A method for removing solutes without losing suspended cells during the cell proliferation process.

Abstract

A method for removing solute while retaining suspended cells in a bioreactor comprises the steps of positioning the cell suspension within a designated area of ​​the bioreactor and generating a flow rate in the suspension within the bioreactor, the flow rate being selected such that the cells are retained in the bioreactor while the solute exits the bioreactor.

Description

Technical Field

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[0001] (Cross - reference to related applications) This application claims priority to U.S. Patent Application No. 18 / 637,782, filed Apr. 17, 2024, and also claims the benefit of U.S. Provisional Patent Application No. 63 / 461,989, filed Apr. 26, 2023. The entire disclosure of the above applications is incorporated herein by reference.

[0002] This disclosure relates to a method for removing high - molecular - weight solutes without loss of suspended cells in a cell growth process.

Background Art

[0003] This section provides background information related to this disclosure, which is not necessarily prior art.

[0004] A cell growth system, or a hollow - fiber cell growth system, also referred to as a hollow - fiber bioreactor, is a cell culture system that includes one or more removable bioreactor cartridges used to grow and differentiate cells, including both adherent and non - adherent cell types. The bioreactor cartridge comprises a hollow - fiber membrane that includes a plurality of semi - permeable hollow fibers. The space inside the hollow fibers (i.e., the lumen) defines the intracapillary space, while the space outside the hollow fibers defines the extracapillary space. Depending on the permeability of the hollow - fiber wall, high - molecular - weight solutes (e.g., having a molecular weight of about 20,000 daltons or more) from the culture medium and / or the growing cell population, etc., may not be able to easily diffuse through the hollow - fiber wall, which results in an undesirable progressive accumulation of solutes (e.g., including proteins, cytokines, and / or waste products) in the intracapillary space (or, alternatively, or in addition to, the extracapillary space). The accumulation of solutes has an undesirable impact on cell viability, growth, and / or phenotype.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, it is desirable to develop a method for removing unwanted solutes from the inner capillary space (or, alternatively, or in addition to, the outer capillary space) without loss of suspended cells. [Means for solving the problem]

[0006] This section provides a general overview of the disclosure and does not constitute a comprehensive disclosure of its entire scope or all features.

[0007] This disclosure provides a method for removing solutes while retaining suspended cells in a bioreactor.

[0008] In at least one exemplary embodiment, the method comprises the steps of positioning a suspension containing cells and solute within a predetermined area of ​​the bioreactor, and generating a flow rate in the suspension within the bioreactor, the flow rate being selected such that the cells are retained within the bioreactor while the solute exits the bioreactor.

[0009] In at least one exemplary embodiment, the step of positioning the suspension comprises the steps of generating a first flow at a first end of the bioreactor and generating a second flow at a second end of the bioreactor, wherein the first and second flows cooperate to coordinate a first axial flow from the first end and a second axial flow from the second end so that the suspension is positioned within the predetermined area of ​​the bioreactor.

[0010] In at least one exemplary embodiment, the first flow has a first flow rate, and the second flow has a second flow rate different from the first flow rate.

[0011] In at least one exemplary embodiment, the second flow rate is smaller than the first flow rate.

[0012] In at least one exemplary embodiment, the second flow rate is less than half of the first flow rate.

[0013] In at least one exemplary embodiment, the step of positioning the suspension further includes the step of positioning the bioreactor in a horizontal or substantially horizontal position such that the suspension settles on or near a wall defining the bioreactor.

[0014] In at least one exemplary embodiment, after the step of positioning the suspension and before the step of generating the flow rate in the suspension, the method further includes the step of stopping all flow through the bioreactor.

[0015] In at least one exemplary embodiment, after the step of stopping all flow through the bioreactor and before the step of generating the flow rate in the suspension, the method further includes the step of setting a flow valve to allow the flow to exit the bioreactor.

[0016] In at least one exemplary embodiment, prior to the step of generating the flow rate in the suspension, the method further includes the step of positioning the bioreactor at an angle of 45 degrees or approximately 45 degrees with respect to the horizontal.

[0017] In at least one exemplary embodiment, the predetermined region of the bioreactor is a region located in the center between a first end of the bioreactor and a second end opposite to the first end.

[0018] In at least one exemplary embodiment, the predetermined region is perpendicular to the long axis of the bioreactor.

[0019] This disclosure provides an alternative method for removing solutes while retaining suspended cells within a bioreactor.

[0020] In at least one exemplary embodiment, the method comprises the steps of: positioning the bioreactor at an angle to the horizontal so as to define the upper and lower ends of the bioreactor; positioning a suspension containing cells and solutes in predetermined locations within the bioreactor; initiating flow at a circulating flow rate; and initiating flow at a pre-selected net ultrafiltration flow rate.

[0021] In at least one exemplary embodiment, the step of positioning the suspension comprises the steps of generating a first flow at the lower end of the bioreactor and generating a second flow at the upper end of the bioreactor, wherein the first and second flows cooperate to coordinate a first axial flow from the first end and a second axial flow from the second end so that the suspension is positioned within the predetermined area of ​​the bioreactor.

[0022] In at least one exemplary embodiment, the first flow has a first flow rate, and the second flow has a second flow rate different from the first flow rate.

[0023] In at least one exemplary embodiment, the second flow rate is smaller than the first flow rate.

[0024] In at least one exemplary embodiment, the second flow rate is less than half of the first flow rate.

[0025] In at least one exemplary embodiment, after the step of positioning the suspension in the predetermined location within the bioreactor and before the step of initiating flow at the circulating flow rate, the method further includes the step of stopping all flow.

[0026] Further scope will become apparent from the descriptions provided herein. The descriptions and specific examples in this summary are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0027] The drawings in this specification are for the purpose of exemplifying only the selected embodiments and are not intended to limit the scope of the present disclosure, not all possible embodiments.

Brief Description of the Drawings

[0028] [Figure 1] FIG. 1 is a view of an example of a cell growth system having a bioreactor according to at least one exemplary embodiment. [Figure 2] FIG. 2 is a view of an example of a bioreactor showing a circulation path through the bioreactor incorporated into a cell growth system similar to the cell growth system illustrated in FIG. 1 according to at least one exemplary embodiment. [Figure 3] FIG. 3 is a view of an example of a rocking device configured to be used with a cell growth system similar to the cell growth system shown in FIG. 1 to drive a bioreactor similar to the bioreactor of FIG. 2 according to at least one exemplary embodiment. [Figure 4] FIG. 4 is a schematic view showing an example of a flow path of a cell growth system similar to the cell growth system shown in FIG. 1 according to at least one exemplary embodiment. [Figure 5] FIG. 5 is a flowchart showing an exemplary method of removing solutes from the inner capillary space (or, alternatively, or in addition to, the outer capillary space) of a bioreactor similar to the bioreactor shown in FIG. 2 according to at least one exemplary embodiment. [Figure 6] FIG. 6 is a simplified explanatory view of the exemplary method shown in FIG. 5 applied to a single hollow fiber of a bioreactor according to at least one exemplary embodiment. [Figure 7] FIG. 7 is a flowchart showing another exemplary method of removing solutes from the inner capillary space (or, alternatively, or in addition to, the outer capillary space) of a bioreactor similar to the bioreactor shown in FIG. 2 according to at least one exemplary embodiment. [Figure 8]Figure 8 is a simplified diagram illustrating the exemplary method shown in Figure 7, applied to a single hollow fiber of a bioreactor according to at least one exemplary embodiment. [Figure 9] Figure 9 is a flowchart illustrating another exemplary method for removing solute from the capillary inner space (or, alternatively, or in addition to, the capillary outer space) of a bioreactor similar to the bioreactor shown in Figure 2, according to at least one exemplary embodiment. [Figure 10] Figure 10 is a simplified diagram illustrating the exemplary method shown in Figure 9, applied to a single hollow fiber of a bioreactor according to at least one exemplary embodiment. [Figure 11] Figure 11 is a flowchart illustrating another exemplary method for removing solute from the capillary inner space (or, alternatively, or in addition to, the capillary outer space) of a bioreactor similar to the bioreactor shown in Figure 2, according to at least one exemplary embodiment. [Figure 12A] Figure 12A is a simplified diagram illustrating the exemplary method shown in Figure 11, applied to a single hollow fiber of a bioreactor according to at least one exemplary embodiment. [Figure 12B] Figure 12B is a simplified diagram illustrating the exemplary method shown in Figure 11, applied to a single hollow fiber of a bioreactor according to at least one exemplary embodiment. [Modes for carrying out the invention]

[0029] The corresponding reference numerals indicate the corresponding parts in the above diagram.

