Transduction methods

Abstract

A method of transducing T cells is provided, the method comprising providing a compressible container including a base, a top configured substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an interior volume of the compressible container. A population of T cells in cell treatment medium is added to the interior volume. A transduction agent is added to the interior volume. The base of the compressible container is moved relative to the top, thereby causing turbulence of the contents within the interior volume for a first period of time.

Description

[Technical Field]

[0001] The present invention relates to a method for transducing T cells in a compressible container. More particularly, the present invention relates to a method for transducing T cells, such as CD3+ T cells with a CD4+ marker and / or CD3+ T cells with a CD8+ marker, in a compressible container. The transduction methods described herein may form part of a cell and / or gene therapy manufacturing process. [Background technology]

[0002] Cell and gene therapy manufacturing processes are often complex, involving manual or semi-automated steps across several devices. Cells are "living" entities and are susceptible to even the simplest manipulations (e.g., variations in cell transfer procedures). The role of cell manufacturing equipment in ensuring scalability and reproducibility is a critical factor for cell and gene therapy manufacturing.

[0003] Cell-based therapeutic products (CTPs) have gained significant momentum in recent years, resulting in a need for improved cell manufacturing equipment and methods for various cell manufacturing procedures, such as the generation of chimeric antigen receptor (CAR) T cells, and various cell manufacturing processes such as collection, purification, transduction, incubation / harvesting, washing, patient infusion, and / or freezing.

[0004] Cell transduction is the process of introducing genetic material into cells using a transduction agent. This process confers specificity to T cells for target antigens. Transduction can also confer other favorable properties to T cells, such as improving T cell proliferation, cytokine production, activation signaling, and effector function characteristics. CAR T cells are typically used in cell therapy for the treatment of cancer. CAR T cells are generated by transducing T cells, thereby genetically modifying the T cells to produce a CAR that binds to cancer cells.

[0005] A typical method for transducing T cells involves first isolating a patient's T cells. Then, the T cells and a transduction agent are added to a culture vessel, such as a flask or bag. When the transduction agent interacts with the T cells, genetic material is transferred to the T cells, transducing the T cells. This process can be facilitated by stirring the mixture of T cells and transduction agent, for example, manually or via an impeller. Alternatively, the mixture of T cells and transduction agent can be placed in a rigid culture vessel and mixed manually or by placing the culture vessel on a shaker plate. Generally, cell transduction is typically performed in minimally processed media to facilitate interaction between the transduction agent and the cells contained therein. The transduced CAR T cells are then infused into the patient, where they target antigens present in the cancer cells.

[0006] Cultivation or further processing of transduced cells generally requires the addition of additional medium and nutrients to the processing medium to maintain a desired level of cell growth. However, the devices utilized for transduction, including shaker flasks, roller bottles, T-flasks, and bags, have limited, typically minimal, working volumes. Therefore, transduced cells typically need to be transferred to a vessel with a larger working volume.

[0007] A key limiting factor in the production of cells for medical use or gene therapy is the lack of a compact, automated, closed system for performing unit operations without contamination. Operational systems are largely manual, resulting in high operating costs. Multiple pieces of equipment, particularly vessels, are typically required to cover all non-cell culture steps, which involves numerous transfers, each of which presents an opportunity for operator error and contamination. Furthermore, as manual labor increases, so does the risk of manual error; therefore, current labor-intensive processes lack the robustness required for the production of clinical-grade therapeutics. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, there is a need for improved transduction methods that provide higher transduction efficiencies than conventional transduction methods. In other words, more cells need to be transduced than conventional transduction methods. In particular, current methods for transducing T cells have been noted to generally have low transduction efficiencies. Typically, only a subset of target cells are transduced with the desired genetic material. For example, only 5% to 35% of cells are typically successfully transduced.

[0009] There is also a need for transduction methods, and related apparatus and devices, in which cell transfer between various containers and / or equipment is reduced or completely eliminated.

[0010] There is also a need for automation, or semi-automation, of the transduction method and further cell processing steps. [Means for solving the problem]

[0011] According to a first aspect of the present invention there is provided a method of transducing T cells comprising: providing a compressible container including a base, a top configured substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an interior volume of the compressible container; adding a population of T cells in cell treatment medium into said internal volume; adding a transduction agent into said internal volume; moving the base relative to the top, thereby causing turbulence of contents within the interior volume for a period of time, such as a first period of time; Includes.

[0012] Movement of the compressible vessel base causes turbulence of the contents within the vessel's internal volume. This turbulence suspends and disperses the T cells in the cell treatment medium, increasing contact between the transduction agent and the T cells in solution, thereby enhancing transduction efficiency. Additionally, providing a compressible vessel allows for variable working volumes within a single vessel, thereby reducing the need for additional vessels and associated transfers to such vessels. Furthermore, the compressible vessel allows for fluid manipulation, thus facilitating further processing steps.

[0013] According to a second aspect of the present invention there is provided a method of transducing T cells, the method comprising: i) providing a compressible container including a base, a top configured substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an interior volume of the compressible container; ii) adding a population of T cells in cell treatment medium into said internal volume; iii) continuously moving the base relative to the top, thereby causing turbulent flow of contents within the interior volume at a first rate for a first period of time; iv) optionally adding additional cell treatment medium into said internal volume; v) continuously moving the base relative to the top, thereby causing turbulent flow of contents within the interior volume at a second rate greater than the first rate for a second period of time; vi) optionally adding additional cell treatment medium into said internal volume; vi) continuously moving the base relative to the top, thereby causing turbulence of contents within the interior volume for a third time period; in this order, The method further comprises adding a transduction agent into said internal volume during step ii) or step iii).

[0014] Advantageously, this results in continuous mixing of the contents of the compressible container. Continuous mixing of the compressible container allows the T cells to be continuously suspended in the cell treatment medium, thereby allowing for effective expansion of the T cells, and also disperses the T cells with the transduction agent during the mixing period, enhancing interaction between the T cells and the transduction agent for effective transduction of the T cells. In this way, desired cell expansion and transduction efficiency can be achieved.

[0015] In an example, the method comprises: step iii) includes continuously rotating the base at the first speed about an axis of rotation extending in a horizontal plane defined by the base; step v) includes continuously rotating the base at the second speed about an axis of rotation extending in a horizontal plane defined by the base; Step vi) is further defined as comprising continuously translating the base portion toward the top portion along a central longitudinal axis of the compressible container, thereby compressing the compressible container.

[0016] In examples, the methods are provided as methods for transducing and culturing T cells.

[0017] According to a third aspect of the present invention there is provided a method of transducing T cells, the method comprising: i) providing a compressible container including a base, a top configured substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an interior volume of the compressible container; ii) adding a population of T cells in cell treatment medium into said internal volume; iii) optionally, maintaining said base stationary relative to said apex after adding a transduction agent for a predetermined period of time; iv) moving the base relative to the top, thereby causing turbulence of contents within the interior volume for a first period of time; v) maintaining the base stationary relative to the top for a second period of time; vi) repeating steps iv) and v) for a predetermined period of time; vii) adding a transduction agent into said internal volume in any of steps ii) to vi); , in this order.

[0018] Advantageously, this results in intermittent mixing of the contents of the compressible vessel. Intermittent mixing of the compressible vessel allows the T cells to settle on the base during rest periods, thereby allowing for effective proliferation of the T cells, and also allows the T cells to be resuspended and dispersed with the transduction agent during mixing periods to enhance interaction between the T cells and the transduction agent for effective transduction of the T cells. In this way, an appropriate balance between cell proliferation and transduction efficiency can be achieved.

[0019] According to a fourth aspect of the present invention there is provided a method of transducing and culturing T cells, the method comprising: i) providing a compressible container including a base, a top configured substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an interior volume of the compressible container; ii) adding a population of T cells in cell treatment medium into said internal volume; iii) optionally, maintaining said base stationary relative to said apex after adding a transduction agent for a predetermined period of time; iv) intermittently moving the base relative to the top, thereby intermittently inducing turbulence of contents within the interior volume for a first period of time; v) adding a transduction agent into the internal volume during the step of adding a population of T cells in the cell treatment medium into the internal volume or during the step of intermittently moving the base with respect to the top to allow transduction of a population of T cells; vi) adding an additional amount of cell treatment medium into said internal volume; vii) culturing said transduced population of T cells; , in this order.

[0020] Advantageously, intermittent mixing of the contents of the compressible container during transduction of the T cells allows the cells to settle to the base during rest periods, allowing for effective proliferation of the T cells, and also allows the T cells to be resuspended and dispersed with the transduction agent during mixing periods to enhance interaction between the T cells and the transduction agent for effective transduction of the T cells.

[0021] In examples, the step of culturing the transduced population of T cells includes continuously moving the base relative to the apex for a second period of time, thereby continuously causing turbulence of the contents within the internal volume.

[0022] Advantageously, continuous mixing of the T cells during cell culture creates turbulence in a cell solution comprising a population of T cells in cell treatment medium, encouraging oxygen held within the cell solution and within the headspace of the compressible container to mix with and permeate throughout the cell solution, thereby increasing the amount of dissolved oxygen in the cell solution.

[0023] According to a fifth aspect of the present invention there is provided a method of transducing and culturing T cells, the method comprising: i) providing a compressible container including a base, a top configured substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an interior volume of the compressible container; ii) adding a population of T cells in cell treatment medium into said internal volume; iii) optionally, maintaining said base stationary relative to said apex after adding a transduction agent for a predetermined period of time; iv) rotating the base about an axis of rotation extending in a horizontal plane defined by the base, thereby causing turbulence of contents within the interior volume for a first period of time; v) maintaining the base stationary relative to the top for a second period of time; vi) repeating steps iv) and v) for a predetermined period of time; vii) optionally adding additional cell treatment medium into said internal volume; viii) translating the base portion relative to the top portion along a central longitudinal axis of the compressible container, thereby compressing the compressible container for a third time period; ix) adding a transduction agent into said internal volume in any of steps ii) to viii); , in this order.

[0024] Advantageously, rotating the base for a first period of time, maintaining the base stationary for a second period of time, and repeating such steps allows for efficient mixing in the smaller volumes where transduction typically occurs, while translating the base for a third period of time allows for efficient mixing in the larger volumes following the addition of additional cell treatment medium required to maintain the desired growth profile of the cell culture.

[0025] The following examples are described in relation to any of the aspects of the invention as described herein (such as the first aspect, second aspect, third aspect, fourth aspect, or fifth aspect).

