Expanding cells

Abstract

The cells may grow in a bioreactor, and the cells may be activated by an activator (e.g., a soluble activator complex). For example, nutrient and gas exchange capabilities of a closed and automated cell expansion system may allow for inoculation of cells at a reduced cell inoculation density. Parameters of the cell growth environment can be manipulated to load cells to specific locations in the bioreactor for efficient exchange of nutrients and gases. System parameters may be adjusted to shear any cell colonies that may form during the expansion phase. Metabolic concentrations can be controlled to improve cell growth and viability. And residence of cells in the bioreactor can be controlled. In embodiments, the cells may include T cells. The cells may include subpopulations of T cells, including, for example, regulatory T cells (Treg).

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Patent Application No. 18 / 368,879, filed on September 15, 2023; and the benefit of U.S. Provisional Application No. 63 / 407,987, filed on September 19, 2022. The entire disclosures of the above - mentioned applications are hereby incorporated herein by reference. Technical Field

[0003] The present disclosure relates to expanding cells and cell expansion systems. Background Art

[0004] This section provides background information related to the present disclosure, which is not necessarily prior art.

[0005] Cell Expansion Systems (CES) are used to expand and differentiate cells. Cell expansion systems can be used to expand (e.g., grow) various adherent cells and suspension cells. Both adherent and non - adherent cells can grow in the bioreactor of a cell expansion system. Summary of the Invention

[0006] This section provides an overall overview of the disclosure, but not the full scope of it or a comprehensive disclosure of all its features.

[0007] In various aspects, the present disclosure provides a method for expanding cells, the method including loading cells into a cell expansion system, wherein the cell expansion system includes a bioreactor and an air removal chamber. The bioreactor can include an inner capillary loop and an outer capillary loop. The flow rate in the bioreactor can be less than 0.1 mL / min. The method can also include chasing cells from the air removal chamber, filling the inner capillary loop with a protein - containing medium, and positioning the cells in the bioreactor for a first period of expansion.

[0008] In at least one exemplary embodiment, the protein can include cell signaling molecules.

[0009] In at least one exemplary embodiment, the cell signaling molecules can include cytokines.

[0010] In at least one exemplary embodiment, the cytokines can include recombinant human IL - 2 cytokine.

[0011] In at least one exemplary embodiment, the positioning of the cells can include positioning the cells at a first position, wherein the first position faces the first side of the bioreactor.

[0012] In at least one exemplary embodiment, the first side of the bioreactor can include the outlet side of the bioreactor.

[0013] In at least one exemplary embodiment, the positioning of the cells may further include positioning the cells at a second location. The second location may be towards the central location of the bioreactor.

[0014] In at least one exemplary embodiment, the cells may move towards the second location due to the pressure difference in the bioreactor.

[0015] In at least one exemplary embodiment, the pressure difference may be generated during the operation of the air removal chamber.

[0016] In at least one exemplary embodiment, the method may further include, after a first time period, recycling the cells for a second time period, positioning the cells for a third time period, and feeding the cells.

[0017] In at least one exemplary embodiment, the flow rate in the bioreactor may be less than 0.02 mL / min.

[0018] In at least one exemplary embodiment, the flow rate in the bioreactor may be about 0.01 mL / min.

[0019] In at least one exemplary embodiment, the cells may include suspended cells.

[0020] In at least one exemplary embodiment, the suspended cells may include one or more types of T cells.

[0021] In various aspects, the present invention provides a cell expansion system, the cell expansion system comprising: a first pump configured to circulate a first fluid; a second pump configured to circulate a second fluid; a fluid delivery assembly including a bioreactor, wherein the fluid delivery assembly is in fluid communication with the first pump and the second pump; a processor; and a memory communicatively coupled to and readable by the processor, the memory including a series of instructions that, when executed by the processor, cause the processor to: direct loading of cells into the fluid delivery assembly, wherein the fluid delivery assembly includes an air removal chamber, the bioreactor includes an inner capillary loop and an outer capillary loop, and the flow rate in the bioreactor is less than 0.1 mL / min; direct chasing of the cells from the air removal chamber; direct filling of the inner capillary loop with a protein-containing medium; and direct positioning of the cells in the bioreactor for a first time period of expansion.

[0022] In at least one exemplary embodiment, the fluid delivery assembly may be detachably attached to the cell expansion system.

[0023] In at least one exemplary embodiment, the fluid delivery assembly may include a bioreactor.

[0024] In at least one exemplary embodiment, the fluid delivery assembly may include a first fluid delivery assembly. The first fluid delivery assembly may include a first bioreactor; or a second fluid delivery assembly including a second bioreactor. The second bioreactor may be smaller than the first bioreactor.

[0025] In at least one exemplary embodiment, the cells may include suspended cells.

[0026] In at least one exemplary embodiment, the suspended cells may include one or more types of T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible embodiments, and are not intended to limit the scope of the present disclosure.

[0028] Figure 1A Embodiments of a cell expansion system (CES) according to at least one exemplary embodiment of the present disclosure are depicted.

[0029] Figure 1B A front elevation view of an embodiment of a bioreactor according to at least one exemplary embodiment of the present disclosure is shown, which shows the circulation path through the bioreactor.

[0030] Figure 1C A perspective view of a first bioreactor and a second bioreactor according to at least one exemplary embodiment of the present disclosure is shown.

[0031] Figure 1D A rocking device for rotating or laterally moving a cell growth chamber during operation of a cell expansion system according to at least one exemplary embodiment of the present disclosure is depicted.

[0032] Figure 2A A front perspective view of a cell expansion system according to at least one exemplary embodiment of the present disclosure is shown.

[0033] Figure 2B An internal perspective view of a cell expansion system having a pre-installed fluid delivery device according to at least one exemplary embodiment of the present disclosure is shown. Figure 2A of

[0034] Figure 2C An internal perspective view of a retainer of a cell expansion system according to at least one exemplary embodiment of the present disclosure is shown. Figure 2A of

[0035] Figure 3 A perspective view of a housing of a cell expansion system according to at least one exemplary embodiment of the present disclosure is depicted.

[0036] Figure 4A A perspective view of a pre - installed fluid delivery device in accordance with at least one exemplary embodiment of the present disclosure is shown.

[0037] Figure 4B A pre - installed fluid delivery assembly in accordance with at least one exemplary embodiment of the present disclosure is shown. Figure 4A thereof

[0038] Figure 4C A pre - installed fluid delivery assembly in accordance with at least one exemplary embodiment of the present disclosure is shown. Figure 4A thereof

[0039] Figure 4D A media bag of a pre - installed fluid delivery assembly in accordance with at least one exemplary embodiment of the present disclosure is shown. Figure 4A thereof

[0040] Figure 4E A waste bag of a pre - installed fluid delivery assembly in accordance with at least one exemplary embodiment of the present disclosure is shown. Figure 4A thereof

[0041] Figure 4F A cross - sectional view of a pressure chamber of a pre - installed fluid delivery assembly in accordance with at least one exemplary embodiment of the present disclosure is shown. Figure 4A thereof

[0042] Figure 4G An exploded view of a pressure chamber in accordance with at least one exemplary embodiment of the present disclosure is shown. Figure 4F thereof

[0043] Figure 4H A sampling coil of a pre - installed fluid delivery assembly in accordance with at least one exemplary embodiment of the present disclosure is shown. Figure 4A thereof

[0044] Figure 4I An in - line filter of a pre - installed fluid delivery assembly in accordance with at least one exemplary embodiment of the present disclosure is shown. Figure 4A thereof

[0045] Figure 5A A schematic diagram of a cell expansion system in accordance with at least one exemplary embodiment of the present disclosure is depicted, the cell expansion system including an operating configuration showing fluid movement.

[0046] Figure 5B A schematic diagram of a cell expansion system in accordance with at least one exemplary embodiment of the present disclosure is depicted, the cell expansion system including another operating configuration showing fluid movement.

[0047] Figure 5CDepicts a schematic diagram of a cell expansion system according to at least one exemplary embodiment of the present disclosure, the cell expansion system including another operational configuration showing fluid movement.

[0048] Figure 6 Shows a schematic diagram of a cell expansion system according to at least one exemplary embodiment of the present disclosure.

[0049] Figure 7A Shows a schematic diagram of a cell expansion system according to at least one exemplary embodiment of the present disclosure.

[0050] Figure 7B Shows a schematic diagram of a cell expansion system according to at least one exemplary embodiment of the present disclosure.

[0051] Figure 7C Shows a schematic diagram of a cell expansion system according to at least one exemplary embodiment of the present disclosure.

[0052] Figure 8 Depicts a flowchart according to at least one exemplary embodiment of the present disclosure, showing operational characteristics of a process for expanding cells.

[0053] Figure 9A Shows a flowchart according to at least one exemplary embodiment of the present disclosure, depicting operational characteristics of a process for expanding cells.

[0054] Figure 9B Depicts a schematic diagram of a part of a cell expansion system according to at least one exemplary embodiment of the present disclosure.

[0055] Figure 10A Depicts a flowchart according to at least one exemplary embodiment of the present disclosure, showing operational characteristics of a process for expanding cells.

[0056] Figure 10B Shows a graph of oxygen consumption in a cell expansion system according to at least one exemplary embodiment of the present disclosure.

[0057] Figure 11A Shows a flowchart according to at least one exemplary embodiment of the present disclosure, depicting operational characteristics of a process for expanding cells.

[0058] Figure 11B Shows a table according to at least one exemplary embodiment of the present disclosure, the table showing exemplary pump rates that can be used in a cell expansion system.

[0059] Figure 12A Depicts a flowchart according to at least one exemplary embodiment of the present disclosure, showing operational characteristics of a process for expanding cells.

[0060] Figure 12B A diagram showing the metabolism of amplified cells according to at least one exemplary embodiment of the present disclosure.

[0061] Figure 12C A diagram showing the metabolism of amplified cells according to at least one exemplary embodiment of the present disclosure.

[0062] Figure 13 A flowchart according to at least one exemplary embodiment of the present disclosure, depicting the operating characteristics of a process for amplifying cells.

[0063] Figure 14 A flowchart according to at least one exemplary embodiment of the present disclosure, showing the operating characteristics of a process for amplifying cells.

[0064] Figure 15 A flowchart according to at least one exemplary embodiment of the present disclosure, depicting the operating characteristics of a process for amplifying cells.

[0065] Figure 16A A flowchart according to at least one exemplary embodiment of the present disclosure, depicting the operating characteristics of a process for amplifying cells.

[0066] Figure 16B A diagram showing the number of cells relative to the flow rate during cell amplification according to at least one exemplary embodiment of the present disclosure.

[0067] Figure 17 A flowchart according to at least one exemplary embodiment of the present disclosure, showing the operating characteristics of a process for amplifying cells.

[0068] Figure 18A A flowchart according to at least one exemplary embodiment of the present disclosure, showing the operating characteristics of a process for amplifying cells.

[0069] Figure 18B A diagram depicting cell amplification in a cell amplification system according to at least one exemplary embodiment of the present disclosure.

[0070] Figure 18C A diagram showing the inner diameter of cell dissociation according to at least one exemplary embodiment of the present disclosure.

[0071] Figure 19 A diagram showing the number of cells and the flow rate relative to the number of culture days during cell amplification according to at least one exemplary embodiment of the present disclosure.

[0072] Figure 20A A flowchart according to at least one exemplary embodiment of the present disclosure, depicting the operating characteristics of a process for operating a pump to amplify cells.

[0073] Figure 20B Schematic diagram depicting a part of a cell expansion system according to at least one exemplary embodiment of the present disclosure.

[0074] Figure 20C Schematic diagram depicting a part of a cell expansion system according to at least one exemplary embodiment of the present disclosure.

[0075] Figure 20D Schematic diagram depicting a part of a cell expansion system according to at least one exemplary embodiment of the present disclosure.

[0076] Figure 21 Flowchart according to at least one exemplary embodiment of the present disclosure, depicting operational characteristics of a process for expanding cells.

[0077] Figure 22 Flowchart according to at least one exemplary embodiment of the present disclosure, showing operational characteristics of a process for expanding cells.

[0078] Figure 23 Flowchart according to at least one exemplary embodiment of the present disclosure, depicting operational characteristics of a process for expanding cells.

[0079] Figure 24 Flowchart depicting operational characteristics of a process for expanding cells according to at least one exemplary embodiment of the present disclosure.

[0080] Figure 25 Exemplary processing system of a cell expansion system on which embodiments of the present disclosure can be implemented according to at least one exemplary embodiment of the present disclosure.

[0081] Figure 26 Flowchart according to at least one exemplary embodiment of the present disclosure, depicting an example of the experimental process.

[0082] Figure 27 Graph showing exemplary results of a coating process according to at least one exemplary embodiment of the present disclosure.

[0083] Figure 28 Graph showing exemplary results of cells harvested after a coating process according to at least one exemplary embodiment of the present disclosure.

[0084] Figure 29 Graph showing exemplary results of cells harvested after a coating process according to at least one exemplary embodiment of the present disclosure.

[0085] Figure 30 Graph showing exemplary results of virus particles measured daily according to at least one exemplary embodiment of the present disclosure.

[0086] Figure 31 A diagram showing exemplary results of cells harvested after a coating process according to at least one exemplary embodiment of the present disclosure.

[0087] Figure 32 A diagram showing exemplary results of cells harvested after a coating process according to at least one exemplary embodiment of the present disclosure.

[0088] Figure 33 A table showing an example of setting days 0 - 2 according to at least one exemplary embodiment of the present disclosure.

[0089] Figure 34 A table showing the steps of programming a 10 - minute coating with a CPPT program according to at least one exemplary embodiment of the present disclosure.

[0090] Figure 35 A table showing the steps of virus proliferation according to at least one exemplary embodiment of the present disclosure.

[0091] Figure 36A and Figure 36B are respectively table parts according to at least one exemplary embodiment of the present disclosure, which show exemplary task settings (such as flow rate, angular rotation, outlet, etc.) of different components (such as pumps, rockers, valves, etc.) of a small - cell expansion system.

[0092] Figure 37 A table according to at least one exemplary embodiment of the present disclosure, which shows exemplary task settings (such as flow rate, angular rotation, outlet, etc.) of different components (such as pumps, rockers, valves, etc.) of a standard cell expansion system.

[0093] Figure 38 A table according to at least one exemplary embodiment of the present disclosure, which shows exemplary task settings (such as flow rate, angular rotation, outlet, etc.) of different components (such as pumps, rockers, valves, etc.) of a smaller - sized cell expansion system. Detailed Description

[0094] Exemplary embodiments will now be described more fully with reference to the accompanying drawings.

[0095] Exemplary embodiments are provided so that this disclosure will be thorough 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 this disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that the exemplary embodiments may be embodied in many different forms, and that none of them should be construed as limiting the scope of this disclosure. In some exemplary embodiments, well-known processes, well-known device structures, and well-known technologies have not been described in detail.

[0096] 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 also 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 the stated features, integers, 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. Unless expressly specified to the contrary, the method steps, procedures, and operations described herein should not be construed as necessarily requiring that they be performed in the particular order discussed or illustrated. It should also be understood that additional or alternative steps may be employed.

[0097] When an element or layer is referred to as being "on", "engaged to", "connected to", or "coupled to" another element or layer, it can be directly on, engaged, connected, or coupled to the other element or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being "directly on", "directly engaged to", "directly connected to", or "directly coupled to" another element or layer, there may be no intervening elements or layers. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., "between" relative to "directly between", "adjacent" relative to "directly adjacent", etc.). As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0098] Although the terms "first", "second", "third", etc. may be used herein to describe various elements, components, regions, layers, and / or sections, these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, and / or section from another region, layer, or section. Unless the context clearly indicates otherwise, terms such as "first", "second", and other numerical terms do not imply an order or sequence when used herein. Thus, without departing from the teachings of the exemplary embodiments, the first element, component, region, layer, and / or section discussed below may be referred to as the second element, component, region, layer, and / or section.

[0099] For ease of description, spatially relative terms such as "inner", "outer", "beneath", "below", "lower", "above", "upper", etc. may be used herein to describe the relationship of one element or feature to another element or feature as shown in the figures. In addition to the orientation depicted in the figures, the spatially relative terms are intended to encompass different orientations of the device during use or operation. For example, if the device in the figures is flipped, an element described as "below" or "beneath" another element or feature will be oriented "above" the other element or feature. Thus, the exemplary term "below" can encompass both an upper and a lower orientation. The device may be oriented in other ways (rotated 90 degrees or in other orientations), and the spatially relative descriptors used herein are to be interpreted accordingly.

[0100] Various components are referred to herein as "operatively associated". As used herein, "operatively associated" means components that are connected together in an operative manner, and encompasses embodiments where the components are directly connected, as well as embodiments where additional components are placed between the connected components. "Operatively associated" components may be "fluidly associated". "Fluidly associated" means that the components are connected together such that fluid can be conveyed between them. "Fluidly associated" encompasses embodiments where additional components are disposed between two fluidly associated components, as well as between components that are directly connected. Fluidly associated components may include components that do not contact the fluid but contact other components to manipulate the system (e.g., a peristaltic pump that pumps fluid through a flexible tube by compressing the outside of the tube).

[0101] The term "donor" as used herein may refer to any person who provides fluid (e.g., whole blood) to a apheresis system. A donor may also be a patient who temporarily provides fluid to the apheresis system, where the fluid is returned to the patient after being processed, treated, manipulated, etc.

[0102] As used herein, the term "automated" and variations thereof refer to any process or operation that can be completed without substantial human input. However, even if substantial or insubstantial human input is used during the execution of a process or operation, the process or operation can still be automated if the input was received prior to the execution of the process or operation. Human input is considered substantial if it affects the execution of the process or operation. Human input that merely acknowledges the execution of a process or operation is not considered "substantial".

[0103] 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 a medium can take many forms, including but not limited to non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, NVRAM, or magnetic or optical disks. Volatile media includes dynamic memory, such as main memory. Common forms of computer-readable media include, for example, a floppy disk, flexible disk, hard disk, magnetic tape, or any other magnetic medium, magneto-optical medium, CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, RAM, PROM, and EPROM, flash-EPROM, solid state media such as memory cards, any other memory chip or cartridge, a carrier wave as described below, or any other medium from which a computer can read. Digital file attachments to e-mails or other self-contained information archives or sets of archives are considered distribution media equivalent to tangible storage media. It should be understood that when a computer-readable medium is configured as a database, the database can be any type of database, such as a relational database, hierarchical database, object-oriented database, etc. Thus, the present disclosure is considered to include tangible storage media or distribution media that store software implementations of the present disclosure, as well as equivalent media and successor media recognized by the prior art.

[0104] 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 can perform functions associated with that element.

[0105] As used herein, the terms "determine", "calculate", and "compute" and variations thereof are used interchangeably and include any type of method, process, mathematical operation, or technique.

[0106] Embodiments of the present disclosure will be described more fully with reference to the accompanying drawings in connection with apheresis methods and systems. The following embodiments may be described with respect to separating blood components from whole blood. However, the exemplary processes are provided for illustrative purposes only. It should be noted that the embodiments are not limited to the following description. These embodiments are intended for products, processes, devices, and systems for separating any complex liquid. Thus, the present disclosure is not limited to separating blood components from whole blood.

[0107] Exemplary embodiments of the present disclosure generally relate to systems and methods for expanding cells in a cell expansion system (CES). According to an embodiment, the expansion can occur by using a bioreactor or a cell growth chamber. In an embodiment, the bioreactor or cell growth chamber may include a hollow fiber membrane. The hollow fiber membrane may include a plurality of hollow fibers and may include an extra-capillary (EC) space and / or an intra-capillary (IC) space. Embodiments can provide adherent or non-adherent cells that are grown or expanded in the cell expansion system. For example, non-adherent cells or suspension cells such as T cells, T lymphocytes, or CD3+-selected cells can be expanded in the system. In an embodiment, one or more subsets or subpopulations of T cells can be grown. For example, embodiments can provide methods and systems for expanding regulatory T cells (Tregs) and / or human regulatory T cells (hTregs).

[0108] In some exemplary embodiments, methods and systems for expanding cells in a closed automated cell expansion system can be provided. In an embodiment, the cell expansion system may include a bioreactor or a cell growth chamber. In a further embodiment, the bioreactor or cell growth chamber may include a hollow fiber membrane. The capabilities of the system, such as nutrient and gas exchange capabilities, can allow cells to be seeded at a reduced cell seeding density. Embodiments provide cell growth environment parameters to be manipulated to position cells loaded or introduced into the bioreactor for efficient exchange of nutrients and gases to the growing cells. For example, in an embodiment, centralization of cells in the bioreactor can increase cell density.

[0109] In an exemplary embodiment, a non-adherent cell population (e.g., T cells) can be introduced or loaded into a hollow fiber bioreactor, which may include a plurality of hollow fibers. In an embodiment, the cells can be exposed to an activator to activate cell expansion in the hollow fiber bioreactor. For example, in an embodiment, the "loading cells centrally without circulation" task is used to introduce a plurality of cells into the cell expansion system. According to an exemplary embodiment, this task can be performed on day 0 and days 4 - 8. In other embodiments, other days can be used. In an embodiment, this cell loading task can centralize the cells in the bioreactor to increase cell density. In other embodiments, the cells can be located in other parts or regions of the bioreactor to increase cell density. In an embodiment, by positioning the cells at a first location in the bioreactor (e.g., around the central region), the cells can receive efficient exchange of nutrients and gases.

[0110] In an exemplary embodiment, a reduced 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 expanded at a cell seeding density from 2.54×10 5 cells / mL to 3.69×10 5 cells / mL. In other embodiments, the cell seeding density can be less than about 1×10 6 cells / mL. Additionally, a Treg cell inoculum can be prepared at a cell seeding density of 1.0×10 5 cells / mL. Other methods (e.g., static Treg cell culture methods) can use a cell seeding density of 1.0×10 6 Treg cells / mL for in vitro expansion. For example, in an embodiment, a lower cell seeding density can be used due to the overall efficiency of the system in delivering nutrients to the culture environment. In other embodiments, one or more steps used in the expansion process, combined with the overall efficiency of the system in delivering nutrients to the culture environment, can allow for the use of a lower initial cell seeding density.

[0111] In an exemplary embodiment, a soluble activator complex can be used for automated cell (e.g., Treg) expansion. In other embodiments, other types of activators (such as beads) can be used to stimulate the cells; or incubated with soluble or surface - immobilized antibodies. In other embodiments, cells (e.g., Treg cells) can be expanded without using bead - based stimulation. In one embodiment, Stem cell Technologies soluble ImmunoCult TMCell expansion is carried out with a 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 by an automated cell expansion system. For example, relative to the cost of bead-based protocols, the use of a soluble activator complex can reduce the cost of stimulation. Other types of cell activators can be used in other embodiments, including co-culture of antigen-presenting cells and T cells for biologic antigen stimulation. Further, other types of cytokines or other growth factors can be used in other embodiments.

[0112] Exemplary embodiments can also provide system parameters to be adjusted or managed to control the residence of cells in a bioreactor or cell growth chamber. For example, by controlling the residence of cells in the hollow fibers of a bioreactor during the cell growth phase, the system can provide efficient gas and nutrient exchange to expand the cells. In an embodiment, the bioreactor can be designed to provide gas exchange to the growing cells and, in some embodiments, nutrient exchange. In an exemplary embodiment, a bioreactor including a semi-permeable hollow fiber membrane can provide gas and nutrient exchange through the semi-permeable hollow fiber membrane. In an embodiment, a method for providing medium components that cannot cross the membrane (such as various cytokines, proteins, etc.) to the cells for their growth can use a fluid inlet to the side of the bioreactor (e.g., the in-capillary (IC) side where the cells grow). However, according to embodiments, even a low, declining, or reduced, e.g., minimal inlet flow rate may cause cells to aggregate in the outlet manifold of the bioreactor. Cells residing in the manifold of the bioreactor may not receive proper gas and nutrient exchange, which can lead to cell death and aggregation.

[0113] Exemplary embodiments relate to methods of retaining cells (e.g., a non - adherent cell population) in a bioreactor when feeding the cells using an inlet (e.g., an IC inlet) stream. While embodiments herein may refer to the cells being on the IC side of a membrane when feeding, for example, other embodiments may provide for the cells to be on the EC side of the membrane, where according to the embodiment, the cells may be included in a first circulation path and / or a second circulation path. In an embodiment, the feeding method may provide for pumping a first volume of fluid (e.g., a medium or a cell - growth - formulated medium) into a first port of the bioreactor at, for example, a first volumetric flow rate, volume flow rate, fluid flow rate, flow rate, fluid flow velocity, or volume velocity. For example, volumetric flow rate, volume flow rate, fluid flow rate, flow rate, fluid flow velocity, or volume velocity may be used interchangeably. In some embodiments, the flow rate may be a vector having both speed and direction. A second volume of fluid may be pumped into a second port of the bioreactor at a second volumetric flow rate, volume flow rate, fluid flow rate, flow rate, fluid flow velocity, or volume velocity. In an embodiment, the volumetric flow rate, volume flow rate, fluid flow rate, flow rate, fluid flow velocity, or volume velocity may be controlled by, for example, one or more pump speeds and / or pump flow rates. The pump may act on the fluid, and the pump rate may generate, cause, or affect the volumetric flow rate or flow rate of the fluid. The pump rate used herein may be described in an embodiment as the volumetric flow rate or fluid flow rate generated, caused, or affected by the pump.

[0114] In an exemplary embodiment, the second flow rate of fluid entering the bioreactor may be in a direction opposite to the first flow rate of fluid entering the bioreactor. For example, Figure 5B and Figure 5C show an exemplary operating configuration according to an embodiment of the present disclosure, which shows the flow rates and flow directions that may be used in a cell expansion system, such as the CES 500 (e.g., Figure 5B and Figure 5C ). In an embodiment, a cell expansion system pump (e.g., an IC pump) may be used to control cell residence in the bioreactor. In an embodiment, for example, during the growth expansion phase, cells may be lost from the bioreactor to the IC circulation path or IC loop. In an embodiment, for example, cells closer to the IC inlet port in the bioreactor may receive the freshest growth medium, while, for example, cells in a portion of the IC circulation path outside the bioreactor may receive expended or conditioned medium that may affect their metabolism. Additionally, according to an embodiment, cells in the bioreactor may receive a mixed gas (O2, CO2, N2) input from a gas transfer module (GTM) by circulating and diffusing through the EC loop, while cells in a portion of the IC circulation path outside the bioreactor may not.

[0115] In an exemplary embodiment, by diverting half of the inlet flow through an IC circuit in the opposite direction of the input flow, cell medium is effectively introduced equally into both ends of the bioreactor to retain cells within the hollow fibers, thereby reducing cell loss in a hollow fiber membrane (HFM) bioreactor. For example, in an embodiment, the IC inlet pump rate of +0.2 mL / min can be halved to a complementary IC circulation pump rate of -0.1 mL / min to maintain cells in the bioreactor during the growth phase of cell culture (which can be days 4 to 7). According to an embodiment, such pump adjustment can counteract the forces associated with cell loss at the IC outlet port. In other embodiments, other pump rates can be used. For example, in other embodiments, the pump rates can be different. In an embodiment, other pumps or additional pumps can be used. In an embodiment, fewer pumps can be used. Further, other time periods can be used in other embodiments.

[0116] In an exemplary embodiment, the metabolic activity of a cell population can affect the feeding parameters. For example, the lactate value of a cell culture can be maintained at or below a predetermined level. In an embodiment, the cell culture value can be maintained at or below, for example, about 7 mmol / L. In an embodiment, by using a cell expansion system graphical user interface (GUI) to control the medium addition rate, during cell (e.g., regulatory T cell) expansion, the lactic acid metabolic waste produced by glycolysis can be maintained at or below a predetermined value. In other embodiments, for example, the medium addition rate and / or other settings can be controlled to maintain or attempt to maintain the lactate level, glucose level, or pH value to improve cell growth and viability. In other embodiments, other concentrations can be used.

[0117] Additional embodiments can provide system features to be controlled for breaking down any cell colonies, micro-colonies, or cell clusters that may form during the expansion phase. For example, embodiments provide for breaking down cell colonies (such as micro-colonies) through hollow fiber membranes in a bioreactor to reduce the number of cells in the micro-colonies, colonies, or clusters, where the micro-colonies, colonies, or clusters can be one or more groups of adherent cells. In an embodiment, a cell expansion system (CES) bioreactor architecture can be used to break down cell (such as Treg cell) micro-colonies. In an embodiment, as cells (such as Treg cells) grow, they tend to form micro-colonies, which can limit the diffusion of nutrients to the cells at the center of the colony. This can cause adverse effects during cell culture, such as necrosis. Embodiments can provide a scheme for breaking down colonies by circulating a suspension cell culture through, for example, an in-capillary (IC) circuit through a hollow fiber (such as a hollow fiber with an inner diameter of 215 μm) during the growing expansion phase. In an embodiment, the colonies, micro-colonies, or cell clusters can be broken down to reduce the size of the colonies, micro-colonies, or cell clusters. In an embodiment, the colonies or cell clusters can be broken down to provide a single cell suspension and generate cell growth / vitality. In an embodiment, this ability can contribute to the continuous perfusion growth of cells (such as T cells or Tregs).

[0118] In an exemplary embodiment, a therapeutic dose of cells (such as Tregs) can be expanded and harvested from a cell expansion system. In an embodiment, the number of cells at harvest can be from about 1×10 6 cells to about 1×10 10 cells, such as about 1×10 9 cells. In one embodiment, the number of harvested cells can be from about 1×10 8 to 1×10 10 cells, an example being from about 7×10 8 to about 1.4×10 9 cells. In an embodiment, the harvested cells can have a viability between about 60% and about 100%. For example, the viability of the harvested cells can be above about 65%, above about 70%, above about 75%, above about 80%, above about 85%, above about 90%, or even above about 95%. In some embodiments, the harvested cells can express biomarkers consistent with Tregs. For example, in some embodiments, the cells can express CD4 + , CD25 + and / or FoxP3 + biomarkers. In an embodiment, the harvested cells can include a frequency of CD4 + CD25 +Phenotype. The harvested cells may include CD4 at a frequency of greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or even greater than about 95%. + CD25 + Phenotype. In other embodiments, the cells may include CD4 at a frequency of about 30% to about 100%. + FoxP3 + Phenotype. In some embodiments, the harvested cells may include CD4 at 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%. + FoxP3 + Phenotype. Other embodiments may expand other types of suspended cells, such as T cells with different phenotypes, different cell surface markers, and different transcription factor expressions.

