Cell proliferation system

Abstract

To provide a cell proliferation system, proliferation method, control method, and culture medium bag capable of proliferating various adherent cells and suspension cells. [Solution] Cells are grown in a bioreactor and activated by an activator (e.g., a soluble activator complex). The nutrient supply and gas exchange functions of an automated closed cell proliferation system make it possible to seed at, for example, a low seeding density. By manipulating the parameters of the cell growth environment, cells are placed in specific locations within the bioreactor for efficient exchange of nutrients and gases. System parameters are adjusted to shear any cell colonies that may form during the proliferation phase. Metabolic concentrations are controlled to improve cell growth and viability. Cell retention within the bioreactor is controlled. In some embodiments, the cells include T cells. In other embodiments, the cells include a T cell subpopulation, for example, regulatory T cells (Tregs).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Patent Application No. 18 / 233,146, filed on 11 August 2023, claiming the benefits of U.S. Provisional Patent Application No. 63 / 399,524, filed on 19 August 2022, and U.S. Provisional Patent Application No. 63 / 405,998, filed on 13 September 2022. The entire disclosure of the above application is incorporated herein by reference.

[0002] This disclosure relates to a cell proliferation system. [Background technology]

[0003] This section provides background information relating to this disclosure that is not necessarily prior art.

[0004] Cell proliferation systems (CESs) are used to proliferate and differentiate cells. By using a cell proliferation system, various adherent and suspension cells can be proliferated (e.g., grown). The growth of both adherent and non-adherent cells takes place in the bioreactor of the cell proliferation system. [Overview of the project] [Problems that the invention aims to solve]

[0005] This section provides a general overview of the disclosure and does not constitute a comprehensive disclosure of its entire scope or all features. [Means for solving the problem]

[0006] In at least one exemplary embodiment, the present disclosure provides a method comprising the steps of introducing a first volume of fluid containing a plurality of cells into a cell growth system, the cell growth system comprising a cell growth chamber. The method also comprises the steps of introducing a second volume of fluid containing a culture medium into a portion of a first fluid circulation path to position the first volume of fluid in a first portion of the cell growth chamber; feeding the cells by continuously circulating the culture medium in an inner capillary circulation loop, an outer capillary circulation loop, or a combination thereof; and growing the cells.

[0007] In at least one exemplary embodiment, the method further comprises the step of continuously harvesting the cells.

[0008] In at least one exemplary embodiment, the fluid of the first fluid circulation path flows through the capillary inner space of the cell growth chamber.

[0009] In at least one exemplary embodiment, the fluid of the second fluid circulation path flows through the capillary outer space of the cell growth chamber.

[0010] In at least one exemplary embodiment, the first volume of fluid containing the plurality of cells is introduced without operating the capillary internal circulation pump.

[0011] In at least one exemplary embodiment, the first volume is the same as the second volume.

[0012] In at least one exemplary embodiment, the present disclosure provides a method for controlling a cell proliferation system. The method includes the steps of: receiving data readouts from at least one sensor by a controller; tracking and logging events during a cell proliferation process by the controller; recording the data readouts from the at least one sensor by the controller; and displaying the events and the data readouts on a display by the controller.

[0013] In at least one exemplary embodiment, the Disclosure provides a method for controlling a group of cell growth apparatuses. The method comprises the step of a controller generating a set of instructions for the group of cell growth apparatuses. The group of cell growth apparatuses comprises two or more cell growth apparatuses. The method also comprises the step of the controller simultaneously transmitting the set of instructions for the group of cell growth apparatuses to each of the cell growth apparatuses in the group of cell growth apparatuses, and the controller programming each of the cell growth apparatuses in the group of cell growth apparatuses with the set of instructions.

[0014] In at least one exemplary embodiment, the present disclosure provides a method for controlling a cell proliferation system. The method comprises the steps of: using a controller to activate a first pump to flow a first fluid at a first fluid flow rate; and using a controller to activate a second pump to flow a second fluid at a second fluid flow rate, wherein the first and second fluid flow rates are in opposite directions.

[0015] In at least one exemplary embodiment, the first fluid flow rate and the second fluid flow rate are less than 0.1 mL / min.

[0016] In at least one exemplary embodiment, the first fluid flow rate and the second fluid flow rate are 0.01 mL / min.

[0017] In at least one exemplary embodiment, the disclosure provides a culture medium bag for a cell growth system. The culture medium bag comprises a flexible housing having an internal space configured to contain a fluid, and a pair of ports arranged in parallel within the bottom of the flexible housing. The flexible housing has a hanger portion at the top of the internal space opposite the pair of ports, and the pair of ports include an inlet port and an outlet port.

[0018] In at least one exemplary embodiment, the hanger portion has slots configured to receive one or more hooks for suspending the culture medium bag.

[0019] In at least one exemplary embodiment, the culture medium bag comprises a rod. The hanger portion has a channel in the housing for receiving the rod, and the rod and the hanger portion are configured to fit into an external rail for suspending the culture medium bag.

[0020] In at least one exemplary embodiment, the present disclosure provides a cell proliferation system. The cell proliferation system comprises a cell proliferation apparatus including a housing and a holder. The housing has a receiving portion on its operating surface, and the holder protrudes from the housing. The cell proliferation system also comprises a disposable set configured to engage with the receiving portion of the cell proliferation apparatus, and a culture medium bag configured to be suspended from the holder of the cell proliferation apparatus. The culture medium bag has a flexible housing having an internal space configured to contain a fluid. The flexible housing has a hanger portion at the top of the internal space, the hanger portion including a rod extending longitudinally along the top of the culture medium bag. The rod is configured to be received in a channel of the holder to secure the culture medium bag to the holder of the cell proliferation apparatus.

[0021] In at least one exemplary embodiment, the disposable set comprises a cell growth chamber and a tube, the tube being configured to connect the cell growth chamber to the culture medium bag.

[0022] In at least one exemplary embodiment, the holder has at least one projection, and the hanger portion has a slot configured to receive the projection and suspend the culture medium bag.

[0023] In at least one exemplary embodiment, the present disclosure provides a method for controlling a cell proliferation system. The method includes the steps of: using a controller to activate a first pump to flow a first fluid at a first fluid flow rate; using a controller to activate a second pump to flow a second fluid at a second fluid flow rate; using a sensor to detect parameters related to the first or second fluid; using a controller to receive the parameters from the sensor and determine whether the parameters are outside a predetermined range; and using a controller to transmit a remote alarm to an external device separate from the cell proliferation system.

[0024] In at least one exemplary embodiment, the remote alarm includes email, text message, digital alert, or a combination thereof.

[0025] In at least one exemplary embodiment, the method further comprises the step of triggering an audible alarm, a visual alarm, or a combination thereof.

[0026] In at least one exemplary embodiment, the sensor includes a thermistor, a pressure sensor, a gas sensor, an air detector, or a combination thereof.

[0027] In at least one exemplary embodiment, the present disclosure provides a method for controlling a cell proliferation system. The method includes the steps of detecting parameters related to the cell proliferation system, a controller receiving the parameters and determining whether the parameters are outside a predetermined range, and the controller transmitting a remote alarm to an external device separate from the cell proliferation system.

[0028] In at least one exemplary embodiment, the remote alarm includes email, text message, digital alert, or a combination thereof.

[0029] In at least one exemplary embodiment, the method further comprises the step of triggering an audible alarm, a visual alarm, or a combination thereof.

[0030] In at least one exemplary embodiment, the parameter is one of temperature, door position, pressure, flow rate, and concentration.

[0031] The drawings herein are for illustrative purposes only of selected embodiments and are not intended to limit the scope of this disclosure to all possible embodiments. [Brief explanation of the drawing]

[0032] [Figure 1A] Figure 1A is a diagram of one embodiment of a cell proliferation system (CES) according to at least one exemplary embodiment. [Figure 1B] Figure 1B is a front view of one embodiment of a bioreactor, showing a circulation path through the bioreactor, according to at least one exemplary embodiment. [Figure 1C] Figure 1C is a perspective view of a first bioreactor and a second bioreactor according to at least one exemplary embodiment. [Figure 1D]Figure 1D shows a rocking device for moving a cell growth chamber in a rotational or lateral direction during operation of a cell proliferation system, according to at least one exemplary embodiment. [Figure 2A] Figure 2A is a front perspective view of a cell proliferation system according to at least one exemplary embodiment. [Figure 2B] Figure 2B is an internal perspective view of the cell proliferation system of Figure 2A, equipped with a pre-mounted fluid transfer device, according to at least one exemplary embodiment. [Figure 2C] Figure 2C shows the rail system and hook system of the holder of the cell proliferation system of Figure 2A, according to at least one exemplary embodiment. [Figure 3] Figure 3 is a perspective view of a cell proliferation system housing according to at least one exemplary embodiment. [Figure 4A] Figure 4A is a perspective view of a pre-mounted fluid transfer device according to at least one exemplary embodiment. [Figure 4B] Figure 4B is a diagram of the pre-mounted fluid transfer assembly of Figure 4A, according to at least one exemplary embodiment. [Figure 4C] Figure 4C is a diagram of the pre-mounted fluid transfer assembly of Figure 4A, according to at least one exemplary embodiment. [Figure 4D] Figure 4D is a diagram of the culture medium bag of the pre-mounted fluid transfer assembly of Figure 4A, according to at least one exemplary embodiment. [Figure 4E] Figure 4E is a diagram of the waste bag of the pre-mounted fluid transfer assembly of Figure 4A, according to at least one exemplary embodiment. [Figure 4F] Figure 4F is a cross-sectional view of the pressure pod of the pre-mounted fluid transfer assembly shown in Figure 4A, according to at least one exemplary embodiment. [Figure 4G] Figure 4G is an exploded view of the pressure pod of Figure 4F, according to at least one exemplary embodiment. [Figure 4H]Figure 4H is a diagram of the sampling coil of the pre-mounted fluid transfer assembly shown in Figure 4A, according to at least one exemplary embodiment. [Figure 4I] Figure 4I is a diagram of an inline filter of the pre-mounted fluid transfer assembly of Figure 4A, according to at least one exemplary embodiment. [Figure 5A] Figure 5A is a schematic diagram of a cell proliferation system including an operational configuration that shows fluid movement, according to at least one exemplary embodiment. [Figure 5B] Figure 5B is a schematic diagram of a cell proliferation system, including other operational configurations illustrating fluid motion, according to an embodiment of this disclosure. [Figure 5C] Figure 5C is a schematic diagram of a cell proliferation system including other operational configurations that demonstrate fluid movement, according to at least one exemplary embodiment. [Figure 6] Figure 6 is a schematic diagram of a cell proliferation system according to at least one exemplary embodiment. [Figure 7A] Figure 7A is a schematic diagram of a cell proliferation system according to at least one exemplary embodiment. [Figure 7B] Figure 7B is a schematic diagram of a cell proliferation system according to at least one exemplary embodiment. [Figure 7C] Figure 7C is a schematic diagram of a cell proliferation system according to at least one exemplary embodiment. [Figure 8] Figure 8 is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 9A] Figure 9A is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 9B] Figure 9B is a schematic diagram of a part of a cell proliferation system according to at least one exemplary embodiment. [Figure 10A] Figure 10A is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 10B]Figure 10B is a graph of oxygen consumption in a cell proliferation system according to at least one exemplary embodiment. [Figure 11A] Figure 11A is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 11B] Figure 11B shows a table of exemplary pump flow rates used in a cell proliferation system according to at least one exemplary embodiment. [Figure 12A] Figure 12A is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 12B] Figure 12B is a graph of cell proliferation metabolism according to at least one exemplary embodiment. [Figure 12C] Figure 12C is a graph of cell proliferation metabolism according to at least one exemplary embodiment. [Figure 13] Figure 13 is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 14] Figure 14 is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 15] Figure 15 is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 16A] Figure 16A is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 16B] Figure 16B shows a graph of cell number versus flow rate during cell proliferation according to at least one exemplary embodiment. [Figure 17] Figure 17 is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 18A] Figure 18A is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 18B]Figure 18B shows a diagram of cells growing in a cell proliferation system according to at least one exemplary embodiment. [Figure 18C] Figure 18C shows a graph illustrating the inner diameter of cell dissociation according to at least one exemplary embodiment. [Figure 19] Figure 19 shows a graph of cell number and flow rate per culture day during cell proliferation, according to at least one exemplary embodiment. [Figure 20A] Figure 20A is a flowchart illustrating the operational characteristics of a process in which a pump is operated to grow cells, according to at least one exemplary embodiment. [Figure 20B] Figure 20B is a schematic diagram of a part of a cell proliferation system according to at least one exemplary embodiment. [Figure 20C] Figure 20C is a schematic diagram of a part of a cell proliferation system according to at least one exemplary embodiment. [Figure 20D] Figure 20D is a schematic diagram of a part of a cell proliferation system according to at least one exemplary embodiment. [Figure 21] Figure 21 is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 22] Figure 22 is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 23] Figure 23 is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 24] Figure 24 is a flowchart illustrating the operational characteristics of a cell proliferation process according to at least one exemplary embodiment. [Figure 25] Figure 25 shows an exemplary processing system for a cell proliferation system that can carry out embodiments of the present disclosure according to at least one exemplary embodiment. [Figure 26] Figure 26 is a flowchart showing the flow of an exemplary experiment according to at least one exemplary embodiment. [Figure 27]Figure 27 is a graph showing an example of the results of a coating process according to at least one exemplary embodiment. [Figure 28] Figure 28 is a graph showing an example of the results of harvested cells after the coating process, according to at least one exemplary embodiment. [Figure 29] Figure 29 is a graph showing an example of the results of harvested cells after the coating process, according to at least one exemplary embodiment. [Figure 30] Figure 30 is a graph showing an example of the results for virus particles measured per day, according to at least one exemplary embodiment. [Figure 31] Figure 31 is a graph showing an example of the results of harvested cells after the coating process, according to at least one exemplary embodiment. [Figure 32] Figure 32 is a graph showing an example of the results of harvested cells after the coating process, according to at least one exemplary embodiment. [Modes for carrying out the invention]

[0033] Exemplary embodiments will be described in more detail with reference to the accompanying drawings.

[0034] Exemplary embodiments are provided for the sake of thoroughness of this disclosure and to ensure that the scope is fully conveyed to those skilled in the art. Numerous specific details, such as examples of specific components, apparatus, and methods, are described in order to provide a complete understanding of the embodiments of this disclosure. It will be apparent to those skilled in the art that specific details are not necessary, that the exemplary embodiments may be embodied in many different forms, and that none should be construed as limiting the scope of this disclosure. In some exemplary embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.

[0035] The terms used herein are intended to describe, and not limit, specific exemplary embodiments. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural form unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having” are inclusive and thus specify the existence of the described features, entities, steps, actions, elements, and / or components, but do not exclude the existence or addition of one or more other features, integers, steps, actions, elements, components, and / or sets thereof. The steps, processes, and actions of the methods described herein should not necessarily be construed as requiring execution in a specific order described or illustrated unless specifically identified as the order of execution. It should also be understood that additional or alternative steps may be used.

[0036] When an element or layer is referred to as "on top of," "engaged to," "connected to," or "bonded to" another element or layer, it may be directly on, directly engaged to, directly connected to, or directly bonded to the other element or layer, or an intervening element or layer may exist. On the other hand, when an element is referred to as "directly on top of," "directly engaged to," "directly connected to," or "directly bonded to" another element or layer, there is no intervening element or layer. Other terms used to describe relationships between elements should be interpreted similarly (e.g., "between" vs. "directly between," "adjacent" vs. "directly adjacent," etc.). As used herein, the term "and / or" includes any and all combinations of one or more of the related enumerated items.

[0037] The terms "first," "second," "third," etc., may be used herein to describe various elements, components, regions, layers, and / or sections, but these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are used solely to distinguish one element, component, region, layer, or part from other regions, layers, or parts. The terms "first," "second," etc., and other numerical terms, when used herein, do not imply order or sequence unless explicitly indicated by the context. Accordingly, the first element, first component, first region, first layer, or first section described below may also be called the second element, second component, second region, second layer, or second section, without departing from the teaching of the exemplary embodiments.

[0038] Terms indicating spatial relationships, such as “inside,” “outside,” “directly below,” “downward,” “below,” “above,” and “upwards,” are used herein to facilitate explanation when describing the relationship between one element or feature part and other elements or feature parts, as shown in the figures. Terms indicating spatial relationships may be intended to include different orientations of the device in use or operation, in addition to the orientation shown in the figures. For example, if the device in the figure is inverted, an element described as being “below” or “directly below” another element or feature part will be changed accordingly to being “above” that other element or feature part. Therefore, for example, the term “below” may include both up and down orientations. The device may be oriented in other directions (rotated 90 degrees or in other directions), and descriptions indicating spatial relationships used herein shall be interpreted accordingly.

[0039] Various components are referred to herein as “operably connected.” As used herein, “operably connected” refers to components that are connected together in an operable manner, and includes embodiments in which components are directly connected, as well as embodiments in which other components are positioned between connected components. “Operatively connected” components can be “fluidically connected.” “Fluidally connected” refers to components that are connected together so that fluid can be transported between them. The term “fluidically connected” includes embodiments in which one component is positioned between two fluidly connected components, and embodiments in which components are directly connected, etc. Fluidly connected components may include components that operate the system by contacting other components but not by contacting the fluid (for example, a peristaltic pump that pumps fluid through a flexible tube by compressing the outside of the tube).

[0040] As used herein, the term “donor” may mean any person who provides a fluid, such as whole blood, to an apheresis system. The donor may also be a patient who temporarily provides a fluid to the apheresis system, which is processed, treated, manipulated, etc., before being returned to the patient.

[0041] As used herein, the term “automatic” and its variations refer to any process or action that is performed without substantial human input when it is performed. However, even if significant or unsignificant human input is used in the execution of a process or action, the process or action may be automatic if such input is received before the execution of the process or action. Human input is considered significant if it affects how the process or action is performed. Human input that signifies consent to the execution of a process or action is not considered “significant.”

[0042] As used herein, the term “computer-readable medium” refers to any tangible storage device and / or transmission medium involved in providing instructions to a processor for execution. Such mediums can take many forms, but are not limited to non-volatile media, volatile media, and transmission media. Examples of non-volatile media include NVRAM, magnetic disks, or optical disks. Examples of volatile media include dynamic memory such as main memory. Common forms of computer-readable media include, for example, floppy disks, flexible disks, hard disks, magnetic 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 media such as memory cards, any other memory chips or cartridges, carriers as described below, or any other computer-readable media. Digital files or other embedded information archives or sets of archives attached to email are considered distribution media equivalent to tangible storage media. When a computer-readable medium is configured as a database, it should be understood that the database may be any type of database, such as relational, hierarchical, object-oriented, and / or similar. Accordingly, this disclosure is considered to include tangible storage media or distribution media on which a software implementation of the disclosure is stored, as well as equivalents and successor media recognized as prior art.

[0043] As used herein, the term “module” refers to any known or subsequently developed hardware, software, firmware, artificial intelligence, fuzzy logic, or combination of hardware and software capable of performing functions associated with such elements.

[0044] The terms “determine,” “calculate,” and “operate” as used herein, and their variations thereof, are to be used interchangeably and include any type of methodology, process, mathematical operation, or technique.

[0045] Embodiments of this disclosure will be described more fully with reference to the accompanying drawings, relating to apheresis methods and systems. The following embodiments are described with respect to the separation of blood components from whole blood. However, these are given for illustrative purposes only. The embodiments are not limited to the following description. The embodiments are intended for use in products, processes, apparatus, and systems for separating any complex fluids. Accordingly, this disclosure is not limited to the separation of blood components from whole blood.

[0046] The exemplary embodiments of this disclosure generally relate to systems and methods for cell proliferation in cell proliferation systems (CES). According to the embodiments, such proliferation is carried out through the use of a bioreactor or cell growth chamber. In one embodiment, such a bioreactor or cell growth chamber includes a hollow fiber membrane. Such a hollow fiber membrane includes a plurality of hollow fiber membranes and includes an outer capillary (EC) space and an inner capillary (IC) space. In the embodiments, adherent or non-adherent cells are grown or proliferated in the cell proliferation system. For example, non-adherent cells or suspension cells (e.g., T cells, T lymphocytes, or CD3+ selected cells) are proliferated in the system. In the embodiments, one or more subpopulations or subsets of T cells are grown. For example, the embodiments provide methods and systems for proliferating regulatory T cells (Tregs) and / or human regulatory T cells (hTregs).

[0047] In some exemplary embodiments, methods and systems for growing cells in a closed, automated cell growth system are provided. In one embodiment, such a cell growth system includes a bioreactor or a cell growth chamber. In further embodiments, such a bioreactor or cell growth chamber includes a hollow fiber membrane. The capabilities of such a system (e.g., nutrient supply and gas exchange functions) allow cells to be seeded at a low seeding density. In embodiments, parameters of the cell growth environment are adjusted so that cells are introduced or placed at a given location within the bioreactor for efficient exchange of nutrients and gases to the growing cells. For example, the cell density is increased by concentrating the cells within the bioreactor.

[0048] In exemplary embodiments, a population of non-adherent cells, such as T cells and / or Treg cells, is introduced or fed into a hollow fiber bioreactor, where the hollow fiber bioreactor may include multiple hollow fibers. In embodiments, cells are exposed to an activator to activate cell proliferation within the hollow fiber bioreactor. In one embodiment, multiple cells are introduced into the cell proliferation system using, for example, a task of “feeding cells into the center without circulation.” According to exemplary embodiments, such a task is performed on days 0 and 4-8. In other embodiments, it may be performed on other days. In embodiments, such a cell feeding task results in a concentration of cells within the bioreactor, increasing cell density. In other embodiments, cells may be placed in other parts or regions of the bioreactor to increase cell density. In embodiments, by placing cells in a first location (e.g., the central region) of the bioreactor, the cells can receive efficient exchange of nutrients and gases.

[0049] In exemplary embodiments, a lower cell seeding density can be used compared to the cell seeding density used in static culture methods. In embodiments using a cell proliferation system, cells (e.g., Treg or Treg cells) are 2.54 × 10⁻⁶ 5 Cell count / mL = 3.69 × 10⁴ 5Cells can be propagated at a cell seeding density of cells / mL. In other embodiments, the cell seeding density is approximately 1 × 10⁻⁶. 6 The cell count / mL is less than 1.0 × 10⁶. Furthermore, the Treg cell inoculation material is 1.0 × 10⁶. 5 It can be prepared from a cell seeding density of cell number / mL. In other methods (e.g., static Treg cell culture), in vitro proliferation is 1.0 × 10⁶ 6 A cell seeding density of Treg cells / mL is used. In one embodiment, a lower cell seeding density may be used, for example, for the overall efficiency of the system in delivering nutrients to the culture environment. In other embodiments, the use of a lower initial cell seeding density may be possible due to one or more steps used during proliferation, in combination with the overall efficiency of the system in delivering nutrients to the culture environment.

[0050] In exemplary embodiments, automated cell (e.g., Treg) proliferation is carried out using a soluble activator complex. In other embodiments, other types of activators are used, such as cell stimulation beads. In other embodiments, cells (e.g., Treg cells) are proliferated without the use of bead-based stimulation. In one embodiment, cell proliferation is performed in an automated cell proliferation system using Stem Cell Technologies' soluble ImmunoCult® human CD3 / CD28 / CD2 T cell activator in the presence of 200 IU / mL of cytokine IL-2 to activate and proliferate Treg cells. For example, using a soluble activator complex can reduce the cost of stimulation compared to the cost of a bead-based protocol. In other embodiments, other types of activators may be used. Furthermore, in other embodiments, other types of cytokines or other growth factors may be used.

[0051] In exemplary embodiments, system parameters are also adjusted or controlled to control cell retention within the bioreactor or cell growth chamber. For example, by controlling cell retention within the bioreactor hollow fibers during the cell growth phase, the system can provide efficient gas and nutrient exchange to the growing cells. In embodiments, the bioreactor may be designed to provide gas exchange and, in some embodiments, nutrient exchange to the growing cells. In exemplary embodiments, in a bioreactor having a semipermeable hollow fiber membrane, gas and nutrient exchange takes place through the semipermeable hollow fiber membrane. In embodiments, a method for providing culture medium components (e.g., various cytokines and proteins, etc.) to growing cells that cannot pass through the membrane may be by using a fluid inlet to the side of the bioreactor where the cells are growing (e.g., the inside of the capillary (IC) side). However, according to embodiments, at low, reduced, or decreased (e.g., minimum) inlet flow rates (e.g., 0.1 mL / min), cells may accumulate in the bioreactor outlet header. Cells present in the bioreactor header may not receive adequate gas and nutrient exchange, potentially leading to cell necrosis and aggregation.

[0052] Exemplary embodiments relate to providing a method for retaining cells (e.g., a population of non-adherent cells) within a bioreactor when feeding cells using an inlet (e.g., IC inlet) flow. In embodiments herein, the cells are on the IC side of the membrane during feeding, but in other embodiments, for example, the cells may be on the EC side of the membrane. According to embodiments, the cells may be contained within a first circulation path and / or a second circulation path. In embodiments, the feeding method pumps a first volume of fluid (e.g., culture medium or cell growth medium) into a first port of the bioreactor at a first volumetric flow rate, volumetric flow rate, fluid flow rate, flow rate, fluid flow rate, or volumetric velocity. For example, the terms volumetric flow rate, fluid flow rate, flow rate, fluid flow rate, or volumetric velocity are used interchangeably. In some embodiments, flow rate is a vector having both speed and direction. A second volume of fluid is pumped into a second port of the bioreactor at a second volumetric flow rate, volumetric flow rate, fluid flow rate, flow rate, fluid flow rate, or volumetric velocity. In embodiments, such volumetric flow rate, fluid flow rate, flow rate, fluid flow rate, or volumetric velocity is controlled by one or more pump speeds and / or pump flow rates. For example, the pump flow rate generates, causes, or affects the volumetric flow rate or flow rate of the fluid on which the pump acts. As used herein, pump speed, i.e., pump flow rate, is described in embodiments as the volumetric flow rate or fluid flow rate generated, caused, or affected by the pump.

[0053] In exemplary embodiments, the second flow rate of fluid to the bioreactor is in the opposite direction to the first flow rate of fluid to the bioreactor. For example, Figures 5B and 5C show exemplary operating configurations illustrating the flow rates and flow directions used in a cell proliferation system such as the CES500 (e.g., Figures 5B and 5C) according to embodiments of the present disclosure. In embodiments, a cell proliferation system pump (e.g., an IC pump) can be used to control cell retention within the bioreactor. In embodiments, cells are lost from the bioreactor and enter the IC circulation path or IC loop, for example, during the growth phase of growth. In embodiments, for example, cells in the bioreactor located near the IC inlet port receive the freshest growth medium, while cells in the portion of the IC circulation path outside the bioreactor essentially receive consumption medium or acclimatization medium that affects glycolysis metabolism. Furthermore, according to embodiments, cells in the bioreactor receive input of a mixed gas (O2, CO2, N2) from the gas transport module (GTM) by diffusion from the EC loop circulation, while cells in the portion of the IC circulation path outside the bioreactor do not.

[0054] In exemplary embodiments, reducing cell loss from a hollow fiber membrane (HFM) bioreactor can be achieved by matching, or substantially matching, the IC circulation pump flow rate to the IC inlet pump flow rate, albeit in the opposite direction, during feeding. For example, in embodiments, to maintain cells in the bioreactor during the growth phase of cell culture (e.g., days 4-7), the IC inlet pump flow rate is matched or substantially matched to a complementary IC circulation pump flow rate of -0.1 mL / min. This pump adjustment, according to embodiments, counteracts the forces associated with cell loss from the IC outlet port. In other embodiments, other pump flow rates may be used. For example, in other embodiments, the pump flow rates may be different. In embodiments, other pumps or additional pumps may be used. In one embodiment, fewer pumps may be used. Furthermore, in other embodiments, different periods may be used.

[0055] In exemplary embodiments, the metabolic activity of the cell population may affect the feeding parameters. For example, the cell culture lactate level is maintained below a predefined level. In one embodiment, the cell culture level is maintained below, for example, about 7 mmol / L. In embodiments, the lactate metabolic waste from glycolysis can be maintained below a predetermined level during cell proliferation (e.g., regulatory T cells) by controlling the culture medium flow rate using a graphical user interface (GUI) of the cell proliferation system. In other embodiments, for example, the culture medium flow rate and / or other settings are controlled to maintain, or attempt to maintain, a lactate level below about 5 mmol / L in order to improve, for example, cell proliferation and viability. In other embodiments, other concentrations may be used.

[0056] In additional embodiments, several system functions are utilized to shear cell colonies, micro-colonies, or cell clusters formed during the growth proliferation phase of cell growth. For example, in one embodiment, cell colonies (e.g., micro-colonies) passing through the hollow fiber membrane of a bioreactor are sheared to reduce the number of cells within the micro-colonies, colonies, or clusters. Here, the micro-colonies, colonies, or clusters are groups of one or more adherent cells. In embodiments, a cell expansion system (CES) bioreactor architecture can be used to shear cell (e.g., Treg cell) micro-colonies. In embodiments, as cells (e.g., Treg cells) grow, they tend to form micro-colonies. For cells at the center of such colonies, nutrient diffusion is restricted. This may thereby cause adverse effects such as necrosis during cell culture. In embodiments, during the growth proliferation phase, a protocol for shearing colonies is provided by circulating a suspension cell culture through, for example, the inner (IC) loop of a hollow fiber capillary (e.g., having a hollow fiber with an inner diameter of 215 μm). In embodiments, colonies, micro-colonies, or clusters of cells can be sheared to reduce the size of the colonies, micro-colonies, or clusters of cells. In one embodiment, colonies or clusters of cells can be sheared to provide a single cell suspension and improve cell growth / survival rate. Such an ability contributes to the continuous perfusion growth of cells (e.g., T cells or Tregs).

[0057] In an exemplary embodiment, a therapeutic dose of cells (e.g., Tregs) is expanded within a cell expansion system and harvested from the cell expansion system. In embodiments, the number of cells at harvest is from about 1×10 6 cells to about 1×10 10 cells (e.g., 1×10 9 cells). In one embodiment, the number of cells harvested is from about 1×10 8 to 1×10 10 cells, and in one example, from about 7.0×10 8 to about 1.4×10 9This is between cells. In some embodiments, the harvested cells have a viability of about 60% to about 100%. For example, the viability of the harvested cells is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the harvested cells express biomarkers consistent with Treg. For example, in some embodiments, the cells express CD4 + CD25 + , and / or FoxP3 + The cells express a biomarker. In this embodiment, the harvested cells express CD4 at a frequency of approximately 50% to 100%. + CD25 + This includes phenotype. Harvested cells were CD4 at a frequency of over 75%, over 80%, over 85%, over 90%, or over 95%. + CD25 + This includes the phenotype. In other embodiments, cells have a frequency of about 30% to about 100% CD4 + FoxP3 + This includes the phenotype. In some embodiments, the harvested cells were CD4 at frequencies of over 30%, over 35%, over 40%, over 45%, over 50%, over 55%, over 60%, over 65%, and even over 70%. + FoxP3 + Includes phenotype.