[0026] In examples, the method may further include moving the base relative to the top, thereby causing turbulence of contents within the interior volume for a second period of time, i.e., rather than maintaining the base stationary for the second period of time, the base may be moved for the second period of time.

[0027] In examples, the first time period may be 96 hours, 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 3 hours, or 1 hour.

[0028] In examples, the second time period may be 96 hours, 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 3 hours, or 1 hour. The step of moving the base relative to the apex for the first time period may be of the same or different duration as the step of moving the base relative to the apex for the second time period.

[0029] In examples, the first time period is 72 hours or 96 hours and the second time period is 24 hours.

[0030] In an example, moving the base relative to the apex for the first period of time may include moving the base at a first velocity (e.g., rotating, as described below), and moving the base relative to the apex for the second period of time may include moving the base at a second velocity (e.g., rotating, as described below), where the first velocity may be the same as or different from the second velocity.

[0031] In examples, the second speed is greater than the first speed. This can be particularly advantageous in situations where a first volume of cell treatment medium is dispensed into the internal volume during moving the base at a first speed for a first period of time, and a second volume of cell treatment medium is dispensed into the internal volume during moving the base at a second speed for a second period of time, the second volume being greater than the first volume. In particular, adequate mixing of the two different cell treatment medium volumes is achieved without removing contents from the internal volume.

[0032] In examples, the method may further include moving the base relative to the top, thereby causing turbulence of contents within the interior volume for a third period of time.

[0033] In such examples, the third time period may be 96 hours, 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 3 hours, or 1 hour.

[0034] In examples, the first time period is 72 hours or 96 hours, the second time period is 24 hours, and the third time period is 72 hours or 96 hours.

[0035] In an example, moving the base relative to the apex for the first period of time may include moving the base at a first velocity (e.g., rotating, as described below), moving the base relative to the apex for the second period of time may include moving the base at a second velocity (e.g., rotating, as described below), and moving the base for a third period of time may include moving the base at a third velocity (e.g., compressing or longitudinally translating, as described below). The first, second, and third velocities may be the same or different.

[0036] In an example, moving the base relative to the apex for the first period of time may include rotating the base at a first velocity (as described further below), moving the base relative to the apex for the second period of time may include rotating the base at a second velocity (as described further below), and moving the base for a third period of time may include translating the base longitudinally toward the apex at a third velocity (as described further below). The first, second, and third velocities may be as further defined below.

[0037] In some examples, the first time period may be from 1 minute to 5 days. The first time period may be from 1 to 60 minutes. The first time period may be from 0.5 hours (i.e., 30 minutes) to 5 hours. The first time period may be from 0.5 hours (i.e., 30 minutes). The first time period may be from 1 to 3 hours. The first time period may be 1 minute. The first time period may be 3 hours. The first time period may be from 1 to 5 days. The first time period may be from 1 to 4 days. The first time period may be from 1 to 3 days.

[0038] In examples, the method may further include maintaining the base stationary relative to the top for a second period of time.

[0039] In examples, the second time period may be 1 minute to 5 hours. The second time period may be 1 to 60 minutes. The second time period may be 0.5 to 5 hours. The second time period may be 1 to 3 hours. The second time period may be 3 hours.

[0040] In examples, the steps of moving the base relative to the apex for a first time period and maintaining the base stationary relative to the apex for a second time period may be repeated. In some examples, such steps may be repeated multiple times (i.e., two or more times). In some examples, such steps are repeated continuously (e.g., successive moving and stationary periods) for 96 hours, 72 hours, 48 ​​hours, or 24 hours.

[0041] In examples, the method may further include moving the base relative to the top, thereby causing turbulence of the contents within the interior volume for a third period of time. Advantageously, this results in intermittent mixing of the contents of the compressible container.

[0042] In examples, the third time period may be from 1 minute to 5 hours. The third time period may be from 1 to 60 minutes. The third time period may be from 0.5 hours (i.e., 30 minutes) to 5 hours. The third time period may be from 0.5 hours (i.e., 30 minutes). The third time period may be from 1 to 3 hours. The third time period may be 1 minute. The third time period may be 3 hours. The third time period may be from 1 to 5 days. The third time period may be from 1 to 4 days. The third time period may be from 1 to 3 days. In some examples, the third time period is 4 days.

[0043] In an example, moving the base relative to the top for a first period of time may include rotating the base about an axis of rotation extending in a horizontal plane defined by the base.

[0044] In an example, moving the base relative to the top for a third period of time may include rotating the base about an axis of rotation extending in a horizontal plane defined by the base.

[0045] Advantageously, rotating the base about the axis of rotation within the horizontal plane of the base creates a rocking motion to agitate the contents of the compressible container. This increases the movement of cells and other contents within the cell treatment medium, dispersing the cells and enhancing cell-transduction agent interaction. This can also create turbulence that promotes mixing of oxygen from the headspace of the compressible container or the cell treatment medium, thereby increasing the amount of dissolved oxygen in the cell solution. Such a rocking motion can be particularly advantageous for mixing small volumes of cell treatment medium present within the container (e.g., 200 mL or less, 150 mL or less, 100 mL or less, etc.).

[0046] In an example, rotating the base may include rotating the base in a first direction about an axis of rotation to a first position where the base forms a first angle with respect to a horizontal plane, and rotating the base in a second direction opposite the first direction about an axis of rotation to a second position where the base forms a second angle with respect to a horizontal plane.

[0047] In an example, the base may be rotated uniformly between the first position and the second position.

[0048] In an example, the base can be rotated between the first position and the second position at a variable speed.

[0049] In examples, the base may be maintained in the first position and / or the second position for a predetermined period of time.

[0050] In examples, the base may be rotated between the first and second positions at a speed of 1 to 60 revolutions per minute. The base may be rotated between the first and second positions at a speed of 1 to 30 revolutions per minute. The base may be rotated between the first and second positions at a speed of 5 to 25 revolutions per minute. The base may be rotated between the first and second positions at a speed of 10 to 20 revolutions per minute. The base may be rotated between the first and second positions at a speed of approximately 5, 10, 20, 30, 40, 50, or 60 revolutions per minute. Such speeds of rotation may be considered speeds for a first speed, a second speed, or a third speed as described herein.

[0051] In one particular example, the base may be rocked. In this sense, the base may rotate from the first position to the second position and then rotate back to the first position. The base may be rocked at a rate of 1 to 60 rockings per minute, 1 to 30 rockings per minute, 5 to 25 rockings per minute, or 10 to 20 rockings per minute. Preferably, the base is rocked at a rate of 10 rockings per minute for 0.5 hours (i.e., 30 minutes). Rocking is defined as a rotation from the first position to the second position and back to the first position. Such rocking rates may be considered as first, second, or third speeds as described herein.

[0052] In examples, the first angle and / or the second angle may be 1 to 45 degrees. The first angle and / or the second angle may be 5 to 25 degrees. The first angle and / or the second angle may be about 5 degrees, 10 degrees, 15 degrees, 20 degrees, or 25 degrees. In examples, the second angle may be equal to the first angle. Preferably, the first angle and / or the second angle, most preferably each of the first angle and the second angle, is about 5 degrees, about 10 degrees, or about 15 degrees.

[0053] In an example, moving the base relative to the top during a first period of time may include translating the base toward the top along a central longitudinal axis of a compressible container, thereby compressing the compressible container.

[0054] In an example, moving the base relative to the top for a third period of time may include translating the base toward the top along a central longitudinal axis of a compressible container, thereby compressing the compressible container.

[0055] Advantageously, moving the base along the central longitudinal axis of the compressible container creates a reciprocating compressive motion to agitate the contents of the compressible container. This enhances the movement of cells and other contents of the compressible container. This also promotes mixing of oxygen in the headspace of the compressible container with the contents within the compressible container, creating turbulence that enhances the amount of dissolved oxygen in the cell solution. Such compressive motion can be particularly advantageous for mixing larger volumes of cell treatment medium present within the container (e.g., 200 mL or more, 150 mL or more, 100 mL or more, etc.).

[0056] In examples, the compressible container may be compressed at a rate of 1 to 60 compressions per minute. The compressible container may be compressed at a rate of 1 to 30 compressions per minute. The compressible container may be compressed at a rate of 5 to 25 compressions per minute. The compressible container may be compressed at a rate of 10 to 20 compressions per minute. The compressible container may be compressed at a rate of about 5, 10, 20, 30, 40, 50, or 60 compressions per minute, where compressions per minute are defined as translation of the base from a first position to a second position toward the top along the central longitudinal axis of the compressible container, followed by translation of the base away from the top and from the second position back to the first position. Such compression rates may be considered to be first, second, or third speeds as described herein.

[0057] In an example, moving the base relative to the top over a first period of time may include pivoting the base about an origin disposed at a center of the base.

[0058] In an example, moving the base relative to the top over a third period of time can include pivoting the base about an origin disposed at a center of the base.

[0059] Advantageously, pivoting the base about its central origin provides a pivoting motion for agitating the contents of the compressible container. This increases the movement of cells and other contents within the cell treatment medium, dispersing the cells and enhancing cell-transduction agent interaction. This can also create turbulence that promotes mixing of oxygen from the headspace of the compressible container or the cell treatment medium, increasing the amount of dissolved oxygen in the cell solution. Such pivoting motion can be particularly advantageous for mixing small volumes of cell treatment medium present within the container (e.g., 200 mL or less, 150 mL or less, 100 mL or less, etc.).

[0060] In examples, the base may be pivoted at a rate of 1 to 60 revolutions per minute. The base may be pivoted at a rate of 1 to 30 revolutions per minute. The base may be pivoted at a rate of 5 to 25 revolutions per minute. The base may be pivoted at a rate of 10 to 20 revolutions per minute. The base may be pivoted at a rate of about 5, 10, 20, 30, 40, 50, or 60 revolutions per minute, where rotation is defined as rotating or pivoting the base circumferentially about a central origin, starting from a first position and ending at that same first position, in either a clockwise or counterclockwise direction. Such pivoting speeds may be considered to be first, second, or third speeds as described herein.

[0061] In examples, the method further includes moving the base relative to the apex for an additional, predetermined, time period, such as a fourth. Such a step of moving the base relative to the apex for an additional, predetermined, time period, such as a fourth, may include any one or more of rotating, rocking, translating (i.e., compressing), or pivoting the base as contemplated herein. Any of the previous moving steps may constitute the additional, predetermined, time period, such as a fourth. The additional, predetermined, time period, such as a fourth, may be repeated one or more times.

