Cell expansion system

Abstract

In general, embodiments described herein provide for expanding cells in a cell expansion system. The cells may grow in a bioreactor, and the cells may be activated by an activator (e.g., a soluble live activator complex). For example, the nutrient and gas exchange capabilities of a closed automated cell expansion system enable vaccination of cells at a reduced cell vaccination density. Parameters of the cell growth environment can be manipulated to load cells into 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. In further embodiments, the cells may comprise a subset of T cells, including, for example, regulatory T cells (Treg).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Patent Application No. 18 / 233,146, filed on August 11, 2023, which claims the benefit of U.S. Provisional Application No. 63 / 399,524, filed on August 19, 2022, and U.S. Provisional Application No. 63 / 405,998, filed on September 13, 2022. The entire disclosure of the above application is incorporated herein by reference. Technical Field

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

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

[0005] The Cell Expansion System (CES) is used to expand and differentiate cells. The Cell Expansion System can be used to expand (e.g., grow) a variety of adherent and suspension cells. Both adherent and non-adherent cell types can be grown in a bioreactor in the Cell Expansion System. Summary of the invention

[0006] This section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features.

[0007] In at least one exemplary embodiment, the present disclosure provides a method. The method includes loading a first volume of fluid into a cell expansion system, the first volume of fluid including a plurality of cells. The cell expansion system includes a cell growth chamber. The method also includes loading a second volume of fluid into a portion of the first fluid circulation path to position the first volume of fluid in a first portion of the cell growth chamber, the second volume of fluid including culture medium; feeding cells by continuously circulating culture medium around an intracapillary circulation loop, an extracapillary circulation loop, or a combination thereof; and expanding cells.

[0008] In at least one exemplary embodiment, the method comprises serially harvesting the cells.

[0009] In at least one exemplary embodiment, the fluid in the first fluid circulation path flows through the intracapillary space of the cell growth chamber.

[0010] In at least one exemplary embodiment, the fluid in the second fluid circulation path flows through the extracapillary space of the cell growth chamber.

[0011] In at least one exemplary embodiment, a first volume of fluid including a plurality of cells is loaded without activating an intracapillary circulation pump.

[0012] In at least one exemplary embodiment, the first volume of fluid is the same as the second volume of fluid.

[0013] In at least one exemplary embodiment, the present disclosure provides a method of controlling a cell expansion system. The method includes receiving, by a controller, data readouts from at least one sensor; tracking and recording events with the controller during a cell expansion process; recording, by the controller, the data readouts from at least one sensor; and reporting, by the controller, the events and data readouts on a display.

[0014] In at least one exemplary embodiment, the present disclosure provides a method for controlling a cell expansion device group. The method includes generating, by a controller, an instruction set for a cell expansion device group. The cell expansion device group includes two or more cell expansion devices. The method also includes: the controller simultaneously sends the instruction set for the cell expansion device group to each cell expansion device in the cell expansion device group; and the controller programs each cell expansion device in the cell expansion device group using the instruction set.

[0015] In at least one exemplary embodiment, the present disclosure provides a method of controlling a cell expansion system. The method includes: actuating a first pump by a controller to cause a first fluid to flow at a first fluid flow rate; and actuating a second pump by a controller to cause a second fluid to flow at a second fluid flow rate. The first fluid flow rate and the second fluid flow rate are opposite.

[0016] In at least one exemplary embodiment, the first fluid flow rate and the second fluid flow rate are less than 0.1 mL / min.

[0017] In at least one exemplary embodiment, the first fluid flow rate and the second fluid flow rate are 0.01 mL / min.

[0018] In at least one exemplary embodiment, the present disclosure provides a culture medium bag for a cell expansion system. The culture medium bag includes a flexible shell defining an interior space configured to contain a fluid and a pair of ports disposed in parallel at the bottom of the flexible shell. The flexible shell defines a hanger on the top of the interior space opposite to the pair of ports, and the pair of ports includes an inlet port and an outlet port.

[0019] In at least one exemplary embodiment, the hanger includes a slot configured to receive one or more hooks for hanging the culture medium bag.

[0020] In at least one exemplary embodiment, the culture medium bag includes a rod. The hanger defines a channel in the housing for receiving the rod, and the rod and the hanger are configured to fit within the external rail for suspending the culture medium bag.

[0021] In at least one exemplary embodiment, the present disclosure provides a cell expansion system. The cell expansion system includes a cell expansion device, which includes a housing and a retainer. The housing defines a receiver on the operating surface of the housing, and the retainer protrudes from the housing. The cell expansion system also includes: a disposable kit configured to engage with a receiver on the cell expansion device; and a culture medium bag configured to be suspended on the retainer of the cell expansion device. The culture medium bag has a flexible housing that defines an internal space configured to accommodate a fluid. The flexible housing defines a hanger on the top of the internal space, and the hanger includes a rod extending longitudinally along the top of the culture medium bag. The rod is configured to be accommodated in a channel on the retainer to fix the culture medium bag to the retainer on the cell expansion device.

[0022] In at least one exemplary embodiment, a disposable kit includes a cell growth chamber and tubing. The tubing is configured to connect the cell growth chamber to a culture medium bag.

[0023] In at least one exemplary embodiment, the holder includes at least one protrusion, and the hanger defines a slot configured to receive the protrusion and suspend the culture medium bag.

[0024] In at least one exemplary embodiment, the present disclosure provides a method for controlling a cell expansion system. The method includes: actuating a first pump by a controller to flow a first fluid at a first fluid flow rate; actuating a second pump by a controller to flow a second fluid at a second fluid flow rate; detecting a parameter associated with the first fluid or the second fluid by a sensor; receiving the parameter from the sensor by the controller and determining whether the parameter is outside a predetermined range; and sending a remote alarm by the controller to an external device separated from the cell expansion system.

[0025] In at least one exemplary embodiment, the remote alert comprises an email, a text message, a digital alert, or a combination thereof.

[0026] In at least one exemplary embodiment, the method includes activating an audio alarm, a visual alarm, or a combination thereof.

[0027] In at least one exemplary embodiment, the sensor includes a thermistor, a pressure sensor, a gas sensor, an air detector, or a combination thereof.

[0028] In at least one exemplary embodiment, the present disclosure provides a method for controlling a cell expansion system. The method includes: detecting a parameter associated with a cell expansion device; receiving the parameter by a controller and determining whether the parameter is outside a predetermined range; and sending a remote alarm by the controller to an external device separated from the cell expansion system.

[0029] In at least one exemplary embodiment, the remote alert comprises an email, a text message, a digital alert, or a combination thereof.

[0030] In at least one exemplary embodiment, the method includes activating an audio alarm, a visual alarm, or a combination thereof.

[0031] In at least one exemplary embodiment, the parameter is one of temperature, door position, pressure, flow rate, and concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

[0033] Figure 1A Embodiments of a cell expansion system (CES) according to at least one exemplary embodiment are depicted.

[0034] Figure 1B A front elevation view of an embodiment of a bioreactor according to at least one example embodiment is shown, illustrating a circulation path through the bioreactor.

[0035] Figure 1C A perspective view of a first bioreactor and a second bioreactor is shown according to at least one example embodiment.

[0036] Figure 1D Depicted is 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.

[0037] Figure 2A A front perspective view of a cell expansion system according to at least one example embodiment is shown.

[0038] Figure 2B A device with a pre-installed fluid delivery device according to at least one exemplary embodiment is shown. Figure 2A An internal perspective view of the cell expansion system.

[0039] Figure 2C According to at least one exemplary embodiment, Figure 2A A guide rail system and a hook system of a holder of a cell expansion system.

[0040] Figure 3 Depicted is a perspective view of a housing of a cell expansion system according to at least one example embodiment.

[0041] Figure 4A A perspective view of a pre-installed fluid delivery device according to at least one exemplary embodiment is shown.

[0042] Figure 4B According to at least one exemplary embodiment, Figure 4A Pre-installed fluid transfer components.

[0043] Figure 4C According to at least one exemplary embodiment, Figure 4A Pre-installed fluid transfer components.

[0044] Figure 4D According to at least one exemplary embodiment, Figure 4A A pre-installed fluid transfer assembly for the culture medium bag.

[0045] Figure 4E According to at least one exemplary embodiment, Figure 4A A waste bag of the pre-installed fluid transfer assembly.

[0046] Figure 4F According to at least one exemplary embodiment, Figure 4A Cross-sectional view of the pressure chamber of the pre-installed fluid delivery assembly.

[0047] Figure 4G According to at least one exemplary embodiment, Figure 4F Exploded view of the pressure chamber.

[0048] Figure 4H According to at least one exemplary embodiment, Figure 4A A pre-installed fluid delivery assembly for the sampling coil.

[0049] Fig. 4I According to at least one exemplary embodiment, Figure 4A In-line filters of pre-installed fluid transfer assemblies.

[0050] Figure 5A Depicted is a schematic diagram of a cell expansion system including an operational configuration showing fluid movement according to at least one example embodiment.

[0051] Figure 5B Depicted is a schematic diagram of a cell expansion system including another operational configuration showing fluid movement, according to an embodiment of the present disclosure.

[0052] Figure 5C Depicted is a schematic diagram of a cell expansion system including another operational configuration showing fluid movement according to at least one example embodiment.

[0053] Figure 6 A schematic diagram of a cell expansion system according to at least one exemplary embodiment is shown.

[0054] Fig. 7A A schematic diagram of a cell expansion system according to at least one exemplary embodiment is shown.

[0055] Figure 7B A schematic diagram of a cell expansion system according to at least one exemplary embodiment is shown.

[0056] Figure 7C A schematic diagram of a cell expansion system according to at least one exemplary embodiment is shown.

[0057] Figure 8 A flow chart illustrating operational features of a process for expanding cells is depicted according to at least one exemplary embodiment.

[0058] Fig. 9A A flow chart depicting operational features of a process for expanding cells is shown, according to at least one exemplary embodiment.

[0059] Fig. 9B A schematic diagram of a portion of a cell expansion system is depicted according to at least one exemplary embodiment.

[0060] Fig. 10A A flow chart illustrating operational features of a process for expanding cells is depicted according to at least one exemplary embodiment.

[0061] Fig. 10B A graph showing oxygen consumption in a cell expansion system according to at least one example embodiment.

[0062] Fig.11A A flow chart depicting operational features of a process for expanding cells is shown, according to at least one exemplary embodiment.

[0063] Fig. 11B A table showing exemplary pump rates that may be used in a cell expansion system is shown according to at least one exemplary embodiment.

[0064] Fig. 12A A flow chart illustrating operational features of a process for expanding cells is depicted according to at least one exemplary embodiment.

[0065] Fig. 12B A diagram illustrating metabolism in a cell expansion system according to at least one example embodiment.

[0066] Fig. 12C A diagram illustrating metabolism in a cell expansion system according to at least one example embodiment.

[0067] Fig.13A flow chart depicting operational features of a process for expanding cells is shown, according to at least one exemplary embodiment.

[0068] Fig.14 A flow chart illustrating operational features of a process for expanding cells is depicted according to at least one exemplary embodiment.

[0069] Fig.15 A flow chart depicting operational features of a process for expanding cells is shown, according to at least one exemplary embodiment.

[0070] Fig.16A A flow chart depicting operational features of a process for expanding cells is shown, according to at least one exemplary embodiment.

[0071] Fig. 16B Shown is a graph of cell number versus flow rate during cell expansion according to at least one example embodiment.

[0072] Fig.17 A flow chart illustrating operational features of a process for expanding cells is depicted according to at least one exemplary embodiment.

[0073] Fig.18A A flow chart illustrating operational features of a process for expanding cells is depicted according to at least one exemplary embodiment.

[0074] Fig.18B A diagram depicting cell expansion in a cell expansion system according to at least one example embodiment.

[0075] Fig.18C Shown is a graph showing a cell dissociation inner diameter according to at least one example embodiment.

[0076] Fig.19 Shown is a graph of cell number and flow rate versus days in culture during cell expansion, according to at least one exemplary embodiment.

[0077] Fig. 20A A flow chart depicting operational features of a process for operating a pump to expand cells is shown according to at least one exemplary embodiment.

[0078] Fig. 20B A schematic diagram of a portion of a cell expansion system is depicted according to at least one exemplary embodiment.

[0079] Fig. 20C A schematic diagram of a portion of a cell expansion system is depicted according to at least one exemplary embodiment.

[0080] Fig.20DA schematic diagram of a portion of a cell expansion system is depicted according to at least one exemplary embodiment.

[0081] Fig.21 A flow chart depicting operational features of a process for expanding cells is shown, according to at least one exemplary embodiment.

[0082] Fig. 22 A flow chart illustrating operational features of a process for expanding cells is depicted according to at least one exemplary embodiment.

[0083] Fig.23 A flow chart depicting operational features of a process for expanding cells is shown, according to at least one exemplary embodiment.

[0084] Fig.24 A flow chart illustrating operational features of a process for expanding cells is depicted according to at least one exemplary embodiment.

[0085] Fig.25 Depicted are exemplary processing systems upon which cell expansion systems of embodiments of the present disclosure may be implemented, according to at least one exemplary embodiment.

[0086] Fig.26 A flow chart is shown depicting an example flow of an experiment according to at least one example embodiment.

[0087] Fig. 27 is a graph illustrating an exemplary result of a coating process according to at least one exemplary embodiment.

[0088] Fig.28 is a graph showing example results for cells harvested after a coating process according to at least one example embodiment.

[0089] Fig.29 is a graph showing example results for cells harvested after a coating process according to at least one example embodiment.

[0090] Fig.30 is a graph showing example results of viral particles measured per day according to at least one example embodiment.

[0091] Fig.31 is a graph showing example results for cells harvested after a coating process according to at least one example embodiment.

[0092] Fig.32 is a graph showing example results of cells harvested after a coating process according to at least one example embodiment. DETAILED DESCRIPTION

[0093] Example embodiments will now be described more fully with reference to the accompanying drawings.

[0094] Exemplary embodiments are provided so that the present disclosure is thorough and the scope is fully conveyed to those skilled in the art. Many specific details, such as examples of specific components, devices, and methods, are set forth to provide a thorough understanding of the embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, and that the exemplary embodiments may be implemented in many different forms, and none should be construed as limiting the scope of the present disclosure. In some exemplary embodiments, well-known processes, well-known device structures, and well-known techniques are not described in detail.

[0095] The terms used herein are only used for the purpose of describing specific exemplary embodiments and are not intended to be limiting. As used herein, the singular forms "a", "an", and "the" may also be used to include plural forms unless the context clearly indicates otherwise. The terms "comprises", "comprising", "including", and "having" are inclusive, thus specifying the presence of the features, integers, steps, operations, elements, and / or parts described, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, parts, and / or groups thereof. Unless specifically identified as a sequence of execution, the method steps, processes, and operations described herein should not be interpreted as necessarily requiring them to be performed in the specific order discussed or shown. It should also be understood that additional or alternative steps may be adopted.

[0096] When an element or layer is referred to as being "located at," "engaged to," "connected to," or "coupled to" another element or layer, it may be directly located at, engaged to, connected to, or coupled to another element or layer, or there may be intermediate elements or layers. Conversely, when an element is referred to as being "directly located at," "directly engaged to," "directly connected to," or "directly coupled to" another element or layer, there may be no intermediate elements or layers. Other words used to describe the relationship between elements should be interpreted in a similar manner (e.g., "between" versus "directly between," "adjacent" versus "directly adjacent," etc.). As used herein, the term "and / or" includes any and all combinations of one or more associated listed items.

[0097] Although the terms "first", "second", "third", etc. can be used to describe various elements, components, regions, layers and / or parts in this article, these elements, components, regions, layers and / or parts should not be limited by these terms. These terms can only be used to distinguish an element, component, region, layer and / or part from another region, layer or part. Unless the context clearly indicates, terms such as "first", "second" and other numerical terms do not mean order or sequence when used in this article. Therefore, without departing from the teachings of the exemplary embodiments, the first element, component, region, layer and / or part discussed below may be referred to as the second element, component, region, layer and / or part.

[0098] For ease of description, spatially relative terms such as "inside," "outside," "beneath," "below," "lower," "above," "upper," and the like may be used herein to describe the relationship of one element or feature to another element or feature as shown in the figures. Spatially relative terms may be intended to encompass different orientations of the device in use or operation in addition to the orientations depicted in the accompanying drawings. For example, if the device in the figure is flipped, elements described as being "below" or "beneath" other elements or features will be oriented "above" the other elements or features. Thus, the example term "below" may encompass both above and below orientations. The device may be oriented in other ways (rotated 90 degrees or in other orientations), and the spatially relative descriptors used herein interpreted accordingly.

[0099] Various components are referred to herein as being "operably associated". As used herein, "operably associated" refers to components that are connected together in an operably manner, and encompasses embodiments in which the components are directly connected, as well as embodiments in which additional components are placed between connected components. Components that are "operably associated" may be "fluidically associated". "Fluidically associated" means that the components are connected together so that fluid can be transported between them. "Fluidically associated" encompasses embodiments in which additional components are disposed between two fluidically associated components, as well as between directly connected components. Fluidically associated components may include components that do not contact the fluid but contact other components to operate the system (e.g., a peristaltic pump that pumps fluid through a flexible tube by compressing the outside of the tube).

[0100] As used herein, the term "donor" may mean any person who provides fluid (e.g., whole blood) to a single collection system. A donor may also be a patient who temporarily provides fluid to a single collection system, where the fluid is returned to the patient after being processed, treated, manipulated, etc.

[0101] As used herein, the term "automatic" and variations thereof refer to any process or operation that is performed without the need for human input that is considered substantial in the performance of any process or operation. However, even if human input, whether substantial or insubstantial, is used in the performance of a process or operation, the process or operation may still be automatic if the input is received prior to the performance of the process or operation. Human input is considered substantial if it affects the performance of the process or operation. Human input that consents to the performance of the process or operation is not considered "substantial."

[0102] As used herein, the term "computer-readable medium" refers to any tangible storage and / or transmission medium that participates in providing instructions to a processor for execution. Such media can take a variety of forms, including but not limited to non-volatile media, volatile media, and transmission media. Non-volatile media include, for example, NVRAM or magnetic disks or optical disks. Volatile media include dynamic memory, such as main memory. Common forms of computer-readable media include, for example, floppy disks, flexible disks, hard disks, tapes or any other magnetic media, magneto-optical media, CD-ROMs, any other optical media, punch cards, paper tapes, any other physical media with hole patterns, RAM, PROMs and EPROMs, flash-EPROMs, solid-state media (such as memory cards), any other memory chips or cartridges, carriers as described below, or any other media from which a computer can read. Digital file attachments to emails or other self-contained information archives or archives are considered to be distribution media equivalent to tangible storage media. It should be understood that when the computer-readable medium is configured as a database, the database can be any type of database, such as a relational database, a hierarchical database, an object-oriented database, etc. Therefore, the disclosure is considered to include a tangible storage medium or distribution medium storing the software implementations of the disclosure and prior art-recognized equivalents and successor media.

[0103] The term "module" as used herein refers to any known or later developed hardware, software, firmware, artificial intelligence, fuzzy logic, or combination of hardware and software capable of performing the functions associated with that element.

[0104] As used herein, the terms "determine," "calculate," and "compute" and variations thereof are used interchangeably and include any type of methodology, process, mathematical operation, or technique.

[0105] Embodiments of the present disclosure will be described more fully with reference to the accompanying drawings and in conjunction with single collection methods and systems. The following embodiments may be described with respect to separation of blood components from whole blood. However, 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 to be used for products, processes, devices and systems for separation of any complex liquid. Therefore, the present disclosure is not limited to separation of blood components from whole blood.

[0106] Exemplary embodiments of the present disclosure generally relate to systems and methods for expanding cells in a cell expansion system (CES). According to embodiments, such expansion can occur using a bioreactor or a cell growth chamber. In embodiments, such a bioreactor or a cell growth chamber can include a hollow fiber membrane. Such a hollow fiber membrane can include a plurality of hollow fibers and can include an extracapillary (EC) space and / or an intracapillary (IC) space. Embodiments can provide adherent or non-adherent cells grown or expanded in a cell expansion system. For example, non-adherent or suspended cells, such as T cells, T lymphocytes, or CD3 T cells, can be expanded in the system. + Selected cells. In embodiments, one or more subpopulations or subsets 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).

[0107] In some exemplary embodiments, methods and systems for expanding cells in a closed automated cell expansion system may be provided. In embodiments, such a cell expansion system may include a bioreactor or a cell growth chamber. In further embodiments, such a bioreactor or a cell growth chamber may include a hollow fiber membrane. The capabilities of such a system, such as nutrient and gas exchange capabilities, may allow cells to be inoculated at a reduced cell inoculation density. Embodiments provide cell growth environment parameters to be manipulated to load or introduce cells into a position in a bioreactor for effectively exchanging nutrients and gases to growing cells. For example, in embodiments, centralization of cells in a bioreactor may increase cell density.

[0108] In an exemplary embodiment, a non-adherent cell population (e.g., T cells and / or Treg cells) can be introduced or loaded into a hollow fiber bioreactor, wherein the hollow fiber bioreactor may include a plurality of hollow fibers. In an embodiment, cells may be exposed to an activator to activate the expansion of cells in a hollow fiber bioreactor. For example, in an embodiment, a plurality of cells are introduced into a cell expansion system using a "centrally load cells without circulation" task. According to an exemplary embodiment, such a task can be performed on day 0 and day 4-8. In other embodiments, other days can be used. In an embodiment, such a loading task of cells can centralize cells in a bioreactor to increase cell density. In other embodiments, cells can be located in other parts or regions of a bioreactor to increase cell density. In an embodiment, by positioning cells in a first position (e.g., around a central region) of a bioreactor, cells can receive an effective exchange of nutrients and gases.

[0109] In an exemplary embodiment, a reduced cell seeding density can be used compared to the cell seeding density used in static culture methods. In an embodiment using a cell expansion system, cells, such as Tregs or Treg cells, can be grown from 2.54×10 5 Cells / mL to 3.69×10 5 In other embodiments, the cell seeding density may be less than about 1×10 6 cells / mL. In addition, the Treg cell inoculum can be composed of 1.0×10 5 Cells / mL can be prepared with a cell seeding density of 1.0 × 10 cells / mL. Other methods, such as static Treg cell culture methods, can use 1.0 × 10 6 In vitro expansion can be performed at a cell seeding density of 100 Treg cells / mL. For example, in an embodiment, due to the overall efficiency of the system in delivering nutrients to the culture environment, a lower cell seeding density can be used. In other embodiments, one or more steps used during expansion combined with the overall efficiency of the system in delivering nutrients to the culture environment can allow the use of a lower initial cell seeding density.

[0110] In an exemplary embodiment, automated cell (e.g., Treg) expansion can be performed using a soluble activator complex. For example, in other embodiments, other types of activators (such as beads) can be used to stimulate cells. In other embodiments, cells (e.g., Treg cells) can be expanded without the use of bead-based stimulation. In one embodiment, a Stem Cell Technologies soluble ImmunoCult TMHuman CD3 / CD28 / CD2 T cell activators are used for cell expansion to activate and expand Treg cells using an automated cell expansion system in the presence of 200 IU / mL cytokine IL-2. For example, the use of soluble activator complexes can reduce the cost of stimulation compared to the cost of bead-based solutions. In other embodiments, other types of activators can be used. In addition, in other embodiments, other types of cytokines or other growth factors can be used.

[0111] Exemplary embodiments may also provide system parameters to be adjusted or managed to control the cell residence 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 period, the system may provide effective gas and nutrient exchange to amplify cells. In embodiments, the bioreactor may be designed to provide gas exchange to growing cells, and in some embodiments, nutrient exchange is provided. In exemplary embodiments, a bioreactor including a semi-permeable hollow fiber membrane may provide gas and nutrient exchange by a semi-permeable hollow fiber membrane. In embodiments, a method for providing to cells to allow them to grow without passing through a membrane of a culture medium component (such as, various cytokines, proteins, etc.) may use a fluid inlet to a bioreactor side (e.g., a capillary (IC) side of cell growth). However, according to embodiments, even low, declining or reduced, such as a minimum inlet flow rate (e.g., 0.1 mL / min) may also cause cells to aggregate in the outlet manifold of a bioreactor. Cells residing in the manifold of a bioreactor may not obtain appropriate gas exchange and nutrient exchange, which may result in cell death and aggregation.

[0112] Exemplary embodiments relate to providing a method for retaining cells (e.g., non-adherent cell populations) in a bioreactor when cells are fed using an inlet (e.g., IC inlet) flow. Although embodiments herein may refer to cells being on the IC side of a membrane when fed, for example, other embodiments may provide cells on the EC side of a membrane, wherein, according to embodiments, cells may be included in a first circulation path and / or a second circulation path. In embodiments, a feeding method may provide, for example, a first volume of fluid (e.g., a culture medium or a culture medium prepared for cell growth) pumped into a first port of a bioreactor at a flow rate, volume flow rate, fluid flow rate, flow rate, fluid flow rate, or volume velocity of a first volume. For example, a volume flow rate, a volume flow rate, a fluid flow rate, a flow rate, a fluid flow rate, or a volume velocity of a volume may be used interchangeably. In some embodiments, a flow rate may be a vector having both speed and direction. A second volume of fluid may be pumped into a second port of a bioreactor at a flow rate, a volume flow rate, a fluid flow rate, a flow rate, a fluid flow rate, or a volume velocity of a second volume. For example, in an embodiment, the flow velocity of such volume, volume flow rate, fluid flow rate, flow rate, fluid flow rate or volume velocity can be controlled by one or more pump rates and / or pump flow rates. A pump can act on a fluid, and a pump rate can produce, cause or affect the flow velocity or flow rate of the volume of the fluid. As used herein, a pump rate can be described in an embodiment as a volume flow rate or a fluid flow rate produced, caused or affected by a pump.