[0058] Exemplary embodiments relate to cell proliferation systems, as described above. In embodiments, the cell proliferation system is closed. The closed cell proliferation system contains contents that are not directly exposed to the atmosphere. Such cell proliferation systems may be automated. In embodiments, both adherent and non-adherent or suspension-type cells can be grown in the bioreactor of the cell proliferation system. According to embodiments, the cell proliferation system may include a basal medium or other types of medium. Methods for replenishing the medium are provided for cell growth carried out in the bioreactor of the closed cell proliferation system. In embodiments, the bioreactor used with such a system is a hollow fiber bioreactor. According to embodiments of the present invention, various bioreactors may be used.

[0059] In some embodiments, the system has a bioreactor hydrodynamically associated with a first fluid channel having at least two ends, the first end of the first fluid channel hydrodynamically associated with a first port of a hollow fiber membrane, and the second end of the first fluid channel hydrodynamically associated with a second port of the hollow fiber membrane. In some embodiments, the hollow fiber membrane comprises a plurality of hollow fibers. The system has a fluid inlet passage hydrodynamically associated with the first fluid channel, through which a plurality of cells are introduced into the first fluid channel. In some embodiments, the system further comprises a pump for transferring capillary inlet fluid from a capillary inlet culture bag into the first fluid channel, and a controller for controlling the operation of the pump. In some embodiments, the controller controls the pump to transfer, for example, cells from the cell inlet bag into the first fluid channel. Other pumps for circulating fluid in the first fluid circulation passage are also included. The pump has a controller for controlling the pump operation. In one embodiment, the controller is, for example, a computing system having a processor. In embodiments, one or more controllers control one or more pumps to circulate fluid in the first fluid circulation path at a certain flow rate. In embodiments, multiple controllers (e.g., a first controller, a second controller, a third controller, a fourth controller, a fifth controller, a sixth controller, etc.) may be used. In embodiments of the present disclosure, multiple pumps (e.g., a first pump, a second pump, a third pump, a fourth pump, a fifth pump, a sixth pump, etc.) may be used. Furthermore, although the present disclosure describes culture medium bags, cell loading bags, etc., multiple bags, e.g., a first culture medium bag, a second culture medium bag, a third culture medium bag, a first cell loading bag, a second cell loading bag, a third cell loading bag, etc., and / or other types of containers may be used. In other embodiments, a single culture medium bag, a single cell loading bag, etc., may be used. Also, in embodiments, additional or other flow paths (e.g., a second fluid flow path, a second fluid inlet path, a second fluid circulation path, etc.) may be provided.

[0060] In an exemplary embodiment, the system is controlled by, for example, a processor coupled to a cell proliferation system, a display device that communicates with the processor and displays data, and a memory that communicates with the processor and stores a set of instructions that are readable by the processor. In the embodiment, when an instruction is executed by the processor, the processor receives, for example, an instruction to prime the system. In response to that instruction, the processor performs a series of steps to prime the system and then receives, for example, an instruction to perform IC / EC washing. In response to an instruction to load cells, the processor performs, for example, a series of steps to load cells from a cell loading bag into a bioreactor.

[0061] An example of a cell growth system (CES) according to an embodiment of the present invention is schematically shown in Figure 1A. The CES 10 has a first fluid circulation path 12 and a second fluid circulation path 14. According to the embodiment, the first fluid circulation path 16 has at least two ends 18, 20 that are fluidically associated with a hollow fiber cell growth chamber 24 (also called a "bioreactor"). In detail, the end 18 is fluidically associated with a first inlet 22 of the cell growth chamber 24, and the end 20 is fluidically associated with a first outlet 28 of the cell growth chamber 24. In the first fluid circulation path 12, the fluid passes through the hollow fibers 116 (see Figure 1B) of a hollow fiber membrane 117 (see Figure 1B) located in the cell growth chamber 24 (the cell growth chamber and hollow fiber membrane will be described in more detail below). A first flow rate control device 30 is operably connected to the first fluid circulation path 16 and controls the fluid flow in the first fluid circulation path 12.

[0062] The second fluid circulation path 14 includes a second fluid channel 34, a cell growth chamber 24, and a second flow rate control device 32. According to the embodiment, the second fluid channel 34 has at least two ends 36 and 38. The end 36 of the second fluid channel 34 is fluidically associated with the inlet port 40 of the cell growth chamber 24, and the end 38 is fluidically associated with the outlet port 42. The fluid flowing through the cell growth chamber 24 is in contact with the outside of the hollow fiber membrane 117 (see Figure 1B) of the cell growth chamber 24. The hollow fiber membrane comprises a plurality of hollow fibers. The second fluid circulation path 14 is operably connected to the second flow rate control device 32.

[0063] Thus, the first fluid circulation path 12 and the second fluid circulation path 14 are separated in the cell growth chamber 24 by a hollow fiber membrane 117 (see Figure 1B). The fluid in the first fluid circulation path 12 flows in the inner capillary (IC) space of the hollow fiber in the cell growth chamber 24. Therefore, the first fluid circulation path 12 is called the "IC loop". The fluid in the second fluid circulation path 14 flows in the outer capillary (EC) space of the hollow fiber in the cell growth chamber 24. Therefore, the second fluid circulation path 14 is called the "EC loop". According to the embodiment, the fluid in the first fluid circulation path 12 may flow in either a parallel or reverse direction relative to the fluid flow in the second fluid circulation path 14.

[0064] The fluid inlet passage 44 is fluidically associated with the first fluid circulation passage 12. Through the fluid inlet passage 44, fluid is introduced into the first fluid circulation passage 12, and through the fluid outlet passage 46, the fluid is discharged from the CES 10. The third flow control device 48 is operably associated with the fluid inlet passage 44. Alternatively, the third flow control device 48 may be associated with the fluid outlet passage 46.

[0065] In some embodiments, the flow control device used herein may be a pump, a valve, a clamp, or a combination thereof. Multiple pumps, multiple valves, and multiple clamps may be arranged in any combination. In various embodiments, the flow control device is or includes a peristaltic pump. In some embodiments, the fluid circulation path, inlet port, and outlet port may consist of piping of any material.

[0066] In general, any type of fluid, such as buffers, protein-containing fluids, or cell-containing fluids, can flow through the circulation pathways, inlet pathways, and outlet pathways. The terms “fluid,” “culture medium,” and “fluid culture medium” as used herein are interchangeable.

[0067] Figure 1B is a front view showing an example of a hollow fiber cell growth chamber 100, i.e., a bioreactor 100, used in conjunction with this disclosure. The cell growth chamber 100 has a longitudinal axis LA-LA and comprises a cell growth chamber housing 104. In at least one embodiment, the cell growth chamber housing 104 has four openings or four ports, i.e., an IC inlet port 108, an IC outlet port 120, an EC inlet port 128, and an EC outlet port 132.

[0068] According to embodiments of the present invention, the fluid in the first circulation path enters the cell growth chamber 100 through the IC inlet port 108 at the first longitudinal end 112 of the cell growth chamber 100, enters and passes through the capillary side of the plurality of hollow fibers 116 constituting the hollow fiber membrane 117 (referred to as the capillary side ("IC") or "IC space" in various embodiments), and exits the cell growth chamber 100 through the IC outlet port 120 located at the second longitudinal end 124 of the cell growth chamber 100. The flow path between the IC inlet port 108 and the IC outlet port 120 constitutes the IC portion 126 of the cell growth chamber 100. The fluid in the second circulation path enters the cell growth chamber 100 through the EC inlet port 128, contacts the capillary side or outside (referred to as the "EC side" or "EC space") of the hollow fibers 116, and exits the cell growth chamber 100 through the EC outlet port 132. The flow path between the EC inlet port 128 and the EC outlet port 132 constitutes the EC portion 136 of the cell growth chamber 100. Fluid entering the cell growth chamber 100 through the EC inlet port 128 comes into contact with the outside of the hollow fiber 116. Small molecules (e.g., ions, water, oxygen, lactates, metabolites, nutrients, gases, etc.) can diffuse through the hollow fiber 116 from the inside of the hollow fiber, i.e., the IC space, to the outside, i.e., the EC space, or from the EC space to the IC space (e.g., continuous perfusion). For example, the hollow fiber 116 has a diffusion distance of 200 microns, providing more efficient transport of gases and nutrients compared to flask-type systems. High molecular weight molecules, such as growth factors, are usually too large to pass through the hollow fiber membrane and remain in the IC space of the hollow fiber 116. The hollow fiber 116 exhibits lower shear stress compared to agitated bioreactors and wave-shaking bioreactors. In embodiments, the culture medium may be replaced if necessary. Furthermore, if necessary, the culture medium may be circulated through an oxygenator or gas transfer module to exchange the gas. In the embodiments described below, the cells can be contained within the first and / or second circulation pathways and can be located on the IC side and / or EC side of the membrane.

[0069] The material used to produce the hollow fiber membrane 117 can be any biocompatible polymer material, as long as it can form hollow fibers. According to one embodiment of the present invention, one example of a material used is a synthetic polysulfone-based material.

[0070] According to some embodiments, as shown in Figure 1C, two or more cell growth chambers 100 of different sizes may be provided separately or included in a single disposable kit. For example, a pair of cell growth chambers 100A and 100B may be provided separately or included in a single disposable kit. In at least one exemplary embodiment, cell growth chamber 100A is smaller than cell growth chamber 100B and has a smaller capacity for cells to grow. For example, cell growth chamber 100A is one-tenth the size of cell growth chamber 100B, or 0.1 times the size. In at least one exemplary embodiment, cell growth chamber 100A (i.e., miniature cell growth chamber 100A) is advantageous for experiments, pediatric use, or other events (e.g., skin grafting) using small batches of starter cells. Since miniature cell growth chamber 100A can grow small batches of cells, its use is advantageous when the initial cell source is too small for cell growth chamber 100B. For further growth, small cell batches may be transferred to cell growth chamber 100B. Thus, the small cell growth chamber 100A provides further opportunities to use CES10 when cell growth chamber 100B is not optimal. In some exemplary embodiments, CES10 automatically recognizes whether cell growth chamber 100A or cell growth chamber 100B is installed and identifies appropriate process parameters, as further described below. Including two or more sizes of cell growth chambers 100 increases the applications of CES10. For example, it can handle bone marrow-derived mesenchymal stem cells (MSCs), adipose tissue-derived MSCs, umbilical cord MSCs, fibroblasts, keratinocytes, HEK293 T cells, human embryonic stem cells (ESCs), periosteum-derived cells, induced pluripotent stem cell-derived MSCs (IPS-MSCs), neural stem cells (NSCs), osteochondral progenitor cells, endothelial cells, dendritic cells, induced pluripotent stem cells (iPSCs), T cells, viral vectors, exosome production, and the proliferation of autologous and allogeneic adherent and suspension cell types.For example, multiple cell growth chambers 100 for CES10 provide the ability to grow up to 300 million to 1 billion MSCs, or up to 25 billion T cells, per run.

[0071] In at least one exemplary embodiment, the CES (e.g., CES500 (Figures 5A, 5B, and 5C) and / or CES600 (Figure 6)) has a device configured to move, i.e., "rock" the cell growth chamber relative to other components of the cell growth system by being attached to rotational and / or lateral rocking devices. Figure 1D shows one such exemplary device. In one embodiment, in the device, the cell growth chamber 100 is connected to rotate two rotational rocking components and one lateral rocking component.

[0072] The first rotational oscillation component 138 rotates the cell growth chamber 100 around its central axis 142. The first rotational oscillation component 138 is associated with the cell growth chamber 100 to rotate. In some embodiments, the cell growth chamber 100 can rotate continuously in a single direction (clockwise or counterclockwise) around the central axis 142. Alternatively, the cell growth chamber 100 can rotate alternately around the central axis 142, for example, first clockwise, then counterclockwise.

[0073] The CES also has a second rotational oscillating component that rotates the cell growth chamber 100 around a rotation axis 144. The rotation axis 144 passes through the center point of the cell growth chamber 100 and is perpendicular to the central axis 142. In embodiments, the cell growth chamber 100 can rotate continuously in a single direction, either clockwise or counterclockwise, around the rotation axis 144. Alternatively, the cell growth chamber 100 can rotate alternately around the rotation axis 144, for example, first clockwise and then counterclockwise. In various embodiments, the cell growth chamber 100 can be rotated around the rotation axis 144 and positioned horizontally or vertically relative to gravity.

[0074] In some embodiments, the lateral oscillation component 140 is associated with the cell growth chamber 100 to move laterally. In embodiments, the plane of the lateral oscillation component 140 moves in the x and y directions. This reduces cell sedimentation in the cell growth chamber due to the movement of the cell-containing medium within the hollow fibers.

[0075] The rotational and / or lateral movement of the oscillating device reduces cell sedimentation within the device and also minimizes the possibility of cells being trapped in a portion of the cell growth chamber. The rate of cell sedimentation in the cell growth chamber is proportional to the density difference between the cells and the suspension medium, according to Stokes' equation. In one embodiment, as described above, the suspension of non-adherent erythrocytes is maintained by repeating 180-degree rotations (fast) and pauses (e.g., a total time of 30 seconds). In exemplary embodiments, the cell growth chamber 100 can be rotated from a minimum rotation of about 180 degrees to a maximum rotation of about 290 to about 300 degrees, but rotations up to 360 degrees or more can also be used. Different oscillating components may be used separately or in combination. For example, an oscillating component that rotates the cell growth chamber 100 around the central axis 142 can be combined with an oscillating component that rotates the cell growth chamber 100 around axis 144. Similarly, clockwise and counterclockwise rotations can be independently combined around different axes.

[0076] Figures 2A and 2B show an embodiment of a cell proliferation system 200 having a pre-mounted fluid transfer assembly according to an embodiment of the present invention. The CES200 has a cell proliferation apparatus 202. The cell proliferation apparatus 202 has a hatch or a closable door 204 that engages with a rear portion 206 of the cell proliferation apparatus 202. An internal space 208 within the cell proliferation apparatus 202 has functionally characterized portions that receive, engage, and secure the pre-mounted fluid transfer assembly. The pre-mounted fluid transfer assembly 210 is removablely attached to the cell proliferation apparatus 202, allowing for relatively rapid replacement of a used fluid transfer assembly 210 with a new or unused pre-mounted fluid transfer assembly 210 in the cell proliferation apparatus 202. The operation of a single cell growth apparatus 202 allows for the growth or proliferation of a first set of cells using a first pre-mounted fluid transfer assembly 210, and then, without sterilizing the cell growth apparatus 202 when replacing the first pre-mounted fluid transfer assembly 210 with a second pre-mounted fluid transfer assembly 210, a second set of cells can be grown or proliferated using the second pre-mounted fluid transfer assembly 210. The pre-mounted fluid transfer assembly 210 may include a cell growth chamber 100 and an oxygen supply or gas transfer module 212 (see Figure 4A). A pipe guide slot 214 is shown in an embodiment for receiving various culture medium pipes connected to the pre-mounted fluid transfer assembly 210.

[0077] The front panel 262 of the CES200 includes a user interface 264. The user interface 264 includes a display 268, such as a touchscreen, to enable the user to input data, retrieve data, input test protocols, switch displays, display data, display alarms, etc. Additionally or alternatively, the user interface 264 may include one or more buttons or switches for inputting information, controlling the display, or performing other functions. Input from the user interface 264 is transmitted to the control system as described below. The front panel 262 of the CES200 may include one or more lights or other visual signals for indicating alarms.

[0078] Holders 270, such as bag holders and disposable holders, extend from the upper surface 272 of the CES 200. The holders 270 include vertical legs 274 and horizontal legs 276, arranged, for example, as an L-shaped support 278. Line supports or clamps 280 are attached to the vertical legs 274 of the L-shaped support 278 to support a portion of the disposable assembly, as described below. For example, a line support 280 supports the tubes of the disposable assembly against the vertical legs 274. Multiple racks 282 are supported by the horizontal legs 276 of the L-shaped support 278. Each of the multiple racks 282 supports one or more bags of a disposable set, as described below. For example, as shown in Figure 2C, each of the multiple racks includes a rail system 284, or a hook system 286, or both a rail system 284 and a hook system 286. The rail system 284 includes side walls 288 that extend parallel to each other and define the channel 290 between them. The side walls 288 are formed of metal, plastic, composite material, or other suitable material. In exemplary embodiments, each side wall 288 has a second side wall or bumper on its inner surface that defines the neck 292, i.e., the narrow portion, of the channel 290. For example, the second side wall 293 is formed of an elastic or flexible material such as rubber. Alternatively, the second side wall may be formed of an inelastic material such as metal, plastic, composite material, or other suitable material. For example, the neck 292 is the narrow portion in the channel 290, and the neck 292 holds the top of the bag or disposable item within the channel 290. The rail system 284 functions to distribute the load of the supported bag and reduce rupture, stretching, or tearing of the bag material. The rail system 284 can accommodate various different bag types and sizes. The bag suspended by the rail system 284 includes a rod for receiving and holding the bag within the channel 290, as described below. The rod within the bag has a diameter greater than the width of the neck 292 defined by the second side wall 293.

[0079] A slot 294 is formed on the bottom surface of the second side wall 293. The slot 294 is located on the side of the second side wall 293 adjacent to the side wall 288. The slot 294 is positioned to receive one or more hooks 296 of the hook system 286. For example, the slots 294 on one second side wall 293 of each rail system 284 may receive the hooks 296 of the hook system 286, while the slots 294 on the other second side wall 293 of the rail system 284 may be left empty. The hooks 296 may be U-shaped or J-shaped and are configured to secure the bag at the opening of the bag.

[0080] The cell proliferation system 200 and / or cell proliferation apparatus 202 includes a barcode scanner configured to scan barcodes of disposable items, culture media, formulations, etc., used with the cell proliferation system 200 and / or cell proliferation apparatus 202. The barcode scanner is configured to collect data from scanned barcodes and transfer that data to a computing system 2400 as described herein. The barcode scanner may be wired or wirelessly connected. The barcode scanner may be a handheld device, or it may be configured within a user interface or fixed on the housing of the cell proliferation apparatus 202. The barcode scanner is compatible with reading customer barcodes, general-purpose barcodes, supplier barcodes, or any barcode that is or is not on the market.

[0081] Figure 3 shows the rear portion 206 of the cell growth apparatus 202 before the pre-mounted fluid transfer assembly 210 (see Figure 2B) is detachably mounted according to an embodiment of the present disclosure. The closable door 204 (see Figures 2A and 2B) is omitted in Figure 3. The rear portion 206 of the cell growth apparatus 202 is provided with several different components that work in conjunction with the components of the pre-mounted fluid transfer assembly 210. In detail, the rear portion 206 of the cell growth apparatus 202 has several peristaltic pumps (IC circulation pump 218, EC circulation pump 220, IC inlet pump 222, EC inlet pump 224) that cooperate with the pump loop in the pre-mounted fluid transfer assembly 210. Furthermore, the rear portion 206 of the cell proliferation device 202 has a plurality of valves (IC circulation valve 226, reagent valve 228, IC culture medium valve 230, air removal valve 232, cell inlet valve 234, washing valve 236, distribution valve 238, EC culture medium valve 240, IC waste or outlet valve 242, EC waste valve 244, harvest valve 246). In addition, a plurality of sensors (IC outlet pressure sensor 248, composite type IC inlet pressure / temperature sensor 250, composite type EC inlet pressure / temperature sensor 252, EC outlet pressure sensor 254) are associated with the rear portion 206 of the cell proliferation device 202. According to one embodiment, an optical sensor 256 for the air removal chamber is further shown.

[0082] According to the embodiment, an axis or oscillation control unit 258 for rotating the cell growth chamber 100 is provided. An axis fitting portion 260 associated with the axis or oscillation control unit 258 allows for proper alignment of the axis access opening of the pre-mounted fluid transfer assembly 210 or 400 (e.g., the opening 424 (Figure 4A) of the piping storage portion 300 (Figure 4A)) with respect to the rear portion 206 of the cell growth apparatus 202. The rotation of the axis or oscillation control unit 258 imparts rotational motion to the axis fitting portion 260 and the cell growth chamber 100. Therefore, when a CES200 operator or user attaches a new or unused pre-mounted fluid transfer assembly 400 (Figure 4A) to the cell growth apparatus 202, the alignment becomes a relatively simple task of properly orienting the axis access opening of the pre-mounted fluid transfer assembly 210 or 400 (e.g., the opening 424 in Figure 4A) with respect to the axis fitting portion 260.

[0083] Figure 4A is a perspective view of a removable pre-mounted fluid transfer assembly 400. The pre-mounted fluid transfer assembly 400 is removablely mounted to the cell growth apparatus 202 (Figures 2B and 3), allowing for relatively quick replacement of a used pre-mounted fluid transfer assembly 400 with a new or unused one in the cell growth apparatus 202. As shown in Figure 4A, the cell growth chamber 100 is mounted to a bioreactor coupling including an axial fitting 402. The axial fitting 402 has one or more axial fastening mechanisms (biased arms or spring members 404) for engaging the shaft (258 in Figure 3) of the cell growth apparatus 202.

[0084] According to one embodiment, the pre-mounted fluid transfer assembly 400 includes piping 408A, 408B, 408C, 408D, 408E, etc., and pipe fittings. These provide fluid flow paths as shown in Figures 5A, 5B, 5C, and 6, as described below. Pump loops 406A, 406B, and 406C are also provided for the pump. In one embodiment, various culture media may be provided where the cell growth apparatus 202 is located, but according to one embodiment, the pre-mounted fluid transfer assembly 400 has piping of sufficient length to extend outside the cell growth apparatus 202, so that piping associated with culture medium bags or culture medium containers can be welded to it.

[0085] Figures 4B to 4H show various diagrams of the assembly and components of the pre-mounted fluid transfer assembly 400. In exemplary embodiments, the pre-mounted fluid transfer assembly 400 includes a culture bag 410 (Figure 4D), a waste bag 412 (Figure 4E), a pressure pod 414 (Figures 4F to 4G), a sampling coil 416 (Figure 4H), a cell loading bag, an in-line filter 420 (Figure 4I), tubing, filters, or a combination thereof. For example, the pre-mounted fluid transfer assembly 400A in Figure 4B includes a cell growth chamber 100B. Similarly, the pre-mounted fluid transfer assembly 400B in Figure 4C also includes a cell growth chamber 100A. Except for the difference in size between the cell growth chambers 100A and 100B, the other components of the pre-mounted fluid transfer assemblies 400A and 400B are the same. This enables the efficient manufacturing of the pre-mounted fluid transfer assembly 400.

[0086] As shown in Figures 4D and 4E, the pre-mounted fluid transfer assembly 400 includes one or more culture bags 410 and one or more waste bags 412. In some embodiments, the culture bag 410 is the same as the waste bag 412. Making the culture bag 410 the same as the waste bag 412 allows for more efficient and lower-cost manufacturing. As shown in Figures 4D and 4E, the culture bag 410 and waste bag 412 are formed from a single sheet of material and have a container portion 422 and a hanger portion 425. For example, the material sheet, culture bag 410, and waste bag 412 are formed from three-layer EVA (ethylene vinyl acetate), which is less permeable than PVC, in order to maintain a more stable pH. This makes it "suitable" for the culture medium, and such an impermeable material extends the shelf life of the culture medium. For example, the material sheet is welded together to form the container portion 422 and the hanger portion 425. The container portion 422 may be a pocket within the culture bag 410 or the waste bag 412. The container portion 422 is formed to a size that holds a specific amount of fluid (e.g., culture medium). For example, the container portion 422 is formed to a size that holds 5 L of fluid. Alternatively, the container portion 422 may be formed to a size that holds an appropriate amount of fluid for the desired function.

[0087] The hanger section 425 includes an opening for receiving the hook 296, as described with reference to Figure 2C. The hanger section 425 includes a rod 426 fixed within the longitudinal opening or channel 428 of the culture bag 410 or waste bag 412. The rod 426 may be a cylindrical rod or an extruded polygonal rod that engages with the rail system 284, as described with reference to Figure 2C. For example, the rod 426 rests on the top of the second side wall 293 when the bag is inserted into the channel 290 between the side walls 288. The rod 426 has a larger diameter or width than the neck 292, so that the hanger section 425 does not slip down through the neck 292 and the culture bag 410 or waste bag 412 is suspended within the rail system 284. Additionally or alternatively, the culture bag 410 may be made of a different material, size, or construction than the waste bag 412.

[0088] A pair of ports 430 are located on the container section 422 opposite the hanger section 425. The pair of ports 430 includes an inlet port 430A and an outlet port 430B. The inlet port 430A engages with the flow of tubes or fluids into the container section 422. The outlet port 430B engages with the flow of tubes or fluids out of the container section 422. The dual-port design of the pair of ports 430, i.e., the culture bag 410 or waste bag 412, allows multiple culture bags 410 or multiple waste bags 412 to be connected together or daisy-chained, as described below.

[0089] Referring to Figures 4F and 4G, the pressure pod 414 is located in the housing of the pre-mounted fluid transfer assembly 400. The pressure pod 414 may be configured to detect fluid pressure through a fluid line, tube, or along a fluid flow path. The pressure pod 414 includes a single outlet housing 433, a diaphragm 432, and a retaining ring 434. The single outlet housing 433 includes a tubular inlet port 436 and a closure port 438. The inlet port 436 is a tubular port configured to receive fluid into the single outlet housing 433. The closure port 438 may be similar to the tubular inlet port 436 but is closed with a plug 440 to prevent fluid flow. The plug 440 may be made of plastic or elastomer. The single outlet housing 433 has a body 442 integrally formed with the inlet port 436 and the closure port 438. The body 442 has an internal space 444, for example, a cylindrical internal space 444. The diaphragm 432 may be a circular planar diaphragm housed within the internal space 444 of the body 442. For example, the diaphragm 432 is made of an elastic material that flexes under fluid pressure. The retaining ring 434 is positioned at the end of the body 442 of the single outlet housing 433 opposite the inlet port 436 and the closure port 438 to secure the diaphragm 432 within the body 442. For example, the retaining ring 434 may be press-fitted or screwed into the end of the body 442 opposite the inlet port 436 and the closure port 438.

[0090] Figures 4H and 4I show exemplary tubing included in the pre-mounted fluid transfer assembly 400. In at least one exemplary embodiment, the sampling coil 416 shown in Figure 4H is a tube configured to take a sample of fluid in one of the fluid channels. In at least one exemplary embodiment, the in-line filter 420 shown in Figure 4I includes a filter positioned within the tube to filter substances from the fluid moving through the fluid channel.

[0091] Next, Figures 5A, 5B, and 5C are schematic diagrams of an embodiment of the cell proliferation system 500. Figure 6 is a schematic diagram of another embodiment, the cell proliferation system 600. Furthermore, Figures 7A to 7C show schematic diagrams of yet another embodiment of the cell proliferation system. In the embodiments shown in Figures 5A, 5B, 5C, and 6, cells are grown in an IC space, as will be described below. However, this disclosure is not limited to such examples. In other embodiments, cells may be grown in an EC space.

[0092] As previously mentioned, Figures 5A, 5B, and 5C show the CES500. While Figures 5A, 5B, and 5C show substantially similar components of the CES500, they also show a possible operational configuration for fluid movement in a first fluid circulation path using the structural features of the CES500 according to embodiments of the present disclosure. As illustrated, the CES500 has a first fluid circulation path 502 (also called the “inner capillary loop” or “IC loop”) and a second fluid circulation path 504 (also called the “outer capillary loop” or “EC loop”). The first fluid passage 506 is fluidically associated with the cell growth chamber 501 and constitutes the first fluid circulation path 502. Fluid flows into the cell growth chamber 501 via the IC inlet port 501A and flows out through the hollow fibers within the cell growth chamber 501 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 used to control the culture medium flow rate. The IC circulation pump 512 can pump the fluid in a first direction or a second direction opposite to the first direction. The outlet port 501B can be used as an inlet in the reverse direction. For example, in the first configuration, the IC circulation pump can pump the fluid in the positive direction, and the fluid enters the IC inlet port 501A. In the second configuration, for example, the IC circulation pump can pump the fluid in the negative direction, and the fluid enters the IC outlet port 501B.

[0093] The culture medium flowing into the IC loop can be introduced via valve 514. As those skilled in the art will understand, 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 in certain parts of the fluid path. Thus, the schematic diagram shown should be understood to be one possible configuration for multiple elements of the CES500 and to be modifiable within the scope of one or more embodiments.

[0094] For the IC loop 502, a sample of the culture medium is obtained during operation from the sample port 516 or the sample coil 518. The pressure and temperature of the culture medium can be measured during operation by a pressure / temperature measuring instrument 520 located in the first fluid circulation path 502. The culture medium then returns to the IC inlet port 501A, completing the fluid circulation path 502. Cells grown / proliferated in the cell growth chamber 501 are flushed out of the cell growth chamber 501 and enter the cell harvest bag 599 through the valve 598 or are redistributed into the hollow fibers for further growth.

[0095] In the second fluid circulation path 504, the fluid enters the cell growth chamber 501 via the EC inlet port 501C and leaves the cell growth chamber 501 via the EC outlet port 501D. In the EC loop 504, the culture medium comes into contact with the outside of the hollow fibers of the cell growth chamber 501, thereby enabling the diffusion of small molecules into and from the hollow fibers.

[0096] In one embodiment, the pressure and temperature of the culture medium can be measured by a pressure / temperature measuring instrument 524 located in the second fluid circulation path 504 before the culture medium enters the EC space of the cell growth chamber 501. After the culture medium leaves the cell growth chamber 501, the pressure of the culture medium in the second fluid circulation path 504 can be measured by a pressure measuring instrument 526. For the EC loop, a sample of the culture medium is obtained during operation from the sample port 530 or from the sample coil.

[0097] In one 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 oxygen supply or gas transfer module 532. The EC circulation pump 528 is capable of pumping fluid in both directions. The second fluid circulation path 522 is fluidly associated with the oxygen supply or gas transfer module 532 via the oxygen supply inlet port 534 and the oxygen supply outlet port 536. During operation, the fluid culture medium flows into the oxygen supply or gas transfer module 532 via the oxygen supply inlet port 534 and out of the oxygen supply or gas transfer module 532 via the oxygen supply outlet port 536. The oxygen supply or gas transfer module 532 adds oxygen to the culture medium in, for example, the CES 500 and removes bubbles. In various embodiments, the culture medium in the second fluid circulation path 504 is in equilibrium with the gas entering the oxygen supply or gas transfer module 532. The oxygen supply or gas transfer module 532 may be any oxygen supply or gas transfer device of appropriate size. Air or gas flows into the oxygen supply or gas transfer module 532 via filter 538 and flows out of the oxygen supply or gas transfer module 532 via filter 540. Filters 538 and 540 reduce or prevent contamination of the oxygen supply or gas transfer module 532 and associated culture media. Air or gas purged from the CES 500 during part of the priming process can be vented to the atmosphere via the oxygen supply or gas transfer module 532.