[0062] In an example, the method includes the steps of (a) moving the base relative to the top for a first time period, (b) holding the base stationary relative to the top for a second time period and repeating steps (a) and (b) for a predetermined time period, and (c) moving the base relative to the top for a third time period. In a preferred example, step (a) includes rotating (or rocking) the base about an axis of rotation extending within a horizontal plane defined by the base. In a preferred example, step (c) includes translating the base toward the top along a central longitudinal axis of the compressible container, thereby compressing the compressible container. In such a preferred example, the first time period may be 0.5 hours (i.e., 30 minutes). In such a preferred example, the second time period may be 3 hours. In such a preferred example, steps (a) and (b) may be repeated for 72 hours, 48 ​​hours, or 24 hours, most preferably 72 hours. In such preferred examples, the third time period may be 96 hours, 72 hours, 48 ​​hours or 24 hours, and is most preferably 96 hours.

[0063] In an example, the transduction agent may be added to the interior volume in the step of moving the base relative to the top for the first period of time.

[0064] In an example, the transduction agent may be added to the interior volume in the step of moving the base relative to the top for the third time period.

[0065] In some embodiments, the transfection agent may be a viral vector. The viral vector may be a lentiviral vector. The lentiviral vector may be a CD19 CAR lentiviral vector (LVV). The lentiviral vector may be a green fluorescent protein (GFP) lentiviral vector (LVV).

[0066] In examples, the flexible wall element may include a wall having one or more folds, such as a Z-fold. Advantageously, the walls of the compressible container are easily manipulated for compression and expansion of the compressible container. In examples, the base (and / or top) may be compressible from the top (and / or base) (i.e., configured to translate toward or away from the top).

[0067] In an example, the compressible container may be substantially gas-impermeable. Advantageously, movement of the base relative to the top causes turbulence of the contents within the interior volume of the compressible container, mixing oxygen within the headspace of the compressible container with the cell treatment medium and increasing the level of dissolved oxygen within the cell treatment medium. As such, sufficient oxygenation of the cell treatment medium can be achieved within a non-gas-permeable (i.e., gas-impermeable) container.

[0068] In examples, the compressible container may be at least partially gas permeable.

[0069] In examples, the base and / or the flexible wall element may comprise a gas permeable material, such as silicone or fluorinated ethylene propylene.

[0070] In examples, the compressible container may include a mixing element within the interior volume, such as a baffle, a stationary impeller, or the like. In other examples, the compressible container is provided without a mixing element within the interior volume.

[0071] In an example, the T cells may be selected from CD4+ T cells and CD8+ T cells. In an example, the T cells may be CD3+ T cells or CD34+ hematopoietic stem / progenitor cells (HSPCs). In an example, the T cells may be CD3+ T cells with a CD4+ marker. In an example, the T cells may be CD3+ T cells with a CD8+ marker. In an example, the T cells may be CD3+ T cells with a CD4+ marker and CD3+ T cells with a CD8+ marker.

[0072] In examples, the method may further include adding an activating agent to the internal volume, which may be simultaneously with adding the population of T cells in the cell treatment medium to the internal volume.

[0073] In examples, e.g., with respect to the third aspect, the predetermined time period for repeating steps iv) and v) may be 1 to 5 days. The predetermined time period may be 2 to 3 days. The predetermined time period may be about 3 days. Such examples are not limited to the third aspect and are equally applicable to other aspects as disclosed herein.

[0074] In an example, the step of adding the transduction agent into the internal volume may occur during the step of adding the population of T cells in the cell treatment medium into the internal volume.

[0075] In an example, adding the transduction agent into the interior volume may occur during the step of moving the base relative to the top for the first period of time.

[0076] In an example, the method further comprises maintaining the base stationary relative to the apex after applying the transduction agent for a predetermined period of time.

[0077] In examples, the predetermined time period may be 1 day (ie, 24 hours), 12 hours, 6 hours, 3 hours, 2 hours, or 1 hour.

[0078] In an example, adding the transduction agent into the interior volume may occur while maintaining the base stationary relative to the top for the second period of time.

[0079] In an example, the step of adding the transduction agent into the internal volume may be performed in repetition of either moving the base relative to the apex for the first time period or maintaining the base stationary relative to the apex for the second time period.

[0080] In an example, adding a transduction agent into the interior volume may occur during intermittent movement of the base relative to the top over the first period of time.

[0081] In an example, e.g., with respect to the fourth embodiment, intermittently moving the base relative to the apex for the first period of time and continuously moving the base relative to the apex for the second period of time may include moving the base relative to the apex according to a first motion. Such examples are not limited to the fourth embodiment and are equally applicable to other embodiments as disclosed herein.

[0082] In an example, the first motion can include rotating the base about an axis of rotation extending within a horizontal plane defined by the base. Advantageously, rotating the base about an axis of rotation within the horizontal plane of the base creates a rocking motion to agitate the contents of the compressible vessel. This increases the movement of cells and other contents of the compressible vessel, dispersing the cells and enhancing cell-transduction agent interaction. This can also create turbulence, encouraging mixing from oxygen within the headspace of the compressible vessel or within the cell treatment medium, increasing the amount of dissolved oxygen in the cell treatment medium. This rocking motion is suitable for mixing small volumes of cell treatment medium within the interior volume of the compressible vessel.

[0083] In examples, e.g., with respect to third and fourth aspects, the method of transducing and culturing T cells may further include viii) adding an additional amount of cell treatment medium into said internal volume, and ix) culturing said population of T cells while continuously moving said base relative to said top according to a second motion, thereby continuously causing turbulence of the contents within said internal volume for a third time period. Such examples are not limited to the fourth aspect and are equally applicable to other aspects as disclosed herein.

[0084] In an example, the second motion can include translating the base toward the top along the central longitudinal axis of the compressible container, thereby compressing the compressible container. Advantageously, moving the base along the central longitudinal axis of the compressible container creates a reciprocating compression motion to agitate the contents of the compressible container. This enhances the movement of cells and other contents of the compressible container. This also creates turbulence that promotes mixing of oxygen from the headspace of the compressible container or the cell treatment medium, increasing the amount of dissolved oxygen in the cell treatment medium. This reciprocating compression motion is suitable for mixing large volumes of cell solution within the interior volume of the compressible container.

[0085] In examples, the step of adding the population of T cells in cell treatment medium into the internal volume includes adding a first amount of the population of T cells in cell treatment medium into the internal volume. The first amount may be 200 mL, 150 mL, 100 mL, 50 mL, 25 mL, or 10 mL. The first amount may be 200 mL or less, 150 mL or less, 100 mL or less, 50 mL or less, 25 mL or less, or 10 mL or less.

[0086] In examples, after adding the transduction agent to the internal volume, a second amount of cell treatment medium is added to the internal volume. The second amount of cell treatment medium may be equal to or greater than the first amount of cell treatment medium. Subsequent additions of an amount (e.g., a third amount, a fourth amount, etc.) of cell treatment medium are also contemplated and may be equal to or greater than the first or second amount. In some examples, the second amount may be 200 mL, 150 mL, 100 mL, 50 mL, 25 mL, or 10 mL.

[0087] In examples, one or more additional amounts of cell treatment medium are added into the internal volume after adding the transduction agent to the amount, and the additional amounts of cell treatment medium may be equal to the amount of cell treatment medium present in the internal volume prior to adding such additional amounts. [Brief explanation of the drawings]

[0088] [Figure 1] 1 is a cross-sectional view illustrating a bioreactor according to the present invention. [Figure 2] FIG. 1 is a cross-sectional view illustrating another bioreactor according to the present invention. [Figure 3] FIG. 3 is a perspective view illustrating an agitator for engaging the base of the bioreactor of FIG. 1 or 2. [Figure 4(a)] FIG. 10 is a front view illustrating the agitator plate in a lowered position. [Figure 4(b)] FIG. 10 is a front view illustrating the agitator plate in an elevated position. [Figure 4(c)] FIG. 2 is a front view illustrating the agitator plate in a first angular position. [Figure 4(d)] FIG. 10 is a front view illustrating the agitator plate in a second angular position. [Figure 5] 1 is a flow chart illustrating the "fast mixing" method of transducing cells according to the present invention. [Figure 6] 1 is a flow chart illustrating the "intermittent mixing" method of transducing cells according to the present invention. [Figure 7]1 is a flow chart illustrating the "continuous mixing" method of transducing cells according to the present invention. [Figure 8] FIG. 1 illustrates a line graph of the total number of viable cells in a compressible container, a rigid gas permeable container, and a gas permeable bag according to the present invention over a seven day period. [Figure 9(a)] FIG. 1 is a box plot illustrating the transduction efficiency of a compressible container according to the present invention, a rigid gas-permeable container, a gas-permeable bag, and a rigid container with agitator for CD8+ T cells. [Figure 9(b)] FIG. 1 is a box plot illustrating the transduction efficiency of a compressible container, a rigid gas-permeable container, a gas-permeable bag, and a rigid container with agitator according to the present invention for CD4+ T cells. [Figure 10] 1A-1C illustrate various examples of mixed modes for control of a compressible container according to the present invention and a rigid gas permeable container over days 0 to 3 of the culture process. [Figure 11] 10A-10C illustrate a comparison of cell growth with various mixing modes of a compressible container according to the present invention and with a static mode of control of a rigid gas permeable container. [Figure 12] FIG. 10 illustrates a comparison of the yield of transduced cells with various mixing modes of a compressible container according to the present invention and with a static mode of control of a rigid gas permeable container. [Figure 13] FIG. 10 illustrates a comparison of transduction efficiency with various mixing modes of a compressible container according to the present invention and with a static mode of control of a rigid gas permeable container. DETAILED DESCRIPTION OF THE INVENTION

[0089] The exemplary embodiments described relate to methods of transducing T cells in a compressible container. As will be appreciated by those skilled in the art, references to container, receptacle, bioreactor, or the like are intended to be used synonymously in the detailed description and are not intended to limit the scope of protection. As will be appreciated by those skilled in the art, the containers described herein are merely examples of suitable containers.

[0090] Certain terminology is used in the following description for convenience only and is not limiting. The terms "upper" or "top" and "lower" or "bottom" designate directions in the referenced drawings and relate to the described components when assembled. Similarly, the terms "inner" or "inwardly" and "outer" or "outwardly" refer to directions toward and away from, respectively, a designated centerline or the geometric center (e.g., central axis) of the described element, with the particular meaning being readily apparent from the context herein. Additionally, the terms "proximal" (i.e., closer) and "distal" (i.e., farther) designate location relative to an axis or point of attachment.

[0091] Furthermore, as used herein, the terms "connected," "attached," "coupled," and similar expressions are intended to include a direct connection between two members with no other intervening members, as well as an indirect connection between members where one or more other members are interposed between them. The term specifically includes the words set forth above, derivatives thereof, and words of similar import.