[0119] Exemplary embodiments relate to the cell expansion system as described above. In an embodiment, the cell expansion system is closed, wherein the closed cell expansion system includes contents that are not directly exposed to the atmosphere. The cell expansion system can be automated. In an embodiment, adherent cells and non - adherent cells or suspended cells can grow in a bioreactor in the cell expansion system. According to an embodiment, the cell expansion system may include a basal medium or other types of media. A method for supplementing the medium is provided for cell growth to occur in the bioreactor of the closed cell expansion system. In an embodiment, the bioreactor used with the system is a hollow fiber bioreactor. According to embodiments of the present disclosure, various types of bioreactors can be used.

[0120] In an embodiment, the system can include: a bioreactor fluidly associated with a first fluid flow path having at least opposite ends; a first opposite end of the first fluid flow path fluidly associated with a first port of a hollow fiber membrane; and a second end of the first fluid flow path fluidly associated with a second port of the hollow fiber membrane. In an embodiment, the hollow fiber membrane includes a plurality of hollow fibers. The system can further include a fluid inlet path fluidly associated with the first fluid flow path, wherein a plurality of cells can be introduced into the first fluid flow path through the first fluid inlet path. In some embodiments, a pump for transferring an in-capillary inlet fluid from an in-capillary media bag to the first fluid flow path and a controller for controlling the operation of the pump are also included. In an embodiment, the controller controls the pump to transfer, for example, cells from a cell inlet bag to the first fluid flow path. Another pump for circulating fluid in the first fluid circulation path can also be included, wherein the pump can also include a controller for controlling the operation of the pump. In an embodiment, the controller is, for example, a computing system that includes a processor. In an embodiment, one or more controllers can be configured to control one or more pumps, such as circulating fluid at a flow rate within the first fluid circulation path. According to an embodiment, multiple controllers (e.g., a first controller, a second controller, a third controller, a fourth controller, a fifth controller, a sixth controller, etc.) can be used. Further, according to an embodiment 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.) can be used. Additionally, although the present disclosure can relate to media bags, cell inlet bags, etc., in an embodiment, multiple bags (e.g., a first media bag, a second media bag, a third media bag, a first cell inlet bag, a second cell inlet bag, a third cell inlet bag, etc.) and / or other types of containers can be used. In other embodiments, a single media bag, a single cell inlet bag, etc. can be used. Further, in an embodiment, additional or other fluid paths (e.g., a second fluid flow path, a second fluid inlet path, a second fluid circulation path, etc.) can also be included.

[0121] In an exemplary embodiment, the system is controlled, for example, by: a processor coupled to a cell expansion system; a display device in communication with the processor and operable to display data; and a memory in communication with the processor and readable by the processor and including a series of instructions. In an embodiment, when the instructions are executed by the processor, the processor receives the instructions to, for example, start the system. For example, in response to an instruction to start the system, the processor can execute a series of steps to prepare the system, and then can receive an instruction to perform an IC / EC flush. For example, in response to an instruction to load cells, the processor can perform a series of steps, such as loading cells from a cell inlet bag into the bioreactor.

[0122] Figure 1AFigure showing a schematic diagram of an exemplary cell expansion system (CES) according to an embodiment of the present disclosure. CES 10 includes a first fluid circulation path 12 and a second fluid circulation path 14. According to an embodiment, the first fluid flow path 16 has at least opposite ends 18 and 20 that are fluidly associated with a hollow fiber cell growth chamber 24 (also referred to herein as a "bioreactor"). Specifically, the opposite end 18 may be fluidly associated with a first inlet 22 of the cell growth chamber 24, and the opposite end 20 may be fluidly associated with a first outlet 28 of the cell growth chamber 24. The fluid in the first fluid circulation path 12 flows through the hollow fibers 116 (see Figure 1B ) of the hollow fiber membrane 117 (see Figure 1B ) placed in the cell growth chamber 24 (the cell growth chamber and the hollow fiber membrane are described in more detail below). Further, a first fluid flow control device 30 may be operably connected to the first fluid flow path 16 and may control the flow of the fluid in the first fluid circulation path 12.

[0123] The second fluid circulation path 14 includes a second fluid flow path 34, the cell growth chamber 24, and a second fluid flow control device 32. According to an embodiment, the second fluid flow path 34 has at least opposite ends 36 and 38. The opposite ends 36 and 38 of the second fluid flow path 34 may be fluidly associated with an inlet port 40 and an outlet port 42 of the cell growth chamber 24, respectively. The fluid flowing through the cell growth chamber 24 may contact the outside of the hollow fiber membrane 117 (see Figure 1B ) in the cell growth chamber 24, where the hollow fiber membrane includes a plurality of hollow fibers. The second fluid circulation path 14 may be operably connected to the second fluid flow control device 32.

[0124] Thus, the first fluid circulation path 12 and the second fluid circulation path 14 may be separated in the cell growth chamber 24 by the hollow fiber membrane 117 (see Figure 1B ). The fluid in the first fluid circulation path 12 flows through the capillary inner ("IC") space of the hollow fibers in the cell growth chamber 24. The first fluid circulation path 12 may be referred to as the "IC loop". The fluid in the second circulation path 14 flows through the capillary outer ("EC") space in the cell growth chamber 24. The second fluid circulation path 14 may be referred to as the "EC loop". According to an embodiment, the fluid in the first fluid circulation path 12 may flow in a co-current or counter-current direction relative to the flow of the fluid in the second fluid circulation path 14.

[0125] The fluid inlet path 44 can be fluidly associated with the first fluid circulation path 12. The fluid inlet path 44 allows fluid to enter the first fluid circulation path 12, while the fluid outlet path 46 allows fluid to leave the CES 10. The third fluid flow control device 48 can be operably associated with the fluid inlet path 44. Alternatively, the third fluid flow control device 48 is alternatively associated with the fluid outlet path 46.

[0126] According to an embodiment, the fluid flow control devices used herein can include pumps, valves, clamps, or combinations thereof. Multiple pumps, valves, and clamps can be arranged in any combination. In various embodiments, the fluid flow control device is or includes a peristaltic pump. In an embodiment, the fluid circulation path, inlet port, and outlet port can be formed of tubing of any material.

[0127] Generally, any kind of fluid, such as including buffers, protein-containing fluids, and cell-containing fluids, can flow through the various circulation paths, inlet paths, and outlet paths. The terms "fluid", "medium", and "fluid medium" used herein are used interchangeably.

[0128] Turning Figure 1B , an example of a hollow fiber cell growth chamber 100 or bioreactor 100 that can be used with the present disclosure is shown through a front elevation view. The cell growth chamber 100 has a longitudinal axis LA-LA and includes a cell growth chamber housing 104. In at least one embodiment, the cell growth chamber housing 104 includes four openings or ports: an IC inlet port 108, an IC outlet port 120, an EC inlet port 128, and an EC outlet port 132.

[0129] According to an embodiment of the present disclosure, the fluid in the first circulation path enters the cell growth chamber 100 through the IC inlet port 108 located at the first longitudinal end 112 of the cell growth chamber 100, enters and passes through the capillary inner side (in various embodiments, referred to as the capillary inner side ("IC") or "IC space" of the hollow fiber membrane) of a plurality of hollow fibers 116 (including the hollow fiber membrane 117); and leaves the cell growth chamber 100 through the IC outlet port 120 located at the second longitudinal end 124 of the cell growth chamber 100. The fluid path between the IC inlet port 108 and the IC outlet port 120 defines the IC portion 126 of the cell growth chamber 100. The fluid in the second circulation path flows in the cell growth chamber 100 through the EC inlet port 128, contacts the capillary outer side or the outside (referred to as the "EC side" or "EC space" of the membrane) of the hollow fiber 116, and leaves the cell growth chamber 100 via the EC outlet port 132. The fluid path between the EC inlet port 128 and the EC outlet port 132 includes the EC portion 136 of the cell growth chamber 100. The fluid entering the cell growth chamber 100 through the EC inlet port 128 can contact the outside of the hollow fiber 116. Small molecules (such as ions, water, oxygen, lactic acid, metabolites, nutrients, gases, etc.) can diffuse (such as continuous perfusion) through the hollow fiber 116 from the inside or the IC space of the hollow fiber to the outside or the EC space, or from the EC space to the IC space. For example, compared with a flask-based system, the hollow fiber 116 can include a diffusion distance of 200 microns, thereby providing more efficient gas and nutrient transfer. Macromolecular weight molecules, such as growth factors, are usually too large to pass through the hollow fiber membrane and may remain in the IC space of the hollow fiber 116. Compared with a stirred bioreactor and a wave bioreactor, the hollow fiber 116 generates less shear stress. In an embodiment, the medium can be replaced as needed. The medium can also be circulated through an oxygenator or a gas delivery module to exchange gases as needed. According to an embodiment, as described below, cells can be included in the first circulation path and / or the second circulation path, and can be on the IC side and / or the EC side of the membrane.

[0130] The material for manufacturing the hollow fiber membrane 117 can be any biocompatible polymer material capable of being formed into hollow fibers. According to an embodiment of the present disclosure, one material that can be used is a material based on synthetic polysulfone.

[0131] According to some embodiments, as Figure 1CAs shown, more than one size of cell growth chamber 100 can be included individually or in a disposable kit. For example, a pair of cell growth chambers 100A and 100B can be included individually or in a disposable kit. In at least one exemplary embodiment, cell growth chamber 100A can be smaller than cell growth chamber 100B to accommodate fewer cells for expansion. For example, the size of cell growth chamber 100A can be one-tenth or 0.1 of cell growth chamber 100B. In at least one exemplary embodiment, cell growth chamber 100A (or small cell growth chamber 100A) can be advantageous for pilot runs, pediatric, and other events (such as skin grafts, etc.) with small starting cell batches. Using small cell growth chamber 100A is advantageous when the initial cell source is too small for cell growth chamber 100B because small cell growth chamber 100A can grow small cell batches, and the small cell batches can be transferred to cell growth chamber 100B for additional growth. Thus, small cell growth chamber 100A provides an additional opportunity for the administration of CES10 when cell growth chamber 100B may not be optimal. In some exemplary embodiments, as described below, CES10 can automatically identify which of cell growth chamber 100A and cell growth chamber 100B is installed and identify the appropriate process parameters. By including more than one size of cell growth chamber 100, CES10 may have more applications, such as including bone marrow-derived mesenchymal stem cells (MSC), adipose-derived MSC, umbilical cord MSC, fibroblasts, keratinocytes, HEK293T cells, human embryonic stem cells (ESC), periosteum-derived cells, induced pluripotent stem cell-derived MSC (IPS-MSC), neural stem cells (NSC), osteochondral progenitor cells, endothelial cells, dendritic cells, induced pluripotent stem cells (iPSC), T cells, viral vectors, exosome production, expansion of autologous and allogeneic doses of adherent and suspension cell types, etc. For example, multiple cell growth chambers 100 for CES10 provide the ability to expand in an exemplary range of up to 300 million to 1 billion MSC or up to 25 billion T cells per run.

[0132] In at least one exemplary embodiment, a CES (such as CES 500) (see Figure 5A , Figure 5B and Figure 5C ) and / or CES 600 (see Figure 6 ) can include a device configured to move or "rock" the cell growth chamber relative to other components of the cell expansion system by attaching it to a rotational and / or lateral rocking device. Figure 1D One such exemplary She Bi'e is shown, where, according to the embodiment, the cell growth chamber 100 can be rotatably connected to two rotational rocking components and a lateral rocking component.

[0133] The first rotational rocking member 138 rotates the cell growth chamber 100 about a central axis 142 of the cell growth chamber 100. The first rotational rocking member 138 may be rotationally associated with the cell growth chamber 100. In some exemplary embodiments, the cell growth chamber 100 may continuously rotate about the central axis 142 in a single direction, either clockwise or counterclockwise. Alternatively, according to an embodiment, the cell growth chamber 100 may rotate in an alternating manner, e.g., first clockwise and then counterclockwise about the central axis 142.

[0134] The CES may further include a second rotational rocking member that rotates the cell growth chamber 100 about a rotational axis 144. The rotational axis 144 may pass through the center point of the cell growth chamber 100 and may be perpendicular to the central axis 142. In an embodiment, the cell growth chamber 100 may continuously rotate about the rotational axis 144 in a single direction, either clockwise or counterclockwise. Alternatively, the cell growth chamber 100 may rotate about the rotational axis 144 in an alternating manner, e.g., first clockwise and then counterclockwise. In various embodiments, the cell growth chamber 100 may also rotate about the rotational axis 144 and be positioned in a horizontal or vertical orientation relative to gravity.

[0135] In some exemplary embodiments, the lateral rocking member 140 may be laterally associated with the cell growth chamber 100. In an embodiment, the plane of the lateral rocking member 140 moves laterally in the -x and -y directions. According to an embodiment, the sedimentation of cells in the cell growth chamber may be reduced by the movement of the medium containing cells within the hollow fibers.

[0136] The rotation and / or lateral movement of the rocking device may reduce the sedimentation of cells within the device and reduce the likelihood of cells being trapped within a portion of the cell growth chamber. According to Stokes' law, the sedimentation rate of cells in the cell growth chamber is proportional to the density difference between the cells and the suspension medium. In certain embodiments, a repeated 180-degree rotation (rapid) with pauses as described above, e.g., with a total combined time of 30 seconds, is used to keep, for example, non-adherent red blood cells suspended. Exemplary embodiments may rotate the cell growth chamber 100 through a minimum rotation of about 180 degrees to a maximum rotation of about 290 to 300 degrees; however, rotations up to 360 degrees or greater may be used. Different rocking members may be used alone or in any combination. For example, a rocking member that rotates the cell growth chamber 100 about the central axis 142 may be combined with a rocking member that rotates the cell growth chamber 100 about the axis 144. Similarly, clockwise and counterclockwise rotations about different axes may be performed independently in any combination.

[0137] Steering Figure 2A and Figure 2B, showing an embodiment of a cell expansion system 200 with a pre - installed fluid delivery assembly according to an embodiment of the present disclosure. The CES 200 includes a cell expansion machine 202, which includes a hatch or closable door 204 for engaging with the rear 206 of the cell expansion machine 202. The internal space 208 within the cell expansion machine 202 includes features adapted to receive and engage the pre - installed fluid delivery assembly 210. The pre - installed fluid delivery assembly 210 is detachably attached to the cell expansion machine 202 to facilitate relatively quickly replacing a used pre - installed fluid delivery assembly 210 at the cell expansion machine 202 with one of the new or unused fluid delivery assemblies 210. A single cell expansion machine 202 can be operated to grow or expand a first set of cells using a first of the pre - installed fluid delivery assemblies 210 and can thereafter be used to grow or expand a second set of cells using a second of the pre - installed fluid delivery assemblies 210 without the need for sterilization between swapping the first of the pre - installed fluid delivery assemblies 210 with the second. The pre - installed fluid delivery assembly 210 includes a cell growth chamber 100 and an oxygenator or gas delivery module 212 (also see FIG. 4). According to an embodiment, a conduit guide groove denoted as 214 is provided for receiving various media conduits connected to the pre - installed fluid delivery assembly 210.

[0138] The front face 262 of the CES 200 includes a user interface 264. The user interface 264 may include a display 268, such as a touch screen, for example, to allow a user to input data, retrieve data, input test protocols, switch between views, view data, view alerts, etc. Additionally or alternatively, the user interface 264 may include one or more buttons or switches for inputting information, controlling the display, or performing other functions. As described below, inputs from the user interface 264 can be sent to the control system. The front face 262 of the CES 200 may also include one or more indicator lights or other visual signals for indicating alerts.

[0139] A retainer 270, such as a bag retainer, a disposable retainer, etc., may extend from the top surface 272 of the CES 200. The retainer 270 may include, for example, a vertical leg 274 and a horizontal leg 276 of an L - shaped support 278. A wire support or clamp 280 may be attached to the vertical leg 274 of the L - shaped support 278 to support a portion of a disposable assembly, as described below. For example, the wire support 280 may support a tube of a disposable assembly relative to the vertical leg 274. A plurality of brackets 282 may be supported by the horizontal leg 276 of the L - shaped support 278. Each of the plurality of brackets 282 may support one or more bags in a disposable kit, as described below. For example, as Figure 2CAs shown, each of the plurality of brackets may include a rail system 284, a hook system 286, or both the rail system 284 and the hook system 286. The rail system 284 may include sidewalls 288 that extend parallel to each other and define a channel 290 therebetween. The sidewalls 288 may be formed of metal, plastic, composite material, or another suitable material. In an exemplary embodiment, the sidewalls 288 may each include a secondary sidewall or buffer on their inner surfaces that defines a neck 292 or constricted portion of the channel 290. For example, the secondary sidewall 293 may be formed of an elastic or flexible material such as rubber. In an alternative embodiment, the secondary sidewall may be formed of a non-elastic material such as metal, plastic, composite material, or another suitable material. For example, the neck 292 may be a narrower portion of the channel 290 such that the top of the bag or disposable item is retained in the channel 290 by the neck 292. The rail system 284 may be used to distribute the load of the bag supported therein and reduce tearing, stretching, or peeling of the bag material. The rail system 284 is compatible with different bag types and sizes. The bag suspended by the rail system 284 may include a rod received in the channel 290 to hold the bag therein, as described below. The rod in the bag may have a diameter greater than the width of the neck 292 defined by the secondary sidewall 293.

[0140] The bottom surface of the secondary sidewall 293 may define a groove 294 therein. The groove 294 may be located on a side of the secondary sidewall 293 adjacent to the sidewall 288. The groove 294 may be arranged to receive one or more hooks 296 of the hook system 286 therein. For example, the groove 294 in a single secondary sidewall 293 of each rail system 284 may receive the hook 296 of the hook system 286, respectively, while the groove 294 in another secondary sidewall 293 of the rail system 284 may remain open. The hook 296 may be a U-shaped or J-shaped hook and may be configured to secure the bag at a hole in the bag.

[0141] The cell expansion system 200 and / or the cell expansion machine 202 may include a barcode scanner configured to scan barcodes on disposable items, media, products, etc. used with the cell expansion system 200 and / or the cell expansion machine 202. The barcode scanner is configured to collect data from the scanned barcode and transmit the data to the computing system 2400 described herein. The barcode scanner may be attached by a solid line or wirelessly. The barcode scanner may be a handheld device or may be fixed within a user interface or on the housing of the cell expansion machine 202. The barcode scanner is compatible with reading customer barcodes, universal barcodes, vendor barcodes, or any other barcode inside or outside the market.

[0142] Next, Figure 3Shows the rear portion 206 of the cell expansion machine 202 before being detachably attached to the pre-installed fluid delivery assembly 210( Figure 2B ). Figure 3 The closable door 204 (shown in Figure 2A and Figure 2B ) is omitted. The rear portion 206 of the cell expansion machine 202 includes a plurality of different structures for cooperating with elements of the pre-installed fluid delivery assembly 210. More specifically, the rear portion 206 of the cell expansion machine 202 includes a plurality of peristaltic pumps for cooperating with the pump circuits on the pre-installed fluid delivery assembly 210, including an IC circulation pump 218, an EC circulation pump 220, an IC inlet pump 222, and an EC inlet pump 224. In addition, the rear portion 206 of the cell expansion machine 202 includes a plurality of valves, including an IC circulation valve 226, a reagent valve 228, an IC medium valve 230, an air removal valve 232, a cell inlet valve 234, a wash valve 236, a dispense valve 238, an EC medium valve 240, an IC waste or outlet valve 242, an EC waste valve 244, and a harvest valve 246. A number of sensors are also associated with the rear portion 206 of the cell expansion machine 202, including an IC outlet pressure sensor 248, a combined IC inlet pressure and temperature sensor 250, a combined EC inlet pressure and temperature sensor 252, and an EC outlet pressure sensor 254. According to an embodiment, an optical sensor 256 for the air removal chamber is also shown.

[0143] According to an embodiment, a shaft or rocker controller 258 for rotating the cell growth chamber 100 is shown. A shaft fitting 260 associated with the shaft or rocker controller 258 allows the shaft of the pipe organizer 300 (FIG. 4) of the pre-installed delivery assembly 210 or 400 to enter holes such as 424( Figure 4A ) to be properly aligned with the rear portion 206 of the cell expansion machine 202. Rotation of the shaft or rocker controller 258 causes a rotational movement of the shaft fitting 260 and the cell growth chamber 100. Thus, when an operator or user of the CES 200 attaches a new or unused pre-installed fluid delivery assembly 400( Figure 4A ) to the cell expansion machine 202, alignment is a relatively simple matter of properly orienting the shaft entry hole 424( Figure 4A ) of the pre-installed fluid delivery assembly 210 or 400 with the shaft fitting 260.

[0144] Turning Figure 4A , which shows a perspective view of the detachably attached pre-installed fluid delivery assembly 400. The pre-installed fluid delivery assembly 400 can be detachably attached to the cell expansion machine 202( Figure 2B and Figure 3) thereon to facilitate relatively quickly replacing a used pre - installed fluid delivery assembly 210 at the same cell expansion machine 202 with a new or unused fluid delivery assembly 400 in the pre - installed fluid delivery assembly 400 at the cell expansion machine 202. As Figure 4A shown, the cell growth chamber 100 can be attached to a bioreactor coupler including a fitting 402. The fitting 402 includes one or more shaft fastening mechanisms, such as a biasing arm or spring member 404 for engaging a shaft of the cell expansion machine 202 (see, e.g., 258 as Figure 3 shown).

[0145] According to an embodiment, the pre - installed fluid delivery assembly 400 includes pipes 408A, 408B, 408C, 408D, 408E, etc., and various pipe fittings to provide Figure 5A , Figure 5B , Figure 5C and Figure 6 the fluid paths shown in, as described below. Pump loops 406A, 406B, and 406C can also be provided for pumps. In an embodiment, although various media can be provided at the location where the cell expansion machine 202 is located, according to an embodiment, the pre - installed fluid delivery assembly 400 can include a sufficient pipe length to extend outside the cell expansion machine 202 and be capable of being welded to pipes associated with media bags or containers.

[0146] Referring to Figures 4B to 4H , an alternative view of the components and parts of the pre - installed fluid delivery assembly 400 is shown. In an exemplary embodiment, the pre - installed fluid delivery assembly 400 can include a media bag 410 ( Figure 4D ), a waste bag 412 ( Figure 4E ), a pressure chamber 414 ( Figures 4F to 4G ), a sampling coil 416 ( Figure 4H ), a cell input bag, an in - line filter 420 ( Figure 4I ), additional pipes, additional filters, or a combination thereof. For example, Figure 4B the pre - installed fluid delivery assembly 400A in Figure 4C can include a cell growth chamber 100B, while the pre - installed fluid delivery assembly 400B in

[0147] As Figure 4D and Figure 4EAs shown, the pre-installed fluid delivery assembly 400 can include one or more media bags 410 and one or more waste bags 412. In some embodiments, the media bags 410 can be the same as the waste bags 412. Keeping the media bags 410 the same as the waste bags 412 can improve manufacturing efficiency and reduce costs. As Figure 4D and Figure 4E shown, the media bag 410 and the waste bag 412 are formed from a sheet of material to define a material housing 422 and a hanger 425. For example, the sheet of material, the media bag 410, and the waste bag 412 may be formed of ethylene vinyl acetate (EVA), which is less breathable than PVC, to maintain a more stable pH, so the media is "happier" and the non-breathable material extends the life of the media. For example, the sheet of material can be welded or melted to form the material housing 422 and the hanger 425. The material housing 422 can be a pocket within the media bag 410 or the waste bag 412. The size of the material housing 422 can be set to accommodate a specific amount of fluid (e.g., media). For example, the size of the material housing 422 can be set to accommodate 5L of fluid. Alternatively, the size of the material housing 422 can be set to accommodate any amount of fluid suitable for the desired function.

[0148] The hanger 425 can include a hole for receiving the hook 296, as discussed with respect to Figure 2C The hanger 425 can include a rod 426 fixed within a longitudinal hole or channel 428 of the media bag 410 or the waste bag 412. The rod 426 can be a cylindrical or extruded polygonal rod that engages with the rail system 284, as discussed with respect to Figure 2C For example, when the bag is inserted into the channel 290 between the sidewalls 288, the rod 426 rests on top of the second sidewall 293 and has a diameter or width greater than that of the neck 292, such that the hanger 425 cannot slide back through the neck 292 and the media bag 410 or the waste bag 412 is suspended in the rail system 284. Additionally or alternatively, the media bag 410 can have a different material, size, or configuration than the waste bag 412.

[0149] A pair of ports 430 can be provided on a portion of the material housing 422 opposite the hanger 425. The pair of ports 430 can include an inlet port 430A and an outlet port 430B. The inlet port 430A can be engaged with a tube or fluid entering the material housing 422. The outlet port 430B can be engaged with a tube or fluid leaving the material housing 422. The pair of ports 430, or dual-port design, of the media bag 410 or the waste bag 412 allows multiple media bags 410 or multiple waste bags 412 to be connected together, or daisy-chained together, as described below.

[0150] Refer to Figure 4F and Figure 4G, the pressure chamber 414 can be provided on the housing of the pre-installed fluid delivery assembly 400. The pressure chamber 414 can be configured to detect the fluid pressure through a fluid line, pipe, or along a fluid flow path. The pressure chamber 414 can include a single-outlet housing 433, a diaphragm 432, and a retaining ring 434. The single-outlet housing 433 can include a tubular inlet port 436 and a blocking port 438. The inlet port 436 can be a tubular port configured to receive fluid entering the single-outlet housing 433. The blocking port 438 can be similar to the tubular inlet port 436, but can be blocked with a plug 440 to prevent fluid from flowing therethrough. The plug 440 can be formed of plastic or elastomer. The single-outlet housing 433 can include a body 442 integrally formed with the inlet port 436 and the blocking port 438. The body 442 can define an internal space 444 therein, such as a cylindrical internal space 444. The diaphragm 432 can be a circular planar diaphragm received within the internal space 444 of the body 442. For example, the diaphragm 432 can be an elastic material that flexes with the fluid pressure. The retaining ring 434 can be provided at the end of the body 442 of the single-outlet housing 433 opposite the inlet port 436 and the blocking port 438 to fix the diaphragm 432 within the body 442. For example, the retaining ring 434 can be press-fitted or threaded at the end of the body 442 opposite the inlet port 436 and the blocking port 438.

[0151] Figure 4H and Figure 4I illustrates an exemplary pipe that can be included in the pre-installed fluid delivery assembly 400. In at least one exemplary embodiment, Figure 4H the sampling coil 416 shown can be a pipe configured to collect a fluid sample in one of the fluid flow paths. In at least one exemplary embodiment, Figure 4I the in-line filter 420 shown can include a filter positioned within the pipe to filter materials from the fluid flowing through the fluid flow path.

[0152] Next, Figure 5A 、 Figure 5B and Figure 5C illustrates a schematic diagram of an embodiment cell expansion system 500, Figure 6 illustrates a schematic diagram of another embodiment cell expansion system 600, and Figures 7A to 7C illustrates a schematic diagram of yet another embodiment cell expansion system. In Figure 5A 、 Figure 5B 、 Figure 5C and Figure 6 the embodiments shown, as described below, cells grow in the IC space. However, the present disclosure is not limited to such examples, and in other embodiments, cells that grow in the EC space can be provided.

[0153] As previously described, Figure 5A, Figure 5B and Figure 5C show the CES 500. While Figure 5A , Figure 5B and Figure 5C describe substantially similar structural components of the CES 500, Figure 5A , Figure 5B and Figure 5C show possible operating configurations of fluid movement in a first fluid circulation path using the structural features of the CES 500 according to an embodiment of the present disclosure. As shown, according to an embodiment, the CES 500 includes a first fluid circulation path 502 (also referred to as the “intracapillary loop” or “IC loop”) and a second fluid circulation path 504 (also referred to as the “extracapillary loop” or “EC loop”). A first fluid flow path 506 may be fluidly associated with the cell growth chamber 501 to form the first fluid circulation path 502. Fluid flows into the cell growth chamber 501 through the IC inlet port 501A, through the hollow fibers of the cell growth chamber 501, and exits via the IC outlet port 501B. A manometer 510 measures the pressure of the medium leaving the cell growth chamber or bioreactor 501. The medium flows through the IC circulation pump 512, and the IC circulation pump 512 can be used to control the medium flow rate. The IC circulation pump 512 can pump fluid in a first direction or a second direction opposite to the first direction. The IC outlet port 501B can be used as an inlet in the opposite direction. For example, in a first configuration, the IC circulation pump can pump fluid in a positive direction, in which the fluid enters the IC inlet port 501A. For example, in a second configuration, the IC circulation pump can pump fluid in a negative direction, in which the fluid enters the IC outlet port 501B, for example.

[0154] The medium entering the IC loop can enter through the valve 514. As will be understood by those skilled in the art, additional valves, manometers, pressure / temperature sensors, ports, and / or other devices can be placed at various locations to separate and / or measure the properties of the medium along portions of the fluid path. Accordingly, it should be understood that the schematic diagrams shown represent one possible configuration of the various elements of the CES 500, and modifications to the schematic diagrams shown are within the scope of one or more current embodiments.

[0155] Regarding the IC loop 502, during operation, a sample of the medium can be obtained from the sample port 516 or the sample coil 518. A pressure / thermometer 520 disposed in the first fluid circulation path 502 allows the detection of the medium pressure and temperature during operation. The medium then returns to the IC inlet port 501A to complete the fluid circulation path 502. Cells grown / amplified in the cell growth chamber 501 can be flushed from the cell growth chamber 501 into the cell harvest bag 599 through the valve 598, or redistributed in the hollow fibers for further growth.

[0156] The fluid in the second fluid circulation path 504 enters the cell growth chamber 501 through the EC inlet port 501C and exits the cell growth chamber 501 via the EC outlet port 501D. The medium in the EC circuit 504 can contact the outside of the hollow fibers in the cell growth chamber 501, allowing small molecules to diffuse into and out of the hollow fibers.

[0157] According to an embodiment, the pressure / thermometer 524 provided in the second fluid circulation path 504 allows the pressure and temperature of the medium to be measured before the medium enters the EC space of the cell growth chamber 501. The pressure gauge 526 allows the pressure of the medium in the second fluid circulation path 504 to be measured after leaving the cell growth chamber 501. Regarding the EC circuit, a sample of the medium can be obtained from the sample port 530 or the sample coil during operation.