[0030] Exemplary embodiments will be described in more detail with reference to the accompanying drawings.

[0031] Exemplary embodiments are provided to those skilled in the art so that the disclosure may be complete and its scope fully conveyed. 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 the 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 the disclosure. In some exemplary embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.

[0032] The terms used herein are for illustrative purposes only and are not intended to limit the use of any particular exemplary embodiment. 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.

[0033] 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.

[0034] The terms "first," "second," "third," etc., may be used herein to describe various elements, components, regions, layers, and / or parts, but these elements, components, regions, layers, and / or parts should not be limited by these terms. These terms are used solely to distinguish one element, component, region, layer, or part from other regions, other layers, or other 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 part described below may also be called the second element, second component, second region, second layer, or second part, without departing from the teaching of the exemplary embodiments.

[0035] Terms indicating spatial relationships, such as “inside,” “outside,” “directly below,” “downward,” “below,” “above,” and “up,” 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. These terms indicating spatial relationships may be intended to include cases where the device is oriented in different directions during use or operation, in addition to the orientation shown in the figures. For example, if the device in the figures 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 upward and downward orientations. The device may be oriented in other directions (by being rotated 90 degrees or in other directions), and the descriptions of spatial relationships used herein shall be interpreted accordingly.

[0036] Various components are referred to herein as “operably connected.” As used herein, “operably connected” refers to components that are connected to one another in an operable manner, and includes embodiments in which components are directly connected, as well as embodiments in which other components are arranged between connected components. “Operatively connected” components can be “fluidically connected.” “Fluidally connected” refers to components that are connected together so that a fluid can be transported between them. The term “fluidically connected” includes embodiments in which other components are arranged between two fluidly connected components, and embodiments in which components are directly connected, etc. Fluidly connected components may include components that control 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).

[0037] In this application, which includes the following definitions, the terms “module” or “controller” may be replaced with the term “circuit.” The term “module” may mean, or be part of, or include: application-specific integrated circuits (ASICs); digital, analog, or mixed analog / digital discrete circuits; digital, analog, or mixed analog / digital integrated circuits; combinational logic circuits; field-programmable gate arrays (FPGAs); processor circuits (shared, dedicated, or grouped) that execute code; memory circuits (shared, dedicated, or grouped) that store code executed by the processor circuits; other suitable hardware components that provide the described function; or any combination of some or all of the above, such as a system on a chip.

[0038] A module may include one or more interface circuits. In some examples, the interface circuits include wired or wireless interfaces connected to a local area network (LAN), the internet, a wide area network (WAN), or a combination thereof. The functionality of any given module in this disclosure may be distributed among multiple modules connected via interface circuits. For example, multiple modules enable load balancing. In other examples, a server module (also known as a remote or cloud module) performs some functions on behalf of a client module.

[0039] The term "code" as used above may include software, firmware, and / or microcode, and may refer to programs, routines, functions, classes, data structures, and / or objects. The term "shared processor circuit" encompasses a single processor circuit that executes some or all of the code from multiple modules. The term "group processor circuit" encompasses a processor circuit that, in combination with additional processor circuits, executes some or all of the code from one or more modules. References to multiple processor circuits include multiple processor circuits on separate dies, multiple processor circuits on a single die, multiple cores in a single processor circuit, multiple threads in a single processor circuit, or a combination of the above. The term "shared memory circuit" encompasses a single memory circuit that stores some or all of the code from multiple modules. The term "group memory circuit" encompasses a memory circuit that, in combination with additional memory, stores some or all of the code from one or more modules.

[0040] The term "memory circuit" is included in what the term "computer-readable medium" refers to. As used herein, the term computer-readable medium does not include transient electrical or electromagnetic signals that propagate through a medium (like a carrier wave), and therefore the term computer-readable medium is considered tangible and non-transient. Non-limiting examples of non-transient tangible computer-readable medium include non-volatile memory circuits (such as flash memory circuits, erasable programmable read-only memory circuits, or mask read-only memory circuits), volatile memory circuits (such as static random-access memory circuits or dynamic random-access memory circuits), magnetic storage media (such as analog or digital magnetic tapes or hard disk drives), and optical storage media (such as CDs, DVDs, or Blu-ray discs).

[0041] The apparatus and methods described in this application may be partially or completely implemented by a dedicated computer created by configuring a general-purpose computer to perform one or more specific functions embodied in a computer program. The aforementioned functional blocks, flowchart components, and other elements function as software specifications, which can be converted into a computer program by the routine work of a skilled technician or programmer.

[0042] A computer program includes processor-executable instructions stored in at least one non-transient, tangible, computer-readable medium. A computer program may also include, or depend on, stored data. A computer program may encompass a basic input / output system (BIOS) that interacts with the hardware of a dedicated computer, device drivers that interact with specific devices of the dedicated computer, one or more operating systems, user applications, background services, background applications, and the like.

[0043] A computer program includes (i) descriptive text to be parsed, such as HTML (hypertext markup language), XML (extensible markup language), or JSON (JavaScript Object Notation); (ii) assembly code; (iii) object code generated from source code by a compiler; (iv) source code for execution by an interpreter; and (v) source code for compilation and execution by a runtime compiler. For example, source code is written using the syntax of languages ​​including C, C++, C#, Objective-C, Swift, Haskell, Go, SQL, R, Lisp, Java®, Fortran, Perl, Pascal, Curl, OCaml, JavaScript®, HTML5 (Hypertext Markup Language 5th revision), Ada, ASP (Active Server Pages), PHP (PHP: Hypertext Preprocessor), Scala, Eiffel, Smalltalk, Erlang, Ruby, Flash®, Visual Basic®, Lua, MATLAB, SIMULINK, and Python®.

[0044] Exemplary embodiments will be described in more detail with reference to the accompanying drawings.

[0045] A cell proliferation system is a cell culture system used to proliferate and differentiate cells, including both adherent and non-adherent cell types. This disclosure relates to cell proliferation systems and processes, including, for example, the following documents: U.S. Patent Application No. 15 / 943,536 (title: Cell Proliferation in a Bioreactor), filed on 2 April 2018 and published on 2 October 2018, and / or U.S. Patent No. 10,577,585 (title: Cell Proliferation), issued on 3 March 2020. The entire disclosure of the above application is incorporated herein by reference.

[0046] Figure 1 shows an example of a cell growth system 10. The cell growth system 10 includes a first fluid circulation path 12 and a second fluid circulation path 14. The first fluid circulation path 12 includes, for example, a first fluid channel 16 having opposing ends 18, 20. The first fluid channel 16 may be in fluid communication with a cell growth chamber 24. For example, the first end 18 of the first fluid channel 16 is in fluid communication with a first inlet 22 of the cell growth chamber 24, and the second end 20 is in fluid communication with a first outlet 28 of the cell growth chamber 24. The cell growth chamber 24 includes or is configured to receive a bioreactor (also called a hollow fiber membrane (HFM)) 117 (see Figure 2). For example, fluid in the first fluid circulation path 12 flows through the interior of a plurality of hollow fibers 116 of the bioreactor 117. In at least one exemplary embodiment, the first flow control device 30 may be operably coupled to the first fluid passage 16 to control the flow of fluid in the first fluid circulation path 12.

[0047] The second fluid circulation path 14 includes, for example, a second fluid passage 34 and a second flow control device 32. Similar to the first fluid passage 16, the second fluid passage 34 has opposing ends 36 and 38. The opposing ends 36 and 38 of the second fluid passage 34 may be in fluid communication with the inlet port 40 and outlet port 42 of the cell growth chamber 24. For example, the first end 36 of the second fluid passage 34 is in fluid communication with the inlet port 40 of the cell growth chamber 24, and the second end 38 of the second fluid passage 34 is in fluid communication with the outlet port 42. The fluid in the second fluid circulation path 14 is in contact with the outside of the bioreactor 117 located inside the cell growth chamber 24. In at least one exemplary embodiment, the second flow control device 32 may be operably coupled to the second fluid passage 34 to control the flow of fluid in the second fluid circulation path 14.