[0098] In at least one embodiment, the culture medium containing cells (from bag 562) and the fluid culture medium from bag 546 are introduced into the first fluid circulation path 502 via the first fluid channel 506. The fluid container 562 (e.g., a cell inlet bag or a saline priming fluid for priming air out of the system) is fluidically associated with the first fluid channel 506 and the first fluid circulation path 502 via a valve 564.

[0099] A fluid container, i.e., a culture medium bag 544 (e.g., a reagent), is fluidically connected to a first fluid inlet passage 542 via a valve 548, or to a second fluid inlet passage 574 via a valve 576. Another fluid container 546 (e.g., an IC culture medium) is fluidically connected to the first fluid inlet passage 542 via a valve 550, or to the second fluid inlet passage 574 via a valve 570. Sterilized and sealable first and second input priming passages 508, 509 are also provided. An air removal chamber (ARC) 556 is fluidically connected to a first circulation passage 502. The air removal chamber 556 may have one or more ultrasonic sensors. These sensors include upper and lower sensors for detecting air, fluid deficiency, and / or gas / fluid boundaries, e.g., air / fluid boundaries, at specific measurement points within the air removal chamber 556. For example, ultrasonic sensors can be used near the bottom and / or top of the air removal chamber 556 to detect air, fluid, and / or air / fluid boundaries at those locations. In embodiments, other types of sensors can be used without departing the scope of the present disclosure. For example, optical sensors may be used according to embodiments of the present disclosure. Air or gas purged from the CES500 during part of the priming process or other protocols can be vented to the atmosphere through the air valve 560 via a line 558 fluidly associated with the air removal chamber 556.

[0100] EC culture medium (e.g., from bag 568) or washing solution (e.g., from bag 566) is added to the first or second fluid channel. Fluid container 566 is fluidically associated with valve 570. Valve 570 is fluidically associated with the first fluid circulation channel 502 via the distribution valve 572 and the first fluid inlet channel 542. Also, by opening valve 570 and closing distribution valve 572, fluid container 566 can be fluidically associated with the second fluid circulation channel 504 via the second fluid inlet channel 574 and the EC inlet channel 584. Similarly, fluid container 568 is fluidically associated with valve 576. Valve 576 is fluidically associated with the first fluid circulation channel 502 via the first fluid inlet channel 542 and the distribution valve 572. Also, by opening valve 576 and closing distribution valve 572, fluid container 568 can be fluidically associated with the second fluid inlet channel 574.

[0101] A heat exchanger 552 may be optionally provided for the introduction of culture medium reagents or washing solutions.

[0102] In the IC loop, the fluid is first supplied by the IC inlet pump 554. In the EC loop, the fluid is first supplied by the EC inlet pump 578. An air detector 580, such as an ultrasonic sensor, may be associated with the EC inlet passage 584.

[0103] In at least one embodiment, the first and second fluid circulation lines 502 and 504 are connected to a waste line 588. When valve 590 is opened, the IC medium flows through the waste line 588 to a waste bag or outlet bag 586. Similarly, when valve 582 is opened, the EC medium flows through the waste line 588 to a waste bag or outlet bag 586.

[0104] In this embodiment, cells are harvested through a cell harvesting pathway 596. Here, cells from the cell growth chamber 501 are harvested by pumping the IC medium containing the cells through the cell harvesting pathway 596 and valve 598 and sending it to a cell harvesting bag 599.

[0105] The various components of the CES500 are housed in a device or housing, such as a cell proliferation device 202 (see Figures 2B and 3), which maintains the cells and culture medium at a predetermined temperature, for example.

[0106] In the configuration shown for the CES500 in Figure 5A, the fluid culture medium in the first fluid circulation path 502 and the second fluid circulation path 504 flows through the cell growth chamber 501 in the same direction (parallel flow configuration) in one embodiment. In other embodiments, the CES500 may be configured to flow through a counterflow structure (not shown). In the configuration shown in Figure 5A, the fluid in the first fluid circulation path 502 enters the bioreactor 501 at the IC inlet port 501A and exits the bioreactor 501 at the IC outlet port 501B. In the configurations shown in Figures 5B and 5C, in embodiments, the fluid culture medium in the first fluid circulation path 502 flows in both opposing directions from the connector 517 to enter the IC inlet port at one end of the bioreactor, i.e., the first port 501A, and also to the IC outlet port at the other end of the bioreactor, i.e., the second port 501B, and retains cells in the bioreactor itself. The first fluid flow path may be fluidically associated with the first fluid circulation path via the connector 517. In embodiments, the connection 517 may be a point or location where fluid flows in both opposing directions, for example, based on the direction of the IC inlet pump and the direction of the IC circulation pump. In one embodiment, the connection 517 may be a T-fitting or T-connection. In other embodiments, the connection 517 may be a Y-fitting or Y-connection. The connection 517 may be any type of fitting, coupling, weld, path, pipe, etc., that allows the first fluid passage to be fluidically associated with the first circulation path. It should be understood that the schematic diagrams and operating configurations shown in Figures 5A, 5B, and 5C represent possible configurations of various elements of the cell proliferation system, and modifications to the shown schematic diagrams and operating configurations are within the scope of one or more embodiments.

[0107] Figure 6 is a schematic diagram of another embodiment of the cell growth system 600. The CES600 has a first fluid circulation path 602 (also called the “inner capillary loop” or “IC loop”) and a second fluid circulation path 604 (also called the “outer capillary loop” or “EC loop”). The first fluid flow path 606 is fluidically associated with the cell growth chamber 601 and constitutes the first fluid circulation path 602. Fluid flows into the cell growth chamber 601 via the IC inlet port 601A, flows through the hollow fibers within the cell growth chamber 601, and flows out via the IC outlet port 601B. A pressure sensor 610 measures the pressure of the culture medium leaving the cell growth chamber 601. In addition to pressure, the sensor 610 may be a temperature sensor that detects the culture medium pressure and culture medium temperature during operation. The culture medium flows via an IC circulation pump 612 used to control the culture medium flow rate. The IC circulation pump 612 can pump the fluid in a first direction or a second direction opposite to the first direction. The outlet port 601B can be used as an inlet in the reverse direction. The culture medium flowing into the IC loop can be introduced through valve 614. As those skilled in the art will understand, 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 in certain parts of the fluid path. Thus, the schematic diagram shown should be understood to be one possible configuration for multiple elements of the CES600 and to be modifiable within the scope of one or more embodiments.

[0108] For the IC loop, a sample of the culture medium is obtained from the sample coil 618 during operation. The medium then returns to the IC inlet port 601A, completing the fluid circulation path 602. Cells grown / proliferated in the cell growth chamber 601 are flushed out of the cell growth chamber 601 and enter the harvest bag 699 via valve 698 and cell harvest path 697. Alternatively, if valve 698 is closed, the cells are redistributed back into chamber 601 for further growth.

[0109] In the second fluid circulation path 604, the fluid enters the cell growth chamber 601 via the EC inlet port 601C and leaves the cell growth chamber 601 via the EC outlet port 601D. In the EC loop, the culture medium comes into contact with the outside of the hollow fibers of the cell growth chamber 601, thereby enabling the diffusion of small molecules into and from the hollow fibers within the chamber 601.

[0110] Before the culture medium enters the EC space of the cell growth chamber 601, the pressure and temperature of the culture medium can be measured by a pressure / temperature sensor 624 located in the second fluid circulation path 604. After the culture medium leaves the cell growth chamber 601, the pressure and / or temperature of the culture medium in the second fluid circulation path 604 can be measured by a sensor 626. For the EC loop, a sample of the culture medium is obtained during operation from the sample port 630 or from the sample coil.

[0111] After leaving the EC outlet port 601D of the cell growth chamber 601, the fluid in the second fluid circulation path 604 passes through the EC circulation pump 628 to the oxygen supply or gas transfer module 632. In some embodiments, the EC circulation pump 628 is also capable of pumping fluid in both directions. The second fluid passage 622 is fluidically associated with the oxygen supply or gas transfer module 632 via its inlet port 632A and outlet port 632B. During operation, the fluid culture medium flows into the oxygen supply or gas transfer module 632 via the inlet port 632A and out of the oxygen supply or gas transfer module 632 via the outlet port 632B. The oxygen supply or gas transfer module 632 adds oxygen to the culture medium in, for example, the CES 600 and removes bubbles. In various embodiments, the culture medium in the second fluid circulation path 604 is in equilibrium with the gas entering the oxygen supply or gas transfer module 632. The oxygen supply or gas transfer module 632 may be any device of appropriate size that is useful for supplying oxygen or transferring gas. Air or gas flows into the oxygen supply or gas transfer module 632 via filter 638 and flows out of the oxygen supply or gas transfer module 632 via filter 640. Filters 638 and 640 reduce or prevent contamination of the oxygen supply or gas transfer module 632 and associated culture media. Air or gas purged from the CES 600 during part of the priming process can be vented to the atmosphere via the oxygen supply or gas transfer module 632.

[0112] In the configuration shown as CES600, the fluid culture medium in the first fluid circulation path 602 and the second fluid circulation path 604 flows through the cell growth chamber 601 in the same direction (parallel flow configuration). The CES600 according to this embodiment may be configured to flow in a countercurrent direction.

[0113] In at least one embodiment, a culture medium containing cells (from a source such as a cell container, e.g., a bag) is attached to attachment point 662, and a fluid culture medium from a culture medium source is attached to attachment point 646. The cells and culture medium are introduced into a first fluid circulation path 602 via a first fluid channel 606. Attachment point 662 is fluidically associated with the first fluid channel 606 via a valve 664. Attachment point 646 is fluidically associated with the first fluid channel 606 via a valve 650. A reagent source may be fluidly connected to point 644 and associated with a first fluid inlet path 642 via a valve 648, or with a second fluid inlet path 674 via valves 648, 672.

[0114] An air removal chamber (ARC) 656 is fluidically associated with the first circulation path 602. The air removal chamber 656 may have one or more sensors. These sensors include upper and lower sensors for detecting air, fluid deficiencies, and / or gas / fluid boundaries, e.g., air / fluid boundaries, at specific measurement points within the air removal chamber 656. For example, ultrasonic sensors can be used near the bottom and / or top of the air removal chamber 656 to detect air, fluid, and / or air / fluid boundaries at those locations. In embodiments, other types of sensors can be used without departing the scope of the disclosure. For example, optical sensors may be used according to embodiments of the disclosure. Air or gas purged from the CES 600 during part of the priming process or other protocols can be vented to the atmosphere through an air valve 660 via a line 658 fluidically associated with the air removal chamber 656.

[0115] An EC medium source is attached to the EC medium attachment point 668. A washing solution source is attached to the washing solution attachment point 666. This allows the EC medium and / or washing solution to be added to the first or second fluid channel. Attachment point 666 is fluidically associated with valve 670. Valve 670 is fluidically associated with the first fluid circulation channel 602 via valve 672 and the first fluid inlet passage 642. By opening valve 670 and closing valve 672, attachment point 666 can also be fluidically associated with the second fluid circulation channel 604 via the second fluid inlet passage 674 and the second fluid channel 684. Similarly, attachment point 668 is fluidically associated with valve 676. Valve 676 is fluidically associated with the first fluid circulation channel 602 via the first fluid inlet passage 642 and valve 672. Furthermore, by opening valve 676 and closing distribution valve 672, the mounting point 668 can be fluidly associated with the second fluid inlet passage 674.

[0116] In the IC loop, the fluid is first supplied by the IC inlet pump 654. In the EC loop, the fluid is first supplied by the EC inlet pump 678. An air detector 680, such as an ultrasonic sensor, may be associated with the EC inlet passage 684.

[0117] In at least one embodiment, the first and second fluid circulation lines 602 and 604 are connected to a waste line 688. When valve 690 is opened, the IC medium flows through the waste line 688 to a waste bag or outlet bag 686. Similarly, when valve 692 is opened, the EC medium flows to the waste bag or outlet bag 686.

[0118] After the cells have grown in the cell growth chamber 601, they are harvested via the cell harvesting pathway 697. The cells from the cell growth chamber 601 are harvested into the cell harvesting bag 699 via the cell harvesting pathway 697 by pumping the IC medium containing the cells with the valve 698 open.

[0119] The various components of the CES600 are housed in a device or housing, such as a cell proliferation device 202 (see Figures 2B and 3), which maintains the cells and culture medium at a predetermined temperature, for example. Furthermore, the components of the CES600 and CES500 may be combined. In other embodiments, the CES may contain fewer or more components than those shown in the CES500 and CES600, and this is also within the scope of the present invention. An example of a cell proliferation system incorporating the features of this disclosure is the Quantum® cell proliferation system manufactured by Terumo BCT, Inc. (Lakewood, Colorado).

[0120] The schematic diagram shown in Figure 6 represents possible configurations of various elements of a cell proliferation system, and modifications to the schematic diagram shown are within the scope of one or more embodiments.

[0121] Examples and detailed descriptions of the cell proliferation system are given in U.S. Patent No. 8,309,347, “Cell Proliferation System and Method of Use,” issued November 13, 2012, and U.S. Patent No. 9,057,045, filed December 15, 2010, “Method for Cell Input and Distribution in a Bioreactor for a Cell Proliferation System,” issued June 16, 2015. The entirety of those U.S. patent applications is expressly incorporated herein by this disclosure.

[0122] In the embodiments shown in Figures 7A to 7C, cells are grown in an IC space, as described below. However, the present invention is not limited to such examples. In other embodiments, cells may be grown in an EC space.

[0123] As previously mentioned, Figures 7A to 7C show the CES700. Figures 7A to 7C show substantially similar components of the CES700, and at the same time, Figures 7A to 7C show a possible operational configuration for fluid movement in a first fluid circulation path using the structural features of the CES700 according to embodiments of the present disclosure. As shown, the CES700 has a first fluid circulation path 702 (also called the “inner capillary loop” or “IC loop”) and a second fluid circulation path 704 (also called the “outer capillary loop” or “EC loop”). The first fluid passage 706 is fluidically associated with the cell growth chamber 701 and constitutes the first fluid circulation path 702. The fluid flows into the cell growth chamber 701 via the IC inlet port 701A and flows out through the hollow fibers within the cell growth chamber 701 via the 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 used to control the flow rate of the culture medium. The IC circulation pump 712 is capable of pumping the fluid in a first direction or a second direction opposite to the first direction. In at least one exemplary embodiment, the IC outlet port 701B may be used as an inlet in the reverse direction. For example, in the first configuration, the IC circulation pump 712 can pump the fluid in the positive direction, and the fluid enters the IC inlet port 701A. In the second configuration, for example, the IC circulation pump 712 can pump the fluid in the negative direction, and the fluid enters the IC outlet port 701B.

[0124] The fluid in the IC loop, i.e., the first fluid circulation path 702, passes through the IC circulation valve 714. As those skilled in the art will understand, additional valves, pressure gauges, pressure / temperature sensors, ports, and / or other devices can be placed at various locations to isolate and / or measure the properties of a culture medium in a portion of the fluid path. For example, the first fluid circulation path 702 has an IC inlet pressure sensor 715. In some exemplary embodiments, the IC inlet pressure sensor 715 is located between the IC circulation pump 712 and the IC circulation valve 714. Thus, the schematic diagram shown should be understood to be one possible configuration for multiple elements of the CES700 and to be modifiable within the scope of one or more embodiments.

[0125] For the first fluid circulation path 702, a sample of the culture medium is obtained during operation from the sample port or sample coil 718. The pressure and temperature of the culture medium can be measured during operation by a pressure / temperature measuring instrument 720 located in the first fluid circulation path 702. The culture medium then returns to the IC inlet port 701A, completing the first fluid circulation path 702. Cells grown / proliferated in the cell growth chamber 701 are flushed out of the cell growth chamber 701 and enter the cell harvest bag 799 through the valve 798, or are redistributed into the hollow fibers for further growth.

[0126] In the second fluid circulation path 704, the fluid enters the cell growth chamber 701 via the EC inlet port 701C and leaves the cell growth chamber 701 via the EC outlet port 701D. In the second fluid circulation path 704, the culture medium comes into contact with the outside of the hollow fibers of the cell growth chamber 701, thereby enabling the diffusion of small molecules into and from the hollow fibers.

[0127] In at least one exemplary embodiment, the pressure and temperature of the culture medium can be measured by a pressure / temperature measuring instrument 724 located in the second fluid circulation path 704 before the culture medium enters the EC space of the cell growth chamber 701. After the culture medium leaves the cell growth chamber 701, the pressure of the culture medium in the second fluid circulation path 704 can be measured by a pressure measuring instrument 726. For the EC loop, a sample of the culture medium is obtained during operation from a sample port 730 or from a sample coil.

[0128] 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 to the oxygen supply or gas transfer module 732. The EC circulation pump 728 is capable of pumping fluid in both directions. The second fluid passage 722 is fluidly associated with the oxygen supply or gas transfer module 732 via the oxygen supply inlet port 734 and the oxygen supply outlet port 736. During operation, the fluid culture medium flows into the oxygen supply or gas transfer module 732 via the oxygen supply inlet port 734 and out of the oxygen supply or gas transfer module 732 via the oxygen supply outlet port 736. The oxygen supply or gas transfer module 732 adds oxygen to the culture medium in, for example, CES 700 and removes bubbles. In various exemplary embodiments, the culture medium in the second fluid circulation path 704 is in equilibrium with the gas entering the oxygen supply or gas transfer module 732. The oxygen supply or gas transfer module 732 may be any oxygen supply or gas transfer device of appropriate size. Air or gas flows into the oxygen supply or gas transfer module 732 via filter 738 and flows out of the oxygen supply or gas transfer module 732 via filter 740. Filters 738 and 740 reduce or prevent contamination of the oxygen supply or gas transfer module 732 and associated culture media. Air or gas purged from the CES 700 during part of the priming process can be vented to the atmosphere via the oxygen supply or gas transfer module 732.

[0129] In at least one embodiment, a culture medium containing cells from bag 762 and a fluid culture medium from bag 746 are introduced into a first fluid circulation path 702 via a first fluid channel 706. The fluid container 762 (e.g., a cell inlet bag or a saline priming fluid for priming air out of the system) is fluidically associated with the first fluid channel 706 and the first fluid circulation path 702 via a valve 764.

[0130] Culture medium bags 744 (e.g., reagents) and containers 746 (e.g., IC culture medium) are fluidically associated with the first fluid inlet passage 742 via corresponding valves 748 and 750, respectively, or fluid containers are fluidically associated with the second fluid inlet passage 774 via corresponding valves 770 and 776, respectively. An air removal chamber (ARC) 756 is fluidically associated with the first fluid circulation passage 702. The air removal chamber 756 may have one or more ultrasonic sensors. The sensors include upper and lower sensors for detecting air, fluid deficiencies, and / or gas / fluid boundaries, e.g., air / fluid boundaries, at specific measurement points within the air removal chamber 756. For example, ultrasonic sensors can be used near the bottom and / or top of the air removal chamber 756 to detect air, fluid, and / or air / fluid boundaries at those locations. In exemplary embodiments, other types of sensors can be used without departing from the scope of the present disclosure. For example, optical sensors may be used according to embodiments of the present disclosure. Air or gas purged from the CES700 during part of the priming process or other protocols can be vented to the atmosphere through an air valve 760, which is fluidly associated with the air removal chamber 756.

[0131] EC culture medium (e.g., from bag 768) and / or washing solution (e.g., from bag or fluid container 766) are added to the first fluid inlet passage 742 and / or the second fluid passage 722. Fluid container 766 is fluidically associated with valve 770. Valve 770 is fluidically associated with the first fluid circulation passage 702 via the distribution valve 772 and the first fluid inlet passage 742. Also, by opening valve 770 and closing distribution valve 772, fluid container 766 can be fluidically associated with the second fluid circulation passage 704 via the second fluid inlet passage 774 and the EC inlet passage 784. Similarly, as shown in Figure 7C, fluid containers 768A and 768B are fluidically associated with valve 776. Valve 776 is fluidically associated with the first fluid circulation passage 702 via the first fluid inlet passage 742 and the distribution valve 772. Furthermore, by opening valve 776 and closing distribution valve 772, fluid containers 768A and 768B can be fluidly associated with the second fluid inlet passage 774.

[0132] The fluid containers 768A and 768B may be culture medium containers for distributing culture medium into the first fluid inlet passage 742 or the second fluid inlet passage 774. In some exemplary embodiments, the fluid containers 768A and 768B may be the same as the culture medium bag 410 shown in Figure 4D. The fluid containers 768A and 768B may be part of the EC circulation supply loop 752 when communicating with the second fluid inlet passage 774 (or part of the IC circulation supply loop when communicating with the first fluid inlet passage 742). In the EC circulation supply loop 752, the culture medium is sent from the fluid container 768B to the fluid container 768A, through the EC loop 704, through the EC waste valve 782, and back to the fluid container 768B. By using the EC circulation supply loop 752, the use of pass-through mass feeding, in which fresh culture medium is continuously added to the system and collected in an outlet waste bag which is then discarded when full, is avoided. In pass-through high-volume feeding, the more cells you grow, the higher the flow rate of culture medium to the cells must be. Users typically measure glucose or lactate levels once a day and then increase the flow rate for that day, for example. The flow rate may not be accurate throughout the day, leading to wasted culture medium.

[0133] On the other hand, in the CES700 as specified herein, the EC circulation supply loop 752 expands the capacity of either the IC circulation loop or the EC circulation loop by connecting fluid container 768A to fluid container 768B (via the aforementioned pair of ports) or by daisy-chaining them. In the CES700, the cell growth chamber 701 is supplied manually from outside the CES700. The outlet line 788 returns to fluid container 768B, and the medium is recirculated. The user can calculate how much lactic acid is produced and determine the waste. The user does not need to change bags, only set the supply flow rate and execute the protocol. Thus the CES700 is more efficient and uses less medium overall.

[0134] Two fluid containers 768A and 768B are illustrated and described, but it will be understood that more fluid containers 768N may be added to the system.

[0135] In the exemplary protocol, gas is supplied to the system via a GTM such as GTM732 using EC circulation. The gas is supplied externally to CES700, and each time the medium passes through GTM732, the gas is exchanged to the desired concentration. In this case, fluid containers 768A and 768B serve as exchange containers for the medium returning to the cell growth chamber 701. For example, the accompanying T cell feeding protocol (CFA-CSS-CEdj-036-03) uses approximately 11 L of EC medium and 3.4 L of IC medium over the duration of the growth protocol. Using this new feeding method, the user adds 3.4 L of IC medium to the IC medium line and daisy-chains 11 L (3 medium bags) of EC medium and adds it to the EC inlet and outlet lines. As the oxygen demand of the cell population increases, the EC circulation flow rate needs to be increased, as does the EC inlet flow rate (effectively the EC supply circulation).

[0136] Depending on the cell type and protocol dynamics, either an IC supply cycle or an EC supply cycle is implemented. Alternatively, a dual supply cycle can be used, for example, as shown in Figure 7C.

[0137] This method allows for the implementation of numerous possible feeding techniques. It also improves the ease of use of the CES700, reducing human-to-instrument interaction as there is no need to empty waste bags. New culture medium bags are supplied to the instrument at various intervals during cell proliferation. Furthermore, in many of these situations, there is no need to determine the supply flow rate. Even with continuous perfusion feeding, lactate and glucose concentrations can fluctuate significantly without using very long custom tasks that involve gradually changing the flow rate.

[0138] Depending on the desired application, the EC circulation supply loop 752 may be used to coat the hollow fibers of the cell growth chamber 701. In this case, the fluid circulates and is pushed through the membrane (IC side). Additionally or alternatively, the EC circulation supply loop 752 may be used in countercurrent techniques. In this case, the fluid is pushed at both ends of the cell growth chamber 701.

[0139] The passive coating model used in the CES system requires coating the cell growth surface with a coating agent to promote cell adhesion and subsequent proliferation of adherent cell lines such as human mesenchymal stromal cells (hMSCs). Examples of coating agents include human fibronectin (hFN) or cryoprecipitate (CPPT). The bioreactor coating protocol for the passive coating model involves introducing the coating agent into the capillaries of the bioreactor and circulating the coating agent in the IC circulation loop for a minimum of 16 hours. The passive coating model protocol requires at least two CES systems to immediately initiate further proliferation of the cell population harvested from the CES system (hMSCs can only be stored for a maximum of 16 hours in non-freezing conditions). A new protocol or method for coating the hollow fibers of the cell growth chamber 701 completes the coating of the growth surface 10 minutes after introducing the coating agent into the IC circulation loop. This method (see Appendix B for the protocol) utilizes active ultrafiltration of the fluid (fluid movement from the IC side to the EC side of the bioreactor) to reduce the time required for the proper chemical reaction between the coating agent and the growth surface of the bioreactor. A molecular barrier formed by the specific structure of the hollow fibers within the bioreactor acts as a barrier preventing the coating agent from passing through the hollow fiber walls along with the fluid it is suspended in. By using active ultrafiltration to move the fluid, the movement of the coating agent on the surface of the hollow fibers is "actively" promoted. Enabling users to coat hollow fiber bioreactors and introduce cells on the same day reduces errors, saves time, and reduces the time required for cell proliferation by one day. This allows customers with access to only a single CES instrument (e.g., a Quantum® / Quantum Flex® instrument) to harvest cell populations from that system and subsequently coat new disposable items for subculturing / growing the same cell population without the need for cryopreservation.

[0140] As illustrated with reference to Figures 4D and 4E, a two-port bag can be used, allowing the inlet line (typically a "wash" line) to be connected to one port of the bag and the "discard" line to be connected to the other port simultaneously. This allows the protein medium solution (usually phosphate-buffered saline (PBS)) containing the coating agent to be recirculated within the system for as long as the user desires. Using a high IC inlet flow rate to facilitate contact between the coating solution and the HFB membrane via ultrafiltration (UF) promotes the adsorption of the coating agent and reduces the time required to coat the hollow fiber bioreactor compared to a passive model of coating. Moving the IC fluid across the HFB membrane also promotes the deposition of the coating agent on the IC side of the HFB membrane. For example, the coating solution has a higher molecular weight and cannot pass through the HFB membrane, so it is deposited on the IC side of the HFB membrane.

[0141] Optionally, a heat exchanger may be provided for the introduction of culture medium reagents or washing solution. In some embodiments, a heat exchanger may optionally be provided in the first fluid inlet passage 742 and / or the second fluid inlet passage 774.

[0142] One or more differential pressure sensors may be included for real-time pressure monitoring and alarming. This allows for more efficient and accurate measurements compared to manual measurements. Pressure sensors facilitate the detection of decreased or stopped flow, depleting gas supply, user errors, etc. When a decrease or stoppage in flow is detected, the differential pressure sensor triggers an alarm to warn the user. For example, the alarm may be a remote alarm, an audible alarm on the device, a visual alarm on the device, or a combination thereof. For example, a remote alarm may be an email, text message, or other digital alert sent to the user.

[0143] One or more gas regulators may be provided for gas management. For example, the gas regulator may be located inside the CES700 so as not to be visible to the user. This prevents errors. The gas regulator may have low sensitivity to the manipulated variable. In this case, fine control can be performed. If an error is detected in gas management, the gas regulator may trigger an alarm to warn the user. For example, the alarm may be a remote alarm, an audible alarm on the device, a visual alarm on the device, or a combination thereof. For example, a remote alarm may be an email, text message, or other digital alert sent to the user.

[0144] For highly sensitive temperature detection, one or more temperature thermistors may be provided. A temperature change of 0.5 degrees is significant in cell culture. Thermistors degrade less than resistance temperature detectors (RTDs) and therefore can be used for a longer period before inspection is required. If a temperature change outside a predetermined range, or a temperature above or below a predetermined threshold is detected, the thermistor may trigger an alarm to warn the user. For example, the alarm may be a remote alarm, an audible alarm on the device, a visual alarm on the device, or a combination thereof. For example, a remote alarm may be an email, text message, or other digital alert sent to the user.

[0145] In the IC loop, the fluid is first supplied by the IC inlet pump 754. In the EC loop, the fluid is first supplied by the EC inlet pump 778. An air detector 780, such as an ultrasonic sensor, may be associated with the EC inlet passage 784. When air is detected by the air detector 780, the air detector may trigger an alarm to warn the user. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alarm on the device, or a combination thereof. For example, a remote alarm may be an email, text message, or other digital alert sent to the user.

[0146] In at least one embodiment, the first and second fluid circulation paths 702 and 704 are connected to an outlet line 788. When valve 790 is opened, the IC medium flows through the outlet line 788 and returns to the fluid container 768B. Similarly, when valve 782 is opened, the EC medium returns through the outlet line 788 to the fluid container 768B.

[0147] In an exemplary embodiment, cells are harvested via a cell harvesting pathway 796. Here, cells from the cell growth chamber 701 are harvested by pumping the IC medium containing the cells through the cell harvesting pathway 796 and valve 798 into a cell harvesting bag 799.

[0148] The various components of the CES700 are housed in a device or housing, such as the cell proliferation device 202 (see Figures 2A, 2B, and 3), which maintains the cells and culture medium at, for example, a predetermined temperature.

[0149] In the configuration shown for the CES700 in Figure 7A, the fluid culture 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 (parallel flow configuration) in one embodiment. In other embodiments, the CES700 may be configured to flow through a counterflow structure (not shown). In the configuration shown in Figure 7A, the fluid in the first fluid circulation path 702 enters the cell growth chamber 701 at the IC inlet port 701A and exits the cell growth chamber 701 at the IC outlet port 701B. In other configurations, in embodiments, the fluid culture medium in the first fluid circulation path 702 flows in both opposing directions from the connector 717 to enter the IC inlet port at one end of the cell growth chamber 701, i.e., the first port 701A, and also to the IC outlet port at the other end of the cell growth chamber 701, i.e., the second port 701B, and holds the cells in the cell growth chamber itself. The first fluid flow path may be fluidically associated with the first fluid circulation path via the connector 717. In embodiments, the connection 717 may be a point or location where fluid flows in both opposing directions, for example, based on the direction of the IC inlet pump and the direction of the IC circulation pump. In one embodiment, the connection 717 may be a T-fitting or T-connection. In other embodiments, the connection 717 may be a Y-fitting or Y-connection. The connection 717 may be any type of fitting, coupling, weld, path, tube, etc., that allows the first fluid passage to be fluidically associated with the first circulation path. It should be understood that the schematic diagrams and operating configurations shown in Figures 7A to 7C represent possible configurations of various elements of the cell proliferation system, and modifications to the shown schematic diagrams and operating configurations are within the scope of one or more embodiments.

