or gas transfer module 632. During operation, the fluid medium enters the oxygenator or gas transfer module 632 via the inlet port 632A and exits the oxygenator or gas transfer module 632 via the outlet port 632B. The oxygenator or gas transfer module 632 adds oxygen and removes air bubbles from the medium in the CES 600, for example. In various embodiments, the medium in the second fluid circuit 604 is in equilibrium with the gas entering the oxygenator or gas transfer module 632. Oxygenator or gas transfer module 632 may be any suitably sized device useful for oxygen supply or gas transfer. Air or gas enters oxygenator or gas transfer module 632 through filter 638 and exits oxygenator or gas transfer module 632 through filter 640. Filters 638, 640 reduce or prevent contamination of oxygenator or gas transfer module 632 and associated media. Air or gas purged from CES 600 during part of the priming process can be vented to atmosphere through oxygenator or gas transfer module 632.

[0107] In the configuration shown as CES 600, the fluid media in the first fluid circuit 602 and the second fluid circuit 604 flow in the same direction (co-current configuration) through the cell growth chamber 601. Embodiments of the CES 600 may also be configured for counter-current flow.

[0108] In at least one embodiment, media containing cells (from a source such as a cell container, e.g., a bag) is attached to attachment point 662, and fluid media from a media source is attached to attachment point 646. The cells and media are introduced into first fluid circuit 602 via first fluid flow path 606. Attachment point 662 is fluidly associated with first fluid flow path 606 via valve 664. Attachment point 646 is fluidly associated with first fluid flow path 606 via valve 650. A reagent source may be fluidly connected to point 644 and associated with first fluid inlet path 642 via valve 648, or with second fluid inlet path 674 via valves 648, 672.

[0109] An air elimination chamber (ARC) 656 is fluidly associated with the first circuit 602. The air elimination chamber 656 may include one or more sensors, including upper and lower sensors for detecting air, a lack of fluid, and / or a gas / fluid boundary, e.g., an air / fluid boundary, at specific measurement points within the air elimination chamber 656. For example, ultrasonic sensors may be used near the bottom and / or top of the air elimination chamber 656 to detect air, fluid, and / or an air / fluid boundary at those locations. In embodiments, other types of sensors may be used without departing from the scope of the present disclosure. For example, optical sensors may be used in accordance with embodiments of the present disclosure. Air or gas purged from the CES 600 during part of a priming process or other protocols may be vented to the atmosphere through a line 658 fluidly associated with the air elimination chamber 656 and through an air valve 660.

[0110] An EC medium source is attached to EC medium attachment point 668. A wash fluid source is attached to wash fluid attachment point 666, allowing EC medium and / or wash fluid to be added to the first or second fluid flow path. Attachment point 666 is fluidly associated with valve 670. Valve 670 is fluidly associated with first fluid circuit 602 via valve 672 and first fluid inlet path 642. Additionally, by opening valve 670 and closing valve 672, attachment point 666 can be fluidly associated with second fluid circuit 604 via second fluid inlet path 674 and second fluid flow path 684. Similarly, attachment point 668 is fluidly associated with valve 676. Valve 676 is fluidly associated with first fluid circuit 602 via first fluid inlet path 642 and valve 672. Additionally, attachment point 668 can be fluidly associated with second fluid inlet passage 674 by opening valve 676 and closing dispensing valve 672 .

[0111] In the IC loop, the fluid is first pumped by the IC inlet pump 654. In the EC loop, the fluid is first pumped by the EC inlet pump 678. An air detector 680, such as an ultrasonic sensor, may be associated with the EC inlet line 684.

[0112] In at least one embodiment, the first and second fluid circuits 602, 604 are connected to a waste line 688. When valve 690 is opened, the IC medium flows through the waste line 688 to the waste or outlet bag 686. Similarly, when valve 692 is opened, the EC medium flows to the waste or outlet bag 686.

[0113] After the cells have grown in the cell growth chamber 601, the cells are harvested via cell harvest line 697. Cells from the cell growth chamber 601 are harvested into a cell harvest bag 699 via cell harvest line 697 by pumping the IC medium containing the cells with valve 698 open.

[0114] The various components of the CES600 are housed within a device or housing, such as cell growth device 202 (see FIGS. 2B and 3), which maintains the cells and culture medium, for example, at a predetermined temperature. Additionally, the components of the CES600 and CES500 may be combined. In other embodiments, a CES may include fewer or more components than those shown in the CES500 and CES600, and still be within the scope of the present invention. One example of a cell growth system incorporating features of the present disclosure is the Quantum® Cell Growth System, manufactured by Terumo BCT, Inc. (Lakewood, Colorado).

[0115] It should be understood that the schematic shown in FIG. 6 represents a possible configuration of the various elements of the cell growth system, and that modifications to the schematic shown are within the scope of one or more of the present embodiments.

[0116] Examples and detailed descriptions of cell expansion systems are provided in U.S. Patent No. 8,309,347 ("Cell Expansion System and Method of Use," issued November 13, 2012) and U.S. Patent No. 9,057,045 ("Method for Loading and Distributing Cells in a Bioreactor of a Cell Expansion System," filed December 15, 2010, and issued June 16, 2015), the entire contents of which are expressly incorporated herein by reference.

[0117] In the embodiment shown in Figures 7A-7C, the cells are grown in the IC space, as described below. However, the present invention is not limited to such an example. In other embodiments, the cells may be grown in the EC space.

[0118] As previously mentioned, FIGS. 7A-7C illustrate CES 700. While FIGS. 7A-7C illustrate substantially similar components of CES 700, FIGS. 7A-7C also illustrate possible operational configurations for fluid movement in a first fluid circuit using structural features of CES 700, according to embodiments of the present disclosure. As shown, CES 700 includes a first fluid circuit 702 (also referred to as the "capillary inner loop" or "IC loop") and a second fluid circuit 704 (also referred to as the "capillary outer loop" or "EC loop"). A first fluid flow path 706 is fluidly associated with cell growth chamber 701 to form first fluid circuit 702. Fluid enters cell growth chamber 701 through IC inlet port 701A, passes through hollow fibers within cell growth chamber 701, and exits through IC outlet port 701B. A pressure measuring device 710 measures the pressure of the cell growth chamber 701 or the medium leaving the bioreactor. The medium flows through an IC circulation pump 712, which is used to control the medium flow rate. The IC circulation pump 712 can pump the fluid in a first direction or a second direction opposite the first direction. In at least one exemplary embodiment, the IC outlet port 701B can be used as an inlet in the reverse direction. For example, in a first configuration, the IC circulation pump 712 can pump the fluid in a positive direction, with the fluid entering the IC inlet port 701A. In a second configuration, the IC circulation pump 712 can pump the fluid in a negative direction, with the fluid entering the IC outlet port 701B.

[0119] Fluid in the IC loop, i.e., first fluid circuit 702, passes through IC circulation valve 714. As will be appreciated by those skilled in the art, additional valves, pressure gauges, pressure / temperature sensors, ports, and / or other devices can be placed in various locations to isolate and / or measure characteristics of the medium in certain portions of the fluid path. For example, first fluid circuit 702 includes IC inlet pressure sensor 715, which, in some exemplary embodiments, is located between IC circulation pump 712 and IC circulation valve 714. Therefore, it should be understood that the illustrated schematic is one possible configuration for elements of CES 700 and that variations are possible within the scope of one or more embodiments.

[0120] For the first fluid circuit 702, samples of the medium are obtained during operation from a sample port or sample coil 718. A pressure / temperature measuring device 720 located in the first fluid circuit 702 allows the pressure and temperature of the medium to be measured during operation. The medium then returns to the IC inlet port 701A, completing the first fluid circuit 702. Cells grown / expanded in the cell growth chamber 701 are flushed out of the cell growth chamber 701 and passed through valve 798 into a cell harvest bag 799 or redistributed into the hollow fibers for further growth.

[0121] In second fluid circuit 704, fluid enters cell growth chamber 701 through EC inlet port 701C and leaves cell growth chamber 701 through EC outlet port 701D. In second fluid circuit 704, medium contacts the outside of the hollow fibers of cell growth chamber 701, thereby allowing diffusion of small molecules into and out of the hollow fibers.

[0122] In at least one exemplary embodiment, the pressure and temperature of the medium can be measured by a pressure / temperature measuring device 724 located in the second fluid circuit 704 before the medium enters the EC space of the cell growth chamber 701. After the medium leaves the cell growth chamber 701, the pressure of the medium in the second fluid circuit 704 can be measured by a pressure measuring device 726. For the EC loop, a sample of the medium is obtained from a sample port 730 or from a sample coil during operation.

[0123] In at least one exemplary embodiment, after leaving the EC outlet port 701D of the cell growth chamber 701, the fluid in the second fluid circuit 704 passes through an EC circulation pump 728 to an oxygenator or gas transfer module 732. The EC circulation pump 728 is capable of pumping fluid in both directions. The second fluid flow path 722 is fluidly associated with the oxygenator or gas transfer module 732 via an oxygenator inlet port 734 and an oxygenator outlet port 736. During operation, the fluid medium enters the oxygenator or gas transfer module 732 via the oxygenator inlet port 734 and exits the oxygenator or gas transfer module 732 via the oxygenator outlet port 736. The oxygenator or gas transfer module 732 adds oxygen and removes air bubbles from the medium in the CES 700, for example. In various exemplary embodiments, the medium in the second fluid circuit 704 is in equilibrium with the gas entering the oxygenator or gas transfer module 732. Oxygenator or gas transfer module 732 can be any oxygenator or gas transfer device of appropriate size. Air or gas enters oxygenator or gas transfer module 732 through filter 738 and exits oxygenator or gas transfer module 732 through filter 740. Filters 738, 740 reduce or prevent contamination of oxygenator or gas transfer module 732 and associated media. Air or gas purged from CES 700 during part of the priming process can be vented to atmosphere through oxygenator or gas transfer module 732.

[0124] In at least one embodiment, cell-containing media from bag 762 and fluid media from bag 746 are introduced into first fluid circuit 702 via first fluid flow path 706. A fluid container 762 (e.g., a cell inlet bag or saline priming fluid for priming air out of the system) is fluidly associated with first fluid flow path 706 and first fluid circuit 702 via valve 764.

[0125] A medium bag 744 (e.g., a reagent), a container 746 (e.g., IC medium), or a fluid container is fluidly associated with the first fluid inlet path 742 via corresponding valves 748 and 750, respectively, or with the second fluid inlet path 774 via corresponding valves 770 and 776, respectively. An air removal chamber (ARC) 756 is fluidly associated with the first fluid circuit 702. The air removal chamber 756 may include one or more ultrasonic sensors, including upper and lower sensors for detecting air, a lack of fluid, and / or a gas / fluid boundary, e.g., an air / fluid boundary, at specific measurement points within the air removal chamber 756. For example, ultrasonic sensors may be used near the bottom and / or top of the air removal chamber 756 to detect air, fluid, and / or an air / fluid boundary at those locations. In exemplary embodiments, other types of sensors may be used without departing from the scope of the present disclosure. For example, optical sensors may be used in accordance with embodiments of the present disclosure. Air or gas purged from CES 700 during part of the priming process or other protocols can be vented to atmosphere through air valve 760 fluidly associated with air removal chamber 756 .

[0126] EC medium (e.g., from bag 768) and / or wash solution (e.g., from bag or fluid container 766) are applied to first fluid inlet path 742 and / or second fluid flow path 722. Fluid container 766 is fluidly associated with valve 770. Valve 770 is fluidly associated with first fluid circuit 702 via distribution valve 772 and first fluid inlet path 742. Also, by opening valve 770 and closing distribution valve 772, fluid container 766 can be fluidly associated with second fluid circuit 704 via second fluid inlet path 774 and EC inlet path 784. Similarly, as shown in FIG. 7C , fluid containers 768A, 768B are fluidly associated with valve 776. Valve 776 is fluidly associated with first fluid circuit 702 via first fluid inlet path 742 and distribution valve 772. Additionally, fluid containers 768A, 768B can be fluidly associated with second fluid inlet passage 774 by opening valve 776 and closing dispensing valve 772 .

[0127] Fluid containers 768A, 768B may be medium containers for distributing medium to first fluid inlet line 742 or second fluid inlet line 774. In some exemplary embodiments, fluid containers 768A, 768B may be the same as medium bags 410 shown in FIG. 4D . Fluid containers 768A, 768B may be part of an EC circulatory supply loop 752 when in communication with second fluid inlet line 774 (or part of an IC circulatory supply loop when in communication with first fluid inlet line 742). In EC circulatory supply loop 752, medium is pumped from fluid container 768B to fluid container 768A, through EC loop 704, through EC waste valve 782, and back to fluid container 768B. Use of EC circulatory supply loop 752 avoids the use of pass-through bulk feeding, in which fresh medium is continuously added to the system and collected in an outlet waste bag, which is discarded when full. In pass-through mass feeding, the more cells you grow, the higher the supply flow rate of medium to the cells must be. Users typically measure glucose or lactate once a day, then increase the supply flow rate for that day, for example. The supply flow rate may not be accurate throughout the day, leading to medium waste.

[0128] In contrast, in the CES700 herein, the EC circulation supply loop 752 expands the capacity of either the IC or EC circulation loop by connecting or daisy-chaining fluid container 768A with fluid container 768B (via the aforementioned port pair). In the CES700, the cell growth chamber 701 is manually fed from outside the CES700. The outlet line 788 returns to fluid container 768B, and the medium is recirculated. The user can calculate how much lactic acid is produced and determine waste. The user does not need to change bags; they simply set the feed flow rate and run the protocol. Thus, the CES700 is more efficient and uses less medium overall.

[0129] Although two fluid containers 768A, 768B are shown and described, it will be appreciated that more fluid containers 768N may be added to the system.

[0130] In the exemplary protocol, gas is supplied to the system through a GTM, such as GTM 732, using an EC circulation. The gas is supplied externally to the CES 700, and the gas is exchanged to the desired concentration each time the medium passes through the GTM 732. In this case, fluid containers 768A and 768B serve as exchange containers for the medium returning to the cell growth chamber 701. For example, the accompanying T cell feeding protocol (CFA-CSS-CEdj-036-03) uses approximately 11 L of EC medium and 3.4 L of IC medium over the duration of the growth protocol. Using this new supply approach, the user adds 3.4 L of IC medium to the IC medium line and daisy-chains 11 L (three media bags) of EC medium to the EC inlet and outlet lines. As the oxygen demand of the cell population increases, the EC circulation flow rate must increase, as does the EC inlet flow rate (effectively the EC supply circulation).

[0131] Depending on the cell type and protocol kinetics, either an IC or an EC supply circulation loop is implemented, or a dual supply circulation loop can be used, for example, as shown in Figure 7C.

[0132] This approach allows for a large number of possible feeding strategies to be implemented. This approach also improves the ease of use of the CES700, reducing human interaction with the device as there is no need to empty the waste bag. Fresh media bags are fed to the device at various intervals during cell growth. Furthermore, in many of these situations, there is no need to determine the feed flow rate. Even when using continuous perfusion feeding, lactate and glucose concentrations can fluctuate significantly unless very long custom tasks with small flow rate changes are used.

[0133] Depending on the desired application, EC circulation supply loop 752 may be used in coating the hollow fibers of cell growth chamber 701, in which case fluid is circulated and forced through the membrane (IC side). Additionally or alternatively, EC circulation supply loop 752 may be used in a counter-flow technique, in which case fluid is forced at both ends of cell growth chamber 701.

[0134] The passive coating model used in the CES system requires the application of a coating agent to the cell growth surface to promote cell attachment and subsequent proliferation of adherent cell lines, such as human mesenchymal stromal cells (hMSCs). Examples of coating agents include human fibronectin (hFN) or cryoprecipitate (CPPT). The bioreactor coating protocol for the passive coating model involves introducing the coating agent into the inside of the bioreactor's capillaries and circulating the coating agent in the IC circulation loop for a minimum of 16 hours. The passive coating model protocol requires at least two CES systems to immediately begin further proliferation of cell populations harvested from the CES system (hMSCs can only be stored for a maximum of 16 hours in a non-cryopreserved state). The new protocol or method for coating the hollow fibers of the cell growth chamber 701 completes coating of the growth surface 10 minutes after introducing the coating agent into the IC circulation loop. This method (see Appendix B for the protocol) utilizes active ultrafiltration of fluid (movement of fluid from the IC side of the bioreactor to the EC side of the bioreactor) to reduce the time required for proper chemical reaction between the coating and the bioreactor growth surface. The molecular barrier formed by the specific structure of the hollow fibers within the bioreactor prevents the coating from passing through the hollow fiber walls along with the fluid in which it is suspended. Using active ultrafiltration to move fluid "actively" drives the movement of the coating across the surface of the hollow fibers. Enabling users to coat a hollow fiber bioreactor and load cells on the same day reduces errors, saves time, and reduces cell growth time by one day. This allows customers with access to only a single CES device (e.g., the Quantum® / Quantum Flex® device) to harvest a cell population from that system and coat new disposables for subsequent passage / expansion of that same cell population without the need for cryopreservation.

[0135] As described with reference to Figures 4D and 4E, a two-port bag can be used, connecting an inlet line (typically a "wash" line) to one port of the bag while simultaneously connecting a "waste" line to the other port. This allows the protein medium solution (usually phosphate-buffered saline (PBS)) containing the coating agent to be recirculated within the system for as long as the user desires. Using a high IC inlet flow rate to promote contact between the coating solution and the HFB membrane via ultrafiltration (UF) promotes coating agent adsorption and reduces the time required to coat a hollow fiber bioreactor compared to passive coating models. Moving the IC fluid across the HFB membrane also promotes deposition of the coating agent on the IC side of the HFB membrane. For example, the coating solution, which has a higher molecular weight and cannot pass through the HFB membrane, is deposited on the IC side of the HFB membrane.

[0136] Optionally, a heat exchanger may be provided for the introduction of media reagents or wash fluids. In some embodiments, a heat exchanger may optionally be provided in the first fluid inlet passage 742 and / or the second fluid inlet passage 774.

[0137] One or more differential pressure sensors may be included for real-time pressure monitoring and alarms. This allows for more efficient and accurate measurements as opposed to manual measurements. The pressure sensors facilitate detection of reduced or stopped flow, a depleting gas supply, user error, etc. Upon detecting a reduced or stopped flow, the differential pressure sensor triggers an alarm to alert the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alarm on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0138] One or more gas regulators may be provided for gas management. For example, the gas regulators may be internal to the CES700 so that they do not face the user. This prevents errors. The gas regulators may be less sensitive to the amount of control. This allows for finer control. If an error in gas management is detected, the gas regulator may trigger an alarm to alert the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alarm on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0139] One or more temperature thermistors may be provided for highly sensitive temperature detection. A temperature change of 0.5 degrees is important in cell culture. Thermistors degrade less than resistance temperature detectors (RTDs), thus allowing for longer use before service is required. Upon detecting a temperature change outside a predetermined range or a temperature above or below a predetermined threshold, the thermistor may trigger an alarm to alert the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alarm on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0140] In the IC loop, fluid is first pumped by the IC inlet pump 754. In the EC loop, fluid is first pumped by the EC inlet pump 778. An air detector 780, such as an ultrasonic sensor, may be associated with the EC inlet line 784. When air is detected by the air detector 780, the air detector may trigger an alarm to alert a user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alarm on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0141] In at least one embodiment, first and second fluid circuits 702 and 704 are connected to an outlet line 788. When valve 790 is opened, IC medium flows through outlet line 788 and returns to fluid reservoir 768B. Similarly, when valve 782 is opened, EC medium flows through outlet line 788 and returns to fluid reservoir 768B.

[0142] In the exemplary embodiment, cells are harvested via cell harvest channel 796. Here, cells from the cell growth chamber 701 are harvested by pumping the IC medium containing the cells through cell harvest channel 796 and valve 798 into a cell harvest bag 799.

[0143] The various components of the CES 700 are housed within a device or housing, such as cell growth device 202 (see FIGS. 2A, 2B, 3), which maintains the cells and culture medium, for example, at a predetermined temperature.

[0144] In the configuration shown for CES 700 in FIG. 7A , the fluid medium in first fluid circuit 702 and second fluid circuit 704, in one embodiment, flows through cell growth chamber 701 in the same direction (co-current configuration). In other embodiments, CES 700 may be configured to flow in a counter-current configuration (not shown). In the configuration shown in FIG. 7A , the fluid in first fluid circuit 702 enters cell growth chamber 701 at IC inlet port 701A and exits cell growth chamber 701 at IC outlet port 701B. In other configurations, in embodiments, the fluid medium in first fluid circuit 702 flows in opposite directions from connection 717, entering an IC inlet port at one end of cell growth chamber 701, i.e., first port 701A, and entering an IC outlet port at the other end of cell growth chamber 701, i.e., second port 701B, to retain cells within the cell growth chamber itself. The first fluid flow path may be fluidly associated with the first fluid circuit via connection 717. In embodiments, connection 717 is a point or location where fluid flows in opposite directions, for example, based on the direction of the IC inlet pump and the direction of the IC circulation pump. In one embodiment, connection 717 may be a T-piece or T-junction. In other embodiments, connection 717 may be a Y-piece or Y-junction. Connection 717 may be any type of fitting, coupling, weld, passage, tubing, etc. that allows the first fluid flow path to be fluidly associated with the first circulation path. It should be understood that the schematics and operational configurations shown in FIGS. 7A-7C represent possible configurations of the various elements of the cell growth system, and that modifications to the illustrated schematics and operational configurations are within the scope of one or more of the present embodiments.

[0145] FIG. 7B shows CES 700B. CES 700B may be similar to CES 700A and may include the same or similar components as CES 700A, in which case like reference numerals are used. CES 700B includes an IC circulatory supply loop 753. IC circulatory supply loop 753 may be similar to EC circulatory supply loop 752 described above. IC circulatory supply loop 753 may include multiple fluid containers, such as fluid container 746A and fluid container 746B. Fluid container 746A and fluid container 746B may be the same as medium bag 410 shown in FIG. 4D.

