Enhanced expansion and differentiation of t-cells through indirect agitation

Abstract

A method of T-cell expansion which directs differentiation towards central memory T-cells (Tcm). Also provided is a device to promote Tcm directed T-cell differentiation and high yield expansion, which may be employed in the presently disclosed method of T-cell expansion; and a method for screening to identify a process that modulates differentiation of T cells into Central Memory T cells (Tcm) subpopulation ex-vivo.

Description

[0001] ENHANCED EXPANSION AND DIFFERENTIATION OF T-CELLS THROUGH INDIRECT AGITATION

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to a method of T-cell expansion which directs differentiation towards central memory T-cells (Tern). Also provided is a device to promote Tern directed T-cell differentiation and high yield expansion, which may be employed in the presently disclosed method of T-cell expansion; and a method for screening to identify a process that modulates differentiation of T cells into Central Memory T cells (Tern) subpopulation ex-vivo.

[0004] BACKGROUND

[0005] The expansion process in a cell therapy manufacturing workflow is a crucial step in determining the efficacy of the therapy as the quality and quantity of the cells. Cell therapy requires GMP-grade reagents and reagent validation has to be performed extensively to reduce the risk of failure during manufacturing.

[0006] Central memory T-cells (Tern) are a subset of differentiated T-cells that have been shown to be more efficacious than effector memory T-cells (Tern) in cell therapy [36,37,38]. In particular, the enrichment of Tern has been correlated with improved clinical outcomes and T cell persistence [7,25-28], Hence, the improved efficacy of Tern can reduce the cell dosage per therapy and accelerate the manufacturing of T cell therapy products, both of which help to reduce the overall cost of T-cell therapy.

[0007] Studies have shown that it is possible to drive T-cell expansion towards the Tern phenotype by supplementing culture media with cytokines [9,10] and also by employing low DO2 levels (hypoxia) [16,17], However, the use of cytokines and specialized equipment necessary for hypoxia-based methods would significantly increase the complexity and cost of the culturing process, thereby increasing the cost of goods (COGS).

[0008] Hence, there is a need for an improved method of cell culture that is able to specifically enhance the expansion of Tern.

[0009] SUMMARY

[0010] In one aspect, there is provided a method of T-cell expansion which directs differentiation towards central memory T-cells (Tern), comprising:

[0011] (i) culturing undifferentiated T-cells in a cell culture device comprising a cell chamber and a media chamber, wherein the chambers are separated by a semi-permeable membrane, which permits nutrients and waste, but not T-cells to diffuse between the cell and media chambers, and wherein the cell and media chambers both contain media, but only the cell chamber contains the T-cells; and

[0012] (ii) employing indirect agitation to promote diffusion of nutrients and waste across the semi-permeable membrane, wherein only the media in the media chamber is agitated.

[0013] In another aspect, there is provided a device to promote central memory T-cell (Tern) - directed T-cell differentiation and high yield expansion during T cell manufacturing comprising:

[0014] (i) a double chamber vessel comprising a cell chamber holding the cells and a media chamber holding the bulk excess media, wherein both chambers are separated by a porous membrane, enabling diffusion of nutrients and waste between the two chambers, while retaining cells in the cell chamber; and

[0015] (ii) a homogenization module in the media chamber to promote homogenization of fluid between the two chambers.

[0016] In one embodiment, a supplement for directing differentiation towards central memory T-cells (Tern) is not added to the media.

[0017] In one embodiment, the supplement is a cytokine that directs differentiation towards Tern, for example selected from the group comprising: IL-7, IL-15 and IL-21.

[0018] In one embodiment, the levels of one or more of the following in the media are not adjusted: glucose, lactate, arginine and / or glutamine.

[0019] In one embodiment, the undifferentiated T-cells are selected from the group comprising naive T-cells and memory T-cells, such as naive T-cells.

[0020] In one embodiment, the source of undifferentiated T-cells is peripheral blood mononuclear cells (PBMCs), for example wherein PBMCs are cultured in the device.

[0021] In one embodiment, the differentiation towards Tern increases the total Tern yield and / or increases the proportion of Tom vs other types of cells, i.e. enriches for Tcm.

[0022] In one embodiment, the increase in Tcm yield and / or proportion of Tcm is associated with an increase in CDR7+CD45RA- expression levels.

[0023] In one embodiment, the diffusion of nutrients and waste across the semi- permeable / porous membrane homogenises the nutrient and waste distribution between the cell and media chambers.

[0024] In one embodiment, the method or device further comprises: monitoring / measuring the levels of one or more metabolites in the cell and media chambers, wherein similar levels of metabolites in both chambers indicates thorough homogenisation of nutrients and / or waste between the chambers, for example wherein the one or more metabolites are selected from the group comprising glucose, lactate, glutamine, ammonia and IL-2, in particular selected from the group comprising glucose and lactate.

[0025] In one embodiment, the indirect agitation is selected from the group comprising: stirring, shaking, vortexing, rolling and swirling, in particular stirring.

[0026] In one embodiment, the indirect agitation is employed by introducing a homogenization module in the media chamber, for example wherein the homogenization module improves the diffusion of nutrients and waste across the porous / semi-permeable membrane.

[0027] In one embodiment, the homogenization module comprises a stirrer, for example a mechanical or a magnetic stirrer, in particular a magnetic stirrer.

[0028] In one embodiment, the homogenization module further comprises an external mechanical controller to control the magnetic stirrer, thereby allowing the agitation / homogenization rate to be controlled.

[0029] In one embodiment, the agitation / homogenization rate is modulated such that thorough homogenisation of nutrients and waste is achieved with minimal disruption and / or turbulence to the contents in the cell chamber, for example such that the T-cells remain the first / cell chamber during the method.

[0030] In one embodiment, the stirrer is set to stir at 40 to 80 rpm, for example 40, 45, 50, 55, 60, 65, 70, 75 or 80 rpm, such as 55 to 65 rpm, in particular 60 rpm.

[0031] In one embodiment, the method or device further comprises activating the T- cells, for example by exposing the T-cells to one or more T-cell activators, for example wherein the one or more T-cell activators are selected from the group comprising anti- CD3, anti-CD28 and anti-CD2.

[0032] In one embodiment, the T-cells are exposed to the one or more T-cell activators on Day 0 and / or Day 7, in particular on Day 0.

[0033] In one embodiment, the agitation is first employed 10 minutes or more after T- cell activation, such as 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours or 24 hours.

[0034] In one embodiment, the agitation is first employed a day after the T-cells are activated.

[0035] In one embodiment, the agitation is employed throughout the entire T-cell expansion process.

[0036] In one embodiment, the agitation is employed intermittently during the T-cell expansion process, i.e. the agitation is only employed on some days.

[0037] In one embodiment, the agitation is employed for 2 or 3 consecutive days.

[0038] In one embodiment, the agitation is employed on one or more days selected from the group comprising: Days 1 , 2, 8 and 9, such as on Days 1 and 2, on Days 8 and 9 or on Days 1 , 2, 8 and 9; or one or more days selected from the group comprising: Days 5, 6, 7, 12, 13 and 14, such as on Days 5 to 7, on Days 12 to 14, or on Days 5 to 7 and Days 12 to 14.

[0039] In one embodiment, the T-cells are cultured for 6 to 14 days, such as 6, 7, 8, 9, 10, 11 , 12, 13 or 14 days; for example, wherein the T-cells are cultured for 6 or 7 days, in particular 6 days.

[0040] In one embodiment, the method or device further comprises harvesting the T- cells between Days 6 and 14, for example between Days 6 and 9, such as on Day 9, Day 8, Day 7, or Day 6, in particular on Days 6 or 7, for example on Day 6.

[0041] In one embodiment, the T-cells are activated on Day 0, agitation is first employed between 10 minutes to 24 hours after T-cell activation, and the T-cells are harvested between Days 6 and 10, such as on Day 7.

[0042] In one embodiment, the T-cells are activated on Day 0, agitation is first employed 10 minutes after T-cell activation, and the T-cells are harvested on Day 7.

[0043] In one embodiment, the T-cells are cultured in the presence of IL-2.

[0044] In one embodiment, the volume of the cell chamber is equal to or less than the volume of the media chamber.

[0045] In one embodiment, the culture media in the media chamber is equal to or in excess of the media in the cell chamber.

[0046] In one embodiment, the volume of the cell chamber is less than half the volume of the media chamber, for example is 0.5, 0.4, 0.3, 0.2 0.1 , or 0.05X the volume of the media chamber.

[0047] In one embodiment, the fluid height in the cell chamber is maintained at a low fluid height, for example 0.4 - 0.6 cm.

[0048] In one embodiment, the device comprises a well which contains the cell and media chambers.

[0049] In one embodiment, the device further comprises a lid.

[0050] In one embodiment, the semi-permeable / porous membrane rests on a support or is attached to the support, for example using adhesive tape, such as silicon doublesided adhesive tape.

[0051] In one embodiment, the semi-permeable / porous membrane is sandwiched between two gaskets, for example two biocompatible silicon gaskets, which are in turn sandwiched between the support and the first chamber.

[0052] In one embodiment, the semi-permeable / porous membrane is welded to the support using heat or ultrasonic welding methods.

[0053] In one embodiment, the method or device further comprises one or more securing components for ensuring an adequate seal of the first / cell chamber and / or ensuring that the semi-permeable / porous membrane remains flat during the method, for example wherein the securing components are selected from the group comprising clamps and screws, for example made of titanium or stainless steel, such as Stainless Steel 316.

[0054] In one embodiment, the device further comprises a sealant, such as a biocompatible silicon sealant, to seal any gaps between one or more of the following:

[0055] • Between the media chamber and the well;

[0056] • Between the cell and media chambers;

[0057] • Between the support and the cell chamber, media chamber or both chambers; and

[0058] • Between the lid and the well. In one embodiment, the semi-permeable membrane / porous has a pore size ranging from 0.2 to 5.0 pm, for example 0.2, 0.5, 1 , 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 pm, in particular a pore size of 1.0 pm.

[0059] In one embodiment, the device further comprises a gaseous exchange means which facilitates gas exchange between the device and the environment, for example wherein the gaseous exchange means is a tube, for example a silicon or rigid tube made of biocompatible materials

[0060] In one embodiment, the gaseous exchange means comprises a gas permeable membrane., for example wherein the gas permeable membrane has a pore size ranging from 0.1 to 0.3 pm, such as 0.1 , 0.15, 0.2, 0.25 or 0.3 pm, in particular a pore size of 0.2 pm.

[0061] In one embodiment, the well, cell chamber, media chamber, lid, semi- permeable / porous membrane, support, gaseous exchange means and / or gas permeable membrane is made of a biocompatible material, for example a material selected from the group comprising medical grade polycarbonate (PC), polystyrene, polypropylene (PP), nylon, and Teflon.

[0062] In one embodiment, the cell chamber, media chamber, lid and / or support are made of polycarbonate (PC) plastic.

[0063] In one embodiment, the lid and / or well are made of polypropylene (PP) plastic.

[0064] In one embodiment, the semi-permeable / porous membrane is made of a material selected from the group comprising polyethylene terephthalate (PET), polycarbonate (PC) plastic and polypropylene (PP), such as polyethylene terephthalate (PET).

[0065] In one embodiment, the gas permeable membrane is a PTFE hydrophobic membrane.

[0066] DEFINITIONS

[0067] As used herein the term “expansion” or “T-cell expansion” refers to process of artificially stimulating and growing T-cells in vitro to increase their numbers, i.e. to increase T-cell yield.

[0068] As used herein the term “differentiation” refers to the process by which naive T- cells develop into specialized T-cell subsets with distinct functions. This multi-step process is highly regulated and is crucial for the establishment of immunological memory and effector functions, thereby allowing the adaptive immune system to effectively respond to infections and malignancies. The differentiation of T cells is driven by a complex interplay of signalling pathways, such as cytokine signalling for example IL-2 and IL-12 signalling; transcription factors, for example T-bet, GATA3, RORyt, and FoxP3and epigenetic modifications, including DNA methylation and histone modifications.

[0069] The main stages of T-cell differentiation are:

[0070] 1. Activation and Clonal Expansion: Upon encountering an antigen, naive T cells become activated and transform into large blast cells. These blast cells then proliferate through clonal expansion.

[0071] 2. Differentiation into Effector and Memory T Cells: The cloned daughter cells differentiate into either effector T cells or memory T cells. Effector T cells are responsible for immediate immune responses, while memory T cells provide long-term immunity.

[0072] 3. Further Differentiation of Helper T Cells: Helper T cells continue to differentiate into various subsets, such as Th1 , Th2, Th17, and Treg cells, each with specific roles in immune regulation.

[0073] Key molecular mechanisms which drive T-cell differentiation include:

[0074] • Cytokine Signalling: Cytokines such as IL-2, IL-12, IL-4, and TGF-p play crucial roles in guiding the differentiation of T cells into specific subsets.

[0075] • Transcription Factors: Transcription factors like T-bet, GATA3, RORyt, and FoxP3 are pivotal in determining the fate of differentiating T cells.

