Transduction method

Abstract

A method of transducing T cells includes providing a compressible container including a base, a top disposed 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 a cell treatment medium is added to the internal volume. A transduction agent is added to the interior volume. The base of the compressible container moves relative to the top, causing turbulence of the contents in the interior volume during a first period of time.

Description

TECHNICAL FIELD

[0001] The present invention relates to methods of transducing T cells within a compressible container. More particularly, the present invention relates to methods of transducing T cells, such as CD3+ T cells having a CD4+ marker and / or CD3+ T cells having a CD8+ marker, within a compressible container. The transduction methods discussed herein can form part of a cell and / or gene therapy manufacturing process. BACKGROUND

[0002] Manufacturing processes for cell and gene therapies are often complex, involving several manual or semi-automated steps of devices. Cells are“living” entities, which are sensitive to even the simplest manipulations, such as differences in cell transfer procedures. The role of cell manufacturing equipment in ensuring scalability and repeatability is an important factor in cell and gene therapy manufacturing.

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

[0004] Cell transduction is the process of introducing genetic material into a cell through the use of a transduction agent. This process confers specificity to a T cell for a target antigen. Transduction can also confer other advantageous features to a T cell, such as improved proliferation, cytokine production, activation signaling, and effector function features of the T cell. CAR T cells are commonly used in cell therapy in cancer treatment. CAR T cells are produced by transducing a T cell, thereby genetically altering the T cell to produce a CAR that binds to a cancer cell.

[0005] A typical method of transducing T cells involves first isolating T cells from a patient. The T cells and a transduction agent are then added to a culture vessel, such as a flask or a bag. As the transduction agent interacts with the T cells, genetic material is transferred to the T cells to transduce the T cells. This process can be facilitated by mixing the T cells and the transduction agent mixture, such as by hand or by impeller agitation of the mixture. Alternatively, the T cells and the transduction agent mixture can be provided in a rigid culture vessel, and can be mixed manually or by placing the culture vessel on a shaking plate. Typically, the transduction of the cells is performed in a minimal amount of processing media to facilitate the interaction between the transduction agent and the cells contained therein. The transduced CAR T cells are then infused into a patient, where they will target an antigen present in a cancer cell.

[0006] The culturing or further processing of transduced cells often requires the addition of more media and nutrients to the processing media to maintain the desired level of cell growth. However, the devices used for transduction, including shake flasks, roller bottles, T-flasks, and bags, have limited, and often minimal, working volumes. Therefore, it is often necessary to transfer the transduced cells to a vessel having a larger working volume.

[0007] In the production of cell or gene therapies for medical use, a key limiting factor is the lack of compact, automated, closed systems to perform contamination-free unit operations. The operating systems are largely manual and therefore expensive to operate. Multiple pieces of equipment, in particular vessels, are often required to cover all non-cell culture steps, which involves many transfers, each of which is an opportunity for operator error and contamination. Furthermore, as the number of manual operations increases, so does the risk of human error, and therefore the current labor-intensive processes lack the robustness required to manufacture clinical grade therapeutic drugs.

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

[0009] There is also a need for transduction methods, and associated instruments and devices, in which the transfer of cells between various vessels and / or equipment is mitigated or eliminated altogether.

[0010] There is also a need for automation or semi-automation of transduction methods and other cell processing steps. SUMMARY

[0011] According to a first aspect of the application, there is provided a method of transducing T cells, comprising:

[0012] providing a compressible container comprising a base, a top arranged 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;

[0013] adding a population of T cells in a cell processing medium to the interior volume;

[0014] adding a transduction agent to the interior volume; and

[0015] moving the base relative to the top, thereby causing turbulence of the contents in the interior volume for a period of time, such as a first period of time.

[0016] The movement of the base of the compressible container induces turbulence within its internal volume. This turbulence suspends and disperses T cells in the cell treatment medium, increasing contact between the transduction agent and the T cells in solution, thereby improving transduction efficiency. Furthermore, the compressible container configuration allows for a variable working volume within a single container, reducing the need for additional containers and the associated transfers to them. Additionally, the compressible container enables fluid manipulation, thus facilitating further processing steps.

[0017] According to a second aspect of the present invention, a method for transducing T cells is provided, comprising the following sequential steps:

[0018] i) Provide a compressible container, the compressible container including a base, a top arranged substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an internal volume of the compressible container;

[0019] ii) Add the T cell population from the cell treatment medium to the internal volume;

[0020] iii) The base is continuously moved relative to the top, thereby causing turbulence in the contents of the internal volume at a first rate during a first time period;

[0021] iv) Optionally, other cell treatment media may be added to the internal volume;

[0022] v) The base is continuously moved relative to the top, thereby causing turbulence in the contents of the internal volume at a second rate during a second time period, the second rate being greater than the first rate;

[0023] vi) Optionally, other cell treatment media may be added to the internal volume;

[0024] vi) The base is continuously moved relative to the top, thereby causing turbulence in the contents of the internal volume during the third time period.

[0025] The method further includes the step of adding a transducer to the internal volume during step ii) or step iii).

[0026] Advantageously, this provides for continuous mixing of the contents of the compressible container. Continuous mixing of the compressible container allows T cells to remain continuously suspended in the cell treatment medium, thereby promoting efficient T cell growth, and also enables the dispersion of T cells and transduction agents during the mixing phase to increase the interaction between T cells and transduction agents for efficient T cell transduction. In this way, the desired cell growth and transduction efficiency can be achieved.

[0027] In this example, the method is further defined, wherein:

[0028] Step iii) includes continuously rotating the base at the first rate about a rotation axis extending in a horizontal plane defined by the base;

[0029] Step v) includes continuously rotating the base at the second rate about a rotation axis extending within a horizontal plane defined by the base; and

[0030] Step vi) includes continuously translating the base along the central longitudinal axis of the compressible container toward the top, thereby compressing the compressible container.

[0031] In this example, the method is provided as a method for transducing and culturing T cells.

[0032] According to a third aspect of the present invention, a method for transducing T cells is provided, comprising the following sequential steps:

[0033] i) Provide a compressible container, the compressible container including a base, a top arranged substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an internal volume of the compressible container;

[0034] ii) Add the T cell population from the cell treatment medium to the internal volume;

[0035] iii) Optionally, after the addition of the transconductant, the base is kept stationary relative to the top for a predetermined time period;

[0036] iv) Moving the base relative to the top, thereby causing turbulence in the contents of the internal volume during a first time period;

[0037] v) Keep the base stationary relative to the top during the second time period;

[0038] vi) Repeat steps iv) and v) within the predetermined time period; and

[0039] vii) During any of steps ii) to vi), a transductant is added to the internal volume.

[0040] Advantageously, this provides intermittent mixing of the contents of the compressible container. Intermittent mixing of the compressible container allows T cells to remain at the base during the resting phase, thereby promoting efficient T cell growth, and also allows for the resuspension and dispersion of T cells and the transduction agent during the mixing phase, increasing the interaction between T cells and the transduction agent for efficient T cell transduction. In this way, an appropriate balance between cell growth and transduction efficiency can be achieved.

[0041] According to a fourth aspect of the present invention, a method for transducing and culturing T cells is provided, comprising the following sequential steps:

[0042] i) Provide a compressible container, the compressible container including a base, a top arranged substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an internal volume of the compressible container;

[0043] ii) Add the T cell population from the cell treatment medium to the internal volume;

[0044] iii) Optionally, after the addition of the transconductant, the base is kept stationary relative to the top for a predetermined time period;

[0045] iv) The base is moved intermittently relative to the top, thereby intermittently causing turbulence in the contents of the internal volume during a first time period;

[0046] v) During the step of adding the T cell population from the cell treatment medium to the internal field of view, or during the step of intermittently moving the base relative to the top to enable transduction of the T cell population, a transduction agent is added to the internal volume;

[0047] vi) Add an additional volume of cell treatment culture medium to the internal volume; and

[0048] vii) Culture the transduced T cell population.

[0049] Advantageously, during T cell transduction, intermittent mixing of the contents of the compressible container allows cells to remain at the base during rest, promoting efficient T cell growth, and also enables resuspension and dispersion of T cells and transducers during mixing to increase the interaction between T cells and transducers for efficient T cell transduction.

[0050] In one example, the step of culturing the transduced T cell population includes continuously moving the base relative to the top, thereby continuously causing turbulence in the contents of the internal volume during a second time period.

[0051] Advantageously, during cell culture, the continuous mixing of T cells creates turbulence in the cell solution consisting of the T cell population in the cell treatment medium, which promotes the mixing of oxygen in the cell solution and the oxygen contained in the top space of the compressible container with the cell solution and permeates into the entire cell solution, thereby increasing the amount of dissolved oxygen in the cell solution.

[0052] According to a fifth aspect of the present invention, a method for transducing and culturing T cells is provided, comprising the following sequential steps:

[0053] i) Provide a compressible container, the compressible container including a base, a top arranged substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an internal volume of the compressible container;

[0054] ii) Add the T cell population from the cell treatment medium to the internal volume;

[0055] iii) Optionally, after the addition of the transconductant, the base is kept stationary relative to the top for a predetermined time period;

[0056] iv) Rotate the base about a rotation axis extending within a horizontal plane defined by the base, thereby causing turbulence in the contents of the internal volume during a first time period;

[0057] v) Keep the base stationary relative to the top during the second time period;

[0058] vi) Repeat steps iv) and v) within the predetermined time period;

[0059] vii) Optionally, additional cell treatment culture medium is added to the internal volume;

[0060] viii) The base is translated relative to the top along the central longitudinal axis of the compressible container, thereby compressing the compressible container in the third time period; and

[0061] ix) During any of steps ii) to viii), the transductant is added to the internal volume.

[0062] Advantageously, the base is rotated in a first time period, kept stationary in a second time period, and these steps are repeated to enable efficient mixing at a lower volume where transduction typically occurs, while the base is translated in a third time period to enable efficient mixing at a higher volume, followed by the addition of other cell treatment media required to maintain the desired growth profile of the cell culture.