[0146] Fluid containers 746A and 746B may be media containers for distributing media to first fluid flow path 706 via IC media line 746. In IC circulation supply loop 753, media is pumped from fluid container 746B to fluid container 746A, through IC loop 702, through cell growth chamber 701, and IC waste valve 790, and back to fluid container 746B. The use of IC circulation supply loop 753 avoids the use of pass-through mass feeding, in which fresh media is continuously added to the system and collected in an outlet waste bag, which is discarded when full. With pass-through mass feeding, the more cells grown, the higher the media feed rate to the cells must be. Users typically measure glucose or lactate once a day and then increase the feed rate for that day. The feed rate may not be accurate throughout the day, resulting in media waste.

[0147] In contrast, in the CES700 herein, the IC circulation supply loop 753 expands the capacity of the IC circulation loop by connecting or daisy-chaining fluid container 746A with fluid container 746B (via the aforementioned port pair). In the CES700, the cell growth chamber 701 is manually fed from outside the CES700. The outlet line 788 returns to fluid container 746B, and the medium is recirculated. The user can calculate how much lactic acid is produced and determine waste. The user does not need to change bags; they simply set the feed flow rate and run the protocol. Thus, the CES700 is more efficient and uses less medium overall.

[0148] Although two fluid reservoirs 746A, 746B are shown and described, it will be appreciated that more fluid reservoirs 746N may be added to the system.

[0149] This approach allows for a large number of possible feeding strategies to be implemented. This approach improves the ease of use of the CES700 and reduces manual intervention by eliminating the need to empty waste bags and feed new media bags to the device at various time intervals during cell growth. Additionally, in many of these situations, there is no need to determine the feed flow rate. Even when using continuous perfusion feeding, lactate and glucose concentrations can fluctuate significantly unless very long custom tasks with small flow rate changes are used.

[0150] Depending on the desired application, IC circulation supply loop 753 may be used in coating the hollow fibers of cell growth chamber 701. In this case, fluid is circulated and forced across the membrane (IC side). Additionally or alternatively, IC circulation supply loop 753 may be used in a counter-flow technique. In this case, fluid is forced across both ends of cell growth chamber 701.

[0151] One or more differential pressure sensors may be included for real-time pressure monitoring and alarms. This allows for more efficient and accurate measurements as opposed to manual measurements. The pressure sensors facilitate detection of reduced or stopped flow, a depleting gas supply, user error, etc. Upon detecting a reduced or stopped flow, the differential pressure sensor triggers an alarm to alert the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alarm on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0152] One or more gas regulators may be provided for gas management. For example, the gas regulators may be internal to the CES700 so that they do not face the user. This prevents errors. The gas regulators may be less sensitive to the amount of control. This allows for finer control. If an error in gas management is detected, the gas regulator may trigger an alarm to alert the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alarm on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0153] One or more temperature thermistors may be provided for highly sensitive temperature detection. A temperature change of 0.5 degrees is important in cell culture. Thermistors degrade less than resistance temperature detectors (RTDs), thus allowing for longer use before service is required. Upon detecting a temperature change outside a predetermined range or a temperature above or below a predetermined threshold, the thermistor may trigger an alarm to alert the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alarm on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0154] 7C shows a CES 700C with dual supply circulation loops. CES 700C may be similar to CES 700A and CES 700B and may include the same or similar components as CES 700A and CES 700B, in which case like reference numerals are used. CES 700C includes an EC circulation supply loop 752 from CES 700A and an IC circulation supply loop 753 from CES 700B. IC circulation supply loop 753 passes fluid from fluid reservoir 746B to fluid reservoir 746A, through first fluid inlet passage 742, through cell growth chamber 701, through harvest valve 798, and back to fluid reservoir 746B. EC circulation supply loop 752 directs fluid from fluid reservoir 768B to fluid reservoir 768A, through EC medium valve 776, second fluid inlet line 774, GTM 732, cell growth chamber 701, EC waste valve 782, and back to fluid reservoir 768B.

[0155] Having described various exemplary embodiments of cell growth systems and associated methods, FIG. 8 illustrates exemplary operational steps of a process 756 for growing non-adherent or suspension cells in a cell growth system, such as the CES500, CES600, or CES700A-700C, according to an embodiment of the present disclosure.

[0156] START operation 758 begins, and process 756 proceeds with cell preparation (step 760). In embodiments, cell preparation 760 may involve several different optional steps. For example, in step 762, cells are collected. Cell collection step 762 may include isolating and collecting cells from a source or blood source or donor or patient or subject (these terms are used interchangeably herein). In some embodiments, an apheresis procedure is performed to collect a quantity of lymphocytes from the peripheral blood of a donor (e.g., leukapheresis). Such lymphocytes comprise the target cell population that is expanded by process 756. In other embodiments, cells may be collected from umbilical cord blood.

[0157] After collection step 762, optionally as part of preparation step 760, the cells are separated in step 764. The quantity of cells collected in step 762 may include many different cell types, including cells targeted for proliferation. Optional step 764 may be performed to isolate target cells. By way of example, the target cells are T cells, e.g., regulatory T cells. In one embodiment, regulatory T cells are CD4 + CD25 + The cells are T cells. The cells are separated using any suitable separation technique. For example, the cells can be isolated using immunomagnetic separation, in which magnetic beads functionalized with antibodies are contacted with the cells collected in step 762. The functionalized beads preferentially attach to the target cell population. A magnetic field can then be used to retain the beads attached to the target cell population while removing other cells.

[0158] After separation step 764, the cells may optionally be resuspended in step 766. In some embodiments, the cells are resuspended in a medium containing numerous nutrients and / or reagents that help maintain cell viability. In some embodiments, the medium includes reagents such as at least serum albumin and a cytokine. The cytokine, in some embodiments, is recombinant human IL-2 cytokine. In one embodiment, the medium may include the cytokine at a concentration of 200 IU / ml.

[0159] Following cell preparation step 760, process 756 proceeds to cell exposure step 768, where the cells are activated and expanded. The cells are optionally exposed to a soluble activator in step 770. The activator, including a soluble antibody, antibody conjugate, or antibody bead conjugate, is added to the medium in which the cells are resuspended. In embodiments, the activator is a human antibody CD3 / CD28 / CD2 cell activator conjugate. In some embodiments, the activator may be included in the medium used in cell resuspension step 766. Optionally, in step 772, the cells are exposed to beads having an activator on their surface. In embodiments, exposing the cells to the beads includes adding a predetermined amount of beads to the resuspended cells. The beads may be added at different ratios relative to the number of cells. For example, the beads may be added at a ratio of 1 bead:2 cells. In other embodiments, the beads are added at different ratios, such as 1 bead:1 cell or 1 bead:3 cells. The beads can have antibodies on their surface to activate and proliferate the cells. In embodiments, the beads may include antibodies CD3 / CD28 on their surface. In other embodiments, the antibodies used for activation may be coated onto the surface of the bioreactor.

[0160] Process 756 proceeds to cell growth step 774. As part of cell growth step 774, cells may be introduced into a cell growth chamber, e.g., a hollow fiber membrane bioreactor, where the cells are grown. In step 776, the cells are nourished to promote cell growth. For example, media may be delivered to the cell growth chamber to provide nutrients for growth. For example, media may be delivered via IC circulatory supply loop 753, EC circulatory supply loop 752, or a combination thereof, as described with reference to Figures 7A-7C. Cell growth step 774 may include periodically adding a reagent to the cell growth chamber in step 778 to continue promoting cell growth. For example, in some embodiments, a reagent (e.g., a cytokine) may be added to the cell growth chamber to promote cell growth. In one embodiment, the reagent may be additional IL-2 cytokine, e.g., recombinant human IL-2 cytokine.

[0161] For example, media, reagents, or combinations thereof may be delivered to cells from one or more connected fluid containers, as described with reference to Figures 7A-7C, and the media, reagents, or combinations thereof may be circulated and recirculated through IC circulating supply loop 753 or EC circulating supply loop 752.

[0162] Also as part of cell growth step 774, the environment within the cell growth chamber is controlled in step 780. For example, gases may be continuously delivered and exchanged to provide a balance of, for example, carbon dioxide and oxygen to the cells growing within the cell growth chamber. Additionally, the temperature may be controlled to be within a range optimized for cell growth. Cell growth step 774 may also include step 782 of monitoring metabolic products. For example, lactate and glucose levels may be monitored periodically. Lactate and glucose levels, among other metabolic products and nutrients, may be monitored by one or more sensors. The one or more sensors may also be configured to measure gas levels, such as oxygen. An increase or decrease in metabolic products may prompt changes to the control of the environment within the cell growth chamber in step 780 (e.g., additional feeding, additional reagent addition, additional gas exchange, etc.).

[0163] When various parameters are monitored, data readings that fall outside of predetermined thresholds may trigger an alarm to alert a user. For example, the data reading may be a temperature, a door position, a pressure, a flow rate, or a concentration. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alarm on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0164] Process 756 then proceeds to cell harvest step 784. Further processing or other analysis of the removed cells may optionally be performed in step 786. For example, the cells may be characterized to determine cell phenotype. Further processing or other analysis step 786 may include, for example, performing flow cytometry to characterize cell phenotype. Process 756 then ends at END operation 788. If no further processing / analysis is desired to be performed, process 756 ends at END operation 788.

[0165] FIG. 9A illustrates operational steps of a process 800 that may be used to place cells or other materials (e.g., proteins, nutrients, growth factors) into a cell growth chamber, according to embodiments of the present disclosure. In embodiments, process 800 may be performed as part of a "Central Cell Loading Without Circulation" task. Initiating operation 802 initiates process 800, which proceeds to step 804, where a first volume of fluid containing cells is loaded into a cell growth chamber of a cell expansion system. In embodiments, the cells include one or more types of non-adherent cells, such as T cells. In one embodiment, the plurality of cells includes Tregs. As can be appreciated, loading the first volume of fluid with cells can be performed by components of a cell expansion system, such as the systems CES500 (e.g., FIG. 5A), CES600 (FIG. 6), and CES700 (FIGS. 7A-7C) described above. 9B illustrates a portion of a cell growth system including a first fluid inlet pump 840, a first fluid flow path 860, a first fluid circulation pump 848, a first fluid circuit 852, a cell growth chamber 844, and a second fluid circuit 854. The first fluid flow path 860 is fluidly associated with the first fluid circuit 852 via connection 860B. In an embodiment, in step 804, a first volume of fluid having cells is introduced into the first fluid circuit 852 through the first fluid flow path 860 using the first fluid inlet pump 840. In an embodiment, the first volume of fluid is introduced without operating the first fluid circulation pump 848. The first volume of fluid may be provided, at least in part, from a fluid reservoir 746A, a fluid reservoir 746B, an IC fluid reservoir 746, or a combination thereof. For example, the first volume of fluid may be circulated through an IC circulation supply loop 753.

[0166] 9B, the volume of first fluid circuit 852 is made up of multiple partial volumes. For example, a first portion of the volume is the space inside the capillaries of cell growth chamber 844 (if the cell growth chamber is a hollow fiber membrane bioreactor). A second portion of the volume is from connection 860B to inlet port 844A of cell growth chamber 844. A third portion of the volume is from connection 860B to outlet port 844B of cell growth chamber 844.

[0167] Process 800 proceeds to step 806 with inputting a second volume of fluid. The second volume of fluid may include culture medium and is introduced into a portion of first fluid flow path 860. In some embodiments, the second volume is a predetermined amount selected for placing the first volume in a first portion of cell growth chamber 844 (step 808). In embodiments, the first volume of fluid and the second volume of fluid may be the same. In other embodiments, the first volume of fluid and the second volume of fluid may be different. In still other embodiments, the sum of the first volume of fluid and the second volume of fluid may be equal to a percentage of the volume of first fluid circuit 852, e.g., pathway (FIG. 9B).

[0168] The second volume of fluid may be provided, at least in part, from fluid reservoir 768A, fluid reservoir 768B, EC fluid reservoir 768, or a combination thereof. For example, the second volume of fluid may be circulated through IC circulation supply loop 753. For example, the second volume of fluid may be provided from two or more connected fluid reservoirs, as described with respect to Figures 7A-7C.

[0169] To position the first volume of fluid, the second volume of fluid must be sufficient to urge the first volume to a desired location within cell growth chamber 844. Thus, in an embodiment, the second volume of fluid is approximately the same size as the volume of first fluid circuit 852 between connection 860B and inlet port 844A. As can be appreciated, this will urge the first volume of fluid containing the cells to a location within cell growth chamber 844.

[0170] In other embodiments, in step 808, the first volume of fluid (including the cells) is placed about a central region 866 of the cell growth chamber 844. In these embodiments, the second volume is approximately equal to the sum of the volume of the first fluid circuit 852 between connection 860B and inlet port 844A and the volume of the portion of the first fluid circuit (e.g., the volume of the capillary interior) defined by the cell growth chamber 844 that is not occupied by the first volume of fluid when the first volume of fluid is placed in the cell growth chamber. For example, in one embodiment, the first volume of fluid (including the cells) is 50 ml. The cell growth chamber has a volume of, for example, 124 ml. When the first volume is placed about the central region 866, the 50 ml volume occupies the central region 866, leaving 74 ml on either side of the central region 866. Thus, to position the first 50 ml volume about central region 866, 50% of the 74 ml (ie, 37 ml) may be added to the volume between connection 860B and inlet port 844A.

[0171] In embodiments, positioning the first volume step 808 includes adding fluid to position the first volume of fluid with cells within the cell growth chamber. For example, if the desired positioning of the first volume is not achieved with the second volume, additional fluid can be added to position and position the first volume (step 808).

[0172] Process 800 proceeds to query step 810 to determine whether the first volume should be relocated. For example, in one embodiment, the first volume is located closer to inlet port 844A. If it is desired to move the first volume closer to central region 866 of cell growth chamber 844, process 800 returns to step 808 and adds additional fluid to the first fluid circuit to position and position the first volume of fluid.

[0173] If query step 810 determines that the first volume does not need to be repositioned, process 800 proceeds to cell feeding step 812. In some embodiments, the cells are fed using a medium containing multiple elements, such as glucose, proteins, growth factors, reagents, or other nutrients. In some embodiments, cell feeding step 812 includes activating an inlet pump and a circulation pump (e.g., pumps 840, 848) to deliver nutrient-containing medium to the cells in cell growth chamber 844. In some embodiments, as described below, the cells are maintained in cell growth chamber 844 during step 812. These embodiments include activating pumps (e.g., an inlet pump and a circulation pump (e.g., pumps 840, 848, described below)) such that fluid flow into cell growth chamber 844 is bidirectional (from inlet port 844A and outlet port 844B).

[0174] For example, medium is delivered to the cells from one or more connected fluid reservoirs, as described with respect to Figures 7A-7C. Inlet and circulation pumps can circulate and recirculate medium from either the IC circulatory supply loop 753 or the EC circulatory supply loop 752 in both directions, from the inlet port 844A and the outlet port 844B into the cell growth chamber 844.

[0175] Process 800 then proceeds to cell growth step 814, where the cells are grown or expanded. Although step 814 is shown after step 812, according to some embodiments, step 814 may occur before or simultaneously with step 812. The cells are then removed from the cell growth chamber (step 816) and collected in a storage container. In embodiments, step 816 involves several substeps. For example, the cells may be circulated by a circulation pump (e.g., pump 848) before being collected and stored in a container. Process 800 ends at END operation 830.

[0176] Next, FIG. 10A illustrates exemplary operational steps of a process 900 for retaining cells within a bioreactor of a cell growth system, such as the CES500 (e.g., FIGS. 5B and 5C) or the CES700 (e.g., FIGS. 7A-7C), according to an embodiment of the present disclosure. As previously discussed, cells in the header of the bioreactor or in the IC loop outside the bioreactor may not receive adequate gas and nutrient exchange, potentially resulting in cell clumping and death. In one embodiment, the bioreactor performs gas and nutrient exchange through semipermeable hollow fiber membranes. Because the surface area-to-volume ratio of a cell growth system including hollow fiber membranes is significantly greater than that of other cell culture methods (e.g., approximately 15 times the surface area of ​​a cell culture flask), it is important that such exchange be efficient. Such efficiency is achieved by minimizing the diffusion distance of media components across the membrane surface through which the exchange occurs.

[0177] For example, FIG. 10B shows a graph 936 of oxygen consumption in a cell expansion system, such as the Quantum® Cell Expansion System or the Quantum Flex® Cell Expansion System, during cell expansion (approximately 3E+09 T cells in the bioreactor). FIG. 10B also shows the percentage (%) of oxygen (O2) at the bioreactor outlet (938). For example, according to one embodiment, a sensor measuring oxygen levels can be placed at the EC outlet port of the bioreactor. In other embodiments, a sensor measuring oxygen delivery can be placed at the IC outlet port of the bioreactor. The percentage of O2 is measured versus run time (min) (940). To maximize oxygen delivery to the cells, in one embodiment, the EC circulation flow rate, Q EC Circis set to 300 mL / min (942). Figure 10B shows the change in oxygen supply when the EC circulation flow rate is reduced from 300 mL / min (942) to 50 mL / min (944). For example, as the medium passes through the bioreactor, the cells consume oxygen (O2) in the medium. Because fluid at 50 mL / min (944) moves through the bioreactor more slowly than fluid at 300 mL / min (942), at an EC circulation flow rate of 50 mL / min, the cells may have more time to deprive the medium of oxygen, i.e., more opportunities for the cells to deprive the medium of oxygen. When the EC circulation flow rate is returned to 300 mL / min, the oxygen supply is restored (946). Figure 10B illustrates the potential advantage of retaining the cells in the hollow fibers themselves, where gas transfer occurs, as opposed to, for example, retaining them in the portion of the IC circuit outside the bioreactor where oxygen may be deprived from the cells. Therefore, it is beneficial to maintain a population of cells (e.g., non-adherent cells) within the hollow fibers of the bioreactor during feeding by directing media flow to both sides of the bioreactor, e.g., the IC inlet and outlet ports. In one embodiment, a technique can be used that evenly distributes flow to the IC inlet and outlet ports. In other embodiments, the flow to the IC inlet port can be greater or less than that to the IC outlet port, depending, for example, on the desired location of the cells within the bioreactor.

[0178] Returning to FIG. 10A, starting operation 902, process 900 proceeds to step 904, where a disposable set or pre-mounted fluid transfer assembly (e.g., 210, 400) is loaded onto a cell growth system. The disposable set can include cell growth chamber 100A or cell growth chamber 100B, as shown in FIGS. 1C, 4B, and 4C. In step 906, the disposable set is primed. Here, for example, a Lonza Ca 2+ / Mg 2+The disposable set is primed with PBS containing no phosphate-buffered saline. In preparation for cell loading and seeding, the priming fluid may be replaced in IC / EC wash step 908. In one embodiment, the PBS in the system is replaced with, for example, TexMACS GMP basal medium.

[0179] For example, during priming, fluid is delivered through the disposable set from one or more connected fluid containers, as described with respect to Figures 7A-7C, and the fluid may be circulated and recirculated using the fluid containers for additional volume within the fluid loop.

[0180] Next, process 900 proceeds to step 910, which involves closing the IC outlet valve. In an embodiment, the EC outlet valve is opened to allow ultrafiltration of fluid added to the hollow fibers of the bioreactor, including the hollow fiber membranes. Next, in step 912, the medium is adjusted. Next, process 900 proceeds to step 914, which involves adding cells (e.g., suspension or non-adherent cells, such as T cells or Tregs). In one embodiment, in step 914, such cells are added via the "Add cells to center without circulation" task. In another embodiment, in step 914, such cells are added via the "Add cells in uniform suspension" task. In other embodiments, other adding tasks and / or adding procedures may be used.

[0181] In one embodiment, the cells being introduced in step 914 may be suspended in a solution containing, for example, a medium for feeding the cells during and after the introduction. In other embodiments, such a solution may contain both a medium for cell feeding and a soluble activator complex for stimulating the cells (e.g., T cells). The introduction step 914 may occur, for example, on day 0.

[0182] Following cell input step 914, the cells are further fed in step 916. In an exemplary embodiment, the cells are fed through an EC circulation supply loop 752, an IC circulation supply loop 753, or a combination thereof (step 916), as described with reference to Figures 7A-7C. During feeding step 916, it may be desirable to control cell retention within the bioreactor. Adjusting flow control parameters in step 918 can retain cells within the bioreactor, for example, during the proliferation phase of growth, without losing cells from the bioreactor to the IC loop portion outside the bioreactor. According to embodiments, retaining cells within the bioreactor can bring cells within the bioreactor closer to the IC inlet port, allowing such cells to receive the freshest growth medium. Meanwhile, cells within the IC loop may be receiving spent or conditioned medium, which may affect, for example, glycolytic metabolism. Additionally, cells within the bioreactor receive a gas mixture (e.g., oxygen, carbon dioxide, nitrogen) input from the gas transfer module (GTM) by diffusion from the EC loop circulation, while cells in other portions of the IC loop do not receive such a gas mixture. In certain embodiments, the cells are retained in the bioreactor itself, while in other embodiments, the cells may be maintained in any location that allows for improved nutrient delivery and / or gas exchange. Thus, in embodiments, other locations are used to retain cells or control cell retention without departing from the spirit and scope of the present disclosure.

[0183] Returning to FIG. 10A and process 900, in one embodiment, cell loss from a hollow fiber membrane bioreactor can be reduced by matching, or approximately or substantially matching, the IC circulation pump flow rate to the IC inlet pump flow rate (albeit in opposite directions). In step 920, the IC inlet pump is adjusted to generate a first flow rate or volumetric flow rate, and in step 922, the IC circulation pump is adjusted to generate a second, opposing flow rate or second opposing volumetric flow rate. Here, volumetric flow rate or fluid flow rate or fluid rate or flow rate can be thought of as the volume of fluid passing per unit time (represented by the symbol "Q"). For example, for an IC inlet pump flow rate of 0.1 mL / min, a complementary IC circulation pump rate of -0.1 mL / min can be matched or approximately matched to maintain cells in the bioreactor during the growth phase of the cell culture (e.g., in embodiments, days 4-7). Alternatively, an IC inlet pump flow rate of 0.01 mL / min may be matched or approximately matched with a complementary IC circulation pump rate of −0.01 mL / min to maintain cells in the bioreactor during the growth phase of the cell culture (e.g., in embodiments, days 4-7). Such pump adjustment step 918 may, for example, counter any forces associated with cell loss from the bioreactor's IC outlet port.