[0092] Furthermore, unless otherwise specified, the use of ordinal numbers such as "first," "second," "third," etc., merely indicates that similar objects or different instances of words are being referred to and is not intended to imply that the objects so described must be in a given order, either temporally, spatially, manner-wise, in rank, or in any other manner. Like reference numerals are used throughout to denote like features.

[0093] The bioreactor 1 shown in FIG. 1 comprises a cell culture vessel 2 and an interface plate 3. During use, the cell culture vessel 2 holds a fluid in which cell processing takes place. Specifically, the fluid is a cell suspension 4 containing a population of cells, e.g., T cells, present in a cell processing medium. Additionally, a headspace exists within the cell culture vessel 2 above the upper surface of the cell processing medium. The headspace fills the remaining volume of the bioreactor not otherwise occupied by the cell processing medium. The cell suspension may also contain a transduction agent. The cells are transduced within the cell culture vessel by the introduced transduction agent to produce genetically modified cells. The cells may also be cultured, reproduced, or otherwise processed within the cell culture vessel 2 to produce a cell-based therapeutic product.

[0094] The interface plate 3 is attached to the top of the cell culture vessel 2, thereby serving as a lid or closure. The interface plate 3 includes at least one connector interface 5 for connecting to an external component, such as an external vessel configured to supply fluid to or remove fluid from the cell culture vessel 2. In some examples, the interface plate 3 includes multiple connector interfaces 5, each connecting to one of multiple external components. Each connector interface 5 can be used one or more times to add or remove liquid. The connector interfaces 5 can be distributed around the interface plate 3. Thus, the interface plate 3 is provided for adding cell treatment media and other fluids to the cell culture vessel 2 during cell processing and / or removing fluids from the cell culture vessel 2 during processing, for example, to remove sample or waste fluids.

[0095] An external container (not shown) may be connectable to the connector interface 5 in the interface plate 3 to add or remove materials from the cell culture vessel 2. The external container may contain substances to be added to the cell culture vessel 2 during cell processing. For example, the external container may contain cell treatment media, transduction agents, activating agents, cytokines, growth factors, magnetic beads, or the like.

[0096] The cell culture vessel 2 is a compressible vessel. Therefore, the cell culture vessel has flexibility, such that it is extensible and compressible. In particular, the cell culture vessel 2 has a compressible wall element 6, e.g., a bellows wall. The cell culture vessel 2 has a base 7 disposed opposite the interface plate 3 and the compressible wall element 6, which defines the sidewall of the cell culture vessel 2. The top of the compressible wall element 6 is attached to the interface plate 3. The top of the compressible wall element 6 may include a rigid ring 8 or the like for attachment to the interface plate 3. The rigid ring 8 is threaded to engage and couple with corresponding threads on the interface plate 3. The compressible wall element 6 is compressible and / or extensible such that the base 7 can move toward and away from the interface plate 3, changing the internal volume of the cell culture vessel 2. The base 7 can be moved relative to the interface plate 3 to stir or mix the cell suspension 4 within the cell culture vessel 2.

[0097] The compressible wall element 6 may be a bellows wall having a concertina-like arrangement that allows the compressible wall element 6 to collapse for compression. In particular, as illustrated, the compressible wall element 6 may include a series of alternatingly arranged deformable portions 9 a, 9 b. Leaf segments 10 extend between, i.e., are interleaved with, the deformable portions 9 a, 9 b. The leaf segments 10 are stiffer than the deformable portions 9 a, 9 b. The deformable portions 9 a, 9 b function as hinges that allow the compressible wall element 6 to collapse like a bellows or concertina, while the leaf segments 10 do not substantially deform.

[0098] The compressible wall element 6 may comprise at least one inwardly deformable portion 9 a and at least one outwardly deformable portion 9 b, for example at least two inwardly deformable portions 9 a and at least two outwardly deformable portions 9 b. The compressible wall element 6 may comprise three, four or more inwardly deformable portions 9 a and three, four or more outwardly deformable portions 9 b.

[0099] The inwardly deformable portion 9a and the outwardly deformable portion 9b may be formed by thinned sections in the compressible wall element 6. The inwardly deformable portion 9a may comprise a thinned section arranged on an outer surface of the compressible wall element 6 so as to be deformable in an inward direction. The outwardly deformable portion 9b may comprise a thinned section arranged on an inner surface of the compressible wall element 6 so as to be deformable in an outward direction.

[0100] The compressible wall element 6 may be formed from a gas-permeable or gas-impermeable material. In examples, the compressible wall element 6 includes silicone, particularly liquid silicone rubber, or fluorinated ethylene propylene (FEP). In other examples, the compressible wall element 6 includes low-density polyethylene (LDPE). In other examples, the compressible wall element 6 includes a thermoplastic elastomer (TPE), such as a gas-permeable TPE. In examples, as described further below, the compressible wall element 6 may be coated, laminated, or otherwise treated to reduce the gas permeability of the compressible wall element 6 or to make the compressible wall element 6 impermeable to gases, particularly oxygen. In some examples, the compressible wall element 6 includes an inner portion and an outer sheath, jacket, or coating. For example, the compressible wall element 6 may include an inner portion and a jacket overmolded on the inner portion. The inner portion may include LDPE, and the jacket may include TPE. In another example, the compressible wall element 6 may comprise an elastomeric outer sheath, e.g., a TPE outer sheath, and a liner. The sheath may be in the form of the vessel and the general shape of the cell culture vessel 2. For example, an LDPE liner may be blow-mounted onto the inner surface of the TPE outer sheath to form the liner. In another example, the liner may be an insert, e.g., an LDPE insert, that is received within the elastomeric outer sheath but is not co-molded with the TPE outer sheath. In such an example, it may be preferable for the liner to include a base sheet and define a closed vessel (excluding the top) that holds the cell suspension 4.

[0101] The cell culture vessel 2 can therefore expand and contract, or be expanded and contracted, according to the material held in the cell culture vessel 2. In particular, the cell culture vessel 2 can expand as the amount of cell suspension 4 in the cell culture vessel 2 increases and / or as additional material is added.

[0102] As illustrated, interface plate 3 also includes expansion vessel 11. Expansion vessel 11 allows cell culture vessel 2 to expand and contract without significantly changing the pressure within cell culture vessel 2. Alternatively or additionally, expansion vessel 11 may be operable to expand or retract compressible wall 6 of cell culture vessel 2, for example, by being mechanically or manually compressed or expanded, thereby changing the volume of cell culture vessel 2. Alternatively or additionally, expansion vessel 11 may be operable to change the pressure within cell culture vessel 2, for example, by being mechanically or manually compressed or expanded.

[0103] In various examples, the cell culture vessel 2 includes a base 7 coupled thereto. The base 7 is generally planar, i.e., flat, and rigid. The base 7 is attached to or co-molded with the compressible wall element 6.

[0104] The base 7 is substantially planar, thereby defining a rigid, substantially flat bottom of the cell culture vessel 2. The flat base 7 of the cell culture vessel 2 may result in improved cell culture, particularly mixing and control of the cell culture. Thus, the base 7 of the cell culture vessel 2 helps ensure that the cells are spread substantially evenly across the cross-section of the cell culture vessel 2 as they settle to the bottom of the cell culture vessel 2. This mitigates cell "piling" at isolated locations on the base 7, which can cause reduced oxygen levels at such isolated locations. The flat base 7 of the cell culture vessel 2 also helps prevent the cell suspension 4 from becoming trapped within the cell culture vessel 2 when the cells are harvested or extracted at the end of the cell culture process.

[0105] In various examples, the base 7 comprises a thermoplastic, such as high density polyethylene (HDPE), polycarbonate (PC), or another rigid polymer.

[0106] Additionally, as shown in Figure 1, an agitator plate 12 is provided for abutting engagement with the base 7. The agitator plate 12 is described in more detail with respect to Figure 3. The agitator plate 12 may, in some instances, be coupled, for example directly coupled, to the base 7.

[0107] In the illustrated example, the cell culture vessel 2 is generally cylindrical, having a generally circular base 7 and a generally cylindrical compressible wall element 6. An axial direction is therefore defined between the base 7 and the end of the compressible wall element 6 to which the interface plate 3 is attached. However, it will be appreciated that the cell culture vessel 2 may take alternative forms, such as having a generally triangular or square cross-sectional shape.

[0108] As shown in FIG. 2 , which shows a cross section of another example of a bioreactor 1 according to the present invention, the bioreactor 1 includes a mixing element, such as a baffle 22. The baffle 22 is attached to an interface plate 3 such that the baffle 22 is suspended within the cell culture vessel 2. The baffle 22 includes a mounting portion 23 that is attachable to the interface plate 3. In the example, the mounting portion 23 can be attached to the interface plate 3 by a threaded connector, or a clip or clamp. In the example shown, the mounting portion 23 is attached to the center of the interface plate 3 such that the baffle 22 is centered within the cell culture vessel 2. However, it will be understood that the baffle 22 can be positioned off-center within the cell culture vessel 2. The mounting portion 23 extends from the interface plate 3 toward the base portion 7, and the baffle member 24 is attached to or integrally formed with the mounting portion 23. In this example, the baffle member 24 includes a substantially flat bottom surface 25 that faces the base 7 of the cell culture vessel 2. The baffle member 24 also has a conical upper surface 26 facing the interface plate 3, although this may alternatively be substantially flat.

[0109] Baffle member 24 is circular and sized to fit within cell culture vessel 2. In an example, baffle member 24 is sized to be spaced apart from compressible wall element 6 of cell culture vessel 2. Baffle member 24 may be sized to be spaced apart from compressible wall element 6 of cell culture vessel 2 by allowing sampling tube 17 to pass between compressible wall element 6 and baffle member 24. Sampling tube 17 provides a fluid sampling path from cell culture vessel 2 to interface plate 3. In an example, baffle member 24 may be spaced apart from compressible wall element 6 by a distance between about 5 millimeters and about 20 millimeters.

[0110] Baffles 22 are provided for mixing the contents of bioreactor 1 during use. In particular, base portion 7 of bioreactor 1 can be moved relative to interface plate 3 and baffles 22 such that baffles 22 contact and mix cell suspension 4 within cell culture vessel 2. In examples, as described in more detail below, base 7 can be raised or lowered relative to interface plate 3 (i.e., changing the distance between base 7 and interface plate 3), and / or base 7 can be tilted relative to interface plate 3, and / or base 7 can be rotated relative to interface plate 3.