[0158] In an embodiment, after leaving the EC outlet port 501D of the cell growth chamber 501, the fluid in the second fluid circulation path 504 reaches the oxygenator or gas transfer module 532 through the EC circulation pump 528. The EC circulation pump 528 can also pump the fluid in the opposite direction. The second fluid flow path 522 can be fluidly associated with the oxygenator or gas transfer module 532 via the oxygenator inlet port 534 and the oxygenator outlet port 536. In operation, the fluid medium flows into the oxygenator or gas transfer module 532 via the oxygenator inlet port 534 and flows out of the oxygenator or gas transfer module 532 via the oxygenator outlet port 536. For example, the oxygenator or gas transfer module 532 adds oxygen to the medium in the CES 500 and removes bubbles from the medium. In various embodiments, the medium in the second fluid circulation path 504 can be in equilibrium with the gas entering the oxygenator or gas transfer module 532. The oxygenator or gas transfer module 532 can be any appropriately sized oxygenator or gas transfer device. Air or gas flows into the oxygenator or gas transfer module 532 via the filter 538 and flows out of the oxygenator or gas transfer module 532 via the filter 540. The filters 538 and 540 reduce or prevent contamination of the oxygenator or gas transfer module 532 and the associated medium. During a portion of the priming sequence, the air or gas purged from the CES 500 can be discharged to the atmosphere via the oxygenator or gas transfer module 532.

[0159] According to at least one embodiment, the medium, including cells (from the bag 562) and the fluid medium from the bag 546, can be introduced into the first fluid circulation path 502 via the first fluid flow path 506. The fluid container 562 (e.g., a cell inlet bag or a saline priming fluid for priming gases outside the system) can be fluidly associated with the first fluid flow path 506 and the first fluid circulation path 502 via the valve 564.

[0160] Fluid containers or media bags 544 (e.g., reagents) and 546 (e.g., IC media) can be fluidly associated with the first fluid inlet path 542 via valves 548 and 550 respectively, or with the second fluid inlet path 574 via valves 570 and 576 respectively. First and second sterile-sealable input prefill paths 508 and 509 are also provided. An air removal chamber (ARC) 556 can be fluidly associated with the first circulation path 502. The air removal chamber 556 can include one or more ultrasonic sensors (including an upper sensor and a lower sensor) for detecting air, fluid deficiency, and / or gas / fluid interfaces (e.g., air / fluid interfaces) at certain measurement locations within the air removal chamber 556. For example, ultrasonic sensors can be used near the bottom and / or near the top of the air removal chamber 556 to detect air, fluid, and / or air / fluid interfaces at these locations. Without departing from the spirit and scope of the present disclosure, embodiments provide for the application of many other types of sensors. For example, according to embodiments of the present disclosure, optical sensors can be used. During a portion of the prefill sequence or other scenarios, air or gas purged from the CES 500 can be discharged to the atmosphere via line 558 from air valve 560, and line 558 can be fluidly associated with the air removal chamber 556 during air removal operations.

[0161] EC media (e.g., from bag 568) or wash fluid (e.g., from bag 566) can be added to the first or second fluid flow paths. Fluid container 566 can be fluidly associated with valve 570, and valve 570 can be fluidly associated with the first fluid circulation path 502 via dispensing valve 572 and the first fluid inlet path 542. Alternatively, by opening valve 570 and closing dispensing valve 572, fluid container 566 can be fluidly associated with the second fluid circulation path 504 via the second fluid inlet path 574 and the EC inlet path 584. Similarly, fluid container 568 can be fluidly associated with valve 576, and valve 576 can be fluidly associated with the first fluid circulation path 502 via the first fluid inlet path 542 and dispensing valve 572. Alternatively, by opening valve 576 and closing dispensing valve 572, fluid container 568 can be associated with the second fluid inlet path 574.

[0162] An optional heat exchanger 552 can be provided for introducing media reagents or wash fluid.

[0163] In the IC loop, fluid can initially be propelled by the IC inlet pump 554. In the EC loop, fluid can initially be propelled by the EC inlet pump 578. An air detector 580, such as an ultrasonic sensor, can also be associated with the EC inlet path 584.

[0164] In at least one embodiment, the first fluid circulation path 502 and the second fluid circulation path 504 are connected to the waste line 588. When the valve 590 is opened, the IC medium can flow through the waste line 588 and into the waste or outlet bag 586. Similarly, when the valve 582 is opened, the EC medium can flow through the waste line 588 into the waste or outlet bag 586.

[0165] In an embodiment, cells can be harvested via the cell harvest path 596. Here, cells from the cell growth chamber 501 can be harvested by pumping the cell-containing IC medium through the cell harvest path 596 and the valve 598 to the cell harvest bag 599.

[0166] Various components of the CES 500, such as the cell expansion machine 202, can be housed or disposed within a machine or housing (Figures 2 and Figure 3 ), where the machine, for example, maintains the cells and the medium at a predetermined temperature.

[0167] In Figure 5A the configuration depicted for the CES 500, in an embodiment, the fluid medium in the first fluid circulation path 502 and the second fluid circulation path 504 flows through the cell growth chamber 501 in the same direction (co-current configuration). In another embodiment, the CES 500 can also be configured to flow in a counter-current configuration (not shown). In Figure 5A the configuration shown, the fluid in the first fluid circulation path 502 enters the bioreactor 501 from the IC inlet port 501A and exits the bioreactor 501 from the IC outlet port 501B. In Figure 5B and Figure 5C the configuration described, according to an embodiment, the fluid medium in the first circulation path 502 can flow in an opposite or relative direction from the connector 517 such that the fluid can enter the IC inlet port, the first port 501A, at one end of the bioreactor, and the fluid can enter the IC outlet port, the second port 501B, at the other end of the bioreactor to hold the cells within the bioreactor itself. The first fluid flow path can be fluidly associated with the first fluid circulation path through the connector 517. In an embodiment, the connector 517 can be a point or location through which the fluid can flow in opposite directions (e.g., based on the direction of the IC inlet pump and the direction of the IC circulation pump). In an embodiment, the connector 517 can be a T-shaped fitting or a T-shaped coupling. In another embodiment, the connector 517 can be a Y-shaped fitting or a Y-shaped coupling. The connector 517 can be any type of fitting, coupling, fusion, passage, pipe, etc. that allows the first fluid flow path to be fluidly associated with the first circulation path. It should be understood that Figure 5A 、 Figure 5B and Figure 5CThe schematic diagrams and operational configurations shown represent possible configurations of the various elements of the cell expansion system, and within the scope of one or more current embodiments, changes can be made to the schematic diagrams and operational configurations shown.

[0168] Turning Figure 6 , a schematic diagram of another embodiment of the cell expansion system 600 is shown. The CES 600 includes a first fluid circulation path 602 (also referred to as the "intracapillary loop" or "IC loop") and a second fluid circulation path 604 (also referred to as the "extracapillary loop" or "EC loop"). The first fluid flow path 606 can be fluidly associated with the cell growth chamber 601 to form the first fluid circulation path 602. Fluid flows into the cell growth chamber 601 through the IC inlet port 601A, through the hollow fibers of the cell growth chamber 601, and exits via the IC outlet port 601B. The pressure sensor 610 measures the media pressure leaving the cell growth chamber 601. In addition to pressure, in an embodiment, the sensor 610 can also be a temperature sensor that detects the media pressure and temperature during operation. The media flows through the IC circulation pump 612 that can be used to control the media flow rate. The IC circulation pump 612 can pump fluid in a first direction or a second direction opposite to the first direction. The outlet port 601B can be used as an inlet in the opposite direction. The media entering the IC loop can enter through the valve 614. As will be understood by those skilled in the art, additional valves, pressure gauges, pressure / temperature sensors, ports, and / or other devices can be placed at different locations to isolate and / or measure the properties of the media along portions of the fluid path. Thus, it should be understood that the schematic diagram shown represents one possible configuration of the various elements for the CES 600, and within the scope of one or more current embodiments, changes can be made to the schematic diagram shown.

[0169] Regarding the IC loop, during operation, a sample of the media can be obtained from the sample coil 618. The media then returns to the IC inlet port 601A to complete the fluid circulation path 602. The cells grown / expanded in the cell growth chamber 601 can be flushed from the cell growth chamber 601 through the valve 698 and the cell harvest path 697 into the cell harvest bag 699. Alternatively, when the valve 698 is closed, the cells can be re-distributed into the chamber 601 for further growth.

[0170] The fluid in the second fluid circulation path 604 enters the cell growth chamber 601 through the EC inlet port 601C and exits the cell growth chamber 601 via the EC outlet port 601D. According to an embodiment, the media in the EC loop can be in contact with the outside of the hollow fibers in the cell growth chamber 601, thereby allowing small molecules to diffuse into and out of the hollow fibers that can be located within the chamber 601.

[0171] A pressure / temperature sensor 624 disposed in the second fluid circulation path 604 allows the pressure and temperature of the medium to be measured before the medium enters the EC space of the cell growth chamber 601. A sensor 626 allows the pressure and / or temperature of the medium in the second fluid circulation path 604 to be measured after leaving the cell growth chamber 601. Regarding the EC circuit, during operation of the device, a sample of the medium can be obtained from the sample port 630 or the sample coil.

[0172] After leaving the EC outlet port 601D of the cell growth chamber 601, the fluid in the second fluid circulation path 604 reaches the oxygenator or gas transfer module 632 via the EC circulation pump 628. According to an embodiment, the EC circulation pump 628 can also pump the fluid in the opposite direction. The second fluid flow path 622 can be fluidly associated with the oxygenator or gas transfer module 632 via the inlet port 632A and the outlet port 632B of the oxygenator or gas transfer module 632. In operation, the fluid medium flows into the oxygenator or gas transfer module 632 via the inlet port 632A and leaves the oxygenator or gas transfer module 632 via the outlet port 632B. For example, the oxygenator or gas transfer module 632 adds oxygen to the medium in the CES 600 and removes bubbles from the medium in the CES 600. In various embodiments, the medium in the second fluid circulation path 604 can be balanced with the gas entering the oxygenator or gas transfer module 632. The oxygenator or gas transfer module 632 can be any appropriately sized device useful for oxygenation or gas transfer. Air or gas flows into the oxygenator or gas transfer module 632 via the filter 638 and flows out of the oxygenator or gas transfer module 632 via the filter 640. The filters 638 and 640 reduce or prevent contamination of the oxygenator or gas transfer module 632 and the associated medium. During a portion of the priming sequence, air or gas purged from the CES 600 can be vented to the atmosphere via the oxygenator or gas transfer module 632.

[0173] In the configuration described for the CES 600, the fluid media in the first fluid circulation path 602 and the second fluid circulation path 604 flow through the cell growth chamber 601 in the same direction (co-current configuration). According to an embodiment, the CES 600 can also be configured to flow in a counter-current configuration.

[0174] According to at least one embodiment, a medium including cells (from a source such as a cell container e.g., a bag) can be attached at attachment point 662, and a fluid medium from the medium source can be attached at attachment point 646. The cells and the medium can be introduced into the first fluid circulation path 602 via the first fluid flow path 606. The attachment point 662 can be fluidly associated with the first fluid flow path 606 via valve 664, and the attachment point 646 can be fluidly associated with the first fluid flow path 606 via valve 650. A reagent source can be fluidly connected to point 644 and associated with the first fluid inlet path 642 via valve 648, or associated with the second fluid inlet path 674 via valves 648 and 672.

[0175] An air removal chamber (ARC) 656 can be fluidly associated with the first circulation path 602. The air removal chamber 656 can include one or more sensors (including an upper sensor and a lower sensor) to detect the absence of air, fluid, and / or a gas / fluid interface (e.g., an air / fluid interface) at certain measurement locations within the air removal chamber 656. For example, ultrasonic sensors can be used near the bottom and / or near the top of the air removal chamber 656 to detect air, fluid, and / or the air / fluid interface at these locations. Embodiments provide for the use of many other types of sensors without departing from the spirit and scope of the present disclosure. For example, according to embodiments of the present disclosure, optical sensors can be used. During a portion of a priming sequence or other scenarios, air or gas purged from the CES 600 can be discharged to the atmosphere via line 658 from air valve 660, which can be fluidly associated with the air removal chamber 656.

[0176] An EC medium source can be attached to the EC medium attachment point 668, and a wash solution source can be attached to the wash solution attachment point 666 to add the EC medium and / or the wash solution to the first or second fluid flow path. The attachment point 666 can be fluidly associated with valve 670, which can be fluidly associated with the first fluid circulation path 602 via valve 672 and the first fluid inlet path 642. Alternatively, the attachment point 666 can be fluidly associated with the second fluid circulation path 604 via the second fluid inlet path 674 and the second fluid flow path 684 by opening valve 670 and closing valve 672. Similarly, the attachment point 668 can be fluidly associated with valve 676, which can be fluidly associated with the first fluid circulation path 602 via the first fluid inlet path 642 and valve 672. Alternatively, by opening valve 676 and closing distribution valve 672, the attachment point 668 can be fluidly associated with the second fluid inlet path 674.

[0177] In the IC circuit, fluid can initially be propelled by IC inlet pump 654. In the EC circuit, fluid can initially be propelled by EC inlet pump 678. An air detector 680, such as an ultrasonic sensor, can also be associated with the EC inlet path 684.

[0178] In at least one embodiment, the first fluid circulation path 602 and the second fluid circulation path 604 are connected to the waste line 688. When valve 690 is opened, the IC medium can flow through the waste line 688 and into the waste or outlet bag 686. Similarly, when valve 692 is opened, the EC medium can flow into the waste or outlet bag 686.

[0179] After the cells have grown in the cell growth chamber 601, they can be harvested via the cell harvest path 697. Here, the cells from the cell growth chamber 601 can be harvested by pumping the IC medium containing the cells through the cell harvest path 697 to the cell harvest bag 699 with valve 698 open.

[0180] Various components of the CES 600, such as the cell expansion machine 202, can be housed or disposed in a machine or housing, (Figure 2 and Figure 3 ), where the machine, for example, maintains the cells and the medium at a predetermined temperature. Further note that, in an embodiment, the components of the CES 600 and the CES 500 can be combined. In other embodiments, the CES can include fewer or additional components than those shown in the CES 500 and / or the CES 600 and still be within the scope of the present disclosure. An example of a cell expansion system that can incorporate the features of the present disclosure is manufactured by Terumo BCT, Inc. of Lakewood, Colorado cell expansion system.

[0181] It should be understood that Figure 6 the schematic diagrams shown represent possible configurations of the various elements for a cell expansion system, and within the scope of one or more embodiments, the shown schematic diagrams can be altered.

[0182] Examples and further descriptions of cell expansion systems are provided in U.S. Patent No. 8,309,347 ("Cell Expansion System and Method of Use", published November 13, 2012) and U.S. Patent No. 9,057,045, filed December 15, 2010 ("Method of Loading and Distributing Cells in a Bioreactor of a Cell Expansion System", published June 16, 2015), the entire teachings and all purposes of which are incorporated herein by reference in their entirety.

[0183] In Figures 7A to 7CIn the illustrated embodiments, and as described below, cells grow in the IC space. However, the present disclosure is not limited to such examples, and in other embodiments, cells that grow in the EC space may be provided.

[0184] As previously described, Figures 7A to 7C CES 700 is shown. Although Figures 7A to 7C substantially similar structural components of CES 700 are depicted, Figures 7A to 7C a possible operational configuration of fluid movement in a first fluid circulation path using the structural features of CES 700 according to an embodiment of the present disclosure is shown. As shown, according to an embodiment, CES 700 includes a first fluid circulation path 702 (also referred to as the "intracapillary loop" or "IC loop") and a second fluid circulation path 704 (also referred to as the "extracapillary loop" or "EC loop"). A first fluid flow path 706 may be fluidly associated with a cell growth chamber 701 to form the first fluid circulation path 702. Fluid is configured to flow into the cell growth chamber 701 through an IC inlet port 701A, through hollow fibers in the cell growth chamber 701, and exit via an IC outlet port 701B. A pressure gauge 710 measures the pressure of the medium leaving the cell growth chamber 701 or bioreactor. The medium flows through an IC circulation pump 712, which can be used to control the rate of medium flow. The IC circulation pump 712 can pump fluid in a first direction or a second direction opposite to the first direction. In at least one exemplary embodiment, the IC outlet port 701B can be used as an inlet in the opposite direction. For example, in a first configuration, the IC circulation pump 712 can pump fluid in a positive direction, in which the fluid enters the IC inlet port 701A. In a second configuration, for example, the IC circulation pump 712 can pump fluid in a negative direction, in which the fluid enters the IC outlet port 701B.

[0185] The fluid in the IC loop or the first fluid circulation path 702 can pass through an IC circulation valve 714. As will be understood by those skilled in the art, additional valves, pressure gauges, pressure / temperature sensors, ports, and / or other devices can be placed at various locations to isolate and / or measure the properties of the medium along portions of the fluid path. For example, the first fluid circulation path 702 can include an IC inlet pressure sensor 715. In some exemplary embodiments, the IC inlet pressure sensor 715 can be positioned between the IC circulation pump 712 and the IC circulation valve 714. Thus, it should be understood that the illustrated schematic represents one possible configuration of the various elements for CES 700, and within the scope of one or more exemplary embodiments, changes can be made to the illustrated schematic.

[0186] Regarding the first fluid circulation path 702, a sample of the medium can be obtained from the sample port or the sample loop 718 during operation. The pressure / thermometer 720 provided in the first fluid circulation path 702 allows the detection of the pressure and temperature of the medium during operation. The medium then returns to the IC inlet port 701A to complete the first fluid circulation path 702. Cells grown and / or amplified in the cell growth chamber 701 can be flushed out of the cell growth chamber 701 and enter the harvest bag 799 through the valve 798, or redistributed within the hollow fibers for further growth.

[0187] The fluid in the second fluid circulation path 704 enters the cell growth chamber 701 via the EC inlet port 701C and exits the cell growth chamber 701 via the EC outlet port 701D. The medium in the second fluid circulation path 704 can contact the outside of the hollow fibers in the cell growth chamber 701, allowing small molecules to diffuse into and out of the hollow fibers.

[0188] In at least one exemplary embodiment, the pressure / thermometer 724 provided in the second fluid circulation path 704 allows the measurement of the pressure and / or temperature of the medium before the medium enters the EC space of the cell growth chamber 701. The pressure gauge 726 allows the measurement of the pressure of the medium in the second fluid circulation path 704 after leaving the cell growth chamber 701. Regarding the EC circuit, a sample of the medium can be obtained from the sample port 730 or the sample loop during operation.

[0189] 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 circulation path 704 passes through the EC circulation pump 728 and reaches the oxygenator or gas transfer module 732. The EC circulation pump 728 can also pump the fluid in the opposite direction. The second fluid flow path 722 can be fluidly associated with the oxygenator or gas transfer module 732 via the oxygenator inlet port 734 and the oxygenator outlet port 736. In operation, the fluid medium flows into 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, for example, adds oxygen to the medium in the CES 700 and removes bubbles from the medium. In various exemplary embodiments, the medium in the second fluid circulation path 704 can be in equilibrium with the gas entering the oxygenator or gas transfer module 732. The oxygenator or gas transfer module 732 can be any appropriately sized oxygenator or gas transfer device. Air or gas flows into the oxygenator or gas transfer module 732 through the filter 738 and flows out of the oxygenator or gas transfer device 732 through the filter 740. The filters 738 and 740 reduce or prevent contamination of the oxygenator or gas transfer module 732 and the associated medium. During a portion of the priming sequence, air or gas purged from the CES 700 can be discharged to the atmosphere through the oxygenator or gas transfer module 732.

[0190] According to at least one embodiment, the medium (including cells from the bag 762) and the fluid medium from the bag 746 can be introduced into the first fluid circulation path 702 via the first fluid flow path 706. The fluid container 762 (e.g., a cell inlet bag or a saline priming fluid for priming air out of the system) can be fluidly associated with the first fluid flow path 706 and the first fluid circulation path 702 via the valve 764.

[0191] Fluid containers or media bags 744 (e.g., reagents) and 746 (e.g., IC media) can be fluidly associated with the first fluid inlet path 742 via valves 748 and 750, respectively, or with the second fluid inlet path 774 via valves 770 and 776. An air removal chamber (ARC) 756 can be fluidly associated with the first circulation path 702. The air removal chamber 756 can include one or more ultrasonic sensors (including an upper sensor and a lower sensor) to detect the absence of air, fluid, and / or gas / fluid interfaces (e.g., air / fluid interfaces) at certain measurement locations within the air removal chamber 756. For example, ultrasonic sensors can be used near the bottom and / or near the top of the air removal chamber 756 to detect air, fluid, and / or air / fluid interfaces at these locations. Without departing from the spirit and scope of the present disclosure, exemplary embodiments provide for the use of many other types of sensors. For example, according to embodiments of the present disclosure, optical sensors can be used. During a portion of the priming sequence or other scenarios, air or gas purged from the CES 700 can be discharged to the atmosphere through an air valve 760, which can be fluidly associated with the air removal chamber 756..

[0192] EC media (e.g., from bag 768) and / or a wash solution (e.g., from a bag or fluid container 766) can be added to the first fluid inlet path 742 and / or the second fluid flow path 722. The fluid container 766 can be fluidly associated with a valve 770, which can be fluidly associated with the first fluid circulation path 702 via a dispensing valve 772 and the first fluid inlet path 742. Alternatively, by opening the valve 770 and closing the dispensing valve 772, the fluid container 766 can be fluidly associated with the second fluid circulation path 704 via the second fluid inlet path 774 and an EC inlet path 784. Similarly, as Figure 7C shown, fluid containers 768A and 768B can be fluidly associated with a valve 776, which can be fluidly associated with the first fluid circulation path 702 via the first fluid inlet path 742 and the dispensing valve 772. Alternatively, by opening the valve 776 and closing the dispensing valve 772, the fluid containers 768A and 768B can be fluidly associated with the second fluid inlet path 774.

[0193] Fluid containers 768A and 768B can be media containers for dispensing media into the first fluid inlet path 742 or the second fluid inlet path 774. In some exemplary embodiments, fluid containers 768A and 768B can be associated with Figure 4Dis the same as the media bag 410 shown. The fluid containers 768A and 768B can be part of the EC circulation supply loop 752 when in communication with the second fluid inlet path 774 (or can be part of the IC circulation supply loop when in communication with the first fluid inlet path 742). In the EC circulation supply loop 752, the media is transferred from the fluid container 768B to the fluid container 768A through the EC loop 704, through the EC waste valve 782, and back to the fluid container 768B. Using the EC circulation supply loop 752 avoids the use of pass-through profusion where fresh media is continuously added to the system and collected in an outlet waste bag that is discarded when full. With pass-through profusion, as more cells grow, the media must be introduced at a higher rate to feed the cells. For example, a user typically reads the glucose or lactate readings once a day and then increases the feed rate for that day. The feed rate throughout the day may not be correct, resulting in wasted media.

[0194] In contrast, in the CES 700 herein, the EC circulation supply loop 752 increases the volume of the IC circulation loop or the EC circulation loop by connecting or daisy-chaining the fluid container 768A to the fluid container 768B (through the pair of ports previously discussed). For the CES 700, the cell growth chamber 701 is manually fed outside of the CES 700. The outlet line 788 returns to the fluid container 768B and the media is recycled. The user can calculate how much lactate is produced to determine the waste. The user does not need to change the bag; they only set the feed rate and run the protocol. Thus, the CES 700 is more efficient and uses less total media.

[0195] Although two fluid containers 768A and 768B are shown and discussed, it should be understood that more fluid containers 768N can be added to the system.

[0196] In an exemplary scenario, the gas is supplied to the system via a GTM (such as GTM 732) using an EC loop. The gas is supplied externally to the CES 700, and each time the medium passes through the GTM 732, the gas is exchanged to the desired concentration. In this case, the fluid containers 768A and 768B are used as exchange vessels for the medium returning to the cell growth chamber 701. For example, the attached T-cell feeding protocol (CFA-CSS-CEdj-036-03) uses approximately 11 L of EC medium and 3.4 L of IC medium during the growth protocol. Using this new feeding strategy, the user attaches 3.4 L of IC medium to the IC medium line and a daisy chain of 11 L (3 medium bags) of EC medium and connects it to the EC inlet line and outlet line. Just as the EC loop rate needs to increase as the oxygen demand of the cell population increases, the EC inlet rate (which is actually the EC feeding loop) also increases.

[0197] Depending on the cell type and protocol dynamics, an IC feeding loop or an EC feeding loop can be implemented. Alternatively, a dual feeding loop can be used, such as as Figure 7C shown.

[0198] This strategy allows for the implementation of a large number of possible feeding strategies. This strategy also improves the ease of use of the CES 700, reducing the interaction between the human and the device, as there is no need to empty the waste bag and new medium bags are provided to the machine at different time intervals during cell growth. Additionally, in many cases, there is no need to determine the feeding rate. Even when using continuous perfusion feeding, the lactate and glucose concentrations may fluctuate significantly without the need to use very long custom tasks to change the flow rate in small increments.

[0199] Depending on the desired application, the EC loop feeding circuit 752 can be used to coat the fibers of the cell growth chamber 701 to circulate and push the fluid through the membrane (on the IC side). Additionally or alternatively, the EC loop feeding circuit 752 can be used for countercurrent techniques to push the fluid at opposite ends of the cell growth chamber 701.

[0200] Passive coating models for use in CES systems may require applying a coating agent to the cell growth surface to promote cell adhesion and subsequent expansion of adherent cell lines such as human mesenchymal stromal cells (hMSCs). Example coating agents are human fibronectin (hFN) or cryoprecipitate (CPPT). The bioreactor coating protocol for the passive coating model loads the coating agent inside the capillary of the bioreactor and circulates the coating agent in the IC circulation loop for at least 16 hours. The passive coating model protocol requires at least two CES systems to immediately start additional expansion of the cell population harvested from the CES system (hMSCs cannot be stored in a non-frozen state for up to 16 hours). A new protocol or method for coating the fibers of the cell growth chamber 701 results in successful coating of the growth surface 10 minutes after loading the coating agent into the IC circulation loop. This method (protocol in Appendix B) utilizes forward ultrafiltration of the fluid (moving the fluid from the IC side of the bioreactor to the EC side of the bioreactor) to reduce the time required for the appropriate chemical reaction between the coating agent and the growth surface of the bioreactor. The molecular barrier created by the specific structure of the hollow fibers in the bioreactor prevents the coating agent from passing through the fiber wall along with the fluid in which it is suspended. Using forward ultrafiltration to move the fluid "actively" promotes the coating agent to the surface of the hollow fibers. Allowing the user to coat the hollow fiber bioreactor and load cells on the same day reduces errors, saves time, and reduces the cell expansion duration by one day. This also allows customers who only access a single CES device (such as the device) to harvest the cell population from the system and coat new disposable items for subsequent passage / expansion of the same cell population without cryopreservation.

[0201] Using a dual-port bag, as referenced in Figure 4D and Figure 4E described, the inlet line (usually the "wash" line) can be connected to one port of the bag, while the "waste" line can be connected to the other port. This allows the protein carrier fluid containing the coating reagent (usually phosphate buffered saline (PBS)) to be recycled through the system as long as the user desires. When compared to the passive model of coating, using a high IC inlet rate to drive contact between the coating solution and the HFB membrane via ultrafiltration (UF) drives the adsorption of the coating reagent and reduces the time required to coat the hollow fiber bioreactor. Moving the IC fluid across the HFB membrane also enhances the deposition of the coating reagent on the IC side of the HFB membrane. For example, since the coating solution has a higher molecular weight and cannot pass through the HFB membrane, it deposits on the IC side of the HFB membrane.

[0202] An optional heat exchanger may be provided for introducing media reagents or wash solutions. In some embodiments, the optional heat exchanger may be disposed in the first fluid inlet path 742 and / or the second fluid inlet path 774.

[0203] It may include one or more differential pressure sensors for real-time pressure monitoring and alarming, providing more effective and accurate measurements as opposed to manual measurements. The pressure sensors help detect flow reduction or stoppage, gas supply depletion, user errors, etc. When a flow reduction or stoppage is detected, the differential pressure sensor triggers an alarm to alert the user. For example, the alarm can be a remote alarm, an audio alarm on the device, a visual reminder on the device, or a combination thereof. For example, the remote alarm can be an email, a text message, or other digital reminder sent to the user.

[0204] It may include one or more gas regulators for gas management. For example, the gas regulator can be inside the CES 700 so that it is not facing the user, thus preventing errors. The gas regulator can be less sensitive to movement, thus providing fine control. When an error in gas management is detected, the gas regulator can trigger an alarm to alert the user. For example, the alarm can be a remote alarm, an audio alarm on the device, a visual reminder on the device, or a combination thereof. For example, the remote alarm can be an email, a text message, or other digital reminder sent to the user.

[0205] It may include one or more temperature thermistors for high-sensitivity temperature detection. In cell culture, a 0.5-degree temperature change is crucial. Compared with a resistive temperature detector (RTD), the thermistor has less degradation, so it can allow more use before maintenance is required. When a temperature change outside a predetermined range or higher or lower than a predetermined threshold is detected, the thermistor can trigger an alarm to alert the user. For example, the alarm can be a remote alarm, an audio alarm on the device, a visual reminder on the device, or a combination thereof. For example, the remote alarm can be an email, a text message, or other digital reminder sent to the user.

[0206] In the IC loop, the fluid can be initially propelled by the IC inlet pump 754. In the EC loop, the fluid can be initially propelled by the EC inlet pump 778. An air detector 780, such as an ultrasonic sensor, can also be associated with the EC inlet path 784. When the air detector 780 detects air, the air detector can trigger an alarm to alert the user. For example, the alarm can be a remote alarm, an audio alarm on the device, a visual reminder on the device, or a combination thereof. For example, the remote alarm can be an email, a text message, or other digital reminder sent to the user.

[0207] In at least one embodiment, the first fluid circulation path 702 and the second fluid circulation path 704 are connected to the outlet pipeline 788. When the valve 790 is opened, the IC medium can flow through the outlet pipeline 788 and return to the fluid container 768B. Similarly, when the valve 782 is opened, the EC medium can flow through the outlet pipeline 788 back to the fluid container 768B.

[0208] In an exemplary embodiment, cells can be harvested via a cell harvest path 796. Here, cells from the cell growth chamber 701 can be harvested by pumping the IC medium containing the cells through the cell harvest path 796 and valve 798 into the cell harvest bag 799.

[0209] Various components of the CES 700, such as the cell expansion machine 202, can be included or housed within a machine or housing ( Figure 2A 、 Figure 2B and Figure 3 ), wherein the machine maintains the cells and the medium at a predetermined temperature, for example.

