Systems and methods for producing t cells

Abstract

A method of producing T cells using a cell expansion system includes expanding T cells using a small bioreactor of the cell expansion system, the small bioreactor having a surface area of about 2000 cm2 and an in-capillary volume of about 58 ml. The method further includes flowing the T cells into the mini bioreactor of the cell expansion system. The rate at which the T cells flow into the miniature bioreactor may be greater than or equal to about 0.007 [mu] L / min / fiber to less than or equal to about 0.0281 [mu] L / min / fiber. The cell expansion system may also include a set of conduits in fluid communication with the small bioreactor, and the volume ratio of the set of conduits to the bioreactor may be about 2.96.

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Patent Application No. 18 / 657,020, filed May 7, 2024, and benefit of U.S. Provisional Application No. 63 / 465,909, filed May 12, 2023. The entire disclosures of the above applications are incorporated herein by reference. TECHNICAL FIELD

[0002] The present disclosure relates to systems and methods for producing T cells. BACKGROUND

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

[0004] T cells are white blood cells that play a central role in the adaptive immune response, and each is a candidate of interest for various cell therapies and treatments for various diseases, e.g., where therapies and treatments focus on harnessing and supporting the power of the existing immune system. For tumor immunotherapy, it has been shown that central memory T cells (Tcm) and stem cell memory T cells (Tscm) provide at least in part the best therapeutic benefit, as this cell can serve as a long-lived reservoir or progenitor cell that can maintain homeostasis and also differentiate into effective effector cells that rapidly eliminate cancer cells. Despite their promise, widespread clinical success of T cell therapies and / or treatments is limited due to challenges in readily and efficiently manufacturing large doses of T cells. Current methods for T cell expansion are typically flask-based, which can be time-consuming and expensive, and generally have a relatively low success rate. It is desirable to develop systems and methods for T cell expansion that have improved success rates, are better time- and cost-efficient. SUMMARY

[0005] This section provides a general summary of the disclosure and is not a comprehensive

[0006] In various aspects, the present disclosure provides methods of producing T cells using a cell expansion system.

[0007] In at least one exemplary embodiment, the method can include expanding T cells using a small bioreactor of the cell expansion system, wherein the small bioreactor has a surface area of about 2000 cm2. 2

[0008] In at least one exemplary embodiment, the small bioreactor has an intracapillary volume of about 58 milliliters.

[0009] ​In at least one exemplary embodiment, the cell expansion system can further include a tubing set in fluid communication with the small bioreactor, and a volume ratio of the tubing set to the bioreactor can be about 2.96.

[0010] In at least one exemplary embodiment, the method can further include placing the tubing set in fluid communication with the small bioreactor.

[0011] In at least one exemplary embodiment, the method can further include flowing the T cells into the small bioreactor of the cell expansion system.

[0012] In at least one exemplary embodiment, a rate of the T cells flowing into the small bioreactor can be greater than or equal to about 0.01 mL / min to less than or equal to about 100 mL / min.

[0013] In at least one exemplary embodiment, a rate of the T cells flowing into the small bioreactor can be greater than or equal to about 0.007 μL / min / fiber to less than or equal to about 0.0281 μL / min / fiber.

[0014] In at least one exemplary embodiment, the method can further include retaining the T cells within the small bioreactor using counterflow containment.

[0015] In at least one exemplary embodiment, the counterflow containment can include a first flow rate in a first direction and a second flow rate in a second, opposite direction, wherein the first flow rate is about twice the second flow rate.

[0016] In at least one exemplary embodiment, the first flow rate can be about 0.02 mL / min and the second flow rate can be about 0.01 mL / min.

[0017] In at least one exemplary embodiment, the method can further include separating the T cells from the source.

[0018] In at least one exemplary embodiment, separating the T cells from the source can include contacting magnetic conjugated antibodies with the source, the selected magnetic conjugated antibodies associated with non-target cells or materials or components of the source; contacting non-conjugated antibodies with the source, the selected non-conjugated antibodies associated with the T cells; and moving the source including the magnetic conjugated antibodies and the non-conjugated antibodies through a magnetic column, the selected magnetic column retaining the magnetic conjugated antibodies while allowing the non-conjugated antibodies to pass.

[0019] In at least one exemplary embodiment, the method can further include harvesting the produced T cells from the bioreactor.

[0020] In at least one exemplary embodiment, the total viable cells flowed into the small bioreactor can be greater than or equal to about 20 mL to less than or equal to about 60 mL, the total variable volume of the harvested T cells can be greater than or equal to about 100 mL to less than or equal to about 250 mL, and the percent cell viability at the time of harvest can be greater than or equal to about 70% to less than or equal to about 100%.

[0021] In various aspects, the present disclosure provides methods of producing T cells using a cell expansion system having a tubing set.

[0022] In at least one exemplary embodiment, the method can include expanding T cells using a small bioreactor of a cell expansion system, the small bioreactor in fluid communication with a tubing set, and the tubing set having a volume to bioreactor ratio of about 2.96.

[0023] In at least one exemplary embodiment, the small bioreactor can have a surface area of about 2000 cm 2 and a capillary inner volume of about 58 mL.

[0024] In at least one exemplary embodiment, the method can further include flowing the T cells into the small bioreactor of the cell expansion system at a rate greater than or equal to about 0.007 μL / min / fiber to less than or equal to about 0.0281 μL / min / fiber.

[0025] In at least one exemplary embodiment, the method can further include retaining the T cells within the small bioreactor using a backflush, the backflush including a first flow rate in a first direction and a second flow rate in a second, opposite direction, wherein the first flow rate is about twice the second flow rate.

[0026] In at least one exemplary embodiment, the method can further include separating the T cells from the source, wherein separating the T cells from the source includes: contacting magnetic conjugated antibodies with the source, the selected magnetic conjugated antibodies associated with non-target cells or materials or components of the source; contacting non-conjugated antibodies with the source, the selected non-conjugated antibodies associated with the T cells; and moving the source including the magnetic conjugated antibodies and the non-conjugated antibodies through a magnetic column, the selected magnetic column retaining the magnetic conjugated antibodies while allowing the non-conjugated antibodies to pass.

[0027] In at least one exemplary embodiment, the method can further include harvesting the produced T cells from the bioreactor, wherein the total viable cells flowed into the small bioreactor are greater than or equal to about 20 mL to less than or equal to about 60 mL, the total variable volume of the harvested T cells are greater than or equal to about 100 mL to less than or equal to about 250 mL, and the percent cell viability at the time of harvest is greater than or equal to about 70% to less than or equal to about 100%.