[0111] After mixing, the baffle 22 is removed from the cell suspension 4 via expansion of the cell culture vessel 2, and the conical upper surface 26 of the baffle member 24 ensures that the fluid is not retained on the baffle 22 but instead flows back into the cell culture vessel 2.

[0112] As shown in FIG. 3 , the bioreactor 1 is positioned on an agitator 18. The agitator 18 includes an agitator plate 12 that engages the base 7 of the bioreactor 1 to move the base 7 relative to the interface plate 3, thereby agitating the contents of the bioreactor 1. Such agitation mixes the fluids in the bioreactor 1 and aids in the transduction process. Agitation can also aid in the cell culture process, for example, by mixing the fluids within the bioreactor 1 or encouraging oxygen to dissolve in the cell culture medium. The agitator 18 can be mounted within an incubator housing (not shown).

[0113] 3 illustrates an exemplary agitator 18 having an actuator mechanism configured to move the agitator plate 12 relative to the bioreactor 1. As shown, the agitator plate 12 is movable to engage the bioreactor 1, and in particular the base (i.e., base 7, see FIG. 1). The actuator mechanism is mounted on an agitator base plate 28. Between the agitator base plate 28 and the agitator plate 12 are one or more actuators 29 that operate to raise and lower the agitator plate 12.

[0114] In the illustrated example, the actuator 29 is a motor arranged to rotate an articulated crank arm 33 rotatably connected to the agitator base plate 28 and the agitator plate 12, such that rotation of the articulated crank arm 33 moves the agitator plate 12. In other examples, a linear actuator may be provided to act directly between the agitator base plate 28 and the agitator plate 12.

[0115] The supports and guides may guide the movement of the agitator plate 12 .

[0116] The actuator mechanism further comprises a pivotable rod 30, which allows the agitator plate 12 to pivot about the pivotable rod 30 to tilt the base of the bioreactor 1. The actuator 29 allows the agitator plate 12 to pivot about the pivotable rod 30 to tilt the base of the bioreactor 1. Thus, the agitator plate 12 can be moved relative to the agitator base plate 28, thereby engaging the base of the bioreactor 1 and stirring the contents of the bioreactor 1.

[0117] Figures 4(a) through 4(d) illustrate the agitation motion of agitator plate 12, without showing bioreactor 1 or the actuator mechanism. Figures 4(a) through 4(d) show receiver 13, which supports, holds, and clamps interface plate 3 of bioreactor 1 during use, with the cell culture vessel (i.e., cell culture vessel 2, see Figure 1) suspended below receiver 13. Receiver 13 holds interface plate 3 in a horizontal position such that actuation of base 7 by agitator plate 12 moves base 7 relative to interface plate 3.

[0118] As shown in Figures 4(a) and 4(b), the agitator plate 12 is translated between a low position and a high position along the central longitudinal axis of the cell culture vessel 2. The movement of the agitator plate 12 between the low position and the high position agitates the contents within the interior volume of the cell culture vessel 2. The movement between the low position and the high position can be a reciprocating compression motion to cause agitation or turbulence of the contents of the cell culture vessel 2 over a predetermined period of time, as described in further detail below. The compression motion enables mixing of the large volume of cell suspension 4 within the cell culture vessel 2. In some examples, baffles 22 increase mixing of the cell suspension 4 during compression mixing.

[0119] The compression movement may be at a rate of 1 to 60 cycles per minute (cpm), with each cycle being a compression (i.e., translation of the base 7 toward the interface plate 3) and retraction (i.e., translation of the base 7 away from the interface plate 3) of the cell culture vessel 2. In one example, the compression movement may be at a rate of 1 to 30 cpm. In another example, the compression movement may be at a rate of 5 to 25 cpm. In another example, the compression movement may be at a rate of 10 to 20 cpm. In other examples, the compression movement may be at a rate of 5 cpm, 10 cpm, 20 cpm, 30 cpm, 40 cpm, 50 cpm, or 60 cpm.

[0120] As shown in Figures 4(c) and 4(d), the agitator plate 12 is tilted to agitate the contents of the bioreactor 1. In Figure 4(c), the agitator plate 12 is tilted in a first position where the base forms a first angle with respect to the horizontal plane. The agitator plate can be rotated in the opposite direction to a second position, as shown in Figure 4(d), where the base forms a second angle with respect to the horizontal plane. The first and second angles are between 1 and 45 degrees. Preferably, the first and second angles are between 5 and 25 degrees. The first and second angles may be one of 5, 10, 15, 20, and 25 degrees. The second angle may be equal to the first angle. Movement of the agitator plate 12 between the first and second positions causes a rocking motion. This rocking motion gently agitates the cell suspension 4 within the cell culture vessel 2, creating turbulence. The movement of the agitator plate 12 between the first and second positions can have a variable speed. Alternatively, the movement of the agitator plate 12 between the first and second positions can have a constant speed. In examples utilizing a constant speed, the base can be held at each of the first and second angles for a predetermined period of time, such as 0.1 to 5 seconds. The movement between the first and second positions can be a reciprocating motion to provide agitation of the contents of the bioreactor 1 for a predetermined period of time, as described in more detail below.

[0121] The rocking motion may occur at a rate of 1 to 60 rocking motions per minute (rpm), with each rocking motion starting from a first position (e.g., FIG. 4(c)), rotating to a second position (e.g., FIG. 4(d)), and then rotating back to the first position (e.g., FIG. 4(c)) and returning. In one example, the rocking motion may be at a rate of 1 to 30 rpm. In another example, the rocking motion may be at a rate of 5 to 25 rpm. In another example, the rocking motion may be at a rate of 10 to 20 rpm. In other examples, the rocking motion may be at a rate of 5 rpm, 10 rpm, 20 rpm, 30 rpm, 40 rpm, 50 rpm, or 60 rpm.

[0122] A combination of vertical and tilting motion can be provided to agitate the contents of the bioreactor 1. The agitator plate 12 may be tiltable in different directions, and / or the agitator plate 12 may be tiltable in only one or two directions, but rotation of the bioreactor 1 can change the direction of tilt of the bioreactor 1 itself. This results in a pivoting motion about a center point of the base 7 of the bioreactor 1.

[0123] In a further example, not illustrated, the base 7 can be pivoted about an origin disposed at the center of the base. This creates a vortex motion to agitate the contents within the interior volume of the cell culture vessel 2. The base can be pivoted to a first angle with respect to a horizontal plane. The first angle is between 1 and 45 degrees. Preferably, the first angle is between 5 and 25 degrees. The first angle can be one of 5, 10, 15, 20, and 25 degrees.

[0124] The base may be pivoted at a rate of 1 to 60 revolutions per minute, with each revolution being one circular revolution about the origin. In one example, the pivoting motion may be at a rate of 1 to 30 revolutions per minute. In another example, the pivoting motion may be at a rate of 5 to 25 revolutions per minute. In another example, the pivoting motion may be at a rate of 10 to 20 revolutions per minute. In other examples, the pivoting motion may be at a rate of 5 revolutions per minute, 10 revolutions per minute, 20 revolutions per minute, 30 revolutions per minute, 40 revolutions per minute, 50 revolutions per minute, or 60 revolutions per minute.

[0125] In some examples, the agitator plate 12 may be coupled to the base 7 of the bioreactor 1 such that the base 7 moves with the agitator plate 12. In other examples, the agitator plate 12 is not coupled to the base 7, and the base 7 may be lifted completely or partially off the agitator plate 12 at some positions and / or during some agitation movements. Some agitation movements may result in impact contact, i.e., engagement, between the agitator plate 12 and the base 7 to agitate the contents of the bioreactor. In other examples, the agitator plate 12 may be a vibrating agitator plate configured to vibrate the base 7 of the bioreactor 1.

[0126] Next, a method of transducing cells using bioreactor 1 as described above will be described with reference to Figures 5 to 7. Prior to initiating the transduction method, target cells are isolated from a patient or donor sample. Any suitable target cells requiring transduction may be used. In some examples, the target cells may be T cells, such as CD3+, CD4+, or CD8+ T cells. In other examples, the target cells may be CD34+ hematopoietic stem and progenitor cells (HSPCs).

[0127] In each method 100a, 100b, 100c, T cells are added to the interior volume of a cell culture vessel (i.e., cell culture vessel 2, see Figures 1 and 2) in cell treatment medium in steps 110a, 110b, 110c. In other words, incubation of T cells in the cell culture vessel occurs on day 0.

[0128] The cell treatment medium can be selected from any suitable medium. In some examples, the cell treatment medium can be Dulbecco's Modified Eagle Medium (DMEM) available from Thermo Fisher Scientific, Sigma Aldrich, etc., X-VIVO™ 15 available from Lonza, or TexMACS™ available from Miltenyi Biotec. The cell treatment medium can be any cell treatment medium suitable for T cells.

[0129] Optionally, an activating agent may be added to the cell solution 4 in the cell culture vessel (i.e., cell culture vessel 2, see FIGS. 1 and 2). The activating agent may be added to the interior volume of the cell culture vessel at the same time as the T cells (i.e., day 0). The activating agent may be one of magnetic beads or soluble particles. The contents of the cell culture vessel—including the activating agent—may be agitated according to one of the mixing methods outlined below (steps 130a and 140a, steps 130b, 140b and 145b, or steps 130c and 140c). Agitation allows the activating agent to mix with the cell treatment medium.

[0130] In steps 120a, 120b, and 120c, a transduction agent is added to the interior volume of the cell culture vessel (i.e., cell culture vessel 2, see FIGS. 1 and 2). The transduction agent may be a viral vector. Preferably, the viral vector is a lentiviral vector. The transduction agent may be added 6 hours to 2 days after addition of the activating agent. Preferably, the transduction agent is added 1 day (24 hours) after addition of the T cells and activation of the activating agent (i.e., the transduction agent is added on day 1).

[0131] The addition of the transduction agent, i.e., steps 120a, 120b, 120c, may occur before, during, or after any of steps 130a, 130b, or 130c, steps 140a, 140b, or 140c, or step 145b, as described in further detail below.

[0132] The cell suspension 4, which includes cells, cell treatment medium, and transduction agent, can be mixed in the cell culture vessel by moving the base of the cell culture vessel (i.e., base 7, see Figures 1 and 2) using "fast mixing," "intermittent mixing," and / or "continuous mixing" methods. This mixing agitates the cell suspension and stimulates transduction of the cells in the cell suspension.