[0210] In an embodiment, in the configuration depicted for the CES 700 in Figure 7A , the fluid medium in the first fluid circulation path 702 and the second fluid circulation path 704 flows through the cell growth chamber 701 in the same direction (co-current configuration). In another embodiment, the CES 700 can also be configured to flow in a counter-current configuration (not shown). In the configuration shown in Figure 7A , the fluid in the first fluid circulation path 702 enters the cell growth chamber 701 at the IC inlet port 701A and exits the cell growth chamber 701 at the IC outlet port 701B. According to an embodiment, in an alternative configuration, the fluid medium in the first circulation path 702 can flow in the opposite or relative direction from the connector 717, such that the fluid can enter the IC inlet port, the first port 701A, on one end of the cell growth chamber 701, and the fluid can enter the IC outlet port, the second port 701B, on the opposite end of the cell growth chamber 701 to hold the cells within the bioreactor itself. The first fluid flow path can be fluidly associated with the first fluid circulation path through the connector 717. In an embodiment, the connector 717 can be a point or location from which the fluid can flow in opposite directions (e.g., based on the direction of the IC inlet pump and the direction of the IC circulation pump). In an embodiment, the connector 717 can be a T-shaped fitting or T-shaped coupling. In another embodiment, the connector 717 can be a Y-shaped fitting or Y-shaped coupling. The connector 717 can be any type of fitting, coupling, fusion, passage, pipe, etc. that allows the first fluid flow path to be fluidly associated with the first circulation path. It should be understood that Figures 7A to 7C the schematic diagrams and operating configurations shown in

[0211] Reference Figure 7B, showing the CES 700B. The CES 700B can be similar to the CES 700A and may include components that are the same as or similar to those of the CES 700A, where the same or similar components are designated with the same reference numerals. The CES 700B may include an IC circulation supply loop 753. As previously described, the IC circulation supply loop 753 can be similar to the EC circulation supply loop 752. The IC circulation supply loop 753 may include a plurality of fluid containers, such as fluid containers 746A and 746B. Fluid containers 746A and 746B can be the same as Figure 4D the media bags 410 shown in

[0212] Fluid containers 746A and 746B can be media containers for distributing media into the first fluid flow path 706 through the IC media line 746. In the IC circulation supply loop 753, the media passes through the IC loop 702, through the cell growth chamber 701, through the IC waste valve 790, and returns to the fluid container 746B. Using the IC circulation supply loop 753 avoids using pass-through profusion, in which fresh media is continuously added to the system and collected in an outlet waste bag that is discarded when full. With pass-through profusion, as more cells grow, the media must be introduced at a higher rate to feed the cells. The user typically reads the glucose or lactate readings once a day and then increases the feeding rate for that day. The feeding rate throughout the day may not be correct, resulting in wasted media.

[0213] In contrast, in the CES 700 herein, by connecting or daisy-chaining the fluid container 746A to the fluid container 746B (through the pair of ports previously described), the IC circulation supply loop 753 increases the volume of the IC circulation loop. For the CES 700, the cell growth chamber 701 is manually fed outside the CES 700. The outlet line 788 returns to the fluid container 746B and the media is recycled. The user can calculate how much lactate is produced to determine the waste. The user does not need to change the bags; they only set the feeding rate and run the protocol. Thus, the CES 700 is more efficient and uses less total media.

[0214] Although two fluid containers 746A and 746B are shown and discussed, it should be understood that more fluid containers 746N can be added to the system.

[0215] This strategy allows for the implementation of a large number of possible feeding strategies. This strategy also enhances the ease of use of the CES 700 by reducing human-device interaction, as there is no need to empty waste bags and new media bags are provided to the machine at different time intervals during cell growth. In many cases, there is also no need to determine the feeding rate. Even when using continuous perfusion feeding, lactate and glucose concentrations may fluctuate significantly without the need to use very long custom tasks to change the flow rate in small increments.

[0216] Depending on the desired use, the IC circulation feeding circuit 753 can be used to coat the fibers of the cell growth chamber 701 to circulate and push fluid through the membrane (on the IC side). Additionally or alternatively, the IC circulation feeding circuit 753 can be used in a countercurrent technique to push fluid at opposite ends of the cell growth chamber 701.

[0217] May include one or more differential pressure sensors for real-time pressure monitoring and alarming, providing more effective and accurate measurements as opposed to manual measurements. The pressure sensors help detect reduced or stopped flow, depleted gas supply, user errors, etc. When a reduced or stopped flow is detected, the differential pressure sensor triggers an alarm to alert the user. For example, the alarm can be a remote alarm, an audio alarm on the device, a visual reminder on the device, or a combination thereof. For example, a remote alarm can be an email, text message, or other digital reminder sent to the user.

[0218] May include one or more gas regulators for gas management. For example, the gas regulator can be inside the CES 700 so that it is not facing the user, thus preventing errors. The gas regulator can be less sensitive to movement, providing fine control. When an error in gas management is detected, the gas regulator can trigger an alarm to alert the user. For example, the alarm can be a remote alarm, an audio alarm on the device, a visual reminder on the device, or a combination thereof. For example, a remote alarm can be an email, text message, or other digital reminder sent to the user.

[0219] May include one or more temperature thermistors for high-sensitivity temperature detection. In cell culture, a temperature change of 0.5 degrees is crucial. Compared to a resistive temperature detector (RTD), the thermistor has less degradation and thus allows for more use before maintenance is required. When a temperature change outside a predetermined range or above or below a predetermined threshold is detected, the thermistor can trigger an alarm to alert the user. For example, the alarm can be a remote alarm, an audio alarm on the device, a visual reminder on the device, or a combination thereof. For example, a remote alarm can be an email, text message, or other digital reminder sent to the user.

[0220] Now refer to Figure 7C, showing the CES 700C with a dual supply circulation loop. The CES 700C may be similar to the CES 700A and CES 700B and may include the same or similar components as the CES 700A and CES 700B, where the same or similar components are denoted by the same reference numerals. The CES 700C may include the EC circulation supply loop 752 from the CES 700A and the IC circulation supply loop 753 from the CES 700B. The IC circulation supply loop 753 may transfer fluid from the fluid container 746B to the fluid container 746A, to the first fluid inlet path 742, to the cell growth chamber 701, to the harvest valve 798, and back to the fluid container 746B. The EC circulation supply loop 752 may transfer fluid from the fluid container 768B to the fluid container 768A, to the EC medium valve 776, to the second fluid inlet path 774, to the GTM 732, to the cell growth chamber 701, to the EC waste valve 782, and back to the fluid container 768B.

[0221] Although various exemplary embodiments of the cell expansion system and methods associated therewith have been described, Figure 8 illustrates an example of the operational steps 756 of a process for expanding non - adherent or suspended cells in a cell expansion system, such as a CES 500, CES 600, or CES 700A - CES 700C, according to an embodiment of the present disclosure.

[0222] Start the start operation 758, and the process 756 proceeds to prepare cells 760. In an embodiment, preparing cells 760 may involve multiple different and optional steps. For example, cells may be collected 762. Collecting cells 762 may involve separating and collecting cells from a source or a blood source or a donor or a patient or a subject, where the terms are used interchangeably herein. In some embodiments, a apheresis procedure (e.g., leukapheresis) may be performed to collect a volume of lymphocytes from a donor's peripheral blood. The volume of lymphocytes may include the target cell population to be expanded by the process 756. In other embodiments, cells may be collected from umbilical cord blood.

[0223] After collection 762, optionally, cell separation 764 can be part of preparation 760. The volume of cells collected at step 762 can include multiple different cell types, including the cells that are the target for expansion. Optional step 764 can be performed to isolate the target cells. As an example, the target cells can be T cells, for example, regulatory T cells. In one embodiment, the regulatory T cells can be CD4+CD25+ T cells. Any suitable separation technique can be used to separate the cells. For example, immunomagnetic separation can be used to separate the cells, where magnetic beads functionalized with an antibody are contacted with the cells collected at 762. The functionalized beads can preferentially attach to the target cell population. Then a magnetic field can be used to hold the particles with the attached target cell population while other cells can be removed.

[0224] Optionally, after separation 764, the cells 766 can be resuspended. In an embodiment, the cells can be resuspended in a medium that includes a variety of nutrients and / or reagents that help maintain cell viability. In an embodiment, the medium can include at least serum albumin and a reagent such as a cytokine. In an embodiment, the cytokine can be a recombinant human IL-2 cytokine. In one embodiment, the medium can include the cytokine at a concentration of 200 IU / mL.

[0225] After preparing the cells 760, process 756 proceeds to expose the cells 768 to activate the cells for expansion. Optionally, in 770, the cells can be exposed to a soluble activator. An activator that can include a soluble antibody, an antibody complex, or an antibody-bead conjugate can be added to the medium in which the cells are resuspended. In an embodiment, the activator can be a human antibody CD3 / CD28 / CD2 cell activator complex. In some embodiments, the activator can be included in the medium used to resuspend the cells 766. Optionally, at 772, the cells can be exposed to beads that have an activator on their surface. In an embodiment, exposing the cells to the beads can involve adding a predetermined amount of beads to the resuspended cells. The beads can be added in different ratios relative to the number of cells. For example, the beads can be added at a ratio of 1 bead:2 cells. Other embodiments can be provided where the beads are added in different ratios, such as 1 bead:1 cell, 1 bead:3 cells, etc. The beads can have an antibody on their surface to activate the cells for expansion. In an embodiment, the beads can include antibody CD3 / CD28 on their surface. In other embodiments, the antibody for activation can be coated on the surface of the bioreactor.

[0226] Process 756 proceeds to cell expansion 774. As part of the cell expansion at 774, cells can be loaded into a cell growth chamber (e.g., a hollow fiber membrane bioreactor) where the cells are expanded. Nutrients can be supplied to the cells at 776 to facilitate their expansion. For example, a medium can be delivered into the cell growth chamber to provide nutrients for expansion. For example, as discussed with respect to Figures 7A to 7C the medium can be provided via the IC circulation supply loop 753, the EC circulation supply loop 752, or a combination thereof. The expansion of the cells 774 can also include periodically adding a reagent 778 to the cell growth chamber to continue to facilitate cell expansion. For example, in some embodiments, a reagent (e.g., a cytokine) can be added to the cell growth chamber to promote the expansion of the cells. In one embodiment, the reagent can be an additional IL-2 cytokine (e.g., recombinant human IL-2 cytokine).

[0227] For example, as discussed with respect to Figures 7A to 7C the medium, reagent, or a combination thereof can be delivered to the cells from one or more connected fluid containers, and the medium, reagent, or a combination thereof can be circulated and recycled via the IC circulation supply loop 753 or the EC circulation supply loop 752.

[0228] In addition, as part of the expansion of the cells 774, the environment within the cell growth chamber can be controlled at 780. For example, gases can be continuously delivered and exchanged to provide a balance of, for example, carbon dioxide and oxygen to the cells expanding within the cell growth chamber. Additionally, the temperature can be controlled within a range optimized for cell expansion. The expansion of the cells 774 can also include monitoring metabolites at 782. For example, lactate and glucose levels can be monitored periodically. Lactate and glucose levels, as well as other metabolites and nutrients, can be monitored by one or more sensors. The one or more sensors can also be configured to measure gas (such as oxygen) levels. An increase or decrease in metabolites can prompt a change (e.g., additional feeding, additional reagent addition, additional gas exchange, etc.) to control the environment within the cell growth chamber at 780.

[0229] When monitoring various parameters, a data reading that falls outside a predetermined threshold can trigger an alarm to alert the user. For example, the data reading can be temperature, door position, pressure, flow rate, or concentration. For example, the alarm can be a remote alarm, an audible alarm on the device, a visual reminder on the device, or a combination thereof. For example, the remote alarm can be an email, a text message, or other digital reminder sent to the user.

[0230] Process 756 then proceeds to harvesting cells at 784. Optionally, further processing or other analysis of the removed cells is performed at step 786. For example, the cells can be characterized to determine the cell phenotype. Further processing or other analysis at 786 can include performing flow cytometry, for example to characterize the cell phenotype. Then, process 756 can terminate at end operation 788. If no further processing / analysis is desired, process 756 terminates at end operation 788.

[0231] Figure 9A The operational steps of process 800 according to an embodiment of the present disclosure are shown, which can be used to localize cells or other materials (e.g., proteins, nutrients, growth factors) into a cell growth chamber. In an embodiment, process 800 can be implemented as part of a "loading cells centrally without cycling" task. Start operation 802 is initiated, and process 800 proceeds to step 804, in which a first volume of fluid having cells can be loaded into the cell growth chamber of the cell expansion system. In an embodiment, the cells can include non-adherent cells, such as one or more types of T cells. In one embodiment, the plurality of cells includes Tregs. As can be understood, the loading of the first volume of fluid having cells can be performed by components of a cell expansion system such as the aforementioned system CES 500 (e.g. Figure 5A ), CES 600 ( Figure 6 ), and CES 700 ( Figures 7A to 7C ). Figure 9B A portion of the cell expansion system is shown, which includes a first fluid inlet pump 840, a first fluid flow path 860, a first fluid circulation pump 848, a first fluid circulation path 852, a cell growth chamber 844, and a second fluid circulation path 854. The first fluid flow path 860 is fluidly associated with the fluid circulation path 852 through a connector 860B. An embodiment can utilize the first fluid inlet pump 840 to load a first volume of fluid having cells into the first fluid circulation path 852 through the first fluid flow path 860 at 804. In an embodiment, the first volume of fluid is loaded without activating the first fluid circulation pump 848. The first volume of fluid is provided at least in part from fluid container 746A, fluid container 746B, IC fluid container 746, or a combination thereof. For example, the first volume of fluid can be circulated through the IC circulation supply loop 753.

[0232] As Figure 9BAs shown, the volume of the first fluid circulation path 852 can be composed of a plurality of volumes of its parts. For example, the first part of the volume can be the capillary inner space of the cell growth chamber 844 (when the cell growth chamber is a hollow fiber membrane bioreactor). The second part of the volume can be from the connector 860B to the inlet port 844A of the cell growth chamber 844. The third part can be from the connector 860B to the outlet port 844B of the cell growth chamber 844.

[0233] The process 800 proceeds to loading a second volume of fluid 806. The second volume of fluid can include a medium and can be introduced into a part of the first fluid flow path 860. In an embodiment, the second volume can be a selected predetermined amount to position 808 the first volume into the first part of the cell growth chamber 844. In an embodiment, the first volume of fluid and the second volume of fluid can be the same. In other embodiments, the first volume of fluid and the second volume of fluid can be different. In still other embodiments, the sum of the first volume of fluid and the second volume of fluid can be equal to a percentage of the volume of the first fluid circulation path (e.g., path 852 ( Figure 9B ))).

[0234] The second volume of fluid can be provided at least in part from the fluid container 768A, the fluid container 768B, the EC fluid container 768, or a combination thereof. For example, the second volume of fluid can be circulated through the IC circulation supply loop 753. For example, the second volume of fluid can be provided from two or more connected fluid containers, as referred to Figures 7A to 7C as described.

[0235] To position the first volume of fluid, the second volume of fluid must be sufficient to push the first volume into the desired position in the cell growth chamber 844. Thus, in an embodiment, the second volume of fluid can be approximately as large as the volume of the first fluid circulation path 852 between the connector 860B and the inlet port 844A. As can be understood, this will push the first volume of fluid with cells into the position within the cell growth chamber 844.

[0236] In other embodiments, a first volume of fluid (with cells) can be positioned 808 around a central region 866 of the cell growth chamber 844. In these embodiments, the second volume can be approximately the sum of the volume of the first fluid circulation path 852 between the connector 860B and the inlet port 844A and the volume of the first fluid circulation path formed by the cell growth chamber 844 (e.g., the volume of the intracapillary space), which is not occupied by the first volume of fluid when positioned within the cell growth chamber. For example, in one embodiment, the first volume of fluid (with cells) can be 50 mL. The cell growth chamber can have a volume of, for example, 124 mL. When the first volume is positioned around the central region 866, it will occupy 50 mL around the central region 866, leaving 74 mL on either side of the central region 866. Thus, 50% (or 37 mL) of 74 mL can be added to the volume between the connector 860B and the inlet port 844A to position the 50 mL first volume around the central region 866.

[0237] In embodiments, positioning 818 the first volume can involve adding an additional volume of fluid to position the first volume with cells within the cell growth chamber. For example, if the desired position of the first volume is not achieved using the second volume, additional fluid can be added to position 808 the first volume.

[0238] Process 800 proceeds to query 810 to determine whether the first volume should be repositioned. For example, in embodiments, the first volume can be positioned closer to the inlet port 844A. If it is desired to move the first volume closer to the central region 866 of the cell growth chamber 844, process 800 can return to step 808, where additional fluid can be added to the first fluid circulation path to position the fluid of the first volume.

[0239] If it is determined at query 810 that the first volume does not need to be repositioned, process 800 proceeds to feed the cells 812. In embodiments, a medium including many compounds (such as glucose, proteins, growth factors, reagents, or other nutrients) can be used to feed the cells. In embodiments, feeding the cells 812 can involve activating the inlet pump and the circulation pump (e.g., pumps 840 and 848) to deliver the medium with nutrients to the cells in the cell growth chamber 844. As described below, some embodiments provide for holding the cells within the cell growth chamber 844 during step 812. These embodiments can involve activating the pumps (e.g., the inlet pump and the circulation pump, such as pumps 840 and 848, as described below) such that the fluid entering the cell growth chamber 844 can flow into the cell growth chamber 844 from two directions, such as from the inlet port 844A and the outlet port 844B.

[0240] For example, as referenced Figures 7A to 7CAs described, a medium can be delivered from one or more connected fluid containers to cells. Through the IC circulation supply circuit 753 or the EC circulation supply circuit 752, the inlet pump and the circulation pump can circulate and recirculate the medium into the cell growth chamber 844 from two directions, such as from the inlet port 844A and the outlet port 844B.

[0241] Then, the process 800 proceeds to expand the cells 814, where the cells can be expanded or grown. According to an embodiment, although step 814 is shown after step 812, step 814 can occur before or simultaneously with step 812. Then the cells 816 can be removed from the cell growth chamber and collected in a storage container. In an embodiment, step 816 can involve multiple sub-steps. For example, the cells are circulated by a circulation pump (e.g., pump 848) before being collected and stored in a container. The process 800 terminates at the end operation 830.

[0242] Next, Figure 10A An example operational step of a process 900 according to an embodiment of the present disclosure for maintaining cells in a bioreactor of a cell expansion system, such as CES 500 (e.g., Figure 5B and Figure 5C ) or CES 700 (e.g., Figures 7A to 7C ) is shown. As described above, cells residing in the manifold of the bioreactor or in the IC circuit outside the bioreactor may not receive proper gas exchange and nutrient exchange, which may lead to cell aggregation and death. In an embodiment, the bioreactor provides gas exchange and nutrient exchange through a semi-permeable hollow fiber membrane. Importantly, such an exchange is effective because in a cell expansion system including a hollow fiber membrane, the surface area to volume ratio can be significantly greater than the surface area to volume ratio of other cell culture methods (e.g., about 15 times the surface area to volume ratio of a cell culture flask). Such efficiency can be achieved by minimizing the diffusion distance of the medium components through the membrane surface where the exchange occurs.

[0243] For example, Figure 10B A graph 936 of the oxygen consumption demand on a cell expansion system (such as a cell expansion system or Quantum a cell expansion system) during cell proliferation (about 3E+09 T cells present in the bioreactor) is shown. Figure 10BDepicts the percentage (%) of oxygen (O2) at the bioreactor outlet 938. For example, according to an embodiment, a sensor for measuring the oxygen level can be placed at the EC outlet port of the bioreactor. In another embodiment, a sensor for measuring oxygenation can be placed at the IC outlet port of the bioreactor. The percentage 940 of O2 is measured relative to the run time (e.g., in minutes). According to an embodiment, in order to maximize the supply of oxygen to the cells, the EC circulation flow rate Q EC循环 can be set to 300 mL / min (942). Figure 10B Shows the change in oxygenation when the EC circulation rate is decreased from 300 mL / min (942) to 50 mL / min (944). For example, as the medium passes through the bioreactor, the cells can consume the oxygen (O2) in the medium. Compared to the fluid at 300 mL / min (942), the fluid at 50 mL / min (944) passes through the bioreactor more slowly, so when the EC circulation rate is 50 mL / min, the cells can have a longer period or greater opportunity to remove oxygen from the medium. Then, when the EC circulation rate is restored to 300 mL / min, the oxygenation is restored (946). Figure 10B Shows the possible benefit of retaining the cells within the fibers themselves where gas transfer occurs, rather than in a portion of the IC circulation path external to the bioreactor, e.g., in that portion where the cells may be deoxygenated. Thus, it may be beneficial to keep a population of cells (e.g., non - adherent cells) within the hollow fibers of the bioreactor during oxygen supply by directing the medium flow into both sides of the bioreactor (e.g., the IC inlet port and the IC outlet port). In an embodiment, an equal flow distribution to the IC inlet port and the IC outlet port can be used. For example, in another embodiment, a greater flow can be used to flow into the IC inlet compared to the IC outlet port and vice versa, depending on, for example, the desired location of the cells within the bioreactor.

[0244] Return to Figure 10A , start the start operation 902, and the process 900 proceeds to loading 904 a disposable set or a pre - installed fluid delivery assembly (e.g., 210 or 400) into the cell expansion system. As Figure 1C 、 Figure 4B and Figure 4C shown, the disposable kit can include a cell growth chamber 100A or a cell growth chamber 100B. For example, then the disposable kit can be pre - filled 906, where it can be filled with Lonza Ca - free 2+ / Mg 2+The PBS of the pre - fill kit 906. To prepare for cell loading and seeding, an IC / EC flush 908 can be used to exchange the pre - fill fluid. For example, in an embodiment, the PBS in the system can be exchanged with TexMACS GMP basal medium.

[0245] For example, as referred to Figures 7A to 7C As described, during pre - filling, fluid can be delivered through a disposable kit from one or more connected fluid containers. Fluid containers for additional volume in the fluid circuit can be used to circulate and recirculate the fluid.

[0246] Then, the process 900 proceeds to closing the IC outlet valve 910. In an embodiment, the EC outlet valve can be opened to allow ultrafiltration of the fluid in the hollow fibers added to the bioreactor including the hollow fiber membrane. Next, the medium can be conditioned 912. Next, the process 900 proceeds to converting cells 914, such as suspension or non - adherent cells (such as T cells or Tregs). In an embodiment, this can load the cells 914 through a "concentrate - load cells without recirculation" task. In another embodiment, such cells can be loaded 914 through a "load uniformly - suspended cells" task. In other embodiments, other loading tasks and / or loading procedures can be used.

[0247] For example, in an embodiment, the loaded cells 914 can be suspended in a medium - containing solution to nourish the cells during and after such loading. In another embodiment, such a solution can include a medium for nourishing the cells and a soluble activator complex that stimulates the cells (such as T cells). This loading of 914 can occur, for example, on day 0.

[0248] After loading the cells 914, the cells can be further nourished 916. In an exemplary embodiment, as referenced Figures 7A to 7CAs described, cells 916 can be nourished through the EC circulation nourishment loop 752, the IC circulation nourishment loop 753, or a combination thereof. During such nourishment of 916, it is desirable to control the cell residence in the bioreactor itself. By adjusting the flow control parameter 918, the cells can be retained in the bioreactor itself without, for example, losing cells from the bioreactor to a portion of the IC loop outside the bioreactor during the exponential growth phase of growth. According to an embodiment, by retaining the cells in the bioreactor, the cells in the bioreactor can be closer to the IC inlet port, where such cells can receive the freshest growth medium. On the other hand, for example, the cells in the IC loop may be receiving spent or conditioned media, which may affect their glycolytic metabolism. Additionally, according to an embodiment, through diffusion from the EC loop circulation, the cells in the bioreactor can receive a mixed gas (e.g., oxygen, carbon dioxide, and nitrogen) input from the gas transfer module (GTM), while the cells in other portions of the IC loop may not receive such a mixed gas. It should be noted that while embodiments can provide for retaining the cells in the bioreactor itself, other embodiments can maintain the cells in any position to allow for improved nutrient delivery and / or gas exchange. Thus, without departing from the spirit and scope of the present disclosure, embodiments provide for using other locations to retain the cells or control the cell residence.

[0249] Return to Figure 10A For process 900, according to an embodiment, cell loss in the hollow fiber membrane bioreactor can be reduced by matching, approximating, or substantially matching the IC circulation pump rate with the IC inlet pump rate during nourishment, but in the opposite direction. The IC inlet pump can be adjusted at 920 to produce a first flow rate or volumetric flow rate, and the IC circulation pump can be adjusted at 922 to produce a second reverse flow rate or second reverse volumetric flow rate, where the volumetric flow rate or fluid flow rate or rate or velocity of the fluid can be considered the volume of fluid passing through per unit time (which can be represented by the symbol "Q"). In an embodiment, for example, an IC inlet pump rate of +0.1 mL / min can be matched, approximated, or substantially matched with a complementary IC circulation pump rate of -0.1 mL / min in order to retain the cells in the bioreactor during the growth phase of cell culture (which may be days 4 to 7). Alternatively, for example, an IC inlet pump rate of 0.01 mL / min can be matched, approximated, or substantially matched with a complementary IC circulation pump rate of -0.01 mL / min in order to retain the cells in the bioreactor during the growth phase of cell culture (which may be days 4 to 7). For example, such pump adjustment 918 can allow for counteracting any forces associated with cell loss from the IC outlet port of the bioreactor.

[0250] Next, cell growth or expansion 924 can be allowed. The cells are not limited to growing or expanding in step 924, rather, the cells can also expand during, for example, step 914, step 916, step 918, step 920, step 922. Process 900 can then proceed to harvest operation 926, where the cells can be transferred to a harvest bag or container. Then, at 932, the disposable kit can be unloaded from the cell expansion system, and then process 900 terminates at end operation 934.

[0251] Alternatively, from harvest operation 926, process 900 can optionally proceed to allow further processing / analysis 928. Such further processing 928 can include characterizing, for example, the phenotype of the harvested cells. From the optional further processing / analysis step 928, process 900 can optionally proceed to reload any remaining cells 930. Then process 900 can continue to unload the disposable kit 932, and then process 900 can terminate at end operation 934. Alternatively, process 900 can proceed from the further processing / analysis step 928 to the unloading of the disposable kit 932. Then, process 900 can terminate at end operation 934.

[0252] Next, Figure 11A Exemplary operating steps of a process 1000 for feeding cells according to an embodiment of the present disclosure are shown, and the operating steps can be used with a cell expansion system, such as CES 700 (e.g., Figures 7A to 7C )). For example, start start operation 1002, and process 1000 proceeds to load a disposable kit onto the cell expansion system, pre-fill the kit, perform IC / EC flushing, condition the medium, and load cells (e.g., suspended or non-adherent cells). As Figure 1C , Figure 4B and Figure 4C shown, the disposable kit can include a cell growth chamber 100A or a cell growth chamber 100B. Next, process 1000 proceeds to feed the cells 1004 during a first time period. In an exemplary embodiment, as referenced Figures 7A to 7CAs described, cells 916 are fed through the EC feed loop 752, the IC feed loop 753, or a combination thereof. In an embodiment, a first inlet flow rate and a first circulation flow rate may be used. As an example, a first IC inlet flow rate and a first IC circulation flow rate may be used, where the first IC inlet flow rate may be controlled by an IC inlet pump (e.g., a first pump), and the first IC circulation flow rate may be controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, the IC inlet pump (754) may cause an IC inlet port (701A) of a bioreactor (701) to receive a volume flow rate of 0.1 mL / min, where the IC circulation pump (712) causes a complementary IC circulation volume flow rate or fluid flow rate of -0.1 mL / min to enter an IC outlet port (701B) of the bioreactor (701), where, for example, the negative sign ("-") used in -0.1 mL / min indicates the direction of the IC circulation pump (712) to cause or create a countercurrent rate to maintain the cells in the bioreactor during the growth phase of the cell culture.

[0253] During cell feeding and during feeding using an IC pump to control the residence time of cells in the bioreactor through flow characteristics and countercurrent characteristics, the cells continue to grow and expand. Thus, the cells may require additional media (e.g., glucose and / or cell growth formulated media) to support the expanding population. In an exemplary embodiment, a plurality of fluid containers 768A and 768B may be connected to provide media for circulation and recirculation through the system, thereby obviating the need to control the lactate value. In an alternative embodiment, efforts may be made to control the lactate value of the expanding cell population. In an embodiment, the cell culture lactate value may be maintained at or below about 20 mmol / L, at or below about 15 mmol / L, at or below about 10 mmol / L, or even at or below about 7 mmol / L. In other embodiments, for example, the media addition rate and / or other settings may be controlled to attempt to maintain the lactate level ≤ about 5 mmol / L to, for example, improve cell growth and viability. In other embodiments, other concentrations may also be used.

[0254] In an exemplary embodiment, since the fluid containers 768A and 768B or the fluid containers 746A and 746B are connected, there is no need to control the pump rate to control the lactate value. In an alternative exemplary embodiment, according to the embodiment, efforts may be made to control the lactate value at ≤ about 7 mmol / L by synchronously increasing both the IC inlet (+) pump rate and the IC circulation (-) pump rate in the lumen of the hollow fiber membrane from ±0.1 mL / min to ±0.4 mL / min over a plurality of time periods (e.g., several days (days 4 - 8)). For example, Figure 11BTable 1018 provides example IC pumping rates for use in, for example, a cell expansion system (e.g., CES 500) to feed using a "feed cells" task. Table 1018 provides example time periods 1020 (e.g., days) relative to an example IC pump rate 1022 to produce volumetric flow rates to both sides of a bioreactor to maintain cells in the bioreactor. For example, from day 0 - 4 (1024), an IC inlet or input pump rate of 0.1 mL / min (1026) and an IC circulation pump rate of -0.1 mL / min (1028) may be used; on day 5 (1030), an IC inlet pump rate of 0.2 mL / min (1032) and an IC circulation pump rate of -0.2 mL / min (1034) may be used; on day 6 (1036), an IC inlet pump rate of 0.3 mL / min (1038) and an IC circulation pump rate of -0.3 mL / min (1040) may be used; and on day 7 (1042), an IC inlet pump rate of 0.4 mL / min (1044) and an IC circulation pump rate of -0.4 mL / min (1046) may be used. Although Figure 10B Table 1018 provides example pump rates of ±0.1 to ±0.4 mL / min for feeding cells while maintaining cells in a bioreactor during the growth phase of a cell culture, other pump rates and corresponding flow rates may be used according to embodiments without departing from the spirit and scope of the present disclosure. For example, although an increment of ±0.1 mL / min for increasing the feed flow rate is shown in this example, other increments (e.g., ±0.005 mL / min, ±0.05 mL / min, etc.) may be used in embodiments to increase the feed flow rate. Figure 11B The time periods (e.g., days) and pump rates in Table 1018 of Figure 11B are provided for illustrative purposes only and are not intended to be limiting. For example, the time periods and pump rates in Table 1018 of Figure 11B may be provided for the cell growth chamber 100B. Figure 11B The time periods and pump rates in Table 1018 of Figure 11B . Figure 11B The pump rates in Table 1018 of Figure 11B may be reduced to 10% of the pump rates of the cell growth chamber 100A.