[0113] In an exemplary embodiment, the second flow rate of the fluid entering the bioreactor can be in the opposite direction to the first flow rate of the fluid entering the bioreactor. Figure 5B and Figure 5C An exemplary operational configuration according to an embodiment of the present disclosure is shown, which shows that it can be used in a cell expansion system, such as CES 500 (e.g., Figure 5B and Figure 5C ) flow rate and flow direction. In embodiments, a cell expansion system pump (e.g., an IC pump) may be used to control cell residence in a bioreactor. In embodiments, for example, during the expansion phase of growth, cells may be lost from the bioreactor to the IC circulation path or IC loop. In embodiments, for example, cells in the bioreactor closer to the IC inlet port may receive the freshest growth medium, while, for example, cells in a portion of the IC circulation path outside the bioreactor may substantially receive spent or conditioned medium, which may affect the glycolytic metabolism of the cells. In addition, according to embodiments, cells in the bioreactor may receive a mixed gas (O2, CO2, N2) input from a gas transfer module (GTM) via EC loop circulation diffusion, while cells in a portion of the IC circulation path outside the bioreactor may not.

[0114] In an exemplary embodiment, during the maintenance period, a reduction in cell loss in a hollow fiber membrane (HFM) bioreactor can be achieved by matching, or approximately or substantially matching, the IC circulation pump rate to the IC inlet pump rate, but in the opposite direction. In an embodiment, for example, an IC inlet pump rate of +0.1 mL / min can be matched, or approximately or substantially matched to a complementary IC circulation pump rate of -0.1 mL / min, so as to maintain cells in the bioreactor during the growth period of the cell culture (possibly the 4th to 7th day). According to an embodiment, the pump adjustment can offset the force associated with the loss of cells from the IC outlet port. In other embodiments, other pump rates can be used. For example, in other embodiments, the pump rates can be different. In an embodiment, other pumps or additional pumps can be used. In an embodiment, fewer pumps can be used. In addition, other time periods can be used in other embodiments.

[0115] In an exemplary embodiment, the metabolic activity of the cell mass can affect the feeding parameters. For example, the lactate value of the cell culture can be maintained at or below a predetermined level. In an embodiment, the cell culture value can be maintained at or below about 7mmol / L, for example. In an embodiment, the rate of medium addition is controlled by using a cell expansion system graphical user interface (GUI), and during the expansion of cells (e.g., regulatory T cells), lactate metabolic waste from 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 a lactate level of ≤ about 5mmol / L, for example, to improve cell growth and viability. In other embodiments, other concentrations can be used.

[0116] Additional embodiments can provide system features for shearing any cell colonies, microcolonies or cell clusters that may be formed during the expansion phase. For example, embodiments provide colonies (such as microcolonies) of cells sheared by bioreactor hollow fiber membranes to reduce the number of cells in microcolonies, colonies or clusters, wherein microcolonies, colonies or clusters can be groups of one or more groups of adherent cells. In embodiments, cell expansion system (CES) bioreactor architecture can be used for shearing microcolonies of cells (such as Treg cells). In embodiments, as cells (such as Treg cells) grow, they tend to form microcolonies, which may limit the diffusion of nutrients to the cells in the center of the colony. This may cause adverse effects during cell culture, such as necrosis. Embodiments can provide schemes to circulate suspension cell cultures through, for example, hollow fiber capillary (IC) loops (e.g., hollow fibers with an inner diameter of 215 μm) during the expansion phase of growth, to shear colonies. In embodiments, colonies, microcolonies or clusters of cells can be sheared to reduce the size of colonies, microcolonies or clusters of cells. In embodiments, colonies or clusters of cells may be sheared to provide single cell suspensions and generate cell growth / viability. In embodiments, such a capability may facilitate continuous perfusion growth of cells (eg, T cells or Tregs).

[0117] In an exemplary embodiment, a therapeutic dose of cells (e.g., Tregs) can be expanded in a cell expansion system and harvested from the cell expansion system. In an embodiment, the number of cells at the time of harvest can be about 1×10 6 cells to about 1×10 10 cells, such as about 1×10 9 In one embodiment, the number of cells harvested may be about 1×10 8 Up to 1×10 10 cells, one sample is about 7.0×10 8 About 1.4×10 9 In embodiments, the harvested cells may have a viability between about 60% and about 100%. For example, the viability of the harvested cells may be about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, or even about 95% or more. In some embodiments, the harvested cells may express biomarkers consistent with Tregs. For example, in some embodiments, the cells may express CD4 + 、CD25 + and / or FoxP3 + Biomarkers. In embodiments, the harvested cells may include a frequency of about 50% to about 100% CD4 + CD25 +The harvested cells may include a frequency of more than about 75%, more than about 80%, more than about 85%, more than about 90%, or even more than about 95% CD4 + CD25 + In other embodiments, the cells may include a frequency of about 30% to about 100% CD4 + FoxP3 + In some embodiments, the harvested cells may include a frequency of CD4 at least about 35%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or even at least about 70%. + FoxP3 + Phenotype.

[0118] Exemplary embodiments relate to a cell expansion system as described above. In an embodiment, such a cell expansion system is closed, wherein the closed cell expansion system includes contents that are not directly exposed to the atmosphere. Such a cell expansion system can be automated. In an embodiment, adherent and non-adherent or suspension type cells can be grown 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 culture medium. A method for culture medium supplementation for cell growth occurring in a bioreactor of a closed cell expansion system is provided. In an embodiment, the bioreactor used with such a system is a hollow fiber bioreactor. Many types of bioreactors can be used according to embodiments of the present disclosure.

[0119] In an embodiment, the system may include: a bioreactor fluidically associated with a first fluid flow path having at least opposite ends, a first opposite end of the first fluid flow path, which is fluidically associated with a first port of a hollow fiber membrane, and a second end of the first fluid flow path, which is fluidically 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 may further include a fluid inlet path fluidically associated with the first fluid flow path, wherein a plurality of cells may be introduced into the first fluid flow path through the first fluid inlet path. In some embodiments, a pump and a controller for controlling the operation of the pump are also included, wherein the pump is used to transfer the capillary inlet fluid from the capillary culture medium bag to the first fluid flow path. For example, in an embodiment, the controller controls the pump to transfer cells from the cell inlet bag to the first fluid flow path. Another pump for circulating fluid in the first fluid circulation path may also be included, wherein such a pump may also include a controller for controlling the operation of the pump. In an embodiment, the controller is, for example, a computing system, which includes a processor. In an embodiment, one or more controllers may be configured to control one or more pumps, such as to circulate fluid at a flow rate, for example, in the first fluid circulation path. According to an embodiment, a plurality of controllers may be used, such as a first controller, a second controller, a third controller, a fourth controller, a fifth controller, a sixth controller, etc. In addition, according to an embodiment of the present disclosure, a plurality of pumps may be used, such as a first pump, a second pump, a third pump, a fourth pump, a fifth pump, a sixth pump, etc. In addition, although the present disclosure may relate to a culture medium bag, a cell entry bag, etc., a plurality of bags may be used in an embodiment, such as a first culture medium bag, a second culture medium bag, a third culture medium bag, a first cell entry bag, a second cell entry bag, a third cell entry bag, etc. and / or other types of containers. In other embodiments, a single culture medium bag, a single cell entry bag, etc. may be used. In addition, 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.) may be included.

[0120] In an exemplary embodiment, the system comprises, for example, 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 containing a series of instructions. In an embodiment, when the instructions are executed by the processor, the processor receives instructions to, for example, start the system. In response to the instruction to start the system, the processor may perform a series of steps to prepare the system, and may then receive an instruction to perform an IC / EC flush. For example, in response to an instruction to load cells, the processor may perform a series of steps to load cells from a cell access bag into, for example, a bioreactor.

[0121] Figure 1AA schematic diagram of an exemplary cell expansion system (CES) according to an embodiment of the present disclosure is depicted in FIG. 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 fluidically associated with a hollow fiber cell growth chamber 24 (also referred to herein as a "bioreactor"). Specifically, the opposite end 18 can be fluidically associated with a first inlet 22 of the cell growth chamber 24, and the opposite end 20 can be fluidically 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 fiber membrane 117 (see FIG. 1 ) disposed in the cell growth chamber 24. Figure 1B ) of the hollow fiber 116 (see Figure 1B ) (the cell growth chamber and hollow fiber membrane are described in more detail below). In addition, the first fluid flow control device 30 can be operably connected to the first fluid flow path 16 and can control the fluid flow in the first fluid circulation path 12.

[0122] The second fluid circulation path 14 includes a second fluid flow path 34, a cell growth chamber 24, and a second fluid flow control device 32. According to an embodiment, the second fluid flow path 34 has at least opposite ends 36 and 38. The opposite ends 36 and 38 of the second fluid flow path 34 can be fluidically 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 can be in contact with the hollow fiber membrane 117 (see Figure 1B ), wherein the hollow fiber membrane comprises a plurality of hollow fibers. The second fluid circulation path 14 may be operably connected to a second fluid flow control device 32.

[0123] Therefore, the first fluid circulation path 12 and the second fluid circulation path 14 can 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 intracapillary ("IC") space of the hollow fibers in the cell growth chamber 24. The first fluid circulation path 12 may be referred to as an "IC loop". The fluid in the second circulation path 14 flows through the extracapillary ("EC") space in the cell growth chamber 24. The second fluid circulation path 14 may be referred to as an "EC loop". Depending on the 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.

[0124] The fluid inlet path 44 may 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 exit the CES 10. A third fluid flow control device 48 may be operably associated with the fluid inlet path 44. Alternatively, the third fluid flow control device 48 may alternatively be associated with the fluid outlet path 46.

[0125] According to embodiments, the fluid flow control device as used herein may include a pump, a valve, a fixture, or a combination thereof. A plurality of pumps, valves, and fixtures may be arranged in any combination. In various embodiments, the fluid flow control device is or includes a peristaltic pump. In embodiments, the fluid circulation path, the inlet port, and the outlet port may be formed by a tube of any material.

[0126] Generally, any kind of fluid including, for example, buffers, protein-containing fluids, and cell-containing fluids can flow through the various circulation paths, inlet paths, and outlet paths.As used herein, "fluid," "medium," and "fluid medium" are used interchangeably.

[0127] Go to 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 in a front elevation view. Cell growth chamber 100 has a longitudinal axis LA-LA and includes a cell growth chamber housing 104. In at least one embodiment, cell growth chamber housing 104 includes four openings or ports: IC inlet port 108, IC outlet port 120, EC inlet port 128, and EC outlet port 132.

[0128] According to an embodiment of the present disclosure, a fluid in a first circulation path enters the cell growth chamber 100 through an IC inlet port 108 located at a first longitudinal end 112 of the cell growth chamber 100, enters and passes through the capillary inner side of a plurality of hollow fibers 116 (including hollow fiber membranes 117) (referred to in various embodiments as the inner capillary ("IC") side of the hollow fiber membranes or the "IC space"), and exits the cell growth chamber 100 through an IC outlet port 120 located at a 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 an IC portion 126 of the cell growth chamber 100. A fluid in a second circulation path flows in the cell growth chamber 100 through an EC inlet port 128, contacts the capillary outer side or exterior of the hollow fibers 116 (referred to as the "EC side" or "EC space" of the membranes), and exits the cell growth chamber 100 via an 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 via the EC inlet port 128 can contact the outside of the hollow fiber 116. Small molecules (e.g., ions, water, oxygen, lactic acid, metabolites, nutrients, gases, etc.) can diffuse (e.g., continuously perfuse) from the inside or IC space of the hollow fiber to the outside or EC space, or from the EC space to the IC space through the hollow fiber 116. For example, compared with a flask-based system, the hollow fiber 116 can include a diffusion distance of 200 microns, thereby providing a more efficient transfer of gases and nutrients. Molecules of large molecular weight (such as growth factors) may usually be too large to pass through the hollow fiber membrane and may be retained in the IC space of the hollow fiber 116. Compared with stirred bioreactors and wave bioreactors, the hollow fiber 116 produces less shear stress. In an embodiment, the culture medium may be replaced as needed. The culture medium may also be circulated through an oxygenator or a gas transfer module to exchange gases as needed. Depending on the embodiment, the cells may be contained within the first circulation path and / or the second circulation path, as described below, and may be on the IC side and / or the EC side of the membrane.

[0129] The material used to make the hollow fiber membrane 117 can be any biocompatible polymer material that can be made into a hollow fiber. According to an embodiment of the present disclosure, one material that can be used is a synthetic polysulfone based material.

[0130] According to some embodiments, Figure 1CAs shown, more than one size of cell growth chamber 100 may be included individually or in a disposable kit. For example, a pair of cell growth chambers 100A and 100B may be included individually or in a disposable kit. In at least one exemplary embodiment, cell growth chamber 100A may be smaller than cell growth chamber 100B to accommodate fewer cells for expansion. For example, the size of cell growth chamber 100A may be one-tenth or 0.1 of the size of cell growth chamber 100B. In at least one exemplary embodiment, cell growth chamber 100A (or mini cell growth chamber 100A) may be advantageous for pilot runs, pediatrics, and other events (e.g., skin grafts, etc.) with small batches of starting cells. In cases where the initial cell source is too small for cell growth chamber 100B, using mini cell growth chamber 100A is advantageous because mini 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. Therefore, in cases where cell growth chamber 100B may not be optimal, mini cell growth chamber 100A provides additional opportunities for the use of CES 10. In some exemplary embodiments, the CES 10 can automatically identify which of the cell growth chambers 100A and 100B is installed and identify appropriate process parameters, as further described below. By including more than one size of cell growth chamber 100, the CES 10 can have increased applications, including, for example, bone marrow-derived mesenchymal stem cells (MSCs), adipose-derived MSCs, umbilical cord MSCs, fibroblasts, keratinocytes, HEK293T cells, human embryonic stem cells (ESCs), periosteum-derived cells, induced pluripotent stem cell-derived MSCs (IPS-MSCs), neural stem cells (NSCs), osteochondroprogenitor cells, endothelial cells, dendritic cells, induced pluripotent stem cells (iPSCs), T cells, viral vectors, exosome production, expansion of autologous and allogeneic doses of adherent and suspended cell types, etc. For example, multiple cell growth chambers 100 for the CES 10 provide the ability to expand in an exemplary range of up to 300 million to 1 billion MSCs or up to 25 billion T cells per run.

[0131] In at least one exemplary embodiment, a CES (e.g., such as CES 500 (see Figure 5A , Figure 5B and Figure 5C ) and / or CES 600 (see Figure 6 )) may 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 rotating and / or lateral rocking device. Figure 1D One such exemplary apparatus is shown, wherein a cell growth chamber 100 may be rotationally coupled to two rotational rocking members and a lateral rocking member, according to an embodiment.

[0132] The first rotational rocking member 138 rotates the cell growth chamber 100 about the 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 be continuously rotated in a single direction, either clockwise or counterclockwise, about the central axis 142. Alternatively, the cell growth chamber 100 may be rotated in an alternating manner, such as first clockwise and then counterclockwise about the central axis 142, depending on the embodiment.

[0133] The CES may also include a second rotating rocking member that rotates the cell growth chamber 100 about a rotation axis 144. The rotation axis 144 may pass through a center point of the cell growth chamber 100 and may be perpendicular to the central axis 142. In embodiments, the cell growth chamber 100 may be continuously rotated in a single direction about the rotation axis 144, either clockwise or counterclockwise. Alternatively, the cell growth chamber 100 may be rotated about the rotation axis 144 in an alternating manner, such as first clockwise and then counterclockwise. In various embodiments, the cell growth chamber 100 may also be rotated about the rotation axis 144 and positioned in a horizontal or vertical orientation relative to gravity.

[0134] In some exemplary embodiments, lateral rocking member 140 can be laterally associated with cell growth chamber 100. In embodiments, the plane of lateral rocking member 140 moves laterally in the -x and -y directions. According to embodiments, sedimentation of cells in a cell growth chamber can be reduced by movement of cell culture medium contained within the hollow fibers.

[0135] Rotation and / or lateral movement of the rocking device can reduce cell sedimentation within the device and reduce the likelihood of cells being trapped within a portion of the cell growth chamber. According to Stokes' law, the rate at which cells settle in the cell growth chamber is proportional to the density difference between the cells and the suspension medium. In certain embodiments, repeated 180-degree rotations (rapid) with pauses as described above, for example with a total combined time of 30 seconds, maintain, for example, non-adherent red blood cells in suspension. Exemplary embodiments can rotate the cell growth chamber 100 from a minimum rotation of about 180 degrees to a maximum rotation of about 290 to 300 degrees; however, rotations of up to 360 degrees or more can be used. Different rocking components can be used alone or in any combination. For example, a rocking component that rotates the cell growth chamber 100 around the central axis 142 can be combined with a rocking component that rotates the cell growth chamber 100 around the axis 144. Similarly, clockwise and counterclockwise rotations around different axes can be performed independently in any combination.

[0136] Go to Figure 2A and Figure 2B, shows 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 including a hatch or closable door 204 for engaging with a rear portion 206 of the cell expansion machine 202. An interior space 208 within the cell expansion machine 202 includes features adapted to receive and engage a pre-installed fluid delivery assembly 210. The pre-installed fluid delivery assembly 210 is removably attached to the cell expansion machine 202 to facilitate relatively quick replacement of a used pre-installed fluid delivery assembly 210 at the same cell expansion machine 202 with one of a new or unused fluid delivery assembly 210. The single cell expansion machine 202 can be operated to grow or expand a first set of cells using a first one of the pre-installed fluid delivery assemblies 210, and thereafter can be used to grow or expand a second set of cells using a second one of the pre-installed fluid delivery assemblies 210, without requiring sterilization between interchanging the first one of the pre-installed fluid delivery assemblies 210 with the second one of the pre-installed fluid delivery assemblies 210. The pre-installed fluid delivery assemblies 210 include a cell growth chamber 100 and an oxygenator or gas delivery module 212 (see also FIG. 4 ). According to an embodiment, a conduit guide groove is designated 214 for receiving various culture medium conduits connected to the pre-installed fluid delivery assemblies 210.

[0137] The front 262 of CES200 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 enter data, retrieve data, enter a test plan, switch between views, view data, view alarms, etc. In addition or alternatively, the user interface 264 may include one or more buttons or switches for entering information, controlling the display, or performing other functions. As described below, input from the user interface 264 may be sent to the control system. The front 262 of CES200 may also include one or more lights or other visual signals for indicating an alarm.

[0138] A holder 270, such as a bag holder, disposable holder, etc., can extend from a top surface 272 of the CES 200. The holder 270 can include, for example, a vertical leg 274 and a horizontal leg 276 arranged as an L-shaped support 278. A wire support or clamp 280 can 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 can support a tube of the disposable assembly relative to the vertical leg 274. A plurality of brackets 282 can be supported by the horizontal leg 276 of the L-shaped support 278. Each of the plurality of brackets 282 can 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 a rail system 284 and a hook system 286. The rail system 284 may include sidewalls 288 extending in parallel and defining 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 second sidewall (secondary sidewall) or buffer on its inner surface, which defines a neck 292 or narrow portion of the channel 290. For example, the second sidewall 293 may be formed of an elastic or flexible material (such as rubber, etc.). In an alternative embodiment, the second 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 so that the top of the bag or disposable is held in the channel 290 by the neck 292. The rail system 284 may be used to disperse 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 second sidewall 293.

[0139] The bottom surface of the second side wall 293 may define a slot 294 therein. The slot 294 may be positioned on a side of the second side wall 293 adjacent to the side wall 288. The slot 294 may be arranged to receive one or more hooks 296 of the hook system 286 therein. For example, the slots 294 in a single second side wall 293 of each rail system 284 may each receive a hook 296 of the hook system 286, while the slots 294 in another second side wall 293 of the rail system 284 may remain open. The hooks 296 may be U-shaped or J-shaped hooks and may be configured to secure the bag at a hole in the bag.

[0140] The cell expansion system 200 and / or the cell expansion machine 202 may include a barcode scanner configured to scan barcodes on disposables, culture 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 can be a wire-attached or wireless. The barcode scanner can be a handheld device or fixed within the user interface or on the housing of the cell expansion machine 202. The barcode scanner is compatible with reading customer barcodes, universal barcodes, supplier barcodes, or any other barcodes in or out of the market.

[0141] Next, according to an embodiment of the present disclosure, Figure 3The embodiment of the invention is shown in which a pre-installed fluid delivery assembly 210 ( Figure 2B ) is shown in the rear portion 206 of the cell expansion machine 202 before. Figure 3 The closable door 204 (shown in FIG. Figure 2A and Figure 2B ). The rear portion 206 of the cell expansion machine 202 includes a plurality of different structures for working in combination with the 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 dispensing valve 238, an EC medium valve 240, an IC waste or outlet valve 242, an EC waste valve 244, and a harvest valve 246. Several sensors are also associated with the back 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.

[0142] According to an embodiment, an axis or rocker controller 258 is shown for rotating the cell growth chamber 100. An axis fitting 260 associated with the axis or rocker controller 258 allows an axis of a pre-installed tube organizer such as 300 ( FIG. 4 ) of a transport assembly 210 or 400 to enter a hole such as 424 ( Figure 4A ) is properly aligned with the rear portion 206 of the cell expansion machine 202. Rotation of the shaft or rocker control 258 causes the shaft assembly 260 and the cell growth chamber 100 to rotate. Thus, when the operator or user of the CES 200 places a new or unused pre-installed fluid delivery assembly 400 ( Figure 4A ) is attached to the cell expansion machine 202, alignment is a relatively simple matter of inserting the shaft of the pre-installed fluid delivery assembly 210 or 400 into the hole 424 ( Figure 4A ) is correctly oriented with shaft assembly 260.

[0143] Go to Figure 4A , which shows a perspective view of a removably attached pre-installed fluid delivery assembly 400. The pre-installed fluid delivery assembly 400 can be removably attached to the cell expansion machine 202 ( Figure 2B and Figure 3) to facilitate relatively quick replacement of a used pre-installed fluid delivery assembly 210 at a cell expansion machine 202 with a new or unused fluid delivery assembly 400 at the same cell expansion machine 202. Figure 4A As shown, cell growth chamber 100 may be attached to a bioreactor coupling including shaft assembly 402. Shaft assembly 402 includes one or more shaft fastening mechanisms, such as shaft 258 (e.g., for engaging a cell expansion machine 202). Figure 3 A biasing arm or spring member 404 is provided.

[0144] 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 , as described below. Pump circuits 406A, 406B, and 406C may also be provided for pumps. In embodiments, although various culture media may be provided at the location where the cell expansion machine 202 is located, according to embodiments, the pre-installed fluid delivery assembly 400 may include sufficient tubing length to extend to the exterior of the cell expansion machine 202 and be capable of being welded to tubing associated with a culture medium bag or container.

[0145] refer to FIG. 4B to FIG. 4H , showing an alternative view of the components and parts of the pre-installed fluid delivery assembly 400. In an exemplary embodiment, the pre-installed fluid delivery assembly 400 may include a culture medium bag 410 ( Figure 4D )、waste bag 412( Figure 4E )、pressure chamber 414( Figures 4F to 4G )、Sampling coil 416( Figure 4H )、Cell input bag、Inline filter 420( Fig. 4I ), additional pipes, additional filters, and combinations thereof. For example, Figure 4B The pre-installed fluid delivery assembly 400A may include a cell growth chamber 100B, and Figure 4C The pre-installed fluid delivery assembly 400B in the embodiment of the present invention may include the cell growth chamber 100A. Except for the size change of the cell growth chambers 100A, 100B, the rest of the pre-installed fluid delivery assemblies 400A and 400B may be identical. This allows the pre-installed fluid delivery assembly 400 to be manufactured efficiently.

[0146] like Figure 4D and Figure 4EAs shown, the pre-installed fluid delivery assembly 400 may include one or more culture medium bags 410 and one or more waste bags 412. In some embodiments, the culture medium bags 410 may be identical to the waste bags 412. Keeping the culture medium bags 410 identical to the waste bags 412 allows for manufacturing efficiencies and lower costs. Figure 4D and Figure 4E As shown, the culture medium bag 410 and the waste bag 412 are formed by material sheets to define a material outer seal 422 and a hanger 425. For example, the material sheet, the culture medium bag 410 and the waste bag 412 can be formed by three layers of EVA (ethylene vinyl acetate) whose air permeability is inferior to PVC to keep the pH more stable, so that the culture medium is more "happy", and the airtight material extends the life of the culture medium. For example, the material sheet can be welded or melted to form the material shell 422 and the hanger 425. The material shell 422 can be a pocket in the culture medium bag 410 or the waste bag 412. The size of the material shell 422 can be set to accommodate a specific amount of fluid (e.g., culture medium). For example, the size of the material shell 422 can be set to accommodate 5L of fluid. Alternatively, the size of the material shell 422 can be set to accommodate any amount of fluid suitable for the desired function.

[0147] The hanger 425 may include a hole for receiving the hook 296, such as Figure 2C The hanger 425 may include a rod 426 that is secured within a longitudinal hole or channel 428 of the culture medium bag 410 or the waste bag 412. The rod 426 may be a cylindrical or extruded polygonal rod that engages with the rail system 284, as described with reference to Figure 2C For example, when the bag is inserted into the channel 290 between the side walls 288, the rod 426 rests on top of the second side wall 293 and has a diameter or width greater than the neck 292 so that the hanger 425 cannot slide back through the neck 292 and the media bag 410 or waste bag 412 is suspended in the rail system 284. Additionally or alternatively, the media bag 410 and the waste bag 412 may differ in material, size, or configuration.