[0048] The first and second fluid circulation channels 12 and 14 are maintained within the cell growth chamber 24 by the bioreactor 117. In this case, the fluid in the first fluid circulation channel 12 flows through the inner capillary space ("IC") of the bioreactor 117, and the fluid in the second fluid circulation channel 14 flows through the outer capillary space ("EC") of the cell growth chamber 24. The first fluid circulation channel 12 is also called the "inner capillary loop" or "IC loop." The second fluid circulation channel 14 is also called the "outer capillary loop" or "EC loop." The fluid in the first fluid circulation channel 12 may flow in either a parallel or counter-flow direction relative to the fluid flow in the second fluid circulation channel 14.

[0049] In at least one exemplary embodiment, a fluid inlet passage 44 is fluidically associated with a first fluid circulation passage 12, and a fluid outlet passage 46 is fluidically associated with a second fluid circulation passage 14. The fluid inlet passage 44 allows fluid to enter the first fluid circulation passage 12, while the fluid outlet passage 46 allows fluid to exit the cell proliferation system 10. In at least one exemplary embodiment, a third flow control device 48 is operably associated with the fluid inlet passage 44, as shown. It should be noted that in other exemplary embodiments, a fourth flow control device may be operably associated with the first outlet passage 46, either alternatively or additionally, although not shown. In at least one exemplary embodiment, the flow control devices (including a first flow control device 30, and / or a second flow control device 32, and / or a third flow control device 48, and / or a fourth flow control device) include pumps, valves, clamps, or any combination thereof. For example, multiple pumps, multiple valves, and multiple clamps may be arranged in any combination. In at least one exemplary embodiment, the flow control device may be a peristaltic pump or may include a peristaltic pump. The fluid circulation pathways (including the first fluid circulation pathway 12 and / or the second fluid circulation pathway 14), and / or the inlet port (including the fluid inlet port 44), and / or the outlet port (including the fluid outlet port 46) may include any known tubular material, and any type of fluid, such as buffer, protein-containing fluid, and cell-containing fluid, can flow through the various circulation pathways (including the first fluid circulation pathway 12 and / or the second fluid circulation pathway 14), and / or the inlet pathway (including the fluid inlet port 44), and the outlet pathway (including the fluid outlet port 46). It should be noted that the terms “fluid,” “culture medium,” and “fluid culture medium” are used interchangeably.

[0050] An exemplary cell growth chamber 100 is shown in Figure 2. The cell growth chamber 100 may be used as the cell growth chamber 24 of the cell proliferation system 10 shown in Figure 1. The cell growth chamber 100 has a longitudinal axis (represented by the line LA-LA) and includes a cell growth chamber housing 104. The cell growth chamber housing 104 has four openings or four ports, including, for example, an internal capillary inlet port 108, an internal capillary outlet port 120, an external capillary inlet port 128, and an external capillary outlet port 132. The first fluid (also called capillary-inside fluid or capillary-inside medium) in the first circulation path (such as the first fluid circulation path 12) enters the cell growth chamber 100 through the capillary-inside inlet port 108 at the first fluid manifold end 112 of the cell growth chamber 100, enters the capillary-inside space of a plurality of hollow fibers 116, passes through there, and exits the cell growth chamber 100 through the capillary-inside outlet port 120 located at the second fluid manifold end 124 of the cell growth chamber 100. The flow path between the capillary-inside inlet port 108 and the capillary-inside outlet port 120 constitutes the capillary-inside portion 126 of the cell growth chamber 100. The second fluid (also called capillary-outside medium or capillary-outside fluid) in the second circulation path (such as the second fluid circulation path 14) can enter the cell growth chamber 100 through the capillary-outside inlet port 128. This second fluid comes into contact with the capillary outer space or the outside of the bioreactor 117 and exits the cell growth chamber 100 through the capillary outer outlet port 132. The fluid path between the capillary outer inlet port 128 and the capillary outer outlet port 132 constitutes the capillary outer portion 136 of the cell growth chamber 100.

[0051] When the second fluid comes into contact with the outside of the hollow fiber 116, small molecules (e.g., ions, water, oxygen, lactate, etc.) can diffuse through the hollow fiber 116 from the inside of the hollow fiber 116, i.e., the inner capillary space, to the outside, i.e., the outer capillary space, or, furthermore, from the outer capillary space to the inner capillary space. Molecules with large molecular weights (e.g., growth factors and / or proteins) are often too large to pass through the membrane wall of the hollow fiber 116 and remain in the inner capillary space of the hollow fiber 116 (or, alternatively or additionally, the outer capillary space). The culture media constituting the first and second fluids may be replaced as needed, and, alternatively or in addition, may be circulated through an oxygen supplyer and / or gas transfer module as needed to exchange the gas. As described below, cells for proliferation are housed in the first fluid circulation pathway 12 and / or the second fluid circulation pathway 14 and enter the cell growth chamber 100 in either the inner capillary space or the outer capillary space, or both.

[0052] In at least one exemplary embodiment, cells are seeded in the capillary inner space of the hollow fiber 116 (for example, umbilical cord blood-derived CD34+ hematopoietic stem cells / progenitor cells, monocytes, macrophages, hepatocytes, and / or endothelial cells for proliferation and / or differentiation and / or harvesting), while the cell culture medium is pumped through the capillary outer space of the hollow fiber 116, and nutrients are delivered to the cells via hollow fiber membrane perfusion during proliferation. However, in at least one other exemplary embodiment, cells for proliferation and / or differentiation and / or harvesting may be seeded in the capillary outer space, while the cell culture medium is pumped through the capillary inner space, and nutrients are delivered to the cells via hollow fiber membrane perfusion during proliferation. In at least one other exemplary embodiment, cells for proliferation and / or differentiation and / or harvesting may be seeded in the capillary inner space, while the cell culture medium may be pumped through both the capillary outer and inner spaces. The movement of the cell culture medium in the capillary inner space and / or capillary outer space helps to remove excess cells, such as cells that are not attached to the surface of the hollow fiber membrane. In at least one exemplary embodiment, the material used to construct the bioreactor 117 may be any biocompatible polymer material, as long as it forms the hollow fiber 121. For example, synthetic polysulfone materials (e.g., polyethersulfone (PES)) are often used to form the hollow fiber.

[0053] In at least one exemplary embodiment, the cell growth system 10 has a device configured to move, i.e., "rock" the cell growth chamber 100 relative to other parts of the cell growth system 10. The device may be a rotational and / or lateral rocking device. For example, as shown in Figure 3, the cell growth chamber 100 may be rotatably connected to one or more rotational rocking components 138 and lateral rocking components 140. The first rotational rocking component 138 is associated with the cell growth chamber 100 to rotate. For example, the first rotational rocking component 138 is configured to rotate the cell growth chamber 100 about a first axis or central axis of rotation 142. In at least one exemplary embodiment, the cell growth chamber 100 may rotate alternately, for example, about the central axis 142 in a first direction, which is clockwise, and then in a second direction, which is counterclockwise.

[0054] It should be noted that, although not shown, in at least one exemplary embodiment, the second rotational oscillating component may be configured to move the cell growth chamber 100 about a second rotation axis 144 that passes through the center point of the cell growth chamber 100 perpendicular to the central axis 142. In at least one exemplary embodiment, the cell growth chamber 100 may be rotated alternately, for example, in a first direction, which is clockwise, and then in a second direction, which is counterclockwise, about the second axis 144. In at least one exemplary embodiment, the cell growth chamber 100 may be rotated about the second axis 144 to be oriented horizontally or vertically to gravity. The lateral oscillating component 140 is associated with the cell growth chamber 100 to move laterally. For example, the plane of the lateral oscillating component 140 can move laterally in the x and y directions.