[0255] Returning to Figure 11A , during a first time period 1004, the process 1000 progresses from cell feeding to increasing a first inlet flow rate by a first amount to achieve a second inlet flow rate 1006. For example, as described above, as referenced Figure 11BIn the depicted embodiment, the IC inlet pump rate (+) can be increased from 0.1 mL / min to 0.2 mL / min to produce an IC inlet flow rate of 0.2 mL / min. Additionally, the IC circulation pump rate (−) can be synchronously increased from −0.1 mL / min to −0.2 mL / min to produce an IC circulation flow rate of −0.2 mL / min. (The pump rate can be reduced to 10% of the pump rate of the cell growth chamber 100A). Thus, the first circulation flow rate is increased by a first amount to achieve a second circulation flow rate 1008. For example, then, at 1010, cells can be fed at a second inlet flow rate and a second circulation flow rate during a second time period to maintain cells within the bioreactor and outside the manifold and outside portions of the IC circulation path outside the bioreactor. For example, after the feeding 1010 of the second time period, if it is not desired to continue feeding and / or expanding the cells, the process 1000 can terminate at the end operation 1016. Alternatively, the process 1000 can optionally continue to increase or otherwise change the feeding flow rate 1012. There can be any number of feeding time periods, as indicated by the ellipsis 1014. After a desired number of feeding time periods 1014, the process 1000 then terminates at the end operation 1016. Although Figure 10A and Figure 10B process 1000 shows an “increase” in the flow rate, other adjustments to the flow rate can be made. For example, the flow rate can decrease or remain substantially the same from one feeding segment to the next. Considerations such as metabolic activity can be used to determine how to adjust the flow rate. Figure 10A and Figure 10B The “increase” in the flow rate in

[0256] is provided for illustrative purposes only and is not intended to be limiting. Figure 12A Turning to Figure 5B and Figure 5C)For use together. For example, start operation 1102, and process 1100 proceeds to loading a disposable kit onto the cell expansion system, priming the kit, performing IC / EC flushing, conditioning the medium, and loading cells (e.g., suspended or non-adherent cells). Next, process 1100 proceeds to feeding the cells 1104 during a first time period. In an embodiment, a first inlet rate or flow rate and a first circulation rate or flow rate may be used. By way of example, a first IC inlet flow rate and a first IC circulation flow rate may be used, where the first IC inlet flow rate may be controlled by an IC inlet pump (e.g., a first pump), and the first IC circulation flow rate may be controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, the IC inlet pump 554 may cause a volumetric flow rate or fluid flow rate of 0.1 mL / min to enter the IC inlet port 501A of the bioreactor 501, and the IC circulation pump 512 causes a complementary IC circulation flow rate of -0.1 mL / min to enter the IC outlet port 501B of the bioreactor 501, where, for example, the negative sign (“-”) used in -0.1 mL / min indicates the direction of the IC circulation pump 512 to cause or create a countercurrent rate to maintain the cells in the bioreactor during the growth phase of the cell culture. For example, the described flow rates may be applicable to the cell growth chamber 100B. The described flow rates may be reduced to 10% of the cell growth chamber 100A.

[0257] During feeding the cells and using the IC pumps to control the residence of the cells in the bioreactor through flow characteristics and countercurrent characteristics, the cells continue to grow and expand. Thus, the cells may require additional medium (e.g., glucose and / or cell growth formulated medium) to support the expanding population. In an exemplary embodiment, a plurality of fluid containers 768A and 768B may be connected to provide medium for circulation and recirculation through the system, thereby obviating the need to control the lactate value. In an alternative embodiment, an effort may be made to control the lactate value of the expanding cell population. In an exemplary embodiment, according to the embodiment, for example, by synchronously increasing both the IC inlet (+) pump rate and the IC circulation (-) pump rate in the lumen of the hollow fiber membrane from ±0.1 mL / min to ±0.4 mL / min over several days (e.g., days 4 - 8), an effort may be made to control the lactate value at ≤ about 7 mmol / L. For example, see Figure 11B , Table 1018, and the discussion above, such as the feeding pump rates. As described above, although Figure 11B Table 1018 provides pump rates of ±0.1 to ±0.4 mL / min for feeding the cells while maintaining the cells in the bioreactor during the growth phase of the cell culture, other pump rates and corresponding flow rates may be used according to the embodiment without departing from the spirit and scope of the present disclosure. Figure 11BThe time periods (e.g., number of days) and pump rates in Table 1018 are provided for illustrative purposes only and are not intended to be limiting. For example, the time periods and pump rates in Table 1018 for the cell growth chamber 100B can be provided Figure 11B The pump rate in Table 1018. Figure 11B The pump rate in Table 1018 can be reduced to 10% of the pump rate of the cell growth chamber 100A.

[0258] Return to Figure 12A , during the first time period 1104, the process 1100 proceeds from cell feeding to increasing the first inlet rate or flow rate by a first amount to achieve a second inlet rate or flow rate 1106. For example, as described above, in the embodiment depicted as Figure 11B , the IC inlet pump rate (+) can be increased from 0.1 mL / min to 0.2 mL / min to produce an IC inlet flow rate of 0.2 mL / min. Additionally, the IC circulation pump rate (-) can be synchronously increased from -0.1 mL / min to -0.2 mL / min to produce an IC circulation flow rate of -0.2 mL / min. (The pump rate can be reduced to 10% of the pump rate of the cell growth chamber 100A) Thus, the first circulation rate or flow rate is increased by a first amount to achieve a second circulation rate or flow rate 1108. For example, then at 1110, the cells can be fed at the second inlet rate or flow rate and the second circulation rate or flow rate during the second time period to maintain the cells in the bioreactor and in the portion of the IC circulation path outside the header or outside the bioreactor. For example, after the feeding 1110 in the second time period, if it is not desired to continue feeding and / or expanding the cells, the process 1100 can terminate at the end operation 1116.

[0259] Alternatively, the process 1100 can optionally determine whether to adjust the feeding rate or flow rate based on metabolic activity, where the process 1100 proceeds to an optional query 1112 to determine whether to adjust the feeding based on the metabolic level. For example, monitoring glucose and / or lactate levels can assist in adjusting the cell expansion system media flow rate (e.g., IC media flow rate) to support the expansion of cells (e.g., Tregs) in a bioreactor such as a hollow fiber bioreactor.

[0260] As Figure 12B and Figure 12C shown, the embodiment provides control of lactate values for a cell expansion run or program involving, for example, the expansion of hTregs. Graph 1118 and Graph 1132 show that measuring glucose and lactate levels, for example, can be used to adjust the cell expansion system media flow rate (e.g., IC media flow rate) to support the expansion of, for example, Tregs. For example, Figure 12B provides Curve 1118 showing the metabolism of expanding hTregs, where such cell expansion can occur in a cell expansion system such as in a cell expansion system). Glucose concentration (mg / dL) 1120 and lactate concentration (mmol / L) 1122 of various cell expansion runs 1124 and 1125 over a period of time (e.g., number of days) 1126 are shown. During these Treg runs 1124 and 1125, for example, from day 4 to day 8, efforts can be made to control the lactate value of the expanded cell population to a value less than or equal to (≤) about 7 mmol / L by synchronously increasing the IC inlet (+) pump rate and the IC circulation pump rate from ±0.1 to ±0.4 mL / min, for example, within the lumen of the hollow fiber membrane. In other embodiments, other pump rates can be used. As shown, according to the illustrated embodiment, the lowest glucose level during Treg cell expansion can range from a concentration of 264 mg / dL at day 7 (Q1584) 1128 to a concentration of 279 mg / dL at day 8 (Q1558) 1130. As depicted, the basal glucose concentration 1124 in the cell growth formulation medium for the runs can range, for example, from 325 mg / dL to 335 mg / dL. In other embodiments, it may be desirable to maintain the lactate level ≤ about 5 mmol / L to improve cell growth and viability. In an embodiment, a graphical user interface (GUI) element can be used to control the rate of medium addition and keep the lactate metabolic waste from glycolysis below a defined level during cell expansion.

[0261] Go to Figure 12C , graph 1132 shows the metabolism of expanded hTregs, where such cell expansion may occur in a cell expansion system (such as a cell expansion system). Glucose consumption (mmol / day) 1134 and lactate production (mmol / day) 1136 of various cell expansion runs 1138 and 1139 over a period of time 1140 (e.g., number of days) are shown. To control the lactate value to less than or equal to (≤) about 7 mmol / L, for example, in an embodiment, the IC inlet (+) pump rate and the IC circulation (-) pump rate can be synchronously increased from ±0.1 mL / min to ±0.4 mL / min. For example, graph 1132 shows an IC circulation and IC feeding rate of ±0.1 mL / min (1142); ±0.2 mL / min (1144); ±0.3 mL / min (1146); and ±0.4 mL / min (1148). Other embodiments can use other flow rates. Figure 12B and Figure 12CThe flow rates used and shown are provided for illustrative purposes and are not intended to be limiting. It should be noted that while a positive (+) may be shown for the direction of the IC inlet pump and a negative (-) may be shown for the direction of the IC recirculation pump, such directions are provided for illustrative purposes only, where such directions depend on the configuration of the pumps used.

[0262] Return Figure 12A and optional query 1112, if it is not desired to measure metabolic activity and / or adjust the nutrient supply level based on such a measurement, process 1100 proceeds from "No" to end operation 1116, and process 1100 terminates. For example, in the case of using multiple nutrient containers, as referred to Figures 7A to 7C as described, the medium can be circulated and recycled, thus preventing the need to measure metabolic activity and / or adjust the nutrient supply level. Alternatively, in the case where it is desired to adjust the nutrient supply level based on metabolic activity, process 1100 proceeds from "Yes" to optional step 1114 to continue increasing the rate of medium addition and nourishing the expanding cell population. For example, while step 1114 is shown as one step, this step can involve multiple adjustments to the medium addition rate, such as increasing the IC inlet flow rate and increasing the IC recirculation flow rate. Step 1114 is shown as one step for illustrative purposes only and is not intended to be restrictive. After any adjustment to the medium addition rate, process 1100 proceeds to optional query 1112 to determine whether to continue measuring the metabolic level and / or adjusting the nutrient supply. If it is not desired to continue measuring metabolic activity and / or adjust the nutrient supply level based on metabolic activity, process 1100 advances to "No" and enters end operation 1116, and process 1100 terminates. While Figure 11A and process 1100 show an "increase" in rate or flow rate, other adjustments can be made to the flow rate. For example, the rate or flow rate can decrease or remain substantially the same from one nutrient segment to the next. The type of adjustment that can be made can depend on the assessment of the metabolic activity of the growing cell population. For example, Figure 11A the "increase" in the flow rate in

[0263] Next, Figure 13 illustrates an example operating step of process 1200 according to an embodiment of the present disclosure for maintaining cells in one position during the nutrient supply of a cell expansion system, such as CES 500 (e.g. Figure 5B and Figure 5C ) or CES 700 (e.g. Figures 7A to 7C ). For example, start start operation 1202, and process 1200 proceeds to load a disposable kit onto the cell expansion system, pre-fill the kit, perform IC / EC flushing, condition the medium, and load cells (e.g. suspended or non-adherent cells). As Figure 1C ,Figure 4B and Figure 4C As shown, the disposable kit may include cell growth chamber 100A or cell growth chamber 100B. Next, process 1200 proceeds to feeding cells 1204 during a first time period. In an embodiment, a first inlet flow rate and a first circulation flow rate may be used. By way of example, a first IC inlet flow rate and a first IC circulation flow rate may be used, where the first IC inlet flow rate may be generated and controlled by an IC inlet pump (e.g., a first pump), and the first IC circulation flow rate may be generated and controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, IC inlet pump 554 may cause an IC inlet port 501A of the bioreactor to receive a volume flow rate of 0.1 mL / min, and IC circulation pump 512 may cause an IC outlet port 501B of bioreactor 501 to receive a complementary IC circulation volume flow rate or fluid flow rate of -0.1 mL / min, where, for example, the negative sign ("-") used in -0.1 mL / min indicates the direction of the IC circulation pump to cause or create a countercurrent rate to maintain cells in the bioreactor during the growth phase of the cell culture. In another embodiment, 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 may be another percentage or fraction according to the embodiment. Alternatively, in an exemplary embodiment, the first IC inlet pump may cause an IC inlet port (701A) to receive a volume flow rate of less than 0.1 mL / min or approximately 0.01 mL / min, and the IC circulation pump may cause an IC outlet port (701B) to receive a complementary IC circulation volume flow rate or fluid flow rate of less than -0.1 mL / min or approximately -0.01 mL / min, where, for example, the negative sign ("-") used in -0.01 mL / min indicates the direction of the IC circulation pump to cause or create a countercurrent rate to maintain cells in the bioreactor during the growth phase of the cell culture.

[0264] For example, during the feeding (and expansion) of cells and the use of IC pumps to control the residence of cells in the bioreactor through flow characteristics and countercurrent characteristics, the cells continue to grow and expand. Thus, the cells may require additional media (e.g., glucose and / or cell growth formulated media) to support the expanding population. In an embodiment, an effort may be made to increase the rate of media addition to feed the expanding cell population. In an exemplary embodiment, an increase in the IC inlet pump rate (+) may increase the IC inlet flow rate by a first amount to achieve a second IC inlet flow rate 1206. For example, according to an embodiment, the IC inlet flow rate may be increased by a first amount of 0.1 mL / min to achieve a second IC inlet flow rate of 0.2 mL / min. According to other embodiments, other adjustments may be made to the IC inlet flow rate.

[0265] In an exemplary embodiment, as described with reference to Figures 7A to 7C that described, cells 916 can be fed through an EC circulation feeding loop 752, an IC circulation feeding loop 753, or a combination thereof.

[0266] Next, according to an embodiment, the second IC circulation flow rate can be set or adjusted or configured to be equal to a percentage, portion, or fraction of the second IC inlet flow rate 1208. For example, according to an embodiment, the second IC circulation flow rate can be set or adjusted or configured to be equal to approximately fifty percent (50%) or approximately one-half (1 / 2) of the IC inlet flow rate value, or according to an embodiment, can be another percentage or fraction. Depending on the value of the first IC circulation flow rate, according to an embodiment, an adjustment to the IC circulation pump rate (-) can cause an increase in the second IC circulation flow rate. In another embodiment, the IC circulation pump rate can be adjusted to produce or cause a decrease in the second IC circulation flow rate such that the IC circulation flow rate can be substantially equal to a predetermined percentage or a predetermined fraction of the second IC inlet flow rate. In yet another embodiment, the IC circulation pump rate can be left unadjusted. For example, in a case where 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, if the second IC inlet flow rate increases to 0.2 mL / min, then the second IC circulation flow rate can be set to (-1 / 2)*(the second Q IC入口 )(where Q IC入口 is the IC inlet flow rate) or (-1 / 2)*(0.2 mL / min), which provides a second Q of -0.1 mL / min IC循环 (where Q IC循环 is the IC circulation flow rate), and the IC circulation pump rate can be left unadjusted to achieve such a second Q IC循环 .

[0267] Turning to Figure 5B and Figure 5C , according to embodiments of the present disclosure, these figures depict operating configurations of the cell expansion system 500, showing fluid movement in a first circulation path. Figure 5B and Figure 5CThe configuration shows a split in the flow rate, for example, to an IC inlet port (e.g., the first port) and to an IC outlet port (e.g., the second port), to keep cells in a cell growth chamber or bioreactor. As discussed above with respect to the CES 500, in the IC loop or first circulation path 502, fluid can initially be propelled by the IC inlet pump 554. Such fluid can, for example, travel in a first direction, such as in the forward direction. The fluid can flow into the cell growth chamber or bioreactor 501 through the IC inlet port 501A, through the hollow fibers in the cell growth chamber or bioreactor 501, and can exit via the IC outlet port 501B. The medium can flow through the IC circulation pump 512, which can be used to control the rate of medium flow. The IC circulation pump can pump fluid in a first direction or in a second direction opposite to the first direction. For example, the IC outlet port 501B can be used as an inlet for reverse flow. For example, in an embodiment, the IC circulation pump 512 can pump fluid in a direction opposite to the direction of the IC inlet pump. As an example, the direction of the IC inlet pump can be forward (+), and the direction of the IC circulation pump can be negative (-) to cause a countercurrent so that fluid can enter both sides of the bioreactor to keep cells in the bioreactor.

[0268] In an embodiment, the fluid in the first portion may branch at the connector 517 to flow into the IC inlet port (501A) of the bioreactor 501. In an embodiment, the IC circulation pump 512 may operate at a pump rate that matches, approximates, or is substantially matched but in the opposite direction to the rate of the IC inlet pump 554, such that the second portion of the fluid may branch at the connector 517 to flow into the IC outlet port (501B) of the bioreactor 501. For example, an IC inlet pump rate of +0.1 mL / min may match, approximate, or be substantially matched to a complementary IC circulation pump rate of -0.1 mL / min in order to maintain the cells in the bioreactor during the growth phase of cell culture. Alternatively, an IC inlet pump rate less than +0.1 mL / min or approximately +0.01 mL / min may match, approximate, or be substantially matched to a complementary IC circulation pump rate less than -0.1 mL / min or approximately -0.01 mL / min in order to maintain the cells in the bioreactor during the growth phase of cell culture. Additionally, in an additional embodiment, as described herein, cell harvest synchronization may subsequently be performed during the growth phase of the in-capillary culture. Such a pump adjustment strategy during the feeding period may counteract the forces associated with cell loss from the IC outlet port. According to an embodiment, using pump adjustment to effect such a type of feeding that enters the IC inlet port (501A) and the IC outlet port (501B) of the bioreactor in flow and counterflow, respectively, may be referred to as a modified feeding method. In another exemplary embodiment, the IC circulation pump rate may be adjusted such that the flow rate entering the IC outlet port (501B) may be equal to approximately fifty percent (50%) or approximately one-half (1 / 2) of the IC inlet flow rate, or in other embodiments, may be another percentage or fraction, but in the opposite direction. For example, in the case where the IC inlet pump rate is 0.4 mL / min, the IC circulation pump rate may be set, configured, or adjusted to approximately -0.2 mL / min. In other embodiments, other percentages or fractions may be used.

[0269] According to an embodiment, Figure 5B and Figure 5C shows an operational configuration showing fluid movement in the CES 500, where the flow rates and counterflow rates for holding cells in the cell growth chamber or bioreactor 501 are shown. According to an embodiment, for example, such flow rates are shown as the "X" flow rate 503 in Figure 5B ; the "(-1 / 2)X" flow rate 505 (where the negative sign ("-") is an indication of direction, where the direction of the flow rate 505 is given by Figure 5BThe direction arrows in [figure number] show); and a flow rate of "(1 / 2)X" 507, where approximately 1 / 2 or the first part of the flow rate branches at the connector 517 to enter the IC inlet port (501A) of the bioreactor 501, and approximately 1 / 2 or the second part of the flow rate branches at the connector 517 to enter the IC outlet port (501B) of the bioreactor 501. As shown, the sum of the first part and the second part can be substantially equal to the total flow rate 503, where the total flow rate 503 is pumped out by the IC inlet pump 554. Depending on the flow rate and countercurrent rate available to hold cells in the bioreactor or cell growth chamber 501, other types of fractions or percentages of the IC inlet pump rate can be used to set, configure, or adjust the IC circulation pump. Thus, Figure 5C shows such a flow rate as "X" flow rate 511; "-(y%)*X" flow rate 513 (where the minus sign ("-") is an indication of direction, and the direction of the flow rate 513 is shown by the Figure 5C direction arrows in [figure number]); and "(100%-y%)*X" flow rate 515, where "y" equals a numerical percentage, according to the embodiment. In an embodiment, the sum of the flow rate 513 and the flow rate 515 is substantially equal to the flow rate 511.

[0270] For example, in an embodiment, all or substantially all of the flow from the first fluid flow path 506 can flow from the connector 517 to the IC inlet port 501A of the bioreactor 501. In another embodiment, all or substantially all of the flow from the first fluid flow path 506 can flow from the connector 517 into the IC outlet port 501B of the bioreactor 501. In yet another embodiment, a first part of the flow from the first fluid flow path 506 can flow from the connector 517 to the IC inlet port 501A, and a second part of the flow from the first fluid flow path 506 can flow from the connector 517 to the IC outlet port 501B. In an embodiment, the percentage of the IC inlet flow rate of the IC circulation flow rate can be set in the range of about 0% to about 100%. In other embodiments, the percentage can be between about 25% and about 75%. In other embodiments, the percentage can be between about 40% and about 60%. In other embodiments, the percentage can be between about 45% and about 55%. In an embodiment, the percentage can be approximately 50%. It should be understood that, Figure 5B and Figure 5C the operating configurations shown in [figure number] represent possible configurations for various operations of the cell expansion system, and within the scope of one or more current embodiments, changes can be made to the shown configurations.

[0271] Return Figure 13, at 1210, during a second time period, cells can be fed (and continue to be amplified) at a second inlet flow rate and a second recycle flow rate to maintain the cells in the bioreactor, outside the header, and in a portion of the IC recycle path outside the bioreactor. For example, after the feeding at 1210 during the second time period, if it is not desired to continue feeding and / or amplifying the cells, the process 1200 can terminate at the end operation 1216. Alternatively, the process 1200 can optionally continue to increase or otherwise change the feeding flow rate 1212. There can be any number of feeding time periods, as indicated by the ellipsis 1214. After the desired number of feeding time periods 1214, the process 1200 can then terminate at the end operation 1216. Although Figure 13 and the process 1200 shows an "increase" in the flow rate, other adjustments can be made to the flow rate. For example, the flow rate can decrease or remain substantially the same from one feeding segment to the next. Considerations, such as, for example, metabolic activity, can determine how to adjust the flow rate. Figure 13 The "increase" in the flow rate in

[0272] is provided for illustrative purposes only and is not intended to be limiting. Figure 14 , an example operating step of a process 1300 according to an embodiment of the present disclosure is provided for maintaining cells at a first location (e.g., a bioreactor) while feeding the cells, and the bioreactor can be associated with a cell expansion system such as CES 500 (e.g., Figure 5B and Figure 5C ) or CES 700 (e.g., Figures 7A to 7C)Used together. For example, start operation 1302, and process 1300 proceeds to load a disposable kit onto the cell expansion system, prime the kit, perform IC / EC flushing, condition the medium, and load cells (e.g., suspended or non-adherent cells). Next, process 1300 proceeds to feed the cells 1304 during a first time period. In an embodiment, a first inlet flow rate and a first circulation flow rate can be used. As an example, a first IC inlet flow rate and a first IC circulation flow rate can be used, where the first IC inlet flow rate can be generated and controlled by an IC inlet pump (e.g., a first pump), and the first IC circulation flow rate can be generated and controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, the first IC inlet pump rate can be such that the IC inlet port of the bioreactor is entered at a volumetric flow rate of 0.1 mL / min, where a complementary IC circulation pump rate of -0.1 mL / min causes the IC outlet port of the bioreactor to be entered at a volumetric flow rate of -0.1 mL / min, where, for example, the negative sign ("-") used in -0.1 mL / min indicates the direction of the IC circulation pump (512) to cause or produce a countercurrent rate to maintain the cells in the bioreactor during the growth phase of the cell culture. Alternatively, in an exemplary embodiment, the first IC inlet pump can be such that the IC inlet port (701A) is entered at a volumetric flow rate less than 0.1 mL / min or about 0.01 mL / min, and the IC circulation pump causes a complementary IC circulation volumetric flow rate or fluid flow rate less than -0.1 mL / min or about -0.01 mL / min to enter the IC outlet port (701B), where, for example, the negative sign ("-") used in -0.01 mL / min indicates the direction of the IC circulation pump to cause or produce a countercurrent rate to maintain the cells in the bioreactor during the growth phase of the cell culture. In another embodiment, the first IC circulation flow rate can be a percentage, fraction, or portion of the first IC inlet flow rate. For example, the first IC circulation flow rate can be about fifty percent (50%) or about one-half (1 / 2) of the IC inlet flow rate, or can be another percentage or fraction depending on the embodiment.

[0273] During seeding (and expanding) cells and using an IC pump to control the residence of cells in a bioreactor through flow characteristics and countercurrent characteristics, the cells continue to grow and expand. Thus, the cells may require additional media (such as glucose and / or cell growth formulated media) to support the continuously expanding population. In an embodiment, an effort may be made to increase the rate of media addition to feed the continuously expanding cell population. In an exemplary embodiment, an increase in the IC inlet pump rate (+) can increase the IC inlet flow rate by a first amount to achieve a second IC inlet flow rate 1306. For example, according to an embodiment, the IC inlet flow rate can be increased by a first amount of 0.1 mL / min to achieve a second IC inlet flow rate of 0.2 mL / min. According to other embodiments, other adjustments can be made to the IC inlet flow rate.

[0274] Next, according to an embodiment, the second IC circulation flow rate can be set or configured or adjusted to be equal to a percentage, portion, or fraction of the second IC inlet rate 1308. For example, according to an embodiment, the second IC circulation flow rate can be set or configured or adjusted to be equal to approximately fifty percent (50%) or approximately one-half (1 / 2) of the IC inlet flow rate value, or according to an embodiment, can be another percentage or fraction. For example, in an embodiment, all or substantially all of the flow from the first fluid flow path 506 can flow from the connector 517 into the IC inlet port (501A) of the bioreactor 501. In another embodiment, all or substantially all of the flow from the first fluid flow path 506 can flow from the connector 517 into the IC outlet port (501B) of the bioreactor 501. In yet another embodiment, a first portion of the flow from the first fluid flow path 506 can flow from the connector 517 into the IC inlet port (501A), and a second portion of the flow from the first fluid flow path 506 can flow from the connector 517 to the IC outlet port (501B). In an embodiment, the percentage of the IC inlet flow rate of the IC circulation flow rate can be set in the range of approximately 0% to approximately 100%. In other embodiments, the percentage can be between approximately 25% and approximately 75%. In other embodiments, the percentage can be between approximately 40% and approximately 60%. In other embodiments, the percentage can be between approximately 45% and approximately 55%. In an embodiment, the percentage can be approximately 50%.

[0275] Depending on the value of the first IC recycle flow rate, according to an embodiment, adjustment of the IC recycle pump rate (-) can cause an increase in the second IC recycle flow rate. In another embodiment, the IC recycle pump rate can be adjusted such that the second IC recycle flow rate is decreased, such that the second IC recycle flow rate can be substantially equal to a predetermined percentage or fraction of the second IC inlet flow rate. In yet another embodiment, the IC recycle pump rate may not be adjusted. For example, according to an embodiment, when the first IC inlet flow rate is equal to 0.1 mL / min and the first IC recycle flow rate is equal to -0.1 mL / min, if the second IC inlet flow rate increases to 0.2 mL / min, the second IC recycle flow rate can be set to (-1 / 2)*(the second Q IC入口 ) or (-1 / 2)*(0.2 mL / min), which provides a second Q of -0.1 mL / min IC循环 , and the IC recycle pump rate may not be adjusted to achieve such a second Q IC循环 .

[0276] Then, cells 1310 can be fed (and continue to be amplified) at the second IC inlet flow rate and the second IC recycle flow rate during a second time period to maintain the cells in the bioreactor and in portions of the IC recycle path outside the header and outside the bioreactor. For example, after feeding 1310 in the second time period, if it is not desired to continue feeding and / or amplifying the cells, process 1300 can terminate at end operation 1316.

[0277] In an embodiment, the first time period, the second time period, the third time period, the fourth time period, the fifth time period, etc. can each include one or more days (and / or hours and / or minutes). For example, according to an embodiment, the time period can be from one (1) day to fourteen (14) days. However, in other embodiments, the time period may be less than one (1) day or greater than fourteen (14) days. For example, in an embodiment, the first time period for feeding can include day 0, day 1, day 2, day 3, day 4; the second time period for feeding can include day 5; the third time period for feeding can include day 6; and the fourth time period for feeding can include day 7. In another embodiment, the first time period can include day 0, day 1, and day 2 (e.g., with a duration of about 3 days); the second time period can include day 3, day 4, and day 5 (e.g., lasting about 3 days); the third time period can include day 6, day 7, and day 8 (e.g., lasting about 3 days); the fourth time period can include day 9 and day 10 (e.g., lasting about 2 days); and the fifth time period can include day 11, day 12, and day 13 (e.g., lasting about 3 days). According to an embodiment, the time periods can be of different durations. Each time period can be measured in days, hours, minutes, and / or fractions thereof.

[0278] Return to Figure 14 ,Process 1300 may optionally proceed to optional query 1312 to determine whether to adjust the feeding rate or flow rate based on metabolic activity or metabolic level. For example, for the expansion of Tregs, it may be desirable to control the lactate value of the expanded cell population to a value of ≤ about 7 mmol / L. In cases where it is desired to maintain the cell culture lactate value at about 7 mmol / L or below, the rate of media addition can be controlled, for example, during the expansion of cells (e.g., regulatory T cells). In other embodiments, it may be desirable to maintain the lactate level less than or equal to (≤) about 5 mmol / L to improve cell growth and viability. In an embodiment, a graphical user interface (GUI) element can be used to control the rate of media addition and keep the lactate metabolic waste from glycolysis below a predetermined level during cell expansion.