[0184] Next, the cells are grown or expanded in step 924. The cells are not limited to being grown or expanded in step 924; instead, the cells may be expanded during steps 914, 916, 918, 920, and 922, for example. The process 900 then proceeds to a harvesting step 926, where the cells are transferred to a harvest bag or container. The disposable set is then removed from the cell expansion system (step 932), and the process 900 ends at end operation 934.

[0185] Alternatively, process 900 can optionally proceed from harvesting step 926 to further processing / analysis step 928. Such further processing / analysis step 928 may include, for example, phenotypic characterization of the harvested cells. Process 900 may proceed from optional further processing / analysis step 928 to a step of reloading the remaining cells 930. Process 900 then proceeds to removing the disposable set (step 932), with process 900 ending at end operation 934. Alternatively, process 900 may proceed from further processing / analysis step 928 to removing the disposable set (step 932). Process 900 then ends at end operation 934.

[0186] Next, FIG. 11A illustrates exemplary operational steps of a process 1000 for feeding cells for use with a cell growth system, such as CES 700 (e.g., FIGS. 7A-7C), according to an embodiment of the present disclosure. A start operation 1002 begins, and process 1000 proceeds with loading a disposable set into the cell growth system, priming the set, performing an IC / EC wash, conditioning media, and loading cells (e.g., suspension cells or non-adherent cells). The disposable set may include a cell growth chamber 100A or a cell growth chamber 100B, as shown in FIGS. 1C, 4B, and 4C. Process 1000 then proceeds to step 1004, which involves feeding cells for a first period of time. In an exemplary embodiment, cells are fed through an EC circulation supply loop 752, an IC circulation supply loop 753, or a combination thereof (step 916), as described with reference to FIGS. 7A-7C. In an embodiment, a first inlet flow rate and a first circulation flow rate are used. As an example, a first IC inlet flow rate and a first IC circulation flow rate are used, where the first IC inlet flow rate is controlled by an IC inlet pump (e.g., first pump) and the first IC circulation flow rate is controlled by an IC circulation pump (e.g., second pump). In an exemplary embodiment, the IC inlet pump (754) injects fluid into the IC inlet port (701A) of the bioreactor (701) at a volumetric flow rate of 0.1 mL / min, and the IC circulation pump (712) injects fluid into the IC outlet port (701B) of the bioreactor (701) at a complementary IC circulation volumetric or flow rate of −0.1 mL / min. Here, the negative sign (“−”) used, e.g., −0.1 mL / min, indicates that the direction of the IC circulation pump (712) creates a counterflow so that cells are maintained in the bioreactor during the growth phase of the cell culture.

[0187] During cell feeding and use of an IC pump for feeding while controlling cell retention in the bioreactor via co-flow / counter-flow characteristics, the cells continue to grow and proliferate. As a result, the cells require additional medium (e.g., glucose and / or cell growth medium) to support the growing population. In an exemplary embodiment, multiple fluid containers 768A, 768B are connected to provide medium to circulate and recirculate through the system, eliminating the need to control lactate levels. In other embodiments, steps are also taken to control lactate levels in the growing cell population. In embodiments, cell culture lactate levels are maintained at about 20 mmol / L or less, about 15 mmol / L or less, about 10 mmol / L or less, or about 7 mmol / L or less. In other embodiments, lactate levels are maintained at about 5 mmol / L or less, e.g., by controlling the flow rate of medium addition and / or other settings, e.g., to improve cell growth and viability. In other embodiments, other concentrations may be used.

[0188] In an exemplary embodiment, because fluid containers 768A, 768B or fluid containers 746A, 746B are connected, controlling pump flow rates is not necessary to control lactate levels. In another exemplary embodiment, lactate levels can be controlled to approximately 7 mmol / L or less within the lumen of the hollow fiber membrane by simultaneously increasing both the IC inlet (+) pump flow rate and the IC circulation (-) pump flow rate by ±0.1 to ±0.4 mL / min over multiple time periods, e.g., several days (days 4-8). For example, FIG. 11B shows a table 1018 of exemplary IC pump flow rates for feeding, e.g., using the "Cell Feeding" task in a cell expansion system (e.g., CES500). Table 1018 shows an exemplary relationship between time period 1020 (e.g., day number) and IC pump flow rate 1022 for generating volumetric flow rates to both sides of the bioreactor to maintain cells within the bioreactor. For example, on days 0-4 (1024), use an IC inlet or input pump flow rate of 0.1 mL / min (1026) and an IC circulation pump flow rate of -0.1 mL / min (1028). On day 5 (1030), use an IC inlet pump flow rate of 0.2 mL / min (1032) and an IC circulation pump flow rate of -0.2 mL / min (1034). On day 6 (1036), use an IC inlet pump flow rate of 0.3 mL / min (1038) and an IC circulation pump flow rate of -0.3 mL / min (1040). On day 7 (1042), use an IC inlet pump flow rate of 0.4 mL / min (1044) and an IC circulation pump flow rate of -0.4 mL / min (1046). Although table 1018 in FIG. 11B illustrates a pump flow rate of ±0.1 to ±0.4 mL / min for feeding cells while maintaining the cells in the bioreactor during the growth phase of the cell culture, other pump flow rates and resulting flow rates may be used for embodiments without departing from the spirit and scope of the present disclosure. For example, while this example shows ±0.1 mL / min increments for increasing the feeding flow rate, other increments, e.g., ±0.005 mL / min, ±0.05 mL / min, etc., may be used to increase the feeding flow rate. The time periods (e.g., days) and pump flow rates in table 1018 in FIG. 11B are provided for illustrative purposes only and are not intended to be limiting.For example, the durations and pump flow rates of table 1018 of Figure 11B may be provided for cell growth chamber 100B. The pump flow rates of table 1018 of Figure 11B may be reduced to 10% of the listed pump flow rates for cell growth chamber 100A.

[0189] Returning to FIG. 11A, process 1000 proceeds from step 1004, which involves feeding cells for a first period of time, to step 1006, which involves increasing the first inlet flow rate by a first amount to achieve a second inlet flow rate. As in the embodiment illustrated in FIG. 11B described above, the IC inlet pump flow rate (+) is increased from 0.1 mL / min to 0.2 mL / min to generate an IC inlet flow rate of 0.2 mL / min. Simultaneously, the IC circulation pump flow rate (-) is increased from -0.1 mL / min to -0.2 mL / min to generate an IC circulation flow rate of -0.2 mL / min. (The pump flow rate may be reduced to 10% of the stated pump flow rate for cell growth chamber 100A.) Thus, the first circulation flow rate is increased by a first amount to achieve the second circulation flow rate (step 1008). The cells are then fed for a second period of time at a second inlet flow rate and a second circulation flow rate (step 1010), maintaining the cells within the bioreactor and outside the header and a portion of the IC circuit outside the bioreactor. Following the second period of feeding (step 1010), process 1000 ends at end operation 1016, e.g., if continued cell feeding and / or growth is not desired. Alternatively, optionally, process 1000 can continue by increasing or otherwise changing the feeding flow rate (step 1012). There may be any number of feeding periods, as represented by ellipsis 1014. Following the desired number of feeding periods 1014, process 1000 ends at end operation 1016. While FIGS. 10A and 10B and process 1000 illustrate an "increase" in the flow rate, other adjustments to the flow rate may be made. For example, the flow rate may be decreased from one feeding period to the next, or may remain substantially the same. For example, considerations such as metabolic activity may be taken into account to determine how to adjust flow rates. The "increases" in flow rates in Figures 10A and 10B are provided for illustrative purposes only and are not intended to be limiting.

[0190] 12A illustrates exemplary operational steps of a process 1100 for feeding cells for use with a cell expansion system such as CES 500 (e.g., FIGS. 5B and 5C), according to an embodiment of the present disclosure. A start operation 1102 begins, and the process 1100 proceeds to loading a disposable set into the cell expansion system, priming the set, performing an IC / EC wash, adjusting media, and loading cells (e.g., suspension cells or non-adherent cells). The process 1100 then proceeds to step 1104, which involves feeding the cells for a first period of time. In embodiments, a first inlet flow rate or flow rate and a first circulation flow rate or flow rate are used. By way of example, a first IC inlet flow rate and a first IC circulation flow rate are used, where the first IC inlet flow rate is controlled by an IC inlet pump (e.g., the first pump) and the first IC circulation flow rate is controlled by an IC circulation pump (e.g., the second pump). In the illustrated embodiment, the IC inlet pump (554) pumps a volumetric flow rate or flow rate of 0.1 mL / min into the IC inlet port (501A) of the bioreactor (501), and the IC circulation pump (512) pumps a complementary IC circulation volumetric flow rate of −0.1 mL / min into the IC outlet port (501B) of the bioreactor (501). Here, the negative sign (“−”) used, e.g., −0.1 mL / min, indicates that the direction of the IC circulation pump (512) creates or generates a counterflow rate so that cells are maintained within the bioreactor during the growth phase of the cell culture. For example, the listed flow rates are appropriate for the cell growth chamber 100B. The listed flow rates may be reduced to 10% for the cell growth chamber 100A.

[0191] During cell feeding and the use of an IC pump to control cell retention within the bioreactor via co-flow / counter-flow characteristics, cells continue to grow and proliferate. As a result, cells require additional media (e.g., glucose and / or cell growth media) to support the growing population. In an exemplary embodiment, multiple fluid containers 768A, 768B are connected to provide media for circulation and recirculation within the system, eliminating the need to control lactate levels. In other embodiments, steps are also taken to control lactate levels within the growing cell population. In an exemplary embodiment, lactate levels are controlled to approximately 7 mmol / L or less within the lumen of the hollow fiber membrane by simultaneously increasing both the IC inlet (+) pump flow rate and the IC circulation (-) pump flow rate by ±0.1 to ±0.4 mL / min over several days (days 4-8). See, for example, the table in FIG. 11B and the related discussion above (e.g., pump flow rates for feeding). As mentioned above, while table 1018 of FIG. 11B illustrates pump flow rates of ±0.1 to ±0.4 mL / min for feeding cells while maintaining them in a bioreactor during the growth phase of a cell culture, other pump flow rates and resulting flow rates may be used for embodiments without departing from the spirit and scope of the present disclosure. The time periods (e.g., days) and pump flow rates in table 1018 of FIG. 11B are provided for illustrative purposes only and are not intended to be limiting. For example, the time periods and pump flow rates in table 1018 of FIG. 11B may be provided for cell growth chamber 100B. The pump flow rates in table 1018 of FIG. 11B may be reduced to 10% of the listed pump flow rates for cell growth chamber 100A.

[0192] Returning to FIG. 12A, process 1100 proceeds from step 1104, which involves feeding cells for a first period of time, to step 1106, which involves increasing the first inlet flow rate by a first amount to achieve a second inlet flow rate or flow rate. As in the embodiment illustrated in FIG. 11B described above, the IC inlet pump flow rate (+) is increased from 0.1 mL / min to 0.2 mL / min to generate an IC inlet flow rate of 0.2 mL / min. Furthermore, simultaneously, the IC circulation pump flow rate (-) is simultaneously increased from -0.1 mL / min to -0.2 mL / min to generate an IC circulation flow rate of -0.2 mL / min. (The pump flow rate may be reduced to 10% of the stated pump flow rate for cell growth chamber 100A.) Thus, the first circulation flow rate or flow rate is increased by a first amount to achieve the second circulation flow rate or flow rate (step 1108). The cells are then fed (step 1110) for a second period of time at a second inlet flow rate or flow rate and a second circulation flow rate or flow rate, maintaining the cells within the bioreactor and outside the header or part of the IC circulation outside the bioreactor. Following the second period of feeding (step 1110), for example, if it is not desired to continue feeding and / or growing the cells, process 1100 ends at end operation 1116.

[0193] Alternatively, optionally, process 1100 can determine whether to adjust the feeding rate or flow rate based on metabolic activity. In this case, process 1100 proceeds to optional query step 1112 to determine whether to adjust feeding based on metabolic levels. For example, by monitoring glucose and / or lactate levels, the medium flow rate (e.g., IC medium flow rate) of a cell expansion system can be adjusted to promote expansion of cells (e.g., Tregs) in a bioreactor, such as a hollow fiber bioreactor.

[0194] As shown in Figures 12B and 12C, in embodiments, lactate levels are controlled in cell expansion experiments or procedures involving, for example, hTreg expansion. Graphs 1118, 1132 illustrate using measurements of glucose and lactate levels to adjust media flow rates (e.g., IC media flow rates) in a cell expansion system, for example, to support Treg expansion. For example, Figure 12B provides graph 1118 illustrating the metabolism of hTreg expansion, where cell expansion is performed in a cell expansion system such as the Quantum® Cell Expansion System. Glucose concentration (mg / dL) 1120 and lactate concentration (mmol / L) 1122 are shown for various cell expansion runs 1124, 1125 versus time period (e.g., days 1126). In these Treg runs 1124, 1125, lactate levels in the expanding cell population are controlled to values ​​of approximately 7 mmol / L or less by simultaneously increasing both the IC inlet (+) pump flow rate and the IC circulating pump flow rate within the lumen of the hollow fiber membrane from ±0.1 to ±0.4 mL / min, for example, from day 4 to day 8. In other embodiments, other pump flow rates may be used. As shown, according to embodiments, the lowest glucose levels during Treg cell expansion range from a concentration of 264 mg / dL on day 7 (Q1584) to a concentration of 279 mg / dL on day 8 (Q1558). As depicted, the starting glucose concentration in the cell growth medium for run 1124 ranges, for example, from 325 mg / dL to 335 mg / dL. In other embodiments, it may be desirable to maintain lactate levels at approximately 5 mmol / L or less to improve cell growth and viability. In embodiments, a graphical user interface (GUI) element is used to control the rate of medium addition to maintain lactate metabolic waste products from glycolysis below a prescribed level during cell growth.

[0195] Turning to FIG. 12C, graph 1132 illustrates the metabolism of hTreg expansion, where such cell expansion is performed in a cell expansion system, such as the Quantum® Cell Expansion System. Glucose consumption (mmol / day) 1134 and lactate production (mmol / day) 1136 are shown for various cell expansion runs 1138, 1139 over a period 1140 (e.g., days). In one embodiment, the IC inlet (+) pump flow rate and the IC circulation (-) pump flow rate are simultaneously increased from ±0.1 to ±0.4 mL / min to control lactate levels, e.g., below about 7 mmol / L. For example, graph 1132 illustrates IC circulation and IC feed 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. The flow rates used and displayed in Figures 12B and 12C are provided for illustrative purposes and are not intended to be limiting. While positive (+) may be shown relative to the direction of the IC inlet pump and negative (-) may be shown relative to the direction of the IC circulation pump, such directions are shown for illustrative purposes only and will depend on the pump configuration used.

[0196] Returning to FIG. 12A and optional query step 1112, if it is not desired to measure metabolic activity and / or adjust feeding levels based on that measurement, process 1100 proceeds with a "no" answer, and process 1100 terminates at end operation 1116. For example, as described with respect to FIGS. 7A-7C, if multiple supply vessels are used, medium may be circulated and recirculated, avoiding the need to measure metabolic activity and / or adjust feeding levels. Alternatively, if it is desired to adjust feeding levels based on metabolic activity, process 1100 proceeds with a "yes" answer, and increases the medium addition rate in optional step 1114 to continue feeding the expanding cell population. While step 1114 is shown as a single step, this step may include multiple steps of making adjustments to the medium addition rate, such as, for example, increasing the IC inlet flow rate and increasing the IC circulation flow rate. Step 1114 is shown as a single step for illustrative purposes only and is not intended to be limiting. Following the adjustment of the medium addition rate, process 1100 proceeds to optional query step 1112 to determine whether to continue measuring metabolic levels and / or adjust feeding. If it is not desired to continue measuring metabolic activity and / or adjusting feeding levels based on metabolic activity, process 1100 proceeds to "no" and ends at end operation 1116. While FIG. 11A and process 1100 show an "increase" in rate or flow rate, other adjustments to flow rate may be made. For example, from one feeding period to the next, the rate or flow rate may be decreased or maintained substantially the same. The type of adjustment that may be made depends on the metabolic activity assessment of the growing cell population. For example, the "increase" in flow rate in FIG. 11A is provided merely for illustrative purposes and is not intended to be limiting.

[0197] FIG. 13 next illustrates exemplary operational steps of a process 1200 for holding cells in a given position during cell feeding using a cell growth system such as CES500 (e.g., FIGS. 5B and 5C) or CES700 (e.g., FIGS. 7A-7C), according to embodiments of the present disclosure. Initiating operation 1202, process 1200 proceeds with loading a disposable set into the cell growth system, priming the set, performing IC / EC washes, adjusting media, and loading cells (e.g., suspension or non-adherent cells). The disposable set may include cell growth chamber 100A or cell growth chamber 100B, as shown in FIGS. 1C, 4B, and 4C. Process 1200 then proceeds to step 1204, where cells are fed for a first period of time. In embodiments, a first inlet flow rate and a first circulation flow rate are used. As an example, a first IC inlet flow rate and a first IC circulation flow rate are used, where the first IC inlet flow rate is generated and controlled by an IC inlet pump (e.g., the first pump) and the first IC circulation flow rate is generated and controlled by an IC circulation pump (e.g., the second pump). In an exemplary embodiment, the IC inlet pump (554) provides a volumetric flow rate of 0.1 mL / min into the bioreactor's IC inlet port (501A), and the IC circulation pump (512) provides a complementary IC circulation volumetric flow rate or flow rate of −0.1 mL / min into the bioreactor's IC outlet port (501B). Here, the negative sign (“−”) used, e.g., −0.1 mL / min, indicates that the direction of the IC circulation pump creates or generates a counterflow so that cells are maintained in the bioreactor during the growth phase of the cell culture. In other embodiments, the first IC circulation flow rate may be a percentage of the first IC inlet flow rate. For example, the first IC circulation flow rate may be approximately fifty percent (50%) or approximately one-half (1 / 2) of the first IC inlet flow rate, or in some embodiments, some other percentage or fraction.Alternatively, in an exemplary embodiment, a first IC inlet pump provides a volumetric flow rate of less than 0.1 mL / min, or about 0.01 mL / min, to the IC inlet port (701A), and an IC circulation pump provides a complementary IC circulation volumetric or flow rate fluid flow rate of less than −0.1 mL / min, or about −0.01 mL / min, to the IC outlet port (701B), where the negative sign (“−”) used, for example, at −0.01 mL / min, indicates that the direction of the IC circulation pump creates a counterflow so that cells are maintained in the bioreactor during the growth phase of the cell culture.

[0198] During cell feeding (and expansion) and use of the IC pump to control cell retention in the bioreactor via co-current-countercurrent flow characteristics, the cells continue to grow and proliferate. As a result, the cells require additional medium (e.g., glucose and / or cell growth medium) to support the expanding cell population. In embodiments, an increase in the medium addition rate is performed to feed the expanding cell population. In an exemplary embodiment, increasing the IC inlet pump flow rate (+) increases the IC inlet flow rate by a first amount to achieve a second IC inlet flow rate (step 1206). For example, according to one embodiment, the IC inlet flow rate is 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 to the IC inlet flow rate may be made.

[0199] In an exemplary embodiment, the cells are fed through an EC circulatory supply loop 752, an IC circulatory supply loop 753, or a combination thereof (step 916), as described with reference to Figures 7A-7C.

[0200] Next, according to an embodiment, the second IC circulation flow rate is set, adjusted, or configured to be equal to a percentage, a proportion, or a ratio of the second IC inlet flow rate (step 1208). For example, according to one embodiment, the second IC circulation flow rate is set, adjusted, or configured to be equal to approximately fifty percent (50%), or approximately half (1 / 2), of the value of the IC inlet flow rate, or according to another embodiment, some other percentage or proportion. According to one embodiment, the second IC circulation flow rate is increased by adjusting the IC circulation pump flow rate (-) in response to the value of the first IC circulation flow rate. In another embodiment, the IC circulation pump flow rate is adjusted to decrease the second IC circulation flow rate so that the IC circulation flow rate is substantially equal to a predetermined percentage or a predetermined ratio of the second IC inlet flow rate. In still other embodiments, no adjustment of the IC circulation pump flow rate may be performed. For example, if the first IC inlet flow rate is equal to 0.1 mL / min and the first IC circulation flow rate is equal to -0.1 mL / min, when the second IC inlet flow rate is increased to 0.2 mL / min, the second IC circulation flow rate will be (-1 / 2)*(second Q IC Inlet )(Here Q IC Inlet is set to (IC inlet flow rate), i.e. (-1 / 2)*(0.2mL / min), which results in a second Q of -0.1mL / min. IC Circ is given (where Q IC Circ is the IC circulation flow rate). Therefore, without adjusting the IC circulation pump flow rate, the second Q IC Circ is achieved.

[0201] 5B and 5C illustrate an operational configuration of a cell growth system 500 showing fluid movement within a first circuit, according to an embodiment of the present disclosure. The configurations of FIGS. 5B and 5C illustrate the division of flow to an IC inlet port (e.g., a first port) and an IC outlet port (e.g., a second port), for example, to retain cells within a cell growth chamber or bioreactor. As described with respect to CES 500 above, in IC loop or first circuit 502, fluid is initially pumped by IC inlet pump 554. Such fluid is pumped in a first direction (e.g., forward). Fluid enters cell growth chamber or bioreactor 501 through IC inlet port 501A, flows through the hollow fibers of cell growth chamber or bioreactor 501, and exits through IC outlet port 501B. Media flows through IC circulation pump 512, which is used to control media flow rate. The IC circulation pump pumps fluid in either the first direction or a second direction opposite the first direction. The IC outlet port 501B can be used as an inlet, for example, in the reverse direction. In one embodiment, the IC circulation pump 512 pumps fluid in a direction opposite to that of the IC inlet pump, for example. By way of example, the direction of the IC inlet pump is positive (+) and the direction of the IC circulation pump is negative (-), thus creating a counterflow of fluid into both sides of the bioreactor, retaining cells within the bioreactor.