[0076] • Epigenetic Modifications: Epigenetic changes, including DNA methylation and histone modifications, regulate gene expression patterns essential for T-cell differentiation.

[0077] As used herein the term “undifferentiated T-cells” refers to any T-cells which have not yet differentiated into central memory T-cells (Tern). Thus, within the context of the present disclosure, naive T-cells and memory T-cells are both considered undifferentiated T-cells, since neither of these have fully differentiated into central memory T-cells.

[0078] As used herein the term “memory T-cells” refers to a specialised subset of T-cells that persist for the long-term after an initial immune response, providing the immune system with the ability to respond more rapidly and effectively upon re-exposure to the same antigen. Memory T-cells can be further subclassified into 4 main groups:

[0079] • Central memory T-cells (Tcm), which have several attributes in common with stem cells, in particular the ability to self-renew;

[0080] • Effector memory T-cells (Tern), which are primarily active as CD8 cytotoxic T-cell variants;

[0081] • Tissue-resident memory T-cells (Trm), which are present over long periods in tissues, for example barrier tissues such as the epithelium, allowing these T-cells to rapidly respond to any pathogens which breach the barrier tissue; and

[0082] • Stem cell-like memory T-cells (Tscm), which have the ability to reconstitute the full diversity of memory and effect T-cell subpopulations and also the ability to self-renew;

[0083] In addition to these 4 main subsets, other less common subsets of memory T- cells have also been identified, including virtual memory T-cells (Tvm), which have the memory phenotype but do not have any obvious memory function; and regulatory memory T-cells (Treg) which help maintain immune tolerance by supressing immune responses.

[0084] As used herein the term “central memory T-cells” or Tcm refers to a subset of memory T-cells, characterized by their ability to rapidly expand upon encountering their specific antigen, and to self-renew, thereby providing long term immunity against pathogens. Tcm express homing receptors such as CD62L and CCR7, which allows them to re-circulate through the secondary lymphatic organs in a similar fashion to naive T-cells. This property distinguishes Tcm from other memory T-cell subtypes such as Tern and Trm, which are predominantly found in tissues. While Terns may not immediately produce effector cytokines after stimulation, they can eventually produce cytokines such as IL-2 and can proliferate and differentiate into effector cells. Due to their properties, Terns play a crucial role in preventing the development of chronic infections and cancers.

[0085] As used herein the term “media” refers to any liquid or solution which provides essential nutrients and / or environmental conditions required to support T-cell growth in vitro. Media suitable for T-cell expansion typically includes a buffering system such as sodium bicarbonate or HEPES in order to provide and maintain pH levels within a range of 7.0 to 7.4 which is optimal for T-cell growth. Examples of commonly used T-cell media include but are not limited to RPMI 1640 supplemented with fetal bovine serum (FBS), X-VIVO® 15, AIM-V™, CTS™ OpTmizer™ T cell Expansion SFM and ImmunoCult™- XF.

[0086] As used herein the term “nutrient” refers to any type or class of nutrient which is necessary to support T-cell expansion. Examples of nutrients include but are not limited to carbohydrates, such as glucose and sodium pyruvate; proteins, for example serum, such as fetal bovine serum (FBS) and human serum albumin (HSA), insulin, transferrin; amino acids, such as leucine, glutamine, serine and arginine; vitamins, such as B12, B7 ,B9, D and A; trace elements, such as zinc, copper and selenium; inorganic salts such as sodium chloride or potassium chloride; fatty acids, cholesterol; bile acid.

[0087] As used herein the term “waste” refers to any type or class of waste generated by T-cells during the process of T-cell expansion. Examples of waste include but are not limited to metabolites such as lactate, ammonia, and urea.

[0088] As used herein, the term “cell chamber” used interchangeably with the term “first chamber” refers to a chamber in a cell culture device / vessel which contains T-cells.

[0089] As used herein the term ‘media chamber’ used interchangeably with the term ‘second chamber’ refers to a chamber in the cell culture device / vessel which contains media but does not contain any T-cells.

[0090] As used herein the term “agitation” refers to any type of motion applied to the media in order to promote the movement of nutrients and waste between the first / cell chamber and second / media chamber. Thus, “agitation” includes but is not limited to shaking, stirring, vortexing, swirling, tumbling, rolling, perfusion, etc.

[0091] As used herein, the term “indirect agitation” refers to agitation which does not occur in a chamber containing T-cells. Thus, when ‘indirect agitation’ is employed, only the media in the chamber without T-cells is agitated. This can be achieved for example when the source of the agitation is not in the chamber containing T-cells.

[0092] Conversely, the term “direct agitation” refers to agitation of the media which occurs within the chamber containing T-cells. Thus, when ‘direct agitation’ is employed, the media in the chamber containing T-cells is agitated. This can be achieved for example by introducing a source of agitation in the chamber containing T-cells.

[0093] The term ‘homogenization’ or ‘homogenize’ as used herein refers to the act of making the media uniform between the first / cell chamber and second / media chamber, for example wherein there is a substantially equal distribution of nutrients and waste between the chambers.

[0094] As used herein the term “supplement for directing differentiation towards central memory T-cells (Tern)” refers to any supplement which has the ability to steer the differentiation of T-cells towards the Tern phenotype. Examples of such supplements include but are not limited to specific cytokines, in particular IL-7, IL-15 and IL-21.

[0095] The term “T-cell activation” as used herein refers to the process of stimulating T- cells to proliferate and express their effector functions in vitro, thereby mimicking the natural immune response in vivo.

[0096] Hence, as used herein the term “T-cell activator” refers to any agent or substances used to stimulate the activation and proliferation of T-cells in vitro. These activators mimic the natural signals required for T-cell activation, which typically involve the engagement of the T-cell receptor (TCR) and co-stimulatory molecules.

[0097] Examples of T-cell activators include but are not limited to antibodies, such as anti-CD3 and anti-CD28 antibodies; peptide-MHC complexes, such as synthetic or recombinant complexes that specifically engage the TCR, mimicking antigen presentation by antigen-presenting cells (APCs); B7 molecules, such as B7-1 (CD80) and B7-2 (CD86) which interact with CD28 on T-cells to deliver co-stimulatory signals; and cytokines, such as IL-2, IL-5, IL-7 and IL-21 which are known to promote T-cell growth, differentiation, and memory formation.

[0098] The term "and / or", e.g., "X and / or Y" is understood to mean either "X and Y" or "X or Y" and should be taken to provide explicit support for both meanings or for either meaning.

[0099] Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, "entirely" or “completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements / components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist”, and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of + / - 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1% of the disclosed value.

[0100] Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1% to 2%, 1% to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1%, 2%, 3%, 4% and 5%. It is to be appreciated that the individual numerical values within the range also include integers, fractions and decimals. Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1% to 5% is intended to have specifically disclosed the ranges 1 .00% to 5.00% and also 1 .0% to 5.0% and all their intermediate values (such as 1 .01 %, 1.02% ... 4.98%, 4.99%, 5.00% and 1.1%, 1.2% ... 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth / breadth of a range.

[0101] Additionally, when describing some embodiments, the disclosure may have disclosed a method and / or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and / or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.

[0102] Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.

[0103] DESCRIPTION OF EMBODIMENTS

[0104] It will be appreciated by a person skilled in the art that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

[0105] The present inventors have successfully developed an in vitro method of T-cell expansion which is able to direct differentiation towards the central memory T-cell (Tern) phenotype, i.e. increases the total yield and / or overall proportion of Tern.

[0106] Briefly, undifferentiated T-cells are cultured using a vessel comprising two separate chambers stacked vertically and separated by a porous membrane to allow for flow of aqueous material. Culture media is added to the lower chamber and the cell population suspended in culture media is added to the upper chamber. Agitation of the lower chamber is executed by either placing a stir bar in the lower chamber and setting the culture vessel on a stir plate, or perfusing through the lower chamber. Cell culture is performed for a predetermined period of time whereby the cell population number and phenotype are assessed at the end of the culture period. Media changes are performed periodically as required.

[0107] Thus, in one aspect, there is provided a method of T-cell expansion which directs differentiation towards central memory T-cells (Tern), comprising:

[0108] (i) culturing undifferentiated T-cells in a cell culture device comprising a first chamber and a second chamber, wherein the chambers are separated by a semi-permeable membrane, which permits nutrients and waste, but not T-cells to diffuse between the first and second chambers, and wherein the first and second chambers both contain media, but only the first chamber contains the T-cells; and (ii) employing indirect agitation to promote diffusion of nutrients and waste across the semi-permeable membrane, wherein only the media in the second chamber is agitated.

[0109] Advantageously, the method employs indirect agitation of the culture media, i.e. a purely physical method. Hence, the presently disclosed method is able to direct differentiation towards the Tern phenotype without the usual need to supplement the media with growth factors, cytokines and other drugs that drive differentiation specifically towards Tern; and without the need for specialized equipment (typically required for hypoxia-based methods). This therefore allows for high Tern yield with lower culture process complexity and lower manufacturing costs compared to prior art methods.

[0110] Surprisingly, the present inventors have demonstrated that use of indirect agitation as opposed to other forms of agitation, such as direct agitation, resulted in the highest number of Tern after 7 days of culture. See indirect stirring vs direct stirring in Figures 10, 11 and Table 11. Thus, the novel use of indirect agitation has the technical benefit of producing a high Tern yield within a short period of culture time.

[0111] In another aspect, there is provided a method of T-cell expansion which directs differentiation towards central memory T cells (Tern), comprising:

[0112] (i) culturing undifferentiated T-cells in a cell culture device comprising a first chamber and a second chamber, wherein the chambers are separated by a semi-porous membrane, which permits nutrients and waste, but not T-cells to diffuse between the first and second chambers, and wherein both the first and second chambers contain media but only the first chamber contains the T-cells; and

[0113] (II) agitating the media only in the second chamber to promote diffusion of nutrients and / or waste across the semi-permeable membrane.

[0114] In another aspect, there is provided a method to promote Tern - directed T cell differentiation and high yield expansion during T cell culture in a double chamber vessel with homogenization module and methodology, comprising one or more of the following steps: a. Culturing of T cells in the cell chamber of a double chamber vessel, b. Adding bulk excess T cell culture media in the media chamber of the double chamber vessel c. Separating the two chambers through a porous membrane allowing for diffusion of fluid across both chambers d. Retaining the cells in the cell chamber throughout the culture e. Ensuring low and consistent media height in the cell culture chamber throughout the culture f. Ensuring media chamber is fully filled with media and media can equilibrate easily between cell and media chamber g. Engaging the homogenization module in the media chamber, to homogenize the fluid in both chambers h. The homogenization module is engaged 10 minutes or later after cell activation.

[0115] In another aspect, there is provided a method to promote Tern - directed T cell differentiation and high yield expansion during T cell culture in a double chamber vessel with homogenization module and methodology, comprising: a. Culturing of T cells in the cell chamber of a double chamber vessel, b. Adding bulk excess T cell culture media in the media chamber of the double chamber vessel c. Separating the two chambers through a porous membrane allowing for diffusion of fluid across both chambers d. Retaining the cells in the cell chamber throughout the culture e. Ensuring low and consistent media height in the cell culture chamber throughout the culture f. Ensuring media chamber is fully filled with media and media can equilibrate easily between cell and media chamber g. Engaging the homogenization module in the media chamber, to homogenize the fluid in both chambers h. The homogenization module is engaged at least 1 day after cell activation.

[0116] In one aspect, there is provided a method for screening to identify the process that modulates differentiation of T cells into Central Memory T cells (Tern) ex-vivo, the method comprising, a. Dividing the population of undifferentiated T cells into a first and second subpopulation of undifferentiated T cells b. Incubating a first sub-population of undifferentiated T cells in a cell chamber, and bulk T cell culture media in a separate media chamber, with a porous membrane separating the two chambers allowing for passage of fluid across c. Incubating a second sub-population of undifferentiated T cells in a cell chamber, and bulk T cell culture media in a separate media chamber, with a porous membrane separating the two-chamber allowing for passage of fluid across, and homogenization of the fluid in both chambers with a homogenization module in the media chamber d. Detecting the expression of CD45RA and CCR7 markers in each of the said first and second sub populations of T cells after incubation without or with the homogenization module and methodology e. Comparing the expression of CD45RA and CCR7 markers in each of the said first and second sub populations of differentiated T cells generated following incubation without or with the homogenization module and methodology, wherein a change in the expression of the CD45RA and CCR7 markers following incubation with the homogenization module and methodology relative to the expression of CD45RA and CCR7 markers following incubation without the homogenization module and methodology, indicate that the homogenization module and methodology is a modulator of Tcm - directed T cell differentiation.

[0117] In one embodiment, the increase in CCR7+ CD45RA- and decrease CCR7- CD45RA- markers expression following incubation with the homogenization module and methodology, indicates the module and methodology is a promoter of Tcm - directed T cell differentiation and an inhibitor of Tef-directed T cell differentiation.

[0118] Any one of the methods described above may be performed in vitro. Thus, in one embodiment, the method is an in vitro method.

