Improved apparatus and methods for cell culture

Abstract

An in vitro method of producing a population of cells comprising the steps of introducing a source of cells into the first cell culture chamber of an in vitro cell culture vessel comprising three cell culture chambers arranged in a series, each chamber being separated from the next chamber in the series by a semipermeable barrier; contacting the cells in the first cell culture chamber to a first composition of one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants so as to promote migration of at least a subset of cells from the first cell culture chamber into an intermediate cell culture chamber of the series; and contacting cells in the intermediate cell culture chamber of the series to a further composition of one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants so as to promote migration of at least a subset of cells from the intermediate cell culture chamber to the final cell culture chambers in the series. Also related cell culture vessels, cell populations and methods of using cell populations.

Description

[0001] Improved apparatus and methods for cell culture

[0002] Field of the Invention

[0003] The present invention concerns cell culture vessels and methods of using cell culture vessels. More particularly, but not exclusively, the invention concerns multi chambered cell culture vessels and methods of using multi chambered cell culture vessels whereby cells are able to migrate from one chamber to the next and whereby they may be contacted with different compounds in each chamber. The invention also concerns methods of producing populations of cells including methods of expansion and differentiation of cells, and cell culture vessels adapted for such purposes. The invention also concerns populations of cells and uses thereof, especially in the field of cell therapy.

[0004] Background of the Invention

[0005] A large variety of cell culture vessels are available. They typically share the common features of providing a space in which a suitable cell culture medium may be contained and into which cells may be introduced. Cells are cultured for a variety of purposes, including research, diagnostic and therapeutic purposes. An aim of cell culture may be to obtain a sufficient number of cells of a desired phenotype. A desired phenotype may be obtained by selecting cells having that phenotype from a preexisting mixed-phenotype population of cells. Selecting may be achieved by differential survival, differential expansion, or differential migration of cells of different phenotypes. Alternatively or additionally selecting cells of a desired phenotype may be achieved by inducing differentiation of a pre-existing population of cells into cells of the desired phenotype. Many cell culture methods are available in which a population of cells are exposed in culture to various compounds, for example cell growth factors or differentiation factors or chemoattractants, in order to obtain a sufficient number of cells of the desired phenotype. Such methods include multistep methods, for example methods in which a preexisting population of cells is exposed to a first compound (or a first mixture of compounds) under first cell culture conditions, and then the resultant cells are subsequently exposed to different compounds (or a different mixture of compounds) under subsequent cell culture conditions. Typically, such a method may involve the use of a first cell culture chamber in which the cells are exposed to the first culture conditions, followed by transfer of cells into one or more subsequent cell culture chambers in which cells are exposed to one or more subsequent cell culture conditions. Alternatively, the cell culture conditions may be changed by exchanging the cell culture medium or by supplementing the cell culture medium whilst the cells remain in the name cell culture container. Transfer of cells from one culture chamber to another may be a manual process requiring the use of skilled personnel and / or specialist equipment. For example it may involve use of accurate pipetting and washing / gathering cells by centrifugation. Many labs currently purify cells using magnetic bead separation methods which can be laborious and time consuming. Such methods suffer from a number of disadvantages, for example, cells may be lost or damaged or contaminated during transfer, and the need to use highly-skilled personnel increases costs. Furthermore, manipulation of cell populations may in fact change their phenotype away from that which is required. Every handling step of a cell culture method introduces a risk of failure, including, for example, bacterial or fungal contamination but also simple reduction in viable cell number.