[0063] As described herein, the following examples are used to describe any aspect of the invention (such as the first aspect, second aspect, third aspect, fourth aspect, or fifth aspect).

[0064] In an example, the method may further include moving the base relative to the top, thereby causing turbulence in the contents of the internal volume during a second time period. That is, the base may be moved during the second time period, rather than remaining stationary during the second time period.

[0065] In the example, the first time period can be 96 hours, 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 3 hours, or 1 hour.

[0066] In this example, the second time period can be 96 hours, 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 3 hours, or 1 hour. The duration of the step of moving the base relative to the top during the first time period can be the same as or different from the duration of the step of moving the base relative to the top during the second time period.

[0067] In the example, the first time period is 72 hours or 96 hours, and the second time period is 24 hours.

[0068] In one example, the step of moving the base relative to the top during the first time period may include moving the base at a first rate (as described below, for example, rotating it), and the step of moving the base relative to the top during the second time period may include moving the base at a second rate (as described below, for example, rotating it). The first rate may be the same as or different from the second rate.

[0069] In this example, the second rate is greater than the first rate. This may be particularly advantageous when a first volume of cell treatment medium is provided within the internal volume during the step of moving the base at the first rate in a first time period, and a second volume of cell treatment medium, larger than the first volume, is provided within the internal volume during the step of moving the base at the second rate in a second time period. In particular, proper mixing is achieved in two different volumes of cell treatment medium without having to remove the contents from the internal volume.

[0070] In an example, the method may further include moving the base relative to the top, thereby causing turbulence in the contents of the internal volume during a third time period.

[0071] In such instances, the third time period can be 96 hours, 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 3 hours, or 1 hour.

[0072] In the example, the first time period is 72 or 96 hours, the second time period is 24 hours, and the third time period is 72 or 96 hours.

[0073] In an example, the step of moving the base relative to the top during the first time period may include moving the base at a first rate (as described below, for example, rotation), the step of moving the base relative to the top during the second time period may include moving the base at a second rate (as described below, for example, rotation), and the step of moving the base during the third time period may include moving the base at a third rate (as described below, for example, compression or longitudinal translation). The first, second, and third rates may be the same or different.

[0074] In an example, the step of moving the base relative to the top during the first time period may include rotating the base at a first rate (as discussed further below), the step of moving the base relative to the top during the second time period may include rotating the base at a second rate (as discussed further below), and the step of moving the base during the third time period may include longitudinally translating the base toward the top at a third rate (as discussed further below). The first, second, and third rates may be further defined below.

[0075] In some instances, the first time period can be from 1 minute to 5 days. The first time period can be from 1 to 60 minutes. The first time period can be from 0.5 hours (i.e., 30 minutes) to 5 hours. The first time period can be 0.5 hours (i.e., 30 minutes). The first time period can be 1 to 3 hours. The first time period can be 1 minute. The first time period can be 3 hours. The first time period can be 1 to 5 days. The first time period can be 1 to 4 days. The first time period can be 1 to 3 days.

[0076] In an example, the method may further include keeping the base stationary relative to the top during a second time period.

[0077] In the examples, the second time period can be from 1 minute to 5 hours. The second time period can be from 1 to 60 minutes. The second time period can be from 0.5 to 5 hours. The second time period can be from 1 to 3 hours. The second time period can be 3 hours.

[0078] In some instances, the steps of moving the base relative to the top in a first time period and keeping the base stationary relative to the top in a second time period can be repeated. In some instances, these steps can be repeated multiple times (i.e., twice or more). In some instances, these steps are repeated consecutively over 96 hours, 72 hours, 48 ​​hours, or 24 hours (e.g., successive periods of movement and stillness).

[0079] In an example, the method may further include moving the base relative to the top, thereby causing turbulence in the contents of the internal volume during a third time period. Advantageously, this provides intermittent mixing of the contents of the compressible container.

[0080] In some examples, the third time period can be from 1 minute to 5 hours. The third time period can be from 1 to 60 minutes. The third time period can be from 0.5 hours (30 minutes) to 5 hours. The third time period can be 0.5 hours (30 minutes). The third time period can be 1 to 3 hours. The third time period can be 1 minute. The third time period can be 3 hours. The third time period can be 1 to 5 days. The third time period can be 1 to 4 days. The third time period can be 1 to 3 days. In some examples, the third time period is 4 days.

[0081] In one example, the step of moving the base relative to the top during a first time period may include rotating the base about a rotation axis extending within a horizontal plane defined by the base.

[0082] In one example, the step of moving the base relative to the top during a third time period may include rotating the base about a rotation axis extending within a horizontal plane defined by the base.

[0083] Advantageously, rotating the base around a horizontal axis provides 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 to disperse cells and enhance interactions between cells and transduction agents. It can also generate turbulence to promote mixing of oxygen from the top space of the compressible container or within the cell treatment medium, thereby increasing the amount of dissolved oxygen in the cell solution. This rocking motion may be particularly advantageous for cell treatment media with smaller volumes (e.g., less than or equal to 200 mL, or less than or equal to 150 mL, or less than or equal to 100 mL) present in the mixing container.

[0084] In an example, rotating the base may include rotating the base to a first position in a first direction about a rotation axis, wherein the base forms a first angle relative to a horizontal plane in the first position; and rotating the base to a second position in a second direction opposite to the first direction about the rotation axis, wherein the base forms a second angle relative to a horizontal plane in the second position.

[0085] In this example, the base can rotate at a constant speed between the first position and the second position.

[0086] In this example, the base can rotate between the first position and the second position at varying speeds.

[0087] In an example, the base may remain in the first position and / or the second position for a predetermined period of time.

[0088] In the examples, the base can rotate between the first and second positions at a rate of 1 to 60 revolutions per minute. The base can rotate between the first and second positions at a rate of 1 to 30 revolutions per minute. The base can rotate between the first and second positions at a rate of 5 to 25 revolutions per minute. The base can rotate between the first and second positions at a rate of 10 to 20 revolutions per minute. The base can rotate between the first and second positions at a rate of approximately 5, 10, 20, 30, 40, 50, or 60 revolutions per minute. Such rotation rates can be considered as the first, second, or third rates discussed herein.

[0089] In a specific instance, the base can be oscillating. In this sense, the base can rotate from a first position to a second position and then back to the first position. The base can oscillate at a rate of 1 to 60 oscillations per minute, 1 to 30 oscillations per minute, 5 to 25 oscillations per minute, or 10 to 20 oscillations per minute. Preferably, the base oscillates at a rate of 10 oscillations per minute for 0.5 hours (i.e., 30 minutes). Herein, oscillation is defined as a rotation from a first position to a second position and then back to the first position. Such an oscillation rate can be considered as the first, second, or third rate discussed herein.

[0090] In an example, the first angle and / or the second angle can be from 1 to 45 degrees. The first angle and / or the second angle can be from 5 to 25 degrees. The first angle and / or the second angle can be about 5 degrees, 10 degrees, 15 degrees, 20 degrees, or 25 degrees. In an example, the second angle can be equal to the first angle. Preferably, and most preferably, each of the first angle and the second angle is about 5 degrees, about 10 degrees, or about 15 degrees.

[0091] In one example, moving the base relative to the top during a first time period may include translating the base toward the top along the central longitudinal axis of the compressible container, thereby compressing the compressible container.

[0092] In an example, moving the base relative to the top during a third time period may include translating the base toward the top along the central longitudinal axis of the compressible container, thereby compressing the compressible container.

[0093] Advantageously, the movement of the base along the central longitudinal axis of the compressible container generates a reciprocating compression motion to agitate the contents of the compressible container. This increases the movement of cells and other contents within the compressible container. It also generates turbulence to promote the mixing of oxygen in the headspace of the compressible container with the contents of the container and to increase the amount of dissolved oxygen in the cell solution. This compression motion may be particularly advantageous for cell treatment media with larger volumes (e.g., greater than or equal to 200 mL, or greater than or equal to 150 mL, or greater than or equal to 100 mL) present in the mixing container.

[0094] In examples, the compressible container can be compressed at a rate of 1 to 60 compressions per minute. The compressible container can be compressed at a rate of 1 to 30 compressions per minute. The compressible container can be compressed at a rate of 5 to 25 compressions per minute. The compressible container can be compressed at a rate of 10 to 20 compressions per minute. The compressible container can be compressed at a rate of approximately 5, 10, 20, 30, 40, 50, or 60 compressions per minute. In this document, compression per minute is defined as the base translating along the central longitudinal axis of the compressible container toward the top, from a first position to a second position, and then the base translating away from the top, from the second position back to the first position. Such a compression rate can be considered as the first, second, or third rate discussed herein.

[0095] In an example, moving the base relative to the top during a first time period may include pivoting the base around an origin set at the center of the base.

[0096] In an example, moving the base relative to the top during a third time period may include pivoting the base around an origin set at the center of the base.

[0097] Advantageously, the pivoting of the base around its central origin provides pivoting motion to agitate the contents of the compressible container. This increases the movement of cells and other contents within the cell treatment medium to disperse cells and enhance interactions between cells and transduction agents. It can also generate turbulence to promote mixing of oxygen from the top space of the compressible container or within the cell treatment medium, and increase the amount of dissolved oxygen in the cell solution. This pivoting motion may be particularly advantageous for cell treatment media present in smaller volumes (e.g., less than or equal to 200 mL, or less than or equal to 150 mL, or less than or equal to 100 mL) in the mixing container.

[0098] In examples, the base can be pivoted at a rate of 1 to 60 revolutions per minute. The base can be pivoted at a rate of 1 to 30 revolutions per minute. The base can be pivoted at a rate of 5 to 25 revolutions per minute. The base can be pivoted at a rate of 10 to 20 revolutions per minute. The base can be pivoted at a rate of approximately 5, 10, 20, 30, 40, 50, or 60 revolutions per minute. In this document, revolution is defined as the circular rotation or pivoting of the base around a central origin in a clockwise or counterclockwise direction, beginning at a first position and ending at the same first position. Such a pivoting rate can be considered as the first, second, or third rate discussed herein.