[0119] In one aspect, there is provided a device to promote central memory T-cell (Tcm) - directed T-cell differentiation and high yield expansion during T-cell manufacturing comprising:

[0120] (i) a double chamber vessel comprising a cell chamber holding the cells and a media chamber holding the bulk excess media, wherein both chambers are separated by a porous membrane, enabling diffusion of nutrients and waste between the two chambers, while retaining cells in the cell chamber. In one embodiment, the device further comprises:

[0121] (ii) a homogenization module in the media chamber to promote homogenization of fluid between the two chambers.

[0122] Advantageously, the homogenization module promotes the diffusion of nutrients and waste across the membrane, thereby helping to distribute the nutrients and waste equally between the two chambers.

[0123] Hence, in one aspect, there is provided a device to promote central memory T-cell (Tern) - directed T cell differentiation and high yield expansion during T cell manufacturing comprising:

[0124] (i) a double chamber vessel comprising a cell chamber holding the cells and a media chamber holding the bulk excess media, wherein both chambers are separated by a porous membrane, enabling diffusion of nutrients and waste between the two chambers, while retaining cells in the cell chamber; and

[0125] (ii) a homogenization module in the media chamber to promote homogenization of fluid between the two chambers.

[0126] In one aspect, there is provided a device to direct differentiation towards central memory T-cells (Tern), comprising:

[0127] (i) a double chamber vessel comprising a cell chamber holding the cells and a media chamber holding the bulk excess media, wherein both chambers are separated by a porous membrane, enabling diffusion of nutrients and waste between the two chambers, while retaining cells in the cell chamber; and

[0128] (ii) a homogenization module in the media chamber to promote homogenization of fluid between the two chambers.

[0129] The devices disclosed herein are suitable for use in any one of the methods described herein.

[0130] Thus, in one embodiment, the device is employed in the method of T-cell expansion which directs differentiation towards central memory T-cells (Tern). In one embodiment, the device is employed in the method to promote Tern - directed T cell differentiation and high yield expansion during T cell culture in a double chamber vessel with homogenization module and methodology. In another embodiment, the device is employed in the method for screening to identify the process that modulates differentiation of T cells into Central Memory T cells (Tern) ex-vivo. In one embodiment, a supplement for directing differentiation towards Tern is not added to the media. Advantageously, the presently disclosed method / device does not rely on the use of media supplements such as cytokines to direct differentiation towards the Tern phenotype. This has the benefit of reducing culture complexity and the overall cost of producing Tern.

[0131] In one embodiment, the agent is a cytokine that directs differentiation towards Tern, for example selected from the group comprising: IL-7, IL-15 and IL-21. Thus, in one embodiment, IL-7, IL-15 and IL-21 are not added to the media.

[0132] Studies have shown that levels of glucose, lactate, arginine and / or glutamine levels can be adjusted in order to enrich for Tern. Advantageously, the presently disclosed method does not rely on adjusting or fine-tuning any of these levels. Accordingly, in one embodiment, the glucose, lactate, arginine and / or glutamine levels in the media are not adjusted.

[0133] In one embodiment, the undifferentiated T-cells are selected from the group comprising naive T-cells and memory T-cells. In one embodiment, the undifferentiated T-cells are naive T-cells. In one embodiment, the undifferentiated T-cells are memory T- cells. In one embodiment, the undifferentiated T-cells are naive T-cells and memory T- cells.

[0134] In one embodiment, the source of undifferentiated T-cells is peripheral blood mononuclear cells (PBMCs). Thus, in one embodiment, peripheral blood mononuclear cells (PBMCs) are cultured in the device.

[0135] In one embodiment, the first chamber is a cell chamber and / or the cell chamber is the first chamber.

[0136] In one embodiment, the second chamber is a media chamber and / or the media chamber is the second chamber.

[0137] In one embodiment, the differentiation towards Tern increases the total Tern yield.

[0138] In one embodiment, the differentiation towards Tern increases the proportion of Tern vs other types of T-cells, such effector T-cells (Tern).

[0139] The extent of differentiation towards the Tern phenotype can be assessed by testing the T-cells for the expression of markers associated with the Tern phenotype, for example one or more of the following: CD62L+, CCR7+, CD45RO+, CD27+, CD28+, CD127+ and CD45RA-. Hence, in one embodiment, the increase in Tern yield and / or proportion of Tern is associated with an increase in the expression levels of one or more of the following: CD62L+, CCR7+, CD45RO+, CD27+ and CD28+.

[0140] In one embodiment, the increase in Tern yield and / or proportion of Tern is associated with a decrease in CD45RA expression, i.e. an increase in T-cells that are CD45RA-.

[0141] In one embodiment, the increase in Tern yield and / or proportion of Tern is associated with an increase in CCR7+CD45RA- expression levels.

[0142] In one embodiment, the diffusion of nutrients and waste across the semi- permeable / porous membrane homogenises the nutrient and waste distribution between the first / cell and second / media chambers.

[0143] In one embodiment, the method further comprises: monitoring / measuring the levels of one or more metabolites in the first / cell and second / media chambers, wherein similar levels of metabolites in both chambers indicates thorough homogenisation of nutrients and / or waste between the chambers.

[0144] In one embodiment, the one or more metabolites are selected from the group comprising T-cell activators, glucose, lactate, glutamine, ammonia and IL-2.

[0145] In one embodiment, the one or more metabolites are selected from the group comprising glucose, lactate, glutamine, ammonia and IL-2.

[0146] In one embodiment, the one or more metabolites are selected from the group comprising glucose and lactate.

[0147] In one embodiment, the indirect agitation is selected from the group comprising: stirring, shaking, vortexing, rolling and swirling, in particular stirring.

[0148] In one embodiment, the indirect agitation is employed by introducing a homogenization module in the second / media chamber.

[0149] In one embodiment, the homogenization module and methodology in the second / media chamber improves the diffusion of nutrients and waste across the porous / semi-permeable membrane.

[0150] In one embodiment, the homogenization module comprises a stirrer, for example a mechanical or a magnetic stirrer.

[0151] In one embodiment, the homogenization module comprises a magnetic stirrer.

[0152] In one embodiment, the homogenization module further comprises an external mechanical controller to control the magnetic stirrer, thereby allowing the agitation / homogenization rate to be controlled.

[0153] In one embodiment, the agitation / homogenization rate is modulated such that thorough homogenisation of nutrients and waste is achieved with minimal disruption and / or turbulence to the contents in the cell chamber.

[0154] In one embodiment, the T-cells remain in the first chamber / cell chamber during the method.

[0155] In one embodiment, the stirrer is set to stir at 40 to 80 rpm, for example 40, 45, 50, 55, 60, 65, 70, 75 or 80 rpm.

[0156] In one embodiment, the stirrer is set to stir at 55 to 65 rpm, in particular 60 rpm.

[0157] In one embodiment, the method further comprises activating the T-cells.

[0158] In one embodiment, the T-cells are activated by exposing the T-cells to one or more T-cell activators.

[0159] In one embodiment, the one or more T-cell activators are able to diffuse across the semi-porous / permeable membrane.

[0160] In one embodiment, the one or more T-cell activators are selected from the group comprising anti-CD3, anti-CD28 and anti-CD2.

[0161] In one embodiment, the T-cells are exposed to the one or more T-cell activators on Day 0 and / or Day 7. Advantageously, the present inventors have established that T- cell enrichment occurs by Day 7 if the T-cells are activated on Day 0 and occurs by Day 13 with an activation on Day 0 and an activation on Day 7.

[0162] In one embodiment, the agitation is first employed 10 minutes or more after T- cell activation, such as 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours or 24 hours. Advantageously, starting indirect agitation as soon as 10 minutes after T-cell activation may result in enhanced Tern yield.

[0163] In one embodiment, the agitation is first employed a day after the T-cells are activated.

[0164] In one embodiment, the agitation is employed throughout the entire T-cell expansion process, i.e. constant agitation. The advantage of employing agitation throughout the entire expansion method is that it may result in a higher proportion of Tern, i.e. enriches for Tern compared to intermittent agitation.

[0165] In one embodiment, the agitation is employed intermittently during the T-cell expansion process, i.e. the agitation is only employed on some days. Surprisingly, the present inventors have established that the agitation does not have to be continuously employed to drive Tern generation. Advantageously, intermittent agitation reduces handling and therefore labour costs. Furthermore, intermittent agitation may result in a higher overall total T-cell and Tern yield compared to constant agitation.

[0166] In one embodiment, the agitation is employed for 2 or 3 consecutive days.

[0167] In one embodiment, agitation is employed on one or more of the days selected from the group comprising: Days 1 , 2, 5, 6, 7, 8, 9, 12, 13 and 14.

[0168] In one embodiment, agitation is employed on one or more days selected from the group comprising: Days 1 , 2, 8 and 9, such as on Days 1 and 2, on Days 8 and 9 or on Days 1 , 2, 8 and 9.

[0169] In one embodiment, agitation is employed on one or more days selected from the group comprising: Days 5, 6, 7, 12, 13 and 14, such as on Days 5 to 7, on Days 12 to 14, or on Days 5 to 7 and Days 12 to 14.

[0170] In one embodiment, agitation is employed on Days 0 to 14. In one embodiment agitation is employed on Days 1 to 2 and Days 7 to 8. In one embodiment, agitation is employed on Days 5 to 7 and Days 12 to 14.

[0171] In one embodiment, the T-cells are cultured for 6 to 14 days, such as 6, 7, 8, 9, 10, 11, 12, 13 or 14 days.

[0172] In one embodiment, the T-cells are cultured for 6 or 7 days, for example 6 days. Advantageously, a shorter culture period allows Tern cells to be delivered to patients in a time sensitive manner. Furthermore, cells cultured with a shorter duration have a lower exhaustion phenotype which is detrimental to treatment.

[0173] In one embodiment, the method further comprises harvesting the T-cells between Days 6 and 14, for example between Days 6 and 9, such as on Day 9, Day 8, Day 7, or Day 6.

[0174] In one embodiment, the T-cells are harvested on Days 6 or 7. In one embodiment, the T-cells are harvested on Day 7. In one embodiment, the T-cells are harvested on Day 6. Advantageously, the present inventors have demonstrated that the T-cells can be successfully harvested as early as on Day 6 due to a high Tern yield being achieved by this day.

[0175] In one embodiment, the T-cells are activated on Day 0, agitation is first employed between 10 minutes to 24 hours after T-cell activation, and the T-cells are harvested on between Days 6 and 10, such as on Day 7. In one embodiment, the T-cells are activated on Day 0, agitation is first employed 10 minutes after T-cell activation, and the T-cells are harvested on Day 6. The present inventors have shown that in general a longer culture time will produce a higher Tern yield. However, this optimised protocol balances Tern yield against culture time, thereby producing high quality T-cells enriched for the Tern phenotype within a short period of time. In order to achieve higher yields despite the shortened culture period, the skilled person can for example adjust the starting cell population number and / or culture multiple batches of T-cells in multiple devices in parallel.

[0176] In one embodiment, the T-cells are cultured in the presence of IL-2. The advantage of adding IL-2 is that this cytokine acts as a growth factor to stimulate T-cell proliferation and enhance T-cell yield.

[0177] In one embodiment, the method is performed in an incubator, for example a 36 °C or 37 °C CO2 incubator.

[0178] In one embodiment, the volume of the first / cell chamber is equal to or less than the volume of the second / media chamber. Advantageously, the volume of media in the second / media chamber is at least 1x the volume of the media in the first / cell chamber because this helps to facilitate molecule exchange between the two chambers.

[0179] In one embodiment, the culture media in the media / second chamber is equal to or in excess of the media in the cell / first chamber.

[0180] In one embodiment, the volume of the first / cell chamber is less than half the volume of the second / media chamber, for example is 0.5, 0.4, 0.3, 0.2 0.1 , or 0.05X the volume of the second / media chamber.

[0181] In one embodiment, the cells in the first / cell chamber are maintained at an optimum cell density with respect to culture volume. In one embodiment, the cells in the first / cell chamber are maintained at an optimum cell density with respect to culture area. These can be achieved for example by expanding the culture area in order to maintain optimal cell density.

[0182] In one embodiment, the fluid height in the first / cell chamber is maintained at a low fluid height, for example 0.4 - 0.6 cm. A low fluid height in the cell chamber provides at least the following benefits:

[0183] • better gaseous exchange between the cells in media, and air-media interface, more efficient nutrient exchange / homogenization, and more contact between cells and activators.

[0184] In one embodiment, the device comprises a well which contains the first / cell and second / media chambers.

[0185] In one embodiment, the device further comprises a lid.

[0186] In one embodiment, the semi-permeable / porous membrane rests on a support.

[0187] In one embodiment, the semi-permeable / porous membrane is attached to the support.

[0188] In one embodiment, the semi-permeable membrane / porous is attached to the support using adhesive tape, such as silicon double-sided adhesive tape.

[0189] In one embodiment, the semi-permeable / porous membrane is sandwiched between two gaskets for example two biocompatible silicon gaskets, which are in turn sandwiched between the support and the first / cell chamber.

[0190] In one embodiment, the semi-permeable / porous membrane is welded to the support using heat or ultrasonic welding methods.