[0279] In optional query 1312, if it is not desired to measure metabolic activity and / or adjust the feeding level based on such a measurement, process 1300 proceeds from "No" to end operation 1316, and process 1300 terminates. Alternatively, in cases where it is desired to adjust the feeding level based on metabolic activity, process 1300 proceeds from "Yes" to optional step 1314 to continue increasing the rate of media addition and feeding the expanded cell population. Although step 1314 is shown as one step, for example, this step may involve multiple adjustments to the media addition rate, such as increasing the IC inlet flow rate and increasing the IC recycle flow rate. Step 1314 is shown as one step for illustrative purposes only and is not intended to be limiting. After any adjustment to the media addition rate, process 1300 returns to optional query 1312 to determine whether to continue measuring the metabolic level and / or adjusting the feeding. If it is not desired to adjust the feeding level based on metabolic activity, process 1300 proceeds from "No" to end operation 1316, and process 1300 terminates. Although ​ and process 1300 shows an "increase" in the flow rate, other adjustments can be made to the flow rate. For example, the rate or flow rate can be decreased or kept substantially the same from one feeding segment to the next. The type of adjustment that can be made can depend on the assessment of the metabolic activity of the growing cell population. ​ The "increase" in the flow rate in

[0280] Next, ​ shows an example operating step of process 1400 for retaining cells during feeding according to an embodiment of the present disclosure, which can be used with a cell expansion system such as CES 500 ( ​ and ​ ) or CES 700 ( ​)Used together. Start the start operation 1402, and the process 1400 proceeds to load 1404 a disposable tubing set or a pre-installed fluid delivery assembly (such as 210 or 400) into the cell expansion system. As ​ , ​ and ​ shown, the disposable kit may include a cell growth chamber 100A or a cell growth chamber 100B. Next, the system 1406 can be pre-filled. For example, in an embodiment, for example, by selecting a task for pre-filling, a user or operator can provide instructions to the system to pre-fill. For example, in an embodiment, such a task for pre-filling can be a pre-programmed task. For example, next, an IC / EC flush task 1408 can be performed, in which the fluids on the IC circulation loop and the EC circulation loop can be replaced. According to an embodiment, the replacement volume can be determined by the amounts of the IC volume and the EC volume being exchanged.

[0281] Next, in order to maintain an appropriate or desired gas concentration in the fibers of the bioreactor membrane, a conditioning medium task 1410 can be performed to allow the medium to equilibrate with the provided gas supply before the cells are loaded into the bioreactor. For example, by adjusting the EC circulation rate, contact between the medium and the gas supply provided by a gas transfer module (GTM) or an oxygenator can be provided. Then the system can be held in an appropriate or desired state until the user or operator is ready, for example, to load cells into the bioreactor. For example, in an embodiment, the system can be conditioned with a medium (such as a complete medium). The complete medium can be any medium for cell growth. For example, in an embodiment, the system can be conditioned with a serum-free medium. In an embodiment, the system can be conditioned with a basal medium. Any type of medium understood by those skilled in the art can be used.

[0282] Process 1400 then proceeds to loading 1412 of cells from, for example, a cell inlet bag into the bioreactor. In an embodiment, the cells in the cell inlet bag can be in a solution, for example, with a medium to nourish the cells 1414. In another embodiment, the cells in the cell inlet bag can be in a solution that has both a medium for nourishing the cells 1414 and a soluble activator complex for stimulating the cells (such as T cells or Tregs). In an embodiment, the cells (and the nourishing solution, in an embodiment) can be loaded from the cell inlet bag into the bioreactor until the bag is empty. The cells (and the nourishing solution, in an embodiment) can be chased from the air removal chamber into the bioreactor. In an embodiment, the task of "loading evenly suspended cells" can be performed to load the cells (and the nourishing solution, in an embodiment). In another embodiment, the task of "loading cells concentratedly without circulation" can be performed to load the cells (and the nourishing solution, in an embodiment) into a specific area (such as the central area) of the bioreactor. According to embodiments, other loading methods and / or loading tasks can be used.

[0283] Next, process 1400 proceeds to query 1416 to determine whether to use a modified nourishing method to retain cells, such as non - adherent or suspended cells, such as T cells or Tregs, in the bioreactor (such as, a hollow fiber bioreactor). For example, it may be desirable to position the cells within the bioreactor itself and outside the remainder of the bioreactor's manifolds or IC loops. For example, if it is not desired to use a modified nourishing method to retain the cells in the bioreactor itself, process 1400 proceeds from "no" to amplifying the cells 1426, where the cells can continue to grow / amplify using the medium that initially nourished them in step 1414.

[0284] On the other hand, if it is desired to maintain the cells within the bioreactor itself, process 1400 proceeds from "Yes" to modified feeding 1418, where the cells can be fed using the flow rate into the IC inlet 501A of bioreactor 501 and the flow rate out of the IC outlet 501B of bioreactor 501 to maintain the cells within the bioreactor. In doing so, the inlet volumetric flow rate or inlet flow rate can be introduced into the first fluid flow path 506 at 1420. For example, it can be introduced into the first fluid flow path 506 at the IC inlet flow rate at 1420. The IC inlet pump 554, such as a first peristaltic pump (in an embodiment), can operate at a predetermined revolutions per minute (RPM) such that the fluid in the first fluid flow path 506 has a predetermined IC inlet volumetric flow rate or IC inlet flow rate at 1420. According to an embodiment, a processor and / or controller can direct or control the first pump and / or the second pump, for example, to operate at a predetermined number of RPMs. Depending on the speed and direction of the IC circulation pump 512, a first portion of the modified first flow rate or IC inlet flow rate can enter the IC inlet port (501A) or the first port of bioreactor 501 at 1422. The pumping rate of the pump can depend on the diameter of the pump or the configuration of the pump (such as a peristaltic pump). Other types of pumps can also be used, where the pumping rate can depend on the configuration of the pump used. The IC circulation pump 512, such as a second peristaltic pump (in an embodiment), can operate at a predetermined number of RPMs and in a direction opposite to that of the first pump to cause or produce a predetermined IC circulation flow rate, or a second flow rate, or a second portion of the IC inlet flow rate to enter the IC outlet port 501B or the second port of bioreactor 501 at 1424. For example, an embodiment can provide that approximately half (about 1 / 2) or a first portion of the IC inlet flow rate branches at the connector 517 to enter the IC inlet port (501A) of bioreactor 501; and approximately half (or 1 / 2) or a second portion of the IC inlet flow rate branches at the connector 517 to enter the IC outlet port 501B of bioreactor 501. As shown, the sum of the first portion and the second portion can be substantially equal to the IC inlet flow rate 503 (e.g., ​ ), where the IC inlet flow rate 503 can be pumped by the IC inlet pump 554. According to an embodiment, depending on the flow rate and countercurrent rate available to maintain the cells within the bioreactor or cell growth chamber 501, other types of fractions or percentages of the IC inlet pump rate can be used to set, configure, or adjust the IC circulation pump.

[0285] In an exemplary embodiment, as described with reference to ​ , the cells 916 can be fed through the EC circulation feeding loop 752, the IC circulation feeding loop 753, or a combination thereof.

[0286] After using such flow characteristics and countercurrent characteristics to feed cells to keep the cells in the bioreactor, process 1400 proceeds to grow / amplify cells 1426. For example, although cell amplification is shown at step 1426, the cells can also grow / amplify during one or more other steps (such as 1412, 1414, 1416, 1418, 1420, 1422, 1424). Process 1400 proceeds from the amplification step 1426 to harvest or remove cells 1430. Then, process 1400 can terminate at the end operation 1432. According to an embodiment, if any other steps are desired before harvesting, such as continuing to use a second modified feeding method or other types of feeding methods, process 1400 proceeds to the optional "other" step 1428. Process 1400 proceeds from the optional step 1428 to harvest or remove cells 1430 from the bioreactor, and process 1400 can then terminate at the end operation 1432.

[0287] Go to ​ , an example operating procedure of process 1500 according to an embodiment of the present disclosure is provided for feeding cells to keep the cells in a first position, such as a bioreactor, which can be used in conjunction with a cell expansion system such as CES 500 (e.g., ​ and ​ ) or CES 700 ( ​ ). Start the start operation 1502, and process 1500 proceeds to load 1504 a disposable tubing kit or a pre-installed fluid delivery assembly (such as 210 or 400) into the cell expansion system. As Figure 1C , Figure 4B and Figure 4C show, the disposable kit can include a cell growth chamber 100A or a cell growth chamber 100B. Next, the system can be precharged 1506. For example, in an embodiment, the user or operator can provide instructions to the system to precharge, such as by selecting a task for precharging. In an embodiment, such a task for precharging can be a pre-programmed task. Next, an IC / EC flush task 1508 can be performed, where, for example, the fluids on the IC circulation loop and the EC circulation loop can be replaced. According to an embodiment, the replacement volume can be determined by the amounts of the IC volume and the EC volume exchanged.

[0288] Next, to maintain an appropriate or desired gas concentration in the fibers of the bioreactor membrane, a conditioning media task 1510 can be performed to allow the media to equilibrate with the provided gas supply prior to loading cells into the bioreactor. For example, by adjusting the EC circulation rate, contact can be provided between the media and the gas supply provided by a gas transfer module (GTM) or an oxygenator. The system can then be held in the appropriate or desired state until the user or operator, e.g., is ready to load cells into the bioreactor. For example, in an embodiment, the system can be conditioned with a media such as a complete media. A complete media can be any media used for cell growth. For example, in an embodiment, the system can be conditioned with a serum-free media. In an embodiment, the system can be conditioned with a basal media. Any type of media understood by those skilled in the art can be used.

[0289] Next, process 1500 proceeds to feed cells 1514 according to a first process during a first time period. In an exemplary embodiment, as referenced Figures 7A to 7CAs described, the cells 916 are fed through the EC circulation feed loop 752, the IC circulation feed loop 753, or a combination thereof. In an embodiment, the cells can be fed at a minimal or low feeding rate, for example, where the cell population begins to grow / expand, and the minimal or low feeding rate is capable of meeting the feeding requirements of such a population. For example, during such a first time period, an IC inlet pump rate of +0.1 mL / min can be used to cause or produce a first fluid flow rate of 0.1 mL / min. According to an embodiment, although this example provides a low or minimal feeding rate of 0.1 mL / min, the low or minimal feeding rate can be greater than or equal to about 0.01 mL / min and less than or equal to about 0.1 mL / min. In an embodiment, the low or minimal 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 time period, the +0.1 mL / min IC inlet pump rate can be matched, approximated, or substantially matched with a complementary IC circulation pump rate of -0.1 mL / min in order to retain the cells in the bioreactor during the growth phase of cell culture. In other embodiments, other pump rates and corresponding fluid flow rates can be used. Alternatively, in an exemplary embodiment, the first IC inlet pump can be configured such that a volume flow rate of less than 0.1 mL / min or about 0.01 mL / min enters the IC inlet port (701A), and the IC circulation pump can be configured such that a complementary IC circulation volume flow rate or fluid flow rate of less than -0.1 mL / min or about -0.01 mL / min enters the IC outlet port (701B), where, for example, the negative sign ("-") used in -0.01 mL / min indicates the direction of the IC circulation pump to cause or produce a countercurrent rate to retain the cells in the bioreactor during the growth phase of cell culture.

[0290] Next, process 1500 proceeds to query 1516 to determine whether to adjust the feeding rate to retain the cells within the bioreactor itself, taking into account the growing cell population and increasing feeding requirements. For example, Figure 16B shows an increased feeding rate in response to an increased cell population. As Figure 16B shown, graph 1528 shows the feeding rate in a cell expansion system (e.g., The relationship between the number of cells running or programmed on the cell expansion system) and the IC flow rate. In an embodiment, the IC flow rate includes the medium for feeding cells and can thus also be referred to as the IC medium flow rate. The number of cells 1530 is shown relative to the IC flow rate (mL / min) 1532. As shown, according to the embodiment, as the cells increase and the feeding requirements of the expanding cell population grow, the IC flow rate increases from 0.1 mL / min to 0.2 mL / min to 0.3 mL / min. In the shown embodiment, as shown by line 1534, there can be a substantially linear relationship between the number of cells and the IC flow rate.

[0291] Return Figure 16A and query 1516, if it is not desired to adjust the feeding rate, the process 1500 proceeds from "No" to expanding cells 1520, where, for example, during a first time period, the cells can continue to grow / expand 1514 through the feeding medium. For example, although the cell expansion in step 1520 is shown, the cells can also grow / expand during one or more other steps (such as 1512, 1514, 1516, 1518). On the other hand, if it is desired to adjust the feeding rate while maintaining the cells in the bioreactor, the process 1500 proceeds from "Yes" to feeding the cells 1518 according to a second process during a second time period. In an embodiment, this second process can, for example, involve feeding the cells at a feeding rate that is substantially the same as that during the first time period. In another embodiment, the second process can involve feeding the cells at a feeding rate that is different from the feeding rate used during the first time period. In an embodiment, the IC inlet flow rate can be increased, and the IC recycle flow rate can be set, configured, or adjusted to be equal to a percentage or portion or fraction of the IC inlet flow rate. For example, according to an embodiment, the IC recycle flow rate can be set to be equal to approximately fifty percent (50%) or approximately one-half (1 / 2) of the IC inlet flow rate value, or according to an embodiment, it can be another percentage or fraction. According to an embodiment, determining whether to set, configure, or adjust the IC recycle flow rate to a percentage or fraction or portion of the IC inlet flow rate can be based on the value of the IC inlet flow rate. For example, an embodiment provides the following method for retaining cells in a bioreactor when using the IC inlet flow to feed the cells (where Q IC循环 = IC recycle flow rate (mL / min); Q IC入口 = IC inlet flow rate (mL / min)):

[0292] When Q IC入口 ≥0.2 mL / min, Q IC循环 = (-)1 / 2*Q IC入口 ,

[0293] and

[0294] When QIC入口 When ≥ 0.1 mL / min, Q IC循环 = (-)*Q IC入口 .

[0295] Although the above equations provide different calculations of the IC recycle flow rate based on values of the IC inlet flow rate (e.g., 0.2 mL / min or 0.1 mL / min), other values of the IC inlet flow rate can be used according to other embodiments for making such quantitative calculations. Additionally, although approximately fifty percent (50%) or approximately one-half (1 / 2) is used in this example, other percentages, ratios, fractions, and / or portions can be used according to the embodiments. Returning to process 1500, after using such flow characteristics and countercurrent characteristics as part of a second process to feed cells 1518 to maintain the cells in the bioreactor, process 1500 proceeds to grow / amplify cells 1520. Although cell amplification is shown at step 1520, the cells can also grow / amplify during, for example, one or more other steps such as 1512, 1514, 1516, 1518. Process 1500 proceeds from the amplification step 1520 to harvest or remove cells 1524. Then, process 1500 can terminate at the end operation 1526. According to the embodiments, if any other steps are desired before harvesting the cells, process 1500 proceeds to the optional "other" step 1522. Process 1500 proceeds from the optional step 1522 to harvest or remove cells 1524 from the bioreactor, and process 1500 can then terminate at the end operation 1526.

[0296] Next, Figure 17 An example operational step of a process 1600 for feeding cells according to an embodiment of the present disclosure is shown, which can be used in conjunction with a cell amplification system such as CES 500 (e.g., Figure 5B and Figure 5C ) or CES 700 ( Figures 7A to 7C ). For example, start the start operation 1602, where a disposable kit can be loaded into the cell amplification system, the system can be pre-filled, IC / EC flushing can be performed, the medium can be adjusted, and cells can be loaded. As Figure 1C , Figure 4B and Figure 4C shown, the disposable kit can include a cell growth chamber 100A or a cell growth chamber 100B. Next, process 1600 proceeds to feed cells 1604 according to a first process during a first time period. In an exemplary embodiment, as referenced Figures 7A to 7CAs described, cells 916 can be fed through the EC circulation feed loop 752, the IC circulation feed loop 753, or a combination thereof. In embodiments, the cells can be fed at a minimal or low feed rate, e.g., where a cell population begins to grow / expand and the minimal or low feed rate is capable of meeting the feeding requirements of such a population. For example, a +0.1 mL / min IC inlet pump rate can be used during this first time period. According to embodiments, although the example provides a low or minimal feed rate of 0.1 mL / min, the low or minimal feed rate can be greater than or equal to about 0.01 mL / min and less than or equal to about 0.1 mL / min. In embodiments, the low or minimal feed rate can 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 time period, the +0.1 mL / min IC inlet pump rate can match, approximate, or substantially match a complementary IC circulation pump rate of -0.1 mL / min in order to maintain cells in the bioreactor during the growth phase of the cell culture. Alternatively, in an exemplary embodiment, a first IC inlet pump can cause a volume flow rate of less than 0.1 mL / min or about 0.01 mL / min to enter the IC inlet port (701A), and an IC circulation pump can cause a complementary IC circulation volume flow rate or fluid flow rate of less than -0.1 mL / min or about -0.01 mL / min to enter the IC outlet port (701B), where, for example, the negative sign ("-") used in -0.01 mL / min indicates the direction of the IC circulation pump to cause or create a countercurrent rate to maintain cells in the bioreactor during the growth phase of the cell culture.

[0297] Next, process 1600 proceeds to query 1606 to determine whether the oxygen supply rate can be adjusted to account for the growing cell population and / or continue efforts to keep the cells in the bioreactor. If it is desired to adjust the oxygen supply rate to retain the cells in the bioreactor, process 1600 proceeds from "Yes" to supply the cells 1608 according to a second process during a second time period. In an embodiment, this second process may involve supplying the cells at an oxygen supply rate that is, for example, substantially the same as during the first time period. In another embodiment, the second process may involve supplying the cells at an oxygen supply rate that is different from the oxygen supply rate used during the first time period. In an embodiment, the IC inlet flow rate may be increased, and the IC recycle flow rate may be set, configured, or adjusted to be equal to a percentage or fraction or portion of the IC inlet flow rate. For example, according to an embodiment, the IC recycle flow rate may be set to be equal to about fifty percent (50%) or about one-half (1 / 2) of the IC inlet flow rate value, or according to an embodiment, it may be another percentage or fraction. According to an embodiment, it may be determined whether to set the IC recycle flow rate to a percentage of the IC inlet flow rate based on the value of the IC inlet flow rate. For example, an embodiment provides the following: (where Q IC循环 = IC recycle flow rate (mL / min); Q IC入口 = IC inlet flow rate (mL / min))

[0298] When Q IC入口 ≥ 0.2 mL / min, Q IC循环 = (-)1 / 2 * Q IC入口

[0299] and

[0300] When Q IC入口 ≥ 0.1 mL / min, Q IC循环 = (-) * Q IC入口 .

[0301] While the above equations provide different calculations of the IC recycle flow rate based on values of the IC inlet flow rate (e.g., 0.2 mL / min or 0.1 mL / min), other values of such IC inlet flow rates can be used according to other embodiments for performing such quantitative calculations. Additionally, while approximately fifty percent (50%) or approximately one-half (1 / 2) is used in this example, other percentages, ratios, fractions, and / or portions can be used according to embodiments. Returning to process 1600, after using such flow characteristics and countercurrent characteristics as part of a second process to feed cells 1608 to maintain the cells in the bioreactor, process 1600 proceeds to query 1610 to determine whether to monitor or measure the metabolic activity of the growing cell population, such as glucose consumption and / or lactate production. For example, if monitoring of metabolic activity is not desired, process 1600 proceeds from "No" to expanding cells 1616, where during the first time period 1604 and / or the second time period 1608, the cells can continue to grow / expand by feeding them the medium for them. Although cell expansion at step 1616 is shown, the cells can also grow / expand during, for example, one or more other steps (such as 1604, 1606, 1608, 1610, 1612, 1614).

[0302] Returning to query 1610, if monitoring or measuring the metabolic activity of the growing cell species is desired, based on the metabolic activity and / or its measured values, process 1600 proceeds from "Yes" to continuing to feed the cells according to the second process or to adjusting the feeding rate. In embodiments, monitoring glucose and / or lactate levels can help adjust the medium flow rate (e.g., IC flow rate) to support cell (e.g., T cell or Treg) expansion in a bioreactor (such as a hollow fiber bioreactor). In embodiments, the lactate value of the cell culture can be maintained, for example, below approximately 7 mmol / L. In embodiments, by using a cell expansion system graphical user interface (GUI) to control, for example, the rate of medium addition, the lactate metabolic waste from glycolysis can be maintained, for example, below approximately 7 mmol / L during cell (e.g., regulatory T cell) expansion. In other embodiments, for example, the rate of medium addition and / or other settings can be controlled to attempt to maintain the lactate level, for example, less than or equal to (≤) approximately 5 mmol / L to improve cell growth and viability. In other embodiments, other concentrations can be used.

[0303] Based on the metabolic measurements and the desired lactate level, process 1600 proceeds to feed the cells 1612 according to a second process or to adjust the feeding rate 1614. For example, according to an embodiment, when the measurement of the metabolic activity shows that the lactate level ≤ about 5 mmol / L, the cells 1612 can be fed continuously according to the second process. For example, in another embodiment, when the measurement of the metabolic activity shows that the lactate level ≤ about 7 mmol / L, the cells 1612 can be fed continuously according to the second process. Process 1600 returns from continuously feeding the cells 1612 according to the second process to query 1610 to continue monitoring the metabolic activity of the growing cell population.

[0304] Depending on the metabolic measurements and their desired levels, process 1600 proceeds to adjust the feeding rate 1614, where the cells can be fed according to an additional process during an additional time period. According to an embodiment, such an additional process and additional time period can include, for example, according to a third process during a third time period, according to a fourth process during a fourth time period, according to a fifth process during a fifth time period, and so on. For example, in an embodiment, such an additional process can involve feeding the cells at a feeding rate that is substantially the same as that during the first and / or second time periods. In another embodiment, the additional process can involve feeding the cells at a feeding rate that is different from the feeding rate used during the first and / or second time periods. For example, in an embodiment, the IC inlet flow rate can be increased, and the IC recycle flow rate can match, approximate, or be substantially the same as the IC inlet flow rate, but in the opposite direction. In another embodiment, the IC inlet flow rate can be increased, and the IC recycle flow rate can be set, configured, or adjusted to be equal to a percentage, fraction, or portion of the IC inlet flow rate, but in the opposite direction. Although adjusting the feeding rate 1614 in step 1614 shows an "additional" process and an "additional" time period, any number of processes and time periods can be used to adjust the feeding rate based on the metabolic activity.

[0305] Process 1600 returns from adjusting the feeding rate 1614 to query 1610. For example, if no adjustment or further adjustment is desired, process 1600 proceeds from "No" to amplifying cells 1616, where the cells can continue to grow / amplify by supplying them with a medium during the first time period 1604, the second time period 1608, and / or additional time periods 1614. Although cell amplification at step 1616 is shown, the cells can also grow / amplify during, for example, one or more other steps (such as steps 1604, 1606, 1608, 1610, 1612, 1614). Process 1600 proceeds from the amplification step 1616 to harvesting or removing cells 1618 from the bioreactor and transferring them into, for example, a harvest bag or container. Then, process 1600 can terminate at the end operation 1622. Alternatively, process 1600 can optionally proceed from the harvest operation 1618 to allow further processing / analysis 1620. This optional further processing / analysis 1620 can include characterizing the phenotype of, for example, the harvested cells (such as T cells or Tregs). From the optional further processing / analysis step 1620, process 1600 can then terminate at the end operation 1622.

[0306] Figure 18A The operational steps of a process 1700 for amplifying cells in an embodiment of the present disclosure are shown, which can be used with a cell amplification system. As described below, according to an embodiment of the present disclosure, process 1700 can include a step of shearing cells that have been amplified in a cell growth chamber. In an embodiment, these steps can be implemented as part of a "modified cycle" task. Start the start operation 1702, and process 1700 proceeds to loading 1704 a fluid having cells into the cell growth chamber in the cell amplification system. In an embodiment, the cells can include non-adherent cells, such as one or more types of T cells. In one embodiment, the cells include Tregs.

[0307] Process 1700 proceeds to exposing the cells to an activator 1706. The activator, which can include an antibody complex, can be added to the fluid loaded in step 1704. In an embodiment, the activator can be a soluble human antibody CD3 / CD28 / CD2 cell activator complex. Process 1700 proceeds to amplifying the cells 1708 within a first time period. Step 1708 can include feeding the cells 1710. Nutrients can be fed to the cells to facilitate their amplification. For example, a medium containing glucose, protein, and reagents can be delivered to the cell growth chamber to provide nutrients for cell amplification.

[0308] The first time period for expanding cells 1708 can be based on the time required to form cell colonies, micro-colonies, or clusters. A cell colony, micro-colony, or cluster can be a group of one or more attached cells. In embodiments, cells (such as Tregs) may benefit from cell contact. Cell contact can stimulate signals that promote expansion and growth. However, after a period of expansion, the cells can attach to each other and form cell colonies, micro-colonies, or clusters. Without being bound by theory, it is believed that after the time period of cell expansion 1708, the cells can form relatively large cell colonies, micro-colonies, or clusters that continue to grow. The cell colonies, micro-colonies, or clusters can produce necrotic centers where nutrients (such as glucose), gases (such as oxygen), and reagents (such as activators) cannot reach the cells at the center of the cell colony, micro-colony, or cluster. Thus, the conditions for cell expansion of the cells at the center of these cell colonies, micro-colonies, or clusters may be such that the expansion rate may slow down (such as increasing the doubling time) or the conditions may result in cell necrosis.

[0309] In embodiments, to allow for the expansion of cells 1708, the first time period can be between about 5 hours and about 48 hours. In some embodiments, the first time period can be greater than about 6 hours, greater than about 12 hours, greater than about 24 hours, or even greater than about 48 hours. In other embodiments, the first time period can be less than about 72 hours, less than about 60 hours, less than about 48 hours, less than about 36 hours, less than about 24 hours, or even less than about 12 hours. After the first time period, process 1700 proceeds to cycle 1712 to break down the cell colonies, micro-colonies, or clusters during a second time period. Step 1712 can be performed to reduce the size of the cell colonies, micro-colonies, or clusters. In embodiments, the second time period can be less than about 120 minutes, such as can be between about 60 minutes and about 0.5 minutes. In other embodiments, the second time period can be based on the volume of fluid introduced into the first circulation path.

[0310] Figure 18B Multiple illustrations 1750, 1760, and 1770 of cells in a volume of fluid (1752) are shown, where the cells can be expanded in a cell growth chamber as part of process 1700. For example, in some embodiments, the cell growth chamber can be a hollow fiber bioreactor. For example, in these embodiments, illustrations 1750, 1760, and 1770 can show the cells in the fibers of the hollow fiber bioreactor. 1750A, 1760A, and 1770A are magnified portions of illustrations 1750, 1760, and 1770, respectively. Referring to illustration 1750, the cells can be shown after they have been loaded 1704, exposed to an activator 1706, and expanded 1710 for a period of time (such as the first time period). As shown in illustration 1750, multiple cell colonies 1754A to 1754E have formed.

[0311] To reduce the size of cell colonies, micro-colonies, or clusters from 1754A to 1754E and the number of cells therein, step 1712 can circulate the fluid and cells through a first fluid circulation path. Without being bound by theory, it is believed that the circulation can generate some forces (including shear stress) acting on the cell colonies, as shown by arrow 1756 in the enlarged portion 1760A. The shear stress 1756 can provide sufficient force to separate the cells in the cell colony. As the circulation continues, the cell colonies can begin to break down into smaller sizes, as shown in illustration 1760. Illustration 1770 shows the cells after the circulation has been carried out for a second time period. As shown in illustration 1770, the size of the cell colonies is reduced, and some of the colonies are completely separated into individual cells. In some embodiments, the circulation to shear step 1712 can be carried out until the cells and fluid comprise a single cell suspension.

[0312] In other embodiments, cell colonies, micro-colonies, or clusters of cells may still exist after the circulation to shear 1712. For example, colony 1754F in the enlarged portion 1770A shows that some reduced-size colonies can remain after step 1712. In an embodiment, the remaining cell colonies, micro-colonies, or clusters (e.g., 1754F) can be between about 25 microns and about 300 microns. In other embodiments, the circulation to shear 1812 can reduce the size of the cell colonies, micro-colonies, or clusters (e.g., 1754F), such that the cell colonies, micro-colonies, or clusters can be between about 50 microns and about 250 microns. In still other embodiments, step 1712 can reduce the size of the cell colonies, micro-colonies, or clusters to between about 75 microns and about 200 microns. In some embodiments, after step 1712, the size of the cell colonies, micro-colonies, or clusters can be less than about 200 microns, e.g., about 100 microns.

[0313] In an embodiment, the size of the remaining cell colonies, micro-colonies, or clusters may depend to some extent on some structural features of the cell growth chamber. As described above, in some embodiments, the cell growth chamber can be a hollow fiber bioreactor having hollow fibers. As can be understood, when the cell colonies, micro-colonies, or clusters are circulating, they may be affected by shear stress each time they contact the side wall of the hollow fiber. This contact can more effectively reduce the size of the cell colonies. When the inner diameter is large, such as in a conventional process where a pipette can be used to induce shear stress to reduce the colony size, the contact with the side wall may not occur frequently. Figure 18CShows the inner diameter size difference between a hollow fiber (e.g., 215 microns) 1772 and a pipette tip 1774 (762 microns) in one embodiment, which can be used to dissociate attached cells in cell colonies, micro-colonies or clusters. In an embodiment, the hollow fiber with a smaller inner diameter is considered to be more efficient and effective in reducing the size of cell colonies during the cycling shear step 1712.

[0314] In an embodiment, the second time period for cycling shear 1712 can be less than about 120 minutes, less than about 90 minutes, less than about 60 minutes, less than about 30 minutes or even less than about 15 minutes. In some embodiments, the second time period can be between about 15 minutes and about 1 minute, such as about 4 minutes.

[0315] After the second time period, the process 1700 proceeds to move the cells to the cell growth chamber 1714 during a third time period. In step 1714, as a result of the cycling shear step 1712, the cells not located in the cell growth chamber are moved back into the cell growth chamber during the third time period. In an embodiment, this can involve activating one or more pumps to introduce fluid into the fluid circulation path. For example, the fluid can be introduced from the fluid inlet path into the first fluid flow path, and then into the cell growth chamber from the inlet port and the outlet port of the cell growth chamber. The movement of the fluid from the inlet port and the outlet port into the cell growth chamber can move the cells back into the cell growth chamber.

[0316] In some embodiments, the fluid used in the step of moving the cells back to the cell growth chamber at 1714 can include reagents that promote cell growth. For example, in an embodiment, the fluid can be a medium including glucose, protein or other reagents. In one embodiment, the fluid can include one or more supplements. In one embodiment, the fluid is a complete medium and includes cytokines (e.g., human IL-2 cytokine supplement). The addition of the fluid can be referred to as a bolus addition. The combination of steps 1712 and 1714 can be referred to as cycling and bolus addition in an embodiment.

[0317] In other embodiments, the third time period can be based on the volume of fluid introduced into the cell growth chamber during the cycling shear step 1712. For example, in an embodiment, step 1712 can be performed until about 300 mL, about 250 mL, about 200 mL or about 150 mL has been introduced into the fluid circulation path.