[0055] The rotation and / or lateral movement of the bioreactor 100 reduces cell sedimentation and also reduces the likelihood of cells becoming trapped in a portion of the bioreactor 117 located in the cell growth chamber 100. In at least one exemplary embodiment, the cell sedimentation rate in the cell growth chamber 100 is proportional to the density difference between the cells and the suspension medium, according to Stokes' equation. In at least one exemplary embodiment, as described above, repeating 180-degree rotations with pauses (high speed) (e.g., a total of 30 seconds of combined pause and rotation time) helps maintain the suspension of non-adherent cells (e.g., T cells). A minimum rotation of about 180 degrees is preferred, but various rotation angles including up to 360 degrees or more can be used. Different rocking components may be used separately or in combination. For example, a rocking component that rotates the cell growth chamber 100 around the central axis 142 can be combined with a rocking component that rotates the cell growth chamber 100 around axis 144. Similarly, clockwise and counterclockwise rotations can be performed independently around different axes.

[0056] Figure 4 is a schematic diagram of an exemplary cell proliferation system 500, similar to the cell proliferation chamber 100 shown in Figure 1, showing exemplary flow paths. In at least one exemplary embodiment, cells are placed in the capillary inner space, while the cell culture medium is pumped in the capillary outer space to deliver nutrients to the cells via hollow fiber membrane perfusion during proliferation. However, it should be noted that in at least one other exemplary embodiment, cells may be placed in the capillary outer space, while the cell culture medium may be pumped in the capillary inner space to deliver nutrients to the cells via hollow fiber membrane perfusion during proliferation. In at least one other exemplary embodiment, cells are placed in the capillary inner space, while the cell culture medium is pumped in both the capillary outer and inner spaces.

[0057] As illustrated, the cell growth system 500 includes 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. The cell growth chamber 501 may be used as the cell growth chamber 24 shown in Figure 1 and / or the cell growth chamber 100 shown in Figure 2. The first fluid flows into the cell growth chamber 501 through the inner capillary inlet port 501A. The first fluid exits the cell growth chamber through the inner capillary outlet port 501B. In at least one exemplary embodiment, the first fluid circulation path 502 is provided with a pressure gauge 510 configured to measure the pressure of the first fluid exiting the cell growth chamber 501. In at least one exemplary embodiment, the first fluid circulation path 502 is provided with an internal capillary circulation pump 512 configured to control the flow rate of the first fluid. For example, the internal capillary circulation pump 512 is configured to pump the fluid in a first direction or a second direction opposite to the first direction. In the latter case, the internal capillary outlet port 501B is used as an inlet, and the internal capillary inlet port 501A is used as an outlet. In at least one exemplary embodiment, the first fluid circulation path 502 includes a sample port 516 and / or a sample coil 518 configured for extracting a sample of the first fluid. In at least one exemplary embodiment, the first fluid circulation path 502 is provided with a pressure / thermometer 520 configured to detect the pressure and / or temperature of the first fluid during operation. In at least one exemplary embodiment, the first fluid can enter the internal capillary loop 502 via a valve 514. In at least one exemplary embodiment, a portion of the cells are flowed from the capillary inner loop 502 into the harvest bag 599, for example, via a valve 598.It should be understood that in at least one other exemplary embodiment, several valves, pressure gauges, pressure sensors, temperature sensors, ports, and / or other devices may be added or removed in the first fluid circulation path 502 to isolate the first fluid along some portion of the capillary inner loop 502 and / or to measure the properties of the first fluid in some portion.

[0058] The second fluid can flow into the cell growth chamber 501 through the capillary outer inlet port 501C. The second fluid can exit the cell growth chamber 501 through the capillary outer outlet port 501D. In at least one exemplary embodiment, the second fluid in the capillary outer loop 504 comes into contact with the outer-facing surfaces of the hollow fibers placed in the cell growth chamber 501, thereby allowing the diffusion of small molecules into and out of the hollow fibers. In at least one exemplary embodiment, the capillary outer loop 504 is provided with a pressure / thermometer 524 configured to measure the pressure and / or temperature of the second fluid before it enters the cell growth chamber 501. In at least one exemplary embodiment, the capillary outer loop 504 is provided with a pressure gauge 526 configured to measure the pressure of the second fluid, for example, when the second fluid exits the cell growth chamber 501. In at least one exemplary embodiment, the capillary outer loop 504 is provided with a sample port 530 configured for a second fluid sample extraction.

[0059] In at least one exemplary embodiment, the capillary outer loop 504 is provided with an outer capillary circulation pump 528 and an oxygen supply or gas transfer module 532. For example, after leaving the cell growth chamber 501, the second fluid passes through the outer capillary circulation pump 528 to the oxygen supply or gas transfer module 532 and through it. In at least one exemplary embodiment, the outer capillary circulation pump 528 is configured to control the flow rate of the second fluid. For example, the outer capillary circulation pump 528 is configured to pump the second fluid in a first direction or in a second direction opposite to the first direction, similar to the inner capillary circulation pump 512. In the latter case, the outer capillary outlet port 501D is used as an inlet and the outer capillary inlet port 501C is used as an outlet.

[0060] In at least one exemplary embodiment, the second fluid passage 522 is fluidly associated with the oxygen supply or gas transfer module 532 via an oxygen supply inlet port 534 and an oxygen supply outlet port 536. For example, the second fluid flows into the oxygen supply or gas transfer module 532 via the oxygen supply inlet port 534 and flows out of or out of the oxygen supply or gas transfer module 532 via the oxygen supply outlet port 536. In at least one exemplary embodiment, the oxygen supply or gas transfer module 532 is configured to add oxygen to the second fluid and / or remove bubbles from the second fluid. For example, air and / or gas flows into the oxygen supply or gas transfer module 532 via a first filter 538 and flows out of or out of (i.e., out of) the oxygen supply or gas transfer module 532 through a second filter 540. The first and second filters 538, 540 are configured to reduce or prevent contaminants from entering the oxygen supply or gas transfer module 532. The second fluid in the second fluid circulation path 504 is in equilibrium with the gas entering the oxygen supply or gas transfer module 532. In at least one exemplary embodiment, air and / or gas is purged from the cell proliferation system 500, for example, during the priming sequence, and the air and / or gas is released into the atmosphere via the oxygen supply or gas transfer module 532. In at least one other exemplary embodiment, it should be understood that in the second fluid circulation path 504, several valves, pressure gauges, pressure sensors, temperature sensors, ports, and / or other devices may be added or removed to isolate the second fluid along several portions of the capillary outer loop 504 and / or to measure the properties of the second fluid in several portions.

[0061] In at least one exemplary embodiment, an air removal chamber (ARC) 556 is fluidically associated with a first fluid circulation path 502. The air removal chamber 556 may include one or more ultrasonic sensors. For example, the air removal chamber 556 may include upper and / or lower sensors configured to detect the absence of air and / or fluid and / or the gas-fluid interface at specific measurement locations within the air removal chamber 556. The upper sensor may be located near a first end (e.g., the top) of the air removal chamber 556. The lower sensor may be located near a second end (e.g., the bottom) of the air removal chamber 556. While ultrasonic sensors are described, it should be understood that the air removal chamber 556 may additionally or alternatively include one or more other sensors, for example, optical sensors. Air and / or gases purged from the cell proliferation system 500 during part of the priming process and / or other protocols can be vented to the atmosphere through an air valve 560 via a line 558 fluidically associated with the air removal chamber 556.

[0062] In at least one exemplary embodiment, the first fluid includes, for example, cells from a first fluid container (also called a first culture bag or first bag) 562 and a fluid culture medium (e.g., capillary culture medium or fluid) from a second fluid container (also called a second culture bag or second bag) 546. The material from the first and second fluid containers 562, 546 (i.e., cells and / or capillary culture medium) can enter a first fluid circulation path 502 via a first fluid channel 506. The first fluid container 562 is fluidically associated with the first fluid channel 506 and the first fluid circulation path 502 via a valve 564. In at least one exemplary embodiment, the second fluid container 546 and the third fluid container (also called the third culture bag or third bag) 544 are associated with the first fluid inlet passage 542, for example, via valves 548 and 550, respectively, or are fluidically associated with the second fluid inlet passage 574, for example, via valves 570 and 576, respectively. In at least one exemplary embodiment, material from the second fluid container 546 and / or the third fluid container 544 may be in fluid communication with the first sterile-sealable input priming passage 508 and / or the second sterile-sealable input priming passage 509.