[0099] In an example, the method further includes the step of moving the base relative to the top during an additional predetermined time period, such as a fourth time period. As contemplated herein, this step of moving the base relative to the top during the additional predetermined time period, such as the fourth time period, may include any one or more steps of rotating, rocking, translating (i.e., compressing), or pivoting the base. Any preceding movement steps may form the additional predetermined time period, such as the fourth time period. The additional predetermined time period, such as the fourth time period, may be repeated once or multiple times.

[0100] In an example, the method includes (a) moving the base relative to the top during a first time period, (b) keeping the base stationary relative to the top during a second time period, repeating steps (a) and (b) for a predetermined time period, and (c) moving the base relative to the top during 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 the 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, with 72 hours being most preferred. In such a preferred example, the third time period may be 96 hours, 72 hours, 48 ​​hours, or 24 hours, with 96 hours being most preferred.

[0101] In one example, the transductant may be added to the internal volume during the step of moving the base relative to the top in the first time period.

[0102] In one example, the transductant may be added to the internal volume during the step of moving the base relative to the top in the third time period.

[0103] In this example, the transducer can be a viral vector. The viral vector can be a lentiviral vector. The lentiviral vector can be a CD19 CAR lentiviral vector (LVV). The lentiviral vector can be a green fluorescent protein (GFP) lentiviral vector (LVV).

[0104] In an example, the flexible wall element may include a wall with one or more folds, such as Z-shaped folds. Advantageously, the wall of the compressible container is easily manipulated for the compression and expansion of the compressible container. In an example, the base (and / or top) can be compressed from the top (and / or base) (i.e., configured to translate toward or away from the top (and / or base)).

[0105] In this example, the compressible container may be substantially airtight. Advantageously, the movement of the base relative to the top causes turbulence in the contents of the internal volume of the compressible container, which mixes oxygen in the top space of the compressible container with the cell treatment medium, increasing the level of dissolved oxygen within the cell treatment medium. Thus, sufficient oxidation of the cell treatment medium can be achieved in a gas-permeable (i.e., airtight) container.

[0106] In this example, the compressible container may be at least partially permeable.

[0107] In an example, the base and / or the flexible wall element may include a breathable material, such as silicone or fluorinated ethylene propylene.

[0108] In one example, the compressible container may include mixing elements within the internal volume, such as baffles, static impellers, etc. In other examples, the compressible container has no mixing elements within the internal volume.

[0109] In this example, the T cells may be selected from CD4+ T cells and CD8+ T cells. In this example, the T cells may be CD3+ T cells or CD34+ hematopoietic stem cells / progenitor cells (HSPCs). In this example, the T cells may be CD3+ T cells with a CD4+ marker. In this example, the T cells may be CD3+ T cells with a CD8+ marker. In this example, the T cells may include both CD3+ T cells with a CD4+ marker and CD3+ T cells with a CD8+ marker.

[0110] In one example, the method may further include adding an activator to the internal volume. Adding the activator to the internal volume may be performed simultaneously with adding the T cell population from the cell treatment culture medium to the internal volume.

[0111] In examples, such as with respect to the third aspect, the predetermined time period for repeating steps iv) and v) can be 1 to 5 days. The predetermined time period can be 2 to 3 days. The predetermined time period can be approximately 3 days. These examples are not limited to the third aspect and are equally applicable to other aspects disclosed herein.

[0112] In one example, the step of adding the transduction agent to the internal volume can occur during the step of adding the T cell population from the cell treatment culture medium to the internal volume.

[0113] In an example, the step of adding the transductant to the internal volume can occur during the step of moving the base relative to the top in the first time period.

[0114] In one example, the method further includes the step of keeping the base stationary relative to the top for a predetermined time period after the addition of the transconductant.

[0115] In this example, the scheduled time period can be 1 day (i.e., 24 hours), 12 hours, 6 hours, 3 hours, 2 hours, or 1 hour.

[0116] In an example, the step of adding the transconductant to the internal volume can occur during the step of keeping the base stationary relative to the top in the second time period.

[0117] In an example, the step of adding the transductant to the internal volume may occur during repetition of either the step of moving the base relative to the top in the first time period or the step of keeping the base stationary relative to the top in the second time period.

[0118] In an example, the step of adding the transductant to the internal volume may occur during the step of intermittently moving the base relative to the top in the first time period.

[0119] In examples, such as with respect to the fourth aspect, intermittently moving the base relative to the top during the first time period and continuously moving the base relative to the top during the second time period may include moving the base relative to the top according to a first movement. These examples are not limited to the fourth aspect and are equally applicable to other aspects disclosed herein.

[0120] In an example, the first motion may 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 provides a rocking motion to agitate the contents of the compressible container. This increases the movement of cells and other contents within the compressible container to disperse cells and increase the interaction between cells and transduction agents. This can also generate turbulence to facilitate the mixing of oxygen from the top space of the compressible container or within the cell treatment medium and increase the amount of dissolved oxygen in the cell treatment medium. This rocking motion is suitable for mixing small amounts of cell treatment medium within the internal volume of the compressible container.

[0121] In examples, such as those relating to the third and fourth aspects, the method for transducing and culturing T cells may further include the steps of: viii) adding an additional volume of cell treatment culture medium to the inner volume; and ix) culturing the T cell population while continuously moving the base relative to the top according to a second movement, thereby continuously causing turbulence in the contents of the inner volume during a third time period. These examples are not limited to the fourth aspect and are equally applicable to other aspects disclosed herein.

[0122] In an example, the second movement may include translating the base along the central longitudinal axis of the compressible container toward the top, thereby compressing the compressible container. Advantageously, moving the base along the central longitudinal axis of the compressible container generates a reciprocating compression motion to agitate the contents of the compressible container. This increases the movement of cells and other contents within the compressible container. It also generates turbulence to facilitate the mixing of oxygen from the top space of the compressible container or within the cell treatment medium, and increases the amount of dissolved oxygen in the cell treatment medium. This reciprocating compression motion is suitable for mixing large quantities of cell solution within the internal volume of the compressible container.

[0123] In this example, the step of adding the T cell population from the cell treatment medium to the internal volume includes adding a first volume of the T cell population from the cell treatment medium to the internal volume. The first volume can be 200 mL, 150 mL, 100 mL, 50 mL, 25 mL, or 10 mL. The first volume can be less than or equal to 200 mL, less than or equal to 150 mL, less than or equal to 100 mL, less than or equal to 50 mL, less than or equal to 25 mL, or less than or equal to 10 mL.

[0124] In this example, after the transduction agent is added to the inner volume, a second volume of cell treatment medium is added to the inner volume. The second volume of cell treatment medium may be equal to or greater than the first volume. Subsequent additions of cell treatment medium (e.g., a third volume, a fourth volume, etc.) are also expected and may be equal to or greater than the first or second volume. In some examples, the second volume may be 200 mL, 150 mL, 100 mL, 50 mL, 25 mL, or 10 mL.

[0125] In this example, after the transduction agent is added to the inner volume, one or more additional volumes of cell treatment medium are added to the inner volume. The additional volume of cell treatment medium may be equal to the volume of cell treatment medium present in the inner volume prior to the addition of this additional volume. Attached Figure Description

[0126] Figure 1 A cross-sectional view of the bioreactor according to the present invention is shown.

[0127] Figure 2 A cross-sectional view of another bioreactor according to the present invention is shown.

[0128] Figure 3 A diagram showing the connection method is provided. Figure 1 or Figure 2 A perspective view of the agitator at the base of the bioreactor.

[0129] Figure 4 The diagram shows a front view of the agitator plate in (a) a lower position, (b) a higher position, (c) a first angular position, and (d) a second angular position.

[0130] Figure 5 A flowchart of a "rapid mixing" method for transduced cells according to the present invention is shown.

[0131] Figure 6 A flowchart of the "intermittent mixing" method for transduced cells according to the present invention is shown.

[0132] Figure 7 A flowchart of a "continuous mixing" method for transduced cells according to the present invention is shown.

[0133] Figure 8 Line graphs showing the total number of live cells in the compressible container, rigid breathable container and breathable bag according to the present invention over a seven-day period are shown.

[0134] Figure 9 shows box plots of transduction efficiency in the compressible container, rigid breathable container, breathable bag and rigid container with stirrer according to the present invention, (a) CD8+ T cells and (b) CD4+ T cells.

[0135] Figure 10 Various examples of mixing modes of the compressible container according to the invention are shown from day 0 to day 3 of the culture process, as well as a rigid, breathable container control.

[0136] Figure 11 A comparison of cell growth in various mixing modes of the compressible container according to the invention and a static mode according to a rigid, breathable container control is shown.

[0137] Figure 12 The diagram illustrates a comparison of transduction cell yields in various mixing modes of the compressible container according to the invention and in a static mode according to a rigid, breathable container control.

[0138] Figure 13 A comparison of the transduction efficiency of various mixing modes of the compressible container according to the invention and a static mode according to a rigid permeable container is shown. Detailed Implementation

[0139] The described example embodiments relate to a method of transducing T cells within a compressible container. As those skilled in the art will recognize, references to containers, reservoirs, bioreactors, etc., in the specific embodiments are intended to be used as synonyms and not to limit the scope of protection. As those skilled in the art will recognize, the containers described herein are merely examples of suitable containers.

[0140] The use of certain terms in the following description is for convenience only and not restrictive. The terms “upper” or “top” and “lower” or “bottom” in the accompanying drawings indicate a direction of reference and are relative to the part described during assembly. Similarly, the terms “inner” or “inward” and “outer” or “outward” refer to directions toward and away from the designated centerline or geometric center (e.g., central axis) of the described element, respectively, the specific meanings of which are self-evident from the context of the description. Furthermore, the terms “proximal” (i.e., closer) and “distal” (i.e., farther) indicate positions relative to an axis or attachment point.