[0133] The "fast mixing" method is shown in FIG. 5. The base of the cell culture vessel (i.e., base 7, see FIGS. 1 and 2) is moved relative to the top (i.e., interface plate 3, see FIGS. 1 and 2) for a first time period in step 130a. The movement of the base may be caused by an agitator (i.e., agitator 18, see FIG. 3). The movement of the base may be a rocking motion, a compressing motion, a pivoting motion, a rotating motion, or a combination thereof, as outlined above. For example, the agitator may provide a tilting motion to rock the base or a vertical motion to compress the cell culture vessel.

[0134] The first time period may be from 1 to 60 minutes.

[0135] In step 140a, the base ceases movement and is maintained in a stationary position for a second period of time, in which the base is held in a horizontal position.

[0136] The second time period may be from 1 minute to 5 hours. In one example, the second time period may be from 1 to 60 minutes. In another example, the second time period may be from 0.5 to 5 hours. In another example, the second time period may be from 1 to 3 hours. In another example, the second time period is 3 hours.

[0137] The "brief mixing" method is shown in FIG. 6. The base of the cell culture vessel (i.e., base 7, see FIGS. 1 and 2) is moved relative to the top (i.e., interface plate 3, see FIGS. 1 and 2) for a first time period in step 130b. The movement of the base may be caused by an agitator (i.e., agitator 18, see FIG. 3). The movement of the base may be a rocking motion, a compressing motion, a pivoting motion, a rotating motion, or a combination thereof, as outlined above. For example, the agitator may provide a tilting motion to rock the base or a vertical motion to compress the cell culture vessel.

[0138] The first time period may be from 1 minute to 5 hours. For example, the first time period may be from 1 to 60 minutes. In another example, the first time period may be from 0.5 to 5 hours. In another example, the first time period may be from 1 to 3 hours. In another example, the first time period may be 1 minute. In another example, the first time period may be 3 hours.

[0139] In step 140b, the agitator suspends movement of the base and maintains the base in a stationary position for a second period of time, in which the base is held in a horizontal position.

[0140] The second time period may be from 1 minute to 5 hours. For example, the second time period may be from 1 to 60 minutes. In another example, the second time period may be from 0.5 to 5 hours. In another example, the second time period may be from 1 to 3 hours. In another example, the second time period may be 3 hours.

[0141] In one example, the first time period can be 1 minute and the second time period can be 3 hours, or in another example, the first time period can be 3 hours and the second time period can be 1 to 5 hours.

[0142] Steps 130b and 140b described above are repeated for a predetermined time period in step 145b. This results in intermittent mixing of the cell suspension in the cell culture vessel (i.e., cell culture vessel 2, see FIGS. 1 and 2). The predetermined time period may be 1 to 5 days. In an example, the predetermined time period may be 1 to 3 days. In another example, the predetermined time period may be 3 days.

[0143] The "continuous mixing" method is shown in FIG. 7. The base of the cell culture vessel (i.e., base 7, see FIGS. 1 and 2) is moved relative to the top (i.e., interface plate 3, see FIGS. 1 and 2) for a first time period in step 130c. The movement of the base may be caused by an agitator (i.e., agitator 18, see FIG. 3). The movement of the base may be a rocking motion, a compressing motion, a pivoting motion, a rotating motion, or a combination thereof, as outlined above. For example, the agitator may provide a tilting motion to rock the base or a vertical motion to compress the cell culture vessel.

[0144] The first time period may be from 1 to 5 days. In an example, the first time period may be from 1 to 3 days.

[0145] The base stops moving at step 140c.

[0146] Any combination of rapid mixing, intermittent mixing, and continuous mixing can be used during cell transduction. For example, the cell solution can be intermittently mixed by rocking for a first predetermined time period, continuously mixed by rocking for a second predetermined time period, and continuously mixed by compressing for a third predetermined time period. To accommodate increasing cell populations, additional medium can be added to the internal volume between or during each of these mixing steps.

[0147] After transduction, a sample of the cell solution can be taken to measure the transduction efficiency. The sample can be taken 2 to 4 days after adding the T cells to the internal volume of the cell culture vessel 2 (day 0). For example, the sample can be taken 3 days after adding the T cells to the internal volume of the cell culture vessel (i.e., the sample is taken on day 3). The transduction efficiency is measured using green fluorescent protein (GFP) or CD19 markers. In particular, the transduction efficiency of CAR T cells is measured using the CD19 marker.

[0148] In each of methods 100a, 100b, and 100c of Figures 5 through 7, cell treatment medium is added to the interior volume of a cell culture vessel (i.e., cell culture vessel 2; see Figures 1 and 2) in steps 150a, 150b, and 150c. The amount of cell treatment medium added may be equal to the amount of cell treatment medium within the interior volume of the cell culture vessel to double the amount of cell treatment medium within the cell culture vessel. Additional substances, such as growth factors, cytokines, magnetic beads, nutrients, or the like, may also be added to the interior volume of the cell culture vessel.

[0149] The additional cell treatment medium may be added to the cell culture vessel 2 to 4 days after adding the T cells to the interior volume of the cell culture vessel 2 (day 0). For example, the additional cell treatment medium may be added 3 days after adding the T cells to the interior volume of the cell culture vessel (i.e., the additional cell treatment medium is added on day 3).

[0150] In each of methods 100a, 100b, and 100c, the transduced T cells are then cultured within the interior volume of the cell culture vessel in steps 160a, 160b, and 160c. During this step, the base of the cell culture vessel (i.e., base 7, see FIGS. 1 and 2) may be moved relative to the top (i.e., interface plate 3, see FIGS. 1 and 2) to mix the cell solution. The cell solution may be mixed continuously or intermittently.

[0151] The T cells may be cultured for up to 10 days after adding the T cells to the interior volume of the cell culture vessel 2 (day 0). In examples, the T cells are cultured for 5 to 10 days after adding the T cells to the interior volume of the cell culture vessel 2. In one example, the T cells are cultured for about 7 days after adding the T cells to the interior volume of the cell culture vessel 2 (i.e., the T cells are cultured until day 7).

[0152] In this cell culture step, cell treatment medium can be added to the interior volume of the cell culture vessel at predetermined intervals. For example, cell treatment medium can be added every 24 hours. In one example, the amount of cell treatment medium doubles at each predetermined interval. In another example, cell treatment medium can be dynamically added in response to an increase in cell density.

[0153] In this step, the base of the cell culture vessel (i.e., base 7, see FIGS. 1 and 2) can be moved relative to the top (i.e., interface plate 3, see FIGS. 1 and 2) to perform continuous mixing. The movement of the base can be caused by an agitator (i.e., agitator 18, see FIG. 3). The movement of the base can be a rocking motion, a compressing motion, a pivoting motion, a rotating motion, or a combination thereof, as outlined above. In one example, the base is continuously moved with a rocking motion for a first predetermined time period, and then the base is continuously moved with a compressing motion. In an example, each of the first and second predetermined periods is 1 to 4 days, preferably about 2 days.

[0154] After a predetermined time period, the T cells are harvested from the cell culture vessel (i.e., cell culture vessel 2, see Figures 1 and 2). In an example, the predetermined time period is 5 to 10 days after adding the T cells to the interior volume of the cell culture vessel (day 0). In one example, the T cells are harvested about 7 days after adding the T cells to the interior volume of the cell culture vessel (i.e., the T cells are harvested on day 7). Alternatively, the T cells can be harvested after reaching a target density or target cell count. The harvested T cells can be prepared for infusion into a patient or frozen for storage or transport. [Example]

[0155] Transduction and culture of CD3+ T cells with CD4+ markers in a compressible container A study was conducted to evaluate the viability, proliferation, and phenotype of primary CD3+ T cells bearing CD4+ marker cells that were activated, transduced, and expanded for 7 days in a compressible vessel (i.e., cell culture vessel 2, see Figure 1) with dynamic mixing. Known bioreactor systems, including a rigid gas-permeable vessel (G-REX® 100M™, Wilson Wolf), a flexible gas-permeable bag (Xuri W25™, Cytiva & VueLife® "C" Series Bags, Saint-Gobain), and a rigid vessel with agitator (Prodigy®, Miltenyi Biotec), were used as controls. For each control, transduction was performed while the respective bioreactor system remained stationary. The rigid gas-permeable vessel remained stationary throughout the study. The gas-permeable bag remained stationary during transduction and then continuously rocked after the transduction period (i.e., from day 3 onwards, during subsequent cultures). The contents of the agitator-equipped rigid vessel remained stationary (i.e., not agitated) during transduction and then were agitated continuously after the transduction period (from day 3 onwards, i.e., during subsequent cultures).

[0156] CD3+ T cells bearing CD4+ markers were negatively selected from whole blood samples of healthy donors and cultured at 1 × 10 cells per mL in a cell culture vessel (i.e., cell culture vessel 2, see Figure 1). 6 CD3+ T cells were seeded on day 0 at a density of 50 × 10 cells in a cell treatment medium with a volume of 50 mL. The cell treatment medium consisted of X-VIVO™ 15 (Lonza), 5% normal human AB serum (Sigma), and rhIL-2 (100 units per mL) (R&D Systems). The total number of cells seeded was 50 × 10 cells. 6The activator, CTS™ Dynabeads™ (ThermoFisher), was added on day 0 at a bead-to-cell ratio of 3:1.

[0157] 24 hours after the cells were seeded in bioreactor 1, a GFP lentiviral vector (multiplicity of infection (MOI) of 1) (manufactured by Takara Bio) was added to the cell solution (i.e., day 1).

[0158] The cells were cultured for 7 days, and the cell solution was harvested at designated intervals before adding fresh cell treatment medium. The amount of additional cell treatment medium (the components of which are described above) added to the cell solution was generally equal to the amount of cell treatment medium in the cell solution, so that the volume doubled each time cell treatment medium was added to the cell culture vessel. Further details regarding adding cell treatment medium are provided below.

[0159] Day 3: 150 mL of cell treatment medium (the components of which are described above) was added after harvesting.

[0160] Day 5: 200 mL of cell treatment medium (the components of which are described above) was added after harvesting.

[0161] Day 6: 400 mL of cell treatment medium (the components of which are described above) was added after harvesting.

[0162] From day 0 to day 3, base 7 of bioreactor 1 was activated to provide an intermittent rocking motion. The base was rocked five times every three hours at a rate of five revolutions per minute (rpm), with the base maintained in a stationary position between each set of rocking. The rocking motion was a trapezoidal rocking motion.

[0163] From day 3 to day 5, the base of the cell culture vessel (ie, base 7, see Figures 1 and 2) was actuated to provide a continuous rocking motion at a speed of 5 rpm.