[0150] Figure 7B shows the CES700B. The CES700B may be the same as the CES700A and may contain the same or similar components as the CES700A, in which case the same reference number is used. The CES700B includes an IC circulation supply loop 753. The IC circulation supply loop 753 may be the same as the EC circulation supply loop 752 described above. The IC circulation supply loop 753 may include a plurality of fluid containers, for example, fluid container 746A and fluid container 746B. Fluid containers 746A and 746B may be the same as the culture medium bag 410 shown in Figure 4D.

[0151] Fluid containers 746A and 746B may be culture medium containers for distributing culture medium to the first fluid channel 706 via the IC culture medium line 746. In the IC circulating supply loop 753, the culture medium is sent from fluid container 746B to fluid container 746A, through the IC loop 702, through the cell growth chamber 701 and the IC waste valve 790, and back to fluid container 746B. By using the IC circulating supply loop 753, the use of pass-through bulk feeding is avoided, in which fresh culture medium is continuously added to the system, collected in an outlet waste bag, and discarded when full. In pass-through bulk feeding, the more cells to grow, the higher the culture medium flow rate to the cells must be. Users typically measure glucose or lactate once a day and then increase the flow rate for that day. The flow rate may not be accurate throughout the day, leading to culture medium waste.

[0152] On the other hand, in the CES700 described herein, the IC circulation supply loop 753 expands the capacity of the IC circulation loop by connecting fluid container 746A to fluid container 746B (via the aforementioned pair of ports) or by daisy-chaining them. In the CES700, the cell growth chamber 701 is supplied manually from outside the CES700. The outlet line 788 returns to fluid container 746B, and the medium is recirculated. The user can calculate how much lactic acid is produced and determine the waste. The user does not need to change bags, only set the supply flow rate and execute the protocol. Thus, the CES700 is more efficient and uses less medium overall.

[0153] Two fluid containers 746A and 746B are illustrated and described, but it will be understood that more fluid containers 746N may be added to the system.

[0154] This method allows for the implementation of numerous possible feeding techniques. It also improves the usability of the CES700, reducing human-to-instrument interaction as there is no need to empty waste bags. New culture medium bags are supplied to the instrument at various intervals during cell growth. Furthermore, in many of these situations, there is no need to determine the supply flow rate. Even with continuous perfusion feeding, lactate and glucose concentrations can fluctuate significantly without using very long custom tasks that involve gradually changing the flow rate.

[0155] Depending on the desired application, the IC circulation supply loop 753 may be used to coat the hollow fibers of the cell growth chamber 701. In this case, the fluid circulates and flows through the membrane (IC side). Additionally, or alternatively, the IC circulation supply loop 753 may be used in countercurrent techniques. In this case, the fluid flows through both ends of the cell growth chamber 701.

[0156] One or more differential pressure sensors may be included for real-time pressure monitoring and alarming. This allows for more efficient and accurate measurements compared to manual measurements. Pressure sensors facilitate the detection of decreased or stopped flow, depleting gas supply, user errors, etc. When a decrease or stoppage in flow is detected, the differential pressure sensor triggers an alarm to warn the user. For example, the alarm may be a remote alarm, an audible alarm on the device, a visual alarm on the device, or a combination thereof. For example, a remote alarm may be an email, text message, or other digital alert sent to the user.

[0157] One or more gas regulators may be provided for gas management. For example, the gas regulator may be located inside the CES700 so as not to be visible to the user. This prevents errors. The gas regulator may have low sensitivity to the manipulated variable. In this case, fine control can be performed. If an error is detected in gas management, the gas regulator may trigger an alarm to warn the user. For example, the alarm may be a remote alarm, an audible alarm on the device, a visual alarm on the device, or a combination thereof. For example, a remote alarm may be an email, text message, or other digital alert sent to the user.

[0158] For highly sensitive temperature detection, one or more temperature thermistors may be provided. A temperature change of 0.5 degrees is significant in cell culture. Thermistors degrade less than resistance temperature detectors (RTDs) and therefore can be used for a longer period before inspection is required. If a temperature change outside a predetermined range, or a temperature above or below a predetermined threshold is detected, the thermistor may trigger an alarm to warn the user. For example, the alarm may be a remote alarm, an audible alarm on the device, a visual alarm on the device, or a combination thereof. For example, a remote alarm may be an email, text message, or other digital alert sent to the user.

[0159] Figure 7C shows the CES700C having a dual supply circulation loop. The CES700C may be the same as the CES700A and CES700B, and may contain the same or similar components as the CES700A and CES700B, in which case the same reference numbers are used. The CES700C includes an EC circulation supply loop 752 from the CES700A and an IC circulation supply loop 753 from the CES700B. The IC circulation supply loop 753 flows the fluid from the fluid container 746B to the fluid container 746A, through the first fluid inlet passage 742, the cell growth chamber 701, the harvest valve 798, and back to the fluid container 746B. The EC circulation supply loop 752 flows the fluid from fluid container 768B to fluid container 768A, then sequentially through the EC culture medium valve 776, second fluid inlet passage 774, GTM 732, cell growth chamber 701, and EC waste valve 782, before returning it to fluid container 768B.

[0160] While various exemplary embodiments of cell proliferation systems and related methods have been described, Figure 8 shows exemplary operating steps of process 756 for growing non-adherent or suspended cells in a cell proliferation system such as CES500, CES600, or CES700A-700C according to embodiments of the present disclosure.

[0161] The START operation 758 is initiated, and process 756 proceeds to cell preparation (step 760). In embodiments, cell preparation 760 may involve several different optional steps. For example, in step 762, cells are collected. Cell collection step 762 may include separating and collecting cells from a donor. In some embodiments, an apheresis procedure is performed to collect a certain amount of lymphocytes from the donor's peripheral blood (e.g., leukapheresis). Such lymphocytes include a target cell population that is proliferated by process 756. In other embodiments, cells may be collected from umbilical cord blood.

[0162] After the collection step 762, optionally, as part of the preparation step 760, the cells are separated in step 764. A predetermined amount of cells collected in step 762 may include many different cell types, including cells to be targeted for proliferation. An optional step 764 may be performed to isolate the target cells. As an example, the target cells are T cells, for example, regulatory T cells. In one embodiment, regulatory T cells are CD4 + CD25 + These are T cells. The cells are isolated using any appropriate isolation technique. For example, the cells can be isolated using immunomagnetic separation, in which antibody-functionalized magnetic beads come into contact with the cells collected in step 762. The functionalized beads preferentially adhere to the target cell population. Then, a magnetic field can be used to remove other cells while the target cell population retains the beads to which they have attached.

[0163] After the separation step 764, the cells may optionally be resuspended in step 766. In some embodiments, the cells are resuspended in a medium containing several nutrients and / or reagents that promote the maintenance of cell viability. In some embodiments, the medium contains at least serum albumin and reagents such as cytokines. In some embodiments, the cytokines are recombinant human IL-2 cytokines. In one embodiment, the medium may contain cytokines at a concentration of 200 IU / ml.

[0164] Following the cell preparation step 760, process 756 proceeds to the cell exposure step 768, in which the cells are activated and proliferated. The cells are optionally exposed to a soluble activator in step 770. The activator, containing an antibody conjugate, is added to the culture medium in which the cells are resuspended. In some embodiments, the activator is a human antibody CD3 / CD28 / CD2 cell activator conjugate. In some embodiments, the activator may be contained in the culture medium used in the cell resuspension step 766. Optionally, in step 772, the cells are exposed to beads having an activator on their surface. In some embodiments, exposing the cells to beads involves adding a predetermined amount of beads to the resuspended cells. The beads may be added in different ratios to the number of cells. For example, the beads may be added in a ratio of 1 bead:2 cells. Other embodiments may add beads in different ratios, such as 1 bead:1 cell or 1 bead:3 cells. The beads, having antibodies on their surface, can activate and proliferate the cells. In the embodiment, the beads may contain the antibody CD3 / CD28 on their surface.

[0165] Process 756 proceeds to the cell proliferation step 774. As part of the cell proliferation step 774, cells may be introduced into a cell growth chamber, such as a hollow fiber membrane bioreactor, in which the cells will be grown. In step 776, the cells are nourished to promote cell proliferation. For example, a culture medium may be delivered to the cell growth chamber to supply nutrients for proliferation. For example, the culture medium may be supplied via an IC circulation supply loop 753, an EC circulation supply loop 752, or a combination thereof, as described with reference to Figures 7A-7C. The cell proliferation step 774 may include periodically adding reagents to the cell growth chamber in step 778 in order to continue promoting cell proliferation. For example, in some embodiments, reagents (e.g., cytokines) may be added to the cell growth chamber to promote cell proliferation. In one embodiment, the reagent may be an additional IL-2 cytokine, such as recombinant human IL-2 cytokine.

[0166] For example, culture media, reagents, or combinations thereof may be delivered to cells from one or more connected fluid containers, as described with reference to Figures 7A to 7C, and the culture media, reagents, or combinations thereof may be circulated and recirculated through the IC circulation supply loop 753 or the EC circulation supply loop 752.

[0167] Furthermore, as part of the cell proliferation step 774, the environment inside the cell growth chamber is controlled in step 780. For example, gases are continuously delivered and exchanged to the cells growing in the cell growth chamber, for example, to bring about a balance of carbon dioxide and oxygen. In addition, the temperature may be controlled to be within a range optimized for cell proliferation. The cell proliferation step 774 may also include a step 782 in which metabolites are monitored. For example, lactate and glucose levels may be monitored periodically. Lactate and glucose levels may be monitored by one or more sensors. One or more sensors may also be configured to measure oxygen levels. An increase or decrease in metabolite levels may prompt changes in the control of the environment inside the cell growth chamber in step 780 (e.g., additional feeding, additional reagent addition, additional gas exchange, etc.).

[0168] When various parameters are monitored, data readings outside a predetermined threshold range may trigger an alarm to warn the user. For example, data readings may be temperature, door position, pressure, flow rate, or concentration. For example, the alarm may be a remote alarm, an audio alarm on the device, a visual alarm on the device, or a combination thereof. For example, a remote alarm may be an email, text message, or other digital alert sent to the user.

[0169] Process 756 then proceeds to the cell harvesting step 784. Further processing or other analysis of the harvested cells may be optionally performed in step 786. For example, cells may be characterized to determine the cell phenotype. Step 786 performing further processing or other analysis may include, for example, performing flow cytometry to characterize the cell phenotype. Then, process 756 terminates with the END operation 788. If further processing / analysis is not desired, process 756 terminates with the END operation 788.

[0170] Figure 9A shows the operational steps of process 800, which may be used to place cells or other materials (e.g., proteins, nutrients, growth factors) into a cell growth chamber according to embodiments of the present disclosure. In embodiments, process 800 may be performed as part of a task of “introducing cells into the center without circulation.” An initiation operation 802 is started, and process 800 proceeds to step 804, where a first volume of fluid containing cells is introduced into the cell growth chamber of the cell proliferation system. In embodiments, the cells include non-adherent cells such as one or more types of T cells. In one embodiment, the multiple cells include Tregs. As can be understood, the introduction of a first volume of fluid containing cells may be performed by components of the cell proliferation system such as the systems CES500 (e.g., Figure 5A), CES600 (Figure 6), and CES700 (Figures 7A-7C) described above. Figure 9B shows a portion of a cell growth system including a first fluid inlet pump 840, a first fluid channel 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 channel 860 is fluidically associated with the first fluid circulation path 852 via a connector 860B. In an embodiment, in step 804, a first volume of fluid containing cells is introduced into the first fluid circulation path 852 through the first fluid channel 860 using the first fluid inlet pump 840. In an embodiment, the first volume of fluid is introduced without operating the first fluid circulation pump 848. The first volume of fluid is supplied at least partially from a fluid container 746A, a fluid container 746B, an IC fluid container 746, or a combination thereof. For example, the first volume of fluid is circulated through an IC circulation supply loop 753.

[0171] As shown in Figure 9B, the volume of the first fluid circulation path 852 is composed of the volumes of several parts. For example, the first part of the volume is the capillary inner space of the cell growth chamber 844 (if the cell growth chamber is a hollow fiber membrane bioreactor). The second part of the volume is the section from the connection 860B to the inlet port 844A of the cell growth chamber 844. The third part is the section from the connection 860B to the outlet port 844B of the cell growth chamber 844.

[0172] Process 800 proceeds to step 806, in which a second volume of fluid is introduced. The second volume of fluid may contain a culture medium and is introduced into a portion of the first fluid channel 860. In one embodiment, the second volume is a predetermined amount selected to place the first volume into a first portion of the cell growth chamber 844 (step 808). In one embodiment, the first volume of fluid and the second volume of fluid may be the same. In another embodiment, the first volume of fluid and the second volume of fluid may be different. In yet another embodiment, the sum of the first volume of fluid and the second volume of fluid may be equal to a certain percentage of the volume of the first fluid circulation path 852, for example, the path (Figure 9B).

[0173] The second volume of fluid is supplied, at least partially, from fluid container 768A, fluid container 768B, EC fluid container 768, or a combination thereof. For example, the second volume of fluid is circulated through the IC circulation supply loop 753. For example, the second volume of fluid is supplied from two or more connected fluid containers, as described with respect to Figures 7A to 7C.

[0174] To position a first volume of fluid, a second volume of fluid must be sufficient to push the first volume to a desired position within the cell growth chamber 844. Thus, in this embodiment, the second volume of fluid is approximately equal in size to the volume between the connection 860B and the inlet port 844A of the first fluid circulation path 852. As can be understood, this will push the first volume of fluid, containing the cells, to its position within the cell growth chamber 844.

[0175] In other embodiments, in step 808, a first volume of fluid (containing cells) is placed in the central region 866 of the cell growth chamber 844. In these embodiments, the second volume is approximately equal to the sum of the volume of the first fluid circulation path 852 between the connection 860B and the inlet port 844A, and the portion of the first fluid circulation path (e.g., the volume of the capillary inner space) that is comprised of the cell growth chamber 844 and not occupied by the first volume of fluid when it is placed in the cell growth chamber. For example, in one embodiment, the first volume of fluid (containing cells) is 50 ml. The cell growth chamber has a volume of, for example, 124 ml. When the first volume is placed in the central region 866, the 50 ml volume occupies the central region 866, leaving 74 ml on either side of the central region 866. Therefore, in order to place the first volume of 50 ml around the central region 866, 50% of 74 ml (i.e., 37 ml) may be added to the volume between the connection part 860 B and the inlet port 844 A.

[0176] In the embodiment, the first volume positioning step 808 includes adding fluid to position the first volume of fluid containing cells within the cell growth chamber. For example, if the desired positioning of the first volume is not achieved with the second volume, the fluid can be added to position the first volume (step 808).

[0177] Process 800 proceeds to query step 810 to determine whether the first volume should be repositioned. For example, in one embodiment, the first volume may be positioned closer to the inlet port 844A. If it is desired to move the first volume closer to the central region 866 of the cell growth chamber 844, process 800 returns to step 808 and adds additional fluid to the first fluid circulation path to position and arrange the fluid of the first volume.

[0178] If the query step 810 determines that the first volume does not need to be rearranged, the process 800 proceeds to the cell feeding step 812. In embodiments, cells are fed using a culture medium containing multiple elements such as glucose, proteins, growth factors, reagents, or other nutrients. In embodiments, the cell feeding step 812 may include operating an inlet pump and a circulation pump (e.g., pumps 840, 848) to deliver the nutrient-containing culture medium to the cells in the cell growth chamber 844. In some embodiments, the cells are maintained in the cell growth chamber 844 during step 812, as described below. These embodiments include operating the pumps (e.g., an inlet pump and a circulation pump (e.g., pumps 840, 848, described below)) so that the fluid flow to the cell growth chamber 844 is from both directions (from the inlet port 844A and the outlet port 844B) into the cell growth chamber 844.

[0179] For example, the culture medium is delivered to the cells from one or more connected fluid containers, as described with respect to Figures 7A to 7C. The inlet pump and circulation pump can circulate and recirculate the culture medium from both directions, such as from the inlet port 844A and the outlet port 844B into the cell growth chamber 844, via the IC circulation supply loop 753 or the EC circulation supply loop 752.

[0180] Next, process 800 proceeds to a cell proliferation step 814, where the cells are proliferated or grown. Although step 814 is shown after step 812, according to one embodiment, step 814 may be performed before or concurrently with step 812. The cells are then removed from the cell growth chamber (step 816) and collected in a storage container. In some embodiments, step 816 involves several substeps. For example, the cells may be circulated by a circulation pump (e.g., pump 848) before being collected and stored in the container. Process 800 ends with an END operation 830.

[0181] Next, Figure 10A shows exemplary operating steps of process 900 for retaining cells in a bioreactor of a cell proliferation system such as CES500 (e.g., Figures 5B and 5C) or CES700 (e.g., Figures 7A-7C) according to embodiments of the present disclosure. As previously mentioned, cells in the header portion of the bioreactor or in the IC loop outside the bioreactor may not receive adequate gas and nutrient exchange, leading to cell aggregation and death. In one embodiment, the bioreactor performs gas and nutrient exchange through a semipermeable hollow fiber membrane. It is important that such exchange is efficient because the surface area-to-volume ratio of a cell proliferation system including a hollow fiber membrane is significantly larger than that of systems in other cell culture methods (e.g., about 15 times the surface area of ​​a cell culture flask). Such efficiency is achieved by minimizing the diffusion distance of culture medium components that can pass through the membrane surface where the exchange takes place.

[0182] For example, Figure 10B shows a graph of oxygen consumption in a cell proliferation system such as the Quantum® Cell Expansion System or the Quantum Flex® Cell Expansion System during cell proliferation (approximately 3E+09 T cells are present in the bioreactor). Figure 10B shows the percentage (%) of oxygen (O2) at the bioreactor outlet. For example, according to one embodiment, a sensor for measuring the oxygen level can be placed at the EC outlet port of the bioreactor. In another embodiment, a sensor for measuring the oxygen supply may be placed at the IC outlet port of the bioreactor. The percentage of O2 is measured against run time (min) (940). To maximize oxygen supply to the cells, in one embodiment, the EC circulation flow rate, Q EC CircThe EC circulation rate is set to 300 mL / min (942). Figure 10B shows the change in oxygen supply 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, cells consume oxygen (O2) in the medium. Since the fluid at 50 mL / min (944) moves through the bioreactor more slowly than the fluid at 300 mL / min (942), at an EC circulation rate of 50 mL / min, cells may have more time to extract oxygen from the medium, i.e., cells may have more opportunities to extract oxygen from the medium. When the EC circulation rate is returned to 300 mL / min, the oxygen supply is restored (946). Figure 10B shows, for example, the possible advantages of retaining cells in the hollow fibers where gas transfer occurs, as opposed to retaining them in the IC circulation path outside the bioreactor (where oxygen may be extracted from cells). Therefore, it is beneficial to retain cell populations (e.g., non-adherent cells) within the hollow fibers of the bioreactor during feeding by directing the flow of culture medium to enter both sides of the bioreactor, for example, the IC inlet port and the IC outlet port. In one embodiment, a method can be used to evenly distribute the flow to the IC inlet port and the IC outlet port. In another embodiment, for example, the flow to the IC inlet port may be made larger or smaller than the flow to the IC outlet port depending on the desired placement of the cells within the bioreactor.

[0183] Returning to Figure 10A, the start operation 902 is initiated, and process 900 proceeds to step 904, in which a disposable set or a pre-mounted fluid transfer assembly (e.g., 210, 400) is loaded into the cell growth system. The disposable set may include a cell growth chamber 100A or a cell growth chamber 100B, as shown in Figures 1C, 4B, and 4C. In step 906, the disposable set is primed. Here, for example, Lonza Ca 2+ / Mg 2+The disposable set is primed with PBS that does not contain [unspecified element]. In preparation for cell ingestion and seeding, the priming fluid is replaced using IC / EC washing step 908. In one embodiment, the PBS in the system is replaced with, for example, TexMACS GMP basal medium.

[0184] For example, during priming, the fluid is delivered from one or more connected fluid containers through a disposable set, as described with respect to Figures 7A to 7C. The fluid may be circulated and recirculated using the fluid containers for additional volume within the fluid loop.

[0185] Next, process 900 proceeds to step 910, which involves closing the IC outlet valve. In embodiments, the EC outlet valve is opened to allow ultrafiltration of the fluid applied to the hollow fibers of the bioreactor, which includes a hollow fiber membrane. Next, in step 912, the culture medium is prepared. Next, process 900 proceeds to step 914, which involves introducing cells (e.g., suspended or non-adherent cells such as T cells or Tregs). In one embodiment, in step 914, such cells are introduced by the task of “introducing cells into the center without circulation.” In other embodiments, in step 914, such cells are introduced by the task of “introducing cells in a homogeneous suspension.” In other embodiments, other introduction tasks and / or introduction procedures may be used.

[0186] In one embodiment, the cells being introduced in step 914 may be suspended in a solution containing, for example, a culture medium for feeding the cells during and after introduction. In other embodiments, such a solution may contain both a culture medium for feeding the cells and a soluble activator complex for stimulating the cells (e.g., T cells). The introduction step 914 is performed, for example, on day 0.

[0187] In step 916, following the cell introduction step 914, the cells are further fed. In exemplary embodiments, the cells are fed through the EC circulation supply loop 752, the IC circulation supply loop 753, or a combination thereof, as described with reference to Figures 7A-7C (step 916). During the feeding step 916, it is desirable to control the retention of cells within the bioreactor. By adjusting the flow control parameters in step 918, the cells can be retained within the bioreactor without being lost from the bioreactor to the IC loop portion outside the bioreactor, for example, during the growth phase of growth. According to the embodiment, by retaining the cells within the bioreactor, the cells within the bioreactor can be brought closer to the IC inlet port, and such cells can receive the freshest growth medium. On the other hand, cells in the IC loop may receive consumed or adjusted medium, which may affect, for example, glycolytic metabolism. Furthermore, cells within the bioreactor receive a mixed gas (e.g., oxygen, carbon dioxide, nitrogen) introduced from the gas transport module (GTM) by diffusion from the EC loop circulation, while cells in other parts of the IC loop do not receive such a mixed gas. In some embodiments, the cells are retained in the bioreactor itself, while in other embodiments, the cells may be maintained in any location that allows for improved nutrient delivery and / or gas exchange. Thus, in embodiments, other locations may be used to retain or control cell retention without departing from the spirit and scope of this disclosure.

[0188] Returning to Figure 10A and process 900, in one embodiment, cell loss from the hollow fiber membrane bioreactor can be reduced by matching, or substantially matching, the IC circulation pump flow rate to the IC inlet pump flow rate (in the opposite direction). In step 920, the IC inlet pump is adjusted to produce a first flow rate or volumetric flow rate, and in step 922, the IC circulation pump is adjusted to produce a second counter-flow rate or second counter-volumetric flow rate. Here, the volumetric flow rate or fluid flow rate or fluid rate or flow rate can be thought of as the volume of fluid passing per unit time (represented by the symbol "Q"). For example, an IC inlet pump flow rate of 0.1 mL / min is matched, 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 cell culture (e.g., days 4-7 in the embodiment). Alternatively, the IC circulation pump rate is matched, or nearly matched, to a complementary IC circulation pump rate of -0.01 mL / min for an IC inlet pump flow rate of 0.01 mL / min to maintain cells in the bioreactor during the growth phase of cell culture (e.g., days 4–7 in the embodiment). Such a pump adjustment step 918 makes it possible to counteract, for example, any forces associated with cell loss from the IC outlet port of the bioreactor.

[0189] Next, in step 924, the cells are grown or proliferated. The cells are not limited to growing or proliferating in step 924, but may instead be proliferated during steps 914, 916, 918, 920, and 922, for example. Process 900 then proceeds to harvest step 926, where the cells are transferred to harvest bags or containers. The disposable set is then removed from the cell proliferation system (step 932), and process 900 ends with termination operation 934.

[0190] Alternatively, process 900 may optionally proceed from the harvesting step 926 to perform a further processing / analysis step 928. Such a further processing / analysis step 928 may include, for example, characterization of the phenotype of the harvested cells. Process 900 may proceed from the optional further processing / analysis step 928 to the step of reintroducing the remaining cells 930. Next, process 900 proceeds to the removal of the disposable set (step 932), and process 900 terminates with a termination operation 934. Alternatively, process 900 may proceed from the further processing / analysis step 928 to the removal of the disposable set (step 932). Process 900 then terminates with a termination operation 934.

[0191] Next, Figure 11A shows exemplary operating steps of process 1000 for feeding cells to be used with a cell proliferation system such as CES700 (e.g., Figures 7A-7C) according to embodiments of the present disclosure. Starting operation 1002 is initiated, and process 1000 proceeds to the steps of loading a disposable set into the cell proliferation system, priming the set, performing IC / EC washing, preparing the culture medium, and introducing cells (e.g., suspension cells or non-adherent cells). The disposable set may include a cell growth chamber 100A or a cell growth chamber 100B, as shown in Figures 1C, 4B, and 4C. Next, process 1000 proceeds to step 1004, which involves feeding the cells for a first period. In exemplary embodiments, the cells are fed through an EC circulation supply loop 752, an IC circulation supply loop 753, or a combination thereof, as described with reference to Figures 7A-7C (step 916). In embodiments, a first inlet flow rate and a first circulation flow rate are used. As an example, a first IC inlet flow rate and a first IC circulation flow rate are used, where the first IC inlet flow rate is controlled by an IC inlet pump (e.g., a first pump), and the first IC circulation flow rate is controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, the IC inlet pump (754) flows into the IC inlet port (701A) of the bioreactor (701) at a volumetric flow rate of 0.1 mL / min, and the IC circulation pump (712) flows into the IC outlet port (701B) of the bioreactor (701) at a complementary IC circulation volumetric flow rate or flow-fluid flow rate of -0.1 mL / min. Here, the negative sign ("-") used, for example, in -0.1 mL / min, indicates that the direction of the IC circulation pump (712) creates a counterflow so that cells are maintained in the bioreactor during the growth phase of cell culture.

[0192] During cell feeding and while using an IC pump for feeding, with control over cell retention in the bioreactor due to forward / counterflow characteristics, cells continue to grow and proliferate. As a result, cells require additional culture medium (e.g., glucose and / or cell growth medium) to support the growing population. In exemplary embodiments, multiple fluid vessels 768A, 768B are connected to provide the culture medium for circulation and recirculation within the system. This eliminates the need to control lactate levels. In other embodiments, measures are also taken to control the lactate levels of the growing cell population. In embodiments, cell culture lactate levels are maintained at approximately 20 mmol / L or less, approximately 15 mmol / L or less, approximately 10 mmol / L or less, or approximately 7 mmol / L or less. In other embodiments, lactate levels are maintained at approximately 5 mmol / L or less, for example, by controlling the flow rate of culture medium addition and / or other settings to improve cell growth and viability. In other embodiments, other concentrations may be used.

[0193] In exemplary embodiments, fluid vessels 768A, 768B, or fluid vessels 746A, 746B are connected, so it is not necessary to control the pump flow rate to control the lactate level. In other exemplary embodiments, the lactate level can be controlled to approximately 7 mmol / L or less by simultaneously increasing both the IC inlet (+) pump flow rate and the IC circulation (-) pump flow rate within the lumen of the hollow fiber membrane from ±0.1 to ±0.4 mL / min over a period of several days, e.g., several days (days 4-8). For example, Figure 11B shows Table 1018 of exemplary IC pump flow rates for feeding using a "cell feeding" task in a cell proliferation system (e.g., CES500). Table 1018 shows an exemplary relationship between the period 1020 (e.g., which day) and the IC pump flow rate 1022 for generating volumetric flow to both sides of the bioreactor to maintain cells within the bioreactor. For example, on days 0-4 (1024), use an IC inlet flow rate or input pump flow rate of 0.1 mL / min (1026), and an IC circulation pump flow rate of -0.1 mL / min (1028). On day 5 (1030), use an IC inlet pump flow rate of 0.2 mL / min (1032) and an IC circulation pump flow rate of -0.2 mL / min (1034). On day 6 (1036), use an IC inlet pump flow rate of 0.3 mL / min (1038) and an IC circulation pump flow rate of -0.3 mL / min (1040). On day 7 (1042), use an IC inlet pump flow rate of 0.4 mL / min (1044) and an IC circulation pump flow rate of -0.4 mL / min (1046). Table 1018 in Figure 10B illustrates the use of a pump flow rate of ±0.1 to ±0.4 mL / min to feed cells while retaining them in a bioreactor during the growth phase of cell culture; however, other pump flow rates and resulting flow rates may be used for embodiments without departing from the spirit and scope of this disclosure. For example, while this example shows an increment of ±0.1 mL / min to increase the feeding flow rate, other increments, such as ±0.005 mL / min, ±0.05 mL / min, etc., may be used to increase the feeding flow rate. The periods (e.g., days) and pump flow rates in Table 1018 in Figure 11B are provided for illustrative purposes only and are not intended to limit the scope of the invention.For example, the duration and pump flow rates in Table 1018 of Figure 11B may be provided for cell growth chamber 100B. The pump flow rates in Table 1018 of Figure 11B may be reduced to 10% of the stated pump flow rates for cell growth chamber 100A.

[0194] Returning to Figure 11A, process 1000 proceeds from step 1004, in which cells are fed for a first period, to step 1006, in which the first inlet flow rate is increased by a first amount to achieve the second inlet flow rate. As in the embodiment shown in Figure 11B described above, the IC inlet pump flow rate (+) is increased from 0.1 mL / min to 0.2 mL / min to produce an IC inlet flow rate of 0.2 mL / min. Furthermore, simultaneously, the IC circulation pump flow rate (-) is 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 flow rate may be reduced to 10% of the described pump flow rate for the cell growth chamber 100A.) Thus, the first circulation flow rate is increased by a first amount to achieve the second circulation flow rate (step 1008). Next, the cells are fed for a second period at a second inlet flow rate and a second circulation flow rate (step 1010), and the cells are maintained inside the bioreactor and outside the header and outside a portion of the IC circulation path outside the bioreactor. Following the second period of feeding (step 1010), if it is not desired to continue feeding and / or proliferation of the cells, for example, process 1000 terminates with termination operation 1016. Alternatively, process 1000 may optionally continue increasing or otherwise changing the feeding flow rate (step 1012). There may be any number of feeding periods, as indicated by the ellipsis 1014. Following the desired number of feeding periods 1014, process 1000 terminates with termination operation 1016. Figures 10A and 10B and process 1000 show an "increase" in the flow rate, but other adjustments to the flow rate may be made. For example, the flow rate may be decreased from one feeding period to the next, or may remain substantially the same. For example, metabolic activity may be taken into consideration when deciding how to adjust the flow rate. The "increase" in flow rates in Figures 10A and 10B is provided for illustrative purposes only and is not intended to limit the flow rate.