[0063] In at least one exemplary embodiment, a fourth fluid container (also called a fourth culture bag or fourth bag) 568 contains capillary outer culture medium, and a fifth fluid container (also called a fifth culture bag or fifth bag) 566 contains a washing solution. Substances from the fourth and fifth fluid containers 568, 566 (i.e., capillary outer culture medium and / or washing solution) can enter a first fluid circulation path 502 and / or a second fluid circulation path 504. For example, in at least one exemplary embodiment, the fifth fluid container 566 is fluidically associated with a valve 570. The valve 570 is fluidically associated with the first fluid circulation path 502, for example, via a distribution valve 572 and a first fluid inlet path 542. In at least one exemplary embodiment, a fifth fluid vessel 566 can be fluidically associated with a second fluid circulation passage 504 via a second fluid inlet passage 574 and a capillary outer inlet passage 584, for example, by opening valve 570 and closing distribution valve 572. A fourth fluid vessel 568 is fluidically associated with valve 576. Valve 576 is fluidically associated with a first fluid circulation passage 502, for example, via a first fluid inlet passage 542 and distribution valve 572. In at least one exemplary embodiment, a fourth fluid vessel 568 can be fluidically associated with a second fluid inlet passage 574 by opening valve 576 and closing distribution valve 572. In at least one exemplary embodiment, the first fluid inlet passage 542 and / or the second fluid inlet passage 574 may optionally be fluidically associated with a heat exchanger 552.

[0064] In at least one exemplary embodiment, fluid is transferred from a first fluid inlet passage 542 and / or a second fluid inlet passage 574 to an inner capillary loop 502 via an inner capillary inlet pump 554, and fluid is also transferred to an outer capillary loop 504 via an outer capillary inlet pump 578. In at least one exemplary embodiment, an air detector 580 may also be associated with an outer capillary inlet passage 584. The air detector 580 may include, for example, an ultrasonic sensor. In at least one exemplary embodiment, the first fluid circulation passage 502 and the second fluid circulation passage 504 may be fluidically associated with a waste line 588. For example, when valve 590 is open or in the open position, the inner capillary culture medium flows through the waste line 588 to a waste bag (also called an outlet bag) 586. When valve 582 is opened, the outer capillary culture medium flows through the waste line 588 to the waste bag 586. In at least one exemplary embodiment, cells are harvested, for example, via a cell harvesting pathway 596. For example, cells can be harvested from the cell growth chamber 501 by pumping the capillary culture medium containing the cells into a cell harvesting bag 599 via the cell harvesting pathway 596 and valve 598.

[0065] In at least one exemplary embodiment, as shown, the fluids in the first fluid circulation path 502 and the second fluid circulation path 504 flow in the same direction through the cell growth chamber 501 (i.e., parallel flow configuration). It should be understood that in other exemplary embodiments, though not shown, the cell growth system 500 may be configured to flow in a counterflow configuration. As shown in Figure 4, the fluid in the first fluid circulation path 502 enters the cell growth chamber 501 at the capillary inlet port 501A and leaves or exits the cell growth chamber 501 at the capillary inlet port 501B. In at least one exemplary embodiment, the first fluid passage 506 may be fluidly connected to the first fluid circulation path 502, for example, via a connection 517. The connection 517 may be a point or position where the fluid flows in both opposing directions, for example, based on the direction and flow rate of the capillary inlet pump 554 and the direction and flow rate of the capillary circulation pump 512. The connection portion 517 may be any type of joint, coupling, weld, path, and / or tube that enables the first fluid passage 506 to be fluidly associated with the first fluid circulation path 502. In at least one exemplary embodiment, the connection portion 517 may include a T-joint or T-connection and / or a Y-joint or Y-connection.

[0066] In at least one exemplary embodiment, one or more measuring instruments (e.g., pressure gauge 510, and / or pressure / thermometer 520, and / or pressure / thermometer 524, and / or pressure gauge 526), ​​one or more valves (e.g., valve 514, and / or valve 548, and / or valve 550, and / or valve 560, and / or valve 564, and / or valve 570, and / or valve 572, and / or valve 576, and / or valve 582, and / or valve 590, and / or valve 596, and / or valve 598), one or more ports (e.g., capillary inner inlet port 501A, and / or capillary inner outlet port 501B, and / or capillary outer inlet port 501C, and / or capillary outer outlet port Port 501D, and / or sample port 516, and / or sample port 530, and / or oxygen supply inlet port 534, and / or oxygen supply outlet port 536), one or more pumps (e.g., capillary internal circulation pump 512, and / or capillary external circulation pump 528, and / or capillary internal inlet pump 554, and / or capillary external inlet pump 578), one or more filters (e.g., first filter 538 and / or second filter 540), one or more coils (e.g., sample coil 518), one or more modules (e.g., oxygen supply or gas transfer module 532), and / or one or more other components of the cell proliferation system 500 may communicate electrically with a control system (not shown). The control system may include multiple nodes, each node including various hardware, firmware, and / or software (e.g., controllers and memory) configured to control and / or communicate with the mechanical, electromechanical, and electrical components of the cell proliferation system 500.

[0067] The controller (also called a processor) may be any type of microcontroller, microprocessor, field-programmable gate array (FPGA), application-specific integrated circuit (ASIC), etc. An example of a controller may be the NK10DN512VOK10 microcontroller, manufactured and sold by N9P USA, Incorporated, which is a microcontroller unit with a 32-bit architecture. Other examples of controllers include Qualcomm® Snapdragon® 800 and 801, Qualcomm® Snapdragon® 610 and 615 with 4G LTE Integration and 64-bit computing, Apple® A7 processor with 64-bit architecture, Apple® M7 motion coprocessor, Samsung® Exynos® series, Intel® Core® processor family, Intel® Xeon® processor family, Intel® Atom® processor family, Intel Itanium® processor family, Intel® Core® i5-4670K and i7-4770K 22nm Haswell, Intel® Core® i5-3570K 22nm IvyBridge, AMD® FX® processor family, AMD® FX-4300, FX-6300, and FX-8350 32nm Vishera may include at least one of the following: AMD® Kaveri processors, ARM® Cortex®-M processors, ARM® Cortex-A and ARM926EJ-S® processors, or other industrial-equivalent processors, and may perform computer functions using any known or future-developed standard instruction sets, libraries, and / or architectures.The memory may be any type of memory, including random access memory (RAM), read-only memory (ROM), electrically erasable programmable ROM (EEPROM), portable compact disk read-only memory (CD-ROM), optical memory, magnetic memory, any suitable combination thereof, or any other type of memory or memory device that stores and provides instructions for programming and controlling the controller.

[0068] In at least one exemplary embodiment, the protocol for growing suspended cells (such as T cells) involves confining the cells within the hollow fibers of the bioreactor (e.g., a plurality of hollow fibers 116 located within or defining the cell growth chambers 24, 100 as shown in Figures 1 and 2) by implementing countercurrent flow into the capillary inner space of the bioreactor (e.g., by using a capillary inner circulation pump 512 and / or capillary inner inlet pump 554 as shown in Figure 4) (or, alternatively or in addition, by implementing countercurrent flow into the capillary outer space by using a capillary outer circulation pump 528 and / or capillary outer inlet pump 578). Large molecular weight molecules are often too large to pass through the membrane wall of the hollow fiber and remain trapped in the capillary space inside the hollow fiber (or, alternatively, in addition, the capillary space outside the hollow fiber). The trapping of large molecular weight molecules (referred to herein as “solutes”) can often lead to the accumulation of various proteins, cytokines, and other waste products within the capillary space inside the hollow fiber (or, alternatively, in addition, the capillary space outside the hollow fiber), which can have undesirable effects on cell viability, proliferation, and phenotype. Such solutes originate, for example, from the culture medium and / or the growing cell population.

[0069] This disclosure provides a method for removing solute (i.e., molecules with high molecular weight) from the capillary inner space (or, alternatively, or in addition to, the capillary outer space) of a bioreactor. For example, Figure 5 is a flowchart of a first method 600 for removing solute from the capillary inner space (or, alternatively, or in addition to, the capillary outer space) of a bioreactor. Method 600 includes a step 610 of positioning a suspension containing cells and having a high concentration of solute within a central region located midway along the length of the bioreactor. In at least one exemplary embodiment, step 610 of positioning the suspension within the central region along the length of the bioreactor includes using a counterflow confinement process or sequence, such as simultaneously pumping fluid to both ends of the hollow fibers of the bioreactor.