[0141] Furthermore, as used herein, the terms “connection,” “attachment,” “coupling,” etc., are intended to include both direct connections between two components without any other component inserted between them, and indirect connections between components in which one or more other components are inserted between them. This terminology includes the words specifically mentioned above, their derivatives, and words with similar meanings.

[0142] Furthermore, unless otherwise stated, the use of ordinal adjectives such as “first,” “second,” and “third” merely indicates different instances of similar objects or terms, and is not intended to imply that the objects so described must be in a given order in time, space, method, sequence, or any other way. Similar reference numerals are always used to describe similar features.

[0143] Figure 1 The bioreactor 1 shown includes a cell culture vessel 2 and an interface plate 3. During use, the cell culture vessel 2 contains a fluid in which cell treatment is performed. Specifically, the fluid is a cell suspension 4 containing cell populations, such as T cell populations, present in the cell treatment medium. Furthermore, within the cell culture vessel 2, there is a top space above the upper surface of the cell treatment medium. This top space fills the remaining volume in the bioreactor not occupied by the cell treatment medium. The cell suspension may also contain a transduction agent. Cells are transduced in the cell culture vessel by introducing the transduction agent to produce genetically modified cells. The cells are also cultured and multiplied in the cell culture vessel 2 and can undergo further treatment to produce cell-based therapeutic products.

[0144] An interface plate 3 is attached to the top of the cell culture container 2, thus acting 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 container configured to deliver fluid to or extract fluid from the cell culture container 2. In some instances, the interface plate 3 includes multiple connector interfaces 5, each for connecting to one of multiple external components. Each connector interface 5 can be used for adding or removing fluid once or multiple times. The connector interfaces 5 can be distributed around the interface plate 3. Therefore, the interface plate 3 is used to add cell treatment culture medium and other fluids to the cell culture container 2 during cell treatment, and / or to remove fluid from the cell culture container 2 during treatment, such as removing samples or waste liquid.

[0145] An external container (not shown) can be connected to connector interface 5 in interface plate 3 to add or extract materials into cell culture container 2. The external container may contain materials for addition to cell culture container 2 during cell treatment. For example, the external container may contain cell treatment culture medium, transduction agents, activators, cytokines, growth factors, magnetic beads, etc.

[0146] The cell culture container 2 is a compressible container. Therefore, the cell culture container is flexible, making it both extendable and compressible. Specifically, the cell culture container 2 has a compressible wall element 6, such as a bellows wall. The cell culture container 2 has a base 7 disposed opposite to the interface plate 3, and compressible wall elements 6 defining the sidewalls of the cell culture container 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 with a corresponding thread on the interface plate 3, thereby coupling it thereto. The compressible wall element 6 is compressible and / or extendable, allowing the base 7 to move toward and away from the interface plate 3, thereby changing the internal volume of the cell culture container 2. The base 7 can move relative to the interface plate 3 to agitate or mix the cell suspension 4 in the cell culture container 2.

[0147] The compressible wall element 6 can be a bellows wall with an accordion-like arrangement that allows the compressible wall element 6 to fold onto itself for compression. Specifically, as shown, the compressible wall element 6 may include a series of alternating deformable portions 9a, 9b. Blades 10 extend between the deformable portions 9a, 9b, i.e., staggered. Blades 10 are more rigid than the deformable portions 9a, 9b. The deformable portions 9a, 9b act as hinges, allowing the compressible wall element 6 to fold like a bellows or accordion, while the blades 10 remain substantially undeformed.

[0148] The compressible wall element 6 may include at least one inwardly deformable portion 9a and at least one outwardly deformable portion 9b, for example, at least two inwardly deformable portions 9a and at least two outwardly deformable portions 9b. The compressible wall element 6 may include three, four or more inwardly deformable portions 9a and three, four or more outwardly deformable portions 9b.

[0149] The inwardly deformable portion 9a and the outwardly deformable portion 9b can be formed from the thinned portion in the compressible wall element 6. The inwardly deformable portion 9a may include a thinned portion disposed on the outer surface of the compressible wall element 6, such that it can deform in an inward direction. The outwardly deformable portion 9b may include a thinned portion disposed on the inner surface of the compressible wall element 6, such that it can deform in an outward direction.

[0150] The compressible wall element 6 can be formed of a breathable or 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 thermoplastic elastomers (TPEs), such as impermeable TPEs. In examples, as further described below, the compressible wall element 6 may be coated, laminated, or otherwise treated to reduce the breathability of the compressible wall element 6 or to make it impermeable, particularly to 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 protective sheath molded onto the inner portion. The inner portion may include LDPE, and the protective sheath may include TPE. In another example, the compressible wall element 6 may include an elastomer outer sheath, such as a TPE outer sheath, and a liner. The sheath can take the form of a container and the general shape of the cell culture container 2. For example, an LDPE liner can be blow-molded onto the inner surface of the TPE outer sheath to form a liner. In another example, the liner can be an insert, such as an LDPE insert, which is housed outside the elastomer but not co-molded with the TPE outer sheath. In such an example, it may be preferred that the liner includes a substrate and defines a sealed container (except for the top) to contain the cell suspension 4.

[0151] Therefore, depending on the materials contained in the cell culture container 2, the cell culture container 2 can expand and contract, or be expanded and contracted. Specifically, the cell culture container 2 can expand as the volume of the cell suspension 4 within the cell culture container 2 increases and / or as additional materials are added.

[0152] As shown in the figure, the interface plate 3 also includes an expansion container 11. The expansion container 11 allows the cell culture container 2 to expand and contract without significantly altering the pressure within the cell culture container 2. Alternatively or additionally, the expansion container 11 may be operable, for example, by mechanical or manual compression or expansion, to expand or contract the compressible wall 6 of the cell culture container 2, thereby changing the volume of the cell culture container 2. Alternatively or additionally, the expansion container 11 may be operable, for example, by mechanical or manual compression or expansion, to change the pressure within the cell culture container 2.

[0153] In various instances, the cell culture vessel 2 includes a base 7 coupled thereto. The base 7 is typically planar, i.e., flat, and rigid. The base 7 is attached to or co-molded with the compressible wall element 6.

[0154] The base 7 is substantially planar, thus defining a rigid, substantially flat bottom for the cell culture vessel 2. The flat base 7 of the cell culture vessel 2 provides improved cell culture, particularly for mixing and control of the cell culture. Therefore, the base 7 of the cell culture vessel 2 helps ensure that cells are distributed substantially evenly across the cross-section of the cell culture vessel 2 when they settle at the bottom. This reduces cell “clustering” at isolated sites on the base 7, which could lead to oxygen depletion at these isolated sites. The flat base 7 of the cell culture vessel 2 also helps prevent cell suspension 4 from being trapped in the cell culture vessel 2 when harvesting or extracting cells at the end of the cell culture process.

[0155] In various instances, base 7 comprises a thermoplastic, such as high-density polyethylene (HDPE), or polycarbonate (PC), or another rigid polymer.

[0156] In addition, such as Figure 1 As shown, the agitator plate 12 is configured to abut and engage with the base 7. (Refer to...) Figure 3 The agitator plate 12 is described in more detail. In some instances, the agitator plate 12 may be coupled, for example, directly to the base 7.

[0157] In the illustrated example, the cell culture container 2 is typically cylindrical, having a typically circular base 7 and a typically cylindrical compressible wall element 6. Therefore, the axial direction is defined between the base 7 and the end of the compressible wall element 6 that mounts the interface plate 3. However, it should be understood that the cell culture container 2 can take alternative forms, such as having a typically triangular or square cross-section.

[0158] like Figure 2 The figure illustrates a cross-section of another embodiment of a bioreactor 1 according to the invention, which includes mixing elements such as a baffle 22. The baffle 22 is mounted on an interface plate 3 such that it is suspended within a cell culture vessel 2. The baffle 22 includes a mounting portion 23 attachable to the interface plate 3. In this embodiment, the mounting portion 23 may be attached to the interface plate 3 via a threaded connector or via clips or clamps. In the illustrated embodiment, the mounting portion 23 is attached to the center of the interface plate 3, such that the baffle 22 is located at the center of the cell culture vessel 2. However, it should be understood that the baffle 22 may be positioned off-center within the cell culture vessel 2. The mounting portion 23 extends from the interface plate 3 toward a base 7, and a baffle member 24 is attached to or formed therewith the mounting portion 23. In this embodiment, the baffle member 24 includes a substantially flat bottom surface 25 facing the base 7 of the cell culture vessel 2. The baffle member 24 also has a tapered upper surface 26 facing the interface plate 3, although this may also be substantially flat in alternative embodiments.

[0159] The baffle member 24 is circular and sized to fit within the cell culture vessel 2. In this example, the baffle member 24 is sized to be spaced apart from the compressible wall element 6 of the cell culture vessel 2. The baffle member 24 may be sized to be spaced apart from the compressible wall element 6 of the cell culture vessel 2 by allowing a sampling tube 17 to pass between the compressible wall element 6 and the baffle member 24. The sampling tube 17 provides a fluid sampling path from the cell culture vessel 2 to the interface plate 3. In this example, the baffle member 24 may be spaced apart from the compressible wall element 6 by a distance of approximately 5 mm to approximately 20 mm.

[0160] A baffle 22 is provided to mix the contents of the bioreactor 1 during use. Specifically, the base 7 of the bioreactor 1 is movable relative to the interface plate 3 and the baffle 22, such that the baffle 22 contacts and mixes the cell suspension 4 within the cell culture vessel 2. In examples, the base 7 can be raised and lowered relative to the interface plate 3 (i.e., changing the distance between the base 7 and the interface plate 3), and / or the base 7 can be tilted relative to the interface plate 3, and / or the base 7 can be rotated relative to the interface plate 3, as will be described in further detail below.