[0028] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0029] The drawings described herein are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0030] Figure 1 is a schematic diagram of an exemplary cell expansion system having a bioreactor, in accordance with at least one example embodiment of the present disclosure; Figure 2 is a schematic diagram of an exemplary bioreactor showing a circulation path through the bioreactor that can be incorporated into a cell expansion system as shown in Figure 1 is a schematic diagram of an exemplary bioreactor showing a circulation path through the bioreactor that can be incorporated into a cell expansion system as shown in Figure 3 is a schematic diagram of an exemplary shaking device configured for use with a cell expansion system, such as the cell expansion system shown in Figure 1 is a schematic diagram of an exemplary shaking device configured for use with a cell expansion system, such as the cell expansion system shown in Figure 2 is a schematic diagram of an exemplary shaking device configured for use with a cell expansion system, such as the cell expansion system shown in Figure 4 is a schematic diagram of an exemplary flow path of an exemplary cell expansion system, such as the cell expansion system shown in Figure 1 is a schematic diagram of an exemplary flow path of an exemplary cell expansion system, such as the cell expansion system shown in Figure 5 is a flow diagram showing an exemplary method of producing T cells using a cell expansion system having a small bioreactor, such as the cell expansion system shown in Figure 1 is a flow diagram showing an exemplary method of producing T cells using a cell expansion system having a small bioreactor, such as the cell expansion system shown in Figure 6 is a schematic diagram of exemplary countercurrent inhibition of T cells in a bioreactor, such as the bioreactor shown in Figure 2 is a schematic diagram of exemplary countercurrent inhibition of T cells in a bioreactor, such as the bioreactor shown in Figure 7 is a flow diagram showing an exemplary method for separating T cells from a source, such as the cell expansion system shown in Figure 8 is a flow diagram showing an exemplary method of expanding T cells using a cell expansion system having a small bioreactor, such as the cell expansion system shown in Figure 1 is a flow diagram showing an exemplary method of expanding T cells using a cell expansion system having a small bioreactor, such as the cell expansion system shown in Figure 9is a graph showing the percentage of dissolved gas (y-axis) over time (y-axis) for a small bioreactor compared to a standard bioreactor, in accordance with at least one example embodiment of the present disclosure.

[0031] In the several views of the drawings, like reference numerals designate like parts throughout the several views. DETAILED DESCRIPTION

[0032] Example embodiments will now be described more fully with reference to the accompanying drawings.

[0033] The example embodiments provided make the present disclosure thorough and complete, and convey to those skilled in the art the full scope of the present disclosure. Numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the present disclosure. Those skilled in the art will recognize, however, that the example embodiments can be practiced without the specific details, and that the embodiments are not limited to the particular details described herein. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

[0034] The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" can be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprises," "comprising," "including," and "having," are inclusive and therefore specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The method steps, processes, and operations described herein are not to be construed as necessarily requiring their occurrence in the particular order in which they are described, unless explicitly stated otherwise. It is also to be understood that additional or alternative steps can be employed.

[0035] When an element or layer is referred to as being "on," "engaged to," "connected to" or "coupled to" another element or layer, it can be directly on, engaged, connected or coupled to the other element or layer, or intervening elements or layers can be present. In contrast, when an element is referred to as being "directly on," "directly engaged to," "directly connected to" or "directly coupled to" another element or layer, there are no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., "between" versus "directly between," "adjacent" versus "directly adjacent," etc.). As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0036] Although the terms“first,”“second,”“third,” etc. can be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms can be only used to distinguish one element, component, region, layer or section from another region, layer or section. Terms such as“first,”“second,” and other similar terms are used herein when claimed terms are used in this fashion. Thus, terms such as“first” and“second” discussed below can be termed as“second” and“first,” respectively, without departing from the teaching of the exemplary embodiments.

[0037] For the purposes of this description, spatially relative terms such as“inner,”“outer,”“beneath,”“below,”“lower,”“above,”“upper,” and the like can be used herein for ease of description to describe one element or feature’s relationship to another element(s) or feature(s) as illustrated in the figures. The spatially relative terms can be intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as“below” or“beneath” other elements or features would then be oriented“above” the other elements or features. Thus, the exemplary term“below” can encompass both an orientation of above and below. The device can be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

[0038] In this document, various components are referred to as being“operably connected.” As used herein,“operably connected” refers to components that are connected in an operable manner, and encompasses embodiments in which components are directly connected, as well as embodiments in which additional components are placed between connected components. Components that are“operably connected” can be“fluidly connected.”“Fluidly connected” refers to components that are connected such that fluid can be transported between them.“Fluidly connected” encompasses embodiments in which additional components are provided between two fluidly connected components, as well as embodiments in which components are directly connected. Fluidly connected components can include components that do not contact fluid but contact other components to manipulate the system (e.g., a peristaltic pump, which pumps fluid through a flexible tube by compressing the outside of the tube).

[0039] Exemplary embodiments now will be described more fully hereinafter with reference to the accompanying drawings.

[0040] Cell expansion systems are cell culture systems for expanding and differentiating cells, including adherent and non-adherent cell types. The present disclosure relates to cell expansion systems and methods, including those described, for example, in U.S. Patent Application No. 15 / 943,536, filed April 2, 2018, and published October 2, 2018, entitled “Expanding Cells in a Bioreactor,” and / or U.S. Patent No. 10,577,585, entitled “Cell Expansion,” and issued March 3, 2020, the entire disclosures of which are incorporated herein by reference.

[0041] Figure 1 is a schematic view of an exemplary cell expansion system 10. The cell expansion 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 flow path 16 having opposite ends 18 and 20. The first fluid flow path 16 can be in fluid communication with a cell growth chamber 24. For example, the first opposite end 18 of the first fluid flow path 16 can be in fluid communication with a first inlet 22 of the cell growth chamber 24, and the second opposite end 20 can be in fluid communication with a first outlet 28 of the cell growth chamber 24. The cell growth chamber 24 can include or be configured to accept a bioreactor (which can also be referred to as a hollow fiber membrane (HFM) 117 (see Figure 2 ). The bioreactor 117 can be a standard bioreactor or a small bioreactor. The standard bioreactor and the small bioreactor can be similarly configured and received by the same cell expansion system 10. However, the standard bioreactor and the small bioreactor have different conventional sizes. The standard bioreactor can generally be selected to accommodate larger cell stocks (e.g., greater than 50 M cells) and / or to produce larger cell harvests (e.g., greater than 2 B cells), while the small bioreactor can generally be selected to accommodate smaller cell stocks (e.g., less than or equal to 50 M cells) and / or to produce smaller cell harvests (e.g., less than 3 B cells) and / or to maintain lower costs. In each case, fluid in the first circulation path 12 can flow through the interior of the plurality of hollow fibers 116 of the bioreactor 117. In at least one exemplary embodiment, a first fluid flow control device 30 can be operably connected to the first fluid flow path 16 and can control fluid flow in the first fluid circulation path 12.