[0164] From day 5 to day 7, the base of the cell culture vessel was actuated to perform continuous linear compression motion (expansion and contraction of the cell culture vessel) at a rate of 60 cycles per minute.

[0165] On the seventh day, the test was completed.

[0166] Figure 8 shows the total number of viable cells over the 7-day test period. The total number of viable cells is comparable to the static gas permeable bioreactor vessel control, with higher numbers of viable cells compared to the static gas permeable bag control from days 1 through 3 and the continuously rocked gas permeable bag control from days 4 through 7.

[0167] Figure 9(b) shows a comparison of the transduction efficiency of CD4+ T cells using various devices. In this example, GFP was used as a marker for transduction. As shown in Figure 9(b), 13 to 52% of CD4+ T cells were effectively transduced according to this method. [Example]

[0168] Transduction and culture of CD3+ T cells with CD8+ markers in compressible vessels The same methods and vessels were utilized for transduction and culture of CD3+ T cells bearing the CD8+ marker. The methods and vessels used were identical except that a population of CD3+ T cells bearing the CD8+ marker was utilized and will not be described further.

[0169] As shown in Figure 9(a), 19 to 55% of CD8+ T cells were effectively transduced according to this method.

[0170] Thus, as shown by the results in Figures 9(a) and 9(b), transduction carried out in a bioreactor according to the methods described herein and above had a statistically significant greater transduction efficiency compared to the control system. [Example]

[0171] Transduction and culture of CD3+ T cells with CD4+ markers Studies were conducted to evaluate the viability, proliferation, and transduction efficiency of primary CD3+ T cells bearing the CD4+ marker and CD3+ T cells bearing the CD8+ marker. A compressible vessel with baffles as described herein (see FIG. 2) was compared to a rigid gas-permeable vessel, which served as a control, in three mixing modes (static, mixing mode 1, and mixing mode 2, each defined below). For the control, transduction occurred while the vessel system remained static (i.e., without rocking or any other movement).

[0172] FIG. 10 provides a summary of the mixing modes from day 0 to day 3 for the compressible container and the control, which is described in more detail below.

[0173] compressible container CD3+ T cells bearing the CD4+ marker and CD3+ T cells bearing the CD8+ marker were negatively selected from whole blood samples of healthy donors and cultured at 1.5 × 10 cells per mL in a cell culture vessel (i.e., cell culture vessel 2, see Figure 2). 6 These cells were seeded on day 0 in a cell treatment medium with a volume of 100 mL. The cell treatment medium was supplemented with TexMACS™ (Miltenyi Biotec), 5% normal human AB serum (Sigma-Aldrich), IL-7 (12.5 ng / mL), and IL-1 (100 ng / mL). -1 ) (MiltenyiBiotec), and IL-15 (12.5 ng mL -1 ) (Miltenyi Biotec). The total number of cells seeded was 150 × 10 cells. 6 The activator, TransAct™ (Miltenyi Biotec), was added on day 0 at a TransAct™-media ratio of 1:100.

[0174] 24 hours after the cells were seeded into the compressible container, a GFP lentiviral vector (multiplicity of infection (MOI) of 0.5) (Flash Therapeutics) was added to the cell solution (i.e., day 1).

[0175] The cells were cultured for 8 days, and samples of the cell solution were taken at specified intervals. Fresh cell treatment medium was added. The amount of additional cell treatment medium (whose components are described above) added to the cell solution was generally equal to the amount of cell treatment medium in the cell solution, so that the volume doubled each time cell treatment medium was added to the cell culture vessel. Further details about the addition of cell treatment medium and the process are described below.

[0176] Day 0: Cell seeding and activation.

[0177] Day 1: Transduction reagent was added (green fluorescent protein (GFP) lentiviral vector).

[0178] Day 3: 100 mL of cell treatment medium (the components of which are described above) was added.

[0179] Day 4: 200 mL of cell treatment medium (the components of which are described above) was added.

[0180] Day 5: 400 mL of cell treatment medium (the ingredients of which are described above) was added. A sample was also taken on day 5 before adding the cell treatment medium.

[0181] Day 6: 200 mL of cell treatment medium (the ingredients of which are described above) was added. A sample was also taken on day 6 before adding the cell treatment medium.

[0182] Day 7: Samples were taken. No further cell treatment medium was added.

[0183] Day 8: A final sample was taken, marking the end of the process.

[0184] As described in Figure 10, in the static mode of the compressible vessel, the base 7 of the cell culture vessel 2 (see Figure 2) was arranged to remain stationary in a horizontal position for the duration of the transduction process (i.e., up to day 3), i.e., the base 7 of the cell culture vessel 1 (see Figure 2) was not moved during this time.

[0185] As depicted in FIG. 10, in a first mixing mode ("Mixing Mode 1") of the compressible vessel, the base 7 of the cell culture vessel 2 (see FIG. 2) was actuated to provide a continuous rocking motion from days 0 to 3 (inclusive). The base was rocked continuously at a rate of 10 rocking movements per minute. The rocking motion was a sinusoidal (i.e., smooth or uniform) rocking motion. The base 7 was angled 15 degrees relative to the horizontal during rocking.

[0186] As illustrated in FIG. 10, in a second mixing mode ("mixing mode 2") of the compressible vessel, the base 7 of the cell culture vessel 2 (see FIG. 2) was actuated to provide a continuous rocking motion on days 1 through 3 (inclusive), i.e., after adding the transduction agent, but not before the addition. The base was rocked continuously at a rate of 10 rocking movements per minute. The rocking motion was a sinusoidal (i.e., smooth or uniform) rocking motion. The base 7 was angled 15 degrees relative to the horizontal during rocking.

[0187] On day 4, an additional continuous rocking motion was incorporated for 24 hours in each of static mode, mixed mode 1, and mixed mode 2. The base was rocked at a rate of 30 rocking motions per minute. The rocking motion was sinusoidal (i.e., smooth or constant speed) rocking motion. The base 7 was angled 15 degrees relative to the horizontal during rocking.

[0188] In each of the compressible vessel static mode, mixing mode 1, and mixing mode 2, days 5 through 8 (not shown in Figure 10) incorporated an additional compression mixing regime, as outlined below.

[0189] From day 5 to day 8, the base 7 of the cell culture vessel 2 (see Figure 2) was actuated to provide continuous linear compression motion (expansion and contraction of the cell culture vessel) at a rate of 22 cycles per minute, with each expansion (and subsequent contraction) having a stroke length of 20 mm.

[0190] On day 8, after a final collection of cell samples from the cell culture vessels, the study was terminated.

[0191] Rigid gas permeable control device For the purpose of the control device, a cell culture vessel was utilized having a rigid, gas-impermeable cylindrical wall rising from a gas-permeable base. The gas-permeable base is supported by supports such as feet that allow passive diffusion of gas through the gas-permeable base into the volume of the cell culture vessel. The cell culture vessel was also provided with an access port and a cap coupled to the access port that was removable to allow materials to be added and removed from the vessel. Such a vessel was provided with a 10 cm 2 The container also has a volume of 100 mL.

[0192] CD3+ T cells bearing the CD4+ marker and CD3+ T cells bearing the CD8+ marker were negatively selected from whole blood samples of healthy donors and plated at 1.5 x 10 cells per mL in a rigid gas-permeable container (i.e., a container with a rigid wall and a gas-permeable base as described above). 6 These cells were seeded on day 0 in a cell treatment medium with a volume of 10 mL. The cell treatment medium was supplemented with TexMACS™ (Miltenyi Biotec), 5% normal human AB serum (Sigma-Aldrich), IL-7 (12.5 ng / mL), and IL-1 (100 ng / mL). -1 ) (MiltenyiBiotec), and IL-15 (12.5 ng mL -1 ) (Miltenyi Biotec). The total number of cells seeded was 15 × 10 cells. 6The activator, TransAct™ (Miltenyi Biotec), was added on day 0 at a TransAct™-media ratio of 1:100.

[0193] Twenty-four hours after the cells were seeded into rigid gas-permeable containers, a GFP lentiviral vector (multiplicity of infection (MOI) of 0.5) (Flash Therapeutics) was added to the cell solution (i.e., day 1).

[0194] The cells were cultured for 8 days and samples of the cell solution were taken at designated intervals as described below.

[0195] Day 3: 10 mL of cell treatment medium (the components of which are described above) was added.

[0196] Day 4: 20 mL of cell treatment medium (the components of which are described above) was added.

[0197] Day 5: 40 mL of cell treatment medium (the components of which are described above) was added and samples were taken.

[0198] Day 6: 20 mL of cell treatment medium (the components of which are described above) was added and samples were taken.

[0199] Day 7: Samples were collected and frozen.

[0200] Day 8: After a final collection of cell samples from the device, the study was terminated.

[0201] The rigid gas-permeable container remained stationary (i.e., was not moved) for the entire duration of the experiment (i.e., on each of days 0 through 8), as shown in FIG. 10.

[0202] Comparison of compressible and rigid gas-permeable containers Each experiment was performed four times (N=4 for each of static mode, mixed mode 1, mixed mode 2, and control) on two donors (designated RD037 and RD007 in Figures 12 and 13 described below), and the results were compared as outlined in Figures 11, 12, and 13, which are further described below.

[0203] Figure 11 shows the total number of viable cells over the duration of the test comparing static mode, mixed mode 1, and mixed mode 2 in the compressible vessel with that of the rigid gas permeable vessel, which served as a control. The total viable cells in the compressible vessel were 10 9 cells, whereas the total viable cells in the rigid gas-permeable container are 10 8 This is expressed as cells per mL, since the starting volume between such vessels differs by an order of magnitude due to the volume of the vessel (1000 mL for compressible vessels vs. 100 mL for rigid gas-permeable vessels). However, as outlined above, the same seeding density (1.5 x 10 cells per mL) 6 ) were provided in each container.

[0204] As shown in Figure 11, the total number of viable cells in the compressible vessel exceeds the control of the rigid gas permeable bioreactor vessel under each of the mixed conditions (static mode, mixed mode 1, mixed mode 2). Also shown, the total number of viable cells is increased by providing mixed conditions (mixed mode 1, mixed mode 2) in the compressible vessel compared to static growth conditions (static mode).

[0205] Figure 12 shows a comparison of transduced cell yields from days 6 to 8. Notably, statistically significantly more GFP+ cells (i.e., cells that had taken up the GFP lentiviral vector) were obtained in the compressible bioreactor on days 7 and 8 when utilizing mixed mode 1 and mixed mode 2 compared to the rigid gas-permeable vessel control (P value < 0.0001). Note that mixed mode 1 and mixed mode 2 also outperformed the control on day 6. In addition, as shown in Figure 11, static mode in the compressible vessel also outperformed the control on days 7 and 8.