[0318] After the third time period, process 1700 proceeds to amplification 1716 during a fourth time period. Similar to step 1708, step 1716 may include feeding cells 1718. Nutrients can be fed to the cells to facilitate their amplification. For example, a medium containing glucose, protein, and reagents can be delivered to the cell growth chamber to provide nutrients for cell amplification.

[0319] Similar to the first time period, the fourth time period can be based on the time that may be required for cell colony formation. In an embodiment, the fourth time period can be between about 5 hours and about 48 hours. In some embodiments, the fourth time period can be greater than about 6 hours, greater than about 12 hours, greater than about 24 hours, or even greater than about 48 hours. In other embodiments, the fourth time period can be less than about 72 hours, less than about 60 hours, less than about 48 hours, less than about 36 hours, less than about 24 hours, or even less than about 12 hours. Because more cells may be in the cell growth chamber, in some embodiments, the fourth time period may be shorter than the first time period.

[0320] After the fourth time period, process 1700 proceeds to step 1720 to cycle to shear during a fifth time period to reduce the second cell colony. In an embodiment, step 1720 can use a first cycling rate. However, in other embodiments, the cycling rate used in step 1720 can be different from, greater than, or less than the first cycling rate.

[0321] After the fifth time period, process 1700 proceeds to step 1722, in which cells not located in the cell growth chamber can be moved back into the cell growth chamber during a sixth time period. Fluid can be introduced from the fluid inlet path into the first fluid flow path and into the cell growth chamber from the inlet port and the outlet port of the cell growth chamber. The movement of the fluid from the inlet port and the outlet port into the cell growth chamber can move the cells back into the cell growth chamber. In some embodiments, the fluid used to move the cells back into the cell growth chamber can include reagents that promote cell growth. For example, in an embodiment, the fluid can be a medium containing glucose, protein, or other reagents. In one embodiment, the fluid can include one or more supplements. In one embodiment, the fluid is a complete medium and includes cytokines (such as human IL-2 cytokine).

[0322] Optionally, process 1700 may perform additional rounds of amplification, cycling, and cell movement steps, as shown by optional step 1724 and ellipsis 1726. The steps of amplification, cycling, and movement may be performed sequentially for a period of time. For example, in some embodiments, these 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 days to about twenty days (such as about 10 days). In some embodiments, these steps may be performed at different time intervals. For example, in one embodiment, the steps may be performed after three days and then once every other day. As another example, the steps may be performed after two days and then twice a day. These are merely examples, and other time intervals may be utilized in other embodiments.

[0323] For example, Figure 19 Graph 1800 shows cycling and bolus addition at different time intervals during the process of amplifying cells. Curve 1808 shows the number of cells 1802 relative to the number of days of cell culture 1804 on a cell expansion system (e.g., a cell expansion system). Curve 1806 shows the IC flow rate 1818 relative to the number of days of cell culture 1804 on a cell expansion system (e.g., a cell expansion system). As shown by curve 1806, the IC flow rate remains the same at 0.1 mL / min for the first three days. The flow rate increases to 0.2 mL / min on day 6 and increases to 0.3 mL / min after day 7. The increase in flow rate may be in response to the increasing number of cells as the number of days of culture 1804 increases. Additionally, Figure 19 several cycling and bolus addition steps (e.g., 1810, 1812, 1814, and 1816) are shown. Cycling and bolus addition 1810 is performed after 3.5 days of cell culture. Another cycling and bolus addition 1812 is performed after 4.5 days of cell culture. Another cycling and bolus addition 1814 is performed after 6 days of cell culture, and another cycling and bolus addition 1816 is performed after 6.5 days of cell culture. As can be understood by viewing curves 1806 and 1808, the combination of multiple cycling and bolus additions (1810 to 1816) with an increasing IC flow rate can have a positive effect on the rate of cell expansion (e.g., the number of cells).

[0324] Returning to Figure 18A , process 1700 proceeds to removing the cells from the cell growth chamber 1728. In an embodiment, this may involve harvesting the cells. Step 1728 may include additional steps before or during removing the cells from the cell growth chamber, such as cycling steps (e.g., 1712 and 1720). Process 1700 terminates at end operation 1730.

[0325] Figure 20AShows the operating steps of process 1900 for operating a pump, which can be used in a cell expansion system in embodiments of the present disclosure. As described below, according to embodiments of the present disclosure, process 1900 may include the step of activating a pump in a process of reducing cells in cell clusters that have been expanded in a cell growth chamber. In embodiments, these steps may be implemented as part of a "modified circulation" task. In embodiments, the steps of process 1900 may be performed by a computer processor. Start operation 1902 is initiated, and process 1900 proceeds to step 1904, where a first pump is activated at a first flow rate to introduce a first volume of fluid including cells into a portion within a capillary of a bioreactor of the cell expansion system. As Figure 1C , Figure 4B and Figure 4C shown, for example, the bioreactor may be cell growth chamber 100A or cell growth chamber 100B. In embodiments, the first pump may be an inlet pump.

[0326] After activating the first pump at the first flow rate, process 1900 proceeds to a second pump 1906 activated at a second flow rate to introduce a medium having nutrients into the inner portion of the capillary of the bioreactor during a first time period. The nutrients may include, for example, proteins, glucose, and other compounds for nourishing and promoting cell expansion. For example, referring to Figure 20B , the first pump 1960 may be activated at the second flow rate to introduce the medium into the inlet port 1962A of the bioreactor 1962.

[0327] In embodiments, the first time period may be based on the time required for cell colony formation. In embodiments, the first time period may be between about 5 hours and about 48 hours. In some embodiments, the first time period may be greater than about 6 hours, greater than about 12 hours, greater than about 24 hours, or even greater than about 48 hours. In other embodiments, the first time period may be less than about 72 hours, less than about 60 hours, less than about 48 hours, less than about 36 hours, less than about 24 hours, or even less than about 12 hours.

[0328] Then, process 1900 proceeds to activate the second pump at a third flow rate to direct fluid to bioreactor 1908. Optional step 1908 may be performed to activate pump 1964 to direct a portion of the fluid introduced in step 1906 to the outlet port 1962B of bioreactor 1962 to nourish the cells, for example.

[0329] Then, process 1900 proceeds to activate the second pump 1910 at a fourth flow rate to circulate the cells during a second time period and reduce the number of cells in cell clusters in the bioreactor. In embodiments, the cells are circulated throughout the first fluid circulation path. Referring to Figure 20C, the second pump 1964 can be activated in step 1910 to circulate fluid through the first fluid circulation path 1966, as shown by arrows 1968A to 1968D.

[0330] Without being bound by theory, it is believed that after a period of time, the expanded cells will form cell colonies, microcolonies or clusters. The cell colonies can produce necrotic centers where nutrients and proteins (such as activators) cannot reach the cells at the center of the colony. Thus, the conditions for cell expansion at the center of these cell colonies, microcolonies or clusters may cause the expansion rate to slow down (e.g., the doubling time increases) or may cause cell necrosis. Step 1910 can be performed to reduce the size of the cell colonies, microcolonies or clusters.

[0331] In an embodiment, the fourth flow rate may be high enough to induce shear. For example, in an embodiment, the fourth flow rate can be as high as about 1000 mL / min. In other embodiments, the fourth flow rate can be between about 100 mL / min and about 600 mL / min, such as 300 mL / min.

[0332] After the second time period, the process 1900 proceeds to step 1912, where the first pump is activated at a fifth flow rate to introduce fluid through the first fluid flow path during a third time period. In an embodiment, the first portion of the fluid introduced into the first fluid flow path can cause the first cells in the first fluid flow path (which can be in the first fluid flow path due to step 1910) to move back into the cell growth chamber through the inlet port 1962A. Refer Figure 20D , the pump 1960 can be activated to introduce fluid into the first fluid flow path 1970. As shown by arrows 1968A and 1968B, the fluid flows from the first fluid flow path 1970 into the inlet port 1962A. This moves the first cells outside the bioreactor 1962 back into the bioreactor 1962. It is believed that moving the cells in the first fluid flow path back into the cell growth chamber improves the overall expansion of the cells because the conditions for cell growth are optimized in the cell growth chamber.

[0333] In some embodiments, the fluid introduced into the first fluid flow path in step 1912 can include one or more materials (e.g., reagents) that promote cell expansion. For example, in an embodiment, the fluid can be a medium that includes glucose or other nutrients for feeding the cells. In one embodiment, the fluid can include a reagent that can include an additional activator for continuing to activate the expansion of the cells. Using a fluid with a specific reagent or other material to move the cells back into the cell growth chamber and also expose the cells to additional reagents (such as growth factors, proteins, etc.) that promote expansion can provide improved cell expansion. In an embodiment, the additional fluid used in step 1912 can be referred to as a bolus addition.

[0334] In addition, after the third time period, at step 1914, the second pump can be activated at a sixth flow rate to move a second portion of the fluid introduced through the first fluid flow path and the second cells through the outlet port 1962B to the cell growth chamber. In an embodiment, the second portion of the fluid can cause the second cells in the first fluid flow path (which can be in the first fluid flow path due to step 1910) to move back into the cell growth chamber through the outlet port. Refer to Figure 20D , the pump 1964 can be activated to move the fluid introduced into the first fluid flow path 1970 to the outlet port 1962B. As shown by arrow 1968C, the pump 1964 moves the fluid and cells in the first fluid circulation path through the outlet 1962B into the bioreactor 1962. As Figure 20D shown, the sixth flow rate can be in a direction opposite to the fourth flow rate ( Figure 20C ). This fluid movement moves the cells outside the bioreactor 1962 back into the bioreactor 1962.

[0335] In an embodiment, the sixth flow rate can be less than the fifth flow rate, as described above, which moves the fluid into the first fluid flow path. As can be understood, the fifth flow rate can cause a volume of fluid to be introduced into a portion of the circulation path 1966 based on the fifth flow rate. The sixth rate can be set such that a certain percentage of the volume moves towards the outlet port 1962B.

[0336] In an embodiment, the sixth flow rate can be set as a percentage of the fifth flow rate. For example, the sixth flow rate can be less than or equal to about 90% of the fifth flow rate. In some embodiments, the sixth flow rate can be set to less than or equal to about 80% of the fifth flow rate. In other embodiments, the sixth flow rate can be less than or equal to about 70% of the fifth flow rate. In still other embodiments, the sixth flow rate can be less than or equal to about 60% of the fifth flow rate. In some embodiments, the sixth flow rate can be less than or equal to about 50% of the fifth flow rate.

[0337] In an embodiment, the sixth flow rate is at least partially based 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. Refer to Figure 20D , portions of the first circulation path can have different volumes. For example, in one embodiment, the first volume between the second pump 1964 and the inlet port 1962A can have a first volume, and the second volume between the second pump 1964 and the outlet port 1962B can have a second volume different from the first volume (e.g., larger). In these embodiments, the sixth flow rate can be set at least partially based on the difference in these volumes in order to move the cells from the circulation path back into the bioreactor such that the cells generally arrive at the bioreactor at approximately the same time.

[0338] As can be understood, as the fluid enters the fluid circulation path 1966 from the first fluid flow path 1970, the fluid moves towards the inlet port 1962A at the flow rate set by the first pump 1960. When the pump 1964 is activated, it will redirect at least a portion of the fluid towards the outlet port 1962B. In an embodiment, the second volume (the volume between 1964 and the outlet port 1962B) may be larger than the first volume. Thus, to move more fluid into the second volume, the second pump 1964 can be set as a percentage of the rate of pump 1960.

[0339] In one embodiment, at step 1912, the pump 1960 can be set to 100 mL / min. In this embodiment, the second volume (from the pump 1964 to the outlet port 1962B) may be larger than the first volume (from the pump 1964 to the inlet port 1962A). To account for the additional volume, the embodiment can set the pump 1964 to 70 mL / min during step 1914. This embodiment can provide for the cells to arrive at the bioreactor 1962 approximately simultaneously during a third time period.

[0340] In an embodiment, optionally, the process 1900 can perform the steps 1906 to 1914 one or more additional times, as indicated by the ellipsis 1916 and the optional step 1918. The activation steps: the steps of the first pump (at a second rate) 1906, the second pump (at a fourth rate) 1910, the first pump (at a fifth rate) 1912, and the second pump (at a sixth rate) 1914 can be performed continuously for a period of time. As described above, these steps can be performed to feed the cells, circulate the cells to break down cell colonies, micro-colonies, or clusters, and move the cells back to the cell growth chamber. For example, in some embodiments, these steps can be performed once every three days, once every two days, once a day, twice a day, or three times a day for a time period of about two days to about twenty days (such as 10 days). In some embodiments, these steps can be performed at different time periods. For example, in one embodiment, the steps can be performed after three days and then once every other day. As another example, the steps can be performed after two days and then twice a day. This is merely an example, and other embodiments can utilize other time periods. The process 1900 terminates at the end operation 1920.

[0341] It should be noted that, in some embodiments, the process 1900 can include additional steps. For example, a rocking device can be connected to the bioreactor, and after the first time period (and during the second time period), when the first pump is activated at step 1910, the rocking device can be activated to rotate the bioreactor as part of circulating the cells to reduce the number of cells in the cell clusters. This is merely an example, and other embodiments of the process 1900 are not limited thereto.

[0342] In an alternative embodiment, as Figure 20A shown, the steps of Process 1900 may be followed, where the flow rate is reduced through fine motor control. For example, the IC and EC pumps are allowed to operate within a torque range from 0.005 RPM to 600 RPM to prevent stalling across the entire range. Thus, in both directions, the large pump has a flow rate range as low as 0.01 mL / min at 500 RPM, and the small pump has a flow rate range as low as 0.01 mL / min at 300 RPM. The work done to prevent low-speed stalling is due to improved low-speed stall detection methods and control loops with differential control at low speeds. In an exemplary embodiment, as described with reference to Figures 7A to 7C the fluid may be pumped through the EC circulation supply loop 752, the IC circulation supply loop 753, or a combination thereof.

[0343] As previously described, the IC and EC pumps move continuously at nearly continuous speeds to provide strong countercurrent containment at ultra-low flow rates. If two matching pumps are duty cycling, they cannot operate relative to each other to hold the cells in the bioreactor without moving their positions within the bioreactor. With the continuous operation of the IC and EC pumps over a long period of time, the cells are held in an optimized position within the bioreactor, which overall saves cell media.

[0344] As described below, harvest synchronization is provided through alternating pump control that can operate for a long time. The low flow rate (about 1 / 10 of the flow rate used during the steps in the previous strategy) reaches approximately 0.01 mL / min. To obtain these speeds, an RPM of 0.005 is required at continuous speeds. Thus, a constant motion of the pump at 0.005 RPM is achieved without stalling. See the data in D0000038948, D0000038355, D0000044793, D0000033725 (and all attached appendices), and D0000041290.

[0345] In an alternative embodiment, as Figure 20AAs shown, the steps of process 1900 can be followed, which significantly reduces the time period compared to the above description. For example, the IC inlet pump and the EC inlet pump can be activated during alternating time periods, where the time period is less than 10 minutes or approximately 5 minutes. The pumps can be activated at a low flow rate or an ultra-low flow rate within each time period. For example, for a standard bioreactor or a standard cell growth chamber, the low flow rate or ultra-low flow rate can be a flow rate within approximately 0.005 RPM to 600 RPM, or within approximately 0.01 mL / min to 500 RPM, and for a small bioreactor or a small cell growth chamber, it can be within approximately 0.01 mL / min to 300 RPM. Alternately activating the IC inlet pump and the EC inlet pump during low time periods allows for synchronization of the harvest within the capillary. During process 1900, the harvest valve and the EC outlet valve are opened respectively as the IC inlet pump is activated and the EC inlet pump is activated. When the IC and EC inlet pumps are running, they run at twice the commanded speed, unless twice the commanded speed exceeds the allowable range. Then, the pumps run at the maximum allowable flow rate for 5 minutes. If the stop condition depends on volume, the remaining time displayed on the screen will only be updated while the corresponding pump is running.

[0346] Creating an option to cycle between the harvest outlet and the waste outlet allows the user to select to collect cell culture products of interest (such as cells, viral vectors, exosomes, conditioned media, etc.) at a continuous, user-selected slow flow rate into the harvest bag over a long period of time, while still being able to feed and maintain the cell culture. This option can be used for tasks in standard bioreactors or standard cell growth chambers and small bioreactors or small cell growth chambers (disposable type).

[0347] Synchronization of the harvest within the capillary (IC) provides a continuous medium over a long period of time while still feeding the cells. It allows for the regulated medium to feed the cells, which may be advantageous over fresh medium.

[0348] In an exemplary embodiment, as referenced Figures 7A to 7C as described, fluid can be pumped through the EC circulation feeding loop 752, the IC circulation feeding loop 753, or a combination thereof.

[0349] Figure 21 An example operational step of process 2000 for cell expansion according to an embodiment of the present disclosure is shown, which can be used with a cell expansion system, such as the CES 500 (e.g., Figure 5A ), the CES 600 ( Figure 6 ), or the CES 700 ( Figures 7A to 7C ). The start start operation 2002 is initiated, and process 2000 proceeds to load a disposable tubing kit into the cell expansion system 2004. As Figure 1C , Figure 4B and Figure 4CAs shown, the disposable tubing set 2004 can include a cell growth chamber 100A or a cell growth chamber 100B. Next, the system 2006 can be primed. In an embodiment, a user or operator can, for example, provide instructions to the system to prime, such as by selecting a task for priming. In an embodiment, the task for priming can be a pre-programmed task. Then, the process 2000 proceeds to the IC / EC flush task 2008, where the fluids on the IC loop and the EC loop are replaced. The replacement volume is determined by the amount of the IC volume and the EC volume exchanged.

[0350] Next, in order to maintain an appropriate or desired gas concentration in the fibers of the bioreactor membrane, a conditioning media task 2010 can be performed to allow the media to equilibrate with the supplied gas supply before loading cells into the bioreactor. For example, by using a high EC circulation rate, rapid contact between the media and the gas supply provided by a gas transfer module (GTM) or an oxygenator is provided. Then the system can be maintained in an appropriate or desired state until a user or operator is, for example, ready to load cells into the bioreactor. For example, in an embodiment, the system can be conditioned with a complete media. Complete media is any media that can be used for cell growth. In an embodiment, the complete media can include, for example, alpha-MEM (α-MEM) and fetal bovine serum (FBS). Any type of media understood by those skilled in the art can be used.

[0351] For example, process 2000 then proceeds to concentrate and load cells 2012 from the cell inlet bag into the bioreactor without recirculation. In an embodiment, a "concentrate and load cells without recirculation" task can be used, where a first volume of fluid comprising a plurality of cells can be loaded into the cell expansion system at a first flow rate, where the cell expansion system includes a cell growth chamber. For example, a second volume of fluid comprising a medium can then be loaded into a portion of the first fluid circulation path at a second flow rate to position the first volume of fluid in a first portion of the cell growth chamber. In an embodiment, the first portion of the cell growth chamber or bioreactor can include an approximately central region of the bioreactor. In an embodiment, the first volume is the same as the second volume. In an embodiment, the first flow rate is the same as the second flow rate. In another embodiment, the first volume is different from the second volume. In another embodiment, the first flow rate is different from the second flow rate. For example, in an embodiment, the sum of the first volume and the second volume is equal to a percentage or proportion of the volume (e.g., total volume) of the first fluid circulation path. For example, the sum of the first volume and the second volume can be, for example, approximately 50% of the volume (e.g., total volume) of the first fluid circulation path. In an embodiment, the fluid in the first fluid circulation path flows through the intra-capillary (IC) space of the bioreactor or cell growth chamber. For example, in an embodiment, the fluid in the second fluid circulation path flows through the extra-capillary (EC) space of the bioreactor or cell growth chamber. In an exemplary embodiment, as referenced Figures 7A to 7C as described, fluid 916 can be nourished by the EC circulation nourishment loop 752, the IC circulation nourishment loop 753, or a combination thereof. In an embodiment, the sum of the first volume and the second volume can be, for example, approximately 50% of the volume of the intra-capillary (IC) loop, or can be another percentage or proportion according to the embodiment. For example, in an embodiment, the sum of the first volume and the second volume can be approximately 50% of the volume of another fluid path, loop, etc. (if applicable), or can be another percentage or proportion according to the embodiment. According to the embodiment, other percentages or proportions can be used, including, for example, any percentage between approximately 1% and 100%.

[0352] After loading cells 2012, process 2000 then proceeds to feed cells 2014. Cells 2016 can be grown / amplified. According to an embodiment, although step 2016 is shown after step 2014, step 2016 can occur before or simultaneously with step 2014. Next, process 2000 proceeds to query 2018 to determine whether any cell colonies, micro-colonies, or clusters have formed. A cell colony, micro-colony, or cluster can be a group of one or more attached cells. If a cell colony, micro-colony, or cluster has formed, process 2000 proceeds from "Yes" to shear 2020 any cell colonies, micro-colonies, or clusters. For example, after amplifying a plurality of cells in a first time period, the cells can be cycled at a first cycling rate during a second time period to reduce the number of cells in the cell colony, micro-colony, or cluster. In an embodiment, cycling the cells at the first cycling rate may result in shear stress on the cell colony, where one or more cells in the cell colony can separate from the cell colony. For example, in an embodiment, reducing the number of cells in the cell colony, micro-colony, or cluster can provide a single cell suspension. In an embodiment, cycling the cells to shear any colonies, micro-colonies, or clusters 2020 can be used once every two (2) days, for example, during cell culture, to maintain a uniform cell density and nutrient diffusion. According to an embodiment, other time periods can also be used. For example, in an embodiment, such a breakdown of any micro-colonies, colonies, or clusters can start on day 4 or later. According to an embodiment, at other days or time periods, such shearing can be initiated. After shear 2020, process 2000 can then return to feed cells 2014.

[0353] For example, if query 2018 determines not to shear, or if there are no cell colonies or clusters, process 2000 proceeds from "No" to resuspend cells 2022. In an embodiment, cycling the cells can be used to uniformly resuspend those cells that may loosely adhere during culture. In an embodiment, step 2022 can include cycling the cells to uniformly resuspend those cells that may loosely adhere before initiating a harvest task or other task to remove cells from the bioreactor. After resuspending cells 2022, process 2000 then proceeds to harvest cells 2024. Optionally, further processing or other analysis of the removed cells can be performed at step 2026, and process 2000 can then terminate at end operation 2028. If no further processing / analysis is desired, process 2000 terminates at end operation 2030.

[0354] Go to Figure 22 And process 2100, according to an embodiment, starts start operation 2102, and at 2104, process 2100 proceeds to load a disposable kit onto the cell expansion system. As Figure 1C 、Figure 4B and Figure 4C As shown, the disposable kit may include a cell growth chamber 100A or a cell growth chamber 100B. The disposable kit can then be primed 2106, and an IC / EC flush step 2108 can occur. Next, the medium can be conditioned 2110. Next, the process 2100 proceeds to loading cells 2112, such as suspended or non-adherent cells (such as T cells or Tregs). In an embodiment, such cells can be loaded 2112 by the "loading cells concentrated without recirculation" task. In another embodiment, such cells can be loaded 2112 by the "loading uniformly suspended cells" task.

[0355] According to an embodiment, next, the process 2100 proceeds to start feeding the cells 2114, which may start on day 0. In an exemplary embodiment, as referenced Figures 7A to 7C as described, the fluid 916 can be fed through the EC recirculation feed loop 752, the IC recirculation feed loop 753, or a combination thereof. The cells can grow and expand 2116, and for example, on day 3, it may be desirable to add a bolus to the IC loop and redistribute the cells 2118. In an embodiment, such bolus fluid may include reagents, such as cytokines or other growth factors. In another embodiment, such bolus fluid may include, for example, reagents and a basal medium.

[0356] After such a bolus addition and redistribution of cells, process 2100 then proceeds again to feeding the cells 2120, which may occur, for example, on day 3. With such feeding 2120, parameters of the system (such as one or more pumps controlling flow rate) can be controlled 2122 to achieve a complementary flow setting and a countercurrent flow setting for fluid movement from the IC inlet port and the IC outlet port of the bioreactor into the bioreactor. For example, the IC inlet pump 2124 can be adjusted or directed to create flow, and the IC circulation pump 2126 can be adjusted or directed to create countercurrent flow. In an embodiment, for example, an IC inlet pump rate of +0.1 mL / min can match, or approximate or substantially match, a complementary IC circulation pump rate of -0.1 mL / min in order to maintain the cells in the bioreactor during the growth phase of the cell culture (which may be days 4 to 7). Alternatively, in an exemplary embodiment, the first IC inlet pump can cause a volumetric flow rate of less than 0.1 mL / min or about 0.01 mL / min to enter the IC inlet port (701A), and the IC circulation pump can cause a complementary IC circulation volumetric flow rate or fluid flow rate of less than -0.1 mL / min or about -0.01 mL / min to enter the IC outlet port (701B), where, for example, the negative sign ("-") used in -0.01 mL / min indicates the direction of the IC circulation pump to cause or create a countercurrent rate to maintain the cells in the bioreactor during the growth phase of the cell culture. Such a setting control 2122 can allow offsetting any forces associated with cell loss from the IC outlet port of the bioreactor.

[0357] For example, process 2100 then proceeds to a query 2128, in which it is determined 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 2118. For example, according to an embodiment, such bolus addition and redistribution of the cells may then occur on days 6 and 9.

[0358] If, or once, it is not desired to continue adding boluses (such as reagents) and redistributing the cells, process 2100 proceeds from "no" to harvesting the cells 2130, where the cells can be transferred to a harvest bag or container. Then, process 2100 terminates at an end operation 2136.

[0359] Alternatively, process 2100 can optionally proceed from harvest operation 2130 to allow for further processing / analysis 2132. Such further processing 2132 can include characterizing the phenotype of harvested cells such as, for example, T cells or Tregs. Process 2100 can optionally proceed from the optional further processing / analysis step 2132 to reload any remaining cells 2134. Then, process 2100 can terminate at end operation 2136.

[0360] According to an embodiment of the present disclosure, process 2200 illustrates the operational steps of a process for expanding cells in a cell expansion system. In some embodiments, process 2200 can be used to expand T cells. As shown, various steps can be carried out over the course of a 14-day protocol to expand the cells. Initiate start operation 2202, and process 2200 proceeds to day 0, where a disposable kit is loaded onto the cell expansion system 2206. As Figure 1C 、 Figure 4B and Figure 4C shown, the disposable kit can comprise cell growth chamber 100A or cell growth chamber 100B. For example, the disposable kit can then be pre-charged 2208, where the kit can be pre-charged 2208 with PBS (e.g., Lonza, without Ca2+ / Mg2+). To prepare for loading of the cells, an IC / EC flush 2210 can be used to exchange the pre-charge fluid. For example, according to an embodiment, the PBS in the system can be exchanged with TexMACS GMP basal medium. Next, the medium can be conditioned 2212. Conditioning the medium 2212 can be carried out to allow the medium to equilibrate with the provided gas supply prior to loading the cells into the bioreactor.

[0361] Next, on day 0, process 2200 proceeds to loading of the cells 2214, such as suspension or non-adherent cells (such as T cells or Tregs). In an embodiment, such cells can be loaded 2214 by a "loading cells concentrated without cycling" task. In another embodiment, such cells can be loaded 2214 by a "loading uniformly suspended cells" task.

[0362] On day 3, a bolus addition 2216 of cytokines can be added while redistributing the cells 2218. In an embodiment, the redistribution of the cells can be combined with the bolus addition to mix the cells and expose the cells more thoroughly to the cytokines (such as IL-2) that may be present in the bolus addition. In an embodiment, the redistribution may also break up cell colonies or clusters that may have formed. In an embodiment, the redistribution can occur first by circulating the cells in a fluid circulation path. Then, in the process of pushing the cells back into the bioreactor, the bolus addition is added, such as by introducing fluid into the fluid circulation path to push the cells back into the bioreactor. After the redistribution and the bolus addition 2218, the process 2000 proceeds to feeding the cells 2220.

[0363] The cells can be redistributed 2224 again on day 6 by another bolus addition 2222. The redistribution may also break up cell colonies or clusters that may have formed between days 3 - 5. The bolus addition can expose the cells to additional reagents that promote expansion. The process 2000 proceeds to feeding the cells 2226 on day 6. On day 9, the cells 2230 can be redistributed again by a bolus addition 2228. The redistribution may also break up cell colonies or clusters that may have formed between days 6 - 8. The bolus addition can expose the cells to additional supplements that promote expansion. The process 2000 proceeds to feeding the cells 2232 on day 9.

[0364] On days 11 - 13, the cells 2236 can be redistributed again by a bolus addition 2234. The redistribution may also break up cell colonies or clusters that may have formed between days 9 - 10. The bolus addition can expose the cells to additional reagents that promote expansion. The process 2000 then proceeds to feeding the cells 2238. In an embodiment, steps 2236 and 2238 can be performed on each of days 11, 12, and 13. This may be due to the expansion of the cells during days 0 - 10 and the presence of a large number of cells in the bioreactor. By more frequently breaking up the cell colonies and clusters and mixing them with the reagents that promote cell expansion in the bolus addition, performing the redistribution and bolus addition of the cells can promote cell expansion. The process 2200 terminates at the end operation 2240.

[0365] According to an embodiment of the present disclosure, the process 2300 illustrates the operating steps of a process for expanding cells (such as, suspension or non - adherent cells) in a cell expansion system. In some embodiments, the process 2300 can be used to expand T cells, such as Tregs. The combination of the steps of the process 2300 can allow for the expansion of cells to a useful clinical amount using an initial low seeding density.

[0366] Initiate start operation 2302, and process 2300 proceeds to loading a disposable kit into cell expansion system 2304. As Figure 1C , Figure 4B and Figure 4C shown, the disposable kit can include cell growth chamber 100A or cell growth chamber 100B. For example, the disposable kit can then be precharged 2206, where the kit can be precharged 2206 with PBS (e.g., Lonza, without Ca2+ / Mg2+). To prepare for cell loading, IC / EC flushing 2308 can be used to exchange the precharge fluid. For example, according to one embodiment, the PBS in the system can be exchanged with TexMACS GMP basal medium. Next, the medium can be conditioned 2310. The medium can be conditioned 2310 to allow the medium to equilibrate with the provided gas supply before loading the cells into the bioreactor.

[0367] Process 2300 proceeds to loading a fluid with cells at the inlet volume 2312. In an embodiment, the cells can include non-adherent cells, such as one or more types of T cells, e.g., Tregs. In one embodiment, the cells include Tregs. The embodiment can provide a fluid with cells at the inlet volume, which is loaded through the IC inlet path and into the IC circulation path using an IC inlet pump. In an embodiment, the inlet volume 2312 is loaded without activating the IC circulation pump.