[0195] Next, Figure 12A shows exemplary operating steps of process 1100 for feeding cells to be used with a cell proliferation system such as CES500 (e.g., Figures 5B and 5C) according to embodiments of the present disclosure. Starting operation 1102 is initiated, and process 1100 proceeds to the steps of loading a disposable set into the cell proliferation system, priming the set, performing IC / EC washing, preparing the culture medium, and introducing cells (e.g., suspension cells or non-adherent cells). Next, process 1100 proceeds to step 1104, which feeds the cells for a first period. In embodiments, a first inlet flow rate or flow rate and a first circulation flow rate or flow rate are used. As an example, a first IC inlet flow rate and a first IC circulation flow rate are used, where the first IC inlet flow rate is controlled by an IC inlet pump (e.g., a first pump) and the first IC circulation flow rate is controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, the IC inlet pump (554) flows into the IC inlet port (501A) of the bioreactor (501) at a volumetric flow rate or flow rate of 0.1 mL / min, and the IC circulation pump (512) flows into the IC outlet port (501B) of the bioreactor (501) at a complementary IC circulation volumetric flow rate of -0.1 mL / min. Here, the negative sign ("-") used, for example, in -0.1 mL / min, indicates that the direction of the IC circulation pump (512) generates, i.e., creates, a counterflow so that cells are maintained in the bioreactor during the growth phase of cell culture. For example, the described flow rates are suitable for cell growth chamber 100B. For cell growth chamber 100A, the described flow rates may be reduced to 10%.

[0196] During cell feeding and while using IC pumps to control cell retention in the bioreactor due to forward / counterflow characteristics, cells continue to grow and proliferate. As a result, cells require additional culture medium (e.g., glucose and / or cell growth medium) to support the growing population. In exemplary embodiments, multiple fluid vessels 768A, 768B are connected to provide culture medium for circulation and recirculation in the system. This eliminates the need to control lactate levels. In other embodiments, measures are also taken to control the lactate levels of the growing cell population. In exemplary embodiments, lactate levels are controlled to be below approximately 7 mmol / L by simultaneously increasing both the IC inlet (+) pump flow rate and the IC circulation (-) pump flow rate from ±0.1 to ±0.4 mL / min within the lumen of the hollow fiber membrane, for example, over several days (days 4-8). See, for example, the table in Figure 11B and its description above (e.g., pump flow rate for feeding). As described above, Table 1018 in Figure 11B illustrates setting the pump flow rate to ±0.1 to ±0.4 mL / min to feed cells while retaining them in the bioreactor during the growth phase of cell culture; however, other pump flow rates and resulting flow rates may be used for embodiments without departing from the spirit and scope of this disclosure. The periods (e.g., days) and pump flow rates in Table 1018 of Figure 11B are provided for illustrative purposes only and are not intended to limit. For example, the periods and pump flow rates in Table 1018 of Figure 11B may be provided for cell growth chamber 100B. The pump flow rates in Table 1018 of Figure 11B may be reduced to 10% of the stated pump flow rates for cell growth chamber 100A.

[0197] Returning to Figure 12A, process 1100 proceeds from step 1104, in which cells are fed for a first period, to step 1106, in which the first inlet flow rate is increased by a first amount to achieve a second inlet flow rate or flow rate. As in the embodiment shown in Figure 11B described above, the IC inlet pump flow rate (+) is increased from 0.1 mL / min to 0.2 mL / min to produce an IC inlet flow rate of 0.2 mL / min. Furthermore, simultaneously, the IC circulation pump flow rate (-) is 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 flow rate may be reduced to 10% of the described pump flow rate for the cell growth chamber 100A.) Thus, the first circulation flow rate or flow rate is increased by a first amount to achieve the second circulation flow rate or flow rate (step 1108). Next, the cells are fed for a second period at a second inlet flow rate or flow rate and a second circulation flow rate or flow rate (step 1110), and the cells are maintained inside the bioreactor and outside the header or in part of the IC circulation path outside the bioreactor. If, for example, it is not desired to continue feeding and / or proliferation of the cells following the second period of feeding (step 1110), process 1100 is terminated by termination operation 1116.

[0198] Alternatively, process 1100 may optionally determine whether to adjust the feeding flow rate or flow rate based on metabolic activity. In this case, process 1100 proceeds to an optional query step 1112 to determine whether to adjust the feeding based on metabolic levels. For example, by monitoring glucose and / or lactate levels, the culture medium flow rate of the cell proliferation system (e.g., IC medium flow rate) can be adjusted to promote cell growth (e.g., Treg) in a bioreactor such as a hollow fiber bioreactor.

[0199] As shown in Figures 12B and 12C, embodiments demonstrate the control of lactate levels in cell proliferation experiments or procedures involving the proliferation of hTregs, for example. Graphs 1118 and 1132 illustrate the use of glucose and lactate level measurements to adjust the medium flow rate of a cell proliferation system (e.g., IC medium flow rate) to support the proliferation of Tregs, for example. For example, Figure 12B provides graph 1118 showing the metabolism of hTreg proliferation, where cell proliferation is carried out in a cell proliferation system such as the Quantum® cell proliferation system. Glucose concentration (mg / dL) 1120 and lactate concentration (mmol / L) 1122 are shown for various cell proliferation runs 1124 and 1125 over a period (e.g., number of days 1126). In these Treg runs 1124 and 1125, for example, during the period from day 4 to day 8, the lactate level of the growing cell population is controlled to a value of approximately 7 mmol / L or less by simultaneously increasing both the IC inlet (+) pump flow rate and the IC circulation pump flow rate within the lumen of the hollow fiber membrane by ±0.1 to ±0.4 mL / min. In other embodiments, other pump flow rates may be used. As shown, according to the embodiment, the minimum glucose level during Treg cell proliferation is in the range of 264 mg / dL concentration 1128 (Q1584) on day 7 to 279 mg / dL concentration 1130 (Q1558) on day 8. As depicted, the starting glucose concentration in the cell growth medium for run 1124 is, for example, in the range of 325 mg / dL to 335 mg / dL. In other embodiments, it may be desirable to maintain the lactate level at approximately 5 mmol / L or less to improve cell growth and viability. In the embodiment, graphical user interface (GUI) elements can be used to control the rate of culture medium addition and maintain lactate metabolic waste products from glycolysis below a specified level during cell proliferation.

[0200] Turning to Figure 12C, Graph 1132 shows the metabolism of hTreg proliferation, such cell proliferation occurring in a cell proliferation system such as the Quantum® cell proliferation system. Glucose consumption (mmol / day) 1134 and lactate production (millimoles / day) 1136 are shown for various cell proliferation runs 1138, 1139 over a period 1140 (e.g., days). In one embodiment, the IC inlet (+) pump flow rate and IC circulation (-) pump flow rate are simultaneously increased from ±0.1 to ±0.4 mL / min to control the lactate level, for example, to below approximately 7 mmol / L. For example, Graph 1132 shows IC circulation rates and IC feed rates of ±0.1 mL / min (1142), ±0.2 mL / min (1144), ±0.3 mL / min (1146), and ±0.4 mL / min (1148). Other embodiments may use other flow rates. The flow rates used and shown in Figures 12B and 12C are provided for illustrative purposes only and are not intended to limit the flow. Positive (+) may be indicated for the IC inlet pump direction and negative (-) for the IC circulation pump direction, but such directions are shown for illustrative purposes only and depend on the configuration of the pumps used.

[0201] Returning to Figure 12A and the optional query step 1112, if it is not desired to measure metabolic activity and / or adjust the feeding level based on that measurement, process 1100 proceeds to "no", and process 1100 terminates in termination operation 1116. For example, if multiple supply containers are used, as described with respect to Figures 7A-7C, the medium is circulated and recirculated, avoiding the need to measure metabolic activity and / or adjust the feeding level. Alternatively, if it is desired to adjust the feeding level based on metabolic activity, process 1100 proceeds to "yes", and in the optional step 1114, the medium addition rate is increased to continue feeding the growing cell population. Although step 1114 is shown as a single step, this step may include multiple steps that make adjustments to the medium addition rate, such as increasing the IC inlet flow rate or increasing the IC circulation flow rate. Step 1114 is shown as a single step for illustrative purposes only, but is not intended to be limiting. Following the adjustment of the culture medium addition rate, process 1100 proceeds to an optional query step 1112 to determine whether to continue measuring metabolic levels and / or adjust feeding. If it is not desired to continue measuring metabolic activity and / or adjusting feeding levels based on metabolic activity, process 1100 proceeds to “no,” and process 1100 terminates in termination action 1116. Figure 11A and process 1100 show an “increase” in rate or flow rate, but other adjustments to flow rate may be made. For example, from one feeding period to the next, the rate or flow rate may be decreased or kept substantially the same. The type of adjustment that may be made depends on the assessment of the metabolic activity of the growing cell population. For example, the “increase” in flow rate in Figure 11A is provided for illustrative purposes only and is not intended to limit it.

[0202] Next, Figure 13 shows exemplary operating steps of process 1200 for holding cells in a given position during cell feeding using a cell growth system such as CES500 (e.g., Figures 5B and 5C) or CES700 (e.g., Figures 7A-7C) according to embodiments of the present disclosure. Starting operation 1202 is initiated, and process 1200 proceeds to the steps of loading a disposable set into the cell growth system, priming the set, performing IC / EC washing, preparing the culture medium, and introducing cells (e.g., suspension cells i.e., non-adherent cells). The disposable set may include a cell growth chamber 100A or a cell growth chamber 100B, as shown in Figures 1C, 4B, and 4C. Next, process 1200 proceeds to step 1204 of feeding the cells for a first period. In embodiments, a first inlet flow rate and a first circulation flow rate are used. As an example, a first IC inlet flow rate and a first IC circulation flow rate are used, where the first IC inlet flow rate is generated and controlled by an IC inlet pump (e.g., a first pump), and the first IC circulation flow rate is generated and controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, the IC inlet pump (554) flows into the IC inlet port (501A) of the bioreactor at a volumetric flow rate of 0.1 mL / min, and the IC circulation pump (512) flows into the IC outlet port (501B) of the bioreactor (501) at a complementary IC circulation volumetric flow rate or flow rate of -0.1 mL / min. Here, the negative sign ("-") used, for example, in -0.1 mL / min, indicates that the direction of the IC circulation pumps generates, i.e., creates, a counterflow so that cells are maintained in the bioreactor during the growth phase of cell culture. In other embodiments, the first IC circulation flow rate may be a percentage of the first IC inlet flow rate. For example, the first IC circulation flow rate may be about 50 percent (50%), or about half (1 / 2), of the first IC inlet flow rate, or in some embodiments, it may be any other percentage, or any other proportion.Alternatively, in an exemplary embodiment, the first IC inlet pump flows into the IC inlet port (701A) at a volumetric flow rate of less than 0.1 mL / min or about 0.01 mL / min, and the IC circulation pump flows into the IC outlet port (701B) at a complementary IC circulation volumetric flow rate or flow fluid flow rate of less than -0.1 mL / min or about -0.01 mL / min. Here, the negative sign ("-") used, for example, in -0.01 mL / min, indicates that the direction of the IC circulation pump creates a counterflow so that cells are maintained in the bioreactor during the growth phase of cell culture.

[0203] During cell feeding (and proliferation) and while using the IC pump to control cell retention in the bioreactor due to its forward-counterflow characteristics, the cells continue to grow and proliferate. As a result, the cells require additional culture medium (e.g., glucose and / or cell growth medium) to support the growing population. In embodiments, the culture medium flow rate is increased to feed the growing cell population. In exemplary embodiments, increasing the IC inlet pump flow rate (+) increases the IC inlet flow rate by a first amount to achieve a second IC inlet flow rate (step 1206). For example, according to one embodiment, the IC inlet flow rate is increased by a first amount of 0.1 mL / min to achieve a second IC inlet flow rate of 0.2 mL / min. According to other embodiments, other adjustments may be made to the IC inlet flow rate.

[0204] In an exemplary embodiment, cells are fed through the EC circulation supply loop 752, the IC circulation supply loop 753, or a combination thereof, as described with reference to Figures 7A–7C (step 916).

[0205] Next, according to the embodiment, the second IC circulation flow rate is set, adjusted, or configured to be equal to a certain percentage, proportion, or ratio of the second IC inlet flow rate (step 1208). For example, according to one embodiment, the second IC circulation flow rate is set, adjusted, or configured to be equal to about 50 percent (50%), or about half (1 / 2), of the value of the IC inlet flow rate, or, in other embodiments, to a different percentage or proportion. According to one embodiment, the second IC circulation flow rate is increased by adjusting the IC circulation pump flow rate (-) in accordance with the value of the first IC circulation flow rate. In other embodiments, the IC circulation pump flow rate is adjusted to reduce the second IC circulation flow rate so that the IC circulation flow rate is substantially equal to a predetermined percentage or ratio of the second IC inlet flow rate. In yet another embodiment, no adjustment of the IC circulation pump flow rate is required. For example, if the first IC inlet flow rate is equal to 0.1 mL / min and the first IC circulation flow rate is equal to -0.1 mL / min, then when the second IC inlet flow rate increases to 0.2 mL / min, the second IC circulation flow rate becomes (-1 / 2) * (second Q IC Inlet )(Q here) IC Inlet The IC inlet flow rate is set to (-1 / 2)*(0.2 mL / min), which in turn sets the second Q to -0.1 mL / min. IC Circ (where Q IC Circ (This is the IC circulation flow rate). Therefore, without adjusting the IC circulation pump flow rate, the second Q IC Circ This will be achieved.

[0206] Figures 5B and 5C show the operational configuration of a cell growth system 500 illustrating the movement of fluid in a first circulation path according to an embodiment of the present disclosure. The configuration in Figures 5B and 5C illustrates the division of flow to an IC inlet port (e.g., a first port) and an IC outlet port (e.g., a second port) for, for example, retaining cells in a cell growth chamber or bioreactor. In the IC loop or first circulation path 502 as described above with respect to the CES 500, the fluid is initially delivered by an IC inlet pump 554. Such fluid is delivered in a first direction (e.g., the positive direction). The fluid flows into the cell growth chamber or bioreactor 501 via the IC inlet port 501A, flows through the hollow fibers of the cell growth chamber or bioreactor 501, and flows out via the IC outlet port 501B. The culture medium flows through an IC circulation pump 512 used to control the culture medium flow rate. The IC circulation pump pumps the fluid in a first direction or a second direction opposite to the first direction. The IC outlet port 501B can be used as an inlet, for example, in the reverse direction. In one embodiment, the IC circulation pump 512 pumps fluid, for example, in the opposite direction to the IC inlet pump. For example, the direction of the IC inlet pump is positive (+) and the direction of the IC circulation pump is negative (-), so that counterflow occurs so that fluid enters both sides of the bioreactor and cells are retained within the bioreactor.

[0207] In one embodiment, the first portion of the fluid branches off at connection 517 and flows into the IC inlet port (501A) of the bioreactor 501. In another embodiment, the IC circulation pump 512 operates at a pump flow rate that matches, or is substantially matched to, the flow rate of the IC inlet pump 554 (but in the opposite direction), so that the second portion of the fluid branches off at connection 517 and flows into the IC outlet port (501B) of the bioreactor 501. For example, the IC circulation pump flow rate is matched or substantially matched to a complementary IC inlet pump flow rate of -0.1 mL / min to maintain cells in the bioreactor during the growth phase of cell culture. Alternatively, the IC circulation pump flow rate is matched or substantially matched to a complementary IC inlet pump flow rate of less than +0.1 mL / min or about -0.01 mL / min to a complementary IC inlet pump flow 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. Furthermore, in a further embodiment, intracapillary harvest synchronization, as described herein, may be followed during the growth phase of the cell culture. Such pump adjustment techniques during feeding counteract forces associated with cell loss from the IC outlet port. According to one embodiment, a type of feeding in which pump adjustment causes a forward flow to the IC inlet port (501A) and a counterflow to the IC outlet port (501B) of the bioreactor is called a modified feeding method. In another exemplary embodiment, the IC circulation pump flow rate is adjusted to equal the flow rate to the IC outlet port (501B) to about 50 percent (50%), or about half (1 / 2), of the IC inlet flow rate, or in other embodiments, other percentages or proportions (but in the opposite direction). For example, if the IC inlet pump flow rate is 0.4 mL / min, the IC circulation pump flow rate is set, configured, or adjusted to about -0.2 mL / min. In other embodiments, other percentages or proportions may be used.

[0208] Figures 5B and 5C show an operational configuration illustrating the fluid movement in the CES500, which, according to an embodiment, indicates forward and counter-forward flow rates for maintaining cells in the cell growth chamber or bioreactor 501. For example, such flow rates are shown in Figure 5B as "X" flow rate 503, "(-1 / 2)X" flow rate 505 (the negative sign ("-") indicates direction, and the direction of flow rate 505 is indicated by the directional arrow in Figure 5B), and "(1 / 2)X" flow rate 507. Here, approximately half of the flow rate, i.e., the first portion, branches off at connection 517 and enters the IC inlet port (501A) of the bioreactor 501, and approximately half of the flow rate, i.e., the second portion, branches off at connection 517 and enters the IC outlet port (501B) of the bioreactor 501. As shown, the sum of the first and second portions is substantially equal to the total flow rate 503, which is pumped by the IC inlet pump 554. Depending on the forward-counterflow rate used to retain cells in the bioreactor or cell growth chamber 501, the IC circulation pump may be set, configured, or adjusted using other types of percentages or fractions of the IC inlet pump flow rate. As such, Figure 5C shows the following flow rates: namely, "X" flow rate 511, "-(y%)*X" flow rate 513 (where the negative sign ("-") indicates direction, and the direction of flow rate 513 is indicated by the directional arrow in Figure 5C), and "(100%-y%)*X" flow rate 515, where "y" is equal to some percentage. In this embodiment, the sum of flow rates 513 and 515 is substantially equal to flow rate 511.

[0209] In one embodiment, all or substantially all of the flow from the first fluid channel 506 flows, for example, from the connection 517 to the IC inlet port (501A) of the bioreactor 501. In another embodiment, all or substantially all of the flow from the first fluid channel 506 flows from the connection 517 to the IC outlet port (501B) of the bioreactor 501. In yet another embodiment, a first portion of the flow from the first fluid channel 506 flows from the connection 517 to the IC inlet port (501A), and a second portion of the flow from the first fluid channel 506 flows from the connection 517 to the IC outlet port (501B). In the embodiment, the percentage of the IC inlet flow rate for which the IC circulation flow rate can be set is in the range of about 0 percent to about 100 percent. In another embodiment, that percentage is between about 25 percent and about 75 percent. In yet another embodiment, that percentage is between about 40 percent and about 60 percent. In other embodiments, the percentage is between approximately 45 percent and approximately 55 percent. In other embodiments, the percentage is approximately 50 percent. The operating configurations shown in Figures 5B and 5C represent possible configurations for various operations of the cell proliferation system, and modifications to the configurations shown should be understood to be within the scope of one or more embodiments.

[0210] Returning to FIG. 13, the cells are fed (and continuously grown) for a second period at a second inlet flow rate and a second circulation flow rate (step 1210), and the cells are maintained within the bioreactor and outside the header and in a portion of the IC circulation path outside the bioreactor. Following the feeding during the second period (step 1210), for example, if it is not desired to continue feeding and / or growing the cells, process 1200 ends with an end operation 1216. Alternatively, although optional, process 1200 can continue to increase the feeding flow rate or make other types of changes (step 1212). As represented by ellipsis 1214, there can be any number of feeding periods. Following the desired number of feeding periods 1214, process 1200 ends with an end operation 1216. FIG. 13 and process 1200 show an “increase” in flow rate, but other adjustments to the flow rate may be made. For example, the flow rate may be decreased from one feeding period to the next or may remain substantially the same. For example, the flow rate may be determined based on considerations such as metabolic activity. The “increase” in flow rate in FIG. 13 is provided for illustrative purposes only and is not intended to be limiting.

[0211] Next, Figure 14 shows exemplary operating steps of process 1300 for feeding cells while holding them in a first location (e.g., a bioreactor) using a cell proliferation system such as CES500 (e.g., Figures 5B and 5C) or CES700 (e.g., Figures 7A-7C) according to embodiments of the present disclosure. Start operation 1302 is initiated, and process 1300 proceeds to the steps of loading a disposable set into the cell proliferation system, priming the set, performing IC / EC washing, preparing the culture medium, and introducing cells (e.g., suspension cells i.e., non-adherent cells). Next, process 1300 proceeds to step 1304, feeding the cells for a first period. In embodiments, a first inlet flow rate and a first circulation flow rate are used. As an example, a first IC inlet flow rate and a first IC circulation flow rate are used, where the first IC inlet flow rate is generated and controlled by an IC inlet pump (e.g., a first pump), and the first IC circulation flow rate is generated and controlled by an IC circulation pump (e.g., a second pump). In an exemplary embodiment, the first IC inlet pump causes a volumetric flow rate of 0.1 mL / min to flow into the IC inlet port of the bioreactor, and a complementary IC circulation pump flow rate of -0.1 mL / min causes a volumetric flow rate of -0.1 mL / min to flow into the IC outlet port (501B) of the bioreactor at that flow rate. Here, the negative sign ("-") used, for example, in -0.1 mL / min indicates that the direction of the IC circulation pump (512) produces, i.e., generates, a counterflow so that cells are maintained in the bioreactor during the growth phase of cell culture. Alternatively, in an exemplary embodiment, the first IC inlet pump flows into the IC inlet port (701A) at a volumetric flow rate of less than 0.1 mL / min or about 0.01 mL / min, and the IC circulation pump flows into the IC outlet port (701B) at a complementary IC circulation volumetric flow rate or flow fluid flow rate of less than -0.1 mL / min or about -0.01 mL / min. Here, the negative sign ("-") used, for example, in -0.01 mL / min, indicates that the direction of the IC circulation pump creates a counterflow so that cells are maintained in the bioreactor during the growth phase of cell culture.In other embodiments, the first IC recycle flow rate may be a percentage or ratio of the first IC inlet flow rate. For example, the first IC recycle flow rate may be about 50 percent (50%) or about half (1 / 2) of the first IC inlet flow rate, or in certain embodiments, other percentages or ratios.

[0212] During cell feeding (and growth) and during the use of the IC pump to control cell retention in the bioreactor by the co-current / counter-current characteristics, the cells continue to grow and proliferate. As a result, the cells require additional media (e.g., glucose and / or cell growth media) to support the growing population. In embodiments, to feed the growing cell population, the media addition flow rate is increased. In an exemplary embodiment, an increase in the IC inlet pump flow rate (+) increases the IC inlet flow rate by a first amount to achieve a second IC inlet flow rate (step 1306). For example, according to one embodiment, the IC inlet flow rate is increased by a first amount of 0.1 mL / min to achieve a second IC inlet flow rate of 0.2 mL / min. According to other embodiments, other adjustments to the IC inlet flow rate may be made.

[0213] Next, according to the embodiment, the second IC circulation flow rate is set, configured, or adjusted to be equal to a certain percentage, portion, or ratio of the second IC inlet flow rate (step 1308). For example, according to one embodiment, the second IC circulation flow rate is set, configured, or adjusted to be equal to about 50 percent (50%), or about half (1 / 2), of the value of the IC inlet flow rate, or, in other embodiments, to another percentage or ratio. In one embodiment, all or substantially all of the flow from the first fluid channel 506 flows, for example, from the connection 517 to the IC inlet port (501A) of the bioreactor 501. In other embodiments, all or substantially all of the flow from the first fluid channel 506 flows from the connection 517 to the IC outlet port (501B) of the bioreactor 501. In yet another embodiment, a first portion of the flow from the first fluid channel 506 flows into the IC inlet port (501A) through the connection 517, and a second portion of the flow from the first fluid channel 506 flows into the IC outlet port (501B) through the connection 517. In this embodiment, the percentage of the IC inlet flow rate that can be set for the IC circulation flow rate is in the range of about 0 percent to about 100 percent. In another embodiment, the percentage is between about 25 percent and about 75 percent. In another embodiment, the percentage is between about 40 percent and about 60 percent. In another embodiment, the percentage is between about 45 percent and about 55 percent. In this embodiment, the percentage is about 50 percent.

[0214] In one embodiment, the second IC circulation flow rate is increased by adjusting the IC circulation pump flow rate (-) according to the value of the first IC circulation flow rate. In another embodiment, the IC circulation pump flow rate is adjusted to reduce the second IC circulation flow rate so that the second IC circulation flow rate is substantially equal to a predetermined ratio or predetermined proportion of the second IC inlet flow rate. In yet another embodiment, adjustment of the IC circulation pump flow rate is not required. For example, if the first IC inlet flow rate is equal to 0.1 mL / min and the first IC circulation flow rate is equal to -0.1 mL / min, then when the second IC inlet flow rate is increased to 0.2 mL / min, the second IC circulation flow rate becomes (-1 / 2)*(second Q IC Inlet ) That is, it is set to (-1 / 2)*(0.2 mL / min), which results in a second Q of -0.1 mL / min IC Circ Therefore, without adjusting the IC circulation pump flow rate, the second Q IC Circ This will be achieved.

[0215] Then, the cells are fed (and continuously proliferated) for a second period at a second inlet flow rate and a second circulation flow rate (step 1310), and the cells are maintained inside the bioreactor and outside the header or in part of the IC circulation path outside the bioreactor. If, for example, it is not desired to continue feeding and / or proliferating the cells following the second period of feeding (step 1310), process 1300 is terminated by termination operation 1316.

[0216] In the embodiments, the first period, second period, third period, fourth period, fifth period, etc., are each one day or longer (and / or one hour or longer and / or one minute or longer). For example, according to the embodiment, the period is from one to fourteen days. However, in other embodiments, the period may be less than one day or more than fourteen days. In one embodiment, for example, the first feeding period includes day 0, day 1, day 2, day 3, and day 4, the second feeding period includes day 5, the third feeding period includes day 6, and the fourth feeding period includes day 7. In other embodiments, the first period includes day 0, day 1, and day 2 (e.g., a duration of about 3 days), the second period includes day 3, day 4, and day 5 (e.g., a duration of about 3 days), the third period includes day 6, day 7, and day 8 (e.g., a duration of about 3 days), the fourth period includes day 9 and day 10 (e.g., a duration of about 2 days), and the fifth period includes day 11, day 12, and day 13 (e.g., a duration of about 3 days). Depending on the embodiment, the periods may be different. Each period is measured in units of days, hours, minutes, and / or some of these.

[0217] Returning to Figure 14, process 1300 optionally proceeds to query step 1312 to determine whether to adjust the feeding rate or flow rate based on metabolic activity or metabolic level. For example, in Treg proliferation, it may be desirable to control the lactate level of the growing cell population to a value of approximately 7 mmol / L or less. For example, if it is desirable to maintain the cell culture lactate level at approximately 7 mmol / L or less, the flow rate of medium addition is controlled during cell proliferation (e.g., regulatory T cells). In other embodiments, it may be desirable to maintain the lactate level at approximately 5 mmol / L or less to improve cell growth and viability. In embodiments, graphical user interface (GUI) elements are used to control the rate of medium addition and maintain lactate metabolic waste from glycolysis below a specified level during cell proliferation.

[0218] If it is not desired to measure metabolic activity and / or adjust the feeding level based on that measurement in the optional query step 1312, process 1300 proceeds to "no" and terminates in the termination operation 1316. Alternatively, if it is desired to adjust the feeding level based on metabolic activity, process 1300 proceeds to "yes" and continues feeding the growing cell population by increasing the medium addition rate in the optional step 1314. Although step 1314 is shown as a single step, this step may include multiple steps that make adjustments to the medium addition rate, such as increasing the IC inlet flow rate or increasing the IC circulation flow rate. Step 1314 is shown as a single step for illustrative purposes only and is not intended to limit it. Following the adjustment of the medium addition rate, process 1300 returns to the optional query step 1312 to decide whether to continue measuring metabolic levels and / or adjust the feeding. If adjustment of the feeding level based on metabolic activity is not desired, process 1300 proceeds to "no," and process 1300 terminates in termination operation 1316. Figure 13 and process 1300 show an "increase" in rate or flow rate, but other adjustments to the flow rate may be made. For example, the rate or flow rate may decrease from one feeding period to the next, or it may be kept substantially the same. The type of adjustment that may be made depends on the assessment of the metabolic activity of the growing cell population. For example, the "increase" in flow rate in Figure 13 is provided for illustrative purposes only and is not intended to limit the possibilities.

[0219] Next, Figure 15 shows exemplary operating steps of process 1400 for holding cells during feeding, which may be used in a cell proliferation system such as CES500 (Figures 5B and 5C) or CES700 (Figures 7A-7C) according to embodiments of the present disclosure. Start operation 1402 is initiated, and process 1400 proceeds to step 1404, in which a disposable tube set or pre-mounted fluid transfer assembly (e.g., 210 or 400) is mounted on the cell proliferation system. The disposable set may include a cell growth chamber 100A or a cell growth chamber 100B, as shown in Figures 1C, 4B, and 4C. Next, in step 1406, the system is primed. In one embodiment, for example, a user or operator instructs the system to prime by selecting a task for priming, for example. In one embodiment, such a task for priming is, for example, a pre-programmed task. Next, in step 1408, an IC / EC washing task is performed, in which the fluids in the IC circulation loop and the EC circulation loop are, for example, exchanged. According to one embodiment, the exchange volume is determined by the number of IC volumes and EC volumes to be exchanged.

[0220] Next, the medium preparation step 1410 is performed to maintain an appropriate or desired gas concentration across the hollow fibers of the bioreactor membrane so that the medium reaches equilibrium with the gas supply before the cells are introduced into the bioreactor. For example, contact between the medium and the gas supply provided by a gas transfer module (GTM) or oxygen supplier can be made by adjusting the EC circulation flow rate. The system is then maintained in an appropriate or desired state until, for example, the user or operator is ready to introduce the cells into the bioreactor. In one embodiment, the system may be prepared with a medium such as complete medium. Complete medium may be any medium source used for cell growth. In one embodiment, the system is prepared with serum-free medium, for example. In one embodiment, the system is prepared with basal medium. Any type of medium as understood by those skilled in the art may be used.

[0221] Process 1400 then proceeds to step 1412, for example, introducing cells from a cell inlet bag into a bioreactor. In one embodiment, the cells in the cell inlet bag are in a solution containing, for example, a culture medium for performing the cell feeding step 1414. In another embodiment, the cells in the cell inlet bag are in a solution containing both a culture medium for performing the cell feeding step 1414 and a soluble activator complex that stimulates the cells (e.g., T cells or Tregs). In one embodiment, the cells (and, in embodiment, the feeding solution) are introduced from the cell inlet bag into the bioreactor until the cell inlet bag is empty. The cells (and, in embodiment, the feeding solution) may be moved from an air removal chamber into the bioreactor. In one embodiment, the cells (and, in embodiment, the feeding solution) are introduced by performing the task of “introducing cells in a homogeneous suspension”. In another embodiment, the cells (and, in embodiment, the feeding solution) are introduced into a specific area (e.g., the center) of the bioreactor by performing the task of “introducing cells to the center without circulation”. According to embodiments, other introduction methods and / or introduction tasks may be used.