[0206] Figure 12 also illustrates that end-to-end process time can be reduced by as much as 2 days. In particular, utilizing mixed mode 1 or mixed mode 2 in the compressible vessel disclosed herein achieves transduced cell yields in day 6 compared to those achieved in day 8 in a rigid gas permeable bioreactor.

[0207] Figure 13 shows a comparison of transduction efficiency between the compressible vessel and the rigid gas permeable vessel control, and also compares various mixing profiles in the compressible vessel. As can be seen, the compressible vessel is comparable to the rigid gas permeable bioreactor when utilized in static mode. As can also be seen, the compressible vessel demonstrates a statistically significant improvement in transduction efficiency when utilized in mixing mode 1 or mixing mode 2 compared to the rigid gas permeable vessel control and static mode of transduction in the compressible vessel. Transduction efficiency for each experiment was assessed on each of days 6 through 8 (inclusive) and was evaluated using a flow cytometer (specifically, a BD Biosciences FACSLyric™ device).

[0208] Thus, the provision and use of compressible vessels has been shown to increase transduction efficiency, shorten end-to-end cell processing times, and increase total viable cell numbers.

[0209] Throughout the description and claims of this specification, the words "comprise," "comprise," and their conjugations mean "including but not limited to," and are not intended to (and do not) exclude other elements, integers, or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used (in the English original), the specification should be understood to contemplate the plural as well as the singular unless the context otherwise requires.

[0210] It should be understood that features, integers, properties, or groups described with respect to a particular aspect, embodiment, or example of the invention are applicable to other aspects, embodiments, or examples described herein, unless incompatible. All features disclosed herein (including the accompanying claims, abstract, and drawings) and / or all steps of the methods or processes so disclosed may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention extends to any novel feature or novel combination of features disclosed herein (including the accompanying claims, abstract, and drawings) or to any novel or novel combination of steps of the methods or processes so disclosed. [Explanation of symbols]

[0211] 1. Bioreactor 2 Cell culture vessel 3 Interface Plate 4. Cell Suspension 5 Connector Interface 6 Compressible Wall Elements 7 base 8 Hard Ring 9a, 9b A series of alternatingly arranged deformable sections 10 leaf segments 11 Expansion vessel 12 Stirrer plate 13 Receiving part 17 Sampling tube 18 Mixer 22 Baffle 23 Mounting part 24 Baffle member 25 Substantially flat bottom surface 26 Conical upper surface 28 Stirrer base plate 29 Actuators 30 Pivotable Rod 33 Articulated crank arm 100a, 100b, 100c method

Claims

1. 1. A method of transducing T cells, comprising: providing a compressible container including a base, a top configured substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an interior volume of the compressible container; adding a population of T cells in cell treatment medium into said internal volume; adding a transduction agent into said internal volume; moving the base relative to the top, thereby causing turbulence of contents within the interior volume for a first period of time; A method comprising:

2. The method of claim 1 , further comprising moving the base relative to the top, thereby causing turbulence of contents within the interior volume for a second period of time.

3. 3. The method of claim 2, wherein moving the base relative to the top for the first period of time and / or moving the base relative to the top for the second period of time comprises rotating the base about an axis of rotation extending in a horizontal plane defined by the base.

4. 3. The method of claim 1, wherein the base is moved relative to the apex at a first speed for the first period of time, and the base is moved relative to the apex at a second speed that is faster than the first speed for the second period of time.

5. 5. The method of claim 2, further comprising moving the base relative to the top, thereby causing turbulence of contents within the interior volume for a third period of time.

6. 6. The method of claim 5, wherein moving the base relative to the top for the third period of time comprises translating the base toward the top along a central longitudinal axis of the compressible container, thereby compressing the compressible container.

7. The method of claim 1 , further comprising maintaining the base stationary relative to the top for a second period of time.

8. 8. The method of claim 7, wherein the second period of time is from 1 to 5 hours, preferably about 3 hours.

9. 9. The method of claim 7 or 8, further comprising moving the base relative to the top, thereby causing turbulence of contents within the interior volume for a third period of time.

10. 10. The method of claim 9, wherein the third time period is from 1 to 60 minutes.

11. 11. The method of claim 7, wherein either or both of the steps of moving the base relative to the top includes rotating the base about an axis of rotation extending in a horizontal plane defined by the base.

12. The step of rotating the base includes: rotating the base in a first direction about the axis of rotation to a first position where the base is at a first angle with respect to the horizontal plane; rotating the base in a second direction opposite the first direction about the axis of rotation to a second position where the base forms a second angle with respect to the horizontal plane; 12. The method of claim 3 or 11, comprising:

13. The method of claim 12 , wherein the base is rotated at a uniform speed between the first position and the second position.

14. 13. The method of claim 12, wherein the base is rotated between the first position and the second position at a variable speed.

15. 15. The method of any one of claims 12 to 14, wherein the base is maintained in the first position and / or the second position for a predetermined period of time.

16. 16. The method of any one of claims 12 to 15, wherein the base is rotated between the first position and the second position at a speed of 1 to 30 revolutions per minute, preferably 5 to 25 revolutions per minute, more preferably 10 to 20 revolutions per minute.

17. 17. The method according to any one of claims 12 to 16, wherein the first angle and / or the second angle is between 1 and 45 degrees, preferably between 5 and 25 degrees.

18. 18. The method of claim 1, wherein either or both of the steps of moving the base relative to the top comprises translating the base toward the top along a central longitudinal axis of the compressible container, thereby compressing the compressible container.

19. 20. The method of claim 6 or 18, wherein the compressible container is compressed at a rate of 1 to 60 compressions per minute, preferably 20 to 40 compressions per minute.

20. 20. The method of claim 1, wherein either or both of the steps of moving the base relative to the top includes pivoting the base about an origin disposed at a center of the base.

21. 21. The method of claim 20, wherein the base is pivoted at a speed of 1 to 30 revolutions per minute, preferably 5 to 25 revolutions per minute, more preferably 10 to 20 revolutions per minute.

22. 22. The method of any one of claims 1 to 21, wherein the transduction agent is added to the interior volume in the step of moving the base relative to the top for the first period of time.

23. 22. The method of any one of claims 1 to 21, further comprising the step of maintaining the base stationary relative to the apex after adding the transduction agent for a predetermined period of time.

24. 24. The method of any one of claims 9 to 23, wherein the transduction agent is added to the interior volume in the step of moving the base relative to the top for the third time period.

25. 25. The method of any one of claims 1 to 24, wherein the transduction agent is a viral vector, such as a lentiviral vector.

26. 26. The method of any one of claims 1 to 25, wherein the flexible wall element comprises a wall having one or more folds, such as a Z-fold.

27. 27. The method of any one of claims 1 to 26, wherein the compressible container is substantially gas impermeable.

28. 27. The method of any one of claims 1 to 26, wherein the compressible container is at least partially gas permeable.

29. 29. The method of claim 28, wherein the base and / or the flexible wall element comprises a gas-permeable material, such as silicone or fluorinated ethylene propylene.

30. 30. The method of any one of claims 1 to 29, wherein the T cells are selected from CD3+ T cells with a CD4+ marker and / or CD3+ T cells with a CD8+ marker.

31. 1. A method of transducing T cells, comprising: i) providing a compressible container including a base, a top configured substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an interior volume of the compressible container; ii) adding a population of T cells in cell treatment medium into said internal volume; iii) continuously moving the base relative to the top, thereby causing turbulent flow of contents within the interior volume at a first rate for a first period of time; iv) optionally adding additional cell treatment medium into said internal volume; v) continuously moving the base relative to the top, thereby causing turbulent flow of contents within the interior volume at a second velocity greater than the first velocity for a second period of time; vi) optionally adding additional cell treatment medium into said internal volume; vi) continuously moving the base relative to the top, thereby causing turbulence of contents within the interior volume for a third time period; in this order, The method further comprises the step of adding a transduction agent into the internal volume during step ii) or step iii).

32. step iii) includes continuously rotating the base at the first speed about an axis of rotation extending in a horizontal plane defined by the base; step v) includes continuously rotating the base at the second speed about an axis of rotation extending in a horizontal plane defined by the base; 32. The method of claim 31 , wherein step vi) comprises continuously translating the base toward the top along a central longitudinal axis of the compressible container, thereby compressing the compressible container.

33. 1. A method of transducing T cells, comprising: i) providing a compressible container including a base, a top configured substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an interior volume of the compressible container; ii) adding a population of T cells in cell treatment medium into said internal volume; iii) optionally, maintaining said base stationary relative to said apex after adding a transduction agent for a predetermined period of time; iv) moving the base relative to the top, thereby causing turbulence of contents within the interior volume for a first period of time; v) maintaining the base stationary relative to the top for a second period of time; vi) repeating steps iv) and v) for a predetermined period of time; vii) adding a transduction agent into said internal volume in any of steps ii) to vi); A method comprising, in this order:

34. 1. A method for transducing and culturing T cells, comprising: i) providing a compressible container including a base, a top configured substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an interior volume of the compressible container; ii) adding a population of T cells in cell treatment medium into said internal volume; iii) optionally, maintaining said base stationary relative to said apex after adding a transduction agent for a predetermined period of time; iv) intermittently moving the base relative to the top, thereby intermittently inducing turbulence of contents within the interior volume for a first period of time; v) adding a transduction agent into the interior volume during the step of adding a population of T cells in the cell treatment medium into the interior volume or during the step of intermittently moving the base with respect to the top to allow transduction of the population of T cells; vi) adding an additional amount of cell treatment medium into the internal volume; vii) culturing said population of T cells; A method comprising, in this order:

35. 1. A method for transducing and culturing T cells, comprising: i) providing a compressible container including a base, a top configured substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an interior volume of the compressible container; ii) adding a population of T cells in cell treatment medium into said internal volume; iii) optionally, maintaining said base stationary relative to said apex after adding a transduction agent for a predetermined period of time; iv) rotating the base about an axis of rotation extending in a horizontal plane defined by the base, thereby causing turbulence of contents within the interior volume for a first period of time; v) maintaining the base stationary relative to the top for a second period of time; vi) repeating steps iv) and v) for a predetermined period of time; vii) optionally adding additional cell treatment medium into said internal volume; viii) translating the base relative to the top along a central longitudinal axis of the compressible container, thereby compressing the compressible container for a third time period; ix) adding a transduction agent into said internal volume in any of steps ii) to viii); A method comprising, in this order:

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