[0042] The total length of the small bioreactor can be less than the total length of the standard bioreactor. For example, in at least one exemplary embodiment, the small bioreactor can be one-half the length of the standard bioreactor. The surface area of the small bioreactor can be less than the surface area of the standard bioreactor. For example, in at least one exemplary embodiment, the surface area of the small bioreactor can be about one-tenth the surface area of the standard bioreactor. In at least one exemplary embodiment, the standard bioreactor can have a surface area of greater than or equal to about 17,000 cm 2 and less than or equal to about 21,000 cm 2 , while the small bioreactor can have a surface area of about 2,000 cm 2 . The intracapillary volume of the small bioreactor can be less than the intracapillary volume of the standard bioreactor. For example, in at least one exemplary embodiment, the intracapillary volume of the small bioreactor can be about one-tenth the intracapillary volume of the standard bioreactor. In at least one exemplary embodiment, the standard bioreactor can have an intracapillary volume of greater than or equal to about 158 mL and less than or equal to about 190 mL, while the small bioreactor has an intracapillary volume of about 58 mL. In at least one exemplary embodiment, the small bioreactor can have a ratio of tubing to bioreactor volume of about 2.96.

[0043] The second fluid circulation path 14 includes, for example, a second fluid flow path 34 and a second fluid flow control device 32. Like the first fluid flow path 16, the second fluid flow path 34 can have opposite ends 36 and 38. The opposite ends 36 and 38 of the second fluid flow path 34 can be in fluid communication with the inlet port 40 and the outlet port 42 of the cell growth chamber 24. For example, the first opposite end 36 of the second fluid flow path 34 can be in fluid communication with the inlet port 40 of the cell growth chamber 24, and the second opposite end 38 of the second fluid flow path 34 can be in fluid communication with the outlet port 42. Fluid in the second circulation path 14 can be in contact with the exterior of the bioreactor 117 disposed in the cell growth chamber 24. In at least one exemplary embodiment, the second fluid flow control device 32 can be operably connected to the second fluid flow path 34 and can control fluid flow in the second fluid circulation path 14.

[0044] The first fluid circulation path 12 and the second fluid circulation path 14 can be maintained in the cell growth chamber 24 through a bioreactor 117, where fluid in the first fluid circulation path 12 flows through an intracapillary (IC) space of the bioreactor 117, and fluid in the second circulation path 14 flows through an extracapillary (EC) space of the cell growth chamber 24. The first circulation path 12 can be referred to as an "intracapillary loop" or "IC loop." The second fluid circulation path 14 can be referred to as an "extracapillary loop" or "EC loop." Fluid flow in the first fluid circulation path 12 can flow in a co-current or counter-current direction relative to fluid flow in the second fluid circulation path 14.

[0045] In at least one example embodiment, a fluid inlet path 44 can be fluidly associated with the first fluid circulation path 12, and a fluid outlet path 46 can be fluidly associated with the second fluid circulation path 14. The fluid inlet path 44 can allow fluid to enter the first fluid circulation path 12, and the fluid outlet path 46 can allow fluid to exit the cell expansion system 10. In at least one example embodiment, as shown, a third fluid flow control device 48 can be operatively associated with the fluid inlet path 44. Although not shown, it should be appreciated that in various other example embodiments, a fourth fluid flow control device can be alternatively or additionally operatively associated with the first outlet path 46. In at least one example embodiment, the fluid flow control devices (including the first fluid flow control device 30 and / or the second fluid flow control device 32 and / or the third fluid flow control device 48 and / or the fourth fluid flow control device) can include pumps, valves, clamps, or any combination thereof. For example, multiple pumps, valves, and clamps can be arranged in any combination. In at least one example embodiment, the fluid flow control devices can be or include peristaltic pumps. The fluid circulation paths (including the first fluid circulation path 12 and / or the second fluid circulation path 14) and / or the inlet ports (including the fluid inlet port 44) and / or the outlet ports (including the fluid outlet port 46) can include any known tubing material, and any type of fluid - including, for example, buffers, protein-containing fluids, and cell-containing fluids - can flow through the various circulation paths (including the first fluid circulation path 12 and / or the second fluid circulation path 14) and / or the inlet paths (including the fluid inlet port 44) and outlet paths (including the fluid outlet port 46). It should be appreciated that the terms "fluid," "medium," and "fluid medium" can be used interchangeably.

[0046] Figure 2 An example cell growth chamber 100 is shown in FIG. 1. The cell growth chamber 100 can be used as the cell growth chamber 24 in the cell expansion system 10 as described in Figure 1The cell growth chamber 24 of the cell expansion system 10 shown in FIG. 1. The cell growth chamber 100 can have a longitudinal axis (shown by line LA-LA) and can include a cell growth chamber housing 104. The cell growth chamber housing 104 can have four openings or ports, including, for example, an intracapillary inlet port 108, an intracapillary outlet port 120, an extracapillary inlet port 128, and an extracapillary outlet port 132. A first fluid (which can also be referred to as an intracapillary fluid or medium) in a first circulation path (such as the first fluid circulation path 12) can enter the cell growth chamber 100 through the intracapillary inlet port 108 at a first fluid manifold end 112 of the cell growth chamber 100, and into and through the intracapillary spaces of a plurality of hollow fibers 116, and exit the cell growth chamber 100 through the intracapillary outlet port 120 located at a second fluid manifold end 124 of the cell growth chamber 100. The fluid path between the intracapillary inlet port 108 and the intracapillary outlet port 120 can define an intracapillary portion 126 of the cell growth chamber 100. A second fluid (which can also be referred to as an extracapillary medium or fluid) in a second circulation path (such as the second fluid circulation path 14) can enter the cell growth chamber 100 through the extracapillary inlet port 128. This second fluid contacts the extracapillary spaces or exterior of the bioreactor 117 and exits the cell growth chamber 100 via the extracapillary outlet port 132. The fluid path between the extracapillary inlet port 128 and the extracapillary outlet port 132 can define an extracapillary portion 136 of the cell growth chamber 100.

[0047] When the second fluid contacts the exterior of the hollow fibers 116, small molecules (e.g., ions, water, oxygen, lactate, etc.) can diffuse from the interior or intracapillary space of the hollow fibers 116 through the hollow fibers 116 to the exterior or extracapillary space, or alternatively or additionally, from the extracapillary space to the intracapillary space. Large molecular weight molecules (e.g., growth factors and / or proteins) are typically too large to pass through the membrane wall of the hollow fibers 116 and remain in the intracapillary space (or alternatively or additionally, the extracapillary space) of the hollow fibers 116. The media defining the first and second fluids can be replaced as needed, and can alternatively or additionally, be circulated through an oxygenator and / or gas transfer module to exchange gases as needed. Cells (e.g., T cells) for expansion can be housed within the first fluid circulation path 12 and / or the second fluid circulation path 14, and can enter the cell growth chamber 100 in one or both of the intracapillary space or extracapillary space, as discussed below.