[0222] Next, process 1400 proceeds to query step 1416 to determine whether to use a modified feeding method to retain cells (e.g., non-adherent cells or suspension cells such as T cells or Tregs) in the bioreactor (e.g., a hollow fiber bioreactor). For example, it may be desirable to place the cells outside the bioreactor itself and the bioreactor header or outside the rest of the IC loop. If it is not desired to use a modified feeding method to retain cells in the bioreactor itself, process 1400 proceeds to "no" and the cells are grown in step 1426. Thereafter, the cells continue to grow / proliferate using the medium initially supplied with the cells in step 1414.

[0223] On the other hand, if it is desired to retain the cells in the bioreactor itself, process 1400 proceeds to "yes" and a modified feeding step 1418 is performed, in which the cells are fed by retaining them in the bioreactor using the flow rate to the IC inlet port (501A) and the flow rate to the IC outlet port (501B) of the bioreactor (501). At this time, an inlet volumetric flow rate or an inlet flow rate can be introduced into the first fluid channel (506) (step 1420). For example, an IC inlet flow rate can be introduced into the first fluid channel (506) (step 1420). An IC inlet pump (554) (e.g., a first peristaltic pump (in one embodiment)) can operate at a predetermined rotational speed (RPM) to generate a predetermined IC inlet volumetric flow rate or IC inlet flow rate into the first fluid channel (506) (step 1420). According to one embodiment, the processor and / or controller directs or controls the first pump and / or second pump to operate, for example, at a predetermined number of RPMs. Depending on the flow rate and direction of the IC circulation pump (512), a modified first flow rate or a first portion of the IC inlet flow rate enters the IC inlet port (501A) or first port of the bioreactor (501) (step 1422). The pump flow rate of the pump (e.g., peristaltic pump) depends on the diameter of the pump or the configuration of the pump. Other types of pumps may be used. In this case, the pump flow rate depends on the configuration of the pump used. The IC circulation pump (512) (e.g., second peristaltic pump (in one embodiment)) operates at a predetermined rotational speed (RPM) and in the opposite direction to the first pump to generate or produce a predetermined IC circulation flow rate, or second flow rate, or second portion of the IC inlet flow rate, which enters the IC outlet port (501B) or second port of the bioreactor (501) (step 1424). For example, in this embodiment, approximately half of the IC inlet flow rate, i.e., the first portion, is branched off at the connection part 517 and enters the IC inlet port (501A) of the bioreactor 501, and approximately half of the IC inlet flow rate, i.e., the second portion, is branched off at the connection part 517 and enters the IC outlet port (501B) of the bioreactor 501.As shown, the sum of the first and second parts is substantially equal to the IC inlet flow rate 503 (e.g., Figure 5B), which is pumped by the IC inlet pump 554. Depending on the forward and counterflow rates used to retain cells in the bioreactor or cell growth chamber 501, the IC circulation pump may be set, configured, or adjusted using other types of percentages or other percentage fractions of the IC inlet pump flow rate.

[0224] In an exemplary embodiment, cells are fed through the EC circulation supply loop 752, the IC circulation supply loop 753, or a combination thereof, as described with reference to Figures 7A–7C (step 916).

[0225] After feeding the cells using such forward-flow / counter-flow characteristics to retain them in the bioreactor, process 1400 proceeds to the cell growth / proliferation step 1426. Although cell proliferation is shown in step 1426, the cells may grow / proliferate during one or more other steps, such as steps 1412, 1414, 1416, 1418, 1420, 1422, 1424, etc. From the proliferation step 1426, process 1400 proceeds to the cell harvesting or removal step 1430. Then process 1400 terminates with a termination operation 1432. According to the embodiment, if other steps are desired before harvesting (e.g., continuing with a second modified feeding method or another type of feeding method), process 1400 proceeds to an optional “other” step 1428. From the optional step 1428, process 1400 proceeds to step 1430, in which cells are harvested or removed from the bioreactor, and process 1400 terminates with termination operation 1432.

[0226] Next, Figure 16A shows exemplary operating steps of process 1500, which feeds cells and holds them in a first position (e.g., a bioreactor), which may be used in a cell proliferation system such as the CES500 (Figures 5B and 5C) or CES700 (Figures 7A-7C) according to embodiments of the present disclosure. Start operation 1502 is initiated, and process 1500 proceeds to step 1504, in which a disposable tube set or a pre-mounted fluid transfer assembly (e.g., 210 or 400) is mounted on the cell proliferation system. The disposable set may include a cell growth chamber 100A or a cell growth chamber 100B, as shown in Figures 1C, 4B, and 4C. Next, in step 1506, the system is primed. In one embodiment, for example, a user or operator instructs the system to prime by selecting a task for priming, for example. In one embodiment, such a task for priming is, for example, a pre-programmed task. Next, in step 1508, an IC / EC washing task is performed. Here, the fluids in the IC circulation loop and the EC circulation loop are exchanged, for example. According to one embodiment, the exchange volume is determined by the number of IC volumes and EC volumes being exchanged.

[0227] Next, the medium preparation step 1510 is performed to maintain an appropriate or desired gas concentration across the hollow fibers of the bioreactor membrane so that the medium reaches equilibrium with the gas supply before the cells are introduced into the bioreactor. For example, contact between the medium and the gas supply provided by a gas transfer module (GTM) or oxygen supplier can be made by adjusting the EC circulation flow rate. The system is then maintained in an appropriate or desired state until, for example, the user or operator is ready to introduce the cells into the bioreactor. In one embodiment, the system may be prepared with a medium such as complete medium. Complete medium may be any medium source used for cell growth. In one embodiment, the system is prepared with serum-free medium, for example. In one embodiment, the system is prepared with basal medium. Any type of medium as understood by those skilled in the art may be used.

[0228] Process 1500 then proceeds to step 1512, for example, loading cells from a cell inlet bag into a bioreactor. In one embodiment, the cells in the cell inlet bag are in a solution containing, for example, a culture medium for cell feeding. In another embodiment, the cells in the cell inlet bag are in a solution containing both a culture medium and a soluble activator complex that stimulates the cells (e.g., T cells or Tregs). In one embodiment, the cells are loaded from the cell inlet bag into the bioreactor until the cell inlet bag is empty. The cells may be moved from an air removal chamber into the bioreactor. In one embodiment, the cells are loaded by performing the task of "loading cells in a homogeneous suspension". In another embodiment, the cells are loaded into a specific area (e.g., the center) of the bioreactor by performing the task of "loading cells to the center without circulation". Other loading methods and / or loading tasks may be used according to the embodiment.

[0229] Next, process 1500 proceeds to step 1514, which feeds cells according to the first process during the first period. In exemplary embodiments, cells are fed through the EC circulating supply loop 752, the IC circulating supply loop 753, or a combination thereof, as described with reference to Figures 7A–7C (step 916). In one embodiment, for example, if the cell population is just beginning to grow / proliferate and a minimum or low feeding rate can satisfy the feeding requirements of such a cell population, cells are fed at a minimum or low feeding rate. For example, an IC inlet pump flow rate of +0.1 mL / min that causes or generates a first fluid flow rate of 0.1 mL / min may be used during such a first period. In this example, a low or minimum feeding rate such as 0.1 mL / min is used, but according to embodiments, 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 desirable to reduce cell loss from the hollow fiber membrane bioreactor during such a first period, the complementary IC circulation pump flow rate is matched to -0.1 mL / min or nearly matched to the IC inlet pump flow rate of +0.1 mL / min to maintain cells in the bioreactor during the growth phase of cell culture. In other embodiments, other pump flow rates and the resulting fluid flow rates may be used. Alternatively, in an exemplary embodiment, the first IC inlet pump flows into the IC inlet port (701A) at a volumetric flow rate of less than 0.1 mL / min or about 0.01 mL / min, and the IC circulation pump flows into the IC outlet port (701B) at a complementary IC circulation volumetric flow rate or flow-fluid flow rate of less than -0.1 mL / min or about -0.01 mL / min. Here, the negative sign ("-") used, for example, in -0.01 mL / min indicates that the direction of the IC circulation pump produces a counterflow so that cells are maintained in the bioreactor during the growth phase of cell culture.

[0230] Next, process 1500 proceeds to query step 1516, which, according to the embodiment, determines whether to adjust the feeding rate to retain cells in the bioreactor itself, taking into account the growing cell population and increasing feeding demands. For example, Figure 16B shows an increasing feeding rate in response to a growing cell population. In Figure 16B, graph 1528 shows the number of cells against the IC flow rate for a run or procedure in a cell proliferation system, such as the Quantum® cell proliferation system. In one embodiment, the IC flow rate includes the culture medium for feeding the cells and is therefore also called the IC medium flow rate. The number of cells 1530 is shown against the IC flow rate (mL / min) 1532. As shown, according to the embodiment, the IC flow rate increases from 0.1 mL / min to 0.2 mL / min and further to 0.3 mL / min as the number of cells increases and the feeding demands of the growing cell population increase. In the embodiment shown, there is a substantially linear relationship between the number of cells and the IC flow rate, as shown by line 1534.

[0231] Returning to Figure 16A and query step 1516, if adjustment of the feeding rate is not desired, process 1500 proceeds to "no", and the cells are grown in step 1520. In this case, cell growth / proliferation continues using the medium used for feeding in the first period in step 1514. Although cell proliferation is shown in step 1520, the cells may grow / proliferate during one or more other steps, such as steps 1512, 1514, 1516, and 1518. On the other hand, if it is desirable to adjust the feeding rate while retaining the cells in the bioreactor, process 1500 proceeds to "yes", and the cells are fed in step 1518 according to a second process during a second period. In one embodiment, such a second process includes, for example, feeding the cells at approximately the same feeding rate as during the first period. In other embodiments, the second process includes feeding the cells at a different feeding rate compared to the feeding rate used during the first period. In one embodiment, the IC inlet flow rate is increased, and the IC circulation flow rate is set, configured, or adjusted to be equal to a certain percentage, proportion, or ratio of the IC inlet flow rate. For example, according to one embodiment, the IC circulation flow rate may be set to be equal to about 50 percent (50%), or about half (1 / 2), of the value of the IC inlet flow rate, or to any other percentage or proportion. According to one embodiment, the decision of whether to set, configure, or adjust the IC circulation flow rate to be a certain percentage, proportion, or ratio of the IC inlet flow rate is made based on the value of the IC inlet flow rate. For example, in one embodiment, the following method is provided for retaining cells in a bioreactor when feeding cells using the IC inlet flow rate (where Q IC Circ =IC circulation flow rate (mL / min);Q IC Inlet =IC inlet flow rate (mL / min)). : Q IC Inlet If ≥ 0.2 mL / min, Q IC Circ = (-)1 / 2*Q IC Inlet and Q IC InletWhen = 0.1 mL / min, Q IC Circ = (-)Q IC Inlet

[0232] In the above formula, the calculation of the IC circulation flow rate varies depending on the value of the IC inlet flow rate (e.g., 0.2 mL / min or 0.1 mL / min). However, in other embodiments, different values of the IC inlet flow rate may be used in such calculations. Further, in this example, approximately 50 percent (50%) or approximately half (1 / 2) is used, but other percentages, other ratios, other fractions, and / or other proportions may be used according to the embodiments. Returning to process 1500, as part of the second process of step 1518, after feeding the cells using such forward flow and counterflow characteristics to hold the cells within the bioreactor, process 1500 proceeds to the cell growth / proliferation step 1520. The growth of the cells is shown in step 1520, but the cells may grow / proliferate during one or more other steps, such as steps 1512, 1514, 1516, 1518. From the proliferation step 1520, process 1500 proceeds to the step 1524 of harvesting or withdrawing the cells. Then, process 1500 ends with the end operation 1526. According to an embodiment, if other steps are desired before harvesting, process 1500 proceeds to the optional "other" step 1522. From the optional step 1522, process 1500 proceeds to the step 1524 of harvesting or withdrawing the cells from the bioreactor, and process 1500 ends with the end operation 1526.

[0233] Next, Figure 17 shows exemplary operating steps of process 1600 for feeding cells to be used with a cell proliferation system such as CES500 (e.g., Figures 5B and 5C) or CES700 (e.g., Figures 7A-7C) according to embodiments of the present disclosure. A START operation 1602 is initiated, a disposable set is loaded into the cell proliferation system, the system is primed, IC / EC washing is performed, the culture medium is prepared, and cells are introduced. The disposable set may include a cell growth chamber 100A or a cell growth chamber 100B, as shown in Figures 1C, 4B, and 4C. Next, process 1600 proceeds to step 1604, which feeds cells according to a first process during a first period. In exemplary embodiments, cells are fed through an EC circulation supply loop 752, an IC circulation supply loop 753, or a combination thereof, as described with reference to Figures 7A-7C (step 916). In one embodiment, for example, if a cell population is just beginning to grow / proliferate and a minimum or low feeding rate can satisfy the feeding requirements of such a cell population, the cells are fed at a minimum or low feeding rate. For example, an IC inlet pump flow rate of +0.1 mL / min may be used during such a first period. In this example, a low or minimum feeding rate such as 0.1 mL / min is used, but according to embodiments, 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 desirable to reduce cell loss from the hollow fiber membrane bioreactor during such a first period, a complementary IC circulation pump flow rate of -0.1 mL / min is matched to or nearly matched to the IC inlet pump flow rate of +0.1 mL / min to maintain cells in the bioreactor during the growth phase of cell culture.Alternatively, in an exemplary embodiment, the first IC inlet pump flows into the IC inlet port (701A) at a volumetric flow rate of less than 0.1 mL / min or about 0.01 mL / min, and the IC circulation pump flows into the IC outlet port (701B) at a complementary IC circulation volumetric flow rate or flow fluid flow rate of less than -0.1 mL / min or about -0.01 mL / min. Here, the negative sign ("-") used, for example, in -0.01 mL / min, indicates that the direction of the IC circulation pump creates a counterflow so that cells are maintained in the bioreactor during the growth phase of cell culture.

[0234] Next, process 1600 proceeds to query step 1606 to determine whether the feeding rate should be adjusted to take into account the growing cell population and / or to keep the cells in the bioreactor. If it is desirable to adjust the feeding rate while keeping the cells in the bioreactor, process 1600 proceeds to "yes" and feeds the cells according to a second process during a second period in step 1608. In one embodiment, such a second process includes, for example, feeding the cells at substantially the same feeding rate as during the first period. In other embodiments, the second process includes feeding the cells at a different feeding rate compared to the feeding rate used during the first period. In one embodiment, the IC inlet flow rate is increased and the IC circulation flow rate is set, configured or adjusted to be equal to a certain percentage or a certain proportion or ratio of that IC inlet flow rate. For example, according to the embodiment, the IC circulation flow rate may be set to be equal to about 50 percent (50%), i.e., about half (1 / 2), or other percentage or ratio of the value of the IC inlet flow rate. According to one embodiment, the decision of whether to set the IC circulation flow rate to a certain percentage of the IC inlet flow rate is made based on the value of the IC inlet flow rate. For example, in the embodiment, the following method is provided (where Q IC Circ =IC circulation flow rate (mL / min);Q IC Inlet =IC inlet flow rate (mL / min)). : Q IC InletIf ≥ 0.2 mL / min, Q IC Circ = (-)1 / 2*Q IC Inlet and Q IC Inlet = If 0.1 mL / min, Q IC Circ =(-)Q IC Inlet

[0235] The above formula differs in the calculation of the IC circulation flow rate depending on the IC inlet flow rate value (e.g., 0.2 mL / min or 0.1 mL / min), but in other embodiments, different IC inlet flow rate values ​​may be used in such calculations. Furthermore, in this example, about 50 percent (50%), or about half (1 / 2), is used, but other percentages, other ratios, other fractions and / or other proportions may be used according to the embodiment. Returning to process 1600, after feeding the cells using such forward-flow / counter-flow characteristics to retain the cells in the bioreactor as part of the second process in step 1608, process 1600 proceeds to step 1610, which determines whether to monitor or measure the metabolic activity of the growing cell population (e.g., glucose consumption and / or lactate production). If monitoring metabolic activity is not desired, process 1600 proceeds to “no” and the cells are grown in step 1616. Therefore, the cells can continue to grow / proliferate using the medium supplied during feeding in the first period (step 1604) and / or the second period (step 1608). Although cell proliferation is shown in step 1616, the cells may grow / proliferate during one or more other steps, such as 1604, 1606, 1608, 1610, 1612, 1614, etc.

[0236] Returning to query step 1610, if it is desired to monitor or measure the metabolic activity of the growing cell population, process 1600 proceeds to "yes" and proceeds to continue feeding cells or adjust the feeding rate according to a second process based on the metabolic activity and / or its measurement. In one embodiment, monitoring glucose and / or lactate levels can facilitate adjustment of the medium flow rate (e.g., IC flow rate) to support the proliferation of cells (e.g., T cells or Tregs) in a bioreactor (e.g., a hollow fiber bioreactor). In an embodiment, the cell culture lactate level is maintained at, for example, less than about 7 mmol / L. In an embodiment, lactate metabolic waste from glycolysis is maintained at, for example, less than about 7 mmol / L during the proliferation of cells (e.g., regulatory T cells) by controlling the flow rate of medium addition, for example, using a graphical user interface (GUI) of the cell proliferation system. In another embodiment, the lactate level is maintained at, for example, less than about 5 mmol / L to improve, for example, cell growth and viability, by controlling the flow rate of medium addition and / or other settings, for example. In another embodiment, other concentrations may be used.

[0237] For example, depending on the metabolic measurement and the desired level of lactate, process 1600 proceeds to either continue feeding cells according to a second process (step 1612) or adjust the feeding rate (step 1614). For example, in one embodiment, if the measurement of metabolic activity indicates a lactate level of less than approximately 5 mmol / L, cell feeding is continued according to the second process (step 1612). In another embodiment, if the measurement of metabolic activity indicates a lactate level of less than approximately 7 mmol / L, cell feeding is continued according to the second process (step 1612). From step 1612, in which cell feeding is continued according to the second process, process 1600 returns to query step 1610 to continue monitoring the metabolic activity of the growing cell population.

[0238] Depending on the metabolic measurement and its desired level, process 1600 proceeds to step 1614, which involves adjusting the feeding rate. Here, cells are fed according to an additional process for an additional period. According to embodiments, such additional processes and additional periods include, for example, a third process in a third period, a fourth process in a fourth period, a fifth process in a fifth period, and so on. In one embodiment, such an additional process includes, for example, feeding cells at substantially the same feeding rate as in the first and / or second period. In other embodiments, the additional process includes feeding cells at a different feeding rate compared to the feeding rate used in the first and / or second period. For example, in one embodiment, the IC inlet flow rate is increased and the IC circulation flow rate is matched to, or substantially matched to, the IC inlet flow rate, but in the opposite direction. In other embodiments, the IC inlet flow rate is increased and the IC circulation flow rate is set, configured, or adjusted to be equal to and in the opposite direction to a certain percentage or proportion or part of the IC inlet flow rate. Although step 1614 of adjusting the feeding rate indicates “additional” processes and “additional” periods, any number of processes and periods may be used to adjust the feeding rate based on metabolic activity.

[0239] From the feeding rate adjustment step 1614, process 1600 returns to query step 1610. If it is not desired to adjust or further adjust the feeding rate, process 1600 proceeds to “no” and the cells are grown in step 1616, where the cells can continue to grow / proliferate using the medium supplied when feeding in the first period (1604), second period (1608), and / or additional period (1614). Although cell proliferation is shown in step 1616, the cells may grow / proliferate during one or more other steps, for example, steps 1604, 1606, 1608, 1610, 1612, 1614, etc. From the proliferation step 1616, process 1600 proceeds to step 1618, for example, harvesting or removing the cells from the bioreactor into harvest bags or containers. Then process 1600 terminates with END operation 1622. Alternatively, from the harvesting step 1618, process 1600 may optionally proceed to a further processing / analysis step 1620. Such optional further processing / analysis step 1620 includes, for example, characterizing the phenotype of the harvested cells (e.g., T cells or Tregs). Then, from the optional further processing / analysis step 1620, process 1600 terminates with an END operation 1622.

[0240] Figure 18A shows the operational steps of process 1700 for growing cells, which may be used with a cell proliferation system in embodiments of the present disclosure. Process 1700 includes the step of shearing cells grown in a cell growth chamber, according to embodiments of the present disclosure, as described below. In embodiments, these steps may be performed as part of a “modified circulation” task. The START operation 1702 is initiated, and process 1700 proceeds to step 1704, in which a fluid containing cells is introduced into the cell growth chamber in the cell proliferation system. In embodiments, the cells include non-adherent cells such as one or more types of T cells. In one embodiment, the cells include Tregs.

[0241] Process 1700 proceeds to the step of exposing the cells to the activator 1706. The activator, which may contain an antibody complex, is added to the fluid introduced in step 1704. In embodiments, the activator is a soluble human antibody CD3 / CD28 / CD2 cell activator (activator) complex. Process 1700 proceeds to the first period cell proliferation step 1708. Step 1708 includes a cell feeding step 1710. The cells are nourished to promote cell proliferation. For example, a culture medium containing glucose, protein, and reagents is delivered to the cell growth chamber to provide nutrients for cell proliferation.

[0242] The first period for cell proliferation (step 1708) may be based on the time required for the formation of cell colonies, microcolonies, or clusters. A cell colony, microcolony, or cluster is a group of one or more adherent cells. In embodiments, cells (e.g., Tregs) benefit from cell contact. Cell contact stimulates signaling that promotes proliferation and growth. However, after a period of proliferation, cells may adhere to each other to form cell colonies, microcolonies, or clusters. While not bound by theory, it is thought that after the cell proliferation period (1708), cells form relatively large cell colonies, microcolonies, or clusters that continue to grow. Cell colonies, microcolonies, or clusters may form necrotic centers, where nutrients (e.g., glucose), gases (e.g., oxygen), and reagents (e.g., activators) do not reach the cells at the center of the cell colony, microcolony, or cluster. As a result, the state of cell proliferation at the center of these cell colonies, microcolonies, or clusters may be slower in terms of growth rate (e.g., increased doubling time), or the state may lead to cell necrosis.

[0243] In some embodiments, the first period is between approximately 5 hours and approximately 48 hours in order to allow cell proliferation (step 1708). In some embodiments, the first period is longer than approximately 6 hours, or longer than approximately 12 hours, or longer than approximately 24 hours, or longer than approximately 48 hours. In other embodiments, the first period is less than approximately 72 hours, less than approximately 60 hours, less than approximately 48 hours, less than approximately 36 hours, less than approximately 24 hours, or less than approximately 12 hours. After the first period, process 1700 proceeds to step 1712, during which the cell colonies, microcolonies, or clusters are circulated to shear. Step 1712 is performed to reduce the size of the cell colonies, microcolonies, or clusters. In some embodiments, the second period is less than approximately 120 minutes, such as between approximately 60 minutes and approximately 0.5 minutes. In other embodiments, the second period may be based on the amount of fluid introduced into the first circulation path.

[0244] Figure 18B shows multiple Figures 1750, 1760, and 1770 of cells in a volume of fluid (1752) that can be grown in a cell growth chamber as part of process 1700. For example, in some embodiments, the cell growth chamber is a hollow fiber bioreactor. In these embodiments, Figures 1750, 1760, and 1770 show cells in one thread of a hollow fiber bioreactor, for example. 1750A, 1760A, and 1770A are enlarged portions of Figures 1750, 1760, and 1770, respectively. Figure 1750 shows cells after they have been introduced (step 1704), exposed to an activator (step 1706), grown for a period of time (e.g., a first period) (step 1710). As shown in Figure 1750, numerous cell colonies 1754A–1754E have been formed.

[0245] To reduce the number of cells in cell colonies, microcolonies, or clusters 1754A-1754E and the size of those colonies or clusters, step 1712 circulates the fluid and cells in a first fluid circulation path. Although not bound by theory, it is thought that this circulation generates forces (including shear stress) acting on the cell colonies, as indicated by arrow 1756 in enlarged portion 1760A. The shear stress 1756 provides enough force to divide and separate the cells within the cell colonies. As the circulation continues, the cell colonies begin to divide into smaller sizes, as shown in Figure 1760. Figure 1770 shows the cells after circulation over a second period. As shown in Figure 1770, the size of the cell colonies has decreased, and some colonies have been completely separated into individual cells. In some embodiments, the circulating shear step 1712 is performed until the cells and fluid become a single-cell suspension.

[0246] In other embodiments, cell colonies, microcolonies, or clusters of cells may remain after the cyclic shear step 1712. For example, colony 1754F in the enlarged portion 1770A shows that some colonies remain after step 1712, but have been reduced in size. In embodiments, the remaining cell colonies, microcolonies, or clusters (e.g., 1754F) are between approximately 25 μm and approximately 300 μm. In other embodiments, the cyclic shear step 1712 reduces the size of the cell colonies, microcolonies, or clusters (e.g., 1754F) to between approximately 50 μm and approximately 250 μm. In yet another embodiment, step 1712 reduces the size of the cell colonies, microcolonies, or clusters to between approximately 75 μm and approximately 200 μm. In some embodiments, the size of the cell colonies, microcolonies, or clusters becomes less than approximately 200 μm (e.g., approximately 100 μm) after step 1712.

[0247] In embodiments, the size of the remaining cell colonies, microcolonies, or clusters is, to some extent, a function of several structural features of the cell growth chamber. As described above, the cell growth chamber may, in some embodiments, be a hollow fiber bioreactor equipped with hollow fibers. As understood, cell colonies, microcolonies, or clusters are affected by shear stress each time they come into contact with the sidewalls of the hollow fibers as they circulate. This contact reduces the size of the cell colonies more efficiently. If the inner diameter is large, as in conventional processes that use a pipette to induce shear stress and reduce the size of colonies, contact with the sidewalls may not occur as frequently. Figure 18C shows the difference in inner diameter size between a hollow fiber (e.g., 215 microns) 1772 according to one embodiment and a pipette tip 1774 (762 microns) used to divide and separate adherent cells of cell colonies, microcolonies, or clusters. In embodiments, a smaller inner diameter of the hollow fiber is considered to reduce the size of the cell colonies more efficiently and effectively in the circulating shear step 1712.

[0248] The second period of the cyclic shear step 1712 is, in embodiments, less than about 120 minutes, less than about 90 minutes, less than about 60 minutes, less than about 30 minutes, or less than about 15 minutes. In some embodiments, the second period can be between about 1 minute and about 15 minutes, such as about 4 minutes.

[0249] After the second period, process 1700 proceeds to step 1714, which moves the cells into the cell growth chamber over a third period. In step 1714, cells that are not located in the cell growth chamber as a result of the circulating shear step 1712 are returned to the cell growth chamber during the third period. In embodiments, this includes operating one or more pumps to introduce fluid into the fluid circulation path. For example, the fluid is introduced into a first fluid flow path from a fluid inlet path and into the cell growth chamber from both the inlet and outlet ports of the cell growth chamber. The movement of the fluid from the inlet and outlet ports to the cell growth chamber returns the cells to the cell growth chamber.

[0250] In some embodiments, the fluid used in step 1714 to return the cells to the cell growth chamber contains reagents that promote cell growth. For example, in some embodiments, the fluid is a culture medium containing glucose, protein, or other reagents. In one embodiment, the fluid may contain one or more supplements. In one embodiment, the fluid is a complete culture medium containing cytokines (e.g., human IL-2 cytokine supplements). The addition of the fluid may be referred to as a bolus addition. The combination of steps 1712 and 1714 may, in some embodiments, be referred to as a circulating bolus addition.

[0251] In other embodiments, the third period may be based on the volume of fluid introduced into the cell growth chamber during the circulating shear step 1712. For example, in the embodiment, step 1712 is performed until about 300 ml, about 250 ml, about 200 ml, or about 150 ml has been introduced into the fluid circulation path.

[0252] After the third period, process 1700 proceeds to the fourth period, the proliferation step 1716. Similar to step 1708, step 1716 may include a cell feeding step 1718. The cells are nourished to promote cell proliferation. For example, a culture medium containing glucose, protein, and reagents is delivered to the cell growth chamber to provide nutrients for cell proliferation.

[0253] Similar to the first period, the fourth period may be based on the time it takes for the cell colony to form. In some embodiments, the fourth period is between approximately 5 hours and approximately 48 hours. In some embodiments, the fourth period is longer than approximately 6 hours, or longer than approximately 12 hours, or longer than approximately 24 hours, or longer than approximately 48 hours. In other embodiments, the fourth period is less than approximately 72 hours, less than approximately 60 hours, less than approximately 48 hours, less than approximately 36 hours, less than approximately 24 hours, or less than approximately 12 hours. In some embodiments, the fourth period may be shorter than the first period because more cells are likely to be in the cell growth chamber.

[0254] After the fourth period, process 1700 proceeds to step 1720, which circulates and shears over a fifth period, reducing the second cell colony. In some embodiments, step 1720 may use a first circulation rate. However, in other embodiments, the circulation rate used in step 1720 may be different, such as being greater than or less than the first circulation rate.

[0255] After the fifth period, process 1700 proceeds to step 1722, in which cells not located in the cell growth chamber are returned to the cell growth chamber during the sixth period. Fluid is introduced from the fluid inlet passage into the first fluid channel and into the cell growth chamber from both the inlet and outlet ports of the cell growth chamber. Cells are returned to the cell growth chamber by the movement of fluid from the inlet and outlet ports to the cell growth chamber. In some embodiments, the fluid used in the step of returning cells to the cell growth chamber contains reagents that promote cell growth. For example, in an embodiment, the fluid is a culture medium containing glucose, protein, or other reagents. In one embodiment, the fluid may contain one or more supplements. In one embodiment, the fluid is a complete culture medium containing cytokines (e.g., human IL-2 cytokines).

[0256] Process 1700 may optionally perform steps of cell proliferation, circulation, and migration, with additional repetitions, as indicated by optional steps 1724 and ellipsis 1726. Each of the proliferation, circulation, and migration steps may be performed consecutively over a period of time. For example, in some embodiments, these steps may be performed once every four days, once every three days, once every two days, once a day, twice a day, or three times a day over a period of time from about two days to about twenty days (e.g., about ten days). In some embodiments, these steps may be performed over a variety of periods. For example, in one embodiment, these steps may be performed after three days and then every other day thereafter. In another example, these steps may be performed after two days and then twice a day thereafter. These are merely examples, and other embodiments may utilize other periods.