[0148] A pair of ports 430 may be provided on a portion of the material housing 422 opposite the hanger 425. The pair of ports 430 may include an inlet port 430A and an outlet port 430B. The inlet port 430A may be engaged with a tube or fluid stream entering the material housing 422. The outlet port 430B may be engaged with a tube or fluid stream exiting the material housing 422. The pair of ports 430 or dual port design of the culture medium bag 410 or waste bag 412 allows multiple culture medium bags 410 or multiple waste bags 412 to be connected together, or to be connected together in a daisy-chain, as described below.

[0149] refer to Figure 4F and Figure 4G , the pressure chamber 414 may be disposed on the housing of the pre-installed fluid delivery assembly 400. The pressure chamber 414 may be configured to detect the pressure of the fluid passing through the fluid line, the pipeline or along the fluid flow path. The pressure chamber 414 may include a single outlet housing 433, a diaphragm 432 and a retaining ring 434. The single outlet housing 433 may include a tubular inlet port 436 and a blocking port 438. The inlet port 436 may be a tubular port configured to receive the fluid into the single outlet housing 433. The blocking port 438 may be similar to the tubular inlet port 436, but may be blocked with a plug 440 to prevent the fluid from flowing therethrough. The plug 440 may be formed of plastic or an elastomer. The single outlet housing 433 may include a body 442 integrally formed with the inlet port 436 and the blocking port 438. The body 442 may define an internal space 444 therein, such as a cylindrical internal space 444. The diaphragm 432 may be a circular planar diaphragm accommodated in the internal space 444 in the body 442. For example, the diaphragm 432 may be an elastic material that flexes with the pressure of the fluid. A retaining ring 434 may be provided at the end of the body 442 of the single outlet housing 433 opposite to the inlet port 436 and the blocking port 438 to fix the diaphragm 432 in the body 442. For example, the retaining ring 434 may be press-fitted or threadedly connected at the end of the body 442 opposite to the inlet port 436 and the blocking port 438.

[0150] Figure 4H and Fig. 4I Exemplary conduits that may be included in pre-installed fluid delivery assembly 400 are shown. In at least one exemplary embodiment, Figure 4H The sampling coil 416 shown in the figure can be a conduit configured to collect a fluid sample in one of the fluid flow paths. In at least one exemplary embodiment, Fig. 4I The illustrated in-line filter 420 may include a filter positioned within a conduit to filter matter from a fluid flowing through a fluid flow path.

[0151] Next, Figure 5A , Figure 5B and Figure 5C Schematic diagram of an embodiment cell expansion system 500 is shown, Figure 6 A schematic diagram of another embodiment of a cell expansion system 600 is shown, and FIG. 7A to FIG. 7C A schematic diagram of a cell expansion system according to another embodiment is shown. Figure 5A , Figure 5B , Figure 5C and Figure 6 In the illustrated embodiment, and as described below, cells are grown in the IC space. However, the present disclosure is not limited to such examples, and other embodiments may provide for cells to be grown in the EC space.

[0152] As mentioned above, Figure 5A , Figure 5B and Figure 5C CES 500 is shown. Although Figure 5A , Figure 5B and Figure 5C depicts a substantially similar structural component to CES500, but Figure 5A , Figure 5B and Figure 5C Possible operational configurations of fluid movement in a first fluid circulation path using structural features of CES 500 according to embodiments of the present disclosure are shown. As shown, according to embodiments, CES 500 includes a first fluid circulation path 502 (also referred to as an "intra-capillary loop" or "IC loop") and a second fluid circulation path 504 (also referred to as an "extra-capillary loop" or "EC loop"). A first fluid flow path 506 can be fluidically associated with a 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, passes through the hollow fibers in the cell growth chamber 501, and exits via the IC outlet port 501B. A pressure gauge 510 measures the pressure of the culture medium leaving the cell growth chamber or bioreactor 501. The culture medium flows through an IC circulation pump 512, which can be used to control the rate at which the culture medium flows. The IC circulation pump 512 can pump fluid in a first direction or in a second direction opposite to the first direction. The IC outlet port 501B can be used as a reverse inlet. For example, in a first configuration, the IC circulation pump may pump fluid in a positive direction where fluid enters IC inlet port 501 A. In a second configuration, for example, the IC circulation pump may pump fluid in a negative direction where fluid enters IC outlet port 501B, for example.

[0153] Culture medium entering the IC loop may enter through valve 514. As will be appreciated by those skilled in the art, additional valves, pressure gauges, pressure / temperature sensors, ports, and / or other devices may be placed at various locations to separate and / or measure characteristics of culture medium along portions of the fluid path. Thus, it should be appreciated that the schematic diagram shown represents one possible configuration for various elements of CES 500 and that the schematic diagram shown may be modified within the scope of one or more current embodiments.

[0154] With respect to the IC loop 502, a sample of the culture medium can be obtained from the sample port 516 or the sample coil 518 during operation. The pressure / temperature gauge 520 disposed in the first fluid circulation path 502 allows the culture medium pressure and temperature to be detected during operation. The culture medium is then returned to the IC inlet port 501A to complete the fluid circulation path 502. Cells grown / expanded in the cell growth chamber 501 can be flushed from the cell growth chamber 501 into the cell harvesting bag 599 through the valve 598, or redistributed within the hollow fibers for further growth.

[0155] Fluid in second fluid circulation path 504 enters cell growth chamber 501 via EC inlet port 501C and exits cell growth chamber 501 via EC outlet port 501D. The culture medium in EC loop 504 can contact the exterior of the hollow fibers in cell growth chamber 501, allowing small molecules to diffuse into and out of the hollow fibers.

[0156] According to an embodiment, a pressure / temperature gauge 524 disposed in the second fluid circulation path 504 allows for the measurement of the pressure and temperature of the culture medium before the culture medium enters the EC space of the cell growth chamber 501. A pressure gauge 526 allows for the measurement of the pressure of the culture medium in the second fluid circulation path 504 after leaving the cell growth chamber 501. With respect to the EC loop, a sample of the culture medium may be obtained from the sample port 530 or the sample coil during operation.

[0157] 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 passes through the EC circulation pump 528 to the oxygenator or gas delivery module 532. The EC circulation pump 528 can also pump fluid in the opposite direction. The second fluid flow path 522 can be fluidically associated with the oxygenator or gas delivery module 532 via the oxygenator inlet port 534 and the oxygenator outlet port 536. In operation, the fluid culture medium flows into the oxygenator or gas delivery module 532 via the oxygenator inlet port 534 and leaves the oxygenator or gas delivery module 532 via the oxygenator outlet port 536. For example, the oxygenator or gas delivery module 532 adds oxygen to the culture medium in the CES 500 and removes bubbles from the culture medium in the CES 500. In various embodiments, the culture medium in the second fluid circulation path 504 can be balanced with the gas entering the oxygenator or gas delivery module 532. The oxygenator or gas delivery module 532 can be an oxygenator or gas delivery device of any appropriate size. Air or gas flows into the oxygenator or gas delivery module 532 via filter 538 and flows out of the oxygenator or gas delivery module 532 via filter 540. Filters 538 and 540 reduce or prevent contamination of the oxygenator or gas delivery module 532 and associated culture medium. During portions of the priming sequence, air or gas purged from the CES 500 may be exhausted to atmosphere via the oxygenator or gas delivery module 532.

[0158] According to at least one embodiment, culture medium including cells (from bag 562) and fluid culture medium from bag 546 can be introduced into first fluid circulation path 502 via first fluid flow path 506. Fluid container 562 (e.g., a cell entry bag or saline priming fluid for priming air out of the system) can be fluidly associated with first fluid flow path 506 and first fluid circulation path 502 via valve 564.

[0159] Fluid containers or culture medium bags 544 (e.g., reagents) and 546 (e.g., IC culture medium) can be fluidically associated with the first fluid inlet path 542 via valves 548 and 550, respectively, or fluidically associated with the second fluid inlet path 574 via valves 570 and 576. Also provided are first and second sterile sealable input pre-fill paths 508 and 509. An air removal chamber (ARC) 556 can be fluidically associated with the first circulation path 502. The air removal chamber 556 can include one or more ultrasonic sensors (including upper sensors and lower sensors) to detect air, lack of fluid and / or gas / fluid interface (e.g., air / fluid interface) at certain measurement locations within the air removal chamber 556. For example, an ultrasonic sensor 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 interface at these locations. Without departing from the spirit and scope of the present disclosure, embodiments provide the use of many other types of sensors. For example, according to embodiments of the present disclosure, optical sensors can be used. During portions of a priming sequence or other protocols, air or gas purged from the CES 500 may be exhausted to atmosphere via line 558 from an air valve 560 , which may be fluidly associated with the air removal chamber 556 .

[0160] EC culture medium (e.g., from bag 568) or wash solution (e.g., from bag 566) can be added to the first or second fluid flow path. Fluid container 566 can be fluidly associated with valve 570, which can be fluidly associated with first fluid circulation path 502 via dispensing valve 572 and first fluid inlet path 542. Alternatively, by opening valve 570 and closing dispensing valve 572, fluid container 566 can be fluidly associated with second fluid circulation path 504 via second fluid inlet path 574 and EC inlet path 584. Similarly, fluid container 568 can be fluidly associated with valve 576, which can be fluidly associated with first fluid circulation path 502 via first fluid inlet path 542 and dispensing valve 572. Alternatively, by opening valve 576 and closing dispensing valve 572, fluid container 568 can be fluidly associated with second fluid inlet path 574.

[0161] An optional heat exchanger 552 may be provided for the introduction of culture medium reagents or wash solutions.

[0162] In the IC loop, fluid may be initially propelled by the IC inlet pump 554. In the EC loop, fluid may be initially propelled by the EC inlet pump 578. An air detector 580, such as an ultrasonic sensor, may also be associated with the EC inlet path 584.

[0163] In at least one embodiment, the first fluid circulation path 502 and the second fluid circulation path 504 are connected to a waste line 588. When valve 590 is open, IC medium can flow through the waste line 588 and into a waste or outlet bag 586. Similarly, when valve 582 is open, EC medium can flow through the waste line 588 and into a waste or outlet bag 586.

[0164] In embodiments, cells may be harvested via cell harvest path 596. Here, cells from cell growth chamber 501 may be harvested by pumping IC medium containing cells through cell harvest path 596 and valve 598 to cell harvest bag 599.

[0165] The various components of CES 500, cell expansion machine 202 may be included or housed in a machine or housing (FIG. 2 and Figure 3 ) within a chamber, wherein machinery maintains cells and culture medium at, for example, a predetermined temperature.

[0166] In an embodiment, Figure 5A In the configuration depicted for CES 500 in FIG. 5 , the fluid medium in first fluid circulation path 502 and second fluid circulation path 504 flows through cell growth chamber 501 in the same direction (co-current configuration). In another embodiment, CES 500 can also be configured to flow in a counter-current configuration (not shown). Figure 5A In the configuration shown, fluid in the first fluid circulation path 502 enters the bioreactor 501 at the IC inlet port 501A and leaves the bioreactor 501 at the IC outlet port 501B. Figure 5B and Figure 5C In the configuration depicted in , the fluid culture medium in the first circulation path 502 can flow in opposite or opposing directions from the connector 517 so that the fluid can enter the IC inlet port, the first port 501A, on one end of the bioreactor, and the fluid can enter the IC outlet port, the second port 501B, on the opposite end of the bioreactor to maintain the cells in the bioreactor itself. The first fluid flow path can be associated with the first fluid circulation path fluid via the connector 517. In an embodiment, the connector 517 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 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, fuse, passage, conduit, etc. that allows the first fluid flow path to be associated with the first circulation path fluid. It should be understood that Figure 5A , Figure 5B and Figure 5CThe schematic diagrams and operational configurations shown in the drawings represent possible configurations for various elements of a cell expansion system and may be modified within the scope of one or more present embodiments.

[0167] Go to Figure 6 , a schematic diagram of another embodiment of a cell expansion system 600 is shown. CES 600 includes a first fluid circulation path 602 (also referred to as an "intra-capillary loop" or "IC loop") and a second fluid circulation path 604 (also referred to as an "extra-capillary loop" or "EC loop"). The first fluid flow path 606 can be fluidically associated with the cell growth chamber 601 to form the first fluid circulation path 602. The fluid flows into the cell growth chamber 601 through the IC inlet port 601A, passes through the hollow fibers in the cell growth chamber 601, and leaves via the IC outlet port 601B. The pressure sensor 610 measures the pressure of the culture medium 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 pressure and temperature of the culture medium during operation. The culture medium flows through the IC circulation pump 612, which can be used to control the rate at which the culture medium flows. The IC circulation pump 612 can pump fluid in a first direction or in a second direction opposite to the first direction. The exit port 601B can be used as an inlet in the opposite direction. The culture medium entering the IC loop can enter through the valve 614. As will be appreciated by those skilled in the art, additional valves, pressure gauges, pressure / temperature sensors, ports, and / or other devices may be placed at various locations to isolate and / or measure characteristics of the culture medium along portions of the fluid path. Thus, it should be understood that the schematic diagram shown represents one possible configuration for the various elements of the CES 600 and that modifications may be made to the schematic diagram shown within the scope of one or more of the present embodiments.

[0168] With respect to the IC loop, during operation, a sample of the culture medium may be obtained from the sample coil 618. The culture medium is then returned to the IC inlet port 601A to complete the fluid circulation path 602. Cells grown / expanded in the cell growth chamber 601 may be flushed from the cell growth chamber 601 into the cell harvest bag 699 via the valve 698 and the cell harvest path 697. Alternatively, when the valve 698 is closed, the cells may be redistributed within the chamber 601 for further growth.

[0169] Fluid in second fluid circulation path 604 enters cell growth chamber 601 via EC inlet port 601C and exits cell growth chamber 601 via EC outlet port 601D. According to embodiments, the culture medium in the EC loop may be in contact with the exterior of the hollow fibers in cell growth chamber 601, thereby allowing small molecules to diffuse into and out of the hollow fibers that may be within chamber 601.

[0170] A pressure / temperature sensor 624 disposed in the second fluid circulation path 604 allows for the measurement of the pressure and temperature of the culture medium before the culture medium enters the EC space of the cell growth chamber 601. A sensor 626 allows for the measurement of the pressure and / or temperature of the culture medium in the second fluid circulation path 604 after exiting the cell growth chamber 601. With respect to the EC loop, during operation, a sample of the culture medium may be obtained from the sample port 630 or the sample coil.

[0171] 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 delivery module 632 through the EC circulation pump 628. According to an embodiment, the EC circulation pump 628 can also pump fluid in the opposite direction. The second fluid flow path 622 can be fluidically associated with the oxygenator or gas delivery module 632 via the inlet port 632A and the outlet port 632B of the oxygenator or gas delivery module 632. In operation, the fluid culture medium flows into the oxygenator or gas delivery module 632 via the inlet port 632A and leaves the oxygenator or gas delivery module 632 via the outlet port 632B. For example, the oxygenator or gas delivery module 632 adds oxygen to the culture medium in the CES 600 and removes bubbles in the culture medium in the CES 600. In various embodiments, the culture medium in the second fluid circulation path 604 can be balanced with the gas entering the oxygenator or gas delivery module 632. The oxygenator or gas delivery module 632 can be a device of any suitable size that can be used for oxygenation or gas delivery. Air or gas flows into the oxygenator or gas delivery module 632 via filter 638 and flows out of the oxygenator or gas delivery module 632 via filter 640. Filters 638 and 640 reduce or prevent contamination of the oxygenator or gas delivery module 632 and associated culture medium. During portions of the priming sequence, air or gas purged from the CES 600 may be exhausted to atmosphere via the oxygenator or gas delivery module 632.

[0172] In the configuration depicted for CES 600, fluid medium in first fluid circulation path 602 and second fluid circulation path 604 flows in the same direction (co-current configuration) through cell growth chamber 601. According to embodiments, CES 600 may also be configured for flow in a counter-current configuration.

[0173] According to at least one embodiment, culture medium including cells (from a source such as a cell container, e.g., a bag) can be attached at attachment point 662, and fluid culture medium from a culture medium source can be attached at attachment point 646. Cells and culture medium can be introduced into first fluid circulation path 602 via first fluid flow path 606. Attachment point 662 can be fluidly associated with first fluid flow path 606 via valve 664, and attachment point 646 can be fluidly associated with first fluid flow path 606 via valve 650. A reagent source can be fluidly connected to point 644 and associated with first fluid inlet path 642 via valve 648, or associated with second fluid inlet path 674 via valve 648 and valve 672.

[0174] Air removal chamber (ARC) 656 can be associated with the first circulation path 602 fluid. The air removal chamber 656 may include one or more sensors (including upper sensors and lower sensors) to detect the lack of air, fluid and / or gas / fluid interface (e.g., air / fluid interface) at certain measurement locations within the air removal chamber 656. For example, an ultrasonic sensor can be used near the bottom and / or near the top of the air removal chamber 656 to detect air, fluid and / or air / fluid interface at these locations. Without departing from the spirit and scope of the present disclosure, embodiments provide the use of many other types of sensors. For example, according to embodiments of the present disclosure, optical sensors can be used. During parts of the pre-fill sequence or other schemes, the air or gas purged from the CES 600 can be discharged to the atmosphere from the air valve 660 via pipeline 658, and the air valve 660 can be associated with the air removal chamber 656 fluid.

[0175] The EC culture medium source can be attached to the EC culture medium attachment point 668, and the washing solution source can be attached to the washing solution attachment point 666 to add EC culture medium and / or washing solution to the first or second fluid flow path. The attachment point 666 can be fluidly associated with the valve 670, which can be fluidly associated with the first fluid circulation path 602 via the 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 the valve 670 and closing the valve 672. Similarly, the attachment point 668 can be fluidly associated with the valve 676, which can be fluidly associated with the first fluid circulation path 602 via the first fluid inlet path 642 and the valve 672. Alternatively, the attachment point 668 can be fluidly associated with the second fluid inlet path 674 by opening the valve 676 and closing the dispensing valve 672.

[0176] In the IC loop, fluid may be initially propelled by the IC inlet pump 654. In the EC loop, fluid may be initially propelled by the EC inlet pump 678. An air detector 680, such as an ultrasonic sensor, may also be associated with the EC inlet path 684.

[0177] In at least one embodiment, the first fluid circulation path 602 and the second fluid circulation path 604 are connected to a waste line 688. When valve 690 is open, IC medium can flow through the waste line 688 and into a waste or outlet bag 686. Similarly, when valve 692 is open, EC medium can flow into the waste or outlet bag 686.

[0178] After cells have been grown in cell growth chamber 601, they can be harvested via cell harvest path 697. Here, cells from cell growth chamber 601 can be harvested by pumping IC medium containing the cells through cell harvest path 697 to cell harvest bag 699 with valve 698 open.

[0179] Various components of CES 600, such as cell expansion machine 202, may be contained or accommodated in a machine or housing (FIG. 2 and Figure 3 ) wherein the machine maintains the cells and culture medium at, for example, a predetermined temperature. It should be further noted that, in embodiments, components of CES 600 and CES 500 may be combined. In other embodiments, a CES may include fewer or additional components than those shown in CES 500 and / or CES 600 and still be within the scope of the present disclosure. An example of a cell expansion system that may incorporate features of the present disclosure is manufactured by Terumo BCT, Inc., located in Lakewood, Colorado. Cell Expansion System.

[0180] It should be understood that Figure 6 The schematic diagram shown represents one possible configuration of various elements for a cell expansion system and may be modified within the scope of one or more embodiments.

[0181] Examples and further description of cell expansion systems are provided in U.S. Pat. No. 8,309,347 (“Cell Expansion Systems and Methods of Use,” published on November 13, 2012) and U.S. Pat. No. 9,057,045, filed on December 15, 2010 (“Methods of Loading and Distributing Cells in a Bioreactor of a Cell Expansion System,” published on June 16, 2015), the entire teachings of which are incorporated herein by reference for all purposes.

[0182] exist FIG. 7A to FIG. 7CIn the illustrated embodiment, and as described below, cells are grown in the IC space. However, the present disclosure is not limited to such examples, and in other embodiments cells grown in the EC space may be provided.

[0183] As mentioned above, FIG. 7A to FIG. 7C CES 700 is shown. Although FIG. 7A to FIG. 7C depicts substantially similar structural components of CES 700, but FIG. 7A to FIG. 7C Possible operational configurations of fluid movement in a first fluid circulation path using structural features of CES 700 according to embodiments of the present disclosure are shown. As shown, according to embodiments, CES 700 includes a first fluid circulation path 702 (also referred to as an "intra-capillary loop" or "IC loop") and a second fluid circulation path 704 (also referred to as an "extra-capillary loop" or "EC loop"). A first fluid flow path 706 can be fluidically associated with a cell growth chamber 701 to form a first fluid circulation path 702. The fluid is configured to flow into the cell growth chamber 701 through an IC inlet port 701A, through a hollow fiber in the cell growth chamber 701, and exit via an IC outlet port 701B. A pressure gauge 710 measures the pressure of the culture medium leaving the cell growth chamber 701 or bioreactor. The culture medium flows through an IC circulation pump 712, which can be used to control the rate at which the culture medium flows. The IC circulation pump 712 can pump fluid in a first direction or in 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 where fluid enters the IC inlet port 701 A. In a second configuration, for example, the IC circulation pump 712 can pump fluid in a negative direction where fluid enters the IC outlet port 701B.

[0184] The fluid in the IC loop or first fluid circulation path 702 can pass through the IC circulation valve 714. As will be appreciated by those skilled in the art, additional valves, pressure gauges, pressure / temperature sensors, ports and / or other devices can be placed at various locations to isolate and / or measure the characteristics of the culture medium along the portion of the fluid path. For example, the first fluid circulation path 702 may 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. Therefore, it should be understood that the schematic diagram shown represents one possible configuration for the various elements of the CES 700, and within the scope of one or more exemplary embodiments, the schematic diagram shown can be modified.

[0185] With respect to the first fluid circulation path 702, a sample of the culture medium can be obtained from the sample port or sample coil 718 during operation. A pressure / temperature gauge 720 disposed in the first fluid circulation path 702 allows for detection of the culture medium pressure and temperature during operation. The culture medium is then returned to the IC inlet port 701A to complete the first fluid circulation path 702. Cells grown and / or expanded in the cell growth chamber 701 can be flushed out of the cell growth chamber 701 and into the harvest bag 799 through the valve 798, or redistributed within the hollow fibers for further growth.

[0186] Fluid in second fluid circulation path 704 enters cell growth chamber 701 via EC inlet port 701C and exits cell growth chamber 701 via EC outlet port 701D. The culture medium in second fluid circulation path 704 can contact the exterior of the hollow fibers in cell growth chamber 701, thereby allowing small molecules to diffuse into and out of the hollow fibers.

[0187] In at least one exemplary embodiment, a pressure / temperature gauge 724 disposed in the second fluid circulation path 704 allows for the measurement of the pressure and / or temperature of the medium before the medium enters the EC space of the cell growth chamber 701. A pressure gauge 726 allows for the measurement of the pressure of the medium in the second fluid circulation path 704 after leaving the cell growth chamber 701. With respect to the EC loop, a sample of the medium may be obtained from the sample port 730 or the sample coil during operation.

[0188] 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 delivery module 732. The EC circulation pump 728 can also pump fluid in the opposite direction. The second fluid flow path 722 can be fluidically associated with the oxygenator or gas delivery module 732 via the oxygenator inlet port 734 and the oxygenator outlet port 736. In operation, the fluid culture medium flows into the oxygenator or gas delivery module 732 via the oxygenator inlet port 734 and leaves the oxygenator or gas delivery module 732 via the oxygenator outlet port 736. The oxygenator or gas delivery module 732, for example, adds oxygen to the culture medium in the CES 700 and removes bubbles from the culture medium in the CES 700. In various exemplary embodiments, the culture medium in the second fluid circulation path 704 can be balanced with the gas entering the oxygenator or gas delivery module 732. The oxygenator or gas delivery module 732 can be any appropriately sized oxygenator or gas delivery device. Air or gas flows into the oxygenator or gas delivery module 732 via filter 738 and flows out of the oxygenator or gas delivery device 732 via filter 740. Filter 738 and filter 740 reduce or prevent contamination of the oxygenator or gas delivery module 732 and associated culture medium. During portions of the priming sequence, air or gas purged from the CES 700 can be exhausted to the atmosphere via the oxygenator or gas delivery module 732.

[0189] According to at least one embodiment, culture medium (including cells from bag 762) and fluid culture medium from bag 746 can be introduced into first fluid circulation path 702 via first fluid flow path 706. Fluid container 762 (e.g., a cell entry bag or saline priming fluid for priming air out of the system) can be fluidly associated with first fluid flow path 706 and first fluid circulation path 702 via valve 764.

[0190] Fluid containers or culture medium bags 744 (e.g., reagents) and 746 (e.g., IC culture medium) can be associated with the first fluid inlet path 742 fluid via valves 748 and valves 750, respectively, or associated with the second fluid inlet path 774 fluid via valves 770 and valves 776. An air removal chamber (ARC) 756 can be associated with the first circulation path 702 fluid. The air removal chamber 756 may include one or more ultrasonic sensors (including upper sensors and lower sensors) to detect the lack of air, fluid and / or gas / fluid interface (e.g., air gas / fluid interface) at certain measurement positions in the air removal chamber 756. For example, an ultrasonic sensor 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 interface at these positions. Without departing from the spirit and scope of the present disclosure, exemplary embodiments provide the use of many other types of sensors. For example, according to embodiments of the present disclosure, optical sensors can be used. During portions of a priming sequence or other protocols, air or gas purged from the CES 700 may be exhausted to atmosphere from an air valve 760 , which may be fluidly associated with the air removal chamber 756 .