[0006] Cell culture equipment known as “transwells” are well known and available in several variations from a number of suppliers including ThermoFisher and Nunc. In general terms a transwell can be thought of as an “add on” to a cell culture vessel into which it is placed. A transwell typically consists of a substantially cylindrical container having plastic side walls and a permeable base. In use they may be placed into a standard cell culture plate, thus defining a cell culture space inside the transwell (the “transwell”) and a cell culture space outside of the transwell (the “well”). Communication between the two spaces may be possible across the permeable transwell base. Depending on the nature of the base, it may be arranged so as to be permeable to small molecules but impermeable to cells. One such use for this arrangement cultures a first population of cells in the transwell space and a second population of cells in the well space. Because the cell culture spaces are in close proximity, chemical communication between the two populations can be studied without the possibility of the two populations mixing and thus contaminating each other. In an alternative arrangement, the base may be made to be permeable to cells (or a subset of cells used in the study). In such arrangements, a different chemical environment can be maintained between the well and the transwell such that cells can migrate between those different environments. In an alternative arrangement, the permeable base of the transwell is seeded with cells which grow in the base. Such an arrangement can be used to study transport across a layer of cells, for example in studies of epithelial transport of drugs. EP0735134A (Becton Dickinson and Co.) discloses culture ware for transmembrane coculture. That document discloses a transwell-type insert for a standard cell culture plate, the insert comprising a hollow passageway which tapers to a central section where it is blocked by a porous membrane. The insert may be used in a first orientation in which cells are grown adhering to one side of the porous membrane. The insert may then be inverted, and cells added to the other side of the membrane. Accordingly, two populations of cells may be grown separately from each other but in close proximity. This permits interactions between the two populations of cells to be studied.

[0007] Various other complex cell culture devices exist. They may sometimes be termed “artificial organs” because they attempt to place the cells being cultured in an environment which provides cells with conditions mimicking the environment found in an in vivo organ, including an environment provided by other cells and also by cell free chemical signals.

[0008] W02004 / 101773 describes a method of cell culture in which a cell culture space is separated from supplies of cell free media and / or cell containing media in order to provide a desired environment for growth and differentiation. By means of varying the media over time, the cells being cultured can be exposed to different environments in a temporal sequence.

[0009] US5,656,492 discloses a device in which cells are cultured in a permeable chamber which can be placed in contact with a tissue (for example by implanting it into a live human) in order to provide a suitable environment for differentiation and / or expansion of the cells in the chamber.

[0010] US8, 163,537 discloses what is in effect a 96-well plate for use in pharmaceutical testing wherein each well contains two nested transwells. In this set-up cells are seeded onto the transwell membrane which acts as a support for the growth of cells. The transwell membranes permit the passage of “a chemical entity”, but “restrict movement therethrough of cells”, such that cells do not migrate through the transwell membrane.

[0011] Cell therapy, and research thereon, relies heavily on suitable cell culture vessels and methods and there is a pressing need for improvements which are able to produce therapeutic cells in sufficient quantities, with sufficient purity and with the correct phenotype, at an acceptable cost and with acceptable safety. For example, in immunotherapy, there is a need to provide immune effector cells which have the correct antigen specificity, are correctly activated and are in sufficient quantities to be of therapeutic utility. Such cells may be obtained from a patient’s own cells which have been manipulated in a multistage culture method. For example, the cells may need to be expanded, selected, and activated. Each of those requirements may be carried out as a separate cell culture step using a separate cell culture vessel. As such the overall method becomes complex and this complexity introduces an increased risk of cell loss and contamination.

[0012] Cell therapy offers tremendous scope for improvements in clinical outcomes. It is held back by sub-optimal cell culture methods and equipment. For example in chimeric antigen receptor T-cell (CAR-T) therapy a defined homogenous effector cell population must be purified from a complex mixture of a patient’s cells. This is typically done using magnetic beads to isolate the cells followed by a period of culture in flasks or bags to expand and differentiate the required cell type by use of an expansion signal and supportive cytokines. Recent technical advances such as the CliniMACS Prodigy ™ system have automated the process of cell expansion and purification, thus allowing it to be used in a semi-routine manufacturing pathway. That has been achieved by the use of a complex system of syringe pumps and tubing which adds expense and means that it is typically only carried out in a few centralised manufacturing facilities. Existing methods of producing cells for CAR-T therapy and cell therapy in general suffer from one or more of the following disadvantages:

[0013] • the need for extensive handling of the cells with concomitant risk to cell contamination and viability,

[0014] • a highly complex system requiring highly trained specialist staff and with multiple points of possible failure,

[0015] • high cost,

[0016] • the use of centralised facilities which may be located far from patients,

[0017] • dead cell contamination,

[0018] • the use of a single selection parameter based on the expression of a certain surface protein, and few variables to modulate potency may also result in an end-product which is sub-optimal because the selection parameter is not sufficiently clinically relevant, for example, in antitumour therapy, phenotypic selection based on protein expression may not be sufficient for selecting cells which have the clinically useful property of the ability to migrate into tumours.