[0161] After mixing, the baffle 22 can be removed from the cell suspension 4 by expanding the cell culture container 2. The conical upper surface 26 of the baffle member 24 ensures that the fluid does not remain on the baffle 22, but flows back into the cell culture container 2.

[0162] like Figure 3 As shown, bioreactor 1 is located on a stirrer 18. The stirrer 18 includes a stirrer plate 12 that engages with the base 7 of bioreactor 1 to move the base 7 relative to the interface plate 3, thereby agitating the contents of bioreactor 1. This agitation mixes the fluids of bioreactor 1, facilitating transduction processes. Agitation can also aid in cell culture processes, for example, by mixing the fluids within bioreactor 1 or by promoting the dissolution of oxygen into the cell culture. The stirrer 18 can be mounted within an incubator housing (not shown).

[0163] Figure 3 An example agitator 18 with an actuator mechanism is shown, which is arranged to move an agitator plate 12 relative to the bioreactor 1. As shown, the agitator plate 12 is movable to engage the bioreactor 1, particularly the base (i.e., base 7, see figure). Figure 1 The actuator mechanism is mounted on the agitator base plate 28. Between the agitator base plate 28 and the agitator plate 12 are one or more actuators 29 for raising and lowering the agitator plate 12.

[0164] In the illustrated example, actuator 29 is a motor with a crank arm 33 arranged in a rotary hinge, which is rotatably connected to agitator base plate 28 and agitator plate 12, such that rotation of the hinged crank arm 33 moves agitator plate 12. In other examples, a linear actuator may be configured to act directly between agitator base plate 28 and agitator plate 12.

[0165] The support and guide members can guide the movement of the agitator plate 12.

[0166] The actuator mechanism may further include a pivotable rod 30, allowing the agitator plate 12 to pivot about the pivotable rod 30 to tilt the base of the bioreactor 1. The actuator 29 enables the agitator plate 12 to pivot about the pivotable rod 30 to tilt the base of the bioreactor 1. Therefore, the agitator plate 12 can move relative to the agitator substrate 28 to engage the base of the bioreactor 1 and agitate the contents of the bioreactor 1.

[0167] Figure 4 (a) through 4(d) show the agitation movement of the agitator plate 12, but do not show the bioreactor 1 or the actuator mechanism. Figure 4 (a) through 4(d) show the receiving portion 13 of the interface plate 3 that supports, holds, and clamps the bioreactor 1 during use, wherein the cell culture vessel (i.e., cell culture vessel 2, see...) Figure 1 The interface plate 3 is suspended below the receiving section 13. The receiving section 13 holds the interface plate 3 in a horizontal position, so that the actuation of the agitator plate 12 on the base 7 causes the base 7 to move relative to the interface plate 3.

[0168] like Figure 4 As shown in (a) and 4(b), the agitator plate 12 translates between lower and higher positions along the central longitudinal axis of the cell culture vessel 2. This movement of the agitator plate 12 between the lower and higher positions agitates the contents within the internal volume of the cell culture vessel 2. The movement between the lower and higher positions can be a reciprocating compression motion to provide agitation or turbulence to the contents of the cell culture vessel 2 for a predetermined period of time, as will be discussed in further detail below. The compression motion allows for the mixing of large quantities of cell suspension 4 within the cell culture vessel 2. In some instances, the baffle 22 increases the mixing of the cell suspension 4 during compression mixing.

[0169] The compression motion can be performed at a rate of 1 to 60 cycles per minute (cpm), where each cycle consists of 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 motion can be at a rate of 1 to 30 cpm. In another example, the compression motion can be at a rate of 5 to 25 cpm. In yet another example, the compression motion can be at a rate of 10 to 20 cpm. In still other examples, the compression motion can be at a rate of 5 cpm, 10 cpm, 20 cpm, 30 cpm, 40 cpm, 50 cpm, or 60 cpm.

[0170] like Figure 4 As shown in (c) and 4(d), the agitator plate 12 is tilted to agitate the contents of the bioreactor 1. Figure 4 In (c), the agitator plate 12 is tilted in a first position, where the base forms a first angle with respect to the horizontal plane. Figure 4 As shown in (d), the agitator plate can rotate in opposite directions to a second position, in which the base forms a second angle relative to the horizontal plane. The first angle and the second angle are from 1 degree to 45 degrees, and include 1 degree and 45 degrees. Preferably, the first angle and the second angle are from 5 degrees to 25 degrees, and include 5 to 25 degrees. The first angle and the second angle can be one of 5 degrees, 10 degrees, 15 degrees, 20 degrees, and 25 degrees. The second angle can be equal to the first angle. The movement of the agitator plate 12 between the first position and the second position produces a rocking motion. This rocking motion gently agitates the cell suspension 4 in the cell culture vessel 2 and causes turbulence in the cell suspension 4. The movement of the agitator plate 12 between the first and second positions can have a varying speed. Alternatively, the movement of the agitator plate 12 between the first and second positions can have a constant speed. In an example using a constant speed, the base can be held at each of the first and second angles for a predetermined time period, such as from 0.1 seconds to 5 seconds. The movement between the first and second positions can be a reciprocating motion to agitate the contents of bioreactor 1 for a predetermined time period, as will be discussed in further detail below.

[0171] The swinging motion can be performed at a rate of 1 to 60 swings per minute (rpm), with each swing starting from the first position (e.g., Figure 4 (c) Starting with a rotation, rotate to the second position (e.g.) Figure 4 (d)), then rotate back and return to the first position (e.g. Figure 4(c)). In one example, the swaying motion can be at a rate of 1 to 30 rpm. In another example, the swaying motion can be at a rate of 5 to 25 rpm. In yet another example, the swaying motion can be at a rate of 10 to 20 rpm. In still other examples, the swaying motion can be at a rate of 5 rpm, 10 rpm, 20 rpm, 30 rpm, 40 rpm, 50 rpm, or 60 rpm.

[0172] A combination of vertical and tilting movements can be provided to agitate the contents of bioreactor 1. The agitator plate 12 can tilt in different directions, and / or the agitator plate 12 can tilt in only one or two directions, but rotation of bioreactor 1 can change the tilt direction of bioreactor 1 itself. This provides pivoting motion around the center point of the base 7 of bioreactor 1.

[0173] In another example, not shown, the base 7 can pivot about an origin set at the center of the base. This generates a vortex motion to agitate the contents within the internal volume of the cell culture vessel 2. The base can pivot relative to a horizontal plane to a first angle. The first angle is from 1 degree to 45 degrees, and includes both 1 degree and 45 degrees. Preferably, the first angle is from 5 degrees to 25 degrees, and includes both 5 degrees and 25 degrees. The first angle can be one of 5 degrees, 10 degrees, 15 degrees, 20 degrees, and 25 degrees.

[0174] The base can be pivoted at a rate of 1 to 60 revolutions per minute, each revolution being one circular rotation around the origin. In one example, the pivoting motion can be at a rate of 1 to 30 revolutions per minute. In another example, the pivoting motion can be at a rate of 5 to 25 revolutions per minute. In yet another example, the pivoting motion can be at a rate of 10 to 20 revolutions per minute. In still other examples, the pivoting motion can 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.

[0175] In some instances, the agitator plate 12 may be coupled to the base 7 of the bioreactor 1, such that the base 7 will move together with the agitator plate 12. In other instances, the agitator plate 12 may not be coupled to the base 7, and at some locations and / or during some agitation movements, the base 7 may be completely or partially lifted away from the agitator plate 12. Some agitation movements may provide impact contact, i.e., engagement, between the agitator plate 12 and the base 7 to agitate the contents of the bioreactor. In other instances, the agitator plate 12 may be a vibratory agitator plate configured to vibrate the base 7 of the bioreactor 1.

[0176] Now refer to Figure 5 to 7This describes a method for transducing cells using bioreactor 1 as described above. Target cells are isolated from a patient or donor sample before initiating the transduction method. Any suitable target cells required for transduction can be used. In some instances, the target cells can be T cells, such as CD3+, CD4+, or CD8+ T cells. In other instances, the target cells can be CD34+ hematopoietic stem cells and progenitor cells (HSPCs).

[0177] In each of methods 100a, 100b, and 100c, in steps 110a, 110b, and 110c, T cells are added to a cell culture container (i.e., cell culture container 2, see...) in the cell treatment culture medium. Figure 1 and 2 The T cells are seeded into the cell culture vessel within the internal volume of the cell culture vessel. In other words, the seeding of T cells into the cell culture vessel occurs on day 0.

[0178] The cell treatment medium can be selected from any suitable medium. In some instances, the cell treatment medium may be Dulbecco's modified Eagle medium (DMEM), available from Thermo Fisher Scientific, Sigma Aldrich, etc., or X-VIVO, available from Lonza. TM 15, or TexMACS available from Miltenyi Biotec. TM The cell treatment medium can be any cell treatment medium suitable for T cells.

[0179] Optionally, the activator can be added to the cell culture vessel (i.e., cell culture vessel 2, see...) Figure 1 and 2 The cell solution 4 is added simultaneously with the T cells to the internal volume of the cell culture vessel (i.e., on day 0). The activator can be either magnetic beads or soluble particles. The contents of the cell culture vessel (including the activator) can 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 activator to mix with the cell treatment medium.

[0180] In steps 120a, 120b, and 120c, the transduction agent is added to the cell culture vessel (i.e., cell culture vessel 2, see...). Figure 1 and 2 The transduction agent is located within the internal volume of the cell. The transduction agent can be a viral vector. Preferably, the viral vector is a lentiviral vector. The transduction agent can be added 6 hours to 2 days after the addition of the activator. Preferably, the transduction agent is added 1 day (24 hours) after the addition of T cells and the activator (i.e., the transduction agent is added on day 1).

[0181] The addition of the transducer, i.e., steps 120a, 120b, 120c, may be before, during, or after any of the following steps: steps 130a, 130b, or 130c, steps 140a, 140b, or 140c, or step 145b, as discussed in further detail below.