[0048] In at least one example embodiment, cells (e.g., T cells) for expansion and / or differentiation and / or harvesting can be seeded in the intracapillary space of hollow fibers 116, and cell culture media can be pumped through the extracapillary space of hollow fibers 116 to deliver nutrients to the cells via perfusion through the hollow fiber membranes during expansion. However, in at least one other example embodiment, cells (e.g., T cells) for expansion and / or differentiation and / or harvesting can be seeded in the extracapillary space, and cell culture media can be pumped through the intracapillary space to deliver nutrients to the cells via perfusion through the hollow fiber membranes during expansion. In at least one other example embodiment, cells (e.g., T cells) for expansion and / or differentiation and / or harvesting can be seeded in the intracapillary space, and cell culture media can be pumped through both the extracapillary space and the intracapillary space. Movement of the cell culture media through the intracapillary space and / or the extracapillary space can help remove excess cells, e.g., those that do not adhere to the surface of the hollow fiber membranes. In at least one example embodiment, the material used to form bioreactor 117 can be any biocompatible polymeric material capable of being made into hollow fibers 121. For example, synthetic polysulfone-based materials (e.g., polyethersulfones (PES)) are commonly used to form hollow fibers.

[0049] In at least one example embodiment, cell expansion system 10 can also include a device configured to move or "rock" cell growth chamber 100 relative to other components of cell expansion system 10. The device can be a rotational and / or lateral rocking device. For example, as shown, cell growth chamber 100 can be rotationally connected to one or more rotational rocking components 138 and lateral rocking components 140. First rotational rocking components 138 can be rotationally associated with cell growth chamber 100. For example, first rotational rocking components 138 can be configured to rotate cell growth chamber 100 about a first or central rotational axis 142. In at least one example embodiment, cell growth chamber 100 can be rotated in an alternating manner, including, for example, rotating about central axis 142 in a first clockwise direction and then in a second counterclockwise direction. Figure 3

[0050] ​Although not shown, it should be appreciated that in at least one example embodiment, the second rotational shaking component can be configured to move the cell growth chamber 100 about a second rotational axis 144, where the second rotational axis 144 passes through a center point of the cell growth chamber 100, perpendicular to the central axis 142. In at least one example embodiment, the cell growth chamber 100 can be rotated in an alternating fashion, including, for example, rotating about the second axis 144 in a first clockwise direction and then in a second counterclockwise direction. In at least one example embodiment, the cell growth chamber 100 can also be rotated about the second axis 144 and positioned in a horizontal or vertical orientation relative to gravity. The lateral shaking component 140 can be laterally associated with the cell growth chamber 100. For example, a plane of the lateral shaking component 140 can be moved laterally in the x- and y-directions.

[0051] Rotation and / or lateral movement of the cell growth chamber 100 can reduce the likelihood of cell settling and cells becoming trapped within portions of the bioreactor 117 disposed in the cell growth chamber 100. In at least one example embodiment, the rate at which cells settle in the cell growth chamber 100 can be proportional to the difference in density between the cells and the suspension medium, according to Stokes' Law. In at least one example embodiment, repeated 180 degree rotations with pauses (fast) (e.g., with a total combined time of 30 seconds) as described above can help to keep non-adherent cells (e.g., T cells) in suspension. A minimum rotation of about 180 degrees can be preferred, however various degrees of rotation can be used, including up to or greater than 360 degrees. Different shaking components can be used individually, or can be combined in any combination. For example, a shaking component of the cell growth chamber 100 that rotates about the central axis 142 can be combined with a shaking component of the cell growth chamber 100 that rotates about the axis 144. Likewise, clockwise and counterclockwise rotations about different axes can be performed independently in any combination.

[0052] Figure 4 is a schematic illustration of an example cell expansion system 500 (which can be like the cell expansion system 100 shown in Figure 1 FIG. 1), showing an example fluid path. In at least one example embodiment, cells can be positioned in the intracapillary space, and cell culture media can be pumped through the extracapillary space to perfuse nutrients to the cells via the hollow fiber membranes during expansion. However, it should be appreciated that in at least one other example embodiment, cells can be positioned in the extracapillary space, and cell culture media can be pumped through the intracapillary space to perfuse nutrients to the cells via the hollow fiber membranes during expansion. In at least one other example embodiment, cells can be positioned in both the intracapillary and extracapillary spaces, and cell culture media can be pumped through both the intracapillary and extracapillary spaces.

[0053] As shown, the cell expansion system 500 can include a first fluid circulation path 502 (also referred to as an "intracapillary loop" or "IC loop") and a second fluid circulation path 504 (also referred to as an "extracapillary loop" or "EC loop"). A first fluid flow path 506 can be fluidly associated with a cell growth chamber 501 to form the first fluid circulation path 502. The cell growth chamber 501 can be used as Figure 1 the cell growth chamber 24 and / or Figure 2 the cell growth chamber 100. The first fluid can flow into the cell growth chamber 501 through an intracapillary inlet port 501 A. The first fluid can exit the cell growth chamber via an intracapillary outlet port 501B. In at least one example embodiment, the first fluid circulation path 502 can include a pressure gauge 510 configured to measure the pressure of the first fluid exiting the cell growth chamber 501. In at least one example embodiment, the first fluid circulation path 502 can include an intracapillary circulation pump 512 configured to control the flow rate of the first fluid. For example, the intracapillary circulation pump 512 can be configured to pump the first fluid in a first direction or a second direction opposite the first direction. In the latter case, the intracapillary outlet port 501B can be used as an inlet and the intracapillary inlet port 501A can be used as an outlet. In at least one example embodiment, the first fluid circulation path 502 can include a sample port 516 and / or a sample coil 518 configured for sample extraction of the first fluid. In at least one example embodiment, the first fluid circulation path 502 can include a pressure / temperature gauge 520 configured to detect the pressure and / or temperature of the first fluid during operation. In at least one example embodiment, the first fluid can enter the intracapillary loop 502 via a valve 514. In at least one example embodiment, a portion of the cells can be flushed from the intracapillary loop 502 into a harvest bag 599, for example, via a valve 598. It should be appreciated that in at least one other example embodiment, the first fluid circulation path 502 can include additional or fewer valves, pressure gauges, pressure sensors, temperature sensors, ports, and / or other devices configured to isolate and / or measure properties of the first fluid along portions of the intracapillary loop 502.