[0070] For example, referring to the exemplary cell growth system 500 shown in Figure 4, step 610, which positions the suspension within the central region of the bioreactor's length, includes setting the capillary inlet pump 554 to a first flow rate and the capillary loop circulation pump 512 to a second flow rate, the second flow rate being equal to half of the first flow rate. The capillary loop circulation pump 512 is set to pump the fluid to the capillary outlet port 501B of the cell growth chamber 501. Since the capillary loop circulation pump 512 will only take half of the fluid that reaches the connection 517, the remaining half of the flow will flow into the capillary inlet port 501A of the cell growth chamber 501. The fluid entering each end of the capillary inner space (for example, via the capillary inner inlet port 501A and the capillary inner outlet port 501B) crosses the hollow fiber membrane (for example, via ultrafiltration) and thus enters the capillary outer space and exits the cell growth chamber 501 through the capillary outer inlet port 501C and / or the capillary outer outlet port 501D.

[0071] In at least one exemplary embodiment, ultrafiltration is performed uniformly over the cumulative surface area of ​​the hollow fibers. The pumping action of the capillary inlet pump 554 and the capillary loop circulation pump 512 may work together to increase the fluid pressure in the capillary space, applying sufficient pressure to allow water and low molecular weight solutes to cross the membrane. As the fluid escapes across the hollow fiber membrane walls, the axial flow rate within each fiber automatically decreases from a maximum value at the end of each fiber to zero at the midpoint of the length of each fiber (because the flow entering the capillary inlet port 501A and the flow entering the capillary outlet port 501B are equal and in opposite directions, so it becomes zero at the midpoint of the length). This (uniform) decrease in axial flow rate causes both cells and higher molecular weight solutes to accumulate and concentrate at equidistant positions between the capillary inlet port 501A and the capillary outlet port 501B.

[0072] Referring again to Figure 5, once the suspension is positioned in the central region of the bioreactor's length (i.e., the region of the hollow fiber where the first and second flows collide) (step 610), Method 600 performs the step of positioning the suspension against the hollow fiber walls of the hollow fiber (step 620). In at least one exemplary embodiment, step 620 of positioning the suspension against the hollow fiber walls of the hollow fiber includes positioning the hollow fiber in a horizontal orientation to stop the fluid flow from each end of the hollow fiber. In at least one exemplary embodiment, the horizontal orientation is along a central rotation axis 142 perpendicular to gravity, as shown in Figure 3. In at least one exemplary embodiment, the positioning of the hollow fiber from the vertical to the horizontal orientation is performed when the fluid flow is stopped. In at least one exemplary embodiment, the hollow fiber is moved into the horizontal orientation using a rotatable fixture configured to hold the bioreactor. Once the hollow fiber is oriented horizontally and the fluid flow is stopped, the suspension, containing the densified cells and solute, settles against the hollow fiber walls of the hollow fiber. As the suspension settles, the increasingly concentrated solute immediately begins to diffuse away from the cells due to its molecular properties, while the cells, due to their larger size and non-molecular nature (compared to the solute), are unable to diffuse and remain largely stationary.

[0073] Once the suspension is substantially positioned within the hollow fiber walls, Method 600 opens a first waste valve (step 630) and closes a second waste valve (step 640). The first waste valve may be an internal capillary waste valve (such as valve 590 shown in Figure 4), and the second waste valve may be an external capillary waste valve (such as valve 582 shown in Figure 4). Once the first waste valve is open (step 630) and the second waste valve is closed (step 640), Method 600 starts an inlet pump at a flow rate low enough to remove the solute but retain the cells (step 650). For example, in at least one exemplary embodiment, the flow rate is between approximately 0.5 mL / min and approximately 5 mL / min. In at least one exemplary embodiment, the inlet pump is an internal capillary inlet pump, such as internal capillary inlet pump 554 shown in Figure 4. For example, when the capillary inlet pump 554 supplies fluid to the capillary loop 502, the capillary circulation pump 512 remains stationary, and the fluid enters the capillary loop 502 via the connector 517 and then flows into the capillary inlet port 501A via the valve 514. The fluid, carrying away molecular solutes from the cells, can exit the cell growth chamber 501 through the capillary outlet port 501B. Since the waste valve 590 is open, the fluid can move into the waste bag 586. In at least one exemplary embodiment, method 600 is performed after or when the cell growth chamber 501 is positioned at a 45-degree angle to the horizontal position, in which case the capillary outlet port 501B is positioned higher than the capillary inlet port 501A.

[0074] If the inlet pump is an outer capillary inlet pump, such as pump 578 in Figure 4, the inlet pump can generate an outer capillary flow entering the hollow fiber via a reverse ultrafiltration flow that crosses the surface region of the hollow fiber. In this case, the inner capillary inlet pump 554 and the inner capillary circulation pump 512 may each have zero flow rates. The waste valve 590 is open and the waste valve 582 is closed. Since the inner capillary inlet pump 554 and the inner capillary circulation pump 512 are stationary and the flow is blocked, the ultrafiltration fluid entering the outer capillary space from the inner capillary space cannot exit from the inner capillary inlet port 501A. The ultrafiltration fluid must then exit from the inner capillary outlet port 501B. The axial flow rate of the inner capillary space within the hollow fiber is in the direction from the inner capillary inlet port 501A to the inner capillary outlet port 501B. Furthermore, the axial flow rate increases from a minimum value (zero) adjacent to the capillary inner inlet port 501A to a maximum value adjacent to the capillary inner outlet port 501B. This is because the hollow fiber lumen at the capillary inner outlet port 501B can accommodate the entire ultrafiltration volume flow rate, while the hollow fiber lumen between, for example, the capillary inner inlet port 501A and the capillary inner outlet port 501B only needs to accommodate half of the ultrafiltration volume flow rate. As a result, the velocity at the outlet can be twice the velocity between the EC ports. For example, suppose the flow rate generated by the capillary outer inlet pump is approximately 100 mL / min and the membrane surface area is approximately 2.1 square meters. In this example, the uniform radial flow velocity through the hollow fiber wall is approximately 0.005 cm / min. A standard-sized T cell falls at a speed of approximately 0.008 cm / min under the influence of gravity. In such cases, the radial flow velocity is too low to separate the cells from the hollow fiber membrane wall; therefore, the flow velocity through the hollow fiber wall passes through the T cell settlement layer, effectively washing away solutes from between the T cells.

[0075] In at least one exemplary embodiment, the low flow rate may optionally be in the form of periodic pulses, thereby allowing time for the suspension to re-sediment near the hollow fiber wall. In at least one exemplary embodiment, method 600 optionally includes tilting the hollow fiber to move the cell bed (i.e., the suspension) toward the lower end of the hollow fiber, and biasing the hollow fiber outlet flow velocity so that the flow velocity is greater at the raised end of the hollow fiber to cancel the movement of the cell bed and thereby assist in retaining cells within the hollow fiber. This bias can be achieved by forcing all ultrafiltered flow out, for example, from the capillary inner outlet port 501B by preventing the flow from exiting from the capillary inner inlet port 501A. In each case, after at least a portion of the solute has been removed, method 600 includes restarting the normal cell proliferation flow (step 660), as shown in Figure 5. In at least one exemplary embodiment, restarting the normal cell proliferation flow (step 660) includes rearranging the hollow fibers.

[0076] Figure 6 is a simplified diagram of Method 600 when applied to a single hollow fiber 700. For example, as shown, a suspension 710 containing cells 712 and solute 714 is positioned within the central region 720 of the hollow fiber 700 by pumping a first fluid flow 730 through a first end 702 of the hollow fiber 700 and a second fluid flow 732 through a second end 704 of the hollow fiber 700. Figure 6 shows the hollow fiber in a horizontal position during positioning, but it should be understood that in at least one exemplary embodiment, the hollow fiber may be held in a vertical position. Once the suspension 710 is positioned in the center, the flow is stopped (moving the hollow fiber to a horizontal position if the central positioning was performed in a vertical position), a first waste valve is opened (not shown), and a second waste valve is closed (not shown) so that the suspension 710 sinks toward the hollow fiber wall. Subsequently, an inlet pump, such as an external capillary inlet pump, is started to induce a low reverse ultrafiltration flow rate 750. This causes the solute 714 to be pushed out from the first and second ends 702, 704 of the hollow fiber (for example, on the outlet flow 730), while the cells 712 remain mostly within the hollow fiber wall in the central region 720.