[0182] According to the "rapid mixing" method, the "intermittent mixing" method, and / or the "continuous mixing" method, the cell culture vessel is moved by moving the base (i.e., base 7, see...) Figure 1 and 2 A cell suspension comprising cells, cell treatment medium, and transduction agent can be mixed in a cell culture vessel. This mixing agitates the cell suspension to stimulate transduction of the cells within it.

[0183] The "quick mix" method, such as Figure 5 As shown. In step 130a, during the first time period, the base of the cell culture vessel (i.e., base 7, see...) Figure 1 and 2 ) Relative to the top (i.e., interface board 3, see Figure 1 and 2 ) Move. Agitator (i.e., agitator 18, see Figure 3 The agitator can move the base. As mentioned above, the movement of the base can be a rocking motion, a compressive motion, a pivoting motion, a rotational motion, or a combination thereof. For example, an agitator can provide tilting motion to rock the base, or vertical motion to compress the cell culture vessel.

[0184] The first time period can be 1 to 60 minutes.

[0185] In step 140a, the base stops moving and remains stationary for a second time period. During this step, the base remains in a horizontal position.

[0186] The second time period can range from 1 minute to 5 hours. In one instance, the second time period can be 1 to 60 minutes. In another instance, the second time period can be 0.5 to 5 hours. In yet another instance, the second time period can be 1 to 3 hours. In yet another instance, the second time period is 3 hours.

[0187] The "intermittent mixing" method, such as Figure 6 As shown. In step 130b, during the first time period, the base of the cell culture vessel (i.e., base 7, see...) Figure 1 and 2 ) Relative to the top (i.e., interface board 3, see Figure 1 and 2 ) Move. Agitator (i.e., agitator 18, see Figure 3The agitator can move the base. As mentioned above, the movement of the base can be a rocking motion, a compressive motion, a pivoting motion, a rotational motion, or a combination thereof. For example, an agitator can provide tilting motion to rock the base, or vertical motion to compress the cell culture vessel.

[0188] The first time period can be from 1 minute to 5 hours. For example, the first time period can be from 1 to 60 minutes. In another instance, the first time period can be from 0.5 to 5 hours. In another instance, the first time period can be from 1 to 3 hours. In another instance, the first time period can be 1 minute. In another instance, the first time period can be 3 hours.

[0189] In step 140b, the agitator stops the movement of the base and holds the base in a stationary position for a second time period. During this step, the base is held in a horizontal position.

[0190] The second time period can range from 1 minute to 5 hours. For example, the second time period can be 1 to 60 minutes. In another instance, the second time period can be 0.5 to 5 hours. In another instance, the second time period can be 1 to 3 hours. In yet another instance, the second time period can be 3 hours.

[0191] In one instance, the first time period can be 1 minute, and the second time period can be 3 hours. In another instance, the first time period can be 3 hours, and the second time period can be 1 to 5 hours.

[0192] In step 145b, steps 130b and 140b above are repeated for a predetermined time period. This provides a cell culture container (i.e., cell culture container 2, see...). Figure 1 and 2 Intermittent mixing of cell suspensions. The predetermined time period can be 1 to 5 days. In this example, the predetermined time period can be 1 to 3 days. In another example, the predetermined time period can be 3 days.

[0193] The "continuous mixing" method, such as Figure 7 As shown. In step 130c, during the first time period, the base of the cell culture vessel (i.e., base 7, see...) Figure 1 and 2 ) Relative to the top (i.e., interface board 3, see Figure 1 and 2 ) Move. Agitator (i.e., agitator 18, see Figure 3 The agitator can move the base. As mentioned above, the movement of the base can be a rocking motion, a compressive motion, a pivoting motion, a rotational motion, or a combination thereof. For example, an agitator can provide tilting motion to rock the base, or vertical motion to compress the cell culture vessel.

[0194] The first time period can be 1 to 5 days. In this example, the first time period can be 1 to 3 days.

[0195] The base stops moving in step 140c.

[0196] During cell transduction, any combination of rapid mixing, intermittent mixing, and continuous mixing methods can be used. For example, the cell solution can be intermittently mixed by rocking motion during a first predetermined time period, continuously mixed by rocking motion during a second predetermined time period, and continuously mixed by compression motion during a third predetermined time period. Between or during each of these mixing steps, additional culture medium can be added to the internal volume to address the issue of increased cell numbers.

[0197] After transduction, samples of the cell solution can be collected to measure transduction efficacy. Samples can be collected 2 to 4 days after T cells are added to the inner volume of cell culture vessel 2 (day 0). For example, samples can be collected 3 days after T cells are added to the inner volume of cell culture vessel (i.e., on day 3). Transduction efficiency is measured using either green fluorescent protein (GFP) or CD19. Specifically, CD19 is used to measure the transduction efficiency of CAR T cells.

[0198] exist Figure 5 to Figure 7 In each of methods 100a, 100b, and 100c, in steps 150a, 150b, and 150c, cell treatment medium is added to the cell culture vessel (i.e., cell culture vessel 2, see...). Figure 1 and 2 The volume of cell treatment culture medium added can be equal to the volume of the cell treatment culture medium inside the cell culture vessel, thus doubling the volume of the cell treatment culture medium in the cell culture vessel. Additional materials, such as growth factors, cytokines, magnetic beads, and nutrients, can also be added to the internal volume of the cell culture vessel.

[0199] Additional cell treatment medium can be added to the cell culture container 2 two to four days after T cells are added to the internal volume (day 0). For example, additional cell treatment medium can be added three days after T cells are added to the internal volume of the cell culture container (i.e., additional cell treatment medium is added on day 3).

[0200] In each of methods 100a, 100b, and 100c, the transduced T cells are then cultured in the internal volume of a cell culture vessel in steps 160a, 160b, and 160c. In this step, the base of the cell culture vessel (i.e., base 7, see...) Figure 1 and 2 ) can be relative to the top (i.e., interface board 3, seeFigure 1 and 2 The cells are moved to mix the cell solution. The cell solution can be mixed continuously or intermittently.

[0201] After adding T cells to the inner volume of cell culture container 2 (day 0), T cells can be cultured for up to 10 days. In one example, T cells are cultured for 5 to 10 days after being added to the inner volume of cell culture container 2. In another example, T cells are cultured for approximately 7 days after being added to the inner volume of cell culture container 2 (i.e., cultured to day 7).

[0202] In this cell culture step, cell treatment medium can be added to the internal volume of the cell culture vessel at predetermined time intervals. For example, cell treatment medium can be added every 24 hours. In one example, the volume of cell treatment medium is doubled at each predetermined time interval. In other examples, cell treatment medium can be added dynamically in response to an increase in cell density.

[0203] In this step, the base of the cell culture vessel (i.e., base 7, see...) Figure 1 and 2 ) can be relative to the top (i.e., interface board 3, see Figure 1 and 2 The agitator moves to provide continuous mixing. (i.e., agitator 18, see...) Figure 3 The base can be moved. As described above, the movement of the base can be a rocking motion, a compressive motion, a pivoting motion, a rotational motion, or a combination thereof. In one example, the base moves continuously in a rocking motion over a first predetermined time period, and then moves continuously in a compressive motion over a second predetermined time period. In this example, each of the first and second predetermined time periods is 1 to 4 days, preferably about 2 days.

[0204] After the predetermined time period, the cells were removed from the cell culture container (i.e., cell culture container 2, see...). Figure 1 and 2 T cells are harvested from the cell culture vessel. In this example, the planned timeframe is 5 to 10 days after the T cells are added to the internal volume of the cell culture vessel (day 0). In one example, T cells are harvested approximately 7 days after the T cells are added to the internal volume of the cell culture vessel (i.e., harvesting T cells on day 7). Alternatively, T cells can be harvested once the target density or target cell number is reached. The harvested T cells can be prepared for infusion into a patient or can be cryopreserved or transported.

[0205] Example 1

[0206] Transducing and culturing CD3+ T cells with CD4+ markers in a compressible container

[0207] Tests were conducted to evaluate the dynamic mixing in a compressible container (i.e., cell culture container 2, see...) Figure 1 The viability, growth, and phenotype of primary CD3+ T cells with CD4+ markers activated, transduced, and expanded for 7 days were studied. Known bioreactor systems, including rigid aerated containers (G-REX® 100M™, Wilson Wolf), flexible aerated bags (Xuri W25™, Cytiva & VueLife® “C” series bags, Saint-Gobain), and rigid containers with stirrers (Prodigy®, Miltenyi Biotec), were used as controls. For each control, transduction occurred while each bioreactor system remained stationary. Rigid aerated containers remained stationary throughout the test. Aerated bags remained stationary during transduction and then were continuously agitated after the transduction period (starting from day 3, i.e., during subsequent culture periods). The contents of rigid containers with stirrers remained stationary (i.e., unstirred) during transduction and then were continuously agitated after the transduction period (starting from day 3, i.e., during subsequent culture periods).

[0208] CD3+ T cells with CD4+ markers were negatively selected from whole blood samples from healthy donors and were counted at 1x10⁻⁶. 6 cells.mL -1 The density of seeding was carried out in cell culture containers (i.e., cell culture container 2, see...) Figure 1 CD3+ T cells were seeded on day 0 in 50 mL of cell treatment medium. The cell treatment medium consisted of X-VIVO2. TM 15 (Lonza), 5% normal human AB serum (Sigma) and rhIL-2 (100 units / mL) -1 Composed of (R&D Systems). The total number of cells inoculated is 50 × 10⁻⁶. 6 Cells. On day 0, activator CTS was added at a bead-to-cell ratio of 3:1. TM Dynabeads TM (ThermoFisher).

[0209] Twenty-four hours after the cells were seeded into bioreactor 1, the GFP lentiviral vector (multiple of infection (MOI) of 1) (Takarabio) was added to the cell solution (i.e., on day 1).