[0257] For example, Figure 19 shows graph 1800, which describes the process of adding a circulating bolus at different intervals during cell proliferation. Curve 1808 shows the cell number 1802 relative to the cell culture days 1804 in a cell proliferation system, such as the Quantum® cell proliferation system. Curve 1806 shows the IC flow rate 1818 relative to the cell culture days 1804 in a cell proliferation system, such as the Quantum® cell proliferation system. As shown in curve 1806, the IC flow rate is maintained at 0.1 mL / min for the first three days. On day 6, the flow rate increases to 0.2 mL / min, and after day 7, it increases to 0.3 mL / min. The increase in flow rate corresponds to the increase in cell number with the increase in culture days 1804. Also, as shown in Figure 19, several circulating bolus addition steps (1810, 1812, 1814, 1816) are performed. The circulating bolus addition step 1810 is performed from day 3.5 of cell culture. Step 1812, adding another circulating bolus, is performed from day 4.5 of cell culture. Step 1814, adding another circulating bolus, is performed from day 6 of cell culture, and step 1816, adding another circulating bolus, is performed from day 6.5 of cell culture. As can be seen from curves 1806 and 1808, the multiple circulating bolus addition steps (1810-1816) combined with an increase in IC flow rate have a positive effect on the cell proliferation rate (e.g., cell number).

[0258] Returning to Figure 18A, process 1700 proceeds to step 1728, which involves removing cells from the cell growth chamber. In embodiments, this may include cell harvesting. Step 1728 may include additional steps, such as circulation steps (e.g., 1712, 1720), before or during the removal of cells from the cell growth chamber. Process 1700 terminates with termination operation 1730.

[0259] Figure 20A shows the operational steps of process 1900 for operating a pump that may be used in a cell growth system in embodiments of the present disclosure. As described below, process 1900 includes the step of operating the pump in a process of reducing the number of cells in a cell cluster grown in a cell growth chamber, according to embodiments of the present disclosure. In embodiments, these steps may be performed as part of a “modified circulation” task. In embodiments, the steps of process 1900 may be performed by a computer processor. The START operation 1902 is initiated, and process 1900 proceeds to step 1904, where a first pump is operated at a first flow rate to introduce a first volume of fluid containing cells into the capillary inner portion of the bioreactor of the cell growth system. For example, the bioreactor is a cell growth chamber 100A or a cell growth chamber 100B, as shown in Figures 1C, 4B, and 4C. In embodiments, the first pump is an inlet pump.

[0260] After operating the first pump at a first flow rate, process 1900 proceeds to step 1906, in which the first pump is operated at a second flow rate to introduce the nutrient-containing culture medium into the capillary inner portion of the bioreactor for a first period. The nutrients include, for example, proteins, glucose, and other compounds used to promote cell feeding and proliferation. For example, as shown in Figure 20B, the first pump 1960 is operated at a second flow rate to introduce the culture medium into the inlet port 1962A of the bioreactor 1962.

[0261] In some embodiments, the first period may be based on the time it takes for the cell colony to form. In some embodiments, the first period is between approximately 5 hours and approximately 48 hours. In some embodiments, the first period is longer than approximately 6 hours, or longer than approximately 12 hours, or longer than approximately 24 hours, or longer than approximately 48 hours. In other embodiments, the first period is less than approximately 72 hours, less than approximately 60 hours, less than approximately 48 hours, less than approximately 36 hours, less than approximately 24 hours, or less than approximately 12 hours.

[0262] Next, process 1900 proceeds to step 1908, which activates a second pump at a third flow rate to guide fluid into the bioreactor. An optional step 1908 is performed to activate pump 1964, which transfers a portion of the fluid introduced in step 1906 toward the outlet port 1962B of bioreactor 1962 to feed the cells.

[0263] Next, process 1900 proceeds to step 1910, which involves activating a second pump at a fourth flow rate to circulate the cells over a second period, thereby reducing the number of cells in the cell cluster within the bioreactor. In this embodiment, the cells circulate through a first fluid circulation path. Referring to Figure 20C, step 1910 activates a second pump 1964 to circulate the fluid through the first fluid circulation path 1966, as indicated by arrows 1968A to 1968D.

[0264] While not bound by theory, it is thought that after a certain period, proliferating cells form cell colonies, microcolonies, or clusters. Cell colonies may form necrotic centers, where nutrients and proteins (e.g., activators) do not reach the cells at the center of the colony. As a result, the state of cell proliferation at the center of these cell colonies, microcolonies, or clusters may be slower in growth rate (e.g., increased doubling time) and may lead to cell necrosis. Step 1910 is performed to reduce the size of the cell colonies, microcolonies, or clusters.

[0265] In the embodiment, the fourth flow rate is high enough to induce shear. For example, in the embodiment, the fourth flow rate reaches about 1000 ml / min. In other embodiments, the fourth flow rate may be between about 100 mL / min and about 600 mL / min, such as 300 mL / min.

[0266] After the second period, process 1900 proceeds to step 1912, where the first pump is operated at a fifth flow rate to introduce fluid through the first fluid channel over a third period. In this embodiment, the first portion of the fluid introduced into the first fluid channel returns the first cells (which may be in the first fluid channel for step 1910) in the first fluid channel back to the cell growth chamber through the inlet port 1962A. Referring to Figure 20D, pump 1960 is operated to introduce fluid into the first fluid channel 1970. As indicated by arrows 1968A and 1968B, the fluid flows from the first fluid channel 1970 to the inlet port 1962A. This returns the first cells outside the bioreactor 1962 back to the bioreactor 1962. By returning the cells in the first fluid channel to the cell growth chamber, the conditions for cell growth within the cell growth chamber are optimized, thus improving the overall proliferation of the cells.

[0267] In some embodiments, the fluid introduced into the first fluid channel in step 1912 may contain one or more materials (e.g., reagents) that promote cell growth. For example, in one embodiment, the fluid is a culture medium containing glucose or other nutrients for feeding the cells. In one embodiment, the fluid contains a reagent containing an additional activator to keep the cell growth active. Cell growth is improved when the cells are returned to the cell growth chamber using a fluid containing a specific reagent or other material and the cells are exposed to additional reagents (e.g., growth factors, proteins, etc.) that promote growth. In some embodiments, the addition of fluid used in step 1912 may be referred to as bolus addition.

[0268] After the third period, in step 1914, the second pump is operated at a sixth flow rate to transfer the second portion of the fluid introduced through the first fluid channel and the second cells to the cell growth chamber via the outlet port 1962B. In this embodiment, the second portion of the fluid returns the second cells in the first fluid channel (which may be in the first fluid channel for step 1910) to the cell growth chamber via the outlet port. Referring to Figure 20D, the pump 1964 is operated to transfer the fluid introduced into the first fluid channel 1970 to the outlet port 1962B. As indicated by arrow 1968C, the pump 1964 transfers the fluid and cells in the first fluid circulation path to the bioreactor 1962 via the outlet port 1962B. As shown in Figure 20D, the sixth flow rate is in the opposite direction to the fourth flow rate (Figure 20C). This fluid movement causes cells outside bioreactor 1962 to return to bioreactor 1962.

[0269] In this embodiment, the sixth flow rate is less than the fifth flow rate and moves the fluid into the first fluid passage as described above. As can be understood, the fifth flow rate results in the introduction of a portion of the circulation path 1966 of a given volume of fluid based on the fifth flow rate. The sixth flow rate is set so that a certain percentage of that volume moves toward the outlet port 1962B.

[0270] In some embodiments, the sixth flow rate may be set as a predetermined percentage of the fifth flow rate. For example, the sixth flow rate may be about 90% or less of the fifth flow rate. In some embodiments, the sixth flow rate may be set to about 80% or less of the fifth flow rate. In other embodiments, the sixth flow rate may be about 70% or less of the fifth flow rate. In yet another embodiment, the sixth flow rate may be about 60% or less of the fifth flow rate. In some embodiments, the sixth flow rate may be about 50% or less of the fifth flow rate.

[0271] In these embodiments, the sixth flow rate is at least partially based on the difference between a first volume between the second pump and the inlet port and a second volume between the second pump and the outlet port. Referring to Figure 20D, multiple portions of the first circulation path each have different volumes. For example, in one embodiment, the first volume between the second pump 1964 and the inlet port 1962A has a first volume, and the second volume between the second pump 1964 and the outlet port 1962B has a second volume that is different from (e.g., larger than) the first volume. In these embodiments, the sixth flow rate may be set at least partially based on these volume differences so that the cells reach the bioreactor substantially simultaneously in order to return the cells from the circulation path to the bioreactor.

[0272] As can be understood, when the fluid enters the fluid circulation path 1966 from the first fluid passage 1970, the fluid moves toward the inlet port 1962A at a flow rate set by the first pump 1960. When pump 1964 is activated, at least a portion of the fluid is transferred toward the outlet port 1962B. In this embodiment, the second volume (the volume between pump 1964 and the outlet port 1962B) is larger than the first volume. Therefore, in order to move more fluid into the portion of the second volume, the second pump 1964 is set to a certain percentage of the flow rate of pump 1960.

[0273] In one embodiment, in step 1912, pump 1960 is set to 100 ml / min. In this embodiment, the second volume (from pump 1964 to outlet port 1962B) is larger than the first volume (from pump 1964 to inlet port 1962A). Due to this additional volume, in this embodiment, in step 1914, pump 1964 is set to 70 ml / min. In this embodiment, the cells reach bioreactor 1962 almost simultaneously during the third period.

[0274] In some embodiments, process 1900 may be performed by optionally adding steps 1906-1914 multiple times, as indicated by the ellipsis 1916 and the optional step 1918. Steps 1906 (first pump (second flow rate)), 1910 (second pump (fourth flow rate)), 1912 (first pump (fifth flow rate)), and 1914 (second pump (sixth flow rate)) may be performed consecutively for a predetermined period. As described above, steps may be performed to feed the cells, circulate the cells to divide and separate cell colonies, microcolonies, or clusters, and return the cells 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 over a period of about two to about twenty days (e.g., about ten days). In some embodiments, these steps may be performed over varying periods. For example, in one embodiment, these steps may be performed after three days and then every other day thereafter. As another example, those steps may be executed after two days, and then twice a day thereafter. These are merely examples, and other embodiments may utilize other time periods. Process 1900 terminates with termination operation 1920.

[0275] It should be noted that in some embodiments, process 1900 may include additional steps. For example, a oscillator may be connected to the bioreactor and, after the first period (and during the second period), when the first pump is activated in step 1910, the oscillator may be activated as part of a step to circulate the cells and reduce the number of cells in the cell clusters, thereby rotating the bioreactor. This is just one example, and other embodiments of process 1900 are not limited thereto.

[0276] In other embodiments, the steps of process 1900 may proceed as shown in Figure 20A, with reduced flow rates achieved by precision motor control. For example, the IC pump and EC pump can operate from 0.005 RPM to 600 RPM in all ranges, within a torque range that prevents stalling over the entire range. Thus, the flow rate range is low, in both directions, at 500 RPM for the large pump and at 300 RPM for the small pump. Work done to prevent stalling at low speeds has been done by improving the stall detection method at low speeds and by controlling the control loop differently at low speeds. In exemplary embodiments, the fluid is pumped through the EC circulation supply loop 752, the IC circulation supply loop 753, or a combination thereof, as described with reference to Figures 7A-7C.

[0277] The IC and EC pumps operate continuously at nearly continuous speeds, resulting in strong counterflow containment at ultra-low flow rates, as described above. When two matched pumps are performing duty cycles, counter-operating the pumps to retain cells in the bioreactor shifts the position of the cells within the bioreactor. The continuous operation of the IC and EC pumps over extended periods retains the cells in the bioreactor in an optimized position, conserving cell culture medium overall.

[0278] As described below, harvest synchronization is provided by alternating pump control that can be performed over a long period of time. Low flow rates (about 1 / 10 of the flow rates used during the steps in the method described above) achieve approximately 0.01 mL / min. To obtain these rates, a continuous rate of 0.005 RPM is required. Thus, constant operation of the pump at 0.005 RPM is achieved without stalling. See the data in D0000038948, D0000038355, D0000044793, D0000033725 (and all attachments), and D0000041290.

[0279] In other embodiments, the steps of process 1900 may proceed in a significantly shorter period than described above, as shown in Figure 20A. For example, the IC inlet pump and the EC inlet pump are operated in alternating periods. In this case, the periods are less than 10 minutes or about 5 minutes. The pumps are operated at a low or very low flow rate during each period. For example, a low or very low flow rate is a flow rate of about 0.005 RPM to 600 RPM or about 0.01 mL / min at 500 RPM for a standard bioreactor or standard cell growth chamber, and about 0.01 mL / min at 300 RPM for a small bioreactor or small cell growth chamber. By operating the IC inlet pump and the EC inlet pump alternately in short time cycles, synchronization of capillary harvesting is made possible. During process 1900, the harvest valve and the EC outlet valve are opened in response to the operation of the IC inlet pump and the EC inlet pump, respectively. When the IC and EC inlet pumps are operating, they operate at twice the commanded speed, unless twice the commanded speed exceeds the allowable limit. The pumps then operate at the maximum allowable flow rate for the 5 minutes that the pumps are on. If the stop condition is volume-dependent, the remaining time displayed on the screen is updated only when each of those pumps is operating.

[0280] By creating a circulating option between the harvest outlet and the waste outlet, users can continue to supply and maintain cell cultures while continuously collecting target cell culture products (cells, viral vectors, exosomes, conditioned media, etc.) into harvest bags at a slow, user-selected flow rate over extended periods. This option is available for tasks using both standard bioreactors, i.e., standard cell growth chambers, and miniature bioreactors, i.e., miniature cell growth chambers (disposable type).

[0281] Intracapillary (IC) harvest synchronization provides continuous culture medium over a long period while simultaneously feeding cells. This allows for feeding cells with acclimatized medium, offering advantages over fresh medium.

[0282] In exemplary embodiments, the fluid is pumped through the EC circulation supply loop 752, the IC circulation supply loop 753, or a combination thereof, as described with reference to Figures 7A–7C.

[0283] Next, Figure 21 shows exemplary operating steps of a cell growth process 2000 that may be used in a cell growth system such as the CES500 (e.g., Figure 5A), CES600 (Figure 6), or CES700 (Figures 7A-7C) according to embodiments of the present disclosure. Starting operation 2002 is initiated, and process 2000 proceeds to step 2004, in which a disposable tube set is loaded into the cell growth system. The disposable tube set may include a cell growth chamber 100A or a cell growth chamber 100B, as shown in Figures 1C, 4B, and 4C (step 2004). Next, in step 2006, the system is primed. In one embodiment, for example, a user or operator instructs the system to prime by selecting a task for priming, for example. In one embodiment, such a task for priming is, for example, a pre-programmed task. Next, process 2000 proceeds to IC / EC washing task 2008, in which the fluids in the IC circulation loop and the EC circulation loop are exchanged. The exchange volume is determined by the number of IC volumes and EC volumes being exchanged.

[0284] Next, the medium preparation step 2010 is performed to maintain an appropriate or desired gas concentration across the hollow fibers of the bioreactor membrane so that the medium reaches equilibrium with the gas supply before the cells are introduced into the bioreactor. For example, by using a high EC circulation flow rate, contact between the gas supply provided by the gas transfer module or oxygen supply and the medium can be achieved rapidly. The system is then maintained in an appropriate or desired state until, for example, the user or operator is ready to introduce the cells into the bioreactor. In one embodiment, the system may be prepared with, for example, complete medium. Complete medium may be any medium source used for cell growth. In one embodiment, the complete medium includes, for example, alpha-MEM (α-MEM) and fetal bovine serum (FBS). Any type of medium understood by those skilled in the art may be used.

[0285] Process 2000 then proceeds to step 2012, which involves introducing cells, for example, from a cell inlet bag into the center of the bioreactor without circulation. In embodiments, the task of “introducing cells into the center without circulation” may be used, in which a first volume of fluid containing multiple cells is introduced into a cell growth system at a first flow rate, where the cell growth system includes a cell growth chamber. Next, a second volume of fluid containing culture medium is introduced into a portion of the first fluid circulation path at a second flow rate to place the first volume of fluid into, for example, the first portion of the cell growth chamber. In one embodiment, the first portion of the cell growth chamber or bioreactor is the approximate central region of the bioreactor. In one embodiment, the first volume is the same as the second volume. In one embodiment, the first flow rate is the same as the second flow rate. In other embodiments, the first volume is different from the second volume. In other embodiments, the first flow rate is different from the second flow rate. In one embodiment, the sum of the first and second volumes is equal to, for example, a percentage or proportion of the volume of the first fluid circulation path (e.g., total volume). For example, the sum of the first and second volumes is, for example, about 50% of the volume of the first fluid circulation path (e.g., total volume). In one embodiment, the fluid in the first fluid circulation path flows through the inner capillary (IC) space of the bioreactor or cell growth chamber. In one embodiment, the fluid in the second fluid circulation path flows through, for example, the outer capillary (EC) space of the cell growth chamber or bioreactor. In an exemplary embodiment, the fluid is fed through the EC circulation supply loop 752, the IC circulation supply loop 753, or a combination thereof, as described with reference to Figures 7A-7C (step 916). In one embodiment, the sum of the first and second volumes may be, for example, about 50% of the volume of the inner capillary (IC) loop, or other percentages or proportions. In one embodiment, the sum of the first and second volumes may be about 50% of the volume of other fluid paths, other loops, etc., or other percentages or proportions, where applicable. In embodiments, other percentages or proportions may be used, for example, any percentage between about 1% and about 100%.

[0286] Following the cell ingestion step 2012, process 2000 then proceeds to the cell feeding step 2014. The cells are grown / proliferated in step 2016. Although step 2016 is shown after step 2014, according to the embodiment, step 2016 may be performed before or concurrently with step 2014. Next, process 2000 proceeds to the query step 2018 to determine whether a cell colony, microcolony, or cluster has been formed. A cell colony, microcolony, or cluster is a group of one or more adherent cells. If a cell colony, microcolony, or cluster has been formed, process 2000 proceeds to "yes" and then to step 2020 to shear the cell colony, microcolony, or cluster. For example, multiple cells may be proliferated for a first period, and then the cells may be circulated at a first circulation flow rate over a second period to reduce the number of cells in the cell colony, microcolony, or cluster. In one embodiment, circulating cells at a first circulation flow rate generates shear stress in the cell colonies, causing one or more cells within the cell colonies to separate from the colonies. In one embodiment, a single cell suspension can be provided, for example, by reducing the number of cells in a cell colony, microcolony, or cluster. In one embodiment, the step 2020 of circulating cells to shear the colonies, microcolonies, or clusters can be used, for example, every two days during cell culture to maintain uniform cell density and nutrient diffusion. According to the embodiment, other periods may also be used. In one embodiment, such shearing of any microcolony, colony, or cluster may be started, for example, on or after day 4. According to the embodiment, other days or periods may be used to start such shearing. After the shearing step 2020, process 2000 then returns to the cell feeding step 2014.

[0287] If it is determined in query step 2018 that no cell colonies or clusters are to be sheared, or for example, none are present, process 2000 proceeds to “no” and resuspends the cells in step 2022. In embodiments, cells that may be lightly attached during culture are uniformly resuspended by circulating the cells. In embodiments, step 2022 may include circulating the cells to uniformly resuspend any lightly attached cells before initiating a harvesting task or other task for removing cells from a bioreactor. Following the cell resuspending step 2022, process 2000 then proceeds to the cell harvesting step 2024. Further processing or other analysis of the removed cells may be optionally performed in step 2026, and process 2000 terminates in termination operation 2028. If further processing / analysis is not desired, process 2000 terminates in termination operation 2030.

[0288] Turning to Figure 22 and process 2100, according to the embodiment, the start operation is initiated (step 2102), and process 2100 proceeds to step 2104, in which the disposable set is loaded into the cell growth system. The disposable set may include cell growth chamber 100A or cell growth chamber 100B, as shown in Figures 1C, 4B, and 4C. Next, in step 2106, the disposable set is primed and an IC / EC washing step 2108 is performed. Then, in step 2110, the culture medium is prepared. Next, process 2100 proceeds to the cell (suspension cells or non-adherent cells, such as T cells or Tregs) loading step 2112. In one embodiment, such cells are loaded by the task of "loading cells into the center without circulation" (step 2112). In other embodiments, such cells are loaded by the task of "loading cells by homogeneous suspension" (step 2112).

[0289] Next, process 2100 proceeds to step 2114, according to the embodiment, to initiate cell feeding (starting on day 0). In exemplary embodiments, the fluid is fed through the EC circulation supply loop 752, the IC circulation supply loop 753, or a combination thereof, as described with reference to Figures 7A-7C (step 916). The cells are grown and proliferated in step 2116, and by, for example, day 3, it may be desirable to bolus the fluid into the IC loop in step 2118 and redistribute the cells. In one embodiment, such a fluid bolus may contain reagents such as cytokines or other growth factors. In other embodiments, such a fluid bolus may contain, for example, reagents and basal medium.

[0290] Following such bolus addition and cell redistribution, process 2100 then proceeds to step 2120, in which the cells are fed again. Such feeding is performed, for example, on day 3. In the feeding step 2120, in step 2122, system parameters (e.g., one or more pump control flow rates) can be controlled to achieve complementary forward-direction / counter-direction flow settings for moving fluid into the bioreactor from both the IC inlet port and the IC outlet port of the bioreactor. For example, in step 2124, the IC inlet pump is adjusted or directed to generate a predetermined flow, and in step 2126, the IC circulation pump is adjusted or directed to generate counter-direction. For example, the IC inlet pump flow rate is matched to -0.1 mL / min, or nearly matched, to maintain the cells in the bioreactor during the growth phase of the cell culture (e.g., days 4-7 in the embodiment). Alternatively, in an exemplary embodiment, the first IC inlet pump flows into the IC inlet port (701A) at a volumetric flow rate of less than 0.1 mL / min or about 0.01 mL / min, and the IC circulation pump flows into the IC outlet port (701B) at a complementary IC circulation volumetric flow rate or flow fluid flow rate of less than -0.1 mL / min or about -0.01 mL / min. Here, the negative sign ("-") used, for example, in -0.01 mL / min, indicates that the direction of the IC circulation pump creates a counterflow so that cells are maintained in the bioreactor during the growth phase of cell culture. Such control in setting control step 2122 makes it possible to counteract any forces associated with cell loss from the bioreactor's IC outlet port.

[0291] Process 2100 then proceeds to query step 2128, where it is determined whether to continue adding reagents or other bolus additions on other days or at other time intervals. If it is desired to add additional reagents or other bolus additions and redistribute the cells, process 2100 branches to "yes", and in step 2118, the reagents are added and the cells are redistributed. For example, according to the embodiment, such bolus additions and cell redistributions are performed on days 6 and 9.

[0292] If it is not desired to continue adding a bolus (e.g., reagents) and redistributing cells, process 2100 proceeds to "no", and in step 2130 the cells are harvested, where they are transferred to a harvest bag or container. Process 2100 terminates in termination operation 2136.

[0293] Alternatively, from the harvesting step 2130, process 2100 may optionally proceed to a further processing / analysis step 2132. Such a further processing / analysis step 2132 may include characterizing the phenotype of the harvested cells (e.g., T cells or Tregs). From the optional further processing / analysis step 2132, process 2100 may optionally proceed to a step 2134 in which any remaining cells are reintroduced. Then process 2100 terminates with a termination operation 2136.

[0294] Process 2200 illustrates the operational steps of the process for growing cells in a cell proliferation system according to embodiments of the present disclosure. In some embodiments, T cells can be grown using Process 2200. As illustrated, cell proliferation is carried out by performing various steps in the course of a 14-day protocol. The START operation 2202 is initiated and Process 2200 proceeds to Day 0, and a disposable set is loaded into the cell proliferation system (Step 2206). The disposable set may include a cell growth chamber 100A or a cell growth chamber 100B, as shown in Figures 1C, 4B, and 4C. The disposable set is then primed (Step 2208). Here, the set is primed with, for example, PBS (e.g., Lonza Ca 2+ / Mg 2+ The cells are primed (free). In preparation for cell loading, the priming solution is replaced using IC / EC washing (step 2210). For example, according to one embodiment, the PBS in the system may be replaced with TexMACS GMP basal medium. Next, the medium is prepared (step 2212). The medium preparation step 2212 is performed to allow the medium to reach equilibrium with the provided gas supply before the cells are loaded into the bioreactor.

[0295] Next, on day 0, process 2200 proceeds to step 2214, in which cells (e.g., suspended cells or non-adherent cells such as T cells or Tregs) are introduced. In one embodiment, in step 2214, such cells are introduced by the task of “introducing cells into the center without circulation.” In another embodiment, in step 2214, such cells are introduced by the task of “introducing cells in a homogeneous suspension.”

[0296] On day 3 (2216), cytokines may be added as a bolus while the cells are being redistributed (step 2218). In embodiments, combining cell redistribution with bolus addition allows for mixing the cells and more complete exposure to the cytokines (e.g., IL-2) during bolus addition. In embodiments, redistribution can divide and separate the formed cell colonies or clusters. In embodiments, redistribution is first carried out by circulating the cells in a fluid circulation channel. Bolus addition is then carried out in a process of pushing the cells back into the bioreactor (e.g., by introducing fluid into the fluid circulation channel to push the cells back into the bioreactor). Following the redistribution and bolus addition step 2218, process 2000 proceeds to the cell feeding step 2220.

[0297] On day 6 (2222), the cells are redistributed again with another bolus added (2224). Redistribution separates the cell colonies or clusters formed between days 3 and 5. Bolus addition exposes the cells to additional reagents that promote growth. Process 2000 proceeds to cell feeding step 2226 on day 6. On day 9 (2228), the cells are redistributed again with another bolus added (step 2230). Redistribution separates the cell colonies or clusters formed between days 6 and 8. Bolus addition exposes the cells to additional supplements that promote growth. Process 2000 proceeds to cell feeding step 2232 on day 9.

[0298] On days 11–13 (2234), the cells are redistributed again with bolus addition (step 2236). Redistribution separates the cell colonies or clusters formed on days 9–10. Bolus addition exposes the cells to additional reagents that promote growth. Process 2000 then proceeds to the cell feeding step 2238. In embodiments, steps 2236, 2238 may be performed on days 11, 12, and 13, respectively. This may occur as a result of growing the cells on days 0–10 and having more cells in the bioreactor. Cell redistribution and bolus addition promote cell growth by more frequently separating the cell colonies and clusters into smaller pieces and mixing them with the reagents of bolus addition to promote cell growth. Process 2200 ends with END operation 2240.

[0299] Process 2300 illustrates the operational steps of a process for growing cells (e.g., suspended cells or non-adherent cells) in a cell proliferation system according to embodiments of the present disclosure. In some embodiments, process 2300 can be used to grow T cells such as Tregs. Combinations of the steps of process 2300 enable cell proliferation to useful clinical quantities at low initial seeding densities.

[0300] The START operation 2302 is initiated, and process 2300 proceeds to step 2304, in which the disposable set is loaded into the cell growth system. The disposable set may include cell growth chamber 100A or cell growth chamber 100B, as shown in Figures 1C, 4B, and 4C. The disposable set is then primed (step 2306). Here, the set is primed with, for example, PBS (e.g., Lonza Ca 2+ / Mg 2+The cells are primed with (free). In preparation for cell loading, the priming solution is replaced using IC / EC washing (step 2308). For example, according to one embodiment, the PBS in the system may be replaced with TexMACS GMP basal medium. Next, the medium is prepared (step 2310). The medium preparation step 2310 is performed to allow the medium to reach equilibrium with the provided gas supply before the cells are loaded into the bioreactor.

[0301] Process 2300 proceeds to step 2312, in which a fluid inlet volume containing cells is introduced. In embodiments, the cells include non-adherent cells such as one or more types of T cells, such as Tregs. In one embodiment, the cells include Tregs. In embodiments, the fluid inlet volume containing cells is introduced into the IC circulation path via the IC inlet path using an IC inlet pump. In embodiments, in step 2312, the introduced inlet volume is introduced without operating the IC circulation pump.

[0302] Process 2300 proceeds to step 2314, which involves positioning the inlet volume in a first portion of the bioreactor. In an embodiment, the positioning step can be performed by introducing a second volume of fluid containing the culture medium into a portion of the IC circulation path, thereby pushing the inlet volume of fluid containing the cells into the first position of the bioreactor. In an embodiment, the inlet volume of the fluid and the second volume of the fluid may be the same. In another embodiment, the inlet volume of the fluid and the second volume of the fluid may be different. In yet another embodiment, the sum of the inlet volume of the fluid and the second volume of the fluid may be equal to a certain percentage of the volume of the IC circulation path.

[0303] Following the inlet volume placement step 2314, process 2300 proceeds to step 2316, in which cells are exposed to an activator to activate and proliferate them. In some embodiments, cells are exposed to a soluble activator in step 2316. In some embodiments, the activator, which contains an antibody conjugate, is added to the culture medium to include the inlet volume, or is added later, such as to a second volume. In embodiments, the activator is a human antibody CD3 / CD28 / CD2 cell activator conjugate.

[0304] Process 2300 proceeds to step 2318, in which cells are fed according to the first process during the first period. In exemplary embodiments, cells are fed through the EC circulation supply loop 752, the IC circulation supply loop 753, or a combination thereof, as described with reference to Figures 7A–7C (step 916). In one embodiment, for example, if the cell population is beginning to grow / proliferate during the first period in step 2320 and the feeding requirements of such a cell population can be met even with a minimum or low feeding rate, the cells are fed at a minimum or low feeding rate. For example, an IC inlet pump flow rate of +0.1 mL / min may be used during such a first period. If it is desired to reduce cell loss from the hollow fiber membrane bioreactor during such a first period, a complementary IC circulation pump flow rate of -0.1 mL / min is matched to the IC inlet pump flow rate of +0.1 mL / min, or nearly matched, to maintain cells in the bioreactor during the growth phase of the cell culture. Alternatively, in an exemplary embodiment, the first IC inlet pump flows into the IC inlet port (701A) at a volumetric flow rate of less than 0.1 mL / min or about 0.01 mL / min, and the IC circulation pump flows into the IC outlet port (701B) at a complementary IC circulation volumetric flow rate or flow fluid flow rate of less than -0.1 mL / min or -0.01 mL / min. Here, the negative sign ("-") used, for example, about -0.01 mL / min, indicates that the direction of the IC circulation pump creates a counterflow so that cells are maintained in the bioreactor during the growth phase of cell culture.

[0305] From step 2320, process 2300 proceeds to step 2322, which involves growing the cells over a second period. The growth step 2322 of the second period may include feeding the cells (2324) according to a second process for the second period. In one embodiment, such a second process includes, for example, feeding the cells at substantially the same feeding rate as during the first period. In other embodiments, the second process includes feeding the cells at a different feeding rate compared to the feeding rate used during the first period. For example, the feeding rate increases in response to cell growth over the first period.