[0191] In one embodiment, the device further comprises one or more securing components for ensuring an adequate seal of the first / cell chamber and / or ensuring that the semi-permeable / porous membrane remains flat during the method.

[0192] In one embodiment, the securing components are selected from the group comprising clamps and screws, for example made of titanium or stainless steel, such as Stainless Steel 316.

[0193] In one embodiment, the device further comprises a sealant to seal any gaps between one or more of the following:

[0194] • Between the second / media chamber and the well;

[0195] • Between the first / cell and second / media chambers;

[0196] • Between the support and the first / cell chamber, second / media chamber or both chambers; and

[0197] • Between the lid and the well.

[0198] In one embodiment, the sealant is a biocompatible silicon sealant.

[0199] In one embodiment, the semi-permeable membrane / porous has a pore size ranging from 0.2 to 5.0 pm, for example 0.2, 0.5, 1 , 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 pm. In one embodiment, the semi-permeable / porous membrane has a pore size of

[0200] 1.0 pm

[0201] In one embodiment, the device further comprises a gaseous exchange means which facilitates gas exchange between the device and the environment.

[0202] In one embodiment, the gaseous exchange means is a tube, for example a silicon or rigid tube made of biocompatible materials.

[0203] In one embodiment, the gaseous exchange means comprises a gas permeable membrane.

[0204] In one embodiment, the gas permeable membrane has a pore size ranging from 0.1 to 0.3 pm, such as 0.1 , 0.15, 0.2, 0.25 or 0.3 pm.

[0205] In one embodiment, the gas permeable membrane has a pore size of 0.2 pm.

[0206] In one embodiment, the well, first / cell chamber, second / media chamber, lid, semi-permeable / porous membrane, support, gaseous exchange means and / or gas permeable membrane is made of a biocompatible material.

[0207] In one embodiment, the well, first / cell chamber, second / media chamber, lid, support, and / or gaseous exchange means is made of a material selected from the group comprising medical grade polycarbonate (PC), polystyrene, polypropylene (PP), nylon, and Teflon.

[0208] In one embodiment, the first / cell chamber, second / media chamber, lid and / or support are made of polycarbonate (PC) plastic.

[0209] In one embodiment, the lid and / or well are made of polypropylene (PP) plastic.

[0210] In one embodiment, the semi-permeable membrane / porous is made of a material selected from the group comprising polyethylene terephthalate (PET), polycarbonate (PC) plastic and polypropylene (PP).

[0211] In one embodiment, the semi-permeable / porous membrane is made of polyethylene terephthalate (PET).

[0212] In one embodiment, the gas permeable membrane is a PTFE hydrophobic membrane.

[0213] In one embodiment, the device is sterilized prior to use in the method, for example by gamma irradiation, autoclaving and / or ethylene oxide (EtO) sterilization.

[0214] In one embodiment, the device is sterilized by autoclaving.

[0215] In one embodiment, the device is a bioreactor.

[0216] In one aspect, there is provided a method or device substantially as described herein, for example with reference to the Drawings.

[0217] BRIEF DESCRIPTION OF FIGURES

[0218] Figure 1 shows an exploded view of the basic components of a two-chamber vessel of the present disclosure.

[0219] Figure 2 shows schematic diagrams of the cell culture device of the present disclosure with (A) indirect agitation, (B) perfusion and (C) direct agitation.

[0220] Figure 3 shows an Illustration of assisted chemical homogenization between the two chambers of the cell culture device in the presence of indirect agitation.

[0221] Figure 4 shows an illustration of passive and restricted chemical diffusion between the two chambers of the cell culture device in the absence of indirect agitation.

[0222] Figure 5A shows a graph of the mean cell population of five runs. Paired two- tailed t-tests were performed between static and stirring cell population numbers for Day 7, 10 / 11 and 14. * p<0.05.

[0223] Figure 5B shows graphs of the Cell populations on Day 10 / 11 and 14 for the five runs.

[0224] Figure 6A shows a graph of the population of T-cells and NK-cells across five runs. Dotted lines indicates levels of 10% and 90% live lymphocytes. DO indicates population at Day 0.

[0225] Figure 6B shows a graph of the population of helper and cytotoxic T-cells across five runs.

[0226] Figure 6C shows a graph of the population of PD-1 T-cells across five runs.

[0227] Figure 7A shows a graph of the populations of Tem and Tcmacross Runs 2-5. DO indicates population at Day 0. “Static” and “Stirring” indicate population from these two groups on Day 14.

[0228] Figure 7B shows a graph of the average of Temand Tcmpopulation across Runs 2-5. Paired two-tailed t-tests were performed between static and stirring population group. * p<0.05.

[0229] Figure 7C shows a graph of populations of Tnand Temra across Runs 2-5.

[0230] Figure 8A shows a graph of the Levels of %glucose normalized to Day 0 in media change (MC) and cell chamber (CC) in static and stirring bioreactor across Runs 1-5. Paired two-tailed t-tests were performed between levels in MC and CC. * p<0.05, ** p<0.01. Figure 8B shows a graph of the calculated values of MC / CC ratio in static and stirring bioreactor across runs 1-5. Dotted line represents baseline ratio of 15.8.

[0231] Figure 8C shows a graph of the representative data of Run 5 for %glucose level normalized to level in Day 0 (left y-axis) and cell population counted on the Day 0, 4, 11 and 14 (right y-axis). Media change and reactivation of culture was performed in Day 7.

[0232] Figure 9A shows a graph of the Lactate (mM) in MC and CC in static and stirring bioreactor across runs 1-5. Paired two-tailed t-tests were performed between levels in MC and CC. * p<0.05, ** p<0.01.

[0233] Figure 9B shows a graph of the calculated values of MC / CC ratio in static and stirring bioreactor across runs 1-5. Dotted line represents baseline ratio of 15.8.

[0234] Figure 9C shows as graph of representative data of Run 5 for lactate (mM) in Day 0 (left y-axis) and cell population counted on the Day 0, 4, 11 and 14 (right y-axis). Media change and reactivation of culture was performed in Day 7.

[0235] Figure 10 shows a graph of the Cell growth with different agitation methods. All agitated groups (indirect stirring on D1-7 and D8-14, direct stirring on D1-7 and D8-14, and perfusion on D1-7 and D8-14) achieved higher cell yield compared to static culture group.

[0236] Figure 11 shows a graph of the Cell phenotypes with different agitation methods. The indirect stirring group on D1-7 and D8-14 achieved higher % Tern compared to static culture group on D7 and D14. The perfusion group on D1-7 and D8-14 achieved higher % Tern compared to static culture group on D14. The direct stirring group on D1-7 and D8-14 achieved lower % Tern compared to static culture group on both D7 and D14,

[0237] Figure 12 shows a graph of the cell growth with different indirect stirring settings. The group with stirring on D1-2 and D8-9, and group with stirring on D5-7 and D12-14 achieved higher total cell yield compared to the static culture group. The group with stirring on DO-14 achieved lower total cell yield compared to the static culture group.

[0238] Figure 13 shows a graph of the cell phenotypes with different indirect stirring settings. All indirect stirring groups achieved higher % Tcm compared to the static culture group on D14.

[0239] Figure 14 shows a graph of the cell growth comparing indirect stirring from 10 mins to Day 7, indirect stirring from Day 1 to 7 and static culture.

[0240] Figure 15 shows a graph of the cell phenotypes comparing indirect stirring from 10 mins to Day 7, indirect stirring from Day 1 to 7 and static culture. Figure 16 demonstrates that Incorporation of a stirring mechanism in a doublechamber bioreactor improves media exchange within bioreactor and promotes cell proliferation. (A) shows schematic diagrams of bioreactors used in Example 2. The left panel shows the design of the BECA-D bioreactor. Shaded area with white dots indicates area where cells are seeded and cultured. Large arrow indicates direction of expansion of culture area via pull of the internal plunger. The right panel shows the design of doublechamber prototype bioreactor. (B) shows a diagram of the side view of media exchange (vertical arrows) between the cell and media chambers through the porous membrane. Introduction of stirring mechanism via a stirbar enhances media exchange. (C) shows a diagram of the experimental plan for comparison of Static and Stirring double-chamber prototype bioreactors. (D) shows graphs of cell counts (solid lines, left axis) and viability (dashed lines, right axis) for both Static and Stirring cultures across Donor 1 to 4. * indicates significant difference in cell count values between Static and Stirring cultures with p-value <0.05. (E) shows graphs of the glucose and lactate concentrations in the cultures MC (solid lines) and CC (dashed lines). (F) shows graphs of the ratio of glucose and lactate concentrations between MC and CC. Dotted line indicates the ideal mixing ratio of 1.0. * Indicates a p-value of <0.05 and ** indicates a p-value of <0.01 for a one- sample T-test with the determined value of 1.0. Significant p-values are indicated above the data bars. (1.5 column fitting image)

[0241] Figure 17 demonstrates that the incorporation of a stirring mechanism enriched for central memory T cells in culture. (A) shows a graph of the percentage CD3 population of PBMCs on Day 0 and in Static and Stirring cultures on Day 14. Dotted line indicates 90% population of live lymphocytes in culture. (B) shows a graph of percentage CD4 and CD8 population of CD3 PBMCs on Day 0 and in Static and Stirring cultures on Day 14. n.s. not significant (C) shows a representative flow cytometry plot (Donor 2) for assessment of CCR7 and CD45RA markers in the Day 0 PBMC culture and Day 14 Static and Stirring cultures. Representative gating for TN, TCM, TEM, TEM in the CD4 and CD8 population are as shown. (D) shows a graph of the percentage of each T cell memory subtype in CD4 and CD8 population. (E) shows a graph of the percentage of TCM and TEM cells in CD4 and CD8 population. P-values indicated were calculated using paired t-test. * p<0.05 (single column fitting image)

[0242] Figure 18 demonstrates that BECA-DS+ preferentially promoted Central Memory T cell (TCM) proliferation. (A) shows a schematic diagram of the experimental plan for comparison of BECA-D, BECA-DS+, and G-Rex10 cultures. (B) shows graphs of cell counts (solid lines, left axis) and viability (dashed lines, right axis) across Donor 1 to 4. (C) shows graphs of average cell counts across Donor 1 to 4 for each day of culture (3 or 4, 7, 10 or 11 , 14). (D) shows a graph of the percentage CD3 population of PBMCs on Day 0 and in the three bioreactors on Day 14. Dotted line indicates 90% population of live lymphocytes in culture. (E) shows a graph of the percentage CD4 and CD8 population of CD3 PBMCs on Day 0 and in the three bioreactors on Day 14. (F) Representative flow cytometry plot (Donor 4) for assessment of CCR7 and CD45RA markers in the Day 0 PBMC culture and Day 14 cultures. Representative gating for TN, TCM, TEM, TEMRA in the CD4 and CD8 population are as shown. (G) shows a graph of the percentage of each T cell memory subtype in CD4 and CD8 population. (H) shows a graph of the percentage of TCM and TEM cells in CD4 and CD8 population. P-values indicated were calculated using paired t-test. n.s. not significant * p<0.05. (1.5 column fitting image)

[0243] Figure 19 shows supplementary data for Example 2. (A) shows graphs of the culture areas of BECA-D, BECA-DS+, and G-Rex10 across four donors during the 14- day culture period. (B) shows graphs of the culture densities of the three bioreactors across four donors during the 14-day culture period. (C) shows graphs of the glucose and lactate concentrations in the cultures MC (solid lines) and CC (dashed lines) of BECA-D and BECA-DS+, and in G-Rex10. (D) shows graphs of the ratio of glucose and lactate concentrations between MC and CC in BECA-D and BECA-DS+. Dotted line indicates the ideal mixing ratio of 1 .0. * indicates a p-value of <0.05 and ** indicates a p- value of <0.01 for a one-sample T-test with the determined value of 1.0. Significant p- values are indicated above the data bars. (E) shows heatmaps of glucose per cell (mg / cell) in the three bioreactors across four donors during the 14-day culture period. Dots indicate a value of <0.5 mg glucose / cell.

[0244] EXAMPLES

[0245] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications may be made without deviating from the scope of the invention. Example embodiments are not

[0246] 27

[0247] SUBSTITUTE SHEET RULE 26 necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

[0248] Example 1 - Proof of concept study

[0249] A comparison study with a static control on T-cell expansion and differentiation to demonstrate the effects of the presently claimed method. This study was performed using a bioreactor designed in-house, depicted in Figures 1 to 4.

[0250] Cell culture device of the present disclosure

[0251] A two-chamber vessel as illustrated in Figure 1A comprises a cell chamber 1 where the cells 10 are located in, and a bulk media chamber 5. A porous membrane 2 attached to a filter support structure 3, separates the two chambers while allowing for diffusion of nutrients and waste materials. This diffusion and homogenization are made more effective with the presence of an agitation, which can be either a mechanical or magnetic stirrer 4 in the bulk media chamber 5, or a perfusion device 11 and 12 in the bulk media chambers, or direct stirrer 13 in the 1 cell chamber, made from biocompatible materials. See Figures 2A to 2C.