[0191] EC culture medium (e.g., from bag 768) and / or wash solution (e.g., from bag or fluid container 766) can be added to first fluid inlet path 742 and / or second fluid flow path 722. Fluid container 766 can be fluidly associated with valve 770, which can be fluidly associated with first fluid circulation path 702 via dispensing valve 772 and first fluid inlet path 742. Alternatively, by opening valve 770 and closing dispensing valve 772, fluid container 766 can be fluidly associated with second fluid circulation path 704 via second fluid inlet path 774 and EC inlet path 784. Similarly, as shown in FIG. Figure 7C As shown, fluid containers 768A and 768B can be fluidly associated with valve 776, which can be fluidly associated with first fluid circulation path 702 via first fluid inlet path 742 and dispensing valve 772. Alternatively, fluid containers 768A and 768B can be fluidly associated with second fluid inlet path 774 by opening valve 776 and closing dispensing valve 772.

[0192] The fluid containers 768A and 768B may be culture medium containers for distributing culture medium to the first fluid inlet path 742 or the second fluid inlet path 774. In some exemplary embodiments, the fluid containers 768A and 768B may be connected to Figure 4D774 when communicating with the second fluid inlet path 774, can be part of the EC circulation supply loop 752 (or can be part of the IC circulation supply loop when communicating with the first fluid inlet path 742). In the EC circulation supply loop 752, the culture medium is transferred from the fluid container 768B to the fluid container 768A through the EC loop 704, passes through the EC waste valve 782, and returns to the fluid container 768B. The use of the EC circulation supply loop 752 avoids the use of pass-through profusion, in which fresh culture medium is continuously added to the system and collected in the outlet waste bag, which is discarded when full. With pass-through profusion, as more cells grow, culture medium must be introduced at a higher rate to feed the cells. For example, users typically read the reading of glucose or lactate once a day and then increase the supply rate for the day. The supply rate for the whole day may be incorrect, resulting in waste of culture medium.

[0193] In contrast, in the CES 700 herein, the EC circulation feed loop 752 increases the volume of the IC circulation loop or EC circulation loop by connecting or daisy-chaining fluid container 768A with fluid container 768B (through a pair of ports discussed previously). 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 culture medium is recirculated. The user can calculate how much lactic acid is produced to determine waste. The user does not need to change bags; they just set the feed rate and run the protocol. Therefore, the CES 700 is more efficient and uses less total culture medium.

[0194] Although two fluid containers 768A and 768B are shown and discussed, it should be understood that more fluid containers 768N may be added to the system.

[0195] In an exemplary scheme, gas is supplied to the system via a GTM (such as GTM 732) using an EC cycle. Gas is supplied to the CES 700 from the outside, and each time the culture medium passes through the GTM 732, the gas is exchanged to the desired concentration. In this case, fluid containers 768A and 768B are used as exchange vessels for the culture medium returned to the cell growth chamber 701. For example, the attached T-cell feeding scheme (CFA-CSS-CEdj-036-03) uses approximately 11L of EC culture medium and 3.4L of IC culture medium during the growth scheme. Using this new feeding strategy, the user attaches 3.4L of IC culture medium to the IC culture medium line and daisy chains 11L (3 culture medium bags) of EC culture medium and connects it to the EC inlet and outlet lines. Just as the EC circulation rate needs to be increased as the cell population's demand for oxygen increases, the EC inlet rate (actually the EC feeding cycle) will also increase.

[0196] Depending on the cell type and protocol dynamics, either an IC feeding loop or an EC feeding loop can be implemented. Alternatively, a dual feeding loop can be used, such as Figure 7C shown.

[0197] This strategy allows the implementation of a large number of possible feeding strategies. This strategy also improves the ease of use of the CES 700, reducing human interaction with the device, since waste bags do not need to be emptied and new bags of media are provided to the machine at different time intervals during cell growth. In addition, in many cases, it is not necessary to determine the feeding rate. Even with continuous perfusion feeding, lactate and glucose concentrations can fluctuate significantly without the need to use very lengthy custom tasks to change the flow rate in small increments.

[0198] Depending on the desired use, EC circulation supply loop 752 can be used to coat the fibers of cell growth chamber 701 to circulate and push fluid through the membrane (on the IC side). Additionally or alternatively, EC circulation supply loop 752 can be used in a counter-flow technique to push fluid on the opposite end of cell growth chamber 701.

[0199] The passive coating model used in the CES system may require the application of 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 to the inside of 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 begin additional expansion of the cell population harvested from the CES system (hMSCs cannot be stored in a non-cryopreserved state for up to 16 hours). A new protocol or method for coating the fibers of the cell growth chamber 701 results in the coating agent successfully coating the growth surface 10 minutes after being loaded into the IC circulation loop. The method (see Appendix B for the protocol) 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 proper 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 results in "actively" forcing the coating agent to the surface of the hollow fibers. Allowing the user to coat the hollow fiber bioreactor and load the cells on the same day reduces errors, saves time, and reduces the length of cell expansion by one day. This also allows only a single device (such as CES A) to be visited / Quantum Devices) harvest cell populations from the system and coat new disposables for subsequent passaging / expansion of the same cell population without cryopreservation.

[0200] Use a dual-port bag as in reference Figure 4D and Figure 4E As described, the inlet line (usually the "wash" line) can be connected to one port of the bag, and the "waste" line can be connected to the other port. This allows the protein carrier fluid (usually phosphate buffered saline (PBS)) containing the coating reagent to be recirculated through the system as long as the user wishes. When compared to the passive model of coating, a high IC inlet rate is used to drive the contact between the coating solution and the HFB membrane via ultrafiltration (UF), driving the adsorption of the coating reagent and reducing the time required to coat the hollow fiber bioreactor. Moving the IC fluid through the HFB membrane also enhances the deposition of the coating reagent on the IC side of the HFB membrane. For example, because the coating solution has a higher molecular weight and cannot pass through the HFB membrane, it is deposited on the IC side of the HFB membrane.

[0201] An optional heat exchanger may be provided for introducing culture medium reagents or wash solutions. In some embodiments, an optional heat exchanger may be disposed in the first fluid inlet path 742 and / or the second fluid inlet path 774.

[0202] One or more differential pressure sensors may be included for real-time pressure monitoring and alarms, providing more efficient and accurate measurements as opposed to manual measurements. The pressure sensor helps detect a reduction or stop in flow, exhaustion of the gas supply, user error, etc. Upon detecting a reduction or stop in flow, the differential pressure sensor triggers an alarm to alert the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alert on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0203] One or more gas regulators may be included for gas management. For example, the gas regulator may be internal to the CES 700 so that it is not facing the user, thereby preventing errors. The gas regulator may be less sensitive to motion, thereby providing fine control. Upon detecting an error in gas management, the gas regulator may trigger an alarm to alert the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alert on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0204] One or more temperature thermistors may be included for high sensitivity temperature detection. In cell culture, a temperature change of 0.5 degrees is critical. Thermistors degrade less than resistance temperature detectors (RTDs), thus allowing more use before requiring repair. Upon detecting a temperature change outside a predetermined range or above or below a predetermined threshold, the thermistor may trigger an alarm to alert the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual reminder on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital reminder sent to the user.

[0205] In the IC loop, fluid may be initially propelled by the IC inlet pump 754. In the EC loop, fluid may be initially propelled by the EC inlet pump 778. An air detector 780, such as an ultrasonic sensor, may also be associated with the EC inlet path 784. When the air detector 780 detects air, the air detector may trigger an alarm to alert a user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alert on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0206] In at least one embodiment, the first fluid circulation path 702 and the second fluid circulation path 704 are connected to an outlet line 788. When valve 790 is open, IC medium can flow through the outlet line 788 and return to the fluid container 768B. Similarly, when valve 782 is open, EC medium can flow through the outlet line 788 back to the fluid container 768B.

[0207] In an exemplary embodiment, cells may be harvested via cell harvest path 796. Here, cells from cell growth chamber 701 may be harvested by pumping IC medium containing cells through cell harvest path 796 and valve 798 to cell harvest bag 799.

[0208] Various components of CES 700, such as cell expansion machine 202, may be contained or accommodated in a machine or housing ( Figure 2A , Figure 2B and Figure 3 ) within a chamber, wherein machinery maintains cells and culture medium at, for example, a predetermined temperature.

[0209] In an embodiment, Fig. 7A In the configuration depicted for CES 700 in FIG. 1 , 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, CES 700 can also be configured to flow in a counter-current configuration (not shown). Fig. 7A In the configuration shown, the fluid in the first fluid circulation path 702 enters the cell growth chamber 701 at the IC inlet port 701A and leaves the cell growth chamber 701 at the IC outlet port 701B. According to embodiments, in alternative configurations, the fluid culture medium in the first circulation path 702 can flow in opposite or opposite directions from the connector 717, so 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 keep the cells in the bioreactor itself. The first fluid flow path can be fluidically associated with the first fluid circulation path through the connector 717. In embodiments, 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 embodiments, the connector 717 can be a T-shaped fitting or a T-shaped coupling. In another embodiment, the connector 717 can be a Y-shaped fitting or a Y-shaped coupling. The connector 717 may be any type of fitting, coupling, fusion, passage, conduit, etc. that allows the first fluid flow path to be fluidically associated with the first circulation path. It should be understood that FIG. 7A to FIG. 7CThe schematic diagrams and operational configurations shown in the drawings represent possible configurations for various elements of a cell expansion system and modifications may be made to the schematic diagrams and operational configurations shown within the scope of one or more present embodiments.

[0210] refer to Figure 7B , CES 700B is shown. CES 700B can be similar to CES 700A and can include the same or similar components as CES 700A, where the same reference numerals are used for the same or similar components. CES 700B can include an IC circulation supply loop 753. As previously described, IC circulation supply loop 753 can be similar to EC circulation supply loop 752. IC circulation supply loop 753 can include multiple fluid containers, such as fluid container 746A and fluid container 746B. Fluid container 746A and fluid container 746B can be connected to Figure 4D The culture medium bag 410 shown in FIG.

[0211] Fluid containers 746A and 746B can be culture medium containers for distributing culture medium into the first fluid flow path 706 through IC culture medium pipeline 746. In the IC circulation supply loop 753, the culture medium 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. The use of the IC circulation supply loop 753 avoids the use of pass-through profusion supply, in which fresh culture medium is continuously added to the system and collected in the outlet waste bag, which is discarded when full. With the pass-through supply, as more cells grow, culture medium must be introduced at a higher rate to feed the cells. Users usually read the reading of glucose or lactate once a day and then increase the supply rate for the day. The supply rate for the whole day may be incorrect, resulting in waste of culture medium.

[0212] In contrast, in the CES 700 herein, the IC circulation feed loop 753 increases the volume of the IC circulation loop by connecting or daisy-chaining fluid container 746A with fluid container 746B (through a pair of ports previously described). 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 746B, and the culture medium is recirculated. The user can calculate how much lactic acid is produced to determine waste. The user does not need to change bags; they just set the feed rate and run the protocol. Therefore, the CES 700 is more efficient and uses less total culture medium.

[0213] Although two fluid containers 746A and 746B are shown and discussed, it should be understood that more fluid containers 746N may be added to the system.

[0214] This strategy allows the implementation of a large number of possible feeding strategies. This strategy also enhances the ease of use of the CES 700, reducing human interaction with the device, since waste bags do not need to be emptied and new bags of media are provided to the machine at different time intervals during cell growth. In many cases, it is also not necessary to determine the feeding rate. Even with continuous perfusion feeding, lactate and glucose concentrations can fluctuate significantly without the need to use very lengthy custom tasks to change the flow rate in small increments.

[0215] Depending on the desired use, IC circulation supply loop 753 can be used to coat the fibers of cell growth chamber 701 to circulate and push fluid through the membrane (on the IC side). Additionally or alternatively, IC circulation supply loop 753 can be used in a counter-current technique to push fluid on the opposite end of cell growth chamber 701.

[0216] One or more differential pressure sensors may be included for real-time pressure monitoring and alarms, providing more efficient and accurate measurements as opposed to manual measurements. The pressure sensor helps detect a reduction or stop in flow, exhaustion of the gas supply, user error, etc. Upon detecting a reduction or stop in flow, the differential pressure sensor triggers an alarm to alert the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alert on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0217] One or more gas regulators may be included for gas management. For example, the gas regulator may be internal to the CES 700 so that it is not facing the user, thereby preventing errors. The gas regulator may be less sensitive to motion, thereby providing fine control. Upon detecting an error in gas management, the gas regulator may trigger an alarm to alert the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alert on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital alert sent to the user.

[0218] One or more temperature thermistors may be included for high sensitivity temperature detection. In cell culture, a temperature change of 0.5 degrees is critical. Thermistors degrade less than resistance temperature detectors (RTDs), thus allowing more use before requiring repair. Upon detecting a temperature change outside a predetermined range or above or below a predetermined threshold, the thermistor may trigger an alarm to alert the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual reminder on the device, or a combination thereof. For example, the remote alarm may be an email, text message, or other digital reminder sent to the user.

[0219] Reference now Figure 7C, a CES 700C having dual supply circulation loops is shown. CES 700C may be similar to CES 700A and CES 700B, and may include the same or similar components as CES 700A and CES 700B, wherein the same reference numerals are used for the same or similar components. CES 700C may include an EC circulation supply loop 752 from CES 700A and an IC circulation supply loop 753 from CES 700B. IC circulation supply loop 753 may transfer fluid from fluid container 746B to fluid container 746A, to first fluid inlet path 742, to cell growth chamber 701, to harvest valve 798, and back to fluid container 746B. EC circulation supply loop 752 can transfer fluid from fluid container 768B to fluid container 768A, to EC medium valve 776, to second fluid inlet path 774, to GTM 732, to cell growth chamber 701, to EC waste valve 782, and back to fluid container 768B.

[0220] Although various exemplary embodiments of cell expansion systems and methods associated therewith have been described, Figure 8 Example operational steps 756 of a process for expanding non-adherent or suspended cells in a cell expansion system, such as CES 500, CES 600, or CES 700A to CES 700C, are shown according to embodiments of the present disclosure.

[0221] Start operation 758 is initiated, and process 756 proceeds to prepare cells 760. In embodiments, preparing cells 760 may involve a plurality of different and optional steps. For example, cells may be collected 762. Collecting cells 762 may involve separating and collecting cells from a donor. In some embodiments, an apheresis procedure (e.g., leukapheresis) may be performed to collect a volume of lymphocytes from the peripheral blood of a donor. The volume of lymphocytes may include a target cell population to be amplified by process 756. In other embodiments, cells may be collected from umbilical cord blood.

[0222] After collection 762, optionally, cell separation 764 may be performed as part of preparation 760. The volume of cells collected at step 762 may include a plurality of different cell types, including cells targeted for amplification. Optional step 764 may be performed to separate target cells. As an example, the target cells may be T cells, e.g., regulated T cells. In one embodiment, the regulated T cells may be CD4+CD25+T cells. Any suitable separation technique may be used to separate the cells. For example, immunomagnetic separation may be used to separate cells, wherein magnetic beads functionalized with antibodies are contacted with the cells collected at 762. Functionalized beads may preferentially attach to a target cell population. A magnetic field may then be used to retain particles with an attached target cell population, while other cells may be removed.

[0223] Optionally, after separation 764, the cells can be resuspended 766. In embodiments, the cells can be resuspended in a culture medium that includes a variety of nutrients and / or agents that help maintain cell viability. In embodiments, the culture medium can include at least serum albumin and agents, such as cytokines. In embodiments, the cytokine can be a recombinant human IL-2 cytokine. In one embodiment, the culture medium can include a cytokine at a concentration of 200 IU / mL.

[0224] After preparing the cells 760, process 756 proceeds to exposing the cells 768 to activate the cells for expansion. Optionally, in 770, the cells may be exposed to a soluble activator. An activator that may include an antibody complex may be added to the culture medium in which the cells are resuspended. In embodiments, the activator may be a human antibody CD3 / CD28 / CD2 cell activator complex. In some embodiments, the activator may be included in the culture medium used in resuspending the cells 766. Optionally, at 772, the cells may be exposed to beads having an activator on their surface. In embodiments, exposing the cells to the beads may involve adding a predetermined amount of beads to the resuspended cells. Beads may be added at different ratios relative to the number of cells. For example, beads may be added at a ratio of 1 bead: 2 cells. Other embodiments may be provided in which beads are added at different ratios, for example, 1 bead: 1 cell, 1 bead: 3 cells, etc. The beads may have antibodies on their surface to activate the cells for expansion. In embodiments, the beads may include antibodies CD3 / CD28 on their surface.

[0225] Process 756 proceeds to expanding the cells 774. As part of the cell expansion at 774, the 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, culture medium can be delivered to the cell growth chamber to provide nutrients for expansion. For example, as described with respect to Figures 7A-7C As discussed, the culture medium can be provided by the IC circulation supply loop 753, the EC circulation supply loop 752, or a combination thereof. Expansion 774 of the cells can also include periodically adding reagents 778 to the cell growth chamber to continue to promote cell expansion. For example, in some embodiments, reagents (e.g., cytokines) can be added to the cell growth chamber to promote cell expansion. In one embodiment, the reagent can be an additional IL-2 cytokine, such as a recombinant human IL-2 cytokine.

[0226] For example, regarding FIG. 7A to FIG. 7C As described, culture medium, reagents, or a combination thereof may be delivered to cells from one or more connected fluid containers, and the culture medium, reagents, or a combination thereof may be circulated and recycled via the IC circulation supply loop 753 or the EC circulation supply loop 752 .

[0227] In addition, as part of expanding cells 774, the environment in 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 expanded in the cell growth chamber. In addition, the temperature can be controlled within a range optimized for cell expansion. The expansion of cells 774 can also include monitoring metabolites at 782. For example, lactate and glucose levels can be periodically monitored. Lactate and glucose levels can be monitored by one or more sensors. The one or more sensors can also be configured to measure oxygen levels. An increase or decrease in metabolites can prompt changes (e.g., additional feeding, additional reagent addition, additional gas exchange, etc.) to control the environment in the cell growth chamber at 780.

[0228] When monitoring various parameters, data readings that fall outside of predetermined thresholds can trigger an alarm to alert a 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 audio alarm on the device, a visual reminder on the device, or a combination thereof. For example, the remote alarm can be an email, text message, or other digital reminder sent to the user.

[0229] The process 756 then proceeds to harvesting the 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. The further processing or other analysis at 786 can include performing flow cytometry, for example, to characterize the cell phenotype. The process 756 can then terminate at end operation 788. If further processing / analysis is not desired, the process 756 terminates at end operation 788.

[0230] Fig. 9A Operational steps of process 800 according to an embodiment of the present disclosure are shown, which can be used to position 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 "centrally load cells without circulation" task. Start operation 802 is initiated, and process 800 proceeds to step 804, in which a first volume of fluid with cells can be loaded into a cell growth chamber of a cell expansion system. In an embodiment, the cells may include non-adherent cells, such as one or more types of T cells. In one embodiment, a plurality of cells include Tregs. As can be appreciated, the cell growth chamber can be loaded by a system such as the above-described system CES 500 (e.g. Figure 5A )、CES 600( Figure 6 ) and CES 700( FIG. 7A to FIG. 7C ) is used to load a first volume of fluid with cells. Fig. 9B A portion of a 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 fluidically 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 with cells at 804 through the first fluid flow path 860 and allow it to enter the first fluid circulation path 852. 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 the fluid container 746A, the fluid container 746B, the IC fluid container 746, or a combination of these. For example, the first volume of fluid can be circulated through the IC circulation supply loop 753.

[0231] like Fig. 9BAs shown, the volume of first fluid circulation path 852 can be composed of multiple volumes of its parts. For example, the first part of the volume can be the capillary space of cell growth chamber 844 (when the cell growth chamber is a hollow fiber membrane bioreactor). The second part of the volume can be from connector 860B to inlet port 844A of cell growth chamber 844. The third part can be from connector 860B to outlet port 844B of cell growth chamber 844.

[0232] Process 800 proceeds to loading a second volume of fluid 806. The second volume of fluid may include culture medium and may be introduced into a portion of first fluid flow path 860. In embodiments, the second volume may be a predetermined amount selected to position 808 the first volume into a first portion of cell growth chamber 844. In embodiments, the first volume of fluid and the second volume of fluid may be the same. In other embodiments, the first volume of fluid and the second volume of fluid may be different. In yet other embodiments, the sum of the first volume of fluid and the second volume of fluid may be equal to the first fluid circulation path, e.g., path 852 ( Fig. 9B ) by volume.

[0233] The second volume of fluid may be provided at least in part from fluid container 768A, fluid container 768B, EC fluid container 768, or a combination thereof. For example, the second volume of fluid may be circulated through IC circulation supply loop 753. For example, the second volume of fluid may be provided from two or more connected fluid containers, as described with reference to FIG. 7A to FIG. 7C described.

[0234] In order to position the first volume of fluid, the second volume of fluid must be sufficient to push the first volume into the desired location in cell growth chamber 844. Thus, in embodiments, the second volume of fluid may be approximately as large as the volume of first fluid circulation path 852 between connector 860B and inlet port 844A. As can be appreciated, this will push the first volume of fluid with cells into position within cell growth chamber 844.

[0235] In other embodiments, the first volume of fluid (with cells) can be positioned 808 around the 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 intra-capillary space), which is not occupied by the first volume of fluid when positioned in 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 both sides of the central region 866. Therefore, 50% (or 37 mL) of the 74 mL can be added to the volume between the connector 860B and the inlet port 844A to position the 50 mL first volume around the central region 866.

[0236] In embodiments, positioning 818 the first volume may involve adding an additional volume of fluid to position the first volume with cells in the cell growth chamber. For example, if the desired position of the first volume is not achieved using the second volume, additional fluid may be added to position the first volume at 808.

[0237] Process 800 proceeds to query 810 to determine whether the first volume should be repositioned. For example, in an embodiment, the first volume may be positioned closer to inlet port 844A. If it is desired to move the first volume closer to central region 866 of cell growth chamber 844, process 800 may return to step 808 where additional fluid may be added to the first fluid circulation path to position the first volume of fluid.

[0238] If it is determined at query 810 that the first volume does not need to be repositioned, process 800 proceeds to feeding cells 812. In embodiments, a culture medium including a number of compounds (such as glucose, proteins, growth factors, reagents, or other nutrients) may be used to feed cells. In embodiments, feeding cells 812 may involve activating an inlet pump and a circulation pump (e.g., pump 840 and pump 848) to deliver culture medium with nutrients to cells in cell growth chamber 844. As described below, some embodiments provide for maintaining cells in cell growth chamber 844 during step 812. These embodiments may involve activating a pump, such as an inlet pump and a circulation pump (e.g., pump 840 and pump 848, described below), so that fluid entering cell growth chamber 844 can flow into cell growth chamber 844 from two directions, such as from inlet port 844A and outlet port 844B.

[0239] For example, as referenced FIG. 7A to FIG. 7CAs described above, culture medium can be delivered to cells from one or more connected fluid containers. Through IC circulation supply loop 753 or EC circulation supply loop 752, inlet pump and circulation pump can make culture medium circulate and recirculate from two directions, such as from inlet port 844A and outlet port 844B into cell growth chamber 844.

[0240] The process 800 then proceeds to expanding cells 814, where the cells can be expanded or grown. Although step 814 is shown after step 812, step 814 can occur before or simultaneously with step 812, depending on the embodiment. The cells can then be removed from the cell growth chamber 816 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 an end operation 830.

[0241] Next, Fig. 10A Example operational steps of a process 900 for maintaining cells in a cell expansion system, such as a CES 500 (e.g., Figure 5B and Figure 5C ) or CES 700 (for example, FIG. 7A to FIG. 7C ) in a bioreactor. As described above, cells residing in the manifold of the bioreactor or in the IC loop outside the bioreactor may not receive appropriate gas exchange and nutrient exchange, which may cause cell aggregation and death. In an embodiment, the bioreactor provides gas exchange and nutrient exchange through a semipermeable hollow fiber membrane. Importantly, such exchange is effective because in a cell expansion system including a hollow fiber membrane, the ratio of surface area to volume can be significantly greater than the ratio of surface area to volume of other cell culture methods (for example, about 15 times the ratio of surface area to volume of a cell culture bottle). Such efficiency can be achieved by minimizing the diffusion distance of the culture medium components that can pass through the membrane surface where exchange occurs.

[0242] For example, Fig. 10B The results show that during cell proliferation (approximately 3E+09 T cells present in the bioreactor) in a cell expansion system such as Cell Expansion System or Quantum A graph 936 of oxygen demand on a cell expansion system). Fig. 10BThe percentage (%) of oxygen (O2) at the bioreactor outlet 938 is depicted. For example, according to an embodiment, a sensor for measuring oxygen levels 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 of O2 is measured relative to the run time (e.g., in minutes) 940. 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 300mL / min(942). Fig. 10B The change in oxygenation is shown when the EC circulation rate is reduced from 300 mL / min (942) to 50 mL / min (944). For example, as the culture medium passes through the bioreactor, the cells can consume oxygen (O2) in the culture 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 of time or a greater opportunity to remove oxygen from the culture medium. Then, when the EC circulation rate is restored to 300 mL / min, oxygenation is restored (946). Fig. 10B The possible benefit of retaining cells in the fiber itself where gas transfer occurs is shown, rather than in a portion of the IC circulation path outside the bioreactor, for example, where the cells may be deoxygenated. Therefore, by directing the flow of culture medium into both sides of the bioreactor (e.g., the IC inlet port and the IC outlet port), it may be beneficial to maintain a population of cells (e.g., non-adherent cells) within the hollow fibers of the bioreactor during the feeding period. 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 larger flow rate can be used to flow to the IC inlet compared to the IC outlet port, or vice versa, depending on, for example, where the cells are desired to be positioned in the bioreactor.