[0210] Cells were cultured for 7 days, and the cell solution was sampled at specific time intervals before adding fresh cell treatment medium. The volume of additional cell treatment medium (whose composition is as described above) added to the cell solution was typically equal to the volume of the cell treatment medium in the cell solution, so that the volume was doubled each time the cell treatment medium was added to the cell culture vessel. Further details regarding the addition of cell treatment medium are described below.

[0211] Day 3: After sampling, add 150 mL of cell treatment culture medium (the composition of which is as described above).

[0212] Day 5: After sampling, add 200 mL of cell treatment culture medium (the composition of which is as described above).

[0213] Day 6: After sampling, add 400 mL of cell treatment culture medium (the composition of which is as described above).

[0214] From day 0 to day 3, the base 7 of bioreactor 1 was actuated to provide intermittent rocking motion. The base rocked 5 times per minute (rpm) every 3 hours, and remained stationary between each set of rocking motions. The rocking motion was a trapezoidal rocking motion.

[0215] From day 3 to day 5, the base of the cell culture vessel (i.e., base 7, see...) Figure 1 and 2 It is actuated to provide continuous oscillating motion at a rate of 5 rpm.

[0216] From day 5 to day 7, the base of the cell culture vessel was actuated to provide continuous linear compression motion (expansion and contraction of the cell culture vessel) at a rate of 60 cycles per minute.

[0217] The test was terminated on the 7th day.

[0218] Figure 8 The total number of live cells during the seven-day test period is shown. The total number of live cells is comparable to the control of a static ventilated bioreactor container and has a greater number of live cells compared to the control of a ventilated bag that was stationary from day 1 to day 3 and continuously swung from day 4 to day 7.

[0219] Figure 9(b) shows a comparison of CD4+ T cell transduction efficiency using various devices. In this example, GFP was used as a transduction marker. As shown in Figure 9(b), according to this method, 13% to 52% of CD4+ T cells were effectively transduced.

[0220] Example 2

[0221] Transducing and culturing CD3+ T cells with CD8+ markers in a compressible container

[0222] The same methods and containers were used for transducing and culturing CD3+ T cells with CD8+ markers. The methods and containers used were identical, except that a population of CD3+ T cells with CD8+ markers was used, and therefore will not be described further.

[0223] As shown in Figure 9(a), according to this method, 19 to 55% of CD8+ T cells were effectively transduced.

[0224] Therefore, as Figure 9(a) and 9(b) The results show that, compared with the control system, the transduction performed in the bioreactor as described herein and according to the above method has statistically significantly higher transduction efficiency.

[0225] Example 3

[0226] Transducing and culturing CD3+ T cells with CD4+ markers

[0227] Tests were conducted to evaluate the viability, growth, and transduction efficiency of primary CD3+ T cells with CD4+ markers and CD3+ T cells with CD8+ markers. In three hybrid modes (static, hybrid mode 1, and hybrid mode 2, each defined below), a compressible container with the baffles described herein (see [link to document]) was used. Figure 2 This was compared to a rigid, permeable container as a control. For the control, transduction occurred while the container system remained stationary (i.e., without rocking or otherwise moving).

[0228] Figure 10 An overview of the respective mixing patterns for the compressible container and the control for days 0-3 is provided, which will be explained in further detail below.

[0229] Compressible container

[0230] CD3+ T cells with CD4+ markers and CD3+ T cells with CD8+ markers were negatively selected from whole blood samples from healthy donors, and were then used at a concentration of 1.5 x 10⁻⁶ cells / mL. 6 cells.mL -1 The density of seeding was carried out in cell culture containers (i.e., cell culture container 2, see...) Figure 2 Cells were seeded on day 0 in 100 mL of cell treatment medium. The cell treatment medium consisted of TexMACS™ (Miltenyi Biotec), 5% normal human AB serum (Sigma-Aldrich), and IL-7 (12.5 ng / mL). -1 ) (Miltenyi Biotec) and IL-15 (12.5 ng.mL -1Composed of Miltenyi Biotec. The total number of cells inoculated is 150 x 10⁻⁶. 6 Cells. The activator TransAct™ (Miltenyi Biotec) was added on day 0 at a ratio of 1:100 to TransAct™-medium.

[0231] Twenty-four hours after the cells were seeded into a compressible container, the GFP lentiviral vector (multiple of infection (MOI) of 0.5) (Flash Therapeutics) was added to the cell solution (i.e., on day 1).

[0232] Cells were cultured for 8 days, and the cell solution was sampled at specific time intervals. Fresh cell treatment medium was provided for addition. The volume of additional cell treatment medium (whose composition is as described above) added to the cell solution was typically equal to the volume of the cell treatment medium in the cell solution, so that the volume was doubled each time the cell treatment medium was added to the cell culture vessel. Further details regarding the addition and process of the cell treatment medium are noted below.

[0233] Day 0: Cell seeding and activation.

[0234] Day 1: Add transduction agent (green fluorescent protein (GFP) lentiviral vector).

[0235] Day 3: Add 100 mL of cell treatment culture medium (the composition of which is as described above).

[0236] Day 4: Add 200 mL of cell treatment culture medium (the composition of which is as described above).

[0237] Day 5: Add 400 mL of cell treatment medium (the composition of which is as described above). Samples were also collected on day 5 before adding the cell treatment medium.

[0238] Day 6: Add 200 mL of cell treatment medium (the composition of which is as described above). Samples were also collected on day 6 before adding the cell treatment medium.

[0239] Day 7: Collect samples. Do not add cell treatment culture medium again.

[0240] Day 8: The final sample, representing the end of the process, was collected.

[0241] like Figure 10 As shown, in the static mode of the compressible container, during the duration of the transduction process (i.e., until day 3), the base 7 of the cell culture container 2 (see...) Figure 2 The cell culture container 1 is statically positioned in a horizontal position. That is, the base 7 of the cell culture container 1 (see...) Figure 2 Do not move during this period.

[0242] like Figure 10 As shown, in the first mixing mode (“mixing mode 1”) of the compressible container, the base 7 of the cell culture container 2 (see...) Figure 2 The base is actuated to provide continuous oscillating motion from day 0 to day 3 (inclusive). The base oscillates continuously at a rate of ten (10) oscillations per minute. The oscillation motion is sinusoidal (i.e., smooth or constant speed) oscillation motion. During the oscillation, the base 7 forms a 15-degree angle with the horizontal plane.

[0243] like Figure 10 As shown, in the second mixing mode (“mixing mode 2”) of the compressible container, the base 7 of the cell culture container 2 (see...) Figure 2 The base was actuated to provide continuous oscillating motion from day 1 to day 3 (inclusive of day 0 and day 3), i.e., after the addition of the transducer but not before its addition. The base oscillated continuously at a rate of ten (10) oscillations per minute. The oscillation motion was sinusoidal (i.e., smooth or constant speed) oscillation motion. During the oscillation, the base 7 was at a 15-degree angle to the horizontal plane.

[0244] In each of the static mode, mixed mode 1, and mixed mode 2, a further continuous oscillating motion for twenty-four (24) hours was added on day 4. The base oscillated at a rate of thirty (30) oscillations per minute. The oscillation motion was sinusoidal (i.e., smooth or constant speed) oscillation motion. During the oscillation, the base 7 formed a 15-degree angle with the horizontal plane.

[0245] In each of the static mode, hybrid mode 1, and hybrid mode 2 of the compressible container, from day 5 to day 8 ( Figure 10 (Not shown in the image) Further compression and mixing schemes are added as described below.

[0246] From day 5 to day 8 (inclusive), the base 7 of the cell culture vessel 2 is 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.

[0247] The test was terminated on day 8 after a final sample of cells was taken from the cell culture vessel.

[0248] Rigid air permeability control device

[0249] For the purposes of comparison, a cell culture container was used, having a rigid and airtight cylindrical wall standing upright from a ventilated base. The ventilated base is supported by a support such as legs, which allows gas to passively diffuse through the ventilated base into the volume of the cell culture container. The cell culture container also provides an inlet and a removable lid coupled to the inlet to allow for the introduction and removal of material from the container. This container comprises a structure with a surface area of ​​10 cm². 2 The container has a breathable base made of silicone. Its volume capacity is also 100 mL.

[0250] CD3+ T cells with CD4+ markers and CD3+ T cells with CD8+ markers were negatively selected from whole blood samples from healthy donors, and were then used at a concentration of 1.5 x 10⁻⁶ cells / mL. 6 cells.mL -1 Cells were seeded at a density in rigid, breathable containers (i.e., containers with rigid walls and a breathable base as described above). On day 0, cells were seeded in 10 mL of cell treatment medium. The cell treatment medium consisted of TexMACS™ (Miltenyi Biotec), 5% normal human AB serum (Sigma-Aldrich), and IL-7 (12.5 ng / mL). -1 ) (Miltenyi Biotec) and IL-15 (12.5 ng.mL -1 Composed of Miltenyi Biotec. The total number of cells inoculated is 15 x 10⁻⁶. 6 Cells. The activator TransAct™ (Miltenyi Biotec) was added on day 0 at a ratio of 1:100 to TransAct™-medium.

[0251] Twenty-four hours after the cells were seeded into rigid, breathable containers, GFP lentiviral vector (multiple of infection (MOI) of 0.5) (Flash Therapeutics) was added to the cell solution (i.e., on day 1).

[0252] The cells were cultured for 8 days, and the cell solution was sampled at the specific time intervals described below.

[0253] Day 3: Add 10 mL of cell treatment culture medium (the composition of which is as described above).

[0254] Day 4: Add 20 mL of cell treatment culture medium (the composition of which is as described above).

[0255] Day 5: Add 40 mL of cell treatment culture medium (the composition of which is as described above) and collect samples.

[0256] Day 6: Add 20 mL of cell treatment culture medium (the composition of which is as described above) and collect samples.

[0257] Day 7: Collect and freeze samples.

[0258] Day 8: The test is terminated after the last cell sample is taken from the device.