[0054] A second fluid can flow into the cell growth chamber 501 through the extracapillary inlet port 501C. The second fluid can exit the cell growth chamber 501 via the extracapillary outlet port 501D. In at least one example embodiment, the second fluid in the extracapillary circuit 504 can contact the exterior of the surface of the hollow fibers disposed in the cell growth chamber 501, thereby allowing small molecules to diffuse into and out of the hollow fibers. In at least one example embodiment, the extracapillary circuit 504 can include a pressure gauge / thermometer 524 configured to measure the pressure and / or temperature of the second fluid prior to the second fluid entering the cell growth chamber 501. In at least one example embodiment, the extracapillary circuit 504 can include a pressure gauge 526 configured to measure the pressure of the second fluid, for example, as the second fluid exits the cell growth chamber 501. In at least one example embodiment, the extracapillary circuit 504 can include a sample port 530 configured for sample extraction of the second fluid.

[0055] In at least one example embodiment, the extracapillary circuit 504 can include an extracapillary circulation pump 528 and an oxygenator or gas transfer module 532. For example, after exiting the cell growth chamber 501, the second fluid can pass through the extracapillary circulation pump 528 and to and through the oxygenator or gas transfer module 532. In at least one example embodiment, the extracapillary circulation pump 528 can be configured to control the second fluid flow rate. For example, as with the intracapillary circulation pump 512, the extracapillary circulation pump 528 can be configured to pump the second fluid in a first direction or in a second direction opposite the first direction. In the latter case, the extracapillary outlet port 501D can function as an inlet and the extracapillary inlet port 501C can function as an outlet.

[0056] In at least one example embodiment, the second fluid flow path 522 can be fluidly associated with an oxygenator or gas transfer module 532 via an oxygenator inlet port 534 and an oxygenator outlet port 536. For example, the second fluid can flow into the oxygenator or gas transfer module 532 via the oxygenator inlet port 534 and look away or exit the oxygenator or gas transfer module 532 via the oxygenator outlet port 536. In at least one example embodiment, the oxygenator or gas transfer module 532 can be configured to add oxygen to the second fluid and / or remove gas bubbles from the second fluid. For example, air and / or gas can flow into the oxygenator or gas transfer module 532 via a first filter 538 and out of or exit (i.e., flow out of) the oxygenator or gas transfer device 532 through a second filter 540. The first filter 538 and the second filter 540 can be configured to reduce or prevent contaminants from entering the oxygenator or gas transfer module 532. The second fluid in the second fluid circulation path 504 can be in equilibrium with the gas entering the oxygenator or gas transfer module 532. In at least one example embodiment, air and / or gas can be purged from the cell expansion system 500, for example, during a priming sequence, the air and / or gas can be vented to the atmosphere by the oxygenator or gas transfer module 532. It should be appreciated that in at least one other example embodiment, the second fluid circulation path 504 can include additional or fewer valves, pressure gauges, pressure sensors, temperature sensors, ports, and / or other devices configured to isolate and / or measure properties of the second fluid along a portion of the capillary extracorporeal circuit 504.

[0057] In at least one example embodiment, an air removal chamber (ARC) 556 can be fluidly associated with the first circulation path 502. The air removal chamber 556 can include one or more ultrasonic sensors. For example, the air removal chamber 556 can include an upper sensor and / or a lower sensor configured to detect air and / or lack of fluid and / or a gas-liquid interface at certain measured locations within the air removal chamber 556. The upper sensor can be disposed proximate a first end (e.g., a top) of the air removal chamber 556. The lower sensor can be disposed proximate a second end (e.g., a bottom) of the air removal chamber 556. While ultrasonic sensors are discussed, it should be appreciated that the air removal chamber 556 can additionally or alternatively include one or more other sensors, including, for example, optical sensors. Air and / or gas purged from the cell expansion system during portions of a priming sequence and / or other protocols can be vented to the atmosphere from an air valve 560 via a line 558 fluidly associated with the air removal chamber 556.

[0058] In at least one example embodiment, the first fluid can include cells (e.g., from a first fluid container (which can also be referred to as a first media bag or a first bag) 562 and from a second fluid container (which can also be referred to as a second media bag or a second bag) 546) and fluid media (e.g., intracapillary media or fluid). The material (i.e., cells and / or intracapillary media) from the first fluid container 562 and the second fluid container 546 can enter the first fluid circulation path 502 via the first fluid flow path 506. The first fluid container 562 can be fluidly associated with the first fluid flow path 506 and the first fluid circulation path 502 via a valve 564. In at least one example embodiment, the second fluid container 546 and a third fluid container (which can also be referred to as a third media bag or a third bag) 544 can be fluidly associated with the first fluid inlet path 542, for example, via a valve 548 and a valve 550, respectively, or fluidly associated with the second fluid inlet path 574, for example, via a valve 570 and a valve 576, respectively. In at least one example embodiment, the material from the second fluid container 546 and / or the third fluid container 544 can be in fluid communication with the first sterile sealable input priming path 508 and / or the second sterile sealable input priming path 509.

[0059] In at least one example embodiment, a fourth fluid container (which can also be referred to as a fourth media bag or a fourth bag) 568 can include extracapillary media, and a fifth fluid container (which can also be referred to as a fifth media bag or a fifth bag) 566 can include a wash solution. The material (i.e., extracapillary media and / or wash solution) from the fourth fluid container 568 and the fifth fluid container 566 can enter the first fluid circulation path 502 and / or the second fluid circulation path 504. For example, in at least one example embodiment, the fifth fluid container 566 can be fluidly associated with a valve 570, where the valve 570 is fluidly associated with the first fluid circulation path 502, for example, via a distribution valve 572 and the first fluid inlet path 542. In at least one example embodiment, the fifth fluid container 566 can be fluidly associated with the second fluid circulation path 504 via the second fluid inlet path 574 and an extracapillary inlet path 584 by, for example, opening the valve 570 and closing the distribution valve 572. The fourth fluid container 568 can be fluidly associated with a valve 576, where the valve 576 is fluidly associated with the first fluid circulation path 502, for example, via the first fluid inlet path 542 and the distribution valve 572. In at least one example embodiment, the fourth fluid container 568 can be fluidly associated with the second fluid inlet path 574 by opening the valve 576 and closing the distribution valve 572. In at least one example embodiment, the first fluid inlet path 542 and / or the second fluid inlet path 574 can be fluidly associated with an optional heat exchanger 552.