[0243] Return to Fig. 10A , a start operation 902 is initiated, and the process 900 proceeds to loading 904 a disposable kit or pre-installed fluid delivery assembly (e.g., 210 or 400) into the cell expansion system. Figure 1C , Figure 4B and Figure 4C As shown, the disposable kit may include cell growth chamber 100A or cell growth chamber 100B. For example, the disposable kit may then be primed 906, wherein the Lonza Ca-free 2+ / Mg 2+PBS is used to prime the kit 906. In preparation for loading and seeding of cells, the priming fluid can be exchanged using IC / EC flush 908. For example, in an embodiment, the PBS in the system can be exchanged with TexMACS GMP basal medium.

[0244] For example, as referenced FIG. 7A to FIG. 7C As described, during priming, fluid can be delivered from one or more connected fluid containers through the disposable set. Fluid can be circulated and recirculated using a fluid container for additional volume in the fluid circuit.

[0245] Then, process 900 proceeds to closing the IC outlet valve 910. In an embodiment, the EC outlet valve may be opened to allow ultrafiltration of the fluid in the hollow fibers added to the bioreactor including the hollow fiber membrane. The culture medium may then be conditioned 912. Next, process 900 proceeds to converting cells 914, such as suspended or non-adherent cells (such as T cells or Tregs). In an embodiment, this may be loaded with cells 914 via a "load cells centrally without circulation" task. In another embodiment, such cells may be loaded 914 via a "load uniformly suspended cells" task. In other embodiments, other loading tasks and / or loading procedures may be used.

[0246] For example, in an embodiment, the loaded cells 914 may be suspended in a solution containing a culture medium to support the cells during and after such loading. In another embodiment, such a solution may include a culture medium for supporting the cells and a soluble activator complex for stimulating the cells (e.g., T cells). Such loading of 914 may occur, for example, on day 0.

[0247] After loading the cells 914, the cells 916 may be further fed. In an exemplary embodiment, as shown in FIG. FIG. 7A to FIG. 7CAs described, cells 916 can be fed by an EC circulation feeding loop 752, an IC circulation feeding loop 753, or a combination thereof. During such feeding 916, it is desirable to control the cell residence in the bioreactor itself. By adjusting the flow control parameters 918, cells can be kept in the bioreactor itself without, for example, losing cells in the portion of the IC loop from the bioreactor to the outside of the bioreactor during the expansion 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 a consumed or conditioned medium, which may affect their glycolytic metabolism. In addition, according to an embodiment, by 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 a gas transfer module (GTM), while the cells in other parts of the IC loop may not receive such a mixed gas. It should be noted that while embodiments may provide for retaining cells in the bioreactor itself, other embodiments may maintain cells in any location to allow for improved nutrient delivery and / or gas exchange. Thus, embodiments provide for the use of other locations to retain cells or control the residency of cells without departing from the spirit and scope of the present disclosure.

[0248] Return to Fig. 10A and process 900, according to embodiments, can reduce cell loss in a hollow fiber membrane bioreactor by matching, or approximately or substantially matching, an IC circulation pump rate to an IC inlet pump rate during a maintenance period, 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 counterflow rate or a second countervolume flow rate, where the volumetric flow rate or fluid flow rate or rate or flow rate of a fluid can be considered as the volume of fluid passing through per unit time (which can be represented by the symbol "Q"). In embodiments, for example, an IC inlet pump rate of +0.1 mL / min can be matched, or approximately or substantially matched, to a complementary IC circulation pump rate of -0.1 mL / min to maintain cells in the bioreactor during the growth phase of the cell culture (which may be days 4 to 7). Alternatively, for example, an IC inlet pump rate of 0.01 mL / min can be matched, or approximately or substantially matched, with a complementary IC circulation pump rate of -0.01 mL / min to maintain cells in the bioreactor during the growth phase of the cell culture, perhaps days 4 to 7. For example, such pump adjustments 918 can allow for the offsetting of any forces associated with cell loss from the IC outlet port of the bioreactor.

[0249] Next, the cells may be allowed to grow or expand 924. The cells are not limited to growing or expanding in step 924, but rather, the cells may also expand during, for example, steps 914, 916, 918, 920, 922. The process 900 may then proceed to a harvesting operation 926, where the cells may be transferred to a harvesting bag or container. The disposable kit may then be unloaded from the cell expansion system at 932, and the process 900 may then terminate at an end operation 934.

[0250] Alternatively, from the harvesting operation 926, the process 900 can optionally proceed to allow for 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, the process 900 can optionally proceed to reloading any remaining cells 930. The process 900 can then proceed to unloading the disposable set 932, and then the process 900 can terminate at an end operation 934. Alternatively, the process 900 can proceed from the further processing / analysis step 928 to unloading the disposable set 932. Then, the process 900 can terminate at an end operation 934.

[0251] Next, FIG. 11A shows exemplary operating steps of a process 1000 for feeding cells according to an embodiment of the present disclosure, which operating steps can be used with a cell expansion system, such as CES 700 (e.g., FIG. 7A to FIG. 7C For example, start operation 1002 is initiated, and process 1000 proceeds to loading the disposable kit onto the cell expansion system, priming the kit, performing IC / EC flushing, conditioning the medium, and loading cells (e.g., suspension or non-adherent cells). 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 1000 proceeds to feeding cells 1004 during a first time period. In an exemplary embodiment, as shown in reference FIG. 7A to FIG. 7CAs described, cells 916 are fed by an EC circulation feeding loop 752, an IC circulation feeding 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, wherein 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 allow a volumetric flow rate of 0.1 mL / min to enter the IC inlet port (701A) of the bioreactor (701), wherein the IC circulation pump (712) allows a complementary IC circulation volumetric flow rate or fluid flow rate of -0.1 mL / min to enter the IC outlet port (701B) of the bioreactor (701), wherein, for example, the minus sign ("-") used in -0.1 mL / min indicates the direction of the IC circulation pump (712) to cause or generate a countercurrent flow rate to maintain the cells in the bioreactor during the growth phase of the cell culture.

[0252] During the period of feeding cells and during the period of feeding, IC pumps are used to control the residence of cells in the bioreactor by flow characteristics and countercurrent characteristics, and the cells continue to grow and expand. Therefore, cells may require additional culture medium, such as glucose and / or cell growth prepared culture medium, to support the group that is constantly expanding. In an exemplary embodiment, multiple fluid containers 768A and 768B can be connected to provide culture medium for circulation and recirculation through the system, thereby preventing the need for controlling lactic acid value. In an alternative embodiment, the lactic acid value of the cell group expanded can be controlled with effort. In an embodiment, the cell culture lactic acid value can be maintained at or below about 20mmol / L, at or below about 15mmol / L, at or below about 10mmol / L or even at or below about 7mmol / L. In other embodiments, for example, the culture medium addition rate and / or other settings can be controlled to try to keep lactic acid level ≤ about 5mmol / L to, for example, improve cell growth and vigor. In other embodiments, other concentrations can also be used.

[0253] In an exemplary embodiment, because fluid containers 768A and 768B or fluid containers 746A and 746B are connected, it is not necessary to control the pump rate to control the lactate value. In an alternative exemplary embodiment, according to an embodiment, 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 multiple time periods, such as several days (days 4-8), efforts can be made to control the lactate value to ≤ about 7 mmol / L. For example, Fig. 11BA table 1018 of example IC pumping rates is provided for feeding using a "feed cells" task in, for example, a cell expansion system (e.g., CES 500). Table 1018 provides example time periods 1020 (e.g., days) relative to example IC pump rates 1022 to generate volumetric flow rates to both sides of the bioreactor to maintain the cells in the bioreactor. For example, days 0-4 (1024) may use an IC inlet or input pump rate (1026) of 0.1 mL / min and an IC circulation pump rate (1028) of -0.1 mL / min; day 5 (1030) may use an IC inlet pump rate (1032) of 0.2 mL / min and an IC circulation pump rate (1034) of -0.2 mL / min; day 6 (1036) may use an IC inlet pump rate (1038) of 0.3 mL / min and an IC circulation pump rate (1040) of -0.3 mL / min; and day 7 (1042) may use an IC inlet pump rate (1044) of 0.4 mL / min and an IC circulation pump rate (1046) of -0.4 mL / min. Although Fig. 10B Table 1018 provides example pump rates of ±0.1 to ±0.4 mL / min for feeding cells while maintaining the cells in a bioreactor during the growth phase of the cell culture, but other pump rates and corresponding flow rates may be used in accordance with embodiments without departing from the spirit and scope of the present disclosure. For example, while increments of ±0.1 mL / min for increasing the feed flow rate are 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. Fig. 11B The time periods (e.g., days) and pump rates in Table 1018 are provided for illustrative purposes only and are not intended to be limiting. For example, a cell growth chamber 100B may be provided with Fig. 11B The time periods and pump rates in Table 1018 are shown. Fig. 11B The pump rate in table 1018 may be reduced to 10% of the pump rate of cell growth chamber 100A.

[0254] Return to Fig.11A , during the first time period 1004, the process 1000 proceeds from cell feeding to increasing the first inlet flow rate by a first amount to achieve a second inlet flow rate 1006. For example, as described above, as described with reference to Fig. 11BIn the embodiment depicted in , 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. In addition, the IC circulation pump rate (-) can be simultaneously 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). Therefore, 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 the second inlet flow rate and the second circulation flow rate during the second time period to maintain cells outside the bioreactor and outside the manifold and the portion of the IC circulation path outside the bioreactor. For example, after the feeding 1010 for the second time period, if it is not desired to continue to feed and / or expand the cells, the process 1000 can terminate at the end operation 1016. Alternatively, the process 1000 may optionally continue to increase or otherwise change the feed flow rate 1012. There may be any number of feed time periods, as indicated by ellipses 1014. After the desired number of feed time periods 1014, the process 1000 then terminates at an end operation 1016. Although Fig. 10A and Fig. 10B And process 1000 shows an "increase" in flow rate, but other adjustments to flow rate can be made. For example, flow rate can decrease or remain substantially the same from one feeding segment to the next. Consideration of factors, such as metabolic activity, for example, can determine how to adjust flow rate. Fig. 10A and Fig. 10B The “increase” in flow rate is provided for illustrative purposes only and is not intended to be limiting.

[0255] Go to Fig. 12A , provides example operating steps for a process 1100 for feeding cells according to embodiments of the present disclosure, which operating steps can be used with a cell expansion system, such as CES 500 (e.g., Figure 5B and Figure 5C). For example, a start operation 1102 is initiated, and the process 1100 proceeds to loading the disposable kit onto the cell expansion system, priming the kit, performing an IC / EC flush, conditioning the medium, and loading cells (e.g., suspension or non-adherent cells). Next, the process 1100 proceeds to feeding the cells during a first time period 1104. In an embodiment, a first inlet rate or flow rate and a first circulation rate or flow rate can be used. As an example, a first IC inlet flow rate and a first IC circulation flow rate can be used, wherein the first IC inlet flow rate can be controlled by an IC inlet pump (e.g., a first pump), and the first IC circulation flow rate can be controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, the IC inlet pump (554) may provide a volumetric flow rate or fluid flow rate of 0.1 mL / min into the IC inlet port (501A) of the bioreactor (501), and the IC circulation pump (512) provides a complementary IC circulation flow rate of -0.1 mL / min into the IC outlet port (501B) of the bioreactor (501), where, for example, the minus sign ("-") used in -0.1 mL / min indicates the direction of the IC circulation pump (512) to cause or generate a counter-flow rate to maintain the cells in the bioreactor during the growth phase of the cell culture. For example, the flow rates described may be applicable to the cell growth chamber 100B. The flow rates described may be reduced to 10% of the cell growth chamber 100A.

[0256] During the period of feeding the cells and using the IC pump to control the residence of the cells in the bioreactor through flow characteristics and countercurrent characteristics, the cells continue to grow and expand. Therefore, the cells may require additional culture medium, such as glucose and / or culture medium formulated for cell growth, to support the continuously expanding population. In an exemplary embodiment, multiple fluid containers 768A and 768B can be connected to provide culture medium for circulation and recycling through the system, thereby preventing the need to control lactate values. In alternative embodiments, efforts can 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 within 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), efforts can be made to control the lactate value to ≤ about 7 mmol / L. For example, see Fig. 11B , Table 1018 and the discussion above, such as the pump rate of the supply. As mentioned above, although Fig. 11B Table 1018 provides pump rates of ±0.1 to ±0.4 mL / min for maintaining cells in a bioreactor while feeding the cells during the growth phase of the cell culture, but other pump rates and corresponding flow rates may be used depending on the embodiment without departing from the spirit and scope of the present disclosure. Fig. 11BThe time periods (e.g., days) and pump rates in Table 1018 are provided for illustrative purposes only and are not intended to be limiting. For example, a cell growth chamber 100B may be provided with Fig. 11B The time periods and pump rates in Table 1018 are shown. Fig. 11B The pump rate in table 1018 may be reduced to 10% of the pump rate of the cell growth chamber 100A.

[0257] Return to Fig. 12A , during a 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, as described with reference to Fig. 11B In the embodiment depicted in , 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. In addition, the IC circulation pump rate (-) can be simultaneously 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) Therefore, 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 the portion of the IC circulation path outside the manifold or outside the bioreactor. For example, after the feeding 1110 for the second time period, if it is not desired to continue to feed and / or expand the cells, the process 1100 can be terminated at the end operation 1116.

[0258] Alternatively, process 1100 can optionally determine whether to adjust a feed rate or flow rate based on metabolic activity, wherein process 1100 proceeds to optional query 1112 to determine whether to adjust feed based on metabolic levels. For example, monitoring glucose and / or lactate levels can help adjust a cell expansion system medium flow rate, such as an IC medium flow rate, to support expansion of cells (e.g., Tregs) in, for example, a bioreactor (such as a hollow fiber bioreactor).

[0259] like Fig. 12B and Fig. 12C As shown, embodiments provide for controlling lactate values ​​for cell expansion runs or procedures, such as involving expansion of hTregs. Graphs 1118 and 1132 show that, for example, measuring glucose and lactate levels can be used to adjust cell expansion system medium flow rates, such as IC medium flow rates, to support, for example, expansion of Tregs. For example, Fig. 12BA curve 1118 is provided showing the metabolism of expanded hTregs, where such cell expansion may occur in a cell expansion system such as Cell expansion system). Glucose concentration (mg / dL) 1120 and lactate concentration (mmol / L) 1122 of various cell expansion runs 1124 and 1125 across time periods (e.g., days 1126) are shown. Throughout these Treg runs 1124 and 1125, for example, on days 4-8, efforts can be made to control the lactate value of the expanded cell population to a value of ≤ about 7mmol / L, by synchronously increasing the IC inlet (+) pump rate and the IC circulation pumping rate from ± 0.1 to ± 0.4mL / min, for example, in the lumen of a hollow fiber membrane. In other embodiments, other pump rates can be used. As shown, according to the embodiment shown, the minimum glucose level during Treg cell expansion can be within the range of a concentration 1128 of 264mg / dL on the 7th day (Q1584) to a concentration 1130 of 279mg / dL on the 8th day (Q1558). As depicted, the basal glucose concentration 1124 in the medium formulated for cell growth for a run may range, for example, from 325 mg / dL to 335 mg / dL. In other embodiments, it may be desirable to maintain lactate levels ≤ about 5 mmol / L to improve cell growth and viability. In embodiments, a graphical user interface (GUI) element may be used to control the rate of medium addition and maintain lactate metabolic waste from glycolysis below a defined level during cell expansion.

[0260] Go to Fig. 12C , graph 1132 shows the metabolism of expanded hTregs, where such cell expansion may occur in a cell expansion system such as Cell expansion system). Glucose consumption (mmol / day) 1134 and lactate production (mmol / day) 1136 for various cell expansion runs 1138 and 1139 across a time period 1140 (e.g., days) are shown. In order to control the lactate value 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 IC circulation and IC feeding rates (1142) of ±0.1 mL / min; ±0.2 mL / min (1144); ±0.3 mL / min (1146); and ±0.4 mL / min (1148). Other embodiments may use other flow rates. Fig. 12B and Fig. 12CThe flow rates used and shown in are provided for the purpose of illustration and are not intended to be limiting. It should be noted that while positive (+) may be shown for the direction of the IC inlet pump and negative (-) may be shown for the direction of the IC circulation pump, such directions are provided for illustration purposes only, wherein such directions depend on the configuration of the pumps used.

[0261] return Fig. 12A and optional query 1112, if it is not desired to measure metabolic activity and / or adjust the supply level based on such measurement, then process 1100 proceeds from "no" to end operation 1116, and process 1100 terminates. For example, in the case of using multiple supply containers, as shown in reference FIG. 7A to FIG. 7C As described, the culture medium may be circulated and recirculated, thereby preventing the need to measure metabolic activity and / or adjust feed levels. Alternatively, in the event that it is desired to adjust feed levels based on metabolic activity, process 1100 proceeds from "yes" to optional step 1114 to continue to increase the rate of culture medium addition and feed the expanded cell population. For example, while step 1114 is shown as one step, the step may involve multiple adjustments to the culture medium addition rate, such as increasing the IC inlet flow rate and increasing the IC circulation flow rate. Step 1114 is shown as one step for illustrative purposes only and is not intended to be limiting. After any adjustments are made to the culture medium addition rate, process 1100 proceeds to optional query 1112 to determine whether to continue measuring metabolic levels and / or adjusting feed. If it is not desired to continue measuring metabolic activity and / or adjusting feed levels based on metabolic activity, process 1100 proceeds to "no" to end operation 1116, and process 1100 terminates. Although Fig.11A The process 1100 shows an "increase" in the rate or flow rate, but other adjustments to the flow rate can be made. 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. For example, Fig.11A The “increase” in flow rate is provided for illustrative purposes only and is not intended to be limiting.

[0262] Next, Fig.13 Example operational steps of process 1200 according to an embodiment of the present disclosure are shown for using a cell expansion system, such as CES 500 (e.g. Figure 5B and Figure 5C ) or CES700 (e.g. FIG. 7A to FIG. 7C ) during the maintenance period, the cells are kept in one location. For example, the start operation 1202 is initiated, and the process 1200 proceeds to loading the disposable kit onto the cell expansion system, priming the kit, performing IC / EC flushing, conditioning the medium, and loading the cells (e.g., suspension or non-adherent cells). 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. Next, the 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. As an example, a first IC inlet flow rate and a first IC circulation flow rate may be used, wherein 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, the IC inlet pump (554) may allow a volumetric flow rate of 0.1 mL / min to enter the IC inlet port (501A) of the bioreactor, and the IC circulation pump (512) allows a complementary IC circulation volumetric flow rate or fluid flow rate of -0.1 mL / min to enter the IC outlet port (501B) of the bioreactor (501), wherein, for example, the minus sign ("-") used in -0.1 mL / min indicates the direction of the IC circulation pump to cause or generate 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 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 first IC inlet flow rate, or another percentage or fraction depending on the embodiment. Alternatively, in an exemplary embodiment, the first IC inlet pump can allow 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 allows 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 minus sign ("-") used in -0.01 mL / min indicates the direction of the IC circulation pump to cause or generate a countercurrent rate to maintain cells in the bioreactor during the growth phase of the cell culture.

[0263] For example, during the period of feeding (and expanding) the cells and using the IC pump to control the residence of the cells in the bioreactor through flow characteristics and countercurrent characteristics, the cells continue to grow and expand. Therefore, the cells may require additional culture medium, such as glucose and / or culture medium formulated for cell growth, to support the continuously expanding population. In an embodiment, efforts may be made to increase the rate at which culture medium is added to feed the continuously 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.

[0264] In an exemplary embodiment, as shown in reference FIG. 7A to FIG. 7C As described, cells 916 may be supported by EC circulation support circuit 752, IC circulation support circuit 753, or a combination thereof.

[0265] Next, according to an embodiment, the second IC circulation flow rate can be set or adjusted or configured to be equal to a percentage, part or fraction of the second IC inlet 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 about fifty percent (50%) or about one-half (1 / 2) of the IC inlet flow rate value, or according to an embodiment, it 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 the second IC circulation flow rate to increase. In another embodiment, the IC circulation pump rate can be adjusted to produce or cause the second IC circulation flow rate to decrease, so that the IC circulation flow rate can be substantially equal to a predetermined percentage or predetermined fraction of the second IC inlet flow rate. In yet another embodiment, the IC circulation pump rate may not be adjusted. For example, when 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 is increased to 0.2 mL / min, the second IC circulation flow rate can be set to (-1 / 2)*(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 may not be adjusted to achieve such a second Q IC循环 .

[0266] Go to Figure 5B and Figure 5C , these figures depict an operational configuration of a cell expansion system 500 showing fluid movement in a first circulation path, according to an embodiment of the present disclosure. Figure 5B and Figure 5CThe configuration shows, for example, a split in the flow rate to an IC inlet port (e.g., a first port) and to an IC outlet port (e.g., a second port) to keep cells in a cell growth chamber or bioreactor. As discussed above with respect to CES 500, in the IC loop or first circulation path 502, the fluid can be initially propelled by an IC inlet pump 554. Such a fluid can, for example, advance in a first direction, such as in a 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 leave via the IC outlet port 501B. The culture medium can flow through the IC circulation pump 512, which can be used to control the rate at which the culture medium flows. 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 a reverse inlet. 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 positive (+) and the direction of the IC circulation pump can be negative (-) to cause reverse flow so that fluid can enter both sides of the bioreactor to keep cells in the bioreactor.

[0267] In embodiments, a first portion of the fluid may be branched at connector 517 to flow into the IC inlet port (501A) of the bioreactor 501. In embodiments, the IC circulation pump 512 may be operated at a pump rate that matches, or approximately or substantially matches, the rate of the IC inlet pump 554 but in the opposite direction, such that a second portion of the fluid may be branched at 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 be matched, or approximately or substantially matched, with a complementary IC circulation pump rate of -0.1 mL / min to maintain cells in the bioreactor during the growth phase of cell culture. Alternatively, an IC inlet pump rate of less than +0.1 mL / min or about +0.01 mL / min may be matched, or approximately or substantially matched, with a complementary IC circulation pump rate of less than -0.1 mL / min or about -0.01 mL / min to maintain cells in the bioreactor during the growth phase of cell culture. In addition, in additional embodiments, as described herein, during the growth phase of the culture in the capillary, cell harvest synchronization can be performed subsequently. Such a pump adjustment strategy during the feeding period can offset the forces associated with cell loss from the IC outlet port. According to an embodiment, this type of feeding using pump adjustment to allow the IC inlet port (501A) and IC outlet port (501B) to enter the bioreactor in flow and countercurrent, respectively, can be referred to as an improved feeding method. In another exemplary embodiment, the IC circulation pump rate can be adjusted so that the flow rate entering the IC outlet port (501B) can be equal to about fifty percent (50%) or about one-half (1 / 2) of the IC inlet flow rate, or in other embodiments, it can 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 can be set, configured or adjusted to about -0.2 mL / min. In other embodiments, other percentages or fractions can be used.

[0268] According to an embodiment, Figure 5B and Figure 5C 5 shows an operational configuration showing the movement of fluids in CES 500, wherein flow rates and counterflow rates are shown to maintain cells in a cell growth chamber or bioreactor 501. According to an embodiment, for example, such flow rates are Figure 5B 503; "(-1 / 2)X" flow rate 505 (where the minus sign ("-") is an indication of direction, where the direction of flow rate 505 is indicated by Figure 5B501); and a "(1 / 2)X" flow rate 507, wherein approximately 1 / 2 or a first portion of the flow rate branches off at connector 517 to enter the IC inlet port (501A) of the bioreactor 501, and approximately 1 / 2 or a second portion of the flow rate branches off at connector 517 to enter the IC outlet port (501B) of the bioreactor 501. As shown, the sum of the first portion and the second portion can be substantially equal to the total flow rate 503, wherein the total flow rate 503 is pumped by the IC inlet pump 554. Other types of fractions or percentages of the IC inlet pump rate may be used to set, configure, or adjust the IC circulation pump, depending on the flow rate and countercurrent rate that may be used to maintain cells in the bioreactor or cell growth chamber 501. Thus, Figure 5C Such flow rates are shown as "X" flow rate 511; "-(y%)*X" flow rate 513 (where the minus sign ("-") is an indication of direction, where the direction of flow rate 513 is indicated by Figure 5C and "(100%-y%)*X" flow rate 515, where "y" equals a numerical percentage, according to an embodiment. In an embodiment, the sum of flow rate 513 and flow rate 515 is substantially equal to flow rate 511.

[0269] 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 portion 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 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 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 about 50%. It should be understood that, Figure 5B and Figure 5C The operating configurations shown in are representative of possible configurations for various operations of the cell expansion system, and modifications to the configurations shown may be made within the scope of one or more of the present embodiments.

[0270] return Fig.13, at 1210, the cells may be maintained (and continue to be expanded) at a second inlet flow rate and a second circulation flow rate during a second time period to maintain the cells in the bioreactor and outside the manifold and in a portion of the IC circulation path outside the bioreactor. For example, after the second time period of maintenance 1210, if it is not desired to continue to maintain and / or expand the cells, the process 1200 may terminate at an end operation 1216. Alternatively, the process 1200 may optionally continue to increase or otherwise change the supply flow rate 1212. There may be any number of supply time periods, as indicated by the ellipsis 1214. After the desired number of supply time periods 1214, the process 1200 may then terminate at an end operation 1216. Although Fig.13 And process 1200 shows an "increase" in flow rate, but other adjustments to flow rate can be made. For example, flow rate can decrease from one feeding segment to the next or remain substantially the same. Considerations, such as metabolic activity, for example, can determine how to adjust flow rate. Fig.13 The “increase” in flow rate is provided for illustrative purposes only and is not intended to be limiting.