[0259] like Figure 10 As shown, the rigid, breathable container remained stationary (i.e., did not move) throughout the entire duration of the experiment (i.e., every day from day 0 to day 8).

[0260] Comparison of compressible containers and rigid permeable containers

[0261] For two donors (discussed below) Figure 12 and Figure 13 RD037 and RD007 in the table represent the results. Each experiment was performed four times (for each of the static mode, mixed mode 1, mixed mode 2, and control, N=4), and the results were compared, as shown in the table. Figure 11 , Figure 12 and Figure 13 As outlined in the document and discussed further below.

[0262] Figure 11 The total number of live cells during the test is displayed, comparing the static mode, mixed mode 1, and mixed mode 2 in the compressible container with a rigid, breathable container as a control. The total number of live cells in the compressible container is expressed as 10-1. 9 The number of cells is represented by 10, while the total number of live cells in a rigid, breathable container is represented by 10. 8 The number of cells is indicated because the initial volumes of these containers differ by an order of magnitude (1000 mL for compressible containers; 100 mL for rigid, breathable containers). However, as mentioned above, the same seeding density (1.5 x 10⁻⁶ cells / mL) is provided in each container. 6 cells.mL -1 ).

[0263] like Figure 11 As shown, under each mixing condition (static mode, mixing mode 1, mixing mode 2), the total number of viable cells in the compressible container exceeded that of the control in the rigid, permeable bioreactor container. Also as shown in the figure, the total number of viable cells increased by providing mixing conditions (mixing mode 1, mixing mode 2) in the compressible container compared to the static growth conditions (static mode).

[0264] Figure 12The comparison of transduced cell yields from day 6 to day 8 is shown. Specifically, statistically significantly greater numbers of GFP+ cells (i.e., cells taking up the GFP lentiviral vector) were obtained in the compressible bioreactor on days 7 and 8 when using Mix Mode 1 and Mix Mode 2 compared to the rigid aerated container control (P < 0.0001). It was also noted that Mix Mode 1 and Mix Mode 2 were superior to the control on day 6. Furthermore, as... Figure 11 As shown, on days 7 and 8, the static mode of the compressible container was also superior to the control.

[0265] Figure 12 It was also shown that the end-to-end process time could be reduced by as much as two days. Specifically, using either mixing mode 1 or mixing mode 2 in the compressible container disclosed herein, a transduced cell yield comparable to that obtained on day 8 in a rigid, permeable bioreactor was achieved on day 6.

[0266] Figure 13 The transduction efficiency was compared between the compressible container and the rigid aerated container control, and various hybrid profiles within the compressible container were also compared. It can be seen that the compressible container is comparable to the rigid aerated bioreactor when used in static mode. It can also be seen that the compressible container showed a statistically significant improvement in transduction efficiency when used in hybrid mode 1 or hybrid mode 2 compared to the rigid aerated container control and static mode transduction in the compressible container. Transduction efficiency for each experiment was assessed every day from day 6 to day 8 (inclusive) using flow cytometry (specifically, a FACSLyric™ device from BD Biosciences).

[0267] Therefore, it has been shown that providing and using compressible containers can improve transduction efficiency, reduce end-to-end cell processing time, and increase the total number of viable cells.

[0268] Throughout the description and claims of this specification, the words “comprising” and “including” and their variations mean “including, but not limited to”, and they are not intended to (and do not) exclude other parts, integrals, or steps. Throughout the description and claims of this specification, the singular includes the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood to consider both the plural and the singular unless the context requires otherwise.

[0269] Features, integrals, characteristics, or groups described in connection with a particular aspect, embodiment, or example of the invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, unless at least some of such features and / or steps in such combination are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel feature or any combination of novel features disclosed in this specification (including any appended claims, abstract, and drawings), or any novel step or any combination of novel steps in any method or process so disclosed.

Claims

1. A method for transducing T cells, comprising: A compressible container is provided, the compressible container including a base, a top arranged substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an internal volume of the compressible container; The T cell population in the cell treatment culture medium is added to the internal volume; Add the transductant to the internal volume; and The base is moved relative to the top, thereby causing turbulence in the contents of the internal volume during a first time period.

2. The method of claim 1, further comprising moving the base relative to the top to cause turbulence in the contents of the internal volume during a second time period.

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

4. The method of claim 1 or 2, wherein the base moves relative to the top at a first rate during the first time period, and wherein the base moves relative to the top at a second rate during the second time period, the second rate being greater than the first rate.

5. The method according to any one of claims 2 to 4, further comprising moving the base relative to the top, thereby causing turbulence in the contents of the internal volume during a third time period.

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

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

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

9. The method of claim 7 or 8, further comprising moving the base relative to the top to cause turbulence in the contents of the internal volume during a third time period.

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

11. The method according to any one of claims 7 to 10, wherein any step or both of the steps of moving the base relative to the top comprises rotating the base about a rotation axis extending in a horizontal plane defined by the base.

12. The method according to claim 3 or 11, wherein rotating the base comprises: The base is rotated about a rotation axis in a first direction to a first position, where the base forms a first angle relative to the horizontal plane in the first position. and The base is rotated about the axis of rotation in a second direction opposite to the first direction to a second position, in which the base forms a second angle relative to the horizontal plane.

13. The method of claim 12, wherein the base rotates at a constant speed between the first position and the second position.

14. The method of claim 12, wherein the base rotates between the first position and the second position at a varying speed.

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

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

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

18. The method according to any one of the preceding claims, wherein any or both of the steps of moving the base relative to the top include translating the base toward the top along the central longitudinal axis of the compressible container, thereby compressing the compressible container.

19. The method according to 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. The method according to any one of the preceding claims, wherein any step or both of the steps of moving the base relative to the top comprises pivoting the base about an origin set around the center of the base.

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

22. The method according to any one of the preceding claims, wherein the transductant is added to the internal volume during the step of moving the base relative to the top in the first time period.

23. The method according to any one of claims 1 to 21, further comprising the step of keeping the base stationary relative to the top for a predetermined time period after the addition of the transconductant.

24. The method according to any one of claims 9 to 23, wherein the transconductant is added to the internal volume during the step of moving the base relative to the top in the third time period.

25. The method according to any one of the preceding claims, wherein the transducer is a viral vector, such as a lentiviral vector.

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

27. The method according to any one of the preceding claims, wherein the compressible container is substantially airtight.

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

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

30. The method according to any one of the preceding claims, wherein the T cells are selected from CD3+ T cells having CD4+ markers and / or CD3+ T cells having CD8+ markers.

31. A method for transducing T cells, comprising the following sequential steps: i) Provide a compressible container, the compressible container including a base, a top arranged substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an internal volume of the compressible container; ii) Add the T cell population from the cell treatment medium to the internal volume; iii) The base is continuously moved relative to the top, thereby causing turbulence in the contents of the internal volume at a first rate during a first time period; iv) Optionally, other cell treatment media may be added to the internal volume; v) The base is continuously moved relative to the top, thereby causing turbulence in the contents of the internal volume at a second rate during a second time period, the second rate being greater than the first rate; vi) Optionally, other cell treatment media may be added to the internal volume; vi) The base is continuously moved relative to the top, thereby causing turbulence in the contents of the internal volume during the third time period. The method further includes the step of adding a transducer to the internal volume during step ii) or step iii).

32. The method according to claim 31, wherein: Step iii) includes continuously rotating the base at the first rate about a rotation axis extending in a horizontal plane defined by the base; Step v) includes continuously rotating the base at the second rate about a rotation axis extending within a horizontal plane defined by the base; and Step vi) includes continuously translating the base along the central longitudinal axis of the compressible container toward the top, thereby compressing the compressible container.

33. A method for transducing T cells, comprising the following sequential steps: i) Provide a compressible container, the compressible container including a base, a top arranged substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an internal volume of the compressible container; ii) Add the T cell population from the cell treatment medium to the internal volume; iii) Optionally, after the addition of the transconductant, the base is kept stationary relative to the top for a predetermined time period; iv) Moving the base relative to the top, thereby causing turbulence in the contents of the internal volume during a first time period; v) Keep the base stationary relative to the top during the second time period; vi) Repeat steps iv) and v) within the predetermined time period; and vii) During any of steps ii) to vi), a transductant is added to the internal volume.

34. A method for transducing and culturing T cells, comprising the following sequential steps: i) Provide a compressible container, the compressible container including a base, a top arranged substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an internal volume of the compressible container; ii) Add the T cell population from the cell treatment medium to the internal volume; iii) Optionally, after the addition of the transconductant, the base is kept stationary relative to the top for a predetermined time period; iv) The base is moved intermittently relative to the top, thereby intermittently causing turbulence in the contents of the internal volume during a first time period; v) During the step of adding the T cell population from the cell treatment medium to the internal field of view, or during the step of intermittently moving the base relative to the top to enable transduction of the T cell population, a transduction agent is added to the internal volume; vi) Add an additional volume of cell treatment culture medium to the internal volume; and vii) Cultivate the T cell population.

35. A method for transducing and culturing T cells, comprising the following sequential steps: i) Provide a compressible container, the compressible container including a base, a top arranged substantially parallel to the base, and at least one flexible wall element extending between the top and the base and defining an internal volume of the compressible container; ii) Add the T cell population from the cell treatment medium to the internal volume; iii) Optionally, after the addition of the transconductant, the base is kept stationary relative to the top for a predetermined time period; iv) Rotate the base about a rotation axis extending within a horizontal plane defined by the base, thereby causing turbulence in the contents of the internal volume during a first time period; v) Keep the base stationary relative to the top during the second time period; vi) Repeat steps iv) and v) within the predetermined time period; vii) Optionally, additional cell treatment culture medium is added to the internal volume; viii) The base is translated relative to the top along the central longitudinal axis of the compressible container, thereby compressing the compressible container in the third time period; and ix) During any of steps ii) to viii), the transductant is added to the internal volume.

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