[0060] In at least one example embodiment, fluid can travel from the first fluid inlet path 542 and / or the second fluid inlet path 574 to the intracapillary loop 502 via the intracapillary inlet pump 554, and fluid can travel to the extracapillary loop 504 via the capillary inlet pump 578. In at least one example embodiment, an air detector 580 can also be associated with the extracapillary inlet path 584. The air detector 580 can comprise, for example, an ultrasonic sensor. In at least one example embodiment, the first fluid circulation path 502 and the second fluid circulation path 504 can be fluidly associated with a waste line 588. For example, when the valve 590 is in an open state or position, intracapillary media can flow through the waste line 588 to a waste bag (also referred to as an outlet bag) 586. When the valve 582 is open, extracapillary media can flow through the waste line 588 to the waste bag 586. In at least one example embodiment, cells can be harvested, for example, via a cell harvest path 596. For example, cells from the cell growth chamber 501 can be harvested by pumping the intracapillary media containing the cells through the cell harvest path 596 and the valve 598 to a cell harvest bag 599.

[0061] In at least one example embodiment, as shown, fluid in the first fluid circulation path 502 and the second fluid circulation path 504 flow through the cell growth chamber 501 in the same direction (i.e., a co-current configuration). Although not shown, it should be recognized that in various other example embodiments, the cell expansion system 500 can also be configured to flow in a counter-current configuration. As shown, fluid in the first fluid circulation path 502 can enter the cell growth chamber 501 at the intracapillary inlet port 501 A, and can exit or exit the cell growth chamber 501 at the intracapillary outlet port 501B. In at least one example embodiment, the first fluid flow path 506 can be fluidly connected with the first fluid circulation path 502, for example, via a connector 517. The connector 517 can be a point or location at which fluid can flow in opposite directions therefrom, for example, based on the direction and flow rate of the intracapillary inlet pump 554 and the fluid circulation pump 512. The connector 517 can comprise any type of fitting, coupling, fusion, passage, and / or tubing that allows the first fluid flow path 506 to be fluidly associated with the first fluid circulation path 502. In at least one example embodiment, the connector 517 can comprise a T-fitting or coupling and / or a Y-fitting or coupling. Figure 4

[0062] ​In at least one example embodiment, one or more instruments (e.g., pressure gauge 510 and / or pressure / temperature gauge 520 and / or pressure / temperature gauge 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., intracapillary inlet port 501 A and / or intracapillary outlet port 501 B and / or extracapillary inlet port 501 C and / or extracapillary outlet port 501 D and / or sample port 516 and / or sample port 530 and / or oxygenator inlet port 534 and / or oxygenator outlet port 536), one or more pumps (e.g., intracapillary circulation pump 512 and / or extracapillary circulation pump 528 and / or intracapillary inlet pump 554 and / or extracapillary 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., oxygenator or gas transfer module 532), and / or one or more other components of cell expansion system 500 can be in electrical communication with a control system (not shown). The control system can include a plurality of nodes, which can include various hardware, firmware, and / or software configured to control and / or communicate with the mechanical, electromechanical, and electrical components of cell expansion system 500, including, for example, a controller and a memory.

[0063] The controller, which can also be referred to as a processor, can be any type of microcontroller, microprocessor, field-programmable gate array (FPGA), application- specific integrated circuit (ASIC), etc. An exemplary controller can be an NK10DN512VOK10 microcontroller manufactured and sold by N9P USA, Incorporated, which is a microcontroller unit with a 32-bit architecture. Other exemplary controllers can include, for example, at least one of 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™ series processors, Intel® Xeon® series processors, Intel® Atom™ series processors, Intel Itanium® series processors, Intel® Core® i5-4670K and i7-4770K 22nm Haswell, Intel® Core® i5-3570K 22nm Ivy Bridge, AMD® FX™ processor series, AMD® FX-4300, FX-6300, and FX-8350 32nm Vishera, AMD® Kaveri processor, ARM® Cortex™-M processors, ARM® Cortex-A and ARM926EJ-S™ processors, other industry equivalent processors, and can perform computing functions using any known or future developed standard, instruction set, library, and / or architecture. The memory can be any type of memory, including random access memory (RAM), read only memory (ROM), electrically erasable programmable ROM (EEPROM), compact disc read only memory (CD-ROM), optical storage, magnetic storage, any suitable combination of the foregoing, or other types of storage or memory devices that store and provide instructions to program and control the controller.

[0064] The present disclosure provides, for example, exemplary methods of producing T cells using a cell expansion system, such as the cell expansion system 10 shown in Figure 1 The present disclosure provides, for example, exemplary methods of producing T cells using a cell expansion system, such as the cell expansion system 10 shown in 2 The present disclosure provides, for example, exemplary methods of producing T cells using a cell expansion system, such as the cell expansion system 10 shown in Figure 5A flowchart of an exemplary method 600 is shown, which includes flowing 620 the isolated T cells into a small bioreactor of a cell expansion system. In at least one exemplary embodiment, the isolated T cells can be flowed into an intracapillary loop of the small bioreactor. More specifically, the T cells can be flowed into the intracapillary loop via an intracapillary inlet (e.g., first inlet 22 of cell growth chamber 24 as shown in Figure 1 FIG. 2, and / or intracapillary inlet 108 of cell growth chamber 100 as shown in Figure 2 FIG. 3, and / or intracapillary inlet 501A of cell growth chamber 501 as shown in Figure 4 FIG. 5), using or regulating, for example, a unidirectional intracapillary inlet flow from a media bag (e.g., second fluid container 546 of cell expansion system 500 as shown in Figure 4 FIG. 6). In at least one exemplary embodiment, the rate at which the T cells are flowed into the small bioreactor can be greater than or equal to about 0.01 mL / min to less than or equal to about 100 mL / min, optionally greater than or equal to about 1 mL / min to less than or equal to about 100 mL / min, optionally greater than or equal to about 1 mL / min to less than or equal to about 10 mL / min, and optionally about 5 mL / min. In at least one exemplary embodiment, the rate at which the T cells are flowed into the small bioreactor can be greater than or equal to about 0.007 μL / min / fiber to less than or equal to about 0.0281 μL / min / fiber. By comparison, the rate at which T cells are flowed into a standard bioreactor is typically greater than or equal to about 0.017 μL / min / fiber to less than or equal to about 0.0347 μL / min / fiber.

[0065] In at least one exemplary embodiment, the total viable cell mass flowed into the small bioreactor can be greater than or equal to about 20 millimeters to less than or equal to about 60 millimeters, and optionally, greater than or equal to about 30 milliliters to less than or equal to about 50 milliliters.