[0252] The cell and bulk media chamber can be made of biocompatible materials such as medical grade polycarbonate (PC), polystyrene, polypropylene (PP), nylon, or teflon. Polyester (PETE), PC, PP, porous membrane can be used, with hydrophobic, hydrophilic or cell culture treated surfaces depending on the type of cells in culture. Further material consideration also includes the ability to sterilize these setups through gamma irradiation, autoclave, or ethanol (EtO) sterilization. Pore sizes ranging from 0.2pm to 5.0pm in diameter can also be selected depending on the type of cells in culture, namely cell size range.

[0253] In a full two-chamber cell culture device setup as illustrated in Figure 2, the two- chamber assembly as illustrated in Figure 1 sits in a containing device consisting of the well 9, and the lid 8. This ensures that the entire setup remains sealed and sterile during incubation. A structure 7 protruding out of the lid which can be a silicone or rigid tube made of biocompatible materials is fixed with a gas membrane housing 6. This enables continuous gas exchange during incubation in the sealed setup, to allow for pH equilibration to optimum values for cell culture. The gas membrane used are normally 0.2|jm pore size, PTFE hydrophobic membranes. The Inner Diameter (ID) of the tubular structure used can be as large as the gas membrane housing would allow and as much as the lid can hold, to enable sufficient gaseous exchange. This structure is sealed onto the lid by making the hole on the lid smaller than the Outer Diameter (OD) ensuring tight fit. Biocompatible silicone sealant and adhesive is further applied onto the edges of the assembly to ensure proper seal from the external environment.

[0254] The lid and well can be made of biocompatible materials such as medical grade polycarbonate (PC), polystyrene, polypropylene (PP), nylon, or teflon. Further material consideration also includes the ability to sterilize these setups through gamma irradiation, autoclave, or EtO sterilization.

[0255] The membrane 2 can be attached to the filter support 3 using a silicone double sided adhesive tape which is also biocompatible. Another silicone double sided adhesive tape can then be used to join the membrane-filter support assembly, to the cell chamber 1 . Biocompatible silicone sealant can be applied on the edges of the assembly between the filter support and the cell chamber to ensure the cells are retained in the cell chamber during culture. Alternatively, the membrane can be sandwich between two biocompatible silicone gaskets, which is then sandwich between the filter support and the cell chamber. Securing components such as clamps or screws made of titanium or stainless steel (316) can be used to ensure sufficient seal of the cell chamber, while ensuring the membrane remains flat for the cells. Another way is to sandwich the filter support and gasketmembrane-gasket assembly to the cell chamber using similar securing components.

[0256] The agitation setup is switched on during culture to promote diffusion of nutrients and waste across the membrane, while retaining the cells in the chamber as illustrated in Figure 3. The agitation modality (e.g., flow rate, stirring speed) can be tuned so much so that the cells remain relatively undisturbed while optimum diffusion is achieved. Optimum diffusion is achieved when the level of monitored metabolites in both chambers, are observed to be as close to each other, indicating thorough homogenization. Figure 4 illustrates the same two-chamber setup without the agitation setup, relying of passive diffusion across the membrane, which reduces the homogenization of nutrients and metabolites between the membranes limiting the benefits of a bulk media chamber.

[0257] The study was performed with the following device specifications:

[0258] Table 1 - Specifications for presently disclosed cell culture device

[0259] Culture protocol

[0260] Two study groups, Static Bioreactor (Figure 1) and Stirring bioreactor (Figure 2a) were used to activate and culture primary peripheral blood mononuclear cells (PBMCs) from 5 donors to obtain expanded and differentiated T cells at the end of the culture.

[0261] Seeding: Day 0 (Static and Stirring)

[0262] 6 million peripheral blood mononuclear cells (PBMCs) (STEMCELL Technologies) were thawed and seeded into the cell chamber. ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator (STEMCELL Technologies) (ratio of 1 :40) and IL-2 (STEMCELL Technologies) (50IU) was added to the cell chamber. Sample of media was taken for assessment of metabolites. Bioreactors were transferred to a 37°C CO2 incubator.

[0263] Start stirring: Day 1 (Stirring) Stirring bioreactors were transferred to a 36°C CO2 incubator and placed on a stir plate to keep the temperature of the culture media inside the bioreactor at 37°C. The stir plate was turned on to stir at 60rpm.

[0264] Cell Count: Day 3 or 4 (Static and Stirring)

[0265] Cells from the cell chamber were mixed into a homogenous cell solution and a sample was taken to assess cell count. Sample of media was taken from both chambers for assessment of metabolites. Bioreactors were returned to their respective CO2 incubators.

[0266] Re-activation: D7 (Static and Stirring)

[0267] Cells from the cell chamber were mixed into a homogenous cell solution and a sample was taken to assess cell count. Sample of media was taken from both chambers for assessment of metabolites. Half media change was performed. ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator (STEMCELL Technologies) (ratio of 1:40) and IL-2 (STEMCELL Technologies) (50IU) was added to the cell chamber. Sample of media was taken from both chambers for assessment of metabolites after media change. Bioreactors were transferred to a 37°C CO2 incubator.

[0268] Start stirring: Day 8 (Stirring)

[0269] Stirring bioreactors were transferred to a 36°C CO2 incubator and placed on a stir plate. The stir plate was turned on to stir at 60rpm.

[0270] Cell Count: Day 10 or 11 (Static and Stirring)

[0271] Cells from the cell chamber were mixed into a homogenous cell solution and a sample was taken to assess cell count. Sample of media was taken from both chambers for assessment of metabolites. Bioreactors were returned to their respective CO2 incubators.

[0272] Harvest: D14 (Static and Stirring)

[0273] A sample of media was taken from both chambers for assessment of metabolites. Cells were collected from the cell chamber and mixed into a homogenous cell solution. Samples were taken to assess cell count and cell phenotype through flow cytometry. Markers assessed: CD3 (T-cell marker), CD56 (NK cell marker), PD-1 (Exhausted T-cell marker), CD4 (Helper T-cell marker), CD8 (Cytotoxic T-cell marker), CD45RA and CCR7 (Naive T-cell (Tn), Effector memory cells re-expressing CD45RA T-cell (Temra), Central Memory T-cell (Tcm), and Effector Memory T-cell (Tem).

[0274] Effect of culture (Cell Yield and Phenotype)

[0275] Stirring Bioreactor had higher cell yield than Static Bioreactor

[0276] The experiment was repeated five times with different PBMC donors. Cell count was assessed on days 0, 3 / 4, 7, 10 / 11 and 14 (Table 2). The cell population in stirring bioreactor achieved statistically significant higher numbers compared to static bioreactor on Day 7 (51.12±17.20 vs 31.01±5.28 million cells) and Day 11 (93.98±34.00 x 106vs 46.07±10.84 million cells) (Figure 5A). However, the cell population dropped on Day 14 for the stirring bioreactor in 2 out of 5 runs (Figure 5B). The runs with dropped cell population (Run 4 and 5) also had high cell population on Day 11 at >10 million cells per cm2or >20 million cells per ml in the cell chamber (Figure 5B), hence we hypothesized that this decrease in cell number could be due to high cell density leading to nutrient depletion or overcrowding.

[0277] Table 2. Cell counts for five runs.

[0278] Stirring bioreactor has higher population of central memory T-cells than static bioreactor Cell phenotype was assessed via flow cytometry on Day 0 and 14 (Table 3). Cell population in stirring bioreactor has higher levels of T-cell population (>90% CD3+) and lower level of NK-cell population (<10% CD56+) compared to static bioreactor (Figure 6A). Both bioreactors also produced similar ratio of helper (CD3+ / CD4+) vs cytotoxic (CD3+ / CD8+) T-cells across most runs except for Run 4 (Figure 6B). Population in stirring bioreactor has higher percentage of PD-1+cells (49.47±7.70%) compared to static bioreactor (29.71±9.61%) (Figure 6C). This is possibly due to the higher number of cells obtained in the stirring bioreactor from multiple rounds of T-cell proliferation on Day 14.

[0279] Table 3. Population percentages for T-cells, NK-cells, PD-1+, helper and cytotoxic T- cells for five runs for Day 0 and Day 14. “DO” indicates population on Day 0. “Static” and “Stirring” indicate population from these two groups on Day 14.

[0280] Subpopulations of T-cells for central memory T-cell (Tcin), effector memory T-cell (Tem), naive T-cell (Tn) and effector memory cells re-expressing CD45RA T-cell (Temra) were assessed for Runs 2-5 (Table 4). Tnand Tcm populations are considered to be less differentiated and associated with better clinical outcomes compared to Temra and Tem1.

[0281] The analysis revealed that stirring bioreactor has statistically significant higher percentage population of Tc cells (75.63±15.15% vs 52.35±23.02%) and lower percentage population of Tem(22.54±14.39% vs 43.22±21.97%) compared to static bioreactor (Figure 7A and 7B). Both stirring and static bioreactor have low levels of Tnand Temra with stirring bioreactor having lower percentage population of Temra (0.55±0.92% vs 2.28±2.04%) compared to static bioreactor. (Figure 7C).

[0282] Table 4. Population percentages for Tem, Tcm, Temra and Tnas a subset of T-cells for Runs 2-5. “DO” indicates population on Day 0. “Static” and “Stirring” indicate population from these two groups on Day 14.

[0283] Possible mechanism

[0284] Different culture environment in stirring and static bioreactors might be attributed for the change in cell population in phenotype. We analyzed the metabolite (glucose and lactate) levels in the two chambers in both bioreactors to understand the difference in culture environment.

[0285] Stirring bioreactor distributes media components better than Static bioreactor

[0286] Media samples were taken from cell chamber (CC) and media chamber (MC) on Day 3 / 4, 7, 10 / 11 , and 14 to assess levels of glucose and lactate for Runs 1 - 5. Analysis of the glucose revealed that levels in CC and MC chamber are significantly different in static bioreactor on all four days assessed while levels for stirring bioreactor are significantly different on Day 7 and 10 / 11 (Figure 8A, Table 5). Ratio of MC / CC glucose levels were also calculated and demonstrated that ratio in stirring bioreactor is closer to the baseline of 15.8 across all days assessed compared to static bioreactor (Figure 8B, Table 6). To demonstrate this point, a representative analysis of Run 5 is shown (Figure 8C). The difference between MC and CC in stirring bioreactor was lower (6.9±6.12%) in the days assessed than that in static bioreactor (23.72±9.85%) (Figure 8C, Table 7). Large consumption of glucose is expected after T-cell activation - between Day 0 and 4 as well as between Day 7 and 11. The stirring bioreactor was able to mitigate this consumption of glucose by equalizing the levels in CC and MC compared to the static bioreactor where glucose levels decreased more sharply in CC than in MC.

[0287] This phenomenon of glucose being better distributed between CC and MC in the stirring bioreactor was observed across all runs.

[0288] Table 5. Levels of %glucose normalized to Day 0 in MC and CC chambers in static and stirring bioreactor on Days 3 / 4, 7, 10 / 11 and 14 across Runs 1-5. Paired two-tailed t-tests were performed between levels in MC and CC. * p<0.05, ** p<0.01. CC: cell chamber. MC: media chamber.

[0289] Table 6. Ratio of MC / CC for %glucose normalized to Day 0 calculated in static and stirring bioreactor on Days 3 / 4, 7, 10 / 11 and 14 across Runs 1-5. CC: cell chamber. MC: media chamber.

[0290] Table 7. Difference between MC and CC for %glucose normalized to Day 0 calculated in static and stirring bioreactor on Days 3 / 4, 7, 10 / 11 and 14 across Runs 1-5. Average and standard deviation across the days assessed were calculated for each run. CC: cell chamber. MC: media chamber.

[0291] The analyses were repeated for lactate level in both chambers in both bioreactors. As the culture grows, lactate is produced by the cells in the CC, resulting in an increase in lactate levels in the CC which we expect to diffuse into the MC. Results were similar to the glucose analysis. Levels in CC and MC chamber in static bioreactor is significantly different in all four days assessed while the levels in stirring bioreactor are significantly different on Day 7 and 14 (Figure 9A, Table 8). Ratios in stirring bioreactor is closer to the baseline of 15.8 across all days assessed compared to static bioreactor (Figure 9B, Table 9). Representative analysis of run 5 was also repeated and showed the difference between chambers in stirring bioreactor was lower (2.57±1.64mM) compared to static bioreactor (6.55±3.30mM) in the days assessed (Figure 9B, Table 10). Lactate production is expected to increase between Day 4 and 7 as well as Day 11 and 14 due to T-cell proliferation. In the stirring bioreactor, lactate was able to be better distributed between MC and CC to reduce the lactate levels in CC. In the static bioreactor, lactate levels in CC remains high. Between Day 11 and 14, the increase in lactate is not observed due to cell death. The phenomenon of lactate being better distributed between CC and MC in the stirring bioreactor was observed across all runs.

[0292]

[0293] Table 8. Lactate (mM) in MC and CC chambers in static and stirring bioreactor on Days 3 / 4, 7, 10 / 11 and 14 across Runs 1-5. Paired two-tailed t-tests were performed between levels in MC and CC. * p<0.05, ** p<0.01.