[0077] Figure 7 is a flowchart of a second method 800 for removing solute from the capillary inner space (or, alternatively, or in addition to, the capillary outer space) of a bioreactor. Similar to method 600 shown in Figure 5, method 800 includes the steps of first positioning a suspension containing cells and having a high concentration of solute in a central region located midway along the length of the bioreactor, and then positioning the suspension on the hollow fiber walls of the hollow fibers in step 820. Also similar to method 600, method 800 includes the step of opening a first waste valve in step 830 once the suspension is positioned on the hollow fiber walls of the hollow fibers. However, unlike method 600, method 800 does not include the step of closing a second waste valve, but instead includes the step of positioning the hollow fibers at a 45-degree angle to the horizontal before step 850 of starting an inlet pump, such as a capillary inner inlet pump (e.g., a capillary inner inlet pump 554 as shown in Figure 4), at a low flow rate in step 840. The 45-degree angle counteracts the fluid drag acting on the cells generated by the axial flow velocity caused by the capillary inlet pump and / or the capillary circulation pump (such as the capillary inlet pump 512 shown in Figure 4), and therefore the angle helps to hold the cells within the hollow fibers. The force applied to the cells by the low axial flow rate helps to counteract the movement of the cells to slide towards the lower side of the hollow fibers when they are in such an angled position. In at least one exemplary embodiment, method 800 may include briefly increasing the flow rate to help accelerate the removal of solute. In each case, after at least a portion of the solute has been removed, method 800 includes step 860 of resuming the normal cell growth flow. In at least one exemplary embodiment, step 860 of resuming the normal cell growth flow includes repositioning the hollow fibers.

[0078] Figure 8 is a simplified diagram of Method 800 when applied to a single hollow fiber 900. For example, as shown, a suspension 910 containing cells 912 and solute 914 is positioned within the central region 920 of the hollow fiber 900 by pumping a first fluid flow 930 through a first end 902 of the hollow fiber 900 and a second fluid flow 932 through a second end 904 of the hollow fiber 900. Figure 8 shows the hollow fiber in a horizontal position during positioning, but it should be understood that in at least one exemplary embodiment, the hollow fiber may be held in a vertical position. Once the suspension 910 is centrally positioned, the flow is stopped (moving the hollow fiber to a horizontal position if centralization was performed in a vertical position) and a waste valve (not shown) is opened so that the suspension 910 sinks toward the hollow fiber wall. Subsequently, the hollow fiber is positioned at a 45-degree angle to the horizontal such that the second end 904 is higher than the first end 902, and an inlet pump, such as an internal capillary inlet pump, is started at a low flow rate 950. This causes the solute 914 to be pushed out from the upper end 904 of the hollow fiber, while the cells 912 remain mostly within the hollow fiber wall in the central region 920.

[0079] Figure 9 is a flowchart of a third method 1000 for removing solute from the capillary inner space (or, alternatively, or in addition to, the capillary outer space) of a bioreactor. Similar to method 600 shown in Figure 5 and / or method 800 shown in Figure 7, method 1000 first includes step 1010 of positioning a suspension containing cells and having a high concentration of solute within a central region located midway along the length of the bioreactor. Unlike method 600 shown in Figure 5 and / or method 800 shown in Figure 6, method 1000 does not include steps 620, 820 of positioning the suspension within the hollow fiber walls of the hollow fibers. Instead, method 1000 includes step 1010 of positioning the suspension within the central region along the length of the bioreactor, and then step 1020 of positioning the hollow fibers in a vertical orientation that is substantially perpendicular to the horizontal. Once the hollow fiber is positioned vertically (step 1020), method 1000 performs step 1030 to clamp the upper end of the hollow fiber, and then step 1040 to pump the culture medium to the lower end of the hollow fiber. In at least one exemplary embodiment, the upper end of the hollow fiber is clamped by setting the associated pump speed to zero (step 1030). Since the upper end of the hollow fiber is clamped, all fluid entering the hollow fiber must exit through ultrafiltration across the hollow fiber wall. This results in a decreasing axial flow state where the axial velocity is maximum at the inlet of the hollow fiber and zero at the clamped end of the hollow fiber. The axial velocity at the clamped upper end is zero, and the axial velocity changes continuously between the hollow fiber inlet and the clamped end of the hollow fiber, so that the cells find a neutral position along the length of the hollow fiber where the cell sedimentation velocity is equal to the axial velocity, while the solute (which does not settle in essence) moves toward and near the region of zero axial velocity. In at least one exemplary embodiment, the hollow fiber is tilted slightly from the vertical to help keep the cells in the neutral position.Once the cells and solute are separated along the length of the hollow fiber, Method 1000 performs step 1050 to remove the clamp from the upper end, and then, or simultaneously, performs step 1060 to clamp the lower end of the hollow fiber, so that the cells, which had zero axial flow, sink toward the clamped lower end, initiating reverse ultrafiltration (step 1070) and removing the solute from the upper end of the hollow fiber. After at least a portion of the solute has been removed, Method 1000 performs step 1080 to resume the normal cell growth flow. In at least one exemplary embodiment, step 1070 to resume the normal cell growth flow includes removing the clamp from the lower end and / or repositioning the hollow fiber.

[0080] Figure 10 is a simplified diagram of Method 1000 when applied to a single hollow fiber 1100. For example, as shown, a suspension 1110 containing cells 1112 and solute 1114 is positioned within a central region 1120 of the hollow fiber 1100 by pumping a first fluid flow 1130 through a first end 1102 of the hollow fiber 1100 and a second fluid flow 1132 through a second end 1104 of the hollow fiber 1100. Figure 10 shows the hollow fiber in a horizontal position during positioning, but it should be understood that in at least one exemplary embodiment, the hollow fiber may also be held in a vertical position. Furthermore, Figure 10 shows the suspension 1110 positioned centered between the hollow fiber ends 1102 and 1104, but it should be understood that in at least one exemplary embodiment, the suspension may be positioned closer to one side of the first and second ends 1102 and 1104.

[0081] After positioning the suspension 1110 centrally with the hollow fiber in a horizontal position, Method 1000 includes the step of positioning the hollow fiber 1100 in a substantially vertical position with the second end 1104 being the upper end and the first end 1102 being the lower end. The first clamp 1150 is applied to or near the upper end 1104 of the hollow fiber 1100, and the culture medium is pumped from the lower end 1102 to the upper end 1104. In at least one exemplary embodiment, referring to Figure 4, this is achieved by setting the capillary internal circulation pump 512 to zero, setting the capillary internal inlet pump 554 to a desired ultrafiltration flow rate, closing the capillary internal waste valve 590, and opening the capillary external waste valve 582. This flow operation causes the solute 1114 to move from the central region 1120 toward and near the upper end 1004, while the cells 1112 remain in and / or near the lower end 1002. As the solute 1114 moves toward the upper end 1004, the first clamp 1150 is released and a second clamp 1160 is applied to or near the lower end 1102 of the hollow fiber 1100, causing the cells 1112 to fall toward the clamped lower end 1102. The second clamp 1160 may be the same as or different from the first clamp 1150. In at least one exemplary embodiment, referring to Figure 4, this is achieved by stopping the capillary inlet pump 554. Then, reverse ultrafiltration 1170 is started to remove the solute 1114 from the upper end of the hollow fiber 1104. In at least one exemplary embodiment, referring to Figure 4, reverse ultrafiltration 1170 is started by stopping all pumps, closing the capillary outside waste valve 582, opening the capillary inside waste valve 590, and setting the flow rate of the capillary outside inlet pump 578 to a desired reverse ultrafiltration flow rate.