[0368] Process 2300 proceeds to positioning the inlet volume in the first part of bioreactor 2314. In an embodiment, the positioning can be performed by introducing a second volume of fluid, which can include medium and can be introduced into a portion of the IC circulation path to push the inlet volume with cells into the first position in the bioreactor. In an embodiment, the inlet volume of fluid and the second volume of fluid can be the same. In other embodiments, the inlet volume of fluid and the second volume of fluid can be different. In still other embodiments, the sum of the inlet volume of fluid and the second volume of fluid can be equal to a percentage of the volume of the IC circulation path.

[0369] After positioning the inlet volume 2314, process 2300 proceeds to exposing the cells to an activator 2316 to activate the cells for expansion. In some embodiments, at 2316, the cells can be exposed to a soluble activator. In some embodiments, the activator can include an antibody complex, which can be added to the medium and can be included in the inlet volume, or added later, such as together with the second volume. For example, in an embodiment, the activator can be a human antibody CD3 / CD28 / CD2 cell activator complex.

[0370] Process 2300 proceeds to supply cells 2318 according to a first process during a first time period. In an exemplary embodiment, as described with reference to Figures 7A to 7C , cells 916 can be supplied through the EC recycle supply loop 752, the IC recycle supply loop 753, or a combination thereof. In an embodiment, the cells can be supplied at a minimal or low supply rate, for example, where the cell population begins to grow / expand during the first time period 2320, and the minimal or low supply rate can meet the needs of such a population. For example, an IC inlet pump rate of +0.1 mL / min can 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, the +0.1 mL / min IC inlet pump rate can match, approximate, or be substantially matched with a complementary IC recycle pump rate of -0.1 mL / min, so as to keep the cells in the bioreactor during the growth phase of the cell culture. Alternatively, in an exemplary embodiment, the first IC inlet pump can cause a volume flow rate of less than 0.1 mL / min or about 0.01 mL / min to enter the IC inlet port (701A), and the IC recycle pump can cause a complementary IC recycle volume flow rate or fluid flow rate of less than -0.1 mL / min or about -0.01 mL / min to enter the IC outlet port (701B), where, for example, the negative sign ("-") used in -0.01 mL / min indicates the direction of the IC recycle pump to result in or produce a countercurrent rate to keep the cells in the bioreactor during the growth phase of the cell culture.

[0371] Process 2300 proceeds from 2320 to expand cells 2322 during a second time period. Expanding 2322 during the second time period can also involve supplying cells 2324 according to a second process during the second time period. For example, in an embodiment, such a second process can involve supplying the cells at a supply rate that is substantially the same as during the first time period. In another embodiment, the second process can involve supplying the cells at a supply rate that is different from the supply rate used during the first time period. For example, the supply rate may increase due to the expansion of the cells during the first time period.

[0372] While expanding the cells during the second time period 2322, the cells can also be circulated to break up cell colonies or cell clusters 2326. Step 2326 can involve circulating the cells in the IC recycle path to break up any colonies or clusters that may have formed during the first time period. The step of shearing the colonies or clusters 2326 can reduce the number of cells in the cell colonies or cell clusters. In an embodiment, circulating to shear 2326 may cause the cell colonies to incur shear stress, causing one or more cells in the cell colonies to separate from the cell colonies.

[0373] Process 2300 can then proceed to harvest operation 2328, where cells can be transferred to a harvest bag or container. In an embodiment, a therapeutically effective dose of cells can be harvested. In an embodiment, the cells harvested in operation 2328 can be on the order of 1×10 9 cells. In an embodiment, the harvested cells can have a viability between about 75% and about 95%.

[0374] Then, optionally, process 2300 can proceed to allow for further processing / analysis 2330. Such further processing can include characterizing the phenotype of, for example, the harvested cells (such as T cells or Tregs). In one embodiment, the harvested cells can express biomarkers consistent with Tregs. For example, the cells can express CD4+, CD25+, and / or FoxP3+ biomarkers. In an embodiment, the harvested cells can include a CD4+CD25+ phenotype with a frequency greater than about 80%. In other embodiments, the cells can include a CD4+FoxP3+ phenotype with a frequency greater than about 55%. Then, process 2300 can terminate at end operation 2332.

[0375] According to embodiments of the present disclosure, the operational steps depicted in the above figures are provided for illustrative purposes and can be rearranged, combined into other steps, used in parallel with other steps, etc. Fewer or additional steps can be used in embodiments without departing from the spirit and scope of the present disclosure. Also, for example, steps such as priming, conditioning media, loading cells (and any sub-steps) can be automated in some embodiments, such as by a processor executing pre-programmed tasks stored in a memory, where such steps are provided for illustrative purposes only. Additionally, for illustrative purposes, for example, example pump rate settings for feeding cells depicted in Figure 11B are provided. According to embodiments of the present disclosure, other pump rates, flow rates, directions, etc. can be used.

[0376] In U.S. Patent Application Serial No. 13 / 269,323 ("Configurable Methods and Systems for Growing and Harvesting Cells in a Hollow Fiber Bioreactor System", filed October 7, 2011) and U.S. Patent Application Serial No. 13 / 269,351 ("Customizable Methods and Systems for Growing and Harvesting Cells in a Hollow Fiber Bioreactor System", filed October 7, 2011), examples and further descriptions of tasks and protocols (including custom tasks and pre-programmed tasks) for use with a cell expansion system are provided, the entire contents and all purposes of which are incorporated herein by reference in their entirety.

[0377] Next, Figure 25An example component of a computing system 2400 on which embodiments of the present disclosure may be implemented is shown. For example, in an embodiment, the computing system 2400 may be used, where the cell expansion system uses a processor to perform tasks, such as custom tasks or pre-programmed tasks, which are performed as part of a process such as those shown and / or described herein. In an embodiment, the pre-programmed tasks may include, for example, following "IC / EC flushing" and / or "feeding cells".

[0378] The computing system 2400 may include a user interface 2402, a processing system 2404, and / or a storage device 2406. The user interface 2402 may include an output device 2408 and / or an input device 2410, as understood by those skilled in the art. The output device 2408 may include one or more touchscreens, where the touchscreen may include a display area for providing one or more application windows. For example, the touchscreen may also be the input device 2410, which may receive and / or capture physical touch events from a user or operator. As understood by those skilled in the art, the touchscreen may include a liquid crystal display (LCD) having a capacitive structure that allows the processing system 2404 to infer the location of a touch event. The processing system 2404 may then map the location of the touch event to a UI element presented at a predetermined location in the application window. According to an embodiment, the touchscreen may also receive touch events through one or more other electronic structures. Other output devices 2408 may include printers, speakers, etc. As understood by those skilled in the art, other input devices 2410 may include keyboards, other touch input devices, mice, voice input devices, etc. For example, the user interface 2402 may be the user interface 264 described in reference Figure 2A described. The user interface 2402 may include different screens for different parts of a task / scheme / process / method, for different user inputs or requests, etc.

[0379] According to embodiments of the present disclosure, the processing system 2404 may include a processing unit 2412 and / or a memory 2414. The processing unit 2412 may be a general-purpose processor operable to execute instructions stored in the memory 2414. According to an embodiment, the processing unit 2412 may include a single processor or multiple processors. Additionally, in an embodiment, each processor may be a multi-core processor having one or more cores for reading and executing separate instructions. As understood by those skilled in the art, the processor may include general-purpose processors, application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), other integrated circuits, etc.

[0380] According to an embodiment, the memory 2414 may include any short-term or long-term memory for data and / or processor-executable instructions. As understood by those skilled in the art, the memory 2414 may include, for example, random access memory (RAM), read-only memory (ROM), or electrically erasable programmable read-only memory (EEPROM). As understood by those skilled in the art, other storage media may include, for example, CD-ROM, magnetic tape, digital versatile disk (DVD), or other optical storage, magnetic tape, disk storage, magnetic tape, other magnetic storage devices, and the like.

[0381] The storage device 2406 may be any long-term data storage device or component. According to an embodiment, the storage device 2406 may include one or more systems described in connection with the memory 2414. The storage device 2406 may be permanent or removable. In an embodiment, the storage device 2406 stores data generated or provided by the processing system 2404.

[0382] Via a wireless network, a Bluetooth network, or other network systems, the computing system 2400 may communicate with the cloud, network computers, personal computing devices, mobile devices, and the like. Alternatively, via a hardwired connection, the computing system 2400 may communicate with personal computing devices, network computing devices, mobile devices, and the like.

[0383] The processing system 2404 may control pump activation, speed, and fluid flow. The processing system 2404 may control pump activation / speed to utilize the control loop of the pump process to provide an ultra-low feeding rate with continuous motion (instead of step motion). Continuous, consistent motion (compared to periodic / step motion) contributes to finer pump control and a lower feeding rate. The operation may switch between two pumps operating at low flow rates to facilitate continuous harvesting of cells. In an exemplary embodiment, the first IC inlet pump may cause a volume flow rate of less than 0.1 mL / min or about 0.01 mL / min to enter the IC inlet port (701A), and the IC circulation pump may cause a complementary IC circulation volume flow rate or fluid flow rate of less than -0.1 mL / min or about -0.01 mL / min to enter the IC outlet port (701B), where, for example, the negative sign (“-”) used in -0.01 mL / min indicates the direction of the IC circulation pump to cause or generate a countercurrent rate to keep the cells in the bioreactor during the growth phase of cell culture.

[0384] The processing system 2404 may control pump start / speed to contain countercurrent flow. Countercurrent containment at ultra-low flow rates allows cells to concentrate in the desired area. Countercurrent containment saves the medium of the cell culture and allows for a lower protein concentration in the system. The ability to provide continuous motion allows for a lower protein concentration. Opposing pumps pump in opposite directions, causing the cells to cluster.

[0385] The processing system 2404 can be configured to run various tasks / methods / processes / schemes input by the user or stored in the memory 2414 as preset tasks / methods / processes / schemes. For example, the processing system 2404 can be configured to run or coordinate the execution of a cell processing application (CPA). The cell processing application is capable of tracking and recording the user's materials, procedures, user logins, etc. This system can be used to push a scheme to an identical group of machines, thus saving a significant amount of time in setting up a verified scheme to run on the group of machines. Then, all reports can be pulled back into the application, and the real-time readings of the group can be remotely viewed on the application. Temperature and pressure can be updated to the application at a configurable rate, and alerts and warnings can also be sent to the application. For example, the application can push an alert as a remote alert to one or more users for recording or remediation. For example, the remote alert can be an email, text message, or other digital reminder sent to the user. The CPA also connects and Quantum to save all the user's reports in a unified application.

[0386] The CPA is detected in 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: D0000028798, Protocol Task Management Report: D0000044037, Device Setup Protocol: D0000028621, Device Setup Report: D0000045376, EOR Protocol: D0000028620, EOR Report: D0000045378, D0000047097: Network Performance, D0000047098: Multiple Devices, D0000047099: Access and Audit, D0000047100: Data and Reports.

[0387] The cell processing application can include features for tracking and data management. Tracking and logging can be enabled through the CPA. Tracking and recording can be performed periodically (e.g., scheduled) or on demand. The user can schedule the time to be recorded based on a trigger event or periodic time.

[0388] The cell processing application can also perform group control. The CPA unifies modular cell methods, including CES (e.g., Quantum cell expansion system, etc.). Thus, the CESs (such as CES100, 700, etc.) described herein are individual modules in a modular system controlled by a cell processing application. For example, the CPA can control a group of up to 100 or more devices. Using the CPA, the same instructions can be sent to multiple machines instead of a single machine to simplify and ensure operational consistency.

[0389] The cell processing application can include custom tasks / methods / processes / protocols. The user can draft custom protocols or custom tasks. A task can be the detailed steps of a user-defined process. A protocol can be an assemblage of tasks required to complete a test or a cell expansion process, etc., saved in a single file. The memory 2414 can store predefined, pre-written, or stock protocols and tasks. Stock tasks can be compiled by the user into a custom protocol. The protocols in the CPA system can be modified in real time and re-uploaded to the device group. Tasks and protocols can be written, modified, and selected on the user interface or on an external computer or handheld device.

[0390] The cell processing application can include different user profiles, accounts, and access. Each user is assigned a set of predefined roles or a custom role. Each role includes a set of permissions that can be customized by an administrator. Permissions can include the control level of access (e.g., read-only, write, etc.). A single user can have multiple roles. For security measures, the CPA may require user authentication.

[0391] The cell processing application can trigger and send remote notifications and alerts. An email or other notifications (digital notifications, texts, etc.) can be sent to the user, so that the status, etc. can be monitored / checked without the user directly accessing or touching the machine. The user can be able to communicate with the machine remotely, including viewing real-time data through the cell processing application. The user can be able to remotely send commands to the machine, such as ignoring an alert, stopping a test, modifying a protocol or a test, etc. This is more efficient for the user.

[0392] The cell processing application controls network access for software updates. New software can be pushed to the CES or other devices from the cloud, a remote computer, or a remote device.

[0393] The cell processing application can receive data from a barcode scanner. The barcode scanner can be one of the input devices 2410 described previously. Each of the disposable packages, media, cells, etc. includes a barcode with product information. The CPA can check the data from the barcode scanner to ensure that the correct parts or media are being used. The barcode can be stored in the memory 2414 or in the CPA for future reference (such as product recall) or for augmenting data. The CPA can adjust the configuration of tasks / methods / processes / protocols based on the data from the barcode scanner.

[0394] The computing system 2400 can improve process efficiency by driving contact between "reagents of interest" (viral particles, transfection reagents, secondary cell types, differentiation reagents, induction reagents, etc.) and adherent or suspended cells in a hollow fiber bioreactor (HFB). Cells (adherent or suspended) are seeded on one side of the inside of the HFB capillary (IC). The reagent of interest is introduced into the IC side of the HFB. Countercurrent flow (shunted by driving the IC inlet pump and the IC circulation pump in opposite directions; inlet = forward flow, circulation = reverse flow) is used to drive active contact between the reagent of interest and the cell population. This process can continue as long as necessary.

[0395] Many processes in cell culture require exposing a cell population to specific suspended elements to alter the cells or produce secondary products. For example, to produce a viral vector product, the cell population must be exposed to active viral particles that will enter the cells and replicate inside the cells; to transfect cells by introducing a new gene into the cells, the cells must be exposed to the gene of interest (GOI) and the reagents required to bring the GOI into the cells for translation. Based on the environment in which the process occurs, the efficiency of these two example processes is variable. Many passive models of these types of processes rely on highly random chance to drive interaction between the reagent of interest (ROI) and the cell population. These passive models result in lower efficiency of virus integration / plasmid transfection / GOI expression.

[0396] The process described herein is designed to improve the efficiency of these processes by driving active contact between the cell population and the ROI. Cells (adherent or suspended) are seeded in the fibers of the HFB. Once the cells are established, the "reagent of interest" (viral particles, transfection reagents, secondary cell types, differentiation reagents, induction reagents, etc.) is introduced into the HFB. Carrier fluid is withdrawn from the dual-port bag. The inlet pump and the circulation pump shunt the flow. The carrier fluid enters the IC side of the HFB from both sides. The IC waste valve is closed, so the fluid must leave the HFB from the IC and EC sides through the pores in the HFB membrane. The carrier fluid is recycled back to the dual-port bag from the EC side. The "reagent of interest" is contained in the IC circuit because the molecules are larger than the membrane pores and is pressed against the cell layer on the membrane wall, driving contact between the "reagent of interest" and the cell population. Continue as long as needed.

[0397] Examples

[0398] The following description includes examples of some protocols / methods / processes that can be associated with a cell expansion system, such as the CES 500 (e.g., Figure 5A , Figure 5B , Figure 5C ) and / or the CES 600 ( Figure 6) are used together, for example, to implement aspects of the embodiments. Although specific features may be described in the examples, such examples are provided for illustrative and descriptive purposes only. For example, although the examples may provide for the expansion of T cells and / or Treg cells, in other embodiments, other and / or additional cell types and / or combinations thereof may be used. Although specific parameters, features, and / or values (such as CES, such as Cell Expansion System) are described, these parameters, features, and / or values, etc. are provided for illustrative purposes only. The present disclosure is not limited to the examples and / or specific details provided herein.

[0399] In addition, the examples provided herein are not intended to limit other embodiments, which may include different or additional steps, parameters, or other features. In some embodiments, an exemplary method or protocol including steps (and any sub-steps) may be performed automatically, such as by a processor that executes pre-programmed tasks stored in a memory. In other embodiments, the steps (and any sub-steps) may be performed by a combination of automated and manual operations. In further embodiments, the steps (and any sub-steps) may be performed by an operator or user or by other manual means.

[0400] Although example data may be provided in such examples, such example data is provided for illustrative purposes and is not intended to limit other embodiments, which may include different steps, parameters, values, materials, or other features.

[0401] In some embodiments, the protocol package or method for a smaller bioreactor or smaller cell growth chamber may be the same task as for a standard bioreactor or standard cell growth chamber. In some protocol packages or methods for a smaller bioreactor or smaller cell growth chamber, the following differences may be included: 1. When evacuating the ARC during the air removal chamber (ARC) management step and when forcing flow through the membrane, the flow rate is reduced. This is because a smaller bioreactor has a smaller fiber surface area, so the flow rate is reduced to avoid high pressure. 2. Different default selections, which are based on differences in the disposable kit (i.e., in applicable cases, the flow rate and volume stop conditions are scaled down proportionally according to the volume differences of certain parts of the kit). 3. The range is open, allowing the user to select to scale the flow rate down to 1 / 10 of the standard bioreactor task.

[0402] In some exemplary embodiments, the protocol package or method may include: 1. A default (template) adherent cell expansion protocol; 2. A default (template) suspension cell expansion protocol; 3. A default (template) custom one-step protocol; 4. A custom step 1-10 task with more available options compared to traditional Quantum custom tasks; 5. Loading cells with multiple dispense cycles (a task created by the scientific team to optimize MSC seeding); 6. Loading and positioning cells (a task created by the scientific team to optimize T-cell loading); 7. Feeding non-adherent cells (a task created by the scientific team to optimize T-cell feeding); and / or 8. Cycling and positioning cells (a task created by the scientific team to optimize sampling during T-cell expansion).

[0403] Example 1

[0404] Method

[0405] General Treg cell culture

[0406] Immunomagnetically separated CD4 + CD25 + Tregs can be obtained from the peripheral blood of healthy adult donors by leukapheresis (HemaCare Corporation, Van Nuys, CA) and can subsequently be expanded in three T25 flasks (7 mL / flask) at a concentration of 1.0×10 5 cells / mL in sterile-filtered TexMACS TM GMP medium supplemented with high-quality cytokines of recombinant human IL-2 at 200 IU / mL (Miltenyi Biotec GmbH, Bergisch Gladbach) and Gibco PSN 100X antibiotic mixture (ThermoFisher Scientific, Waltham, MA). The actively growing Treg cell suspension can then be used as an inoculum in each of three (3) Quantum cell expansion system experimental runs. In the absence of beads, soluble tetramer Immunocult TM human antibody CD3 / CD28 / CD2 cell activator complex (Stem Cell Technologies, Vancouver, BC) can be used to co-stimulate the inoculum and Quantum system-expanded Tregs at 25 μL / mL. For the Treg inoculum, co-stimulation can be performed on day 0 and day 9, and for Quantum system Treg expansion, co-stimulation can be performed on day 0. The Quantum system HFM bioreactor can be characterized by a capillary inner loop volume of 177.1 mL and 21000 cm2 Surface area.

[0407] Quantum system Treg expansion

[0408] According to an embodiment, two (2L) bags of sterile filtered medium can be prepared using the Quantum Media Bag 4L Kit (Cat. 21021) for large-scale expansion of Tregs in the Quantum system. One 2L bag containing complete medium of TexMACS GMP, IL-2, and PSN antibiotic can be used to supply the IC compartment, and one 2L bag of basal medium containing TexMACS GMP and PSN antibiotic can be used to supply the EC inlet compartment of the bioreactor. After pre-filling the Quantum system with PBS (Lonza cat. 17-516Q, Walkersville, MD), the media bags can be connected to the appropriate IC and EC inlet lines using a TSCD-Q Terumo sterile welder. The complete medium can be protected from exposure to light.

[0409] The total cell load per run (4.5×10 7 to 6.5×10 7 Tregs) can be resuspended in 50 ml of complete medium using sterile techniques in the Quantum Cell Inlet Bag (Cat. 21020) for introduction into the Quantum system bioreactor. Additional disposable kits such as the Quantum CES Media Bag 4L (Cat. 21021) and Waste Bag 4L (Cat. 21023) can also be used during large-scale Treg expansion runs.

[0410] When completing the "Load cells centrally without circulation" task, Quantum system runs (n = 3) can inoculate Tregs at a concentration of 2.5×10 5 cells / mL to 3.7×10 5 cells / mL in 177 mL of complete medium, or at an average of 2.1×10 3 cells / mL to 3.1×10 3 cells / cm 2 in the lumen or capillary (IC) compartment of the hollow fiber membrane bioreactor.

[0411] Days 0 to 4:

[0412] Example Quantum custom tasks

[0413] Feeding cells, improved IC / EC exchange and conditioning media for regulatory T cells, example

[0414] Using the Terumo BCT TSCD-Q aseptic welder, a media bag containing TexMACS GMP complete medium with IL-2 supplement (200 IU / mL) is attached to the IC media pipeline of the Quantum system. The TexMACS basal medium can be attached to the EC media pipeline. IC / EC flushing and conditioning media tasks can be performed separately. The complete medium can be used for IC exchange or flushing, and the basal medium can be used for EC exchange or flushing to conserve the amount of IL-2 and activator complex.

[0415] Before introducing cells, the system can be placed on a modified "feeder cell". The IC inlet rate (Q1) and IC circulation rate (Q2) can be increased to 0.2, 0.3, and 0.4 mL / min at matching rates on days 5, 6, and 7, but in the opposite direction on days 4, 5, and 6, or maintain the lactate level between 5 mmol / L and 8 mmol / L as needed.

[0416] Table 1: Feeder cells, modified, examples

[0417]

[0418] Example Quantum custom tasks:

[0419] Concentrated loading of cells without circulation, examples.

[0420] Purpose: This task can cause suspended cells to be concentrated and distributed within the bioreactor membrane while allowing flow on the extracapillary (EC) circulation loop. The pump flow rate to the IC loop can be set to zero.

[0421] Before loading cells into the Quantum system by using the load cells without circulation, changes to the task can be entered.

[0422] Table 2: Concentrated loading of cells without circulation, changes, examples

[0423]

[0424] Table 3: Concentrated loading of cells without circulation, examples

[0425]

[0426] As needed, the default cell feeding task can be returned, and with the feeder cell task, the amplification protocol can be continued.

[0427] Table 4: Feeder cells, modified, examples

[0428]

[0429]

[0430] Day 4 or later:

[0431] Resuspend Treg cells during cell culture or prior to harvest, Example.

[0432] The purpose of this modified cycling task can be to evenly resuspend those cells that may have loosely adhered during culture or prior to initiating the harvest task.

[0433] In addition, this task can be used to shear Treg cell colonies every two (2) days during cell culture in order to maintain a uniform cell density and nutrient diffusion starting on Day 4 or later. If the task is used to shear colonies during the culture process, the Quantum system can return to the modified "feed cells" task.

[0434] Table 5: Cycling and resuspension of cells, returning cells to bioreactor and feeding, Example

[0435]

[0436] Harvest Quantum harvest task with modifications, Example.

[0437] Table 6: Harvest, modifications, Example

[0438]

[0439]

[0440] Harvested cells can be removed from the Quantum system by RF welding for further evaluation and analysis.

[0441] Post-harvest analysis

[0442] Harvested cells can be counted in the range of 5μm - 50μm using a VI-XR 2.04 cell viability analyzer (Beckman Coulter), and membrane integrity can be quantified by trypan blue dye exclusion.

[0443] Metabolism

[0444] Regulatory T cell metabolism can be monitored daily from the Quantum EC sample port using an i-STAT handheld analyzer (Abbott Point of Care, Princeton, NJ), where i-STAT G Cartridge (Cat.03P83 - 25) and i-STAT CG4+ cartridge (Cat.03P85 - 50) are used to monitor glucose and lactate concentrations, respectively.

[0445] Cell surface biomarker expression

[0446] 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 may be responsible for maintaining immune homeostasis, which can include the regulation of immune tolerance in innate and adoptive responses. Additionally, the expression of the transcription regulatory factor forkhead box P3 (FoxP3) gene product has been associated with CD4 + CD25 + FoxP3 + CD127 lo / - Treg phenotype and is associated with the immunosuppression of antigen-presenting cells (APCs) and effector T cells (T eff ). The binding of IL-2 to CD25 / IL-2 receptor (Rα) and the activation of the STAT5 transcription factor can be used for Foxp3 induction. FoxP3 inhibition may upregulate the activity of several genes (such as CTLA-4, TNFRSF18, and IL2RA) and may downregulate IL-2 through its binding to histone acetyltransferase KAT5 and histone deacetylase HDAC7.

[0447] The frequency of the Treg phenotype of biomarkers on the harvested cell surface can be quantified by flow cytometry. For this purpose, cells can be stained with the following antibody conjugates and gated against viable, unstained cells: FixableViability Dye 780 (eBioscience 65-0865), mouse anti-human CD4-PE (BD Pharmingen 561844), anti-CD4-Alexa Fluor 647 (BD Phammingen 557707), anti-CD44-FITC (BD Pharmengen 561842), anti-CD4-FITC (BD-Pharmangen 555346), anti-CD25-PE (BD Pharmingen 555432), anti-CD127-PE (BD Pharmingen 557938), anti-CD45RO-PE (BD Pharmingen 347967), and anti-FoxP3-Alexa Fluor647 (BD Pharmingen 560045). On a BD Canto II flow cytometer equipped with FACSDiva v6.1.3 software, sample data can be acquired using 1×10 6 cells and 20,000 total events per sample.

[0448] Figure 26 An example of the experimental procedure is shown.

[0449] Possible results

[0450] Preliminary studies of Tregs in static culture may show that these cells tend to form microcolonies with a diameter of approximately 100 μm. Isolating these cells during the medium exchange process every two days may help limit cell necrosis and restore the cells to a high-density single-cell suspension using a 1000 μL pipette tip with an ID of 762 μm. Alternatively, with the help of a pre-programmed daily cycling task, the process of maintaining a single-cell suspension can be more effectively achieved in an automated HFM bioreactor, where the fiber lumen ID is approximately 200 μm, such as in the Quantum system. Additionally, this automated feeding task can reduce the likelihood of contamination while maintaining a continuous nutrient flow for the Treg culture, as it can be carried out in a functionally closed system.

[0451] Possible Treg cell density and viability

[0452] Table 7: Possible harvests from T25 flasks

[0453]

[0454]

[0455] Table 8: Possible harvests from Quantum

[0456] Cell density Harvest bag <![CDATA[3.61×10 6 cells / m]]> Bioreactor <![CDATA[1.24×10 7 cells / mL]]> <![CDATA[≥7.33×10 4 cells / cm 2 > Viability 84.80％ Stimulation cycle 1 Doubling 4.6 Doubling time 41.6 hours

[0457] Preliminary cell seeding density experiment

[0458] When preparing to expand immunomagnetically selected cells from donors in an automated bioreactor, a series of static growth experiments can be conducted to determine whether stimulated Tregs can be cultured at an inoculation density of less than 1.0×10 6 cells / mL. This part of the study can be carried out by inoculating 1.0×10 5 cells / mL or per well in 18 wells of a 24-well tissue culture plate in TexMACS GMP medium supplemented with IL-2 (200 IU / mL) and PSN antibiotics. These cells can also be co-stimulated with a soluble anti-CD3 / CD28 / CD2 mAb complex at 25 μL / mL on day 0 and day 9. ...

Claims

1. A method for expanding cells, the method comprising: Loading cells into a cell expansion system, the cell expansion system comprising: A bioreactor, the bioreactor comprising an inner capillary loop and an outer capillary loop, the flow rate in the bioreactor being less than 0.1 mL / min; and An air removal chamber; Chasing the cells from the air removal chamber; Filling the inner capillary loop with a protein-containing medium; and Positioning the cells in the bioreactor for a first period of expansion.

2. The method according to claim 1, wherein The protein comprises a cell signaling molecule.

3. The method according to claim 2, wherein, The cell signaling molecule comprises a cytokine.

4. The method according to claim 3, wherein, The cytokine comprises a recombinant human IL-2 cytokine.

5. The method according to claim 1, wherein Positioning the cells in the bioreactor comprises: Positioning the cells at a first position, the first position facing a first side of the bioreactor.

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

7. The method according to claim 6, wherein Positioning the cells in the bioreactor further comprises: Positioning the cells at a second position, the second position facing a central position of the bioreactor.

8. The method according to claim 7, wherein The cells move to the second position due to a pressure difference in the bioreactor.

9. The method according to claim 8, wherein, The pressure difference is generated during operation of the air removal chamber.

10. The method according to claim 1, wherein The method further comprises: After the first period, recycling the cells for a second period; Positioning the cells for a third period; and Feeding the cells.

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

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

13. The method according to claim 1, wherein The cells comprise suspension cells.

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

15. 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 delivery assembly, the fluid delivery assembly comprising a bioreactor, the fluid delivery assembly being in fluid communication with the first pump and the second pump; A processor; And A memory, the memory being in communication with the processor and readable by the processor, the memory comprising a series of instructions that, when executed by the processor, cause the processor to: Direct the loading of cells into the fluid delivery assembly, the fluid delivery assembly comprising an air removal chamber, wherein The bioreactor comprises an inner capillary loop and an outer capillary loop, and wherein the flow rate in the bioreactor is less than 0.1 mL / min; Direct the chasing of cells from the air removal chamber; Direct the filling of the inner capillary loop with a protein-containing medium; and Direct the positioning of the cells in the bioreactor for a first period of expansion.

16. The cell expansion system according to claim 15, wherein the fluid delivery assembly is detachably attached to the cell expansion system.

17. The cell expansion system according to claim 15, wherein the fluid delivery assembly comprises a bioreactor.

18. The cell expansion system according to claim 15, wherein: The fluid delivery assembly includes a first fluid delivery assembly or a second fluid delivery assembly. The first fluid delivery assembly includes a first bioreactor, and the second fluid delivery assembly includes a second bioreactor; and The second bioreactor is smaller than the first bioreactor.

19. The cell expansion system according to claim 15, wherein, The cells include suspended cells.

20. The cell expansion system according to claim 19, wherein, The suspended cells include one or more types of T cells.

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