[0202] In one embodiment, a first portion of the fluid branches at connection 517 and flows into the IC inlet port (501A) of the bioreactor 501. In certain embodiments, the IC circulation pump 512 operates at a pump flow rate that matches, or approximately or substantially matches, the flow rate of the IC inlet pump 554 (albeit in the opposite direction), such that a second portion of the fluid branches at connection 517 and flows into the IC outlet port (501B) of the bioreactor 501. For example, for an IC inlet pump flow rate of +0.1 mL / min, a complementary IC circulation pump flow rate may be matched or approximately matched to be −0.1 mL / min to maintain cells in the bioreactor during the growth phase of the cell culture. Alternatively, for an IC inlet pump flow rate of less than +0.1 mL / min or about +0.01 mL / min, a complementary IC circulation pump flow rate may be matched or approximately matched to be less than −0.1 mL / min or about −0.01 mL / min to maintain cells in the bioreactor during the growth phase of the cell culture. Furthermore, in further embodiments, intracapillary harvest synchronization, as described herein, may be followed during the growth phase of the cell culture. Such pump adjustment techniques during feeding counteract forces associated with cell loss from the IC outlet port. According to one embodiment, a type of feeding in which pump adjustments result in a forward flow into the bioreactor's IC inlet port (501A) and a counterflow into the IC outlet port (501B) is referred to as a modified feeding method. In another exemplary embodiment, the IC circulation pump flow rate is adjusted to equal (but in the opposite direction to) the flow rate into the IC outlet port (501B) equal to approximately fifty percent (50%), or approximately half (1 / 2), of the IC inlet flow rate, or in other embodiments, some other percentage or other ratio. For example, if the IC inlet pump flow rate is 0.4 mL / min, the IC circulation pump flow rate is set, configured, or adjusted to approximately −0.2 mL / min. Other percentages or other ratios may be used in other embodiments.

[0203] 5B and 5C illustrate operational configurations showing fluid movement in a CES 500, in which forward and counterflow rates are shown for maintaining cells within a cell growth chamber or bioreactor 501, according to an embodiment. For example, such flow rates are shown in FIG. 5B as "X" flow rate 503, "(-1 / 2)X" flow rate 505 (where the negative sign ("-") indicates direction, and the direction of flow rate 505 is shown by the directional arrow in FIG. 5B), and "(1 / 2)X" flow rate 507. Here, approximately one-half of the flow rate, or a first portion, branches off at connection 517 to enter the IC inlet port (501A) of the bioreactor 501, and approximately one-half of the flow rate, or a second portion, branches off at connection 517 to enter the IC outlet port (501B) of the bioreactor 501. As shown, the sum of the first and second portions is substantially equal to the total flow rate 503, which is pumped by IC inlet pump 554. Depending on the forward flow-counterflow rate used to maintain cells in the bioreactor or cell growth chamber 501, other types of ratios or percentages of the IC inlet pump flow rate may be used to set, configure, or adjust the IC circulation pump. In such cases, FIG. 5C shows the following flow rates: "X" flow rate 511; "-(y%)*X" flow rate 513 (where the negative sign ("-") indicates direction, and the direction of flow rate 513 is indicated by the directional arrow in FIG. 5C); and "(100%-y%)*X" flow rate 515, where "y" equals a percentage. In an embodiment, the sum of flow rate 513 and flow rate 515 is substantially equal to flow rate 511.

[0204] In one embodiment, all or substantially all of the flow from first fluid flow path 506 enters the IC inlet port (501A) of bioreactor 501, for example, at connection 517. In another embodiment, all or substantially all of the flow from first fluid flow path 506 enters the IC outlet port (501B) of bioreactor 501 at connection 517. In yet another embodiment, a first portion of the flow from first fluid flow path 506 enters the IC inlet port (501A) at connection 517, and a second portion of the flow from first fluid flow path 506 enters the IC outlet port (501B) at connection 517. In embodiments, the percentage of the IC inlet flow rate at which the IC circulation flow rate is set ranges from about 0 percent to about 100 percent. In other embodiments, the percentage is between about 25 percent and about 75 percent. In other embodiments, the percentage is between about 40 percent and about 60 percent. In other embodiments, the percentage is between about 45 percent and about 55 percent. In embodiments, the percentage is about 50 percent. It should be understood that the operating configurations shown in Figures 5B and 5C represent various possible operating configurations of the cell growth system, and that modifications to the shown configurations are within the scope of one or more of the present embodiments.

[0205] Returning to FIG. 13 , the cells are fed (and continued to grow) for a second period of time (step 1210) at a second inlet flow rate and a second circulation flow rate, maintaining the cells within the bioreactor and outside the header or a portion of the IC circuit outside the bioreactor. Following the second period of feeding (step 1210), process 1200 ends at end operation 1216, for example, if it is not desired to continue feeding and / or growing the cells. Alternatively, optionally, process 1200 can continue with an increase or other type of change in the feeding flow rate (step 1212). There may be any number of feeding periods, as represented by ellipsis 1214. Following the desired number of feeding periods 1214, process 1200 ends at end operation 1216. While FIG. 13 and process 1200 illustrate an “increase” in the flow rate, other adjustments to the flow rate may be made. For example, the flow rate may decrease from one feeding period to the next, or may remain substantially the same. Considerations, such as metabolic activity, may determine how to adjust the flow rate. The "increase" in flow rate in Figure 13 is provided for illustrative purposes only and is not intended to be limiting.

[0206] Next, Figure 14 illustrates exemplary operational steps of a process 1300 for feeding cells while holding them in a first location (e.g., a bioreactor) using a cell expansion system such as CES500 (e.g., Figures 5B and 5C) or CES700 (e.g., Figures 7A-7C) according to embodiments of the present disclosure. Initiating operation 1302, process 1300 proceeds with loading a disposable set into the cell expansion system, priming the set, performing an IC / EC wash, adjusting media, and loading cells (e.g., suspension or non-adherent cells). Process 1300 then proceeds to step 1304, feeding the cells for a first period of time. In embodiments, a first inlet flow rate and a first circulation flow rate are used. As an example, a first IC inlet flow rate and a first IC circulation flow rate are used, where the first IC inlet flow rate is generated and controlled by an IC inlet pump (e.g., the first pump) and the first IC circulation flow rate is generated and controlled by an IC circulation pump (e.g., the second pump). In an exemplary embodiment, the first IC inlet pump generates a volumetric flow rate of 0.1 mL / min into the IC inlet port of the bioreactor, and a complementary IC circulation pump flow rate of −0.1 mL / min generates a volumetric flow rate of −0.1 mL / min into the IC outlet port (501B) of the bioreactor. Here, the negative sign (“−”) used for, e.g., −0.1 mL / min, indicates that the direction of the IC circulation pump (512) creates or generates a counterflow so that cells are maintained in the bioreactor during the growth phase of the cell culture. Alternatively, in an exemplary embodiment, a first IC inlet pump provides a volumetric flow rate of less than 0.1 mL / min, or about 0.01 mL / min, to the IC inlet port (701A), and an IC circulation pump provides a complementary IC circulation volumetric or flow rate fluid flow rate of less than −0.1 mL / min, or about −0.01 mL / min, to the IC outlet port (701B), where the negative sign (“−”) used, for example, at −0.01 mL / min, indicates that the direction of the IC circulation pump creates a counterflow so that cells are maintained in the bioreactor during the growth phase of the cell culture.In other embodiments, the first IC circulation flow rate may be a percentage or fraction of the first IC inlet flow rate. For example, the first IC circulation flow rate may be approximately fifty percent (50%) or approximately one-half (1 / 2) of the first IC inlet flow rate, or in some embodiments, some other percentage or fraction.

[0207] During cell feeding (and expansion) and the use of the IC pump to control cell retention within the bioreactor via co-flow and counter-flow characteristics, the cells continue to grow and proliferate. As a result, the cells require additional medium (e.g., glucose and / or cell growth medium) to support the expanding cell population. In embodiments, an increase in the medium addition rate is performed to feed the expanding cell population. In an exemplary embodiment, increasing the IC inlet pump flow rate (+) increases the IC inlet flow rate by a first amount to achieve a second IC inlet flow rate (step 1306). For example, according to one embodiment, the IC inlet flow rate is 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 to the IC inlet flow rate may be made.

[0208] Next, according to embodiments, the second IC circulation flow rate is set, configured, or adjusted to be equal to a percentage, portion, or ratio of the second IC inlet flow rate (step 1308). For example, according to one embodiment, the second IC circulation flow rate is set, configured, or adjusted to be equal to about fifty percent (50%), or about half (1 / 2), of the value of the IC inlet flow rate, or in other embodiments, some other percentage or ratio. In one embodiment, all or substantially all of the flow from first fluid flow path 506 flows into the IC inlet port (501A) of bioreactor 501, for example, via connection 517. In other embodiments, all or substantially all of the flow from first fluid flow path 506 flows into the IC outlet port (501B) of bioreactor 501 via connection 517. In yet another embodiment, a first portion of the flow from first fluid flow path 506 flows from connection 517 to IC inlet port (501A), and a second portion of the flow from first fluid flow path 506 flows from connection 517 to IC outlet port (501B). In an embodiment, the percentage of the IC inlet flow at which the IC circulation flow is set ranges from about 0 percent to about 100 percent. In other embodiments, the percentage is between about 25 percent and about 75 percent. In other embodiments, the percentage is between about 40 percent and about 60 percent. In other embodiments, the percentage is between about 45 percent and about 55 percent. In an embodiment, the percentage is about 50 percent.

[0209] According to one embodiment, the second IC circulation flow rate is increased by adjusting the IC circulation pump flow rate (-) in response to the value of the first IC circulation flow rate. In other embodiments, the IC circulation pump flow rate is adjusted to decrease the second IC circulation flow rate so that the second IC circulation flow rate is substantially equal to a predetermined fraction or a predetermined ratio of the second IC inlet flow rate. In still other embodiments, no adjustment of the IC circulation pump flow rate may be made. For example, if the first IC inlet flow rate is equal to 0.1 mL / min and the first IC circulation flow rate is equal to -0.1 mL / min, when the second IC inlet flow rate is increased to 0.2 mL / min, the second IC circulation flow rate will be (-1 / 2)*(second Q IC Inlet ) or (-1 / 2)*(0.2mL / min), which results in a second Q of -0.1mL / min. IC Circ Therefore, without adjusting the IC circulating pump flow rate, the second Q IC Circ is achieved.

[0210] The cells are then fed (and continued to grow) for a second period of time at a second inlet flow rate and a second circulation flow rate (step 1310), maintaining the cells within the bioreactor and outside the header or part of the IC circuit outside the bioreactor. Following the second period of feeding (step 1310), for example, if it is not desired to continue feeding and / or growing the cells, process 1300 ends at end operation 1316.

[0211] In embodiments, the first period, second period, third period, fourth period, fifth period, etc. are each one day or more (and / or one hour or more and / or one minute or more). For example, according to embodiments, the periods are from one to fourteen days. However, in other embodiments, the periods may be less than one day or more than fourteen days. In one embodiment, for example, the first period of feeding includes days 0, 1, 2, 3, and 4; the second period of feeding includes day 5; the third period of feeding includes day 6; and the fourth period of feeding includes day 7. In other embodiments, the first period includes days 0, 1, and 2 (e.g., about 3 days in duration), the second period includes days 3, 4, and 5 (e.g., about 3 days in duration), the third period includes days 6, 7, and 8 (e.g., about 3 days in duration), the fourth period includes days 9 and 10 (e.g., about 2 days in duration), and the fifth period includes days 11, 12, and 13 (e.g., about 3 days in duration). According to embodiments, the periods may be of different durations. Each period may be measured in days, hours, minutes, and / or fractions thereof.

[0212] Returning to FIG. 14 , process 1300 optionally proceeds to query step 1312 to determine whether to adjust the feeding rate or flow rate based on metabolic activity or metabolic level. For example, in the expansion of Tregs, it may be desirable to control the lactate level of the expanding cell population at a value of about 7 mmol / L or less. For example, if it is desirable to maintain cell culture lactate levels at about 7 mmol / L or less, the flow rate of media addition is controlled during expansion of cells (e.g., regulatory T cells). In other embodiments, it may be desirable to maintain lactate levels at about 5 mmol / L or less to improve cell growth and viability. In embodiments, a graphical user interface (GUI) element is used to control the rate of media addition to maintain lactate metabolic waste products from glycolysis below a predetermined level during cell expansion.

[0213] If it is not desired to measure metabolic activity and / or adjust feeding levels based on that measurement at optional query step 1312, process 1300 proceeds to "no" and ends at end operation 1316. Alternatively, if it is desired to adjust feeding levels based on metabolic activity, process 1300 proceeds to "yes" and increases the medium addition rate at optional step 1314 to continue feeding the expanding cell population. While step 1314 is shown as a single step, this step may include multiple steps of making adjustments to the medium addition rate, such as, for example, increasing the IC inlet flow rate, increasing the IC circulation flow rate, etc. Step 1314 is shown as a single step for illustrative purposes only and is not intended to be limiting. Following the adjustment of the medium addition rate, process 1300 returns to optional query step 1312 to determine whether to continue measuring metabolic levels and / or adjust feeding. If no adjustment of feeding levels based on metabolic activity is desired, process 1300 proceeds "no" and process 1300 ends at end operation 1316. While FIG. 13 and process 1300 show an "increase" in rate or flow rate, other adjustments to flow rate may be made. For example, from one feeding period to the next, the rate or flow rate may be decreased or maintained substantially the same. The type of adjustment that may be made depends on the metabolic activity assessment of the growing cell population. For example, the "increase" in flow rate in FIG. 13 is provided for illustrative purposes only and is not intended to be limiting.

[0214] Next, FIG. 15 illustrates exemplary operational steps of a process 1400 for retaining cells during feeding, which may be used in a cell growth system, such as the CES500 (FIGS. 5B and 5C) or the CES700 (FIGS. 7A-7C), according to an embodiment of the present disclosure. Starting with a start operation 1402, the process 1400 proceeds to step 1404, in which a disposable tubing set or pre-mounted fluid transfer assembly (e.g., 210 or 400) is loaded onto the cell growth system. The disposable set may include a cell growth chamber 100A or a cell growth chamber 100B, as shown in FIGS. 1C, 4B, and 4C. Next, in step 1406, the system is primed. In one embodiment, for example, a user or operator instructs the system to prime by selecting a task for priming. In one embodiment, such a task for priming may be, for example, a pre-programmed task. Next, in step 1408, an IC / EC cleaning task is performed, in which fluids in the IC circulation loop and the EC circulation loop are replaced, for example. According to one embodiment, the exchange volume is determined by the number of IC and EC volumes exchanged.

[0215] Next, a medium conditioning step 1410 is performed to maintain the appropriate or desired gas concentration across the hollow fibers of the bioreactor membrane, allowing the medium to reach equilibrium with the gas supply before cells are added to the bioreactor. For example, the EC circulation flow rate can be adjusted to allow contact of the medium with the gas supply provided by a gas transfer module (GTM) or oxygenator. The system is then maintained in the appropriate or desired state, for example, until a user or operator is ready to add cells to the bioreactor. In one embodiment, the system may be conditioned with a medium, such as complete medium. Complete medium can be any medium source used for cell growth. In one embodiment, the system is conditioned with, for example, serum-free medium. In one embodiment, the system is conditioned with basal medium. Any type of medium understood by one of skill in the art may be used.

[0216] Process 1400 then proceeds to step 1412, in which cells are introduced into the bioreactor, e.g., from a cell inlet bag. In one embodiment, the cells in the cell inlet bag are in a solution containing medium, e.g., for performing cell feeding step 1414. In another embodiment, the cells in the cell inlet bag are in a solution containing both medium for performing cell feeding step 1414 and a soluble activator complex that stimulates the cells (e.g., T cells or Tregs). In one embodiment, the cells (and, in embodiments, the feeding solution) are introduced into the bioreactor from the cell inlet bag until the cell inlet bag is empty. The cells (and, in embodiments, the feeding solution) are moved into the bioreactor from an air removal chamber. In one embodiment, a task "introducing cells in a uniform suspension" is performed to introduce the cells (and, in embodiments, the feeding solution). In another embodiment, a task "introducing cells to the center without circulation" is performed to introduce the cells (and, in embodiments, the feeding solution) into a specific region (e.g., the center) of the bioreactor. Other introduction methods and / or introduction tasks may be used, depending on the embodiment.

[0217] Next, process 1400 proceeds to query step 1416 to determine whether to use a modified feeding method to retain cells (e.g., non-adherent or suspension cells such as T cells or Tregs) in the bioreactor (e.g., a hollow fiber bioreactor). For example, it may be desirable to place cells in the bioreactor itself and outside of the bioreactor's header or outside of the remainder of the IC loop. If it is not desired to use a modified feeding method to retain cells in the bioreactor itself, process 1400 proceeds to "no" and expands the cells in step 1426. The cells then continue to grow / expand using the media originally supplied with the cells in step 1414.

[0218] On the other hand, if it is desired to retain the cells in the bioreactor itself, process 1400 proceeds to "yes" and performs a modified feeding step 1418, in which the cells are fed by retaining them in the bioreactor using a flow rate into the IC inlet port (501A) of the bioreactor (501) and a flow rate into the IC outlet port (501B) of the bioreactor (501). An inlet volumetric flow rate or inlet flow rate can then be introduced into the first fluid flow path (506) (step 1420). For example, an IC inlet flow rate can be introduced into the first fluid flow path (506) (step 1420). An IC inlet pump (554) (e.g., a first peristaltic pump (in one embodiment)) can operate at a predetermined revolutions per minute (RPM) to produce a predetermined IC inlet volumetric flow rate or IC inlet flow rate in the first fluid flow path (506) (step 1420). According to one embodiment, the processor and / or controller directs or controls the first pump and / or the second pump to operate, for example, at a predetermined number of RPM. Depending on the flow rate and direction of the IC circulation pump (512), a first portion of the modified first flow rate or IC inlet flow rate enters the IC inlet port (501A) or first port of the bioreactor (501) (step 1422). The pump flow rate of the pump (e.g., a peristaltic pump) depends on the diameter or configuration of the pump. Other types of pumps may also be used, in which case the pump flow rate depends on the configuration of the pump used. The IC circulation pump (512) (e.g., a second peristaltic pump (in one embodiment)) operates at a predetermined number of revolutions per minute (RPM) in a direction opposite to the direction of the first pump to generate or produce a predetermined IC circulation flow rate, or a second flow rate, or a second portion of the IC inlet flow rate, which enters the IC outlet port (501B) or second port of the bioreactor (501) (step 1424). For example, in an embodiment, approximately ½ of the IC inlet flow rate, i.e., a first portion, branches off at connection 517 to enter the IC inlet port (501A) of bioreactor 501, and approximately ½ of the IC inlet flow rate, i.e., a second portion, branches off at connection 517 to enter the IC outlet port (501B) of bioreactor 501.As shown, the sum of the first and second portions is substantially equal to IC inlet flow rate 503 (e.g., FIG. 5B), which is pumped by IC inlet pump 554. Depending on the forward and counterflow rates used to maintain cells in the bioreactor or cell growth chamber 501, other types of rates or percentages of IC inlet pump flow rates may be used and the IC circulation pump may be set, configured, or adjusted accordingly.

[0219] In an exemplary embodiment, the cells are fed through an EC circulatory supply loop 752, an IC circulatory supply loop 753, or a combination thereof (step 916), as described with reference to Figures 7A-7C.

[0220] After feeding the cells using such co-flow and counter-flow characteristics to maintain the cells within the bioreactor, process 1400 proceeds to cell growth / expansion step 1426. Although cell expansion is shown in step 1426, cells may be grown / expanded during one or more other steps, such as steps 1412, 1414, 1416, 1418, 1420, 1422, and 1424. From expansion step 1426, process 1400 proceeds to cell harvest or removal step 1430. Process 1400 then ends at end operation 1432. According to an embodiment, if other steps are desired before harvesting (e.g., continuing with a second modified feeding method or another type of feeding method), process 1400 proceeds to optional "other" step 1428. From optional step 1428, process 1400 proceeds to step 1430 where cells are harvested or removed from the bioreactor and process 1400 ends at end operation 1432.

[0221] Next, FIG. 16A illustrates exemplary operational steps of a process 1500 for feeding cells and maintaining cells in a first location (e.g., a bioreactor) that may be used in a cell growth system, such as the CES500 (FIGS. 5B and 5C) or the CES700 (FIGS. 7A-7C), according to embodiments of the present disclosure. Starting with a start operation 1502, the process 1500 proceeds to step 1504, where a disposable tubing set or pre-mounted fluid transfer assembly (e.g., 210 or 400) is loaded onto the cell growth system. The disposable set may include cell growth chamber 100A or cell growth chamber 100B, as shown in FIGS. 1C, 4B, and 4C. Next, in step 1506, the system is primed. In one embodiment, for example, a user or operator instructs the system to prime by selecting a task for priming. In one embodiment, such a task for priming is, for example, a pre-programmed task. Next, in step 1508, an IC / EC wash task is performed. Here, the fluids on the IC circulation loop and the EC circulation loop are for example exchanged. According to one embodiment, the exchange volume is determined by the number of IC volumes and EC volumes exchanged.

[0222] Next, a medium conditioning step 1510 is performed to maintain the appropriate or desired gas concentration across the hollow fibers of the bioreactor membrane, allowing the medium to reach equilibrium with the gas supply before cells are added to the bioreactor. For example, the EC circulation flow rate can be adjusted to allow contact of the medium with the gas supply provided by a gas transfer module (GTM) or oxygenator. The system is then maintained in the appropriate or desired state, for example, until a user or operator is ready to add cells to the bioreactor. In one embodiment, the system may be conditioned with a medium, such as a complete medium. The complete medium may be any medium source used for cell growth. In one embodiment, the system is conditioned with, for example, a serum-free medium. In one embodiment, the system is conditioned with a basal medium. Any type of medium understood by one of skill in the art may be used.