[0082] Figure 11 is a flowchart of a fourth method 1200 for removing solute from the capillary inner space (or, alternatively, or in addition to, the capillary outer space) of a bioreactor. Method 1200 first involves step 1210 of positioning a hollow fiber at a 45-degree angle to the horizontal, and then step 1220 of positioning a suspension containing cells and a high concentration of solute near the upper end of the hollow fiber. In at least one exemplary embodiment, the suspension is positioned using a countercurrent confinement process or sequence similar to those described in the context of Method 600 and / or Method 800 and / or Method 1000 (step 1220). Once the suspension is positioned near the upper end of the hollow fiber, method 1200 performs the steps of stopping all flow 1230 so that the cells and solutes constituting the suspension begin to descend through the hollow fiber toward the lower end, closing the capillary outside waste valve and the capillary inside waste valve, and then performing the step of starting flow at a high circulation flow rate (e.g., a high capillary outside circulation flow rate) using the capillary outside circulation pump 1240. Referring to Figure 4, for example, the high capillary outside flow rate causes a pressure drop between the capillary outside inlet port 501C and the capillary outside outlet port 501D. The higher pressure near the capillary outside inlet port 501C causes reverse ultrafiltration (from the outside of the capillary to the inside of the capillary) near the inlet. The lower pressure near the capillary outside outlet port 501D causes forward ultrafiltration (from the inside of the capillary to the outside of the capillary) near the outlet. The axial flow within the hollow fiber moves the solute toward the capillary inner outlet, while the cells continue to descend within the hollow fiber toward the capillary inner inlet. Once a high circulation flow rate is established, method 1200 performs step 1250, opening the capillary inner waste valve and starting the inlet pump (e.g., the capillary outer inlet pump) at a pre-selected flow rate. In at least one exemplary embodiment, the capillary outer inlet pump flow rate is set to a flow rate of about 1 mL / min or more and about 5 mL / min or less. This setting results in a net reverse ultrafiltration flow rate (from the capillary outer to the capillary inner) equal to the flow rate set on the capillary outer inlet pump.Since both internal capillary pumps are stopped, the only exit path for the reverse ultrafiltration fluid is through the internal capillary outlet port 501B and the internal capillary waste valve 590. The (uniform) flow of fluid across the entire surface area of ​​the hollow fiber increases the axial flow rate along the length of the hollow fiber, washing away the solute while leaving behind cells accumulated near the internal capillary inlet port 501A where the axial flow is very low. Placing the suspension of unwanted solute near the top of the hollow fiber (i.e., near the outlet of the hollow fiber) means reducing the amount of ultrafiltration required to wash away the solute and shortening the time required. After at least a portion of the solute has been removed, method 1200 performs step 1260 to restart the normal cell proliferation flow.

[0083] Figures 12A and 12B show a simplified diagram of method 1200 when applied to a single hollow fiber 1300. For example, as shown, a suspension 1310 containing cells 1312 and solute 1314 is positioned near the upper end 1304 of the hollow fiber 1300, which is positioned at a 45-degree angle to the horizontal, by pumping a first fluid flow 1330 through the first end 1302 of the hollow fiber 1300 and a second fluid flow 1332 through the second end 1304 of the hollow fiber 1300. Once the suspension 1310 is positioned, the flow is stopped so that the suspension 1310 begins to descend the hollow fiber toward the lower end 1302. A high circulation flow rate 1350 is provided using an outside capillary circulation pump 528, for example, as shown in Figure 5. Such high circulation flow rates induce a pressure drop between the capillary outside inlet port 501C and the capillary outside outlet port 501D, causing reverse ultrafiltration 1370 (from outside to inside the capillary) at the capillary outside inlet port 501C and forward ultrafiltration 1372 (i.e., from inside to outside the capillary) at the capillary outside outlet port 501D. This flow configuration moves the solute to the upper end of the hollow fiber, while sliding the cells toward the lower end of the hollow fiber. The capillary outside inlet pump 578 is set to a pre-selected net ultrafiltration flow rate 1360 to wash away the solute while leaving the cells behind.

[0084] The above description of embodiments is provided for illustrative and explanatory purposes only. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally interchangeable and may be used in selected embodiments, even if not specifically illustrated or described, as applicable, and are not limited to that particular embodiment. They may also be modified in many ways. Such modifications should not be considered deviations from the disclosure, and all such variations are intended to be within the scope of the disclosure.

Claims

1. A method for removing solutes while retaining suspended cells in a bioreactor, the method being: The steps include positioning a suspension containing cells and solute within a predetermined area of ​​the bioreactor, A step of generating a flow rate in the suspension within the bioreactor, wherein the flow rate is selected such that the cells are maintained within the bioreactor while the solute exits the bioreactor. Having, method.

2. In the method described in claim 1, The step of positioning the suspension is: The steps include generating a first flow at the first end of the bioreactor, The steps include generating a second flow at the second end of the bioreactor, It has, The first flow and the second flow work together to adjust the first axial flow from the first end and the second axial flow from the second end so that the suspension is positioned within the predetermined area of ​​the bioreactor. method.

3. In the method described in claim 2, The first flow has a first flow rate, and the second flow has a second flow rate that is different from the first flow rate. method.

4. In the method described in claim 3, The second flow rate is smaller than the first flow rate. method.

5. In the method described in claim 4, The second flow rate is less than half of the first flow rate. method.

6. In the method described in claim 2, The step of positioning the suspension further includes: The step of positioning the bioreactor in a horizontal or substantially horizontal position such that the suspension settles on or near the wall defining the bioreactor, method.

7. In the method described in claim 6, After the step of positioning the suspension and before the step of generating the flow rate in the suspension, the method further: The process includes a step of stopping all flow through the bioreactor. method.

8. In the method described in claim 7, After the step of stopping all flow through the bioreactor and before the step of generating the flow rate in the suspension, the method further: The step of setting a flow valve to allow the flow to exit the bioreactor, method.

9. In the method described in claim 7, Prior to the step of generating the flow rate in the suspension, the method further: The step of positioning the bioreactor at an angle of 45 degrees or approximately 45 degrees with respect to the horizontal, method.

10. In the method described in claim 1, The predetermined region of the bioreactor is a region located in the center between the first end of the bioreactor and the second end opposite to the first end. method.

11. In the method according to claim 10, The predetermined region is perpendicular to the long axis of the bioreactor. method.

12. A method for removing solutes while retaining suspended cells in a bioreactor, the method being: The steps include positioning a suspension containing cells and solute within a predetermined area of ​​the bioreactor, The steps include: positioning the bioreactor so as to define its upper and lower ends; The steps include preventing flow from occurring from the upper end of the bioreactor, The steps include: introducing the culture medium into the lower end of the bioreactor; The steps include enabling flow from the upper end of the bioreactor, The steps include preventing flow from occurring from the lower end of the bioreactor, The steps include: flowing a fluid across the bioreactor to move the solute out of the bioreactor and retain the cells within the bioreactor; Having, method.

13. In the method according to claim 12, The predetermined region of the bioreactor is a region located in the center between the first end of the bioreactor and the second end opposite to the first end. method.

14. In the method according to claim 12, The predetermined region is perpendicular to the long axis of the bioreactor. method.

15. A method for removing solutes while retaining suspended cells in a bioreactor, the method being: The steps include: positioning the bioreactor at a certain angle to the horizontal so as to define the upper and lower ends of the bioreactor; The steps include positioning a suspension containing cells and solute in a predetermined location within the bioreactor, Steps include starting the flow at a circulating flow rate, The steps include: starting the flow at a pre-selected net ultrafiltration flow rate, Having, method.

16. In the method according to claim 15, The step of positioning the suspension is: The steps include generating a first flow at the lower end of the bioreactor, The steps include generating a second flow at the upper end of the bioreactor, It has, The first flow and the second flow work together to adjust the first axial flow from the first end and the second axial flow from the second end so that the suspension is positioned in the predetermined location within the bioreactor. method.

17. In the method according to claim 16, The first flow has a first flow rate, and the second flow has a second flow rate that is different from the first flow rate. method.

18. In the method described in claim 17, The second flow rate is smaller than the first flow rate. method.

19. In the method described in claim 18, The second flow rate is less than half of the first flow rate. method.

20. In the method according to claim 15, After the step of positioning the suspension in the predetermined location within the bioreactor and before the step of starting the flow at the circulating flow rate, the method further: It has a step to stop all flow, method.

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