[0294] Table 9. Ratio of MC / CC for lactate (mM) calculated in static and stirring bioreactor on Days 3 / 4, 7, 10 / 11 and 14 across Runs 1-5.

[0295] Table 10. Difference between MC and CC lactate (mM) calculated in static and stirring bioreactor on Days 3 / 4, 7, 10 / 11 and 14 across Runs 1-5. Average and standard deviation across the days assessed were calculated for each run.

[0296] Other culture protocols and effect of culture

[0297] In other culture protocols, bioreactors are kept static, indirectly stirred (Figure 2a), directly stirred (Figure 2c), or perfused (Figure 2b). Static cell culture in the bioreactor followed the same protocol as that in Culture protocol 1. Cell culture in the indirectly stirred bioreactor (Figure 2a) followed same protocol as that in Culture protocol 1 , except the timing and duration of the stirring are different, on either D1-7 and D8-14, or D1-2 and D8-9, or D5-7 and D12-14, or DO-14 during the 14-day culture. Cell culture in the directly stirred bioreactor (Figure 2c) was stirred on D1-7 and D8-14. Cell culture in the perfused bioreactor (Figure 2b) was perfused on D1-7 and D8-14.

[0298] Effects of different agitation methods

[0299] Figure 10 and 11 , and Table 11 compared various agitation methods ((indirect stirring on D1-7 and D8-14, direct stirring on D1-7 and D8-14, and perfusion on D1-7 and D8-14) with static culture in total cell yield, %Tcm and number of Tern cells respectively.

[0300] Table 11. Number of cell subunits with different agitation methods. ‘Calculation for number of cells: Total number of cells x Percentage of CD3+ T-cells x Percentage of each subunit

[0301] Results show compared to static culture, both indirect stirring and perfusion led to higher total cell yield (Figure 10) and higher %Tcm (Figure 11) on Day 14. Indirect stirring has higher total cell yield (Figure 10) but lower % Tern (Figure 10) compared to perfusion on Day 14. Direct stirring achieved higher total cell yield (Figure 10) but lower %Tcm compared to static culture on Day 14. Among the different groups, indirect stirring achieved the highest number of Tern cells on D7 and direct stirring achieved the highest number of Tern cells on D14 (Table 11).

[0302] Effects of different indirect agitation setting Figure 12 and 13, and Table 12 compared various setting of indirect stirring (stirring on DO-14, stirring on D1-2 and D8-9, and stirring on D5-7 and D12-14) with static culture in total cell yield, %Tcm and number of Tern cells respectively.

[0303] Table 12. Number of cell subunits with different indirect stirring setting ‘Calculation for number of cells: Total number of cells x Percentage of CD3+ T-cells x Percentage of each subunit

[0304] Results show indirect stirring starting 1 day after stimulation (stirring on D1-2 and D8-9, and on D5-7 and D12-14) resulted in higher total cell yield (Figure 12) and higher % Tern (Figure 13) compared to static culture on D7 and D14. The group with stirring on DO-14 resulted in lower total cell yield (Figure 12), but higher %Tcm on D7 and D14. Effect of commencing indirect agitation from 10 mins after activation

[0305] A follow-up experiment was performed to compare the T-cell yields between a static group, a group with stirring from 10 minutes after activation to Day 7 (Stirring 1) and a group with stirring from Day 1 to Day 7 (Stirring 2). Cell counts were assessed Days 0, 4, 6, 10 and 14. Cell phenotype was assessed via flow cytometry on Days 0, 6, 10 and 14.

[0306] The results are shown in Figures 14 and 15. The results indicate that at Day 6, stirring enhances cell yield. In particular, both stirring groups had higher yield compared to static, with Stirring 1 (10 minutes after activation) having the highest cell yield (Figure 14). In addition, both stirring groups had higher % Tern compared to the static group (Figure 15).

[0307] Thus, these results demonstrate that by using the presently disclosed cell culture method, a) T-cells can potentially be harvested as early as Day 6 due to the significant shift towards Tern by this day, and b) introducing agitation as early as 10 minutes after activation may be particularly beneficial for cell growth / yield.

[0308] Overall, the bioreactor with agitation setup compared to the static setup was able to better distribute nutrient (glucose) and waste (lactate) between the two chambers, thereby allowing for sufficient supply of glucose to the cells from the bulk media as well as the dispersion of lactate levels in the cell chamber. This could allow for better cell growth in the stirring bioreactor and affect the differentiation of T-cells into Tcmin. Terumo Quantum, a hollow fiber bioreactor, also demonstrated a preference for differentiation into Tcm cells. However, G-rex, a gas-permeable bioreactor with a bulk media reservoir, did not display such preference (data not shown). Therefore, it is unclear at the moment the mechanism of our bioreactor skewing T-cell differentiation into Tcmpopulation while achieving higher cell yield. More studies would be required to illuminate the exact mechanism.

[0309] Example 2 - Enriching central memory T cells using a novel bioreactor design for T-cell manufacturing

[0310] Introduction In the last decade, autologous T cell therapy has demonstrated its clinical efficacy, earning approvals from multiple regulatory agencies around the world [1-3], Despite advancements in manufacturing technologies, scaled manufacturing of autologous cell therapies remains challenging, limiting their accessibility and driving up costs [4], Early understanding of T cell manufacturing targeted high cell viability and yields. However, recent studies have shown that T cell subtypes are also important for the efficacy and persistence of the therapy [5], The enrichment of less differentiated T cells - naive T cells (TN), stem memory T cells (TSCM), and central memory T cells (TCM) - in a T cell therapy product has been observed to confer similar efficacy at a significantly lower dose than a matched non-enriched product [6], This translates to a shorter culture period, potentially saving manufacturing costs and shortening turnaround times.

[0311] Process controls can be implemented during manufacturing to direct T cell population differentiation and proliferation towards an enrichment of less differentiated T cells. The most common method would be the media supplementation of cytokines, such as IL-7 and IL-15 [7,8], and IL-21 [9,10], due to the ease of implementation and the availability of good manufacturing practice (GMP)-grade reagents. Media supplementation of other metabolites such as lactate [11,12] and amino acids [13,14] have also been observed to affect T cell memory population, however these methods are not as established as compared to cytokine supplementation. Apart from media supplementation, environmental control of the culture, notably control of pH [12,15] and dissolved oxygen (DO2) [16,17] levels of the culture, has shown potential in directing T cell differentiation. Currently, there are no mechanical methods that have been described in literature that can consistently enrich these desired memory T cell populations.

[0312] We have previously reported on our work in developing a novel bioreactor, Bioreactor with Expandable Culture Area - Dual Chamber (BECA-D), to meet the specific culture needs of T cells and simplify the culture process (Figure 16A)

[0018] , This study describes our work in improving the performance of BECA-D with a stirring mechanism to promote the homogenization of media between the chambers within the bioreactor and enrich for central memory T cells in the culture. The incorporation of the stirring mechanism was first tested with a simple double-chamber prototype bioreactor with fixed culture area, which resulted in an improved effect on T cell culture expansion yield and change in heterogeneity of memory subtypes towards the enrichment of TCM cells. The approach was then validated in BECA-D, and results compared with matched culture in G-Rex10, an industry benchmarked culture vessel for T cell culture [19-21], to establish the robustness of the mechanical method in the enrichment of TCM cells.

[0313] Materials and Methods

[0314] Fabrication of a Double-chamber prototype Bioreactor

[0315] The double-chamber prototype bioreactor (Figure 16A) was fabricated from hard plastics (polycarbonate and polypropylene). It consisted of a cell chamber sitting atop support struts, within a containing device with a lid. The cell chamber has a filter support base with polyethylene terephthalate (PET) membrane having pore size of 1.0 pm (Sterlitech), attached to it. The area beneath and surrounding the cell chamber forms the media chamber. Post-assembly sterilization was performed through autoclave at 121“C, for 20 minutes. All materials used in the construction of the prototype were tested inhouse for biocompatibility with Peripheral Blood Mononuclear Cell (PBMC) culture.

[0316] Culture of PBMCs in the Double-Chamber Prototype Bioreactor with (Stirring) and without (Static)

[0317] The complete culture medium consisted of RPMI-1640 (Thermo Fisher Scientific), 10% heat inactivated Fetal Bovine Serum (FBS) (HyClone), and 2 mM L- glutamine (Thermo Fisher Scientific). This complete culture medium was used for all the experiments described in this manuscript.

[0318] For both Static and Stirring culture, 95 mL of complete culture medium was added into the Media Chamber with 50 lU / mL IL-2 (STEMCELL Technologies). PBMCs (STEMCELL technologies) were thawed and seeded at 0.5 x 106cells / cm2in 6 mL of complete culture medium into the Cell Chamber with 50 lU / mL IL-2. 150 pL of activators (ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator (STEMCELL Technologies)) was added to the Cell Chamber. The culture was then incubated at 37°C under 5% CO2. On Day 7, half media change of the Media Chamber was performed. Culture was reactivated by the addition of 150 pL of activators to the Cell Chamber. 50 lU / mL IL-2 was also added on Day 3 or 4, 7, 10 or 11 to the respective chambers based on their media volume.

[0319] For Stirred cultures, an autoclaved polyetrafluoroethylene (PTFE) magnetic stirbar was placed into the Media Chamber on Day 0. The culture was transferred onto a stir plate (60 rpm) one day after activation (Day 1 and Day 8). The cultures were stirred for the periods of Days 1-7 and Days 8-14.

[0320] Fabrication of B EC A-D

[0321] BECA-D (Figure 16A) was fabricated as described in a previous study

[0018] , Briefly, it was made from injection moulded virgin polystyrene and assembled via ultrasonic welding under an ISO 13485 certified environment. The plunger consists of a polycarbonate core with silicone rubber outer wall. The porous membrane was made from PET with a pore size of 1 .0 pm. All materials used in the construction of BECA were tested in-house for biocompatibility with PBMC culture.

[0322] Culture of PBMCs in BECA-D with stirring (BECA-DS+) and without stirring (BECA-D)

[0323] For both BECA-D and BECA-DS+, 228 ml_ of complete culture medium was added to the Media Chamber with 50 lU / mL IL-2. PBMCs were thawed and seeded at 0.5 x 10scells / cm2in 6 mL of complete culture medium into the Cell Chamber with 50 lU / mL of IL-2. 150 pL of activators was added to the Cell Chamber. The culture was then incubated at 37°C under 5% CO2. On Day 7, half media change of the Media Chamber was performed. Culture area of the Cell Chamber was calculated based on the cell population and adjusted to maintain a cell density of 0.5 x 10scells / cm2. Culture was reactivated by the addition of 1 :40 culture volume of activators to the Cell Chamber. 50 lU / mL IL-2 was also added on Day 3 or 4, 7, 10 or 11 to the respective chambers based on their media volume.

[0324] For BECA-DS+, an autoclaved PTFE magnetic stirbar was placed into the Media Chamber on Day 0. BECA-DS+ was transferred onto a stir plate (60 rpm) one day after activation (Day 1 and Day 8). The cultures were stirred for the periods of Days 1 -7 and Days 8-14.

[0325] Culture of PBMCs in G-Rex10

[0326] PBMCs were thawed and seeded at 0.5 x 106cells / cm2in 40 mL of complete culture medium with 50 lU / mL of IL-2 and 1 mL of activators. The culture was then incubated at 37°C under 5% CO2. On Day 7, half media change of the culture was performed. Culture was re-activated by the addition of 1 mL of activators. 50 lU / mL IL-2 was also added on Day 3 or 4, 7, 10 or 11 . Cell Count and Viability

[0327] Cell counts and viability were determined based on Trypan Blue exclusion method by diluting a sample of culture with 1:1 volume of 0.4% Trypan Blue Solution (Thermo Fisher Scientific), loaded onto a hemacytometer, and counted under an inverted microscope. Cell counts reported in this study are based on live cells.

[0328] Glucose and Lactate Measurements

[0329] Media samples were collected and analysed for glucose and lactate levels using the Cedex Bio Analyzer (Roche Diagnostics) with accordance to the manufacturer’s protocol.

[0330] Phenotyping of memory T cells

[0331] On Day 14, cells were harvested and processed for flow cytometry analysis. Cells were stained with Viobility 405 / 520 Fixable Dye, CD3-VioBlue, CD56-PE-Vio770, CD4- APC-Vio770, CD8-FITC, CD45RA-PerCP-Vio 700, and CD197 (CCR7)-PE (Miltenyi Biotec). MACSQuant Analyser 10 (Miltenyi Biotec) was used to acquire the data and MACSQuantify Software 2.13.3 (Miltenyi Biotec) was used for analyses.

[0332] Statistical Analysis

[0333] Statistical analyses were performed using Microsoft Excel (Office 365). Paired Student’s two-tailed t-tests were performed to assess the statistical significance of differences between two groups within each of the donor sets. One sample two-tailed t- test was performed to assess the statistical significance of differences between a group and a defined value. A p-value of < 0.05 obtained from the test indicates a significant difference.