[0223] Process 1500 then proceeds to step 1512, where cells are introduced into the bioreactor, e.g., from a cell inlet bag. In one embodiment, the cells in the cell inlet bag are in a solution containing medium, e.g., for cell feeding. In another embodiment, the cells in the cell inlet bag are in a solution containing both medium and a soluble activator complex that stimulates the cells (e.g., T cells or Tregs). In one embodiment, the cells are introduced into the bioreactor from the cell inlet bag until the cell inlet bag is empty. The cells are transferred to the bioreactor from an air removal chamber. In one embodiment, the cells are introduced by performing a task to "introduce cells in a uniform suspension." In another embodiment, the cells are introduced into a specific region (e.g., the center) of the bioreactor by performing a task to "introduce cells to the center without circulation." Other introduction methods and / or introduction tasks may be used, depending on the embodiment.

[0224] Process 1500 then proceeds to step 1514, which involves feeding cells according to a first process for a first period of time. In an exemplary embodiment, cells are fed through an EC circulatory supply loop 752, an IC circulatory supply loop 753, or a combination thereof (step 916), as described with reference to Figures 7A-7C. In one embodiment, cells are fed at a minimum or low feeding rate, for example, when a cell population is beginning to grow / proliferate and a minimum or low feeding rate can meet the feeding requirements of such a cell population. For example, during such a first period of time, an IC inlet pump flow rate of +0.1 mL / min may be used to cause or generate a first fluid flow rate of 0.1 mL / min. While this example uses a low or minimum feeding rate such as 0.1 mL / min, according to embodiments, a low or minimum feeding rate may be greater than about 0.01 mL / min and less than or equal to about 0.1 mL / min. In embodiments, a low or minimum feeding rate may be greater than 0.1 mL / min. If it is desired to reduce cell loss from the hollow fiber membrane bioreactor during such a first period, an IC inlet pump flow rate of +0.1 mL / min can be matched or approximately matched to a complementary IC circulation pump flow rate of −0.1 mL / min to maintain cells in the bioreactor during the growth phase of the cell culture. In other embodiments, other pump flow rates and resulting fluid flow rates may be used. Alternatively, in an exemplary embodiment, a first IC inlet pump provides fluid flow to the IC inlet port (701A) at a volumetric flow rate of less than 0.1 mL / min, or about 0.01 mL / min, and an IC circulation pump provides fluid flow to the IC outlet port (701B) at a complementary IC circulation volumetric or flow rate of less than −0.1 mL / min, or about −0.01 mL / min. Here, the negative sign (“−”) used, for example, at −0.01 mL / min, indicates that the direction of the IC circulation pump creates a counterflow so that cells are maintained in the bioreactor during the growth phase of the cell culture.

[0225] Next, process 1500 proceeds to query step 1516, which, according to an embodiment, determines whether to adjust the feeding rate to retain cells within the bioreactor, while also taking into account the growing cell population and increasing feeding requirements. For example, FIG. 16B illustrates an increasing feeding rate in response to an increasing cell population. In FIG. 16B, graph 1528 illustrates cell number versus IC flow rate for a run or procedure in a cell expansion system, such as the Quantum® Cell Expansion System. In one embodiment, the IC flow rate includes the medium used to feed the cells and is therefore also referred to as the IC medium flow rate. Number of cells 1530 is shown versus IC flow rate (mL / min) 1532. As shown, according to an embodiment, the IC flow rate increases from 0.1 mL / min to 0.2 mL / min to 0.3 mL / min with increasing cell population and increasing feeding requirements of the expanding cell population. In the illustrated embodiment, there is an approximately linear relationship between the number of cells and the IC flow rate, as indicated by line 1534.

[0226] Returning to FIG. 16A and query step 1516, if an adjustment in the feeding rate is not desired, process 1500 proceeds to "no" and expands the cells in step 1520. In this case, cell growth / expansion continues using the medium used in the first period of feeding in step 1514. Although cell expansion is shown in step 1520, the cells may be grown / expanded during one or more other steps, such as steps 1512, 1514, 1516, and 1518. On the other hand, if an adjustment in the feeding rate is desired while the cells are maintained in the bioreactor, process 1500 proceeds to "yes" and feeds the cells according to a second process during a second period in step 1518. In one embodiment, such a second process includes, for example, feeding the cells at approximately the same feeding rate as during the first period. In other embodiments, the second process includes feeding the cells at a different feeding rate compared to the feeding rate used during the first period. In one embodiment, the IC inlet flow rate is increased and the IC circulation flow rate is set, configured, or adjusted to be equal to a percentage, fraction, or ratio of the IC inlet flow rate. For example, the IC circulation flow rate, according to an embodiment, may be set equal to about fifty percent (50%), or about one-half (1 / 2), of the value of the IC inlet flow rate, or some other percentage or ratio. According to an embodiment, a determination of whether to set, configure, or adjust the IC circulation flow rate to a percentage, fraction, or ratio of the IC inlet flow rate is made based on the value of the IC inlet flow rate. For example, in an embodiment, the following method is provided for retaining cells in a bioreactor when feeding cells using an IC inlet flow rate (wherein Q IC Circ =IC circulation flow rate (mL / min);Q IC Inlet =IC inlet flow rate (mL / min)). : Q IC Inlet If ≥ 0.2 mL / min, Q IC Circ =(-)1 / 2*Q IC Inlet and Q IC Inlet= 0.1 mL / min, Q IC Circ =(-)Q IC Inlet

[0227] While the above formula calculates the IC circulation flow rate differently depending on the IC inlet flow rate (e.g., 0.2 mL / min or 0.1 mL / min), other embodiments may use different IC inlet flow rates in such calculations. Furthermore, while approximately fifty percent (50%), or approximately one-half (1 / 2), is used in this example, other percentages, ratios, fractions, and / or proportions may be used according to embodiments. Returning to process 1500, after feeding the cells using such co-flow and counter-flow characteristics to maintain the cells within the bioreactor as part of the second process in step 1518, process 1500 proceeds to cell growth / expansion step 1520. While cell expansion is shown in step 1520, the cells may be grown / expanded during one or more other steps, such as steps 1512, 1514, 1516, and 1518. From expansion step 1520, process 1500 proceeds to cell harvesting or removal step 1524. Process 1500 then ends at end operation 1526. According to an embodiment, if other steps are desired before harvesting, process 1500 proceeds to optional "other" step 1522. From optional step 1522, process 1500 proceeds to harvest or remove cells from the bioreactor step 1524, and process 1500 ends at end operation 1526.

[0228] FIG. 17 next illustrates exemplary operational steps of a process 1600 for feeding cells for use with a cell growth system, such as CES500 (e.g., FIGS. 5B and 5C) or CES700 (e.g., FIGS. 7A-7C), according to an embodiment of the present disclosure. A START operation 1602 is initiated, in which a disposable set is loaded into the cell growth system, the system is primed, an IC / EC wash is performed, media is adjusted, and cells are loaded. The disposable set may include cell growth chamber 100A or cell growth chamber 100B, as shown in FIGS. 1C, 4B, and 4C. Process 1600 then proceeds to step 1604, which involves feeding cells according to a first process for a first period of time. In an exemplary embodiment, cells are fed through EC circulation supply loop 752, IC circulation supply loop 753, or a combination thereof (step 916), as described with reference to FIGS. 7A-7C. In one embodiment, for example, when a cell population is beginning to grow / proliferate and a minimum or low feeding rate can meet the feeding requirements of such a cell population, cells are fed at a minimum or low feeding rate. For example, an IC inlet pump flow rate of +0.1 mL / min may be used during such a first period. While this example uses a low or minimum feeding rate such as 0.1 mL / min, according to embodiments, a low or minimum feeding rate may be greater than about 0.01 mL / min and less than about 0.1 mL / min. In embodiments, a low or minimum feeding rate may be greater than 0.1 mL / min. If it is desired to reduce cell loss from the hollow fiber membrane bioreactor during such a first period, the IC inlet pump flow rate of +0.1 mL / min may be matched or approximately matched to a complementary IC circulation pump flow rate of −0.1 mL / min to maintain cells in the bioreactor during the growth phase of the cell culture.Alternatively, in an exemplary embodiment, a first IC inlet pump provides a volumetric flow rate of less than 0.1 mL / min, or about 0.01 mL / min, to the IC inlet port (701A), and an IC circulation pump provides a complementary IC circulation volumetric or flow rate fluid flow rate of less than −0.1 mL / min, or about −0.01 mL / min, to the IC outlet port (701B), where the negative sign (“−”) used, for example, at −0.01 mL / min, indicates that the direction of the IC circulation pump creates a counterflow so that cells are maintained in the bioreactor during the growth phase of the cell culture.

[0229] Next, process 1600 proceeds to query step 1606 to determine whether the feeding rate should be adjusted to account for the growing cell population and / or to maintain cells in the bioreactor. If it is desirable to adjust the feeding rate while maintaining the cells in the bioreactor, process 1600 proceeds to "yes" and feeds the cells according to a second process for a second period of time in step 1608. In one embodiment, such a second process includes, for example, feeding the cells at approximately the same feeding rate as during the first period of time. In other embodiments, the second process includes feeding the cells at a different feeding rate compared to the feeding rate used during the first period of time. In one embodiment, the IC inlet flow rate is increased and the IC circulatory flow rate is set, configured, or adjusted to be equal to a percentage, fraction, or ratio of the IC inlet flow rate. For example, the IC circulatory flow rate may be set equal to approximately fifty percent (50%), or approximately half (1 / 2), of the value of the IC inlet flow rate, or some other percentage or ratio, according to an embodiment. According to one embodiment, the determination of whether to set the IC circulation flow rate to a percentage of the IC inlet flow rate is made based on the value of the IC inlet flow rate. For example, in an embodiment, the following method is provided (where Q IC Circ =IC circulation flow rate (mL / min);Q IC Inlet =IC inlet flow rate (mL / min)). : Q IC InletIf ≥ 0.2 mL / min, Q IC Circ =(-)1 / 2*Q IC Inlet and Q IC Inlet = 0.1 mL / min, Q IC Circ =(-)Q IC Inlet

[0230] While the above formula calculates the IC circulation flow rate differently depending on the IC inlet flow rate (e.g., 0.2 mL / min or 0.1 mL / min), other embodiments may use different IC inlet flow rates in such calculations. Furthermore, while approximately fifty percent (50%), or approximately half (1 / 2), is used in this example, other percentages, ratios, fractions, and / or proportions may be used according to embodiments. Returning to process 1600, after feeding the cells using such co-flow and counter-flow characteristics to maintain the cells in the bioreactor as part of the second process in step 1608, process 1600 proceeds to step 1610, where it is determined whether to monitor or measure metabolic activity (e.g., glucose consumption and / or lactate production) of the growing cell population. If monitoring metabolic activity is not desired, process 1600 proceeds to "no" and expands the cells in step 1616. The cells can then continue to grow / expand using the medium provided during feeding during the first time period (step 1604) and / or the second time period (step 1608). Although cell expansion is shown in step 1616, the cells may also grow / expand during one or more other steps, e.g., 1604, 1606, 1608, 1610, 1612, 1614, etc.

[0231] Returning to query step 1610, if it is desired to monitor or measure metabolic activity of the growing cell population, process 1600 proceeds to "yes" and continues feeding the cells or adjusts the feeding rate according to a second process based on metabolic activity and / or its measurement. In one embodiment, monitoring glucose and / or lactate levels can facilitate adjustment of media flow rate (e.g., IC flow rate) to support cell (e.g., T cells or Treg) expansion in a bioreactor (e.g., hollow fiber bioreactor). In an embodiment, cell culture lactate levels are maintained below, e.g., about 7 mmol / L. In an embodiment, lactate metabolic waste product from glycolysis is maintained below, e.g., about 7 mmol / L during expansion of cells (e.g., regulatory T cells) by controlling the media addition flow rate, e.g., using a graphical user interface (GUI) of the cell expansion system. In other embodiments, lactate levels are maintained below about 5 mmol / L, e.g., to improve cell growth and viability, e.g., by controlling media addition flow rate and / or other settings. In other embodiments, other concentrations may be used.

[0232] For example, depending on the metabolic measurements and the desired level of lactate, process 1600 may proceed to either continue feeding cells according to a second process (step 1612) or adjust the feeding rate (step 1614). For example, in one embodiment, if the metabolic activity measurements indicate a lactate level of less than about 5 mmol / L, cell feeding continues according to a second process (step 1612). In another embodiment, if the metabolic activity measurements indicate a lactate level of less than about 7 mmol / L, cell feeding continues according to the second process (step 1612). From step 1612 of continuing to feed cells according to the second process, process 1600 returns to query step 1610 to continue monitoring the metabolic activity of the growing cell population.

[0233] Depending on the metabolic measurements and their desired levels, process 1600 proceeds to step 1614 of adjusting the feeding rate, where cells are fed according to an additional process for an additional period of time. According to embodiments, such additional processes and additional periods include, for example, a third process in a third period, a fourth process in a fourth period, a fifth process in a fifth period, etc. In one embodiment, such additional processes include, for example, feeding cells at approximately the same feeding rate as during the first and / or second period. In other embodiments, the additional processes include feeding cells at a different feeding rate compared to the feeding rate used during the first and / or second period. For example, in one embodiment, the IC inlet flow rate is increased and the IC circulation flow rate is matched, or approximately or substantially matched, to the IC inlet flow rate, but in the opposite direction. In other embodiments, the IC inlet flow rate is increased and the IC circulation flow rate is set, configured, or adjusted to be equal to, and in the opposite direction to, a percentage, fraction, or portion of the IC inlet flow rate. Although the feeding rate adjustment step 1614 shows "additional" processes and "additional" periods in step 1614, any number of processes and periods may be used to adjust the feeding rate based on metabolic activity.

[0234] From adjusting feeding rate step 1614, process 1600 returns to query step 1610. If adjusting or further adjusting the feeding rate is not desired, process 1600 proceeds "no" to expanding the cells in step 1616, where the cells can continue to grow / expand using the media provided during feeding in the first time period (1604), the second time period (1608), and / or additional time periods (1614). Although expanding the cells is shown in step 1616, the cells may also be grown / expanded during one or more other steps, such as, for example, steps 1604, 1606, 1608, 1610, 1612, 1614, etc. From expanding step 1616, process 1600 proceeds to step 1618, where the cells are harvested or removed from the bioreactor, for example, into a harvest bag or container. Process 1600 then ends at END operation 1622. Alternatively, from harvesting step 1618, process 1600 may optionally proceed to further processing / analysis step 1620. Such optional further processing / analysis step 1620 may include, for example, characterizing the phenotype of the harvested cells (e.g., T cells or Tregs). From optional further processing / analysis step 1620, process 1600 then ends at END operation 1622.

[0235] 18A illustrates operational steps of a process 1700 for expanding cells that may be used with a cell expansion system in embodiments of the present disclosure. As described below, process 1700 includes shearing cells expanded within a cell growth chamber in accordance with embodiments of the present disclosure. In embodiments, these steps may be performed as part of a "modified circulation" task. START operation 1702 begins, and process 1700 proceeds to step 1704, which introduces a fluid having cells into a cell growth chamber within a cell expansion system. In embodiments, the cells include one or more types of non-adherent cells, such as T cells. In one embodiment, the cells include Tregs.

[0236] Process 1700 proceeds to exposing the cells to an activator 1706. The activator, which may include an antibody complex, is added to the input fluid in step 1704. In an embodiment, the activator is a soluble human antibody CD3 / CD28 / CD2 cell activator complex. Process 1700 proceeds to step 1708, growing the cells for a first period of time. Step 1708 includes a cell feeding step 1710. The cells are nourished to promote cell growth. For example, media containing glucose, proteins, and reagents is delivered to the cell growth chamber to provide nutrients for cell growth.

[0237] The first period for expanding the cells (step 1708) may be based on the time it takes for a cell colony, microcolony, or cluster to form. A cell colony, microcolony, or cluster is a group of one or more adherent cells. In embodiments, cells (e.g., Tregs) benefit from cell contact. Cell contact stimulates signaling that promotes proliferation and growth. However, after a period of expansion, cells may adhere to each other to form a cell colony, microcolony, or cluster. Without being bound by theory, it is believed that after the cell expansion period (1708), cells form a relatively large cell colony, microcolony, or cluster that continues to grow. The cell colony, microcolony, or cluster may form a necrotic center, where nutrients (e.g., glucose), gases (e.g., oxygen), and reagents (e.g., activators) cannot reach the cells at the center of the cell colony, microcolony, or cluster. As a result, conditions for cell growth at the center of these cell colonies, microcolonies, or clusters can result in slower growth rates (e.g., increased doubling times) or can lead to cell necrosis.

[0238] In embodiments, the first period of time is between about 5 hours and about 48 hours to allow for cell growth (step 1708). In some embodiments, the first period of time is greater than about 6 hours, or greater than about 12 hours, or greater than about 24 hours, or greater than about 48 hours. In other embodiments, the first period of time is 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 less than about 12 hours. After the first period of time, process 1700 proceeds to step 1712, which involves circulating the cell colonies, microcolonies, or clusters for a second period of time to disaggregate them. Step 1712 is performed to reduce the size of the cell colonies, microcolonies, or clusters. The second period of time is, in embodiments, less than about 120 minutes, such as between about 60 minutes and about 0.5 minutes. In other embodiments, the second period of time may be based on the amount of fluid introduced into the first circuit.

[0239] FIG. 18B shows multiple views 1750, 1760, 1770 of cells within a volume of fluid (1752) that may be grown within a cell growth chamber as part of process 1700. For example, in some embodiments, the cell growth chamber is a hollow fiber bioreactor. In these embodiments, views 1750, 1760, 1770 show cells within a single fiber of a hollow fiber bioreactor, for example. Views 1750A, 1760A, and 1770A are enlarged views of portions of views 1750, 1760, and 1770, respectively. View 1750 shows cells after they have been introduced (step 1704), exposed to an activator (step 1706), and grown for a period of time (e.g., a first period) (step 1710). As shown in view 1750, multiple cell colonies 1754A-1754E have formed.

[0240] Step 1712 circulates the fluid and cells in a first fluid circuit to reduce the number of cells in cell colonies, microcolonies, or clusters 1754A-1754E and the size of those colonies or clusters. Without being bound by theory, it is believed that this circulation generates forces (including shear stress) that act on the cell colonies, as indicated by arrows 1756 in enlarged portion 1760A. The shear stress 1756 provides sufficient force to split and dissociate the cells within the cell colonies. As circulation continues, the cell colonies begin to break down into smaller sizes, as shown in diagram 1760. Diagram 1770 shows the cells after a second period of circulation. As shown in diagram 1770, the cell colonies have reduced in size, with some colonies completely split and dissociated into individual cells. In some embodiments, cyclic shear step 1712 is performed until the cells and fluid form a single-cell suspension.

[0241] In other embodiments, cell colonies, microcolonies, or clusters of cells may remain after cyclic shearing step 1712. For example, colony 1754F in enlarged portion 1770A shows that some colonies remain after step 1712, but are reduced in size. In embodiments, the remaining cell colonies, microcolonies, or clusters (e.g., 1754F) are between about 25 μm and about 300 μm. In other embodiments, cyclic shearing step 1712 reduces the size of the cell colonies, microcolonies, or clusters (e.g., 1754F) so that the cell colonies, microcolonies, or clusters are between about 50 μm and about 250 μm. In still other embodiments, step 1712 reduces the size of the cell colonies, microcolonies, or clusters to between about 75 μm and about 200 μm. In some embodiments, the size of the cell colonies, microcolonies, or clusters is less than about 200 μm (e.g., about 100 μm) after step 1712.

[0242] In embodiments, the size of the remaining cell colonies, microcolonies, or clusters is, in part, a function of certain structural features of the cell growth chamber. As noted above, the cell growth chamber may be a hollow fiber bioreactor, in some embodiments, equipped with hollow fibers. As will be appreciated, as cell colonies, microcolonies, or clusters circulate, they are subjected to shear stress each time they contact the sidewalls of the hollow fibers. This contact reduces the size of the cell colonies more efficiently. If the inner diameter is larger, as in traditional processes that use pipettes to induce shear stress to reduce colony size, contact with the sidewalls may occur less frequently. Figure 18C illustrates the difference in inner diameter size between a hollow fiber (e.g., 215 microns) 1872 and a pipette tip 1874 (762 microns) according to one embodiment, used to split and separate adherent cells from cell colonies, microcolonies, or clusters. In embodiments, the smaller inner diameter of the hollow fiber is believed to more efficiently and effectively reduce the size of the cell colonies during the circulating shear step 1712.

[0243] The second period of time for the cyclic shearing step 1712, in embodiments, is less than about 120 minutes, less than about 90 minutes, less than about 60 minutes, less than about 30 minutes, or less than about 15 minutes. In some embodiments, the second period of time can be between about 1 minute and about 15 minutes, such as about 4 minutes.

[0244] After the second time period, process 1700 proceeds to step 1714, which involves moving cells into the cell growth chamber for a third time period. In step 1714, cells that are not located in the cell growth chamber as a result of cyclic shear step 1712 are returned to the cell growth chamber during the third time period. In embodiments, this includes operating one or more pumps to introduce fluid into the fluid circulation path. For example, fluid is introduced into the first fluid flow path through the fluid inlet path and into the cell growth chamber through both the inlet and outlet ports of the cell growth chamber. Movement of fluid from the inlet and outlet ports into the cell growth chamber returns the cells to the cell growth chamber.

[0245] In some embodiments, the fluid used in step 1714 to return the cells to the cell growth chamber includes a reagent that promotes cell growth. For example, in embodiments, the fluid is a medium containing glucose, proteins, or other reagents. In one embodiment, the fluid may include one or more supplements. In one embodiment, the fluid is a complete medium and includes a cytokine (e.g., a human IL-2 cytokine supplement). The addition of the fluid may be referred to as a bolus addition. The combination of steps 1712 and 1714 may, in embodiments, be referred to as a circulating bolus addition.

[0246] In other embodiments, the third period of time may be based on the volume of fluid introduced into the cell growth chamber during circulating shear step 1712. For example, in embodiments, step 1712 is performed until about 300 ml, about 250 ml, about 200 ml, or about 150 ml has been introduced into the fluid circuit.

[0247] After the third time period, process 1700 proceeds to a fourth time period of growth step 1716. Similar to step 1708, step 1716 may include a cell feeding step 1718. Cells are nourished to promote cell growth. For example, media containing glucose, proteins, and reagents may be delivered to the cell growth chamber to provide nutrients for cell growth.