[0306] During the second period, while the cells are growing, the cells may be circulated to shear the cell colonies or clusters (step 2326). Step 2326 may include circulating the cells within the IC circulation path to shear the colonies or clusters formed during the first period. The colony / cluster shearing step 2326 reduces the number of cells in the cell colonies or clusters. In embodiments, the circulating and shearing step 2326 generates shear stress in the cell colonies, causing one or more cells within the cell colonies to separate from the cell colonies.

[0307] Process 2300 then proceeds to harvest step 2328, where the cells are transferred to a harvest bag or container. In embodiments, a therapeutic amount of cells can be harvested. In embodiments, the cells harvested in step 2328 are 1 × 10⁶ 9 These are approximately one cell. In some embodiments, the harvested cells have a viability rate between approximately 75% and 95%.

[0308] Next, process 2300 optionally proceeds to step 2330, which involves further processing / analysis. Such further processing may include, for example, characterizing the phenotype of the harvested cells (e.g., T cells or Tregs). In one embodiment, the harvested cells express biomarkers consistent with Tregs. For example, the cells express CD4 + CD25 + , and / or FoxP3 + The cells express a biomarker. In one embodiment, harvested cells express CD4 at a frequency of over 80%. + CD25 + This includes the phenotype. In other embodiments, cells have a frequency of over 55% CD4 + FoxP3 + This includes the phenotype. Then, process 2300 terminates with END operation 2332.

[0309] In embodiments of this disclosure, the operational steps shown in the figures above are for illustrative purposes only and may be rearranged, combined with other steps, or performed in parallel with other steps. In embodiments, steps may be reduced or added, without departing from the spirit and scope of this disclosure. Furthermore, steps (and substeps) such as priming, culture medium preparation, and cell loading may, in some embodiments, be performed automatically by a processor that performs pre-programmed tasks stored in memory, for example. Such steps are presented here for illustrative purposes only. In addition, the exemplary pump flow rate settings for feeding cells, for example, shown in Figure 11B, are provided for illustrative purposes only. Other pump flow rates, flow rates, directions, etc., may be used according to embodiments of this disclosure.

[0310] Examples and detailed descriptions of tasks and protocols, including custom and pre-programmed tasks used with the cell proliferation system, are presented in U.S. Patent Application No. 13 / 269,323 (filed October 7, 2011, title "Configurable Method and System for Cell Growth and Cell Harvesting in Hollow Fiber Bioreactor Systems") and U.S. Patent Application No. 13 / 269,351 (filed October 7, 2011, title "Customizable Method and System for Cell Growth and Cell Harvesting in Hollow Fiber Bioreactor Systems"). The entirety of those U.S. Patent Applications is expressly incorporated herein by this disclosure.

[0311] Next, Figure 25 shows an example of the components of a computing system 2400 that implements an embodiment of the present invention. The computing system 2400 is used in embodiments in which a cell proliferation system uses a processor to perform tasks such as custom tasks or pre-programmed tasks as part of a process such as the processes shown and / or described herein. In embodiments, pre-programmed tasks may include, for example, IC / EC washing and / or cell feeding.

[0312] The computing system 2400 includes a user interface 2402, a processing system 2404, and / or a storage device 2406. The user interface 2402 includes an output device 2408 and / or an input device 2410, as will be apparent to those skilled in the art. The output device 2408 may have one or more touchscreens. The touchscreen may have a display area for providing one or more application windows. The touchscreen may also be an input device 2410 capable of receiving and / or capturing physical contact from a user or operator, for example. The touchscreen may also be a capacitive liquid crystal display (LCD) that allows the processing system 2404 to estimate the contact location, as will be apparent to those skilled in the art. In this case, the processing system 2404 can map the contact location to a UI element displayed at a predetermined position in the application window. The touchscreen may also receive contact via one or more other electronic structures in this invention. Other output devices 2408 include printers, speakers, and the like. Other input devices 2410 include keyboards, other touch input devices, mice, voice input devices, etc., as will be understood by those skilled in the art. For example, user interface 2402 may be user interface 264 as described with reference to Figure 2A. User interface 2402 may include different screens for different parts of a task / protocol / process / method, different user inputs or requests, etc.

[0313] In embodiments of the present invention, the processing system 2404 may have a processing unit 2412 and / or memory 2414. The processing unit 2412 may be a general-purpose processor capable of executing instructions stored in memory 2414. In embodiments of the present invention, the processing unit 2412 may include a single processor or multiple processors. Furthermore, in embodiments, each processor may be a multi-core processor having one or more cores for independently reading and executing instructions. The processors may include general-purpose processors, application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), and other integrated circuits, as will be understood by those skilled in the art.

[0314] In embodiments of the present invention, the memory 2414 may include any storage device for short-term or long-term storage of data and / or processor-executable instructions. As will be understood by those skilled in the art, the memory 2414 includes, for example, random-access memory (RAM), read-only memory (ROM), or electrically erasable and programmable read-only memory (EEPROM). Other storage media include, for example, CD-ROMs, tapes, digital multipurpose discs (DVDs), or other optical storage devices, tapes, magnetic disk storage devices, magnetic tapes, and other magnetic storage devices.

[0315] The storage device 2406 is any long-term data storage device or component. In embodiments of the present invention, the storage device 2406 may include one or more of the systems described in relation to the memory 2414. The storage device 2406 may be permanent or removable. In one embodiment, the storage device 2406 stores data generated or given by the processing system 2404.

[0316] The computing system 2400 may communicate with clouds, network computers, personal computing devices, mobile devices, etc., via a wireless network, a Bluetooth® network, or other network system. Alternatively, the computing system 2400 may communicate with personal computing devices, network computing devices, mobile devices, etc., via a hardwired connection.

[0317] The processing system 2404 can control the pump start-up, speed, and fluid flow. The processing system 2404 may use a control loop for the pump process to control the pump operation / speed to provide an ultra-low supply rate in continuous operation (instead of step operation). Continuous and consistent operation (compared to periodic / step operation) facilitates finer pump control and the realization of lower supply rates. To facilitate continuous harvesting of cells, operation can be switched between two pumps operating at low flow rates. In an exemplary embodiment, the first IC inlet pump flows into the IC inlet port (701A) at a volumetric flow rate of less than 0.1 mL / min or about 0.01 mL / min, and the IC circulation pump flows into the IC outlet port (701B) at a complementary IC circulation volumetric flow rate or flow fluid flow rate of less than -0.1 mL / min or -0.01 mL / min. Here, for example, the negative sign ("-") used in -0.01 mL / min indicates that the direction of the IC circulation pump creates a counterflow so that the cells are maintained in the bioreactor during the growth phase of the cell culture.

[0318] The processing system 2404 can control the operation / speed of the pumps for counterflow containment. Ultra-low flow rate counterflow containment allows for cell enrichment in the desired region. Counterflow containment conserves cell culture medium and allows for lower protein concentrations in the system. Lower protein concentrations are possible by enabling continuous operation. The counter-operating pumps cluster the cells by pumping in opposite directions.

[0319] The processing system 2404 may be configured to run various tasks / methods / processes / protocols that are entered by the user or stored as preset tasks / methods / processes / protocols in memory 2414. For example, the processing system 2404 may be configured to run or coordinate the execution of a cell processing application (CPA). The cell processing application enables tracking and logging of user components, procedures, user logins, etc. Using this system, protocols can be sent to a group of identical instruments, thus saving a lot of time setting up valid protocols for execution on the group of instruments. All reports can then be pulled back to the application, and real-time readouts from the group of instruments can be viewed remotely on the application. Temperature and pressure can be updated to the application at a configurable rate, and alarms and warnings are also sent to the application. For example, the application may push alarms as remote alarms to one or more users for recording or correction. For example, remote alarms may be emails, text messages, or other digital alerts sent to users. CPA also connects FINIA® and Quantum Flex® to maintain all user reports in a unified application.

[0320] CPA has been tested 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: D000002 8798, Protocol Task Management Report: D0000044037, Device Configuration Protocol: D0000028621, Device Configuration Report: D0000045376, EOR Protocol: D0000028620, EOR Report: D0000045378, D0000047097: Network Performance, D0000047098: Multiple Devices, D0000047099: Access and Auditing, D0000047100: Data and Reports.

[0321] Cell processing applications include tracking and data management functions. Tracking and logging are enabled through CPA. Tracking and logging are performed periodically (e.g., by schedule) or on demand. Users can schedule recording times based on trigger events or regular intervals.

[0322] Cell processing applications can also perform device group control. CPA integrates modular cell therapy components, including, for example, CES (e.g., Quantum Flex® cell proliferation systems) and FINIA®. Therefore, the CES described herein (e.g., CES100, CES700, etc.) is a module within a modular system controlled by a cell processing application. For example, CPA can control a group of 100 or more devices. Using CPA, the same command can be sent to multiple machines rather than individual machines, enabling streamlined and consistent operation.

[0323] Cell processing applications include custom tasks / methods / processes / protocols. Users can create custom protocols or custom tasks. Tasks may be detailed steps for a user-defined process. Protocols may be a collection of tasks necessary to complete a test or cell proliferation process, etc., stored in a single file. Memory 2414 can store predefined, pre-written, or stock protocols and tasks. Stock tasks are compiled into custom protocols by the user. Protocols in the CPA system are modified in real time and re-uploaded to the instrument cluster. Tasks and protocols may be written, modified, and selected both on the user interface and on an external computer or handheld device.

[0324] Cell processing applications may involve different user profiles, accounts, and access levels. Each user may be assigned either a predefined or custom role. Each role includes a set of permissions that can be customized by the administrator. Permissions may include levels of access control (read-only, write, etc.). A single user may have multiple roles. CPA may require user authentication for security purposes.

[0325] The cell processing application may trigger and send remote notifications and alarms. Email or other notifications (digital notifications, texts, etc.) may be sent to the user, allowing them to monitor and inspect the status without direct access to or contact with the instrument. The user can communicate remotely with the instrument, including reviewing data in real time via the cell processing application. The user can remotely send commands to the instrument, such as ignoring alarms, stopping tests, or modifying protocols or tests. This is more efficient for the user.

[0326] The cell processing application controls network access for software updates. New software may be pushed to CES or other devices from the cloud, a remote computer, or a remote device.

[0327] The cell processing application may receive data from a barcode scanner. The barcode scanner may be one of the input devices 2410 described above. Disposable packages, culture media, cells, etc., each contain a barcode with product information. The CPA can check the data from the barcode scanner to verify that the correct components or culture media are being used. The barcodes may be stored in memory 2414 or the CPA for future reference, such as product recall or proliferation data. The CPA can adjust the task / method / process / protocol configuration based on the data from the barcode scanner.

[0328] Computing system 2400 increases process efficiency by promoting contact between the "target reagent" (viral particles, transfection reagent, secondary cell type, differentiation reagent, induction reagent, etc.) and adherent or suspended cells within a hollow fiber bioreactor (HFB). Cells (adherent or suspended cells) are seeded on the inside of the capillaries (IC) of the HFB. The target reagent is introduced into the IC side of the HFB. Counterflow (inlet = positive flow, circulation = negative flow), which is divided by driving the IC inlet pump and IC circulation pump in opposite directions, is used to promote active contact between the target reagent and the cell population. This process can be continued as long as necessary.

[0329] In many processes in cell culture, a population of cells needs to be exposed to specific suspended elements in order to modify cells or produce secondary products. For example, to produce a viral vector product, a population of cells must be exposed to active viral particles that replicate within the cells; to transfect cells by introducing a novel gene, cells must be exposed to both the target gene (GOI) and the reagents required to take up the GOI into the cell for translation. The efficiency of both of these process examples varies depending on the environment in which the process takes place. In many passive models for these types of processes, the facilitation of the interaction between the target reagent (ROI) and the cell population relies to a high degree of chance. These passive models reduce the efficiency of viral integration / plasmid transfection / GOI expression.

[0330] The processes described herein are intended to increase the efficiency of these processes by facilitating active contact between the cell population and the ROI. Cells (adherent or suspended cells) are seeded onto hollow fibers of the HFB. Once the cells are established, the "target reagent" (viral particles, transfection reagent, secondary cell type, differentiation reagent, induction reagent, etc.) is introduced into the HFB. The medium fluid is drawn from a two-port bag. The flow is divided by an inlet pump and a circulation pump. The medium fluid enters the IC side of the HFB from both sides. The IC waste valve is closed, so the fluid must exit the HFB through the pores in the HFB membrane and proceed from the IC side to the EC side. The medium fluid is recirculated back into the two-port bag from the EC side. Because the "target reagent" molecules are larger than the pores in the HFB membrane, they are contained within the IC loop and pressed against the cell layer on the membrane wall, facilitating contact between the "target reagent" and the cell population. The above is continued as long as necessary.

[0331] Examples The following description includes several examples of protocols / methods / processes that can be used in cell proliferation systems such as the CES500 (e.g., Figures 5A, 5B, 5C) and / or CES600 (Figure 6) that implement aspects of the embodiments. While specific features are described in the embodiments, such embodiments are provided solely for illustrative and explanatory purposes. For example, the embodiments describe the proliferation of T cells and / or Treg cells, but other and / or additional cell types and / or combinations thereof may be used in other embodiments. Certain parameters, functions, and / or values ​​are described (in some embodiments, for example, the use of CES such as the Quantum® cell proliferation system), but these parameters, functions, and / or values ​​are provided solely for illustrative purposes. This disclosure is not limited to the embodiments and / or specific details provided herein.

[0332] Furthermore, the examples shown herein are not limited to other embodiments that include different steps, additional steps, different parameters, additional parameters, or other features. In some embodiments, the illustrated methods or protocols including steps (and substeps) are performed automatically, for example, by a processor that performs a pre-programmed task stored in memory. In other embodiments, the steps (and substeps) are performed by a combination of automated and manual operations. In yet another embodiment, the steps (and substeps) are performed by an operator or user, or by other manual means.

[0333] While example data is provided in such embodiments, such illustrative data is provided for illustrative purposes only and is not intended to limit other embodiments, including different steps, parameters, values, materials, or other features.

[0334] In some cases, a protocol package or method for a small bioreactor, i.e., a small cell growth chamber, may perform the same tasks as a standard bioreactor, i.e., a standard cell growth chamber. Some protocol packages or methods for small bioreactors, i.e., small cell growth chambers, have the following differences: 1. When draining to the air removal chamber (ARC) in the ARC control step and when forcing flow across the membrane, the flow rate is reduced. This is because small bioreactors have a smaller hollow fiber surface area, so the flow rate is reduced to avoid high pressure. 2. Different default selections depending on the disposable set (i.e., where applicable, flow rate and volume (volume) stop conditions are reduced due to the volume differences of the various parts of the set). 3. A range is open, allowing the user to select the option to scale down the flow to 1 / 10 of a standard bioreactor task.

[0335] In some exemplary embodiments, the protocol package or method includes: 1. Default (template) adherent cell proliferation protocol; 2. Default (template) suspension cell proliferation protocol; 3. Default (template) custom 1-step protocol; 4. Custom step 1-10 tasks with increased options available compared to LegacyQuantum custom tasks; 5. Cell loading by multiple distribution cycles (task created by the science team to optimize MSC seeding); 6. Cell loading and placement (task created by the science team to optimize T cell loading); 7. Non-adherent cell feeding (task created by the science team to optimize T cell feeding); and / or 8. Cell circulation and placement (task created by the science team to optimize sampling during T cell proliferation).

[0336] Example 1

[0337] method

[0338] General Treg cell culture

[0339] Immunomagnetically separated CD4 + CD25 + Treg cells were obtained from peripheral blood of healthy adult donors by leukapheresis (HemaCare Corporation, Van Nuys, CA), and then 1.0 × 10⁶ cells were collected in three T25 flasks (7 mL / flask) in sterile filtered TexMACS® GMP medium supplemented with 200 IU / mL of recombinant human IL-2 IS premium grade cytokine (Miltenyi Biotec GmbH, Bergisch-Gladbach) and Gibco PSN 100X antibiotic mixture (ThermoFisher Scientific, Waltham, Massachusetts). 5 Treg cells are grown in Terumo BCT at a concentration of cell count / mL. Subsequently, the actively growing Treg cell suspension is used as inoculum in each of three experimental runs using the Quantum cell proliferation system. Treg cells for both inoculum and Quantum system proliferation are co-stimulated with 25 μL / mL of soluble tetrameric Immunocult® human antibody CD3 / CD28 / CD2 cell activation complex (Stem Cell Technologies, Vancouver, BC) without microbeads. Co-stimulation is performed on days 0 and 9 for Treg inoculation and on day 0 for Quantum system Treg proliferation. The Quantum system HFM bioreactor has a capillary inner loop volume of 177.1 mL and a flow rate of 21,000 cm⁻¹. 2 It is characterized by its surface area.

[0340] Treg proliferation in the Quantum system

[0341] According to the embodiment, two 2L bags of sterile filtered medium are prepared using a Quantum medium bag 4L set (Cat.21021) for Treg scale-up expansion in the Quantum system. One 2L bag containing complete medium with TexMACS GMP, IL-2, and PSN antibiotics is supplied to the IC compartment, and the other 2L bag containing basal medium with TexMACS GMP and PSN antibiotics is supplied to the bioreactor's EC inlet compartment. After priming the Quantum system with PBS (Lonza Cat.17-516Q, Walkersville, MD), the medium bags are connected to the appropriate IC and EC inlet lines using a TSCD-Q Terumo sterile welding machine. The complete medium is protected from exposure to light.

[0342] To introduce cells into the Quantum system bioreactor, use a Quantum cell inlet bag (Cat.21020) to add total cells (4.5-6.5 × 10⁶) to 50 mL of complete culture medium in each run using a sterile method. 7 Resuspend the individual Tregs. Additional disposable bags, such as Quantum CES medium bags 4L (Cat.21021) and waste bags 4L (Cat.21023), are also used during large-scale Treg growth runs.

[0343] Upon completion of the task of "centralizing cells without circulation," the Quantum system run (n=3) yielded 2.5–3.7 × 10⁶ cells in 177 mL of complete medium. 5 At a concentration of cell count / mL, or within the lumen or inner capillary (IC) compartment of a hollow fiber membrane bioreactor, the average concentration is 2.1–3.1 × 10⁻¹⁴. 3 number of cells / cm 2 Sow Treg at this concentration.

[0344] Day 0-4

[0345] Quantum Custom Task Example

[0346] Modified cell feeding

[0347] IC / EC exchange and culture medium preparation examples for regulatory T cells

[0348] A TexMACS GMP complete medium bag containing IL-2 supplement (200 IU / mL) is attached to the IC medium line of the Quantum system using a Terumo BCT TSCD-Q sterile welding machine. TexMACS basal medium is attached to the EC medium line. For each, an IC / EC washing task and a medium preparation task are performed. To conserve the amount of IL-2 and activator complex, complete medium is used for IC exchange or washing, and basal medium is used for EC exchange or washing.

[0349] Before introducing cells, set the system to the modified "cell feeding" setting. Increase the IC inlet flow rate (Q1) and IC circulation flow rate (Q2) to 0.2 mL / min, 0.3 mL / min, and 0.4 mL / min on days 5, 6, and 7, respectively, in the opposite direction on days 4, 5, and 6, while maintaining the lactate level at 5-8 mmol / L if necessary, using matched flow rates.

[0350] [Table 1]

[0351] Quantum Custom Task Example

[0352] Example of a task involving introducing cells to the center without circulation.

[0353] Objective: This task aims to distribute suspended cells to the center of the bioreactor membrane while enabling flow in the outer capillary (EC) circulation loop. The pump flow rate to the IC loop is set to zero.

[0354] Enter any task modifications or changes before loading cells into the Quantum system using non-circulating cell loading.

[0355] [Table 2]

[0356] [Table 3]

[0357] If necessary, revert to the default cell feeding task and continue the proliferation protocol using the cell feeding task.

[0358] [Table 4]

[0359] From day 4 onwards

[0360] Examples of resuspension of Treg cells during cell culture or before harvesting.

[0361] The purpose of this modified circulating task is to uniformly resuspend cells that may be loosely attached during culture or before the start of the harvesting task.

[0362] Furthermore, to maintain uniform cell density and nutrient diffusion from day 4 onward, this task is used to shear Treg cell colonies every two days during cell culture. If this task is used to shear colonies during the culture process, the Quantum system is returned to the modified “cell feeding” task.

[0363] [Table 5]

[0364] Example of a Quantum harvesting task with modifications

[0365] [Table 6]

[0366] The harvested cells are removed from the Quantum system by RF welding for further evaluation and analysis.

[0367] Post-harvest analysis

[0368] The harvested cells were enumerated in the 5–50 μm range using a Vi-CELL XR 2.04 Cell Viability Analyzer (Beckman Coulter), and membrane integrity was quantified by trypan blue staining exclusion.

[0369] metabolic effect

[0370] Regulatory T cell metabolism is monitored daily from the Quantum EC sample port using an i-STAT handheld analyzer (Abbott Point of Care, Princeton, New Jersey) with G cartridges (i-STAT G cartridge (Cat.03P83-25) for glucose and i-STAT CG4+ cartridge (Cat.03P85-50) for lactate).

[0371] Cell surface biomarker expression

[0372] Human regulatory T cells (native and induced) constitute a small subset (2–10%) of all T cells in human umbilical cord blood and peripheral blood. Functionally, Tregs are involved in maintaining immune homeostasis, including regulating immune tolerance in both innate and acquired immune responses. Furthermore, the expression of the transcription factor forkhead box P3 (FoxP3) gene product is linked to CD4 + CD25 + FoxP3 + CD127 lo / - Treg phenotype and antigen-presenting cells (APCs) and effector T cells (T effIt is known to correlate with immunosuppression. The binding of IL-2 to the CD25 / IL-2 receptor (Rα) and the activation of the STAT5 transcription factor are used to induce Foxp3. Foxp3 suppression upregulates the activity of several genes, including CTLA-4, TNFRSF18, and IL2RA, and downregulates IL-2 through its association with histone acetylase KAT5 and histone deacetylase HDAC7.

[0373] The frequency of the Treg phenotype, a surface biomarker of harvested cells, is quantified by flow cytometry. For this purpose, cells are stained with the following antibody conjugates and gated to unstained viable cells: FixableViability Dye eFluor® 780 (eBioscience 65-0865), mouse anti-human CD4-PE (BD Pharmingen 561844), anti-CD4-Alexa Fluor 647 (BD Pharmingen 557707), anti-CD4-FITC (BD Pharmingen 561842), anti-CD4-FITC (BD Pharmingen 555346), anti-CD25-PE (BD Pharmingen 555432), anti-CD127-PE (BD Pharmingen 557938), anti-CD45RO-PE (BD Pharmingen 347967), and anti-FoxP3-Alexa Fluor 647 (BD Pharmingen 560045). Sample data can be acquired using a bead-corrected BD Canto II flow cytometer equipped with FACSDiva v6.1.3 software. This uses 1 x 10⁶ cells and 20,000 events per sample.

[0374] An example experimental flow chart is shown in Figure 26.

[0375] result

[0376] Preliminary studies of Treg cells in static culture have shown that these cells tend to form microcolonies with a diameter of approximately 100 μm. Dividing and separating these cells every two days during the medium change process helps limit cell necrosis, and the cells can be returned to a high-density single-cell suspension using a 1,000 μL pipette tip with an ID (inner diameter) of 762 μm. Alternatively, the process of maintaining the single-cell suspension can be achieved more efficiently in an automated HFM bioreactor, such as the Quantum system, with a hollow fiber lumen ID (inner diameter) of approximately 200 μm, through a pre-programmed daily circulation task. Furthermore, this automated feeding task can be performed in a functionally closed system, reducing the possibility of contamination while maintaining a continuous flow of nutrients to the Treg culture.

[0377] Treg cell density and viability

[0378] [Table 7]

[0379] [Table 8]

[0380] Preliminary experiment on cell seeding density

[0381] In preparation for proliferation of immunomagnetically selected cells from a donor in an automated bioreactor, a series of static growth experiments were performed to show that stimulated Treg cells reached 1.0 × 10⁶. 6 To determine whether culture is possible at a seeding density of less than 1.0 × 10¹⁶ cells / mL. This part of the study involved 1.0 × 10¹⁶ cells in 18 wells of a 24-well tissue culture plate. 5 By seeding at a cell count / mL, or by placing 1.0 × 10⁶ cells in TexMACS GMP medium wells supplemented with IL-2 (200 IU / mL) and PSN antibiotics. 5This is carried out by seeding at a cell count / mL. These cells may be co-stimulated with a soluble anti-CD3 / CD28 / CD2 mAb complex at 25 μL / mL on days 0 and 9. The cells are manually harvested and counted on day 14 using Vi-CELL XR.

[0382] [Table 9]

[0383] After harvesting, cell samples are pooled for Treg biomarker analysis by flow cytometry. The results are CD4 + C25 + CD4 + CD127 - , and CD4 + FoxP3 + The phenotypic frequencies in static culture are 90.9%, 79.7%, and 31.6%, respectively. Since the detection of FoxP3+ is highly dependent on the permeabilization method and cell viability, ...

Claims

1. A method for proliferating cells in a cell proliferation system, wherein the method is A step of introducing a first volume of fluid containing multiple cells into the cell proliferation system, wherein the cell proliferation system includes a cell growth chamber. The steps include: introducing a second volume of fluid containing the culture medium into a portion of the first fluid circulation path to position the first volume of fluid in the first portion of the cell growth chamber; A step of feeding the cells by continuously circulating the culture medium in an inner capillary circulation loop, an outer capillary circulation loop, or a combination thereof, The steps of proliferating the aforementioned cells, Having, method.

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

3. In the method according to claim 1, the fluid of the first fluid circulation path flows through the capillary inner space of the cell growth chamber. method.

4. In the method according to claim 3, the fluid of the second fluid circulation path flows through the capillary outer space of the cell growth chamber. method.

5. In the method according to claim 1, the first volume of fluid containing the plurality of cells is introduced without operating the capillary internal circulation pump. method.

6. In the method according to claim 1, the first volume is the same as the second volume. method.

7. The method according to claim 1 further comprises the step of applying a coating agent to the cell growth surface of the cell growth chamber, The coating agent comprises human fibronectin, cryoprecipitate, or a combination thereof. method.

8. The method according to claim 7, further comprising the step of moving a fluid through the hollow fiber membrane in the cell growth chamber into the capillary outer circulation loop using ultrafiltration, method.

9. In the method according to claim 8, the fluid includes the coating agent, method.

10. In the method according to claim 1, the plurality of cells include a plurality of virus-associated cells, a plurality of Vero cells, or a combination thereof. method.

11. The method according to claim 1, further comprising the step of rotating the cell proliferation system around a central axis using at least one rocking device, method.

12. A method for controlling a cell proliferation system, the method being The controller receives data readout values ​​from at least one sensor, The controller tracks and logs events during the cell proliferation process, The controller records the data read value from at least one sensor, The controller performs the steps of displaying the event and the data read value on a display, Having, method.

13. A method for controlling a group of cell proliferation apparatus, the method is: A step of generating a set of instructions for the cell proliferation apparatus by a controller, wherein the cell proliferation apparatus comprises two or more cell proliferation apparatuses, The controller simultaneously transmits the set of commands for the group of cell proliferation devices to each of the cell proliferation devices in the group of cell proliferation devices. The controller performs the step of programming each of the cell proliferation devices of the cell proliferation device group with the set of instructions, Having, method.

14. A method for controlling a cell proliferation system, the method being The controller operates the first pump to flow the first fluid at a first fluid flow rate, The controller operates a second pump to flow a second fluid at a second fluid flow rate, It has, The first fluid flow rate and the second fluid flow rate are in opposite directions. method.

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

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

17. A culture medium bag for a cell proliferation system, the culture medium bag is A flexible housing having an internal space configured to contain a fluid, A pair of ports arranged in parallel within the bottom of the flexible housing, Equipped with, The flexible housing has a hanger portion at the top of the internal space opposite the pair of ports, The pair of ports includes an inlet port and an outlet port. Culture medium bag.

18. In the culture medium bag according to claim 17, the hanger portion has slots configured to receive one or more hooks for suspending the culture medium bag. Culture medium bag.

19. The culture medium bag according to claim 17, the culture medium bag further comprises a rod, The hanger portion has a channel in the housing for receiving the rod, The rod and the hanger portion are configured to fit into an external rail for suspending the culture medium bag. Culture medium bag.

20. A cell proliferation system, wherein the cell proliferation system is A cell proliferation apparatus comprising a housing and a holder, wherein the housing has a receiving portion on its operating surface, and the holder protrudes from the housing, A disposable set configured to engage with the receiving portion of the cell proliferation device, A culture medium bag configured to be suspended from the holder of the cell proliferation apparatus, Equipped with, The culture medium bag has a flexible housing having an internal space configured to contain a fluid, the flexible housing having a hanger portion at the top of the internal space, and the hanger portion includes a rod extending longitudinally along the top of the culture medium bag. The rod is configured to be received within the channel of the holder and to fix the culture medium bag to the holder of the cell proliferation apparatus. Cell proliferation system.

21. In the cell proliferation system according to claim 20, the disposable set comprises a cell growth chamber and a tube, wherein the tube is configured to connect the cell growth chamber to the culture medium bag. Cell proliferation system.

22. In the cell proliferation system according to claim 20, The holder has at least one protrusion, The hanger portion has a slot configured to receive the protruding portion and suspend the culture medium bag. Cell proliferation system.

23. A method for controlling a cell proliferation system, the method being The controller operates the first pump to flow the first fluid at a first fluid flow rate, The controller operates a second pump to flow a second fluid at a second fluid flow rate, The steps include detecting parameters related to the first fluid or the second fluid using a sensor, The controller receives the parameter from the sensor and determines whether the parameter is outside a predetermined range. The controller transmits a remote alarm to an external device separate from the cell proliferation system, Having, method.

24. In the method according to claim 23, the remote alarm includes email, text message, digital alert, or a combination thereof. method.

25. The method according to claim 23, further comprising the step of activating an audible alarm, a visual alarm, or a combination thereof. method.

26. In the method according to claim 23, the sensor includes a thermistor, a pressure sensor, a gas sensor, an air detector, a metabolite sensor, or a combination thereof. method.

27. A method for controlling a cell proliferation system, the method being A step of detecting parameters related to the cell proliferation system, The controller receives the parameter and determines whether the parameter is outside a predetermined range. The controller transmits a remote alarm to an external device separate from the cell proliferation system, Having, method.

28. In the method according to claim 27, the remote alarm includes email, text message, digital alert, or a combination thereof. method.

29. The method according to claim 27, further comprising the step of activating an audible alarm, a visual alarm, or a combination thereof. method.

30. In the method according to claim 27, the parameter is one of temperature, door position, pressure, flow rate, and concentration. method.

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