[0019] The present invention seeks to provide improved cell culture vessels and methods which mitigate one or more challenges of cell culture. Additionally the present invention seeks to provide improved cell culture vessels and methods. Improvements provided by the present invention may optionally include lower cost and simplicity but may also result in the provision of cell therapies with improved therapeutic function.

[0020] Additionally, the present invention seeks to provide improved cell therapy methods and improved populations of cells, including populations of cells for cell therapy.

[0021] Summary of invention

[0022] According to a first aspect, the invention provides an in vitro method of producing a population of cells comprising the steps of:

[0023] A, introducing a source of cells into the first cell culture chamber of an in vitro cell culture vessel comprising three cell culture chambers arranged in a series, each chamber being separated from the next chamber in the series by a semipermeable barrier,

[0024] B, contacting the cells in the first cell culture chamber to a first composition of one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants so as to promote migration of at least a subset of cells from the first cell culture chamber into an intermediate cell culture chamber of the series,

[0025] C, contacting cells in the intermediate cell culture chamber of the series to a further composition of one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants so as to promote migration of at least a subset of cells from the intermediate cell culture chamber to the final cell culture chambers in the series,

[0026] D, optionally withdrawing cells from the final cell culture chamber in the series.

[0027] According to a second aspect, the invention provides a cell culture vessel comprising at least three cell culture chambers arranged in a series comprising a first cell culture chamber, one or more intermediate cell culture chambers and a final cell culture chamber, each chamber being separated from the previous and next cell culture chamber in the series by a semipermeable barrier, wherein each cell culture chamber is nestable within the next cell culture chamber of the series.

[0028] According to a third aspect, the invention provides a population of cells produced in accordance with a method of the first aspect of the invention or produced by culturing a source of cells in a cell culture vessel according to the second aspect of the invention. According to a fourth aspect, the invention provides a method of cell therapy comprising administering to a subject in need thereof, a population of cells according to a third aspect of the invention.

[0029] According to a fifth aspect of the invention, there is provided a population of cells of the third aspect of the invention for use as a medicament.

[0030] According to a sixth aspect of the invention, there is provided a population of cells of the third aspect of the invention for use in cell therapy.

[0031] The invention also provides in a further aspect a SLAS standard plate comprising an array of cell culture chambers each having at their lower end a semipermeable barrier and each cell culture chamber having adjacent to it at least one pipette bypass.

[0032] It will of course be appreciated that features described in relation to one aspect of the present invention may be incorporated into other aspects of the present invention. For example, the method of the invention may incorporate any of the features described with reference to the apparatus of the invention and vice versa; and features of vessels and methods of the invention may be incorporated into populations of cells of the invention.

[0033] Description of the Drawings

[0034] Embodiments of the present invention will now be described, by way of non-limiting example only, with reference to schematic drawings of figures 1 to 4 in which:

[0035] Figure 1 shows a schematic representation of a cell culture vessel illustrating the principle underlying methods of the embodiment.

[0036] Figure 2 shows a schematic representation of a cell culture vessel which is demountable and which is suitable for use in a method of the invention.

[0037] Figure 3 shows a close-up representation of the junction between one cell culture chamber and the next in order to illustrate one possible embodiment of an arrangement for connecting adjacent cell culture chambers in a series together.

[0038] Figures 4 to 9 show the results of experiments described in the Examples.

[0039] Figure 4 shows cell phenotype of cells harvested from CD3 / CD28 coated wells (control, black bars) or collected from the final cell culture chamber of a cell culture vessel of the invention (white bars). Cells are stained and analysed by flow cytometry. The fraction of live cells, B cells and CD4 or CD8 Tccells was calculated (A). The fraction of CD4 Tccells B) and CD8 Tccells (C) with different phenotypes based on their CD44 and CD62L expression was calculated. Results are representative of two independent experiments (+ / - SD; Student t test; n=3).