[0271] Go to Fig.14 , provides example operating steps of a process 1300 according to an embodiment of the present disclosure for feeding cells while maintaining the cells in a first location, such as a bioreactor, which can be connected to a cell expansion system such as CES500 (e.g., Figure 5B and Figure 5C ) or CES700 (for example, FIG. 7A to FIG. 7C). For example, a start operation 1302 is initiated, and the process 1300 proceeds to loading the disposable kit onto the cell expansion system, priming the kit, performing an IC / EC flush, conditioning the medium, and loading cells (e.g., suspension or non-adherent cells). Next, the process 1300 proceeds to feeding the cells during a first time period 1304. 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, wherein 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, a first IC inlet pump rate may result in a volumetric flow rate of 0.1 mL / min into the IC inlet port of the bioreactor, wherein a complementary IC circulation pump rate of -0.1 mL / min results in a volumetric flow rate of -0.1 mL / min into the IC outlet port of the bioreactor, wherein, for example, the minus sign ("-") used in -0.1 mL / min indicates the direction of the IC circulation pump (512) to induce or generate a countercurrent rate to maintain cells in the bioreactor during the growth phase of the cell culture. Alternatively, in an exemplary embodiment, the first IC inlet pump may provide a volumetric flow rate of less than 0.1 mL / min or about 0.01 mL / min into the IC inlet port (701A), and the IC circulation pump provides a complementary IC circulation volumetric flow rate or fluid flow rate of less than -0.1 mL / min or about -0.01 mL / min into the IC outlet port (701B), where, for example, the minus sign ("-") used in -0.01 mL / min indicates the direction of the IC circulation pump to cause or generate a counterflow 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 another percentage or fraction depending on the embodiment.

[0272] During the period of feeding (and expanding) the cells and using the IC pump to control the residence of the cells in the bioreactor through flow characteristics and countercurrent characteristics, the cells continue to grow and expand. Therefore, the cells may require additional culture medium, such as glucose and / or culture medium formulated for cell growth, to support the continuously expanding population. In an embodiment, efforts may be made to increase the rate at which culture medium is added to feed the continuously 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 1306. 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.

[0273] Next, depending on the 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, depending on the embodiment, the second IC circulation flow rate can be set or configured or adjusted to be equal to about fifty percent (50%) or about one-half (1 / 2) of the IC inlet flow rate value, or depending on the embodiment, it 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 embodiments, the percentage of the IC inlet flow rate of the IC circulation flow rate may be set in the range of about 0% to about 100%. In other embodiments, the percentage may be between about 25% to about 75%. In other embodiments, the percentage may be between about 40% to about 60%. In other embodiments, the percentage may be between about 45% to about 55%. In embodiments, the percentage may be about 50%.

[0274] Depending on the value of the first IC circulation flow rate, according to an embodiment, an adjustment of the IC circulation pump rate (-) may cause an increase in the second IC circulation flow rate. In another embodiment, the IC circulation pump rate may be adjusted to reduce the second IC circulation flow rate so that the second IC circulation flow rate may 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 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 circulation flow rate is equal to -0.1 mL / min, if the second IC inlet flow rate is increased to 0.2 mL / min, the second IC circulation flow rate may be set to (-1 / 2)*(second IC inlet flow rate). 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 circulation pump rate may not be adjusted to achieve such a second Q IC循环 .

[0275] Then, the cells 1310 can be maintained (and continued to be expanded) at a second IC inlet flow rate and a second IC circulation flow rate during a second time period to maintain the cells in the bioreactor and outside the manifold and the portion of the IC circulation path outside the bioreactor. For example, after the second time period of maintenance 1310, if it is not desired to continue to maintain and / or expand the cells, the process 1300 can terminate at an end operation 1316.

[0276] In embodiments, the first time period, the second time period, the third time period, the fourth time period, the fifth time period, etc. may each include one or more days (and / or hours and / or minutes). For example, depending on the embodiment, the time period may 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 embodiments, the first time period for feeding may include day 0, day 1, day 2, day 3, day 4; the second time period for feeding may include day 5; the third time period for feeding may include day 6; and the fourth time period for feeding may include day 7. In another embodiment, the first time period may include day 0, day 1, and day 2 (e.g., for a duration of about 3 days); the second time period may include day 3, day 4, and day 5 (e.g., for a duration of about 3 days); the third time period may include day 6, day 7, and day 8 (e.g., for a duration of about 3 days); the fourth time period may include day 9 and day 10 (e.g., for a duration of about 2 days); and the fifth time period may include day 11, day 12, and day 13 (e.g., for a duration of about 3 days). Depending on the embodiment, the time periods may be of different durations. Each time period may be measured in days, hours, minutes, and / or fractions thereof.

[0277] Return to Fig.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 lactic acid value of the expanded cell group to a value of ≤ about 7mmol / L. In the case where it is desired to maintain the cell culture lactic acid value at about 7mmol / L or less, it is possible, for example, to control the rate of culture medium addition during cell (e.g., regulatory T cells) expansion. In other embodiments, it may be desirable to maintain lactic acid levels ≤ about 5mmol / L to improve cell growth and viability. In an embodiment, a graphical user interface (GUI) element may be used to control the rate of culture medium addition, and lactic acid metabolic waste from glycolysis is maintained below a predetermined level during cell expansion.

[0278] In optional query 1312, if it is not desired to measure metabolic activity and / or adjust feed levels based on such measurements, process 1300 proceeds from "No" to end operation 1316 and process 1300 terminates. Alternatively, in the event that it is desired to adjust feed levels 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, the step may involve multiple adjustments to the media addition rate, such as increasing the IC inlet flow rate and increasing the IC circulation flow rate. Step 1314 is shown as one step for illustrative purposes only and is not intended to be limiting. After any adjustments to the media addition rate are made, process 1300 returns to optional query 1312 to determine whether to continue measuring metabolic levels and / or adjusting feed. If it is not desired to adjust feed levels based on metabolic activity, process 1300 proceeds from "No" to end operation 1316 and process 1300 terminates. Although Fig.13 The process 1300 shows an "increase" in flow rate, but other adjustments to the flow rate can be made. For example, the rate or flow rate can be reduced from one feeding segment to the next or remain substantially the same. The type of adjustment that can be made can depend on the assessment of the metabolic activity of the growing cell population. Fig.13 The “increase” in flow rate is provided for illustrative purposes only and is not intended to be limiting.

[0279] Next, Fig.15 1400 for retaining cells during maintenance according to an embodiment of the present disclosure, which can be used with a cell expansion system such as the CES 500 ( Figure 5B and Figure 5C ) or CES 700( FIG. 7A to FIG. 7C) is used together. Start operation 1402 is initiated, and process 1400 proceeds to loading 1404 a disposable tubing set or a pre-installed fluid delivery assembly (e.g., 210 or 400) into a cell expansion system. Figure 1C , Figure 4B and Figure 4C As shown, the disposable kit may include cell growth chamber 100A or cell growth chamber 100B. Next, the system may be primed 1406. For example, in an embodiment, a user or operator may provide instructions to the system to prime, for example, by, for example, selecting a task for primed. For example, in an embodiment, such a task for primed may be a preprogrammed task. For example, next, an IC / EC flush task 1408 may be performed, in which fluids on the IC circulation loop and the EC circulation loop may be replaced. According to an embodiment, the replacement volume may be determined by the amount of IC volume and EC volume exchanged.

[0280] Next, in order to maintain an appropriate or desired gas concentration in the fibers of the bioreactor membrane, an adjust medium task 1410 can be performed to allow the medium to reach equilibrium 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 the gas transfer module (GTM) or oxygenator can be provided. The system can then be maintained in an appropriate or desired state until the user or operator is ready to load the cells into the bioreactor, for example. For example, in an embodiment, the system can be adjusted with a culture medium (such as a complete culture medium). The complete culture medium can be any culture medium for cell growth. For example, in an embodiment, the system can be adjusted with a serum-free culture medium. In an embodiment, the system can be adjusted with a basal culture medium. Any type of culture medium understood by those skilled in the art can be used.

[0281] Process 1400 then proceeds to loading cells 1412 from, for example, a cell entry bag to a bioreactor. In an embodiment, the cells in the cell entry bag may be in a solution, for example, with a culture medium to feed cells 1414. In another embodiment, the cells in the cell entry bag may be in a solution having both a culture medium for feeding cells 1414 and a soluble activator complex for stimulating cells (e.g., T cells or Tregs). In an embodiment, cells (and a feeding solution, in an embodiment) may be loaded into a bioreactor from a cell entry bag until the bag becomes empty. Cells (and a feeding solution, in an embodiment) may be chased from an air removal chamber to a bioreactor. In an embodiment, a "cells uniformly suspended" task may be performed to load cells (and a feeding solution, in an embodiment). In another embodiment, a "cells centrally loaded without circulation" task may be performed to load cells (and a feeding solution, in an embodiment) into a specific area (e.g., a central area) of a bioreactor. According to an embodiment, other loading methods and / or loading tasks may be used.

[0282] Next, process 1400 proceeds to query 1416 to determine whether to use improved feeding methods to retain cells, for example, non-adherent or suspended cells, such as T cells or Tregs in a bioreactor (e.g., a hollow fiber bioreactor). For example, it may be desirable to position cells in the bioreactor itself and outside the rest of the manifold or IC loop of the bioreactor. For example, if it is not desirable to use a modified feeding method to retain cells in the bioreactor itself, process 1400 proceeds from "no" to amplification cells 1426, where cells can continue to grow / amplify using the culture medium that initially supported them in step 1414.

[0283] On the other hand, if it is desired to maintain the cells in the bioreactor itself, the process 1400 proceeds from "yes" to modified feeding 1418, where the cells can be fed using the flow rate of the IC inlet (501A) entering the bioreactor (501) and the flow rate of the IC outlet (501B) entering the bioreactor (501) to maintain the cells in the bioreactor. In doing so, an inlet volumetric flow rate or inlet flow rate can be introduced into the first fluid flow path (506) 1420. For example, an IC inlet flow rate can be introduced into the first fluid flow path (506) 1420. The IC inlet pump (554), such as a first peristaltic pump (in an embodiment), can be operated at a predetermined number of revolutions per minute (RPM) so that the fluid in the first fluid flow path (506) has a predetermined IC inlet volumetric flow rate or IC inlet flow rate 1420. According to an embodiment, the 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 modified first flow rate, or a first portion of the IC inlet flow rate, can enter the IC inlet port (501A) or the first port 1422 of the bioreactor (501). The pump rate of the pump can depend on the diameter of the pump or the configuration of the pump (e.g., a peristaltic pump). Other types of pumps can also be used, where the pump 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 be operated at a predetermined number of RPMs and in a direction opposite to the direction of the first pump to cause or generate 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 1424 of the bioreactor (501). For example, embodiments may provide: about half (about 1 / 2) or a first portion of the IC inlet flow rate to branch at connector 517 to enter the IC inlet port (501A) of the bioreactor 501; and about half (or 1 / 2) or a second portion of the IC inlet flow rate to branch at connector 517 to enter the IC outlet port (501B) of the bioreactor 501. As shown, the sum of the first portion and the second portion may be substantially equal to the IC inlet flow rate 503 (e.g., Figure 5B ), where IC inlet flow rate 503 may be pumped by IC inlet pump 554. Depending on the flow rate and counterflow rate that may be used to maintain cells in bioreactor or cell growth chamber 501, other types of fractions or percentages of the IC inlet pump rate may be used to set, configure or adjust the IC circulation pump, according to embodiments.

[0284] In an exemplary embodiment, as shown in reference FIG. 7A to FIG. 7C As described, cells 916 may be supported by EC circulation support circuit 752, IC circulation support circuit 753, or a combination thereof.

[0285] After feeding the cells using such flow characteristics and counter-flow characteristics to maintain the cells in the bioreactor, the process 1400 proceeds to growing / expanding the cells 1426. For example, although cell expansion is shown at step 1426, the cells may also be grown / expanded during one or more other steps (such as 1412, 1414, 1416, 1418, 1420, 1422, 1424). From the expansion step 1426, the process 1400 proceeds to harvesting or removing the cells 1430. The process 1400 may then terminate at an end operation 1432. According to an embodiment, if any other steps are desired before harvesting, such as continuing to use a second improved feeding method or other type of feeding method, the process 1400 proceeds to an optional "other" step 1428. From the optional step 1428, the process 1400 proceeds to harvesting or removing the cells 1430 from the bioreactor, and the process 1400 may then terminate at an end operation 1432.

[0286] Go to Fig.16A , provides example operating steps of a process 1500 for feeding cells to maintain the cells in a first location, such as a bioreactor, which can be connected to a cell expansion system such as CES 500 (e.g., Figure 5B and Figure 5C ) or CES 700( FIG. 7A to FIG. 7C ) is used together. Start operation 1502 is initiated, and process 1500 proceeds to loading 1504 a disposable tubing set or a pre-installed fluid delivery assembly (e.g., 210 or 400) into a cell expansion system. Figure 1C , Figure 4B and Figure 4C As shown, the disposable kit may include cell growth chamber 100A or cell growth chamber 100B. Next, the system may be primed 1506. For example, in an embodiment, a user or operator may provide instructions to the system to prime, for example, by selecting a task for prime filling. In an embodiment, such a task for prime filling may be a preprogrammed task. Next, an IC / EC flush task 1508 may be performed, in which, for example, fluids on the IC circulation loop and the EC circulation loop may be replaced. According to an embodiment, the replacement volume may be determined by the amount of IC volume and EC volume exchanged.

[0287] Next, in order to maintain an appropriate or desired gas concentration on the fibers in the bioreactor membrane, an adjust medium task 1510 can be performed to allow the medium to reach equilibrium 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 the gas transfer module (GTM) or oxygenator can be provided. The system can then be maintained in an appropriate or desired state until the user or operator, for example, is ready to load the cells into the bioreactor. For example, in an embodiment, the system can be adjusted with a culture medium (such as a complete culture medium). The complete culture medium can be any culture medium used for cell growth. For example, in an embodiment, the system can be adjusted with a serum-free culture medium. In an embodiment, the system can be adjusted with a basal culture medium. Any type of culture medium understood by those skilled in the art can be used.

[0288] Process 1500 then proceeds to loading 1512 cells from, for example, a cell entry bag to a bioreactor. In an embodiment, the cells in the cell entry bag may be in a solution, for example, with a culture medium to feed the cells. In another embodiment, the cells in the cell entry bag may be in a solution having both a culture medium and a soluble activator complex for stimulating cells (e.g., T cells or Tregs). In an embodiment, cells may be loaded from the cell entry bag into the bioreactor until the bag becomes empty. Cells may be chased from the air removal chamber to the bioreactor. In an embodiment, a "loading uniformly suspended cells" task may be performed to load cells. In another embodiment, a "non-circulating centralized loading of cells" task may be performed to load cells into a specific area (e.g., a central area) of the bioreactor. According to an embodiment, other loading methods and / or loading tasks may 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 shown in reference FIG. 7A to FIG. 7CAs described, cells 916 are fed by the EC circulation feeding circuit 752, the IC circulation feeding circuit 753, or a combination thereof. In embodiments, cells may be fed at, for example, a minimum or low feeding rate, where a cell population begins to grow / expand, and the minimum or low feeding rate is able to meet the feeding needs of such a population. For example, during such a first time period, an IC inlet pump rate of +0.1 mL / min may be used to cause or generate a first fluid flow rate of 0.1 mL / min. According to embodiments, although this example provides a low or minimum feeding rate of 0.1 mL / min, the low or minimum feeding rate may 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 minimum feeding rate may be greater than 0.1 mL / min. If it is desired to reduce cell loss from the hollow fiber membrane bioreactor during such a first time period, the IC inlet pump rate of +0.1 mL / min can be matched, or approximately or substantially matched, with the complementary IC circulation pump rate of -0.1 mL / min to maintain the cells in the bioreactor during the growth phase of the 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 allow 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 allows 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 minus sign ("-") used in -0.01 mL / min indicates the direction of the IC circulation pump to cause or generate a countercurrent rate to maintain the cells in the bioreactor during the growth phase of the cell culture.

[0290] Next, process 1500 proceeds to query 1516 to determine whether to adjust the feed rate to retain the cells in the bioreactor itself while also taking into account the growing cell population and increased feed requirements. For example, Fig. 16B The increased feeding rate in response to the increased cell population is shown. Fig. 16B As shown, graph 1528 shows that in a cell expansion system (e.g., The relationship between the number of cells and the IC flow rate for a run or program on a cell expansion system). In an embodiment, the IC flow rate includes the culture medium that feeds the cells and can therefore also be referred to as the IC culture medium flow rate. The number of cells 1530 relative to the IC flow rate (mL / min) 1532 is shown. As shown, according to an embodiment, as the cells increase and the feed requirements of the expanded cell population increase, the IC flow rate increases from 0.1 mL / min to 0.2 mL / min to 0.3 mL / min. In the illustrated 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 Fig.16A and query 1516, if it is not desired to adjust the feed rate, the process 1500 proceeds from "no" to expanding the cells 1520, where the cells can continue to grow / expand 1514, for example, by feeding the medium during the first time period. For example, although the expansion of cells at 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 feed rate while maintaining the cells in the bioreactor, the process 1500 proceeds from "yes" to feeding the cells according to a second process during the second time period 1518. In an embodiment, the second process may involve, for example, feeding the cells at a feed rate that is substantially the same as during the first time period. In another embodiment, the second process may involve feeding the cells at a feed rate that is different than the feed rate used during the first time period. In an embodiment, the IC inlet flow rate may be increased, and the IC circulation flow rate may be set, configured, or adjusted to be equal to a percentage, portion, or fraction of the IC inlet flow rate. For example, according to embodiments, the IC circulation 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 another percentage or fraction, according to embodiments. According to embodiments, determining whether to set, configure, or adjust the IC circulation flow rate to be a percentage or fraction or portion of the IC inlet flow rate may be based on the value of the IC inlet flow rate. For example, embodiments provide the following method to maintain cells in a bioreactor when using an IC inlet flow to feed cells (where Q IC循环 =IC circulation flow rate (mL / min); Q IC入口 =IC inlet flow rate (mL / min)):

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

[0293] and

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

[0295] Although the above equation provides different calculations of the IC circulation flow rate based on the value 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 may be used according to other embodiments for making such quantitative calculations. In addition, although about fifty percent (50%) or about one-half (1 / 2) is used in this example, other percentages, ratios, fractions and / or portions may be used according to the embodiment. Returning to process 1500, after feeding cells 1518 using such flow characteristics and countercurrent characteristics as part of the second process to maintain the cells in the bioreactor, process 1500 proceeds to growing / expanding cells 1520. Although cell expansion at step 1520 is shown, cells may also grow / expand during, for example, one or more other steps (such as 1512, 1514, 1516, 1518). Process 1500 proceeds from the expansion step 1520 to harvesting or removing cells 1524. Then, process 1500 may terminate at end operation 1526. Depending on the embodiment, if any other steps are desired before harvesting the cells, process 1500 proceeds to optional "other" step 1522. Process 1500 proceeds from optional step 1522 to harvesting or removing cells from the bioreactor 1524, and process 1500 may then terminate at end operation 1526.

[0296] Next, Fig.17 1 shows example operational steps for a process 1600 for feeding cells according to an embodiment of the present disclosure, which can be used with a cell expansion system such as CES 500 (e.g., Figure 5B and Figure 5C ) or CES 700( FIG. 7A to FIG. 7C For example, a start operation 1602 is initiated, where the disposable kit can be loaded into the cell expansion system, the system can be primed, IC / EC flushing can be performed, the culture medium can be adjusted, and cells can be loaded. 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 1600 proceeds to feed cells 1604 according to a first process during a first time period. In an exemplary embodiment, as shown in reference FIG. 7A to FIG. 7CAs described, cells 916 can be fed by EC circulation feeding loop 752, IC circulation feeding loop 753, or a combination thereof. In an embodiment, cells can be fed at a minimum or low feeding rate, for example, where a cell population begins to grow / expand, and the minimum or low feeding rate can meet the feeding needs of such a population. For example, an IC inlet pump rate of +0.1 mL / min can be used during the first time period. According to an embodiment, although this example provides a low or minimum feeding rate of 0.1 mL / min, the low or minimum 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 minimum feeding rate may be greater than 0.1 mL / min. If it is desired to reduce cell losses from the hollow fiber membrane bioreactor during such a first time period, the IC inlet pump rate of +0.1 mL / min can be matched, or approximated, or substantially matched with a complementary IC circulation pump rate of -0.1 mL / min so as to maintain the cells in the bioreactor during the growth phase of the cell culture. Alternatively, in an exemplary embodiment, a first IC inlet pump may provide a volumetric flow rate of less than 0.1 mL / min or approximately 0.01 mL / min into the IC inlet port (701A) and an IC circulation pump provides a complementary IC circulation volumetric flow rate or fluid flow rate of less than -0.1 mL / min or approximately -0.01 mL / min into the IC outlet port (701B), where, for example, the minus sign ("-") used in -0.01 mL / min indicates the direction of the IC circulation pump to induce or generate a countercurrent rate to maintain cells in the bioreactor during the growth phase of cell culture.

[0297] Next, process 1600 proceeds to query 1606 to determine whether the feed rate can be adjusted to account for the growing cell population and / or to continue efforts to maintain the cells in the bioreactor. If it is desired to adjust the feed rate to retain the cells in the bioreactor, process 1600 proceeds from "yes" to feeding the cells according to a second process during the second time period 1608. In an embodiment, such a second process may involve feeding the cells at a feed rate that is substantially the same as during the first time period, for example. In another embodiment, the second process may involve feeding the cells at a feed rate that is different from the feed rate used during the first time period. In an embodiment, the IC inlet flow rate may be increased, and the IC circulation 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 circulation 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 circulation 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 circulation flow rate (mL / min); Q IC入口 =IC inlet flow rate (mL / min)

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

[0299] and

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

[0301] Although the above equation provides different calculations of IC circulation flow rate based on values ​​of IC inlet flow rate (e.g., 0.2 mL / min or 0.1 mL / min), other values ​​of such IC inlet flow rate may be used according to other embodiments for making such quantitative calculations. In addition, although about fifty percent (50%) or about one-half (1 / 2) are used in this example, other percentages, ratios, fractions and / or portions may be used according to embodiments. Returning to process 1600, after feeding cells 1608 using such flow characteristics and countercurrent characteristics as part of a second process to maintain the cells in a bioreactor, process 1600 proceeds to query 1610 to determine whether to monitor or measure metabolic activity of the growing cell population, such as glucose consumption and / or lactate production. For example, if it is not desired to monitor metabolic activity, process 1600 proceeds from "No" to expand cells 1616, where the cells can continue to grow / expand by supplying culture medium for them during the first time period 1604 and / or the second time period 1608. Although cell expansion is shown at step 1616 , cells may 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 it is desired to monitor or measure the metabolic activity of the growing cell species, based on the metabolic activity and / or its measured value, process 1600 proceeds from "yes" to continue to feed the cells according to the second process or to adjust the feeding rate. In an embodiment, monitoring glucose and / or lactate levels can help adjust the medium flow rate (e.g., IC flow rate) to support the expansion of cells (e.g., T cells or Treg) in a bioreactor (e.g., a hollow fiber bioreactor). In an embodiment, the lactate value of the cell culture can be maintained, for example, below about 7mmol / L. In an embodiment, the rate of medium addition is controlled by using a cell expansion system graphical user interface (GUI), for example, so that during the expansion of cells (e.g., regulatory T cells), the lactate metabolic waste from glycolysis can be maintained, for example, below about 7mmol / L. In other embodiments, for example, the addition rate and / or other settings of the culture medium can be controlled to try to maintain a lactate level, for example, ≤ about 5mmol / L, to improve cell growth and survival. In other embodiments, other concentrations can be used.

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

[0304] Depending on the metabolic measurement and its desired level, process 1600 proceeds to adjusting the feed rate 1614, where the cells may be fed according to an additional process during the additional time period. According to an embodiment, such additional processes and additional time periods may 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 the like. For example, in an embodiment, such additional processes may involve feeding the cells at a feed rate that is substantially the same as during the first and / or second time periods. In another embodiment, the additional process may involve feeding the cells at a feed rate that is different than the feed rate used during the first and / or second time periods. For example, in an embodiment, the IC inlet flow rate may be increased, and the IC circulation flow rate may match, or approximately or substantially match, the IC inlet flow rate, but in the opposite direction. In another embodiment, the IC inlet flow rate may be increased, and the IC circulation flow rate may 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 feed rate in step 1614 shows one "additional" process and one "additional" time period, any number of processes and time periods may be used to adjust the feed rate based on metabolic activity.

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

[0306] Fig.18A The operation steps of the process 1700 for expanding cells in the embodiment of the present disclosure are shown, which can be used with a cell expansion system. As described below, according to an embodiment of the present disclosure, the process 1700 may include the step of shearing the cells that have been expanded in the cell growth chamber. In an embodiment, these steps can be implemented as part of the "improved circulation" task. Start operation 1702, and process 1700 proceeds to the cell growth chamber in the cell expansion system by loading 1704 with a fluid of cells. In an embodiment, the cell may include non-adherent cells, such as one or more types of T cells. In one embodiment, the cell includes Tregs.

[0307] Process 1700 proceeds to exposing cells to an activator 1706. An activator, which may include an antibody complex, may be added to the fluid loaded at step 1704. In embodiments, the activator may be a soluble human antibody CD3 / CD28 / CD2 cell activator complex. Process 1700 proceeds to expanding cells 1708 within a first time period. Step 1708 may include feeding cells 1710. Nutrients may be fed to the cells to promote their expansion. For example, a culture medium containing glucose, protein, and reagents may be delivered to a cell growth chamber to provide nutrients for cell expansion.