[0248] Similar to the first period of time, the fourth period of time may be based on the time it takes for cell colonies to form. In embodiments, the fourth period of time is between about 5 hours and about 48 hours. In some embodiments, the fourth period of time is greater than about 6 hours, greater than about 12 hours, greater than about 24 hours, or greater than about 48 hours. In other embodiments, the fourth period of time is 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 less than about 12 hours. In some embodiments, the fourth period of time may be shorter than the first period of time because more cells are likely to be present in the cell growth chamber.

[0249] After the fourth period, process 1700 proceeds to step 1720, where the second cell colony is reduced by cycling and shearing for a fifth period. In embodiments, step 1720 may use a first cycling rate. However, in other embodiments, the cycling rate used in step 1720 may be different, such as greater than or less than the first cycling rate.

[0250] After the fifth time period, process 1700 proceeds to step 1722, where cells not located within the cell growth chamber are returned to the cell growth chamber during a sixth time period. Fluid is introduced into the first fluid flow path from the fluid inlet channel and into the cell growth chamber through both the inlet and outlet ports of the cell growth chamber. Movement of fluid from the inlet and outlet ports into the cell growth chamber returns the cells to the cell growth chamber. In some embodiments, the fluid used to return the cells to the cell growth chamber includes a reagent that promotes cell growth. For example, in embodiments, the fluid is a medium containing glucose, proteins, or other reagents. In one embodiment, the fluid may include one or more supplements. In one embodiment, the fluid is a complete medium and includes a cytokine (e.g., human IL-2 cytokine).

[0251] Process 1700 may optionally perform additional steps of expanding, cycling, and transferring cells, as indicated by optional step 1724 and ellipsis 1726. The expansion, cycling, and transfer steps may be performed consecutively for a period of time. For example, in some embodiments, the steps may be performed once every four days, once every three days, once every two days, once a day, twice a day, or three times a day for a period of about two to about twenty days (e.g., about ten days). In some embodiments, the steps may be performed at various time periods. For example, in one embodiment, the steps may be performed after three days and then every other day thereafter. As another example, the steps may be performed after two days and then twice a day thereafter. These are merely examples, and other time periods may be utilized in other embodiments.

[0252] For example, FIG. 19 shows a graph 1800 illustrating the implementation of circulating bolus additions at different time periods during a cell expansion process. Curve 1808 illustrates cell number 1802 versus cell culture days 1804 in a cell expansion system (e.g., a Quantum® Cell Expansion System). Curve 1806 illustrates IC flow rate 1818 versus cell culture days 1804 in a cell expansion system (e.g., a Quantum® Cell Expansion System). As shown in curve 1806, the IC flow rate is maintained at 0.1 mL / min for the first three days. On day 6, the flow rate increases to 0.2 mL / min, and after day 7, it increases to 0.3 mL / min. The increase in flow rate corresponds to an increase in cell number with increasing culture days 1804. Also shown in FIG. 19, several circulating bolus addition steps (1810, 1812, 1814, 1816) are performed. Circulating bolus addition step 1810 is performed beginning on day 3.5 of cell culture. Starting at day 4.5 of cell culture, another circulation bolus addition step 1812 is performed. Starting at day 6 of cell culture, another circulation bolus addition step 1814 is performed, and starting at day 6.5 of cell culture, another circulation bolus addition step 1816 is performed. As can be seen from curves 1806, 1808, the multiple circulation bolus addition steps (1810-1816) combined with increasing IC flow rate have a positive impact on the cell growth rate (e.g., cell number).

[0253] Returning to Figure 18A, process 1700 proceeds to step 1728, which involves removing cells from the cell growth chamber. In embodiments, this may include harvesting the cells. Step 1728 may include additional steps, such as circulation steps (e.g., 1712, 1720), before or during removal of cells from the cell growth chamber. Process 1700 ends at end operation 1730.

[0254] FIG. 20A illustrates operational steps of a process 1900 for operating a pump that may be used in a cell growth system according to an embodiment of the present disclosure. As described below, process 1900 includes steps for operating a pump in a process for reducing cells in cell clusters grown in a cell growth chamber according to an embodiment of the present disclosure. In embodiments, these steps may be performed as part of a "modified circulation" task. In embodiments, the steps of process 1900 may be performed by a computer processor. A START operation 1902 begins, and process 1900 proceeds to step 1904, where a first pump is operated at a first flow rate to introduce a first volume of fluid containing cells into an inner capillary portion of a bioreactor of the cell growth system. For example, the bioreactor may be cell growth chamber 100A or cell growth chamber 100B, as shown in FIGS. 1C, 4B, and 4C. In an embodiment, the first pump is an inlet pump.

[0255] After operating the first pump at the first flow rate, process 1900 proceeds to step 1906, where the first pump is operated at a second flow rate to introduce nutrient-containing medium into the interior capillary portion of the bioreactor for a first period of time. Nutrients include, for example, proteins, glucose, and other compounds used to feed and promote cell growth. For example, as shown in FIG. 20B, first pump 1960 is operated at a second flow rate to introduce medium into inlet port 1962A of bioreactor 1962.

[0256] In embodiments, the first period of time may be based on the time it takes for cell colonies to form. In embodiments, the first period of time is between about 5 hours and about 48 hours. In some embodiments, the first period of time is greater than about 6 hours, greater than about 12 hours, greater than about 24 hours, or greater than about 48 hours. In other embodiments, the first period of time is 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 less than about 12 hours.

[0257] Process 1900 then proceeds to step 1908, which operates a second pump at a third flow rate to direct fluid into the bioreactor. Optional step 1908 is performed to activate pump 1964 to transfer a portion of the fluid introduced in step 1906 toward outlet port 1962B of bioreactor 1962 for feeding the cells.

[0258] Process 1900 then proceeds to step 1910, which involves operating a second pump at a fourth flow rate to circulate the cells for a second period of time and reduce the number of cells in cell clusters within the bioreactor. In an embodiment, the cells circulate through a first fluid circuit. Referring to Figure 20C, in step 1910, a second pump 1964 is operated to circulate fluid in a first fluid circuit 1966, as indicated by arrows 1968A-1968D.

[0259] Without being bound by theory, it is believed that after a period of time, the proliferating cells form cell colonies, microcolonies, or clusters. The cell colonies may form necrotic centers, where nutrients and proteins (e.g., activators) do not reach the cells in the center of the cell colony. As a result, the cell growth conditions in the center of these cell colonies, microcolonies, or clusters may result in slower growth rates (e.g., increased doubling times) and potentially cell necrosis. Step 1910 is performed to reduce the size of the cell colonies, microcolonies, or clusters.

[0260] In embodiments, the fourth flow rate is high enough to induce shear. For example, in embodiments, the fourth flow rate reaches about 1000 mL / min. In other embodiments, the fourth flow rate may be between about 100 mL / min and about 600 mL / min, such as 300 mL / min.

[0261] After the second time period, process 1900 proceeds to step 1912, where the first pump is operated at a fifth flow rate to introduce fluid through the first fluid flow path for a third time period. In embodiments, the first portion of the fluid introduced into the first fluid flow path returns the first cells in the first fluid flow path (which may be in the first fluid flow path due to step 1910) to the cell growth chamber through inlet port 1962A. Referring to FIG. 20D , pump 1960 is operated to introduce fluid into first fluid flow path 1970. As indicated by arrows 1968A, 1968B, fluid flows from first fluid flow path 1970 into inlet port 1962A. This returns the first cells outside of bioreactor 1962 to bioreactor 1962. Returning the cells in the first fluid flow path to the cell growth chamber is believed to optimize cell growth conditions within the cell growth chamber, thereby improving overall cell proliferation.

[0262] In some embodiments, the fluid introduced into the first fluid-flow path in step 1912 may include one or more materials (e.g., reagents) that promote cell growth. For example, in embodiments, the fluid is a medium containing glucose or other nutrients to feed the cells. In one embodiment, the fluid includes a reagent that includes an additional activator to keep the cells activated for growth. Returning the cells to the cell growth chamber using a fluid containing certain reagents or other materials and exposing the cells to additional reagents (e.g., growth factors, proteins, etc.) that promote growth can improve cell growth. In embodiments, the addition of fluid used in step 1912 may be referred to as a bolus addition.

[0263] After the third period of time, in step 1914, the second pump is operated at a sixth flow rate to transport the second portion of the fluid and the second cells introduced through the first fluid flow path into the cell growth chamber via outlet port 1962B. In embodiments, the second portion of the fluid transports the second cells in the first fluid flow path (which may be in the first fluid flow path due to step 1910) back to the cell growth chamber via the outlet port. Referring to FIG. 20D, pump 1964 is operated to transport the fluid introduced into first fluid flow path 1970 to outlet port 1962B. As indicated by arrow 1968C, pump 1964 transports the fluid and cells in the first fluid circuit into bioreactor 1962 via outlet port 1962B. As indicated by FIG. 20D, the sixth flow rate is in the opposite direction to the fourth flow rate (FIG. 20C). This fluid movement causes cells outside of bioreactor 1962 to be returned to bioreactor 1962 .

[0264] In an embodiment, the sixth flow rate is less than the fifth flow rate and, as described above, moves fluid into the first fluid flow path. As can be appreciated, the fifth flow rate introduces a volume of fluid into a portion of the circuit 1966 based on the fifth flow rate. The sixth flow rate is set to move a percentage of that volume toward the outlet port 1962B.

[0265] In embodiments, the sixth flow rate may be set as a percentage of the fifth flow rate. For example, the sixth flow rate is about 90% or less of the fifth flow rate. In some embodiments, the sixth flow rate may be set to about 80% or less of the fifth flow rate. In other embodiments, the sixth flow rate is about 70% or less of the fifth flow rate. In still other embodiments, the sixth flow rate is about 60% or less of the fifth flow rate. In some embodiments, the sixth flow rate may be about 50% or less of the fifth flow rate.

[0266] In embodiments, the sixth flow rate is based, at least in part, on the difference between a first volume between the second pump and the inlet port and a second volume between the second pump and the outlet port. Referring to FIG. 20D , portions of the first circuit have different volumes. For example, in one embodiment, the first volume between the second pump 1964 and the inlet port 1962A has a first volume, and the second volume between the second pump 1964 and the outlet port 1962B has a second volume that is different (e.g., larger) than the first volume. In these embodiments, to return the cells from the circuit to the bioreactor, the sixth flow rate may be set, at least in part, based on these volume differences so that the cells arrive at the bioreactor at approximately the same time.

[0267] As can be seen, when fluid enters the fluid circuit 1966 from the first fluid flow path 1970, the fluid moves toward the inlet port 1962A at a flow rate set by the first pump 1960. When the pump 1964 is activated, at least a portion of the fluid is redirected toward the outlet port 1962B. In an embodiment, the second volume (the volume between the pump 1964 and the outlet port 1962B) is larger than the first volume. Thus, the second pump 1964 is set at a percentage of the flow rate of the pump 1960 to move more fluid into that portion of the second volume.

[0268] In one embodiment, pump 1960 is set to 100 ml / min in step 1912. In this embodiment, the second volume (pump 1964 to outlet port 1962B) is larger than the first volume (pump 1964 to inlet port 1962A). Because of the additional volume, in an embodiment, pump 1964 is set to 70 ml / min in step 1914. In this embodiment, cells may arrive at bioreactor 1962 at approximately the same time during the third time period.

[0269] In embodiments, process 1900 may perform steps 1906-1914, optionally multiple times, as indicated by ellipsis 1916 and optional step 1918. Steps 1906, 1910, 1912, and 1914 may be performed consecutively for a predetermined period of time. As described above, steps may be performed to feed cells, circulate cells to separate and isolate cell colonies, microcolonies, or clusters, and return cells to the cell growth chamber. For example, in some embodiments, the steps may be performed once every three days, once every two days, once a day, twice a day, or three times a day for a period of about two to about twenty days (e.g., about ten days). In some embodiments, the steps may be performed at various time intervals. For example, in one embodiment, the steps may be performed after three days and then every other day thereafter. As another example, the steps may be performed after two days, and then twice a day thereafter. These are merely examples, and other periods may be used in other embodiments. Process 1900 ends at end operation 1920.

[0270] Note that in some embodiments, process 1900 may include additional steps. For example, when connecting a rocking device to the bioreactor and starting the first pump in step 1910 after the first period of time (and during the second period of time), the rocking device may be started to rotate the bioreactor as part of circulating the cells to reduce the number of cells in cell clusters. This is merely one example, and other embodiments of process 1900 are not limited thereto.

[0271] In other embodiments, the steps of process 1900 may proceed as shown in FIG. 20A , with reduced flow rates achieved through precision motor control. For example, the IC and EC pumps can operate from 0.005 RPM to 600 RPM, all within a torque range that prevents stalling throughout the entire range. Thus, the flow range is as low as 0.01 mL / min at 500 RPM for the large pump and as low as 0.01 mL / min at 300 RPM for the small pump, in both directions. Work was done to prevent stalling at low speeds by improving stall detection methods at low speeds and by controlling the control loops differently at low speeds. In an exemplary embodiment, fluid is pumped through EC circulating supply loop 752, IC circulating supply loop 753, or a combination thereof, as described with reference to FIGS. 7A-7C.

[0272] The IC and EC pumps operate continuously at nearly continuous speeds, which, as described above, provides strong counterflow containment at ultra-low flow rates. When two matched pumps are duty cycled, counter-operating the pumps to retain cells within the bioreactor can shift the location of the cells within the bioreactor. Continuous operation of the IC and EC pumps over long periods of time keeps the cells in an optimized position within the bioreactor, conserving cell culture medium overall.

[0273] As explained below, harvest synchronization is provided by alternating pump control that can be run over long periods of time. Low flow rates (approximately 1 / 10 of the flow rates used during the steps in the procedure described above) achieve approximately 0.01 mL / min. To achieve these rates, a continuous rate of 0.005 RPM is required. Thus, constant pump operation at 0.005 RPM without stalling is achieved. See data in D0000038948, D0000038355, D0000044793, D0000033725 (and all attachments), and D0000041290.

[0274] In other embodiments, the steps of process 1900 may proceed as shown in FIG. 20A , but for significantly shorter periods than those described above. For example, the IC inlet pump and the EC inlet pump are operated for alternating periods. In this case, the periods may be less than 10 minutes, or approximately 5 minutes. The pumps are operated at low or very low flow rates during each period. For example, a low or very low flow rate may be within about 0.005 RPM to 600 RPM for a standard bioreactor or standard cell growth chamber, or within about 0.01 mL / min at 500 RPM for a miniature bioreactor or miniature cell growth chamber. Alternating the IC inlet pump and the EC inlet pump for short periods of time allows for synchronized harvesting inside the capillary. During process 1900, the harvest valve and the EC outlet valve are opened in response to the operation of the IC inlet pump and the EC inlet pump, respectively. When the IC and EC inlet pumps are running, they will run at twice the commanded speed unless twice the commanded speed exceeds the tolerance. The pumps will then run at the maximum allowable flow rate for the five minutes the pumps are on. If the stop condition is volumetric (capacity) dependent, the remaining time displayed on the screen will only be updated when their respective pumps are running.

[0275] By creating the option to cycle between the harvest and waste outlets, users have the option to continuously collect the cell culture product of interest (cells, viral vectors, exosomes, conditioned medium, etc.) into a harvest bag at a user-selected slow flow rate over an extended period of time while still being able to feed and maintain the cell culture. This option is available for both standard bioreactor, i.e., standard cell growth chamber, and miniature bioreactor, i.e., miniature cell growth chamber, (disposable type) tasks.

[0276] Inner capillary (IC) harvest synchronization provides continuous medium over an extended period of time while feeding cells, allowing cells to be fed with conditioned medium, which may have advantages over fresh medium.

[0277] In an exemplary embodiment, the fluid is pumped through an EC circulatory supply loop 752, an IC circulatory supply loop 753, or a combination thereof, as described with reference to Figures 7A-7C.

[0278] Next, FIG. 21 illustrates exemplary operational steps of a cell growth process 2000 that may be used in a cell growth system such as the CES500 (e.g., FIG. 5A), CES600 (FIG. 6), or CES700 (FIGS. 7A-7C) according to an embodiment of the present disclosure. A start operation 2002 begins, and process 2000 proceeds to step 2004, where a disposable tubing set is loaded into the cell growth system. The disposable tubing set may include cell growth chamber 100A or cell growth chamber 100B (step 2004), as shown in FIGS. 1C, 4B, and 4C. Next, in step 2006, the system is primed. In one embodiment, for example, a user or operator instructs the system to prime by selecting a task for priming. In one embodiment, such a task for priming is, for example, a pre-programmed task. Next, process 2000 proceeds to IC / EC wash task 2008, where the fluids in the IC circulation loop and the EC circulation loop are exchanged. The exchange volume is determined by the number of IC and EC volumes exchanged.

[0279] Next, a medium conditioning step 2010 is performed to maintain the appropriate or desired gas concentration across the hollow fibers of the bioreactor membrane, allowing the medium to reach equilibrium with the gas supply before cells are added to the bioreactor. For example, a high EC circulation flow rate can be used to quickly achieve contact between the medium and the gas supply provided by the gas transfer module or oxygenator. The system is then maintained in the appropriate or desired state, for example, until a user or operator is ready to add cells to the bioreactor. In one embodiment, the system may be conditioned with, for example, complete medium. The complete medium may be any medium source used for cell growth. In one embodiment, the complete medium includes, for example, alpha-MEM (α-MEM) and fetal bovine serum (FBS). Any type of medium understood by one of skill in the art may be used.

[0280] Process 2000 then proceeds to step 2012, where cells are introduced into the center of the bioreactor, e.g., from a cell inlet bag, without circulation. In embodiments, a "Central Injection of Cells Without Circulation" task can be used, where a first volume of fluid containing a plurality of cells is introduced at a first flow rate into a cell growth system, where the cell growth system includes a cell growth chamber. A second volume of fluid containing medium is then introduced at a second flow rate into a portion of the first fluid circuit, e.g., disposing the first volume of fluid in a first portion of the cell growth chamber. In one embodiment, the first portion of the cell growth chamber or bioreactor is approximately a 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 other embodiments, the first volume is different from the second volume. In other embodiments, the first flow rate is different from the second flow rate. In one embodiment, the sum of the first volume and the second volume is equal to, for example, a percentage or a fraction of the volume (e.g., total volume) of the first fluid circuit. For example, the sum of the first volume and the second volume is, for example, about 50% of the volume (e.g., total volume) of the first fluid circuit. In one embodiment, the fluid in the first fluid circuit flows through an inner capillary (IC) space of a bioreactor or cell growth chamber. In one embodiment, the fluid in the second fluid circuit flows through, for example, an extra-capillary (EC) space of a cell growth chamber or bioreactor. In an exemplary embodiment, the fluid is fed through an EC circulation supply loop 752, an IC circulation supply loop 753, or a combination thereof (step 916), as described with reference to FIGS. 7A-7C. In one embodiment, the sum of the first volume and the second volume can be, for example, about 50% of the volume of the inner capillary (IC) loop, or some other percentage or fraction. In one embodiment, the sum of the first volume and the second volume, if applicable, may be about 50% or some other percentage or fraction of the volume of other fluid paths, other loops, etc. In embodiments, other percentages or fractions may be used, such as any percentage between about 1% and about 100%.

[0281] Following cell input step 2012, process 2000 next proceeds to cell feeding step 2014. The cells are grown / expanded in step 2016. While step 2016 is shown after step 2014, according to embodiments, step 2016 may occur before or simultaneously with step 2014. Process 2000 then proceeds to query step 2018 to determine whether cell colonies, microcolonies, or clusters have formed. A cell colony, microcolony, or cluster is a group of one or more adherent cells. If a cell colony, microcolony, or cluster has formed, process 2000 proceeds to "yes" and to step 2020, shearing the cell colony, microcolony, or cluster. For example, after growing a plurality of cells for a first period of time, the cells are circulated at a first circulation flow rate for a second period of time to reduce the number of cells in the cell colony, microcolony, or cluster. In embodiments, circulating the cells at a first circulation flow rate creates shear stress on the cell colony, causing one or more cells within the cell colony to split off from the cell colony. In one embodiment, for example, the number of cells within a cell colony, microcolony, or cluster can be reduced to provide a single cell suspension. In embodiments, step 2020 of circulating the cells to shear colonies, microcolonies, or clusters can be used, for example, every two days during cell culture to maintain uniform cell density and nutrient diffusion. Other time periods may also be used, according to embodiments. In one embodiment, such disaggregation of any microcolonies, colonies, or clusters may begin, for example, on day four or later. According to embodiments, other days or time periods for the initiation of such shearing may also be used. After shearing step 2020, process 2000 then returns to cell feeding step 2014.

[0282] If query step 2018 determines not to shear cell colonies or clusters, or, for example, if no cell colonies or clusters are present, process 2000 proceeds to a "no" and resuspends the cells in step 2022. In embodiments, cell circulation is performed to uniformly resuspend cells that may be loosely adherent during culture. In embodiments, step 2022 may include cell circulation to uniformly resuspend cells that may be loosely adherent before initiating a harvesting task or other task to remove the cells from the bioreactor. Following cell resuspension step 2022, process 2000 then proceeds to cell harvesting step 2024. Further processing or other analysis of the removed cells may optionally be performed in step 2026, and process 2000 ends at end operation 2028. If no further processing / analysis is desired, process 2000 ends at end operation 2030.

[0283] Turning to FIG. 22 and process 2100, according to an embodiment, an initiation operation is initiated (step 2102), and process 2100 proceeds to step 2104, where a disposable set is loaded into a cell growth system. The disposable set may include cell growth chamber 100A or cell growth chamber 100B, as shown in FIGS. 1C, 4B, and 4C. Then, in step 2106, the disposable set is primed, and an IC / EC wash step 2108 is performed. Next, in step 2110, the medium is adjusted. Next, process 2100 proceeds to step 2112, where cells (e.g., suspension or non-adherent cells, such as T cells or Tregs) are added. In one embodiment, such cells are added by the "Add cells to center without circulation" task (step 2112). In another embodiment, such cells are added by the "Add cells by uniform suspension" task (step 2112).