[0066] Although not shown, it should be appreciated that in various exemplary embodiments, the method 600 can include maintaining the T cells within the small bioreactor, and more specifically, for example, in the intracapillary loop using backflush suppression. By way of example, Figure 6An exemplary illustration of reverse flow containment of T cells is provided. As shown, T cells (and / or other cells) 700 can be positioned (or repositioned) within the intracapillary portion or space 702 of the bioreactor 704 using reverse flow containment. More specifically, as media or fluid media (i.e., fluid) moves from the media bag or container 706, it can be divided between an intracapillary inlet pump (not shown) and an intracapillary circulation pump (not shown) such that the media or fluid moves through both the intracapillary inlet 708 and the intracapillary outlet 710 into the intracapillary portion or space 702 of the bioreactor 704. Conversely, the T cells (and / or other cells) 700 can be seeded and / or recirculated via the intracapillary inlet 708 into the intracapillary portion or space 702 of the bioreactor 704 using unidirectional flow from the media bag or container 706.

[0067] In at least one exemplary embodiment, during the sustentation phase, the intracapillary inlet flow rate is about 2 times the intracapillary circulation pump rate. For example, the intracapillary inlet flow rate can be greater than or equal to about 0.02 mL / min and the intracapillary circulation pump rate can be greater than or equal to about 0.01 mL / min. Arrow 712 illustrates movement of fluid through the pores of the bioreactor 704 from the intracapillary portion or space 702 to the extracapillary portion or space 714 during reverse flow containment. A waste bag 716 can be in fluid communication with the extracapillary portion or space 714, allowing for proper movement from the extracapillary portion 714 to the waste bag 716.

[0068] As discussed above, although the cells (e.g., T cells) 200 are shown seeded within the intracapillary portion or space 702, it should be appreciated that in various other exemplary embodiments, the cells 200 can alternatively be seeded within the extracapillary portion or space 714. In such a case, reverse flow containment can include moving media or fluid from the media bag or container 706 to an extracapillary inlet (not shown) and an extracapillary outlet (not shown).

[0069] Referring again to Figure 5 In at least one exemplary embodiment, the method 600 can include separating or dissociating 610 the target cells (i.e., T cells) from the source. In at least one exemplary embodiment, the target cells and the source can be separated using immunomagnetic positive selection. In other exemplary embodiments, the target cells and the source can be separated using magnetically coupled antibodies and uncoupled antibodies. For example, Figure 7An exemplary method for separating a source from T cells 610 is shown, which may include the use of a magnetically conjugated antibody and an unconjugated antibody. For example, separating a target cell from a source may include contacting the source with a magnetically conjugated antibody 612 and contacting the source with an unconjugated antibody (which may also be referred to as a stimulating antibody) 614. Although shown as separate steps, it should be understood that in various exemplary embodiments, the magnetically conjugated antibody and the unconjugated antibody may be contacted with the source simultaneously or subsequently 612, 614. For example, in at least one exemplary embodiment, the magnetically conjugated antibody may be contacted with the source 612, and then the unconjugated antibody may be contacted with the source 614. In other exemplary embodiments, the unconjugated antibody may be contacted with the source 614, and then the magnetically conjugated antibody may be contacted with the source 614. In yet another exemplary embodiment, the magnetically conjugated antibody may be contacted with the source 614, and the unconjugated antibody may be contacted with the source 614 simultaneously. For example, in at least one exemplary embodiment, contact 612 between the magnetically conjugated antibody and the source, and contact 614 between the non-conjugated antibody and the source, may include moving the source (including non-target cells or materials or components, and target cells) from a first bag (e.g., an inlet bag) to another bag (e.g., a stimulation and selection bag), wherein the other bag contains the magnetically conjugated antibody and the non-conjugated antibody. In each case, the selected magnetically conjugated antibody binds to or associates with the non-target cells or materials or components, and the selected non-conjugated antibody binds to or associates with the target cells, and contacts 612, 614 may occur for a selected duration.

[0070] When the magnetically conjugated antibody associates with a non-target cell and the non-conjugated antibody associates with a target cell, separation 610 may further include moving a source containing both the magnetically conjugated antibody and the non-conjugated antibody through a magnetic column, wherein the magnetic column is configured to retain the magnetically conjugated antibody associated with the non-target cell or material or component, while allowing the non-conjugated antibody associated with the target cell to pass through and reach the capillary loop (e.g., such as...). Figure 1 The first loop path 12 and / or shown Figure 4 The first fluid circulation path 502 shown, or alternatively or additionally, reaches the capillary external loop (e.g., such as...). Figure 1 The second fluid circulation path 14 and / or shown Figure 4 The second fluid circulation path 504 is shown.

[0071] Refer again Figure 5 Method 600 may also include expanding 630 T cells in a small bioreactor. In at least one exemplary embodiment, such as Figure 8As shown, expanding 630 T cells within a miniature bioreactor may include, for example, feeding the expanded T cells 632 by moving cell culture medium through the miniature bioreactor, and more specifically, moving the cell culture medium through the external space of the capillary while the T cells are loaded into the capillary space as described above. Expanding 630 T cells within a miniature bioreactor may also include media recovery 634. In at least one exemplary embodiment, media recovery may be included in a waste bag or container (e.g., such as...) Figure 4 Waste bag 586 of the cell expansion system 500 shown and / or Figure 6 Waste bag 716 shown) and capillary external media bag (e.g., as shown) Figure 4 A fluid connection is established between the fourth fluid container 568 of the cell expansion system 500 shown to create a capillary external media loop through the bioreactor. Expanding 630 T cells within the miniature bioreactor may also include inducing recirculation 636. In at least one exemplary embodiment, media recirculation may include, for example, recirculating the media through the miniature bioreactor prior to backflow inhibition and perfusion culture of cells on a predetermined or selected basis (e.g., daily).

[0072] Refer again Figure 5 Method 600 may further include harvesting T cells prepared in miniature bioreactor 640. In at least one exemplary embodiment, harvesting 640 may include, for example, moving cells from the bioreactor to a harvest bag by opening or causing one or more valves (e.g., as shown in the example). Figure 4 The harvest bag 599 shown is used to transport the cell suspension into the harvest bag. In at least one exemplary embodiment, the total variable number of T cells harvested can be greater than or equal to about 100 ml to less than or equal to about 250 ml. In at least one exemplary embodiment, at harvest, the cell viability percentage can be greater than or equal to about 70% to less than or equal to about 100%.

[0073] The implementation of this disclosure is further illustrated by the following non-limiting examples.

[0074] Example 1 In the first case, primary donor T cells are loaded with a soluble anti-CD2 / anti-CD3 / anti-CD28 antibody activator onto a surface with a volume of approximately 2000 cm⁻¹. 22 to less than or equal to about 21000 cm 2 of surface area, and greater than or equal to about 158 mL to less than or equal to about 190 mL of intracapillary volume. In the first case, the small bioreactor produced about 22.2% to 33.1% Tscm after eight days, while in the second case, the flask culture produced about 1.8% to 3.7% Tscm after the same time period. In the third case, the standard bioreactor produced less than about 4% Tscm after the same time period. Although not fully understood, it is believed that the lower intracapillary inlet flow rate and / or higher levels of concentrated media in the small bioreactor, compared to the standard bioreactor, surprisingly resulted in elevated levels of dissolved oxygen in the small bioreactor compared to the standard bioreactor, which surprisingly resulted in the production of more Tscm. scm scm scm For example, Figure 9 shows the percent dissolved gas of the small bioreactor compared to the standard bioreactor over a selected time period (i.e., eight days).