[0308] The first time period for expanding cells 1708 can be based on the time required for forming cell colonies, microcolonies or clusters.Cell colonies, microcolonies or clusters can be groups of one or more attached cells.In an embodiment, cells (such as Tregs) may benefit from cell contact.Cell contact can stimulate signals that promote expansion and growth.But after a period of expansion, cells can adhere to each other to form cell colonies, microcolonies or clusters.Not bound by theory, it is believed that after the time period of cell expansion 1708, cells can form relatively large cell colonies, microcolonies or clusters that continue to grow.Cell colonies, microcolonies or clusters can produce necrotic centers, wherein nutrients (e.g., glucose), gases (e.g., oxygen) and reagents (e.g., activators) cannot reach the cells in the center of cell colonies, microcolonies or clusters.Therefore, the conditions of cell expansion in the center of these cell colonies, microcolonies or clusters may cause the expansion rate to slow down (e.g., increase the doubling time) or the conditions may cause cell necrosis.

[0309] In an embodiment, in order to allow 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 circulation 1712 to shear cell colonies, microcolonies or clusters during the second time period. Step 1712 can be performed to reduce the size of cell colonies, microcolonies or clusters. In an embodiment, 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] Fig.18B A plurality of illustrations 1750, 1760, and 1770 of cells in a volume of fluid (1752) are shown, wherein the cells may be expanded in a cell growth chamber as part of process 1700. For example, in some embodiments, the cell growth chamber may be a hollow fiber bioreactor. For example, in these embodiments, illustrations 1750, 1760, and 1770 may show cells in fibers of a hollow fiber bioreactor. 1750A, 1760A, and 1770A are enlarged portions of illustrations 1750, 1760, and 1770, respectively. Referring to illustration 1750, cells may be shown after they have been loaded 1704, exposed to an activator 1706, and expanded 1710 for a period of time (e.g., a first period of time). As shown in illustration 1750, a plurality of cell colonies 1754A to 1754E have been formed.

[0311] In order to reduce the size of the cell colony, microcolony or cluster of 1754A to 1754E and the number of cells therein, step 1712 can circulate fluid and cells through the first fluid circulation path. Without being bound by theory, it is believed that circulation can produce some forces (including shear stress) acting on the cell colony, as shown in the arrow 1756 shown in the enlarged part 1760A. Shear stress 1756 can provide enough force to separate the cells in the cell colony. As the circulation continues, the cell colony can begin to decompose into smaller sizes, as shown in diagram 1760. Diagram 1770 shows the cells after the circulation has been carried out for the second time period. As shown in diagram 1770, the size of the cell colony is reduced, and some of the colonies are completely separated into individual cells. In some embodiments, the step 1712 of shearing can be circulated until the cells and fluid include a single cell suspension.

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

[0313] In embodiments, the size of the remaining cell colonies, microcolonies, 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 may be a hollow fiber bioreactor with hollow fibers. As can be appreciated, the cell colonies, microcolonies, or clusters may be subject to shear stress each time they contact the sidewalls of the hollow fibers during circulation. Such contact may more effectively reduce the size of the cell colonies. When the inner diameter is larger, such as in conventional processes where a pipette may be used to induce shear stress to reduce colony size, contact with the sidewalls may not occur frequently. Fig.18CThe difference in inner diameter size between a hollow fiber (e.g., 215 microns) 1772 and a pipette tip 1774 (762 microns) in one embodiment is shown, which can be used to dissociate attached cells in cell colonies, microcolonies, or clusters. In embodiments, the smaller inner diameter hollow fiber is believed to be more efficient and effective in reducing the size of cell colonies during the cycle to shear step 1712.

[0314] In embodiments, the second time period for cycling to shear 1712 may 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 may be between about 15 minutes and about 1 minute, such as about 4 minutes.

[0315] After the second time period, process 1700 proceeds to moving cells to the cell growth chamber 1714 during a third time period. At step 1714, cells that were not positioned in the cell growth chamber during the third time period as a result of the cycle to shear step 1712 are moved back into the cell growth chamber. In embodiments, this may involve activating one or more pumps to introduce fluid into the fluid circulation path. For example, the fluid may be introduced from the fluid inlet path to the first fluid flow path and then introduced into the cell growth chamber from the inlet port and the outlet port of the cell growth chamber. The fluid is moved from the inlet port and the outlet port into the cell growth chamber and the cells may be moved back into the cell growth chamber.

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

[0317] In other embodiments, the third time period may be based on the volume of fluid introduced into the cell growth chamber during circulating to shear step 1712. For example, in embodiments, step 1712 may 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 expansion during a fourth time period 1716. Similar to step 1708, step 1716 may include feeding cells 1718. Nutrients may be fed to the cells to promote their expansion. For example, a culture medium containing glucose, protein, and reagents may be delivered to a cell growth chamber to provide nutrients for cell expansion.

[0319] Similar to the first time period, the fourth time period may be based on the time that the cell colony may need to form. In an embodiment, the fourth time period may be between about 5 hours and about 48 hours. In some embodiments, the fourth 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 fourth 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. 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 cycle rate. However, in other embodiments, the cycle rate used in step 1720 can be different from, greater than, or less than the first cycle rate.

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

[0322] Optionally, process 1700 can perform additional times of amplification, circulation and the steps of moving cells, as shown in optional step 1724 and ellipsis 1726. The steps of amplification, circulation and movement can be performed sequentially for a period of time. For example, in some embodiments, these steps can 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 can be performed in different time periods. For example, in one embodiment, the steps can be performed after three days, and then performed every other day. As another example, the steps can be performed after two days, and then performed twice a day. These are just examples, and other time periods can be utilized in other embodiments.

[0323] For example, Fig.19 Graph 1800 shows cycles and bolus additions at different time periods during a process of expanding cells. Graph 1808 shows the addition of bolus at different time periods during a cell expansion system (e.g. Cell number 1802 relative to cell culture days 1804 on a cell expansion system (e.g., The IC flow rate 1818 on a cell expansion system (cell expansion system) relative to the number of days 1804 of cell culture. As shown in 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 can be in response to the increasing number of cells as the number of days 1804 of culture increases. In addition, Fig.19 1806 and 1808, multiple cycles and bolus additions (1810 to 1816) in combination with an increased IC flow rate can have a positive effect on the rate of cell expansion (e.g., the number of cells).

[0324] Return to reference Fig.18A , process 1700 proceeds to removing cells from the cell growth chamber 1728. In embodiments, this may involve harvesting the cells. Step 1728 may include additional steps before or during the removal of cells from the cell growth chamber, such as recycling steps (e.g., 1712 and 1720). Process 1700 terminates at end operation 1730.

[0325] Fig. 20AOperational steps of process 1900 for operating a pump are shown, which can be used in a cell expansion system in an embodiment of the present disclosure. As described below, according to an embodiment of the present disclosure, process 1900 may include the step of activating a pump in a process of reducing cells in a cell cluster that has been expanded in a cell growth chamber. In an embodiment, these steps can be implemented as part of a "modified circulation" task. In an embodiment, the steps of process 1900 can be performed by a computer processor. Initiation starts operation 1902, and process 1900 proceeds to step 1904, at which 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 a cell expansion system. As shown in FIG. Figure 1C , Figure 4B and Figure 4C As 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, the process 1900 proceeds to activating the second pump 1906 at the second flow rate to introduce culture medium with nutrients into the capillary portion of the bioreactor for a first period of time. The nutrients may include, for example, proteins, glucose, and other compounds for feeding and promoting cell expansion. For example, referring to Fig. 20B , the first pump 1960 can be activated at a second flow rate to introduce culture medium into the inlet port 1962A of the bioreactor 1962.

[0327] In embodiments, the first time period may be based on the time that the cell colony may take to form. 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] Process 1900 then proceeds to activating a 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 at step 1906 into outlet port 1962B of bioreactor 1962 to, for example, feed cells.

[0329] The process 1900 then proceeds to activating 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 the cell clusters in the bioreactor. In embodiments, the cells circulate throughout the first fluid circulation path. Fig. 20C, the second pump 1964 may be activated at step 1910 to circulate fluid through the first fluid circulation path 1966, as indicated by arrows 1968A to 1968D.

[0330] Without being bound by theory, it is believed that after a period of time, the expanded cells form cell colonies, microcolonies, or clusters. Cell colonies can develop necrotic centers, where nutrients and proteins (e.g., activators) cannot reach the cells in the center of the colony. Therefore, the conditions for the expansion of cells in the center of these cell colonies, microcolonies, or clusters may result in a slowed expansion rate (e.g., increased doubling time) or may result in cell necrosis. Step 1910 may be performed to reduce the size of the cell colonies, microcolonies, or clusters.

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

[0332] After the second time period, process 1900 proceeds to step 1912, where the first pump is activated at a fifth flow rate to introduce fluid through the first fluid flow path for a third time period. In embodiments, the first portion of the fluid introduced into the first fluid flow path may cause the first cell in the first fluid flow path (which may be in the first fluid flow path due to step 1910) to move back into the cell growth chamber through inlet port 1962A. Fig.20D , pump 1960 may be activated to introduce fluid into first fluid flow path 1970. As indicated by arrows 1968A and 1968B, fluid flows from first fluid flow path 1970 into inlet port 1962A. This moves the first cells outside of bioreactor 1962 back into bioreactor 1962. It is believed that moving 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 at step 1912 may include one or more materials (e.g., reagents) that promote cell expansion. For example, in an embodiment, the fluid may be a culture medium including glucose or other nutrients that feed the cells. In one embodiment, the fluid may include a reagent, which may 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 exposing the cells to additional reagents (e.g., growth factors, proteins, etc.) that promote expansion can provide improved cell expansion. In an embodiment, the additional fluid used in step 1912 may be referred to as a push addition.

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

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

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

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

[0338] As can be appreciated, as the fluid enters the fluid circulation path 1966 from the first fluid flow path 1970, the fluid moves toward 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 toward 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. Therefore, in order to move more fluid into the second volume, the second pump 1964 can be set to a percentage of the rate of the pump 1960.

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

[0340] In embodiments, optionally, process 1900 may perform steps 1906 to 1914 an additional number of times, as indicated by ellipsis 1916 and optional step 1918. The steps of activating 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 may be performed continuously for a period of time. As described above, these steps may be performed to feed the cells, circulate the cells to break up cell colonies, microcolonies, or clusters, and move the cells back to the cell growth chamber. For example, in some embodiments, these steps may be performed once every three days, once every two days, once a day, twice a day, or three times a day for a period of about two days to about twenty days (such as 10 days). In some embodiments, these steps may be performed at different time periods. 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. This is merely an example, and other embodiments may utilize other time periods. Process 1900 terminates at end operation 1920.

[0341] It should be noted that in some embodiments, process 1900 may include additional steps. For example, a rocking device may 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 may 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 process 1900 are not limited in this regard.

[0342] In an alternative embodiment, if Fig. 20A As shown, the steps of process 1900 can be followed, wherein flow rates are reduced by fine motor control. For example, the IC and EC pumps are allowed to operate from 0.005 RPM to 600 RPM over a torque range to prevent stalling over the entire range. Thus, in both directions, the flow rate range is as low as 0.01 mL / min at 500 RPM for the large pump and as low as 0.01 mL / min at 300 RPM for the small pump. The work performed to prevent low speed stall is due to improved low speed stall detection methods and control loops for differential control at low speeds. In an exemplary embodiment, as shown in reference FIG. 7A to FIG. 7C As depicted, 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 pump and the EC pump move continuously at a nearly continuous speed, providing strong backflow containment at ultra-low flow rates. If the two matched pumps were duty cycled, they could not run relative to each other to keep the cells in the bioreactor without causing them to move their position in the bioreactor. With the IC pump and the EC pump running continuously for long periods of time, the cells are kept in an optimized position within the bioreactor, which saves cell culture medium overall.

[0344] As described below, synchronization of harvesting is provided by alternating pump control that can run for a long time. Low flow rates (approximately 1 / 10 of the flow rates used during the steps in the previous strategy) reach about 0.01 mL / min. To achieve these speeds, an RPM of 0.005 is required at continuous speed. Thus, constant motion of the pump at 0.005 RPM is achieved without stalling. See data in D0000038948, D0000038355, D0000044793, D0000033725 (and all attached attachments), and D0000041290.

[0345] In an alternative embodiment, if Fig. 20AAs shown, the steps of process 1900 can be followed, which greatly reduces the time period compared to the description above. For example, the IC inlet pump and the EC inlet pump can be activated in alternating time periods, wherein the time period is less than 10 minutes or about 5 minutes. The pump can be activated at a low flow rate or an ultra-low flow rate in 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 about 0.005RPM to 600RPM, or a flow rate within about 0.01mL / min to 500RPM, and for a small bioreactor or a small cell growth chamber, it can be within about 0.01mL / min to 300RPM. Alternating activation of the IC inlet pump and the EC inlet pump during the low time period allows synchronization of harvesting in the capillary. During process 1900, the harvest valve and the EC outlet valve are opened as the IC inlet pump is activated and the EC inlet pump is activated, respectively. When the IC and EC inlet pumps are running, they run at twice the command speed unless the twice the command speed is outside the allowed range. Then, the pump runs at the maximum allowed flow rate for 5 minutes. If the stop condition is volume dependent, the remaining time shown on the screen will only be updated when the corresponding pump is running.

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

[0347] Intracapillary (IC) harvest synchronization provides continuous culture medium for a long period of time while still feeding the cells. It allows conditioned culture medium to feed the cells, which may have advantages over fresh culture medium.

[0348] In an exemplary embodiment, as shown in reference Figures 7A to 7C As described, fluid may be pumped through the EC circulation supply loop 752, the IC circulation supply loop 753, or a combination thereof.

[0349] Fig.21 2000 for expanding cells according to an embodiment of the present disclosure, which can be used with a cell expansion system, such as CES 500 (e.g., Figure 5A )、CES 600( Figure 6 ) or CES 700( FIG. 7A to FIG. 7C ) is used together. Start operation 2002 is initiated, and process 2000 proceeds to loading the disposable tubing set into the cell expansion system 2004. Figure 1C , Figure 4B and Figure 4CAs shown, disposable tubing set 2004 may include cell growth chamber 100A or cell growth chamber 100B. Next, the system may be primed 2006. In embodiments, a user or operator may provide instructions to the system to prime, for example, by selecting a task for prime filling. In embodiments, the task for prime filling may be a preprogrammed task. Process 2000 then proceeds to IC / EC flush task 2008, where fluids on the IC circulation loop and the EC circulation loop are replaced. The replacement volume is determined by the amount of IC volume and EC volume exchanged.

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

[0351] For example, process 2000 next proceeds to centrally loading 2012 cells from a cell entry bag to a bioreactor without circulation. In embodiments, a "centrally loading cells without circulation" task may be used, wherein a first volume of fluid including a plurality of cells may be loaded into a cell expansion system at a first flow rate, wherein the cell expansion system includes a cell growth chamber. For example, a second volume of fluid including a culture medium may then be loaded into a portion of a first fluid circulation path at a second flow rate to position the first volume of fluid in a first portion of a cell growth chamber. In embodiments, a first portion of a cell growth chamber or bioreactor may include approximately a central region of the bioreactor. In embodiments, the first volume is the same as the second volume. In embodiments, 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 embodiments, the sum of the first volume and the second volume is equal to a percentage or ratio 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 may be, for example, about 50% of the volume (e.g., total volume) of the first fluid circulation path. In embodiments, the fluid in the first fluid circulation path flows through the intracapillary (IC) space of a bioreactor or cell growth chamber. For example, in embodiments, the fluid in the second fluid circulation path flows through the extracapillary (EC) space of a bioreactor or cell growth chamber. In an exemplary embodiment, as described in reference FIG. 7A to FIG. 7C As described, fluid 916 may be supplied by an EC circulation supply loop 752, an IC circulation supply loop 753, or a combination thereof. In embodiments, the sum of the first volume and the second volume may be, for example, about 50% of the volume of the intra-capillary (IC) loop, or may be another percentage or ratio, depending on the embodiment. For example, in embodiments, the sum of the first volume and the second volume may be about 50% of the volume of another fluid path, loop, etc. (if applicable), or may be another percentage or ratio, depending on the embodiment. Other percentages or ratios may be used, including, for example, any percentage between about 1% and about 100%, depending on the embodiment.

[0352] After loading cells 2012, process 2000 proceeds to feeding cells 2014. Cells 2016 may be grown / expanded. According to embodiments, although step 2016 is shown after step 2014, step 2016 may occur before or simultaneously with step 2014. Next, process 2000 proceeds to query 2018 to determine whether any cell colonies, microcolonies, or clusters have been formed. A cell colony, microcolony, or cluster may be a group of one or more attached cells. If a cell colony, microcolony, or cluster has been formed, process 2000 proceeds from "yes" to shearing 2020 any cell colony, microcolony, or cluster. For example, after amplifying a plurality of cells in a first time period, cells may be circulated at a first cycle rate during a second time period to reduce the number of cells in a cell colony, microcolony, or cluster. In embodiments, circulating cells at a first cycle rate may cause a cell colony to generate shear stress, wherein one or more cells in a cell colony may be separated from the cell colony. For example, in embodiments, reducing the number of cells in a cell colony, microcolony, or cluster can provide a single cell suspension. In embodiments, circulating cells to shear any colony, microcolony, or cluster 2020 can be used once every two (2) days, for example, during cell culture, to maintain uniform cell density and nutrient diffusion. Other time periods may also be used, depending on embodiments. For example, in embodiments, such shearing of any microcolony, colony, or cluster may begin on or after the 4th day. Depending on embodiments, such shearing may be used to begin on other days or time periods. After shearing 2020, process 2000 may then return to feeder cells 2014.

[0353] For example, if it is determined at query 2018 that there is no shearing, or if there are no cell colonies or clusters, the process 2000 proceeds from "No" to resuspending cells 2022. In embodiments, circulating cells can be used to evenly resuspend cells that may have loosely adhered during culture. In embodiments, step 2022 can include circulating cells to evenly resuspend cells that may have loosely adhered before initiating a harvest task or other task to remove cells from the bioreactor. After resuspending cells 2022, the process 2000 next proceeds to harvesting cells 2024. Further processing or other analysis of the removed cells can optionally be performed at step 2026, and the process 2000 can then terminate at end operation 2028. If further processing / analysis is not desired, the process 2000 terminates at end operation 2030.

[0354] Go to Fig. 22 And process 2100, according to an embodiment, initiates start operation 2102, and at 2104, process 2100 proceeds to loading the disposable kit onto the cell expansion system. 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 may then be pre-filled 2106, and an IC / EC flushing step 2108 may occur. The culture medium may then 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 may be loaded 2112 by a "centrally loaded cells without circulation" task. In another embodiment, such cells may be loaded 2112 by a "load uniformly suspended cells" task.

[0355] Depending on the embodiment, the process 2100 then proceeds to starting the feeder cells 2114, which may begin on day 0. In an exemplary embodiment, as shown in reference FIG. 7A to FIG. 7C As described, fluid 916 may be supplied through the EC circulation supply circuit 752, the IC circulation supply circuit 753, or a combination thereof. Cells may grow and expand 2116, and, for example, on day 3, it may be desirable to add a bolus to the IC circuit and redistribute the cells 2118. In an embodiment, such a bolus fluid may include a reagent, such as a cytokine or other growth factor. In another embodiment, such a bolus fluid may, for example, include a reagent and a basal medium.

[0356] After such bolus addition and redistribution of cells, the process 2100 next proceeds to feeding the cells 2120 again, where such feeding may occur, for example, on day 3. With such feeding 2120, parameters of the system (such as one or more pumps controlling flow rates) may be controlled 2122 to achieve complementary flow settings and counter-flow settings for moving fluids from the IC inlet port and IC outlet port of the bioreactor into the bioreactor. For example, the IC inlet pump 2124 may be adjusted or directed to produce flow, and the IC circulation pump 2126 may be adjusted or directed to produce counter-flow. In embodiments, for example, an IC inlet pump rate of +0.1 mL / min may be matched, or approximately or substantially matched, to a complementary IC circulation pump rate of -0.1 mL / min to maintain cells in the bioreactor during the growth phase of the cell culture, which may be days 4 to 7. Alternatively, in an exemplary embodiment, a first IC inlet pump may 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 causes 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 minus sign ("-") used in -0.01 mL / min indicates the direction of the IC circulation pump to cause or generate a counterflow rate to maintain cells in the bioreactor during the growth phase of the cell culture. Such a setting control 2122 can allow for the counteracting of any forces associated with cell loss from the IC outlet port of the bioreactor.

[0357] For example, process 2100 proceeds to query 2128 where it is determined whether to continue to add reagents or other bolus additions at other days or other time intervals. If it is desired to add additional reagents or other boluses and redistribute cells, process 2100 branches "yes" to add reagents and redistribute cells 2118. For example, such bolus additions and redistribution of cells may occur next on days 6 and 9, depending on the embodiment.

[0358] If, or once, it is not desired to continue adding boluses (e.g., reagents) and redistributing cells, the process 2100 proceeds from "No" to harvest cells 2130, where the cells can be transferred to a harvest bag or container. The process 2100 then terminates at end operation 2136.

[0359] Alternatively, the process 2100 can optionally proceed from the harvesting operation 2130 to allow for further processing / analysis 2132. Such further processing 2132 can include characterizing the phenotype of the harvested cells (e.g., T cells or Tregs). The process 2100 can optionally proceed from the optional further processing / analysis step 2132 to reloading any remaining cells 2134. Then, the process 2100 can terminate at an 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 performed over the course of a 14-day protocol to expand cells. Start operation 2202 is initiated, and process 2200 proceeds to day 0, where a disposable kit is loaded onto a cell expansion system 2206. As shown, Figure 1C , Figure 4B and Figure 4C As shown, the disposable kit can include cell growth chamber 100A or cell growth chamber 100B. For example, the disposable kit can then be pre-filled 2208, wherein the kit can be pre-filled 2208 with PBS (e.g., Lonza, without Ca2+ / Mg2+). In preparation for the loading of cells, an IC / EC flush 2210 can be used to exchange the pre-fill fluid. For example, according to an embodiment, the PBS in the system can be exchanged with TexMACS GMP basic medium. Next, the medium can be conditioned 2212. The medium can be conditioned 2212 to allow the medium to reach equilibrium with the provided gas supply before the cells are loaded into the bioreactor.

[0361] Next, on day 0, process 2200 proceeds to loading cells 2214, such as suspended or non-adherent cells (such as T cells or Tregs). In an embodiment, such cells can be loaded 2214 via a "load cells centrally without circulation" task. In another embodiment, such cells can be loaded 2214 via a "load cells uniformly suspended" task.

[0362] On day 3, a bolus of cytokines 2216 may be added while redistributing cells 2218. In embodiments, the redistribution of cells may be performed in conjunction with the bolus addition to mix the cells and expose the cells more thoroughly to the cytokines (e.g., IL-2) that may be present in the bolus addition. In embodiments, the redistribution may also break up cell colonies or clusters that may have formed. In embodiments, the redistribution may first occur 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 a fluid into the fluid circulation path to push the cells back into the bioreactor. After the redistribution and bolus addition 2218, the process 2000 proceeds to feeding the cells 2220.

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

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

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

[0366] Start operation 2302 is initiated, and process 2300 proceeds to loading the disposable kit into the cell expansion system 2304. 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. For example, the disposable kit may then be pre-filled 2206, wherein the kit may be pre-filled 2206 with PBS (e.g., Lonza, without Ca2+ / Mg2+). In preparation for the loading of cells, an IC / EC flush 2308 may be used to exchange the pre-fill fluid. For example, according to one embodiment, the PBS in the system may be exchanged with a TexMACS GMP basal medium. Next, the medium may be conditioned 2310. The medium may be conditioned 2310 to allow the medium to reach equilibrium with the provided gas supply before the cells are loaded into the bioreactor.

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

[0368] Process 2300 proceeds to position the inlet volume in the first portion of bioreactor 2314. In embodiments, the positioning can be performed by introducing a second volume of fluid, which can include culture 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 embodiments, 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 yet 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, the process 2300 proceeds to exposing the cells to an activator 2316 to activate the cells for expansion. In some embodiments, at 2316, the cells may be exposed to a soluble activator. In some embodiments, the activator may include an antibody complex, which may be added to the culture medium and may be included in the inlet volume, or added later, such as with the second volume. For example, in embodiments, the activator may be a human antibody CD3 / CD28 / CD2 cell activator complex.

[0370] Process 2300 proceeds to providing 2318 cells according to a first process during a first time period. In an exemplary embodiment, as shown in reference FIG. 7A to FIG. 7C As described, cells 916 may be fed by an EC circulation feeding loop 752, an IC circulation feeding loop 753, or a combination thereof. In an embodiment, cells may be fed at a minimum or low feeding rate, for example, where a cell population begins to grow / expand during a first time period 2320, and the minimum or low feeding rate is able to meet the needs of such a population. For example, an IC inlet pump rate of +0.1 mL / min may be used during such a first time period. If it is desired to reduce cell losses from the hollow fiber membrane bioreactor during such a first time period, the IC inlet pump rate of +0.1 mL / min may be matched, or approximately or substantially matched, with a complementary IC circulation pump rate of -0.1 mL / min so as to maintain the cells in the bioreactor during the growth phase of the cell culture. Alternatively, in an exemplary embodiment, a first IC inlet pump can enable a volumetric flow rate of less than 0.1 mL / min or approximately 0.01 mL / min into the IC inlet port (701A) and the IC circulation pump enables a complementary IC circulation volumetric flow rate or fluid flow rate of less than -0.1 mL / min or approximately -0.01 mL / min into the IC outlet port (701B), where, for example, the minus sign ("-") used in -0.01 mL / min indicates the direction of the IC circulation pump to cause or generate a countercurrent rate to maintain cells in the bioreactor during the growth phase of the cell culture.