[0284] Process 2100 then proceeds to step 2114 to begin cell feeding (starting as day 0), depending on the embodiment. In an exemplary embodiment, fluid is fed through the EC circulatory supply loop 752, the IC circulatory supply loop 753, or a combination thereof (step 916), as described with reference to FIGS. 7A-7C. Cells are grown and expanded in step 2116, and by day 3, for example, it may be desirable to add a bolus of fluid to the IC loop to redistribute the cells in step 2118. In one embodiment, such a bolus of fluid may include reagents such as cytokines or other growth factors. In other embodiments, such a bolus of fluid may include, for example, reagents and basal medium.

[0285] Following such bolus addition and cell redistribution, process 2100 then proceeds to step 2120, where cells are re-fed. Such feeding may occur, for example, on day 3. During feeding step 2120, system parameters (e.g., one or more pump-controlled flow rates) may be controlled in step 2122 to achieve complementary co-flow and counter-flow settings for moving fluids from both the bioreactor's IC inlet and IC outlet ports into the bioreactor. For example, in step 2124, the IC inlet pump may be adjusted or directed to create a directional flow, and in step 2126, the IC circulation pump may be adjusted or directed to create a counter-flow. For example, an IC inlet pump flow rate of 0.1 mL / min may be matched or approximately matched with a complementary IC circulation pump flow rate of -0.1 mL / min to maintain cells in the bioreactor during the growth phase of the cell culture (e.g., in embodiments, days 4-7). Alternatively, in an exemplary embodiment, a first IC inlet pump provides a volumetric flow rate of less than 0.1 mL / min, or about 0.01 mL / min, to the IC inlet port (701A), and an IC circulation pump provides a complementary IC circulation volumetric or fluid flow rate of less than −0.1 mL / min, or about −0.01 mL / min, to the IC outlet port (701B). Here, the negative sign (“−”) used, for example, at −0.01 mL / min, indicates that the direction of the IC circulation pump creates a counterflow so that cells are maintained within the bioreactor during the cell culture growth phase. Such control in setup control step 2122 allows for countering any forces associated with cell loss from the bioreactor's IC outlet port.

[0286] Process 2100 then proceeds to query step 2128 to determine, for example, whether to continue adding reagents or other bolus additions on other days or other time intervals. If it is desired to add additional reagents or other boluses and redistribute the cells, process 2100 branches "yes" to add reagents and redistribute the cells in step 2118. For example, according to an embodiment, such bolus additions and redistribution of cells occur on days 6 and 9.

[0287] If it is not desired to continue adding boluses (e.g., reagents) and redistributing the cells, process 2100 proceeds to "no" and the cells are harvested in step 2130, where they are transferred to a harvest bag or container. Process 2100 ends at end operation 2136.

[0288] Alternatively, from harvesting step 2130, process 2100 may optionally proceed to further processing / analysis step 2132. Such further processing / analysis step 2132 may include phenotypic characterization of the harvested cells (e.g., T cells or Tregs). From optional further processing / analysis step 2132, process 2100 may optionally proceed to repopulating step 2134 with the remaining cells. Process 2100 then ends at end operation 2136.

[0289] Process 2200 illustrates operational steps for expanding cells in a cell expansion system according to embodiments of the present disclosure. In some embodiments, process 2200 can be used to expand T cells. As shown, cell expansion is accomplished by performing various steps over the course of a 14-day protocol. START operation 2202 is initiated, and process 2200 advances to day 0, where a disposable set is loaded into the cell expansion system (step 2206). The disposable set can include cell growth chamber 100A or cell growth chamber 100B, as shown in FIGS. 1C, 4B, and 4C. The disposable set is then primed (step 2208), where the set is filled with, for example, PBS (e.g., Lonza Ca 2+ / Mg 2+ The bioreactor is primed with PBS (free). In preparation for cell loading, the priming solution is replaced using an IC / EC wash (step 2210). For example, according to one embodiment, the PBS in the system may be replaced with TexMACS GMP basal medium. The medium is then conditioned (step 2212). The medium conditioning step 2212 is performed to allow the medium to reach equilibrium with the provided gas supply before cells are loaded into the bioreactor.

[0290] Next, on day 0, process 2200 proceeds to step 2214, which involves the introduction of cells (e.g., suspension or non-adherent cells, such as T cells or Tregs). In one embodiment, in step 2214, such cells are introduced by the "introduce cells to the center without circulation" task. In another embodiment, in step 2214, such cells are introduced by the "introduce cells in uniform suspension" task.

[0291] On day 3 (2216), a bolus of cytokines is added (step 2218) while the cells are being redistributed. In embodiments, cell redistribution is performed in combination with a bolus addition to mix the cells and more fully expose them to the cytokines (e.g., IL-2) during the bolus addition. In embodiments, redistribution can break up or separate any colonies or clusters of cells that have formed. In embodiments, redistribution is first performed by circulating the cells in a fluid circuit. A bolus addition is then performed in a process that pushes the cells back into the bioreactor (e.g., by introducing a fluid into the fluid circuit to push the cells back into the bioreactor). After redistribution and bolus addition step 2218, process 2000 proceeds to cell feeding step 2220.

[0292] On day 6 (2222), the cells are again redistributed (2224) with another bolus addition. Redistribution splits apart the colonies or clusters of cells that formed on days 3 through 5. Bolus addition exposes the cells to additional reagents that promote proliferation. Process 2000 proceeds to cell feeding step 2226 on day 6. On day 9 (2228), the cells are again redistributed (2230) with another bolus addition. Redistribution splits apart the colonies or clusters of cells that formed on days 6 through 8. Bolus addition exposes the cells to additional supplements that promote proliferation. Process 2000 proceeds to cell feeding step 2232 on day 9.

[0293] On days 11-13 (2234), the cells are again redistributed using bolus additions (step 2236). Redistribution breaks down the cell colonies or clusters that formed on days 9-10. Bolus addition exposes the cells to additional reagents that promote proliferation. Process 2200 then proceeds to cell feeding step 2238. In embodiments, steps 2236 and 2238 may be performed on days 11, 12, and 13, respectively. This may occur as a result of expanding the cells on days 0-10 and having more cells in the bioreactor. Redistribution and bolus addition of cells promotes cell proliferation by more frequently breaking down the cell colonies and clusters and mixing them with bolus-added reagents to promote cell proliferation. Process 2200 ends at END operation 2240.

[0294] Process 2300 shows operational steps for expanding cells (e.g., suspension or non-adherent cells) in a cell expansion system according to embodiments of the present disclosure. In some embodiments, process 2300 can be used to expand T cells, such as Tregs. The combination of steps in process 2300 allows for expansion of cells to useful clinical quantities at low initial seeding densities.

[0295] START operation 2302 begins, and process 2300 proceeds to step 2304, where a disposable set is loaded into the cell growth system. The disposable set may include cell growth chamber 100A or cell growth chamber 100B, as shown in Figures 1C, 4B, and 4C. The disposable set is then primed (step 2306), where the set is primed with, for example, PBS (e.g., Lonza Ca 2+ / Mg 2+The bioreactor is primed with PBS (free). In preparation for cell loading, the priming solution is replaced using an IC / EC wash (step 2308). For example, according to one embodiment, the PBS in the system may be replaced with TexMACS GMP basal medium. The medium is then conditioned (step 2310). The medium conditioning step 2310 is performed to allow the medium to reach equilibrium with the provided gas supply before cells are loaded into the bioreactor.

[0296] Process 2300 proceeds to step 2312, where an inlet volume of fluid containing cells is input. In embodiments, the cells include one or more types of non-adherent cells, such as T cells (e.g., Tregs). In one embodiment, the cells include Tregs. In embodiments, the inlet volume of fluid containing cells is input into the IC circuit via the IC inlet line using an IC inlet pump. In embodiments, at step 2312, the input inlet volume is input without operating the IC circuit pump.

[0297] Process 2300 proceeds to step 2314 of disposing the inlet volume in the first portion of the bioreactor. In embodiments, the disposing step can be performed by introducing a second volume of fluid containing medium into a portion of the IC circuit to force the inlet volume of fluid with the cells into the first portion of the bioreactor. In embodiments, the fluid inlet volume and the second volume of fluid can be the same. In other embodiments, the fluid inlet volume and the second volume of fluid can be different. In still other embodiments, the sum of the fluid inlet volume and the second volume of fluid can be equal to a percentage of the volume of the IC circuit.

[0298] Following inlet volume placement step 2314, process 2300 proceeds to step 2316, where the cells are exposed to an activator to activate the cells for proliferation. In some embodiments, the cells are exposed to a soluble activator in step 2316. In some embodiments, the activator, including an antibody complex, is added to the media and included in the inlet volume, or is added later, such as as a second volume. In embodiments, the activator is a human antibody CD3 / CD28 / CD2 cell activator complex.

[0299] Process 2300 proceeds to step 2318, where cells are fed according to a first process during a first time period. In an exemplary embodiment, cells are fed through an EC circulation feed loop 752, an IC circulation feed loop 753, or a combination thereof (step 916), as described with reference to Figures 7A-7C. In one embodiment, for example, in step 2320, if a cell population is beginning to grow / proliferate during the first time period and a minimum or low feeding rate can meet the feeding requirements of such cell population, cells are fed at a minimum or low feeding rate. For example, an IC inlet pump flow rate of +0.1 mL / min may be used during such a first time period. If it is desired to reduce cell loss from the hollow fiber membrane bioreactor during such a first time period, a complementary IC circulation pump flow rate of -0.1 mL / min may be matched or approximately matched to the +0.1 mL / min IC inlet pump flow rate to maintain cells in the bioreactor during the growth phase of the cell culture. Alternatively, in an exemplary embodiment, a first IC inlet pump provides a volumetric flow rate of less than 0.1 mL / min, or about 0.01 mL / min, to the IC inlet port (701A), and an IC circulation pump provides a complementary IC circulation volumetric or flow rate fluid flow rate of less than −0.1 mL / min, or about −0.01 mL / min, to the IC outlet port (701B), where the negative sign (“−”) used, for example, about −0.01 mL / min, indicates that the direction of the IC circulation pump creates a counterflow so that cells are maintained in the bioreactor during the growth phase of the cell culture.

[0300] From step 2320, process 2300 proceeds to step 2322 of expanding the cells for a second period of time. The second period of expansion step 2322 may include feeding the cells according to a second process for the second period of time (2324). In one embodiment, such a second process includes, for example, feeding the cells at approximately the same feeding rate as during the first period of time. In other embodiments, the second process includes feeding the cells at a different feeding rate compared to the feeding rate used during the first period of time. For example, the feeding rate is increased following cell expansion over the first period of time.

[0301] While growing the cells for a second time period (step 2822), the cells may be circulated to disaggregate the cell colonies or cell clusters (step 2326). Step 2326 may include circulating the cells in an IC circuit to disaggregate the colonies or clusters formed during the first time period. The colony / cluster shearing step 2326 reduces the number of cells within the cell colonies or cell clusters. In embodiments, the circulating shearing step 2326 creates shear stress on the cell colonies, causing one or more cells within the cell colonies to split and separate from the cell colonies.

[0302] Process 2300 then proceeds to a harvesting step 2328, where the cells are transferred to a harvest bag or container. In embodiments, a therapeutic amount of cells can be harvested. In embodiments, the cells harvested in step 2328 are greater than 1×10 9 The harvested cells, in embodiments, have a viability of between about 75% and about 95%.

[0303] Process 2300 then optionally proceeds to step 2330, where further processing / analysis occurs. Such further processing may include, for example, phenotypic characterization of the harvested cells (e.g., T cells or Tregs). In one embodiment, the harvested cells express biomarkers consistent with Tregs. For example, the cells express CD4 + , CD25 + , and / or FoxP3 + In one embodiment, the harvested cells express a biomarker that is greater than about 80% CD4 + CD25 + In other embodiments, the cells have a frequency of greater than about 55% CD4 + FoxP3 + The process 2300 then ends at END operation 2332.

[0304] In accordance with embodiments of the present disclosure, the operational steps depicted in the above figures are for illustrative purposes only and may be rearranged, combined with, or performed in parallel with other steps. In embodiments, fewer steps may be performed or additional steps may be added without departing from the spirit and scope of the present disclosure. Additionally, steps (and substeps), such as priming, adjusting media, and adding cells, may, in some embodiments, be performed automatically, e.g., by a processor executing preprogrammed tasks stored in memory. Such steps are presented here for illustrative purposes only. Furthermore, the exemplary pump flow rate settings for feeding cells, depicted in FIG. 11B, for example, are provided for illustrative purposes. Other pump flow rates, flow rates, directions, etc., may be used in accordance with embodiments of the present disclosure.

[0305] Examples and detailed descriptions of tasks and protocols, including custom and pre-programmed tasks, for use with cell expansion systems are provided in U.S. patent application Ser. No. 13 / 269,323, filed Oct. 7, 2011, entitled "Configurable Method and System for Cell Growth and Cell Harvesting in a Hollow Fiber Bioreactor System," and U.S. patent application Ser. No. 13 / 269,351, filed Oct. 7, 2011, entitled "Customizable Method and System for Cell Growth and Cell Harvesting in a Hollow Fiber Bioreactor System," both of which are expressly incorporated herein by reference in their entireties.

[0306] 25 illustrates an example of components of a computing system 2400 for implementing embodiments of the present invention. The computing system 2400 may be used in embodiments where the cell growth system uses a processor to perform tasks, such as custom tasks or pre-programmed tasks, for example, as part of processes such as those shown and / or described herein. In embodiments, the pre-programmed tasks may include, for example, IC / EC washing and / or cell feeding.

[0307] The computing system 2400 includes a user interface 2402, a processing system 2404, and / or a storage device 2406. The user interface 2402 includes an output device 2408 and / or an input device 2410, as will be appreciated by those skilled in the art. The output device 2408 may include one or more touchscreens. The touchscreen may have a display area for providing one or more application windows. The touchscreen may also be an input device 2410 capable of receiving and / or capturing physical touch from a user or operator, for example. The touchscreen may be a liquid crystal display (LCD) with a capacitive structure that allows the processing system 2404 to estimate the location of the touch, as will be appreciated by those skilled in the art. In this case, the processing system 2404 can map the location of the touch to a UI element displayed at a predetermined location in the application window. The touchscreen may also receive touches through one or more other electronic structures, as is understood by those skilled in the art. Other output devices 2408 include a printer, a speaker, etc. Other input devices 2410 may include a keyboard, other touch input device, a mouse, a voice input device, etc., as would be understood by one skilled in the art. For example, user interface 2402 may be user interface 264 described with reference to Figure 2A. User interface 2402 may include different screens for different portions of a task / protocol / process / method, different user inputs or requests, etc.

[0308] In embodiments of the invention, processing system 2404 may include a processing unit 2412 and / or memory 2414. Processing unit 2412 may be a general-purpose processor operable to execute instructions stored in memory 2414. Processing unit 2412 may include a single processor or multiple processors in embodiments of the invention. Further, in embodiments, each processor may be a multi-core processor having one or more cores for independently loading and executing instructions. Processors may include general-purpose processors, application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), and other integrated circuits, as will be appreciated by those skilled in the art.

[0309] In embodiments of the present invention, memory 2414 may include any storage device for short-term or long-term storage of data and / or processor-executable instructions. Memory 2414 may include, for example, random access memory (RAM), read-only memory (ROM), or electrically erasable programmable read-only memory (EEPROM), as would be understood by one skilled in the art. Other storage media may include, for example, CD-ROM, tape, digital versatile disk (DVD), or other optical storage devices, tape, magnetic disk storage devices, magnetic tape, or other magnetic storage devices, as would be understood by one skilled in the art.

[0310] Storage device 2406 is any long-term data storage device or component. In embodiments of the invention, storage device 2406 may include one or more of the systems described in connection with memory 2414. Storage device 2406 may be permanent or removable. In one embodiment, storage device 2406 stores data generated by or provided to processing system 2404.

[0311] The computing system 2400 may communicate with the cloud, network computers, personal computing devices, mobile devices, etc. through a wireless network, a Bluetooth network, or other network system. Alternatively, the computing system 2400 may communicate with personal computing devices, network computing devices, mobile devices, etc. via a hardwired connection.

[0312] The processing system 2404 can control the activation, speed, and fluid flow of the pumps. Using a pump process control loop, the processing system 2404 can control the pump operation / speed to provide ultra-low delivery rates in continuous operation (instead of step operation). Continuous, consistent operation (compared to periodic / step operation) facilitates finer pump control and lower delivery rates. To facilitate continuous cell harvesting, operation can be switched between two pumps operating at low flow rates. In an exemplary embodiment, a first IC inlet pump provides fluid to the IC inlet port (701A) at a volumetric flow rate of less than 0.1 mL / min or about 0.01 mL / min, and an IC circulation pump provides fluid to the IC outlet port (701B) at a complementary IC circulation volumetric or fluid flow rate of less than -0.1 mL / min or about -0.01 mL / min. Here, the negative sign ("-") used, for example, -0.01 mL / min, indicates that the direction of the IC circulation pump creates a counter flow rate so that cells are maintained within the bioreactor during the growth phase of the cell culture.

[0313] The processing system 2404 can control the operation / speed of the pumps for counter-flow containment. Counter-flow containment at ultra-low flow rates allows for the concentration of cells in a desired area. Counter-flow containment conserves cell culture media and allows for lower protein concentrations in the system. Allowing for continuous operation allows for lower protein concentrations. Counter-acting pumps pump in opposite directions to each other to cluster cells.

[0314] The processing system 2404 may be configured to run various tasks / methods / processes / protocols that are input by a user or stored as preset tasks / methods / processes / protocols in memory 2414. For example, the processing system 2404 may be configured to execute or coordinate the execution of a cell processing application (CPA). The cell processing application allows for tracking and logging of user ingredients, procedures, user logins, etc. Using this system, protocols can be pushed to a fleet of devices that are all identical, thus saving a lot of time setting up valid protocols to run on the fleet. All reports can then be pulled back to the application, and real-time readouts of the fleet can be viewed remotely on the application. Temperatures and pressures can be updated to the application at configurable rates, while alarms and warnings can also be sent to the application. For example, the application may push alarms to one or more users as remote alarms for recording or correction. For example, a remote alarm can be an email, text message, or other digital alert sent to the user. CPA also connects FINIA® with Quantum Flex® to keep all of a user's reports in a unified application.

[0315] CPA has been tested on the following protocols and reports: User Access Control Protocol: D0000028598, User Access Control Report: D0000043953, Remote Alarm Protocol: D0000028623, Remote Alarm Report: D0000043950, Barcode Protocol: D0000028807, Barcode Report: D0000044530, Protocol Task Management Protocol: D000002 8798, Protocol Task Management Report: D0000044037, Device Configuration Protocol: D0000028621, Device Configuration Report: D0000045376, EOR Protocol: D0000028620, EOR Report: D0000045378, D0000047097: Network Performance, D0000047098: Multiple Devices, D0000047099: Access and Audit, D0000047100: Data and Reports.

[0316] The cell processing application includes tracking and data management functionality. Tracking and logging is enabled through the CPA. Tracking and logging can be performed periodically (e.g., scheduled) or on-demand. Users can schedule recording times based on trigger events or periodic times.

[0317] The cell processing application can also perform device cluster control. The CPA integrates modular cell therapy components, including, for example, a CES (e.g., the Quantum Flex® Cell Expansion System) and a FINIA®. Thus, a CES (e.g., CES100, CES700, etc.) described herein is one module in a modular system controlled by the cell processing application. For example, a CPA can control a device cluster of 100 or more devices. Using a CPA, the same command can be sent to multiple machines rather than to each individual machine, streamlining operations and ensuring consistency.

[0318] Cell processing applications include custom tasks / methods / processes / protocols. Users can create custom protocols or tasks. A task can be detailed steps for a user-defined process. A protocol can be a collection of tasks required to complete a test or cell growth process, etc., saved in a single file. Memory 2414 can store predefined, prewritten, or stock protocols and tasks. Stock tasks are compiled into custom protocols by the user. Protocols in the CPA system can be modified in real time and re-uploaded to the instrument cluster. Tasks and protocols can be written, modified, and selected both on the user interface or on an external computer or handheld device.

[0319] A cell processing application may include different user profiles, accounts, and access. Each user is assigned either a predefined or custom role. Each role contains a set of permissions that can be customized by an administrator. Permissions may include a control level of access (read-only, write, etc.). A single user may have multiple roles. The CPA may require user authentication for security purposes.

[0320] The cell processing application may trigger and send remote notifications and alarms. Emails or other notifications (digital notifications, text, etc.) may be sent to the user, allowing the user to monitor or inspect the status without direct access or contact with the device. The user can remotely communicate with the device, including reviewing data in real time through the cell processing application. The user can remotely send commands to the device, such as to ignore alarms, stop tests, or modify protocols or tests, which is more efficient for the user.

[0321] The cell processing application controls network access for software updates. New software may be pushed to the CES or other devices from the cloud, a remote computer, or a remote device.

[0322] The cell processing application may receive data from a barcode scanner. The barcode scanner may be one of the input devices 2410 described above. Disposable packages, media, cells, etc. each include a barcode with product information. The CPA can check the data from the barcode scanner to ensure the correct component or media is being used. The barcode may be stored in memory 2414 or in the CPA for future reference, such as for product recalls or growth data. The CPA can adjust task / method / process / protocol configurations based on the data from the barcode scanner.

[0323] The computing system 2400 increases process efficiency by promoting contact between a "reagent of interest" (such as viral particles, transfection reagents, secondary cell types, differentiation reagents, induction reagents, etc.) and adherent or suspension cells within a hollow fiber bioreactor (HFB). Cells (adherent or suspension) are seeded into the capillary interior (IC) of the HFB. The reagent of interest is introduced into the IC side of the HFB. Counterflow (inlet = positive flow, circulation = negative flow) (split by driving the IC inlet pump and IC circulation pump in opposite directions) is used to promote active contact between the reagent of interest and the cell population. This process can continue for as long as necessary.