[0040] Figure 5 shows cell phenotype of cells harvested from a cell culture vessel of the invention with (white bars) or without (grey bars) the use of chemokines. Cells stained were and analysed by flow cytometry. The fraction of live B cells, CD4 or CD8 Tccells was calculated (A). The fraction of CD4 Tccells (B) and CD8 Tccells (C) with different phenotypes based on their CD44 and CD62L expression was calculated (+ / - SD; n=3).

[0041] Figure 6 shows cell phenotype of cells harvested from CD3 / CD28 coated wells (black bars) or from the final cell culture chamber of a cell culture vessel of the invention (white bars). Cells were stained and analysed by flow cytometry. The proportion of CD4 Tccells (A and B) and CD8 Tccells (C and D) expressing Granzyme B, fFNy, Perforin and TNFa (A and C) or CCR7, CXCR3 and LFA-1 (B and D) was calculated (+ / - SD; n=4).

[0042] Figure 7 shows the phenotype of CD4+Marilyn transgenic T cells harvested from CD3 / CD28 coated wells (black bars) or from the final cell culture chamber of a cell culture vessel of the invention (white bars) and counted, cells were then stained and analysed by flow cytometry. The proportion of CD4+TCR+T cells expressing the relevant proteins is presented (+ / -SD; n=4).

[0043] Figure 8 shows the viability and phenotype of human PBMC cultured in anti-CD3 / CD28 precoated wells or a cell culture vessel of the invention and incubated for 3 days. Cells were harvested from the final cell culture chamber of the cell culture vessel of the invention, or from CD3 / CD28 coated wells. After harvesting cells were analysed by flow cytometry for cell surface expression of lineage markers, CCR7, CD45RA and CD45RO as well as cytokines including TNFa (A-C, n=4, SD and Student’s T tests) or tested for their ability to migrate to chemokine, IP- 10 (D, N=4, SD and Student’s T tests).

[0044] Figure 9 shows the proportion of B cells and T cell subtypes found in each chamber of a cell culture vessel of the invention and, by way of comparison in cells cultured in cell culture wells of the prior art.

[0045] Figure 10 shows a cell culture vessel of an embodiment of the invention in exploded form. Figure 11 shows an alternative view of a cell culture vessel of an embodiment of the invention in exploded form.

[0046] Figure 12 shows some of the features of an intermediate cell culture chamber of a cell culture vessel in accordance with an embodiment of the invention.

[0047] Figure 13 shows some of the features of a first cell culture chamber of a cell culture vessel in accordance with an embodiment of the invention.

[0048] Figure 14 shows a cell culture vessel of a cell culture vessel according to an embodiment of the invention wherein the cell culture chambers are nested within each other in series and a lid is present

[0049] Figure 15 shows a cell culture vessel of a cell culture vessel according to an embodiment of the invention wherein the cell culture chambers are nested within each other in series a lid is present. It can be seen that in figure 14 the membranes at the base of the first cell culture chamber and the intermediate cell culture chambers are higher than they are in the arrangement shown in figure 15. This allows the final cell culture chamber and the intermediate cell culture chamber to be used with a larger volume of cell culture medium than in the arrangement shown in figure 15 wherein the intermediate cell culture chamber is positioned lower within the final cell culture chamber than in the arrangement shown in figure 14 and the first cell culture chamber is positioned lower within the intermediate cell culture chamber than in the arrangement shown in figure 14.

[0050] Figure 16 shows a plan view of a nested cell culture vessel of the invention (lid removed).

[0051] Figure 17 shows a view of a multichamber plate version of a cell culture vessel of the invention in a closed configuration with a lid and a plate comprising multiple first cell culture chambers, a plate comprising multiple intermediate cell culture chambers and a plate comprising multiple final cell culture chambers.

[0052] Figure 18 shows a similar multichamber plate version of a cell culture vessel of the invention in a separated configuration.

[0053] Figure 19 shows a cross section of an assembled multichamber plate version of a cell culture vessel of the invention including the location of an optional spacer element.