[0371] From 2320, process 2300 proceeds to expanding cells 2322 during a second time period. Expanding 2322 during the second time period may also involve feeding cells 2324 according to a second process during the second time period. For example, in an embodiment, such a second process may involve feeding the cells at substantially the same feeding rate as during the first time period. In another embodiment, the second process may involve feeding the cells at a different feeding rate than the feeding rate used during the first time period. For example, the feeding rate may be increased due to the expansion of the cells during the first time period.

[0372] While the cells are being expanded during the second time period 2322, the cells may also be cycled to shear cell colonies or cell clusters 2326. Step 2326 may involve cycling the cells in the IC cycle path to shear any colonies or clusters that may have formed during the first time period. The step of shearing colonies or clusters 2326 may reduce the number of cells in the cell colonies or cell clusters. In embodiments, cycling 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 may then proceed to a harvesting operation 2328, where the cells may be transferred to a harvesting bag or container. In embodiments, a therapeutic dose of cells may be harvested. In embodiments, the cells harvested at operation 2328 may be 1×10 9 In embodiments, the harvested cells may have a viability between about 75% and about 95%.

[0374] Then, optionally, process 2300 can proceed to allow further processing / analysis 2330. Such further processing can include characterizing, for example, the phenotype of the harvested cells (e.g., 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 embodiments, the harvested cells may include a frequency of greater than about 80% CD4 + CD25 + In other embodiments, the cells may include a frequency of greater than about 55% CD4 + FoxP3 + Process 2300 may then 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 may be rearranged, combined into other steps, used in parallel with other steps, etc. Fewer or additional steps may be used in embodiments without departing from the spirit and scope of the present disclosure. Moreover, for example, steps such as pre-filling, conditioning medium, loading cells (and any sub-steps) may be performed automatically 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. In addition, for illustrative purposes, for example, steps are provided Fig. 11B 0046] An example pump rate setting for feeding cells is depicted in 0047. Other pump rates, flow rates, directions, etc. may be used according to embodiments of the present disclosure.

[0376] Examples and further descriptions of tasks and protocols (including custom tasks and pre-programmed tasks) for use with cell expansion systems are provided in U.S. patent application serial number 13 / 269,323 (“Configurable Methods and Systems for Growing and Harvesting Cells in a Hollow Fiber Bioreactor System” filed on October 7, 2011) and U.S. patent application serial number 13 / 269,351 (“Customizable Methods and Systems for Growing and Harvesting Cells in a Hollow Fiber Bioreactor System” filed on October 7, 2011), all contents and all purposes of which are incorporated herein by reference in their entirety.

[0377] Next, Fig.25 Example components of a computing system 2400 on which embodiments of the present disclosure may be implemented are shown. For example, in embodiments, computing system 2400 may be used where a cell expansion system uses a processor to perform tasks, such as custom tasks or pre-programmed tasks, that are performed as part of a process such as shown and / or described herein. In embodiments, 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 touch screens, wherein the touch screen may include a display area for providing one or more application windows. For example, the touch screen may also be an input device 2410 that receives and / or captures physical touch events from a user or operator. As understood by those skilled in the art, the touch screen may include a liquid crystal display (LCD) having a capacitive structure that allows the processing system 2404 to infer the location of the touch event. The processing system 2404 may then map the location of the touch event to a UI element presented in a predetermined location in the application window. Depending on the embodiment, the touch screen may also receive touch events through one or more other electronic structures. Other output devices 2408 may include a printer, a speaker, etc. As understood by those skilled in the art, other input devices 2410 may include a keyboard, other touch input devices, a mouse, a voice input device, etc. For example, the user interface 2402 may be a reference Figure 2A Depicted user interface 264. User interface 2402 may include different screens for different parts of a task / protocol / process / method, for different user inputs or requests, and the like.

[0379] According to an embodiment 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. In addition, in an embodiment, each processor may be a multi-core processor having one or more cores for reading and executing separate instructions. As will be appreciated by those skilled in the art, the processor may include a general-purpose processor, an application-specific integrated circuit (ASICs), a field programmable gate array (FPGAs), other integrated circuits, etc.

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

[0381] Storage 2406 may be any long-term data storage device or component. Depending on the implementation, storage 2406 may include one or more systems described in conjunction with memory 2414. Storage 2406 may be permanent or removable. In an implementation, storage 2406 stores data generated or provided by processing system 2404.

[0382] Through a wireless network, Bluetooth network, or other network system, the computing system 2400 can communicate with the cloud, a network computer, a personal computing device, a mobile device, etc. Alternatively, through a hardwired connection, the computing system 2400 can communicate with a personal computing device, a network computing device, a mobile device, etc.

[0383] The processing system 2404 can control pump activation, speed, and fluid flow. The processing system 2404 can control pump activation / speed to provide ultra-low feed rates with continuous motion (rather than step motion) using a control loop of the pump process. Continuous, consistent motion (compared to periodic / step motion) facilitates finer pump control and lower feed rates. The operation can switch between two pumps operating at a low flow rate to facilitate continuous harvesting of cells. In an exemplary embodiment, the first IC inlet pump can allow 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 allows 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 minus sign ("-") used in -0.01 mL / min indicates the direction of the IC circulation pump to cause or generate a counterflow rate to keep cells in the bioreactor during the growth phase of cell culture.

[0384] The processing system 2404 can control pump activation / speed to contain backflow. Backflow containment at ultra-low flow rates allows cells to be concentrated in desired areas. Backflow containment conserves culture medium for cell cultures and allows for lower protein concentrations in the system. The ability to provide continuous motion allows for lower protein concentrations. Opposite pumps pump in opposite directions, allowing cells to cluster.

[0385] The processing system 2404 can be configured to run various tasks / methods / processes / regimens entered by the user or stored as preset tasks / methods / processes / regimens in the memory 2414. 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 able to track and record the user's materials, procedures, user logins, etc. The system can be used to push protocols to a fleet of identical machines, thus saving a lot of time in setting up a proven protocol to run on a fleet of machines. All reports can then be pulled back to the application and real-time readings of the fleet can be viewed remotely on the application. Temperatures and pressures can be updated to the application at a configurable rate, and alarms and warnings can also be sent to the application. For example, the application can push an alarm as a remote alarm to one or more users for logging or remediation. For example, the remote alarm can be an email, text message, or other digital reminder sent to the user. The CPA is also connected and Quantum to save all the user's reports in a unified application.

[0386] 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 000044037, 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 may include tracking and data management features. Tracking and logging may be enabled through the CPA. Tracking and logging may be performed periodically (e.g., scheduled) or on demand. The user may schedule times to be logged based on trigger events or periodic times.

[0388] Cell processing applications also enable swarm control. CPA unifies modular cell approaches including CES such as Quantum Cell expansion systems and Etc. Thus, the CES described herein (e.g., CES100, 700, etc.) is a single module in a modular system controlled by a cell processing application. For example, a CPA can control a group of up to 100 or more devices. Using a CPA, the same instructions can be sent to multiple machines instead of a single machine to simplify and ensure consistency of operation.

[0389] Cell processing applications may include custom tasks / methods / processes / protocols. Users may draft custom protocols or custom tasks. Tasks may be detailed steps for a process defined by the user. Protocols may be a compilation of tasks required to complete a test or cell expansion process, etc., stored in a single file. Memory 2414 may store predefined, pre-written, or inventory protocols and tasks. Inventory tasks may be compiled into custom protocols by the user. Protocols in the CPA system may be modified in real time and re-uploaded to the fleet. Tasks and protocols may be written, modified, and selected on the user interface or on an external computer or handheld device.

[0390] The cell processing application may 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 the administrator. Permissions may include control levels of access (e.g., read-only, write, etc.). A user may have multiple roles. For security measures, the CPA may require user authentication.

[0391] The Cell Processing Application can trigger and send remote notifications and alarms. Emails or other notifications (digital notifications, texts, etc.) can be sent to the user, allowing monitoring / checking of conditions, etc., without the user having direct access or contact with 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 send commands to the machine remotely, such as ignoring alarms, stopping tests, modifying protocols or tests, 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. Disposable packages, media, cells, etc., each include a barcode with product information. The CPA can check the data from the barcode scanner to ensure that the correct component or media is being used. The barcode can be stored in memory 2414 or in the CPA for future reference (such as product recalls) or amplification data. The CPA can adjust the configuration of the task / method / process / protocol based on the data from the barcode scanner.

[0394] The computing system 2400 can improve process efficiency by driving contact between "agents 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 the inner capillary (IC) side of the HFB. The reagents of interest are introduced into the IC side of the HFB. Counterflow (dividing flow by driving the IC inlet pump and IC circulation pump in opposite directions; inlet = positive flow, circulation = negative flow) is used to drive active contact between the reagents of interest and the cell population. This process can continue as long as necessary.

[0395] Many processes in cell culture require exposing a population of cells to specific suspended elements in order to alter the cells or produce a secondary product. For example, to produce a viral vector product, a population of cells must be exposed to active viral particles that will replicate within 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. Both of these example processes have variable efficiencies based on the environment in which the process occurs. Many passive models of these types of processes rely on highly random chance to drive the interaction between the reagent of interest (ROI) and the cell population. These passive models result in lower efficiencies of viral integration / plasmid transfection / GOI expression.

[0396] The process described herein is intended to improve the efficiency of these processes by driving active contact between cell populations and ROIs. Cells (adherent or suspended) are seeded in the fibers of the HFB. Once the cells are established, the "reagents of interest" (viral particles, transfection reagents, secondary cell types, differentiation reagents, induction reagents, etc.) are introduced into the HFB. The carrier fluid is pumped out of the dual-port bag. The inlet pump and the circulation pump divide 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 holes in the HFB membrane. The carrier fluid is recycled back to the dual-port bag from the EC side. The "reagents of interest" are contained in the IC loop because the molecules are larger than the membrane pores, and are pressed against the cell layer on the membrane wall, driving contact between the "reagents of interest" and the cell population. Continue as long as necessary.

[0397] Example

[0398] The following description includes some examples of protocols / methods / processes that can be used with a cell expansion system, such as CES 500 (e.g., Figure 5A , Figure 5B , Figure 5C ) and / or CES 600( Figure 6 ) together, for example, to implement various aspects of the embodiments. Although specific features may be described in the embodiments, such embodiments are provided for the purpose of illustration and description only. For example, although the embodiments may provide for the expansion of T cells and / or Treg cells, other embodiments may use other and / or additional cell types and / or combinations thereof. According to some embodiments, although specific parameters, features and / or values ​​are described, such as CES (e.g. The use of cell expansion system) is described, but these parameters, characteristics 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 embodiments provided herein are not intended to limit other embodiments, which may include different or additional steps, parameters or other features. In some embodiments, the example method or scheme including step (and any sub-step) may be such as automatically performed by a processor, which performs a pre-programmed task stored in a memory. In other embodiments, the steps (and any sub-step) may be performed by the combined operation of automation and manual execution. In further embodiments, the steps (and any sub-step) may be performed by an operator or user or by other manual devices.

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

[0401] In some embodiments, the protocol package or method for a smaller bioreactor or smaller cell growth chamber can be the same task as that 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. The flow rate is reduced when the air removal chamber (ARC) is emptied in the ARC management step and when the flow is forced through the membrane. This is because the small bioreactor has a smaller fiber surface area, so the flow rate is reduced to avoid high pressure. 2. Different default selections are based on differences in disposable kits (i.e., where applicable, the flow rate and volume stop conditions are proportionally reduced based on the volume differences of certain parts of the kit). 3. The range is open, allowing the user to choose to reduce the flow to 1 / 10 of the standard bioreactor task.

[0402] In some exemplary embodiments, a protocol package or method may include: 1. Default (template) adherent cell expansion protocol; 2. Default (template) suspension cell expansion protocol; 3. Default (template) custom one-step protocol; 4. Custom step 1-10 tasks with more available options compared to traditional Quantum custom tasks; 5. Load cells with multiple distribution cycles (task created by the scientific team to optimize seeding of MSCs); 6. Load and position cells (task created by the scientific team to optimize T-cell loading); 7. Feed non-adherent cells (task created by the scientific team to optimize feeding T-cells); and / or 8. Cycle and position cells (task created by the scientific team to optimize sampling during T-cell expansion)

[0403] Example 1

[0404] method

[0405] Universal Treg cell culture

[0406] Immunomagnetic separation of CD4+ CD25 + Tregs can be obtained from the peripheral blood of healthy adult donors by leukapheresis (HemaCare Corporation, VanNuys, CA) and can be subsequently cultured at 1.0 × 10 5 The concentration of cells / mL was 1.34 μg / mL in three T25 flasks (7 mL / flask) in a sterile-filtered TexMACS TM The cells were expanded in GMP medium supplemented with 200 IU / mL of recombinant human IL-2 premium grade cytokine (Miltenyi Biotec GmbH, Bergisch Gladbach) and Gibco PSN 100X antibiotic cocktail (ThermoFisher Scientific, Waltham, MA). The actively growing Treg cell suspension was then used as an inoculum in each of three (3) Quantum Cell Expansion System experimental runs. In the absence of microbeads, soluble tetrameric Immunocult TM Human antibody CD3 / CD28 / CD2 cell activator complex (Stem Cell Technologies, Vancouver, BC) costimulated inoculum and Quantum system expanded Tregs at 25 μL / mL. For Treg inoculum, costimulation can be performed on days 0 and 9, and for Quantum system Treg expansion, costimulation can be performed on day 0. The Quantum system HFM bioreactor can be characterized by a capillary loop volume of 177.1 mL and a 21000 cm 2 surface area.

[0407] Quantum System Treg Expansion

[0408] According to an embodiment, two (2L) bags of sterile filtered culture medium can be prepared for large-scale expansion of Treg in the Quantum system using Quantum culture medium bag 4L kit (Cat.21021). A 2L bag of complete culture medium containing TexMACS GMP, IL-2 and PSN antibiotics can be used to supply the IC compartment, and a 2L bag of basal culture medium containing TexMACS GMP and PSN antibiotics can be used to supply the EC inlet compartment of the bioreactor. After the Quantum system is pre-filled with PBS (Lonzacat.17-516Q, Walkersville, MD), the TSCD-Q Terumo sterile welder can be used to connect the culture medium bag to the appropriate IC and EC inlet lines. The complete culture medium can be protected from exposure to light.

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

[0410] In the “central loading of cells without cycling” task, the Quantum system runs (n=3) were able to generate 2.5×10 5 Cells / mL to 3.7×10 5 cells / mL, or 2.1×10 cells / mL in the lumen or intracapillary (IC) compartment of a hollow fiber membrane bioreactor. 3 Cells / mL to 3.1×10 3 Cell / em 2 The average value of Treg inoculation.

[0411] Day 0 to 4:

[0412] Example Quantum custom task

[0413] Donor cells, improved

[0414] IC / EC exchange and conditioning medium for regulatory T cells, Examples.

[0415] By Terumo BCT TSCD-Q sterile welder, a medium bag of TexMACS GMP complete medium containing IL-2 supplement (200 IU / mL) is attached to the IC medium line of the Quantum system. TexMACS basal medium can be attached to the EC medium line. IC / EC flushing and conditioning medium tasks can be performed separately. Complete medium can be used for IC exchange or flushing, and basal medium can be used for EC exchange or flushing to save the amount of IL-2 and activator complex.

[0416] The system can be placed on modified "feeder cells" prior to introduction of the cells. The IC inlet rate (Q1) and IC circulation rate (Q2) can be increased to 0.2, 0.3, and 0.4 mL / min on days 5, 6, and 7 at matching rates, but in the opposite direction to days 4, 5, and 6, or as needed to maintain lactate levels between 5 mmol / L and 8 mmol / L.

[0417] Table 1: Support cells, improved, examples.

[0418]

[0419] Example Quantum custom task:

[0420] Centrally load cells without circulation, example.

[0421] Purpose: This task allows the suspended cells to be concentrated 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.

[0422] Modifications to the assignment can be entered prior to loading cells into the Quantum system by using a loading cell without cycling.

[0423] Table 2: Concentrated loading of cells without cycling, modifications, examples.

[0424]

[0425] Table 3: Concentrated loading of cells without cycling, Example

[0426]

[0427]

[0428] If desired, you can return to the default cell support task and continue the expansion protocol using the support cell task.

[0429] Table 4: Support cells, improved, example.

[0430]

[0431] Day 4 or later:

[0432] Resuspend Treg cells during cell culture or prior to harvesting, Example.

[0433] The purpose of this modified cycling task may be to evenly resuspend cells that may have become loosely adhered during the culture period or prior to initiating a harvesting task.

[0434] Additionally, this task can be used to shear Treg cell colonies every two (2) days during cell culture in order to maintain uniform cell density and nutrient diffusion beginning 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 "Feeder Cell" task.

[0435] Table 5: Recycling and resuspension of cells, return of cells to bioreactor and feeding, Example

[0436]

[0437]

[0438] Harvest Quantum harvest task with modifications, example.

[0439] Table 6: Harvest, Modification, Example

[0440]

[0441] Harvested cells can be removed from the Quantum system via RF welding for further evaluation and analysis.

[0442] Post-harvest analysis

[0443] Harvested cells can be counted in the 5 μm-50 μm range using a VI-XR 2.04 cell viability analyzer (Beckman Coulter), and membrane integrity can be quantified by trypan blue dye exclusion.

[0444] metabolism

[0445] 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) using a G Cartridge, with i-STAT GCartridge (Cat. 03P83-25) and i-STAT CG4+ cartridge (Cat. 03P85-50) used to monitor glucose and lactate concentrations, respectively.

[0446] Cell surface biomarker expression

[0447] 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 the maintenance of immune homeostasis, which may include the regulation of immune tolerance in both innate and adoptive responses. In addition, the expression of the gene product of the transcriptional regulator forkhead box P3 (FoxP3) is known to be associated with CD4 + CD25 + FoxP3 + CD127 1o / -Treg Phenotype and antigen presenting cells (APCs) and effector T cells (T eff) is associated with immunosuppression of IL-2. Binding of IL-2 to the CD25 / IL-2 receptor (Rαα) and 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 the histone acetylase KAT5 and the histone deacetylase HDAC7.

[0448] The frequency of Treg phenotypes of biomarkers on the surface of harvested cells can be quantified by flow cytometry. To this end, cells can be stained with the following antibody conjugates and gated for viable, unstained cells: Fixable Viability 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 Pharmingen 561842), anti-CD4-FITC (BD-Pharmangen 555346), anti-CD25-PE (BD Pharmangen 555432), anti-CD127-PE (BD Pharmingen 557938), anti-CD45RO-PE (BD Pharmingen 347967), and anti-FoxP3-Alexa Fluor 647 (BD Pharmingen 560045). 1 × 10 6 Cells and 20,000 total events per sample were collected to acquire sample data.

[0449] Fig.26 An example of the experimental procedure is shown in FIG.

[0450] Possible results

[0451] Initial studies of Tregs in static culture may indicate that these cells tend to form microcolonies with a diameter of approximately 100 μm. Isolating these cells during a two-day medium exchange process may help limit cell necrosis and restore the cells to a high-density single-cell suspension by using a 1000 μL pipette tip with an ID of 762 μm. Alternatively, the process of maintaining a single cell suspension can be more efficiently achieved in an automated HFM bioreactor with the help of a preprogrammed daily cycle task, where the fiber lumen ID is approximately 200 μm, such as in the Quantum system. In addition, this automated feeding task can reduce the possibility of contamination while maintaining a continuous flow of nutrients for the Treg culture, as it can be performed in a functionally closed system.

[0452] Possible Treg cell density and viability

[0453] Table 7: Possible harvests from T25 flasks

[0454] Cell density <![CDATA[3.11×10 6 Cells / mL]]> <![CDATA[8.71×10 5 Cells / cm 2 ]]> vitality 81.90％ Stimulation cycle 2 Multiplication 4.9

[0455] Table 8: Potential gains from Quantum

[0456] Cell density Harvest Bag <![CDATA[3.61×10 6 Cells / mL]]> Bioreactor <![CDATA[1.24×10 7 Cells / mL]]> <![CDATA[≥7.33×10 4 Cells / cm 2 <!-- 65 --> ]]> vitality 84.80％ Stimulation cycle 1 Mu...

Claims

1. A method for expanding cells in a cell expansion system, the method comprising: loading a first volume of fluid into the cell expansion system, the first volume of fluid comprising a plurality of cells, wherein the cell expansion system comprises a cell growth chamber; loading a second volume of fluid into a portion of the first fluid circulation path to position the first volume of fluid in a first portion of the cell growth chamber, the second volume of fluid comprising culture medium; feeding the cells by continuously circulating culture medium around an intracapillary circulation loop, an extracapillary circulation loop, or a combination thereof; and The cells are expanded.

2. The method of claim 1, further comprising continuously harvesting the cells.

3. The method according to claim 1, wherein: The fluid in the first fluid circulation path flows through the capillary space of the cell growth chamber.

4. The method according to claim 3, wherein: Fluid in the second fluid circulation path flows through the extracapillary space of the cell growth chamber.

5. The method according to claim 1, wherein: The first volume of fluid including the plurality of cells is loaded without activating the intracapillary circulation pump.

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

7. The method according to claim 1, further comprising: applying a coating agent to a cell growth surface of the cell growth chamber; Wherein, the coating agent comprises human fibronectin, cold precipitate or a combination thereof.

8. The method according to claim 7, further comprising: Ultrafiltration is used to move fluid through the hollow fiber membranes in the cell growth chamber and into the extracapillary circulation loop.

9. The method according to claim 8, wherein: The fluid includes the coating agent.

10. The method according to claim 1, wherein: The plurality of cells include a plurality of viral cells, a plurality of Vero cells, or a combination thereof.

11. The method according to claim 1, further comprising: The cell expansion system is rotated about a central axis using at least one rocking device.

12. A method for controlling a cell expansion system, comprising: receiving, by a controller, a data readout from at least one sensor; During the cell expansion process, events are tracked and recorded using a controller; recording, by the controller, the data reading from the at least one sensor; and Events and data readouts are reported by the controller on a display.

13. A method for controlling a group of cell expansion devices, comprising: generating, by a controller, a set of instructions for the cell expansion device group, wherein the cell expansion device group includes two or more cell expansion devices; The controller simultaneously sends a set of instructions for the group of cell expansion devices to each cell expansion device in the group of cell expansion devices; and Each cell expansion device in the population of cell expansion devices is programmed by the controller using the instruction set.

14. A method for controlling a cell expansion system, comprising: actuating, by a controller, a first pump to flow a first fluid at a first fluid flow rate; and actuating, by the controller, the second pump to cause the second fluid to flow at a second fluid flow rate; Wherein, the first fluid flow rate and the second fluid flow rate are opposite.

15. The method according to claim 14, wherein: The first fluid flow rate and the second fluid flow rate are less than 0.1 mL / min.

16. The method according to claim 15, wherein: The first fluid flow rate and the second fluid flow rate are 0.01 mL / min.

17. A culture medium bag for a cell expansion system, comprising: a flexible housing defining an interior space configured to contain a fluid; and a pair of ports, the pair of ports being arranged in parallel at the bottom of the flexible housing, wherein the flexible housing defines a hanger on the top of the interior space opposite to the pair of ports, and The pair of ports includes an inlet port and an outlet port.

18. The culture medium bag according to claim 17, wherein: The hanger includes a slot configured to receive one or more hooks for hanging the culture medium bag.

19. The culture medium bag according to claim 17, further comprising a rod, in, The hanger defines a passage in the housing for receiving the rod, and The rod and the hanger are configured to fit within the outer rail for hanging the culture medium bag.

20. A cell expansion system comprising: a cell expansion device comprising a housing and a holder, the housing defining a receptacle on an operating face of the housing and the holder protruding from the housing; a disposable kit configured to engage with a receptacle on the cell expansion device; and a culture medium bag configured to be suspended from a holder of the cell expansion device, wherein The culture medium bag has a flexible shell defining an interior space configured to contain a fluid, the flexible shell defining a hanger on a top of the interior space, the hanger comprising a rod extending longitudinally along the top of the culture medium bag, and The rod is configured to be received within a channel on the holder to secure the culture medium bag to the holder on the cell expansion device.

21. The cell expansion device according to claim 20, wherein: The disposable kit includes a cell growth chamber and tubing configured to connect the cell growth chamber to the culture medium bag.

22. The cell expansion device according to claim 20, wherein The retainer includes at least one protrusion, and The hanger defines a slot configured to receive the protrusion and suspend the culture medium bag.

23. A method for controlling a cell expansion system, comprising: actuating, by a controller, a first pump to flow a first fluid at a first fluid flow rate; actuating, by the controller, the second pump to cause the second fluid to flow at a second fluid flow rate; detecting, by a sensor, a parameter related to the first fluid or the second fluid; receiving, by a controller, a parameter from the sensor and determining whether the parameter is outside a predetermined range; and A remote alarm is sent by the controller to an external device separate from the cell expansion system.

24. The method according to claim 23, wherein: The remote alert includes an email, a text message, a digital alert, or a combination thereof.

25. The method of claim 23, further comprising: Activate an audio alarm, a visual alarm, or a combination thereof.

26. The method of claim 23, wherein: The sensor includes a thermistor, a pressure sensor, a gas sensor, an air detector, a metabolic sensor, or a combination thereof.

27. A method for controlling a cell expansion system, comprising: Detecting parameters related to cell expansion equipment; receiving, by a controller, the parameter and determining whether the parameter is outside a predetermined range; and A remote alarm is sent by the controller to an external device separate from the cell expansion system.

28. The method according to claim 27, wherein: The remote alert includes an email, a text message, a digital alert, or a combination thereof.

29. The method of claim 27, further comprising activating an audio alarm, a visual alarm, or a combination thereof.

30. The method of claim 27, wherein: The parameter is one of temperature, door position, pressure, flow rate and concentration.

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