[0324] Many processes in cell culture require the exposure of a population of cells to specific suspended elements to modify the cells or produce a secondary product. For example, to produce a viral vector product, a population of cells must be exposed to active viral particles that enter and replicate within the cells; to transfect cells by introducing a novel gene into the cells, the cells must be exposed to both the gene of interest (GOI) and the reagents required to incorporate the GOI into the cells for translation. Both of these example processes vary in efficiency depending on the environment in which they are performed. In many passive models for these types of processes, the promotion of interactions between the reagent of interest (ROI) and the cell population is highly dependent on chance. These passive models reduce the efficiency of viral integration, plasmid transfection, and GOI expression.

[0325] The process described herein aims to increase the efficiency of these processes by promoting active contact between the cell population and the ROI. Cells (adherent or suspension) are seeded into the hollow fibers of the HFB. Once the cells are established, a "target reagent" (viral particles, transfection reagent, secondary cell type, differentiation reagent, induction reagent, etc.) is introduced into the HFB. Media fluid is drawn from the two-port bag. Flow is divided by an inlet pump and a circulation pump. Media fluid enters the IC side of the HFB from both sides. The IC waste valve is closed, so the fluid must exit the HFB through the pores in the HFB membrane and proceed from the IC side to the EC side. Media fluid is recirculated from the EC side back to the two-port bag. Because the "target reagent" molecules are larger than the pores in the HFB membrane, they are trapped within the IC loop and pressed against the cell layer on the membrane wall, promoting contact between the "target reagent" and the cell population. This process continues as long as necessary.

[0326] Example The following description includes several example protocols / methods / processes that can be used in a cell expansion system, such as CES500 (e.g., FIGS. 5A, 5B, 5C) and / or CES600 (FIG. 6), implementing aspects of the embodiments. While specific features are described in the examples, such examples are provided solely for purposes of illustration and description. For example, while the examples involve expansion of T cells and / or Treg cells, other and / or additional cell types and / or combinations thereof may be used in other embodiments. While specific parameters, functions, and / or values ​​are described (e.g., use of a CES such as the Quantum® Cell Expansion System), these parameters, functions, and / or values, etc., are provided solely for illustrative purposes. The present disclosure is not limited to the examples and / or specific details provided herein.

[0327] Furthermore, examples provided herein are not intended to be limiting of other embodiments that may include different or additional steps, different or additional parameters, or other features. The illustrated methods or protocols that include steps (and sub-steps) are, in some embodiments, performed automatically, e.g., by a processor executing pre-programmed tasks stored in memory. In other embodiments, the steps (and sub-steps) are performed by a combination of automated and manual operations. In still other embodiments, the steps (and sub-steps) are performed by an operator or user, or by other manual means.

[0328] While example data is provided in such examples, such illustrated data is provided for illustrative purposes and is not intended to limit other embodiments involving different steps, parameters, values, materials, or other features.

[0329] In some examples, a protocol package or method for a miniature bioreactor, i.e., a miniature cell growth chamber, may be the same task as one for a standard bioreactor, i.e., a standard cell growth chamber. Some protocol packages or methods for miniature bioreactors, i.e., a miniature cell growth chamber, have the following differences: 1. The flow rate is reduced when draining the air removal chamber (ARC) and forcing flow across the membrane during the ARC management step. This is because miniature bioreactors have a smaller hollow fiber surface area, so the flow rate is reduced to avoid high pressure. 2. The default selections are different for different disposable sets (i.e., where applicable, flow rate and volumetric stop conditions are scaled down depending on the volume differences in various parts of the set). 3. The range is open, allowing the user the option to scale down the flow down to 1 / 10 of the standard bioreactor task.

[0330] In some exemplary embodiments, the protocol package or method includes: 1. Default (template) adherent cell expansion protocol; 2. Default (template) suspension cell expansion protocol; 3. Default (template) custom 1-step protocol; 4. Custom Step 1-10 tasks with increased options available compared to Legacy Quantum custom tasks; 5. Cell input with multiple distribution cycles (task created by the scientific team to optimize MSC seeding); 6. Cell input and positioning (task created by the scientific team to optimize T cell input); 7. Non-adherent cell feeding (task created by the scientific team to optimize T cell feeding); and / or 8. Cell circulation and positioning (task created by the scientific team to optimize sampling during T cell expansion).

[0331] Example 1

[0332] method

[0333] General Treg cell culture

[0334] Immunomagnetically isolated CD4 + CD25 + Tregs were obtained from peripheral blood of healthy adult donors by leukapheresis (HemaCare Corporation, Van Nuys, CA) and then cultured at 1.0 × 10 cells / ml in three T25 flasks (7 mL / flask) in sterile-filtered TexMACS™ GMP medium supplemented with 200 IU / mL recombinant human IL-2 IS Premium Grade cytokine (Miltenyi Biotec GmbH, Bergisch Gladbach) and Gibco PSN 100X antibiotic mixture (ThermoFisher Scientific, Waltham, MA). 5 Tregs are expanded in a Terumo BCT at a concentration of 100 cells / mL. The actively growing Treg cell suspension is then used as the inoculum in each of three experimental runs using the Quantum Cell Expansion System. Tregs for both the inoculum and Quantum system expansion are costimulated using 25 μL / mL of soluble tetrameric Immunocult™ human antibody CD3 / CD28 / CD2 cell-activating complex (Stem Cell Technologies, Vancouver, BC) without microbeads. Costimulation occurs on days 0 and 9 for Treg inoculation and on day 0 for Quantum system Treg expansion. The Quantum system HFM bioreactor has a capillary inner loop volume of 177.1 mL and a 21,000 cm 2 It is characterized by a surface area of

[0335] Quantum System Treg Expansion

[0336] According to an embodiment, two sterile-filtered media bags (2 L) are prepared for scale-up Treg expansion in the Quantum system using a 4 L Quantum media bag set (Cat. 21021). One 2 L bag containing complete media with TexMACS GMP, IL-2, and PSN antibiotics is used to feed the IC compartment, and the other 2 L bag containing basal media with TexMACS GMP and PSN antibiotics is used to feed the EC inlet compartment of the bioreactor. After priming the Quantum system with PBS (Lonza Cat. 17-516Q, Walkersville, MD), the media bags are connected to the appropriate IC and EC inlet lines using a TSCD-Q Thermo Sterile Welder. The complete media is protected from light exposure.

[0337] To introduce into the Quantum system bioreactor, use aseptic technique to add the total input cells for each run (4.5-6.5 x 10 cells) to 50 mL of complete medium in a Quantum cell inlet bag (Cat. 21020). 7 Additional disposable bags, such as Quantum CES Medium Bags 4L (Cat. 21021) and Waste Bags 4L (Cat. 21023), are also used during large-scale Treg expansion runs.

[0338] Upon completion of the "Put cells in the center without circulation" task, 2.5–3.7 × 10 cells were detected in 177 mL of complete medium for Quantum system runs (n=3). 5 at a concentration of cells / mL, or an average of 2.1–3.1 × 10 in the lumen or capillary inner (IC) compartment of a hollow fiber membrane bioreactor. 3 number of cells / cm 2 Tregs are seeded at a concentration of 1000-10 ...

[0339] Day 0 to Day 4

[0340] Quantum Custom Task Example

[0341] Modified cell feeding

[0342] Example of IC / EC exchange and medium adjustment for regulatory T cells

[0343] A TexMACS GMP complete medium bag containing IL-2 supplement (200 IU / mL) is attached to the IC medium line of the Quantum system using a Terumo BCT TSCD-Q sterile welder. A TexMACS basal medium bag is attached to the EC medium line. For each, perform the IC / EC wash and medium adjustment tasks. To conserve the amount of IL-2 and activator complexes, complete medium is used for IC exchanges or washes, and basal medium is used for EC exchanges or washes.

[0344] Before adding cells, set up the modified "cell feeding" system. Increase the IC inlet flow rate (Q1) and IC circulation flow rate (Q2) to 0.2 mL / min, 0.3 mL / min, and 0.4 mL / min on days 5, 6, and 7, respectively, in the opposite direction on days 4, 5, and 6, but with matching flow rates if necessary, while maintaining lactate levels between 5 and 8 mmol / L.

[0345] [Table 1]

[0346] Quantum Custom Task Example

[0347] Example of a task where cells are injected into the center without circulation

[0348] Purpose: This task distributes the suspended cells centrally within the bioreactor membrane while allowing flow in the outer capillary (EC) circulation loop. The pump flow rate to the IC loop is set to zero.

[0349] Using cell input without cycling, task modifications are entered before cells are input into the Quantum system.

[0350] [Table 2]

[0351] [Table 3]

[0352] Return to the default cell feeding task if necessary and continue the growth protocol using the cell feeding task.

[0353] [Table 4]

[0354] Day 4 and onwards

[0355] Example of resuspension of Treg cells during cell culture or before harvest

[0356] The purpose of this modified circulation task is to uniformly resuspend cells that may be loosely attached during culture or before the start of the harvest task.

[0357] Additionally, this task is used to shear Treg cell colonies every two days during cell culture to maintain uniform cell density and nutrient diffusion after day 4. When this task is used to shear colonies during the culture process, the Quantum system is returned to a modified "Cell Feeding" task.

[0358] [Table 5]

[0359] Quantum Harvesting Task Example with Modifications

[0360] [Table 6]

[0361] Harvested cells are removed from the Quantum system by RF welding for further evaluation and analysis.

[0362] Post-harvest analysis

[0363] Harvested cells are enumerated over a 5–50 μm range using a Vi-CELL XR 2.04 Cell Viability Analyzer (Beckman Coulter), and membrane integrity is quantified by trypan blue dye exclusion.

[0364] metabolic effect

[0365] Regulatory T cell metabolism is monitored daily from the Quantum EC sample port by an i-STAT handheld analyzer (Abbott Point of Care, Princeton, NJ) using G cartridges (i-STAT G cartridge (Cat. 03P83-25) for glucose and i-STAT CG4+ cartridge (Cat. 03P85-50) for lactate, respectively).

[0366] Cell Surface Biomarker Expression

[0367] Human regulatory T cells (natural and induced) constitute a small subset (2-10%) of all T cells in human umbilical cord blood and peripheral blood. Functionally, Tregs are involved in maintaining immune homeostasis, including regulating immune tolerance in both innate and adaptive immune responses. Furthermore, expression of the transcription factor forkhead box P3 (FoxP3) gene product is upregulated by CD4 + CD25 + FoxP3 + CD127 lo / - Treg phenotype and antigen-presenting cells (APCs) and effector T cells (T eff) is known to correlate with immune suppression. Binding of IL-2 to the CD25 / IL-2 receptor (Rα) and activation of the STAT5 transcription factor is used to induce Foxp3. FoxP3 suppression upregulates the activity of several genes, including CTLA-4, TNFRSF18, and IL2RA, and downregulates IL-2 through its association with the histone acetylase KAT5 and histone deacetylase HDAC7.

[0368] The Treg phenotype frequency of surface biomarkers on harvested cells will be quantified by flow cytometry. For this purpose, cells were stained with the following antibody conjugates and gated on unstained viable cells: FixableViability Dye eFluor® 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). Specimen data were acquired on a bead-compensated BD Canto II flow cytometer equipped with FACSDiva v6.1.3 software, using 1x106 cells and 20,000 events per sample.

[0369] An exemplary experimental flow is shown in FIG.

[0370] result

[0371] Preliminary studies of Tregs in static culture have shown that these cells tend to form microcolonies approximately 100 μm in diameter. Splitting these cells every two days during the medium change process helps limit cell necrosis, and a 1,000 μL pipette tip with an ID of 762 μm can be used to return the cells to a dense single-cell suspension. Alternatively, maintaining a single-cell suspension can be more efficiently achieved in an automated HFM bioreactor with a hollow fiber ID of approximately 200 μm, such as the Quantum system, through a preprogrammed daily circulation task. Furthermore, this automated feeding task can be performed in a functionally closed system, reducing the potential for contamination while maintaining a continuous flow of nutrients to the Treg culture.

[0372] Treg cell density and viability

[0373] [Table 7]

[0374] [Table 8]

[0375] Preliminary experiment on cell seeding density

[0376] In preparation for the expansion of immunomagnetically selected cells from donors in an automated bioreactor, a series of static growth experiments were performed to determine whether stimulated Tregs were 1.0 × 10 6 This part of the study was performed to determine whether cells could be cultured at a seeding density of less than 1.0 x 10 cells / mL in 18 wells of a 24-well tissue culture plate. 5 Cells were seeded at 1.0 x 10 cells / mL or 1.0 x 10 cells / mL in wells of TexMACS GMP medium supplemented with IL-2 (200 IU / mL) and PSN antibiotics. 5This is performed by seeding 100 cells / mL of soluble anti-CD3 / CD28 / CD2 mAb complex at 25 μL / mL on days 0 and 9. Cells are manually harvested and counted on day 14 using a Vi-CELL XR.

[0377] [Table 9]

[0378] After harvesting, cell samples were pooled for Treg biomarker analysis by flow cytometry. + C25 + , CD4 + CD127 - , and CD4 + FoxP3 + The phenotypic frequencies were 90.9%, 79.7%, and 31.6%, respectively, in static culture. The detection of FoxP3+ is highly dependent on permeabilization and cell viability, so CD4 + CD25 + Phenotype (>70%) is generally the most reliable determinant in identifying Treg biomarkers.

[0379] This static plate test data shows that when cultured in the presence of soluble costimulatory anti-CD3 / CD28 / CD2 mAb complexes and serum-free medium, 10 5 This suggests that human Tregs may be able to proliferate at a cell seeding density of approximately several cells / mL.

[0380] Treg metabolism

[0381] Regulatory T cells rely on mitochondrial metabolism and have the ability to oxidize multiple carbon sources, i.e., lipids or glucose. As a result of mTOR regulation of glycolysis and fatty acid metabolism, Tregs are known to shift their metabolism from fatty acid oxidation (FAO) to glycolysis upon entering a highly proliferative state. Furthermore, glycolysis has been shown to be necessary for the generation and suppressive function of human inducible Tregs by regulating FoxP3 variant expression through studies of IL-2 / STAT5 / enolase-1 promoter signaling and 2-deoxy-D-glucose inhibition. Therefore, monitoring glucose and lactate levels facilitates the adjustment of medium flow rates in the Quantum system to support Treg proliferation in hollow fiber bioreactors. Initially, Tregs are thought to transiently reduce their metabolic rate before entering the cell cycle and expanding. This is supported by the transient decrease in glycolysis and mTOR activity in freshly isolated human Tregs prior to TCR stimulation. Specifically, mTORC1 is thought to increase the expression of glucose transporters, such as Glut-1-mediated glucose transport, as a result of upregulation of the mTOR pathway.

[0382] Three expansion results from three separate Treg cell aliquots indicate that glucose consumption and lactate production appear to correlate within each Quantum System run. All three in vitro expansion runs demonstrate that Treg glucose consumption exceeds background levels by day 1, and two of the three runs demonstrate that lactate production levels increase above background levels by day 2. In one run with reduced cell viability upon thawing, the lactate production rate may be delayed, which may be reflected in cell yield. In the most actively growing Treg cultures, the maximum glucose consumption rates for two of the three runs are 1.618 mmol / day and 2.342 mmol / day on days 8 and 7, respectively. Maximum lactate production rates are 2.406 mmol / day and 3.156 mmol / day at the same time points.

[0383] Throughout the Treg expansion run, lactate levels were controlled below 7 mmol / L within the hollow fiber lumen on days 4–8 by simultaneously increasing both the IC input (+) and IC circulation (-) pump flow rates (±0.1 to ±0.4 mL / min). Minimum glucose levels during the Treg cell expansion ranged from 264 mg / dL on day 7 (Q1584) to 279 mg / dL on day 8 (Q1558). In the cell growth medium for these viable expansions, the basal glucose concentration was 325–335 mg / dL, which, when used in conjunction with flow modulation in the Quantum system, was found to support expansion.

[0384] Regulatory T cell biomarker expression

[0385] Assessment of Treg cell harvest by flow cytometry was performed in this feasibility study using CD4 + CD25 + FoxP3 +Emphasis is placed on T cell subsets. In T lymphocytes, the human CD4 gene on chromosome 12 encodes a membrane glycoprotein that interacts with major histocompatibility complex class II and functions to initiate the early stages of T cell activation. In regulatory T cells, the human CD25 (IL2R) gene on chromosome 10 encodes the IL-2 receptor and functions by sequestering the cytokine IL-2. In regulatory T cells, the human forkhead / winged helix box P3 gene on chromosome X encodes the FoxP3 transcription factor, which is essential for Treg suppressor function. The FoxP3 gene product binds to the promoter regions of the CD25, CTLA-4, IL-2, IL7R, and IFN-γ genes, upregulating CD25 and CTLA-4 and suppressing the transcription of the IL-2, IL7R, and IFN-γ genes. The CD127 gene encodes the IL-7 receptor, and Tregs are generally characterized by low CD127 (IL-7R) expression compared to conventional T cells. However, certain Treg subsets are known to express high CD127 levels during in vitro and in vivo activation, which correlates with increased Treg survival when cells are incubated with IL-7. The CD45RO gene product is expressed on naive thymus-derived Tregs, which lose CD45RA upon activation and express CD45RO.

[0386] [Table 10]

[0387] CD4 + CD25 + The mean expression of Treg phenotype frequency was 85.5% in cells harvested from the Quantum system, exceeding the published CD4 + CD25 + This is advantageous compared to the >70% release criterion. Q1567 Treg proliferation is associated with CD4 + CD127 lowThe increased frequency of the CD4 population (74.2%) is a reflection of the low cell viability in this particular thawed cell sample, since, according to one embodiment, these cells are cultured with only IL-2 as a cytokine supplement. Cells expanded in two Quantum system runs with seeding and harvest viability exceeding 80% were CD4 + FoxP3 + The expression frequency of FoxP3 is 61.6%. + In addition, the 2 billion cell expansion results are consistent with the published release specifications of over 60% of the CD3 Treg cells in the original donor Treg cell specimens available from HemaCareBioResearch Products. + CD45 + (87.30%), CD25 + (47.76%), and FoxP3+ (59.64%) biomarker expression.

[0388] Additional flow cytometry analysis is performed on cryopreserved Treg cells from Q1584 expansions run by a third-party laboratory, for example, using fluorescence minus one (FMO) gating, different stains, and different equipment. FMO controls are a type of gating control used to interpret cell populations by accounting for the spread of fluorochromes used to quantify the frequency of a specific marker relative to all fluorochromes in the data plot. For example, flow results from a third-party laboratory show only CD4 + CD25 + The Treg cell population frequency from the Q1584 run was 95.4%, which compares favorably to the 90.5% detected in the Terumo BCT CES laboratory. While incomplete staining with the alternative anti-FoxP3-PE clone stain may limit quantification of this internal biomarker in a third-party laboratory, the dot plots demonstrate high FoxP3 expression in the Q1584 specimen, which is not observed in the control Treg cell reference sample. + These findings suggest that there is a subpopulation of Tregs. Although these observations are intriguing, additional studies w...

Claims

1. 1. A method for expanding cells, the method comprising: introducing cells into a cell expansion system comprising a bioreactor containing an inner capillary loop and an outer capillary loop and an air removal chamber; removing the cells from the air removal chamber; filling the inner capillary loop with a medium containing a protein; placing the cells in the bioreactor for growth for a first period of time; and The flow rate in the bioreactor is less than 0.1 mL / min. method.

2. 10. The method of claim 1, wherein the protein comprises a cell signaling molecule. method.

3. 3. The method of claim 2, wherein the cell signaling molecule comprises a cytokine. method.

4. 4. The method of claim 3, wherein the cytokine comprises recombinant human IL-2 cytokine. method.

5. 10. The method of claim 1, wherein the step of placing the cells in the bioreactor comprises: placing the cells at a first location on a first side of the bioreactor; method.

6. 6. The method of claim 5, wherein the first side of the bioreactor comprises an outlet side of the bioreactor. method.

7. 10. The method of claim 6, wherein the step of placing the cells in the bioreactor further comprises: placing the cells in a second location toward a central location of the bioreactor. method.

8. 8. The method of claim 7, wherein the cells migrate toward the second location due to a pressure differential within the bioreactor. method.

9. 9. The method of claim 8, wherein the pressure differential is created during operation of the air removal chamber. method.

10. 10. The method of claim 1, further comprising: after the first period of time, recirculating the cells for a second period of time; disposing the cells for a third period of time; feeding the cells; having method.

11. 10. The method of claim 1, wherein the flow rate in the bioreactor is less than 0.02 mL / min. method.

12. 12. The method of claim 11, wherein the flow rate in the bioreactor is about 0.01 mL / min. method.

13. 10. The method of claim 1, wherein the cells comprise suspension cells. method.

14. 14. The method of claim 13, wherein the suspension cells comprise one or more types of T cells. method.

15. A cell growth system, comprising: a first pump configured to circulate a first fluid; a second pump configured to circulate a second fluid; a fluid transfer assembly including a bioreactor, the fluid transfer assembly fluidly coupled to the first pump and the second pump; a processor; a memory in communication with and readable by said processor, said memory having a set of instructions; Equipped with the fluid transfer assembly has an air removal chamber, the bioreactor has an inner capillary loop and an outer capillary loop, and the flow rate within the bioreactor is less than 0.1 mL / min; When the processor executes the sequence of instructions, the processor: directing the introduction of cells into the fluid transfer assembly; directing the cells to be removed from the air removal chamber; directing the capillary inner loop to be filled with a medium containing a protein; directing the cells to be placed in the bioreactor for growth for a first period of time; Cell proliferation system.

16. 16. The cell growth system of claim 15, wherein the fluid transfer assembly is removably attached to the cell growth system. Cell proliferation system.

17. 16. The cell growth system of claim 15, wherein the fluid transfer assembly comprises a bioreactor. Cell proliferation system.

18. 16. The cell growth system of claim 15, the fluid transfer assembly comprises a first fluid transfer assembly, the first fluid transfer assembly comprising a first bioreactor, or the first fluid transfer assembly comprises a second fluid transfer assembly comprising a second bioreactor; the second bioreactor is smaller than the first bioreactor; Cell proliferation system.

19. 16. The cell expansion system of claim 15, wherein the cells comprise suspension cells. Cell proliferation system.

20. 20. The cell expansion system of claim 19, wherein the suspension cells comprise one or more types of T cells. Cell proliferation system.

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