[0334] Results

[0335] Incorporation of a stirring mechanism within the double-chamber prototype bioreactor improved media exchange and cell yield

[0336] We previously developed a novel bioreactor, BECA-D, consisting of two separate chambers - the Cell Chamber (CC) with an expandable culture area, and the Media Chamber (MC), separated by a porous membrane (Figure 16A). Its performance in the culture of virus specific-T cells was validated in a previous study

[0022] , In that study, it was observed that the diffusion of glucose and lactate across the porous membrane to be inefficient, possibly hindering cell proliferation. We hypothesised that the incorporation of a stirring mechanism in the MC could improve homogenisation of glucose and lactate between the chambers (Figure 16B). This mechanism is expected to allow for glucose in the MC to be more available to the cells in the CC and to dilute lactate from the CC more efficiently to facilitate continuous cell growth leading to a higher cell yield. To study this, we fabricated a pair of simple double-chamber prototype bioreactors with a fixed 12 cm2culture area (Figure 16A) - one prototype with a stirbar added into the MC (Stirring) and one prototype without a stirbar as a control (Static).

[0337] The prototypes were tested with four different sets of donor PBMCs (Donor 1-4) in a 14-day culture. Cells were seeded at 0.50 x 106cells / cm2with activation on Day 0 and Day 7, and a 50% media change on Day 7 (Figure 16C). The stirring mechanism was switched on from Day 1 to Day 7 and Day 8 to Day 14 (Figure 1C). In all donors, the Stirring culture achieved higher cell yield compared to Static on Day 14 for Donor 1 and Day 10 / 11 for Donor 2-4 (Figure 16D). Paired T-test analysis indicated a significant difference (p-value = 0.024) on Day 10 / 11 cell numbers between Stirring (101.18±34.60 x 106cells) and Static (46.81±12.37 x 106cells) cultures. Viability in the cultures remained high (>80%) in all four donors across the 14-day culture with the exception of Donor 4 Day 14 where the viability dropped to 66%, mirroring the drop in cell population between Day 10 / 11 to 14 (Figure 16D).

[0338] Lactate and glucose levels in both chambers were measured on days 0, 3 or 4, 7, 10 or 11 , and 14, showing a steady decrease of glucose levels and increase of lactate levels in the cultures, indicative of T cell proliferation (Figure 16E). Levels of glucose and lactate in the chambers was observed to be more closely matched in Stirring cultures compared to Static cultures. One sample t-test was performed to compare the ratio of glucose and lactate concentration in the chambers to the ideal value of 1.0 (indicating perfect homogenization) (Figure 16F). Both the ratio of glucose and lactate levels in the Static cultures were observed to be significantly different from the ideal value of 1.0 on three days (Day 7, 10 / 11 , and 14 for glucose and Day 3 / 4, 7, and 10 for lactate) while those in Stirring cultures were observed to be significantly different on two days (Day 10 / 11 and 14 for glucose and Day 3 / 4 and 10 / 11 for lactate). In all statistical analyses performed, the p-value calculated from comparing the chambers in Static cultures were lower than those from Stirring cultures. It was also observed that the glucose level in Stirring for Donors 2, 3, and 4 fell to below 0.5 mg / cell on Day 10 / 11, indicating that the glucose levels in these cultures might have been insufficient to support the growing culture resulting in the sharp drop in cell numbers observed on Day 14,

[0339] Stirring mechanism within the double-chamber prototype bioreactor enriched both CD4 and CD8 Central Memory T cells (TCM)

[0340] The percentage of CD3 population in the cultures were assessed on Day 0 and Day 14, showing high percentage of CD3 cells in the culture (>90%) in both Stirring and Static cultures and no significant difference between the two types of cultures (Figure 17A). The same observation was made for the levels of CD4 helper and CD8 cytotoxic T cells indicating that the stirring mechanism did not influence the development and proliferation of bulk CD4 and CD8 T cells (Figure 17B). CD45RA and CCR7 expression for T cell memory subtypes in CD4 and CD8 T cell populations were assessed on Day 0 and Day 14 with flow cytometry. These memory subtypes include naive (TN), central memory (TCM), effector memory (TEM), and terminally differentiated effector memory (TEMRA) T cells (Figure 17C). In all cultures, TN and TEMRA populations decreased from Day 0 to Day 14 and T cells primarily expressed TEM and TCM markers on Day 14 (Figure 17D). Stirring cultures were observed to have significantly higher percentage of TCM cells compared to Static cultures in both CD4 (p-value = 0.031) and CD8 (p-value = 0.010) subpopulations. The opposite trend was observed for TEM population where Stirring cultures had significantly lower percentage of TEM cells compared to Static cultures in both CD4 (p-value = 0.028) and CD8 (p-value = 0.012) subpopulations (Figure 17E).

[0341] Stirring mechanism in BECA-D improved cell proliferation and media homogenization

[0342] To validate the robustness of the method, we repeated the experiment with the same four donor PBMCs (Donor 1 - 4) in the BECA-D bioreactor with a stirbar placed into the Media Chamber of one bioreactor (BECA-DS+) and its static control without the stirbar (BECA-D). Cell cultures were also evaluated with another T cell bioreactor (G- Rex10) that does not have a double-chamber design.

[0343] The cells were seeded at 0.50 x 105cells / cm2and cultured using the same protocol with an additional step of expanding the culture area in BECA-D and BECA- DS+ on Day 7 (Figure 18A). The culture areas of BECA-D bioreactors were adjusted based on the cell numbers on Day 7 to decrease the cell density back to 0.5 x 10scells / cm2(Figure 19A and 19B). Across all four donors, BECA-DS+ achieved the highest maximum yield compared to BECA-D and G-Rex10 (Figure 18B). Similar to the Stirred culture in the double-chamber prototype bioreactor (Figure 16D), BECA-DS+ experienced a decrease in culture growth for Donor 2-4 from Day 10 / 11 to 14. BECA- DS+ was able to improve cell proliferation with a significantly higher yield on Day 10 / 11 compared to BECA-D (p-value = 0.013) and G-rex10 (p-value = 0.037) (Figure 18C). Viability in the cultures remained high (>80%) in all four donors across the 14-day culture in all bioreactors (Figure 18B).

[0344] The stirring mechanism in BECA-DS+ allowed for improved homogenization of media between the two chambers (Figure 19C). The ratios of glucose and lactate levels in BECA-D were determined to be significantly different from the ideal value of 1.0 on Day 7 and 10 / 11 (glucose) and Day 3 / 4, 7, 10 / 11 and 14 (lactate). In BECA-DS+, the ratios were significantly different for only one day for glucose (Day 7) and two days for lactate (Day 3 / 4, 7) (Figure 19D). Glucose availability per cell was calculated to determine whether the level of glucose of media could explain the slowing of cell growth in BECA-DS+ and G-Rex10 cultures from Day 10 / 11 to 14 in Donor 2, 3, and 4 (Figure 19E). Surprisingly, glucose availability in this study did not correlate to the culture growth in BECA-DS+. The only instance between Day 0 and 14 where the glucose / cell fell below 0.5 mg / cell in BECA-D cultures was in Donor 2 on Day 11 , however, this did not result in a drop in cell population as observed in the double-chamber cultures. In addition, the culture in Donor 4 on Day 10 had higher glucose availability and yet had a decrease in cell population. The G-Rex10 cultures consistently had lower glucose availability due to G-Rex10’s lower media capacity (40 ml_, starting glucose level: 78.5±0.3 mg for 5.0 x 106cells) compared to BECA-D (234 mL, starting glucose level: 458.9±1.8 mg for 6.0 x 106cells) but this did not hinder the culture growth. For these cultures, it appears that glucose availability does not sufficiently explain the slowing down of cell proliferation from Day 10 / 11 to 14.

[0345] Stirring mechanism in BECA-D enriched both CD4 and CD8 Central Memory T cells (TCM)

[0346] The cultures were assessed for their T cell population phenotype via flow cytometry analysis on Day 14. The percentage of CD3 cells in the cultures for three bioreactors was high (>85%) with cultures in BECA-DS+ and G-Rex10 having >90% of live lymphocytes being CD3+ cells (Figure 18D). Previously, we observed subtle but not statistically significant difference in CD4 and CD8 population in the Stirring and Static cultures in the double-chamber prototype bioreactor. In this experiment, the difference was more pronounced. There was a statistically significant difference between BECA-D and BECA-DS+ CD4 (p-value = 0.04) and CD8 (p-value = 0.03) populations and same was observed between BECA-DS+ and G-Rex10 CD4 (p-value = 0.05) and CD8 (p- value = 0.04) population (Figure 18E). Interestingly, there was no significant difference in the CD4 and CD8 populations between BECA-D and G-Rex10 (CD4 p-value = 0.61 , CD8 p-value = 0.63) suggesting that the stirring mechanism in BECA-DS+ did skew the T cell population to have higher CD4 and lower CD8 population in culture.

[0347] In line with the findings from the double-chamber prototype bioreactor study, assessment of CD45RA and CCR7 expression revealed that the T cells primarily expressed TCM and TEM markers on Day 14 and that the stirred culture, BECA-DS+, has enriched TCM population and diminished TEM population compared to static cultures - BECA-D and G-Rex10 (Figure 18F and 18G). The enrichment of CD8 TCM population in BECA-DS+ cultures was statistically significant compared to BECA-D (p-value = 0.015) and G-Rex10 (p-value = 0.045). BECA-DS+ also has statistically significant lower percentage of CD8 TEM population compared to BECA-D (p-value = 0.013) and G-Rex10 (p-value = 0.044). However, the same was not true for CD4 subpopulation. The higher percentage of TCM and lower percentage of TEM cells were only statistically significant when comparing BECA-DS+ with G-Rex10 cultures (TCM p-value = 0.012, TEM p-value = 0.012) and not when comparing BECA-DS+ with BECA-D cultures (TCM p-value = 0.071 , TEM p-value = 0.071) (Figure 18H).

[0348] Discussion

[0349] Our previous study demonstrated the effectiveness and ease of use of BECA-D in the culture of Epstein-Barr Virus-Specific T cells

[0010] , We hypothesized that enhancing the media exchange between the cell and media chambers could improve culture outcomes. In this study, we explored our hypothesis with a double-chamber prototype bioreactor and developed an iteration of BECA-D, BECA-DS+, which improved media exchange between the cell and media chambers through a stirring mechanism. The performances of the stirred cultures in both the double-chamber prototype bioreactor and BECA-DS+ was compared to static controls across a 14-day PBMC culture. As expected, the stirring mechanism did improve the glucose and lactate distribution between the chambers and resulted in a higher maximum cell yield on Day 14 for Donor 1 and Day 10 / 11 for Donors 2, 3, and 4. The cell yield in BECA-DS+ was observed to be higher than that in G-Rex10, however, the performance of G-Rex10 could have been impaired by the non-ideal feeding scheme executed in this study. The glucose availability of G-Rex10 was measured to be consistently low at <0.5 mg / mL from Day 10 / 11 to 14. It is possible that adjustment of the feeding scheme with more frequent and higher volume media changes might result in an equivalent cell yield as BECA-DS+.

[0350] Analysis of the T cell subtypes within the cultures revealed stark differences between the stirred cultures (Stirring and BECA-DS+) and static cultures (Static, BECA- D, and G-Rex10). Surprisingly, the CD4 and CD8 ratios (higher levels of CD8 cells) were consistent within the static cultures, whereas, the stirred cultures had a more balanced distribution of CD4 and CD8 cells. Assessment of T cell memory subtypes showed an enrichment of TCM cells in stirred cultures compared to static cultures. The observation that T cell subtype distributions differ between bioreactors is not novel as bioreactors have key features that may affect T cell metabolism and proliferation, such as improved gaseous exchange in gas permeable bioreactors

[0023] and high cell density in hollow fibre reactors

[0024] . It was unexpected to us that the static cultures had a more similar T cell subtype distribution considering that there are major design differences between the two static bioreactors, BECA-D and G-Rex10. The results reinforced the dominant effect of an ‘indirect mixing’ of a separate media chamber on the T cell culture, both in CD4 CD8 distribution as well as enrichment of TCM.

[0351] The enrichment of TCM cells, a less-differentiated T cell memory subtype, has been correlated with improved clinical outcomes and T cell persistence [7,25-28], The improved efficacy of the less-differentiated T cell population could possibly reduce the cell dosage per therapy and accelerate the manufacturing of T cell therapy products. Supplementation of cytokines in media (typically a cocktail involving permutations of IL2, IL7, IL-15, and IL-21) is a common practice to drive T cell expansion towards an enriched TCM population (personal communication)

[0029] , Other culture parameters have also been observed to affect TCM population including other media components [13,14], pH [12,15], and low DO2 levels (hypoxia) [16,17], Compared to these methods, especially in those that requires temporal control, the double-chamber bioreactors (including BECA-DS+) adopt a direct distinct mechanical approach which does not require the use of additional GMP-grade reagents (in contrast to cytokine and media components methods) or specialized equipment (in contrast to hypoxia methods). In addition, cultures in the double-chamber bioreactors did not experience a decrease in proliferative capacity compared to cultures under hypoxia [16,17,30], The use of the double-chamber bioreactor compared to the above methods for the enrichment of TCM population in culture can possibly reduce culture process complexity and cost for the manufacturing of less-differentiated but more efficacious T cell therapy products.