[0075] Example 2 In the fourth case, the primary donor T cells were loaded with a soluble anti-CD2 / anti-CD3 / anti-CD28 antibody activator into a small bioreactor having, for example, a surface area of about 2000 cm 2 and an intracapillary volume of about 58 mL, and expanded with X-Vivo 15 (with modifications to the number of cycles to accommodate the higher protein concentration in the media compared to Example 1). In the fifth case, the primary donor T cells were activated using the same activator (i.e., soluble anti-CD2 / anti-CD3 / anti-CD28 antibody activator), but grown using flask culture. In the sixth case, the primary donor T cells were loaded with the same activator (i.e., soluble anti-CD2 / anti-CD3 / anti-CD28 antibody activator) into a standard bioreactor, and expanded with the same media (i.e., X-Vivo 15), where the standard bioreactor had, for example, a surface area of greater than or equal to about 17000 cm​​​2 to less than or equal to approximately 21,000 cm 2 The surface area, and the capillary volume greater than or equal to about 158 ​​mL to less than or equal to about 190 mL. In the fourth case, after nine days, the small bioreactor produced approximately 44.4% to approximately 46.8% of the T. scm In the fifth scenario, after the same time period, flask culture produced approximately 10.3% to approximately 14.9% of T. scm In the sixth scenario, after the same time period, the standard bioreactor produces less than approximately 15% of T. scm .

[0076] The description of the above embodiments is provided for illustrative and descriptive purposes. It is not intended to be exhaustive or limiting of this disclosure. A single element or feature of a particular embodiment is generally not limited to that particular embodiment, but may be interchanged and used in selected embodiments where applicable, even if not specifically shown or described. This may also vary in many ways. These variations should not be considered as departing from this disclosure, and all such modifications are intended to be included within the scope of this disclosure.

Claims

1. A method for producing T cells using a cell expansion system, the method comprising: T cells were amplified using a small bioreactor of the cell expansion system, the small bioreactor having a diameter of approximately 2000 cm². 2 Surface area.

2. The method according to claim 1, wherein, The small bioreactor has an intracapillary volume of approximately 58 ml.

3. The method according to claim 1, wherein, The cell expansion system also includes a pipeline assembly in fluid communication with the small bioreactor, and the volume ratio of the pipeline assembly to the bioreactor is approximately 2.

96.

4. The method according to claim 3, wherein, The method further includes: The piping assembly is positioned in fluid communication with the small bioreactor.

5. The method according to claim 1, wherein, The method further includes: A small bioreactor that allows T cells to flow into the cell expansion system.

6. The method according to claim 5, wherein, The rate at which the T cells flow into the small bioreactor is greater than or equal to about 0.01 mL / min and less than or equal to about 100 mL / min.

7. The method according to claim 5, wherein, The rate at which the T cells flow into the miniature bioreactor is greater than or equal to about 0.007 μL / min / fiber to less than or equal to about 0.0281 μL / min / fiber.

8. The method according to claim 1, wherein, The method further includes: The T cells are held within the miniature bioreactor using countercurrent inhibition.

9. The method according to claim 8, wherein, The backflow suppression includes a first flow velocity along a first direction and a second flow velocity along the opposite second direction, and The first flow rate is approximately twice the second flow rate.

10. The method according to claim 9, wherein, The first flow rate is about 0.02 mL / min, and the second flow rate is about 0.01 mL / min.

11. The method according to claim 3, wherein, The method further includes: This separates T cells from their source.

12. The method according to claim 11, wherein, Separating T cells from their source includes: The magnetically coupled antibody is brought into contact with the source, and the selected magnetically coupled antibody associates with non-target cells, materials, or components of the source; Contact the source with the unconjugated antibody, and the selected unconjugated antibody associates with the T cells; and The source, comprising the magnetically conjugated antibody and the unconjugated antibody, is moved through a magnetic column, the selected column retaining the magnetically conjugated antibody while allowing the unconjugated antibody to pass through.

13. The method according to claim 1, wherein, The method further includes: The T cells produced are harvested from the bioreactor.

14. The method according to claim 13, wherein, The total number of live cells flowing into the small bioreactor is greater than or equal to about 20 ml and less than or equal to about 60 ml. The total variable number of T cells harvested was greater than or equal to approximately 100 ml and less than or equal to approximately 250 ml, and At harvest, the cell viability percentage is greater than or equal to about 70% to less than or equal to about 100%.

15. A method for producing T cells using a cell expansion system with tubular groups, the method comprising: T cells are amplified using a miniature bioreactor of the cell amplification system, the miniature bioreactor being in fluid communication with the tubing assembly, and the volume ratio of the tubing assembly to the bioreactor being approximately 2.

96.

16. The method according to claim 15, wherein, The small bioreactor has a diameter of approximately 2000 cm. 2 The surface area and the internal volume of the capillary are approximately 58 ml.

17. The method according to claim 15, wherein, The method further includes: T cells are introduced into a small bioreactor of the cell expansion system at a rate greater than or equal to about 0.007 μL / min / fibril to less than or equal to about 0.0281 μL / min / fibril.

18. The method according to claim 15, wherein, The method further includes: The T cells are held in a small bioreactor using countercurrent inhibition, which includes a first flow rate in a first direction and a second flow rate in an opposite second direction, the first flow rate being approximately twice the second flow rate.

19. The method according to claim 15, wherein, The method further includes: Separating T cells from their source, wherein separating T cells from their source includes: The magnetically coupled antibody is brought into contact with the source, and the selected magnetically coupled antibody associates with non-target cells, materials, or components of the source; Contact the source with the unconjugated antibody, and the selected unconjugated antibody associates with the T cells; and The source, comprising the magnetically conjugated antibody and the unconjugated antibody, is moved through a magnetic column, the selected column retaining the magnetically conjugated antibody while allowing the unconjugated antibody to pass through.

20. The method of claim 15, wherein, The method further includes: T cells produced from the bioreactor are harvested, wherein... The total number of live cells flowing into the small bioreactor is greater than or equal to about 20 ml and less than or equal to about 60 ml. The total variable number of T cells harvested was greater than or equal to approximately 100 ml and less than or equal to approximately 250 ml, and At harvest, the cell viability percentage is greater than or equal to about 70% to less than or equal to about 100%.

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