[0352] The stirring mechanism affects the availability of multiple culture media components across the chambers. This study reported only on the glucose and lactate levels in the media, and the improved diffusion of these metabolites does not sufficiently explain the effect on T cell differentiation observed. The availabilities of other media components have been reported to affect T cell differentiation, especially IL-2 [31 ,32] and activators [33-35], Additional studies measuring these would be crucial in the elucidation of mechanism behind the TCM enrichment. Assessment of T cell subpopulations in earlier timepoints in the 14-day culture can also facilitate to determine whether the enrichment was due to an increase in proliferation potential of TCM cells, a decrease in proliferative potential of TEM cells, or a decrease in differentiation potential of the culture. Understanding these details in the mechanism would assist in creating a simpler process which could shorten the culture duration to enable shorter turnaround times in manufacturing.

[0353] This study has demonstrated that a mechanical stirring mechanism introduced to a double-chamber bioreactor was able to consistently enrich less-differentiated T cell subpopulations while achieving yields similar to the static controls and commonly used industry T cell bioreactor, G-Rex10. The current execution for this method relies on a magnetic stirbar to engage the stirring mechanism which would not be compatible with processes involving the use of magnetic bead-based reagents such as the Dynabeads™ activators. The current chamber designs also have a substantially large media reservoir compared to G-Rex10 (101 and 234 mL vs 40 mL) hence increasing the cost for media and reagents consumed per run. As we elucidate the mechanism of action for this method, the design of the bioreactors will be improved upon to circumvent these restrictions and facilitate more workflows at a lower cost.

[0354] As more cell therapies are expected to be approved in the next decade and the indications for T cell therapy expand, there is an urgent need to lower the cost and improve accessibility of these therapies. Recently, multiple bioreactor systems with advanced culture parameter controls have been launched to help address the manufacturing hurdles for the field (e.g. AVATAR from Xcell Biosciences, I RO from Ori Biotech, and Sefia expansion system from Cytiva). Towards the same goal, we report our bioreactor system, BECA-DS+, as a potential and effective manufacturing solution with unique advantage of enriching TCM cell production. Cell therapy developers and manufacturers can consider the use of these novel bioreactors to cater not only to the increase demand in manufacturing, but also the variety of culture needs for different therapies (e.g. low seed bioreactors for tumour-infiltrating T cells, small bioreactor capacity of highly efficacious cells). Bioreactors with different tuneable features available for T cell culture would be crucial in the endeavour to evaluate critical process parameters (CPPs) and critical quality attributes (CQAs) during therapy process and product development. The expansion in the variety of bioreactors in the market will hopefully result in a more vibrant industry where cell therapy developers and manufacturers have an arsenal of tools available to produce the most efficacious therapy at the lowest cost that can be easily accessed by patients.

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[0392] APPLICATIONS

[0393] The presently disclosed methods / devices have various applications, such as the following:

[0394] • T-cell manufacturing, in particular for high yield production of Tern;

[0395] • Directing T-cell differentiation towards the Tern phenotype

[0396] • Cell-based product manufacturing, for example for clinical applications, such as T-cell therapy; and

[0397] • As a research / product development tool for cell therapy and / or cell biology.

Claims

CLAIMS1. A method of T-cell expansion which directs differentiation towards central memory T-cells (Tcm), comprising:(i) culturing undifferentiated T-cells in a cell culture device comprising a cell chamber and a media chamber, wherein the chambers are separated by a semi-permeable membrane, which permits nutrients and waste, but not T-cells to diffuse between the cell and media chambers, and wherein the cell and media chambers both contain media, but only the cell chamber contains the T-cells; and(ii) employing indirect agitation to promote diffusion of nutrients and waste across the semi-permeable membrane, wherein only the media in the media chamber is agitated.

2. A device to promote central memory T-cell (T cm) - directed T-cell differentiation and high yield expansion during T cell manufacturing comprising:(i) a double chamber vessel comprising a cell chamber holding the cells and a media chamber holding the bulk excess media, wherein both chambers are separated by a porous membrane, enabling diffusion of nutrients and waste between the two chambers, while retaining cells in the cell chamber; and(II) a homogenization module in the media chamber to promote homogenization of fluid between the two chambers.

3. The method or device according to any preceding claim, wherein a supplement for directing differentiation towards central memory T-cells (Tcm) is not added to the media.

4. The method or device according to claim 3, wherein the supplement is a cytokine that directs differentiation towards Tcm, for example selected from the group comprising: IL-7, IL-15 and IL-21.

5. The method or device according to any preceding claim, wherein the levels of one or more of the following in the media are not adjusted: glucose, lactate, arginine and / or glutamine.

6. The method or device according to any preceding claim, wherein the undifferentiated T-cells are selected from the group comprising naive T-cells and memory T-cells, such as naive T-cells.

7. The method or device according to any preceding claim, wherein the source of undifferentiated T-cells is peripheral blood mononuclear cells (PBMCs), for example wherein PBMCs are cultured in the device.

8. The method or device according to any preceding claim, wherein the differentiation towards Tern increases the total Tern yield and / or increases the proportion of Tern vs other types of cells, i.e. enriches for Tern.

9. The method or device according to any preceding claim, wherein the increase in Tern yield and / or proportion of Tern is associated with an increase in CDR7+CD45RA- expression levels.

10. The method or device according to any preceding claim, wherein the diffusion of nutrients and waste across the semi-permeable / porous membrane homogenises the nutrient and waste distribution between the cell and media chambers.

11. The method or device according to any preceding claim, further comprising: monitoring / measuring the levels of one or more metabolites in the cell and media chambers, wherein similar levels of metabolites in both chambers indicates thorough homogenisation of nutrients and / or waste between the chambers, for example wherein the one or more metabolites are selected from the group comprising glucose, lactate, glutamine, ammonia and IL-2, in particular selected from the group comprising glucose and lactate.

12. The method or device according to any preceding claim, wherein the indirect agitation is selected from the group comprising: stirring, shaking, vortexing, rolling and swirling, in particular stirring.

13. The method or device according to any preceding claim, wherein the indirect agitation is employed by introducing a homogenization module in the media chamber, for example wherein the homogenization module improves the diffusion of nutrients and waste across the porous / semi-permeable membrane.

14. The method or device according to any preceding claim, wherein the homogenization module comprises a stirrer, for example a mechanical or a magnetic stirrer, in particular a magnetic stirrer.

15. The method or device according to claim 14, wherein the homogenization module further comprises an external mechanical controller to control the magnetic stirrer, thereby allowing the agitation / homogenization rate to be controlled.

16. The method or device according to any preceding claim, wherein the agitation / homogenization rate is modulated such that thorough homogenisation of nutrients and waste is achieved with minimal disruption and / or turbulence to the contents in the cell chamber, for example such that the T-cells remain the first / cell chamber during the method.

17. The method or device according to any one of claims 14 to 16, wherein the stirrer is set to stir at 40 to 80 rpm, for example 40, 45, 50, 55, 60, 65, 70, 75 or 80 rpm, such as 55 to 65 rpm, in particular 60 rpm.

18. The method or device according to any preceding claim, further comprising activating the T-cells, for example by exposing the T-cells to one or more T-cell activators, for example wherein the one or more T-cell activators are selected from the group comprising anti-CD3, anti-CD28 and anti-CD2.

19. The method according to claim 18, wherein the T-cells are exposed to the one or more T-cell activators on Day 0 and / or Day 7, in particular on Day 0.

20. The method or device according to any preceding claim, wherein the agitation is first employed 10 minutes or more after T-cell activation, such as 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours or 24 hours.

21. The method or device according to any one of claims 1 to 20, wherein the agitation is first employed a day after the T-cells are activated.

22. The method or device according to any preceding claim, wherein the agitation is employed throughout the entire T-cell expansion process.

23. The method or device according to any preceding claim, wherein the agitation is employed intermittently during the T-cell expansion process, i.e. the agitation is only employed on some days.

24. The method or device according to any preceding claim, wherein the agitation is employed for 2 or 3 consecutive days.

25. The method or device according to any preceding claim, wherein the agitation is employed on one or more days selected from the group comprising: Days 1 , 2, 8 and 9, such as on Days 1 and 2, on Days 8 and 9 or on Days 1, 2, 8 and 9; or one or more days selected from the group comprising: Days 5, 6, 7, 12, 13 and 14, such as on Days 5 to 7, on Days 12 to 14, or on Days 5 to 7 and Days 12 to 14.

26. The method or device according to any preceding claim, wherein the T-cells are cultured for 6 to 14 days, such as 6, 7, 8, 9, 10, 11 , 12, 13 or 14 days; for example, wherein the T-cells are cultured for 6 or 7 days, in particular 6 days.

27. The method or device according to any preceding claim, further comprising harvesting the T-cells between Days 6 and 14, for example between Days 6 and 9, such as on Day 9, Day 8, Day 7, or Day 6, in particular on Days 6 or 7, for example on Day 6.

28. The method or device according to any preceding claim, wherein the T-cells are activated on Day 0, agitation is first employed between 10 minutes to 24 hours after T-cell activation, and the T-cells are harvested between Days 6 and 10, such as on Day 7.

29. The method or device according to any preceding claim, wherein the T-cells are activated on Day 0, agitation is first employed 10 minutes after T-cell activation, and the T-cells are harvested on Day 7.

30. The method or device according to any preceding claim, wherein the T-cells are cultured in the presence of IL-2.31 . The method or device according to any preceding claim, wherein the volume of the cell chamber is equal to or less than the volume of the media chamber.

32. The method or device according to any preceding claim, wherein the culture media in the media chamber is equal to or in excess of the media in the cell chamber.

33. The method or device according to any preceding claim, wherein the volume of the cell chamber is less than half the volume of the media chamber, for example is 0.5, 0.4, 0.3, 0.2 0.1 , or 0.05X the volume of the media chamber.

34. The method or device according to any preceding claim, wherein the fluid height in the cell chamber is maintained at a low fluid height, for example 0.4 - 0.6 cm.

35. The method or device according to any preceding claim, wherein the device comprises a well which contains the cell and media chambers.

36. The method or device according to any preceding claim, wherein the device further comprises a lid.

37. The method or device according to any preceding claim, wherein the semi- permeable / porous membrane rests on a support or is attached to the support, for example using adhesive tape, such as silicon double-sided adhesive tape.

38. The method or device according to any preceding claim, wherein the semi- permeable / porous membrane is sandwiched between two gaskets, for example two biocompatible silicon gaskets, which are in turn sandwiched between the support and the first chamber.

39. The method or device according to any preceding claim, wherein the semi- permeable / porous membrane is welded to the support using heat or ultrasonic welding methods.

40. The method or device according to any preceding claim, further comprising one or more securing components for ensuring an adequate seal of the first / cell chamber and / or ensuring that the semi-permeable / porous membrane remains flat during the method, for example wherein the securing components are selected from the group comprising clamps and screws, for example made of titanium or stainless steel, such as Stainless Steel 316.

41. The method or device according to any preceding claim, wherein the device further comprises a sealant, such as a biocompatible silicon sealant, to seal any gaps between one or more of the following:• Between the media chamber and the well;• Between the cell and media chambers;• Between the support and the cell chamber, media chamber or both chambers; and• Between the lid and the well.

42. The method or device according to any preceding claim, wherein the semi- permeable membrane / porous has a pore size ranging from 0.2 to 5.0 pm, for example 0.2, 0.5, 1 , 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 pm, in particular a pore size of 1.0 pm.

43. The method or device according to any preceding claim, wherein the device further comprises a gaseous exchange means which facilitates gas exchange between the device and the environment, for example wherein the gaseous exchange means is a tube, for example a silicon or rigid tube made of biocompatible materials44. The method or device according to claim 43, wherein the gaseous exchange means comprises a gas permeable membrane, for example wherein the gas permeable membrane has a pore size ranging from 0.1 to 0.3 pm, such as 0.1, 0.15, 0.2, 0.25 or 0.3 pm, in particular a pore size of 0.2 pm.

45. The method or device according to any preceding claim, wherein the well, cell chamber, media chamber, lid, semi-permeable / porous membrane, support, gaseous exchange means and / or gas permeable membrane is made of a biocompatible material, for example a material selected from the group comprising medical grade polycarbonate (PC), polystyrene, polypropylene (PP), nylon, and Teflon.

46. The method or device according to any preceding claim, wherein the cell chamber, media chamber, lid and / or support are made of polycarbonate (PC) plastic.

47. The method or device according to any preceding claim, wherein the lid and / or well are made of polypropylene (PP) plastic.

48. The method or device according to any preceding claim, wherein the semi- permeable / porous membrane is made of a material selected from the groupcomprising polyethylene terephthalate (PET), polycarbonate (PC) plastic and polypropylene (PP), such as polyethylene terephthalate (PET).

49. The method or device according to any preceding claim, wherein the gas permeable membrane is a PTFE hydrophobic membrane.

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