[0054] Figures 20 to 23 show each part of a multichamber plate version of a cell culture vessel of the invention separately. Figure 24 shows a view of a multichamber plate version of a cell culture vessel of the invention in a separated configuration with the addition of an optional spacer.

[0055] Figure 25 shows a view of a multichamber plate version of a cell culture vessel of the invention in an assembled configuration with a lid.

[0056] Figure 26 shoes a view of a section of a multichamber plate version of a cell culture vessel of the invention in an assembled configuration with a lid.

[0057] Figure 27 shows a close-up view of part of a multichamber plate comprising multiple first cell culture chambers.

[0058] Figure 28 shows view of a multichamber plate comprising multiple final cell culture chambers.

[0059] Detailed Description

[0060] Whilst the present invention has been described and illustrated with reference to particular embodiments, it will be appreciated by those of ordinary skill in the art that the invention lends itself to many different variations not specifically illustrated herein. By way of example only, certain possible variations of each aspect of the invention will now be described.

[0061] Cell culture vessels of the invention

[0062] According to a second aspect, the invention provides a cell culture vessel comprising at least three cell culture chambers arranged in a series comprising a first cell culture chamber, one or more intermediate cell culture chambers and a final cell culture chamber, each cell culture chamber being separated from the previous and next cell culture chamber in the series by a semipermeable barrier, wherein each cell culture chamber is nestable within the next cell culture chamber of the series. Preferably the semipermeable barrier is configured to permit selective passage of cells, for example to permit selective passage of human immune cells.

[0063] Other aspects of the invention also relate to use of cell culture vessels which may optionally be as defined in the first or other aspects of the invention. Alternatively, and in accordance with other aspects of the invention, for example methods of the invention, the cell culture vessel may simply be a cell culture vessel, for example an in vitro cell culture vessel, comprising at least three cell culture chambers arranged in a series comprising a first cell culture chamber, one or more intermediate cell culture chambers and a final cell culture chamber, each cell culture chamber being separated from the previous and next cell culture chamber in the series by a semipermeable barrier.

[0064] For example other embodiments of the invention may relate to use of cell culture vessels or products produced in cell culture vessels. In certain embodiments, of or relating to all aspects of the invention, the cell culture vessel has three cell culture chambers arrange in a series. In other embodiments, of or relating to all aspects of the invention, the cell culture vessel has more than three cell culture chambers arranged in a series. Cell culture vessels having only two cell culture chambers, an input chamber and an output chamber are known in the prior art and lie outside the scope of the present invention. The present invention is based in part on the realisation that cell culture vessels having at least one additional intermediate cell culture chamber, in addition to an input cell culture chamber and an output cell culture chamber, are advantageous, because an increase in the number of chambers allows signals to be given to the cells in a temporal sequence, for example a signal which requires a particular differentiation step to have previously taken place to be effective may be provided in a later chamber of the series of cell culture chambers, and a signal which promotes the required differentiation step may be provided in an early chamber of the series of cell culture chambers.

[0065] Construction of vessel - material of walls

[0066] The cell culture vessel of the second aspect of the invention (and also cell culture vessels relating to other aspects of the invention) can be made from any suitable material. In many embodiments the cell culture chamber walls (not including the semipermeable barrier) of the cell culture vessel of the invention is made from a rigid plastic material, for example polystyrene (PS). Other suitable plastic materials include polyethylene (PE) and polypropylene (PP). Because there may be advantage in the cell culture vessel being at least partially transparent, it may be preferred to make the walls from a substantially clear plastic material. In addition to the plastics mentioned above, acrylic (PMMA), polycarbonate, amorphous copolyester (such as PETG), polyethylene terephthalate (PET), polyvinyl chlorine (PVC) liquid silicone rubber (LSR), cyclic olefin copolymers (COCs), ionomer resins, fluorinated ethylene propylene (FEP), styrene methyl methacrylate (SMMA), and styrene acrylonitrile resin (SAN) may also be used. PET, PE, PS and PP may be especially preferred in certain embodiments. According to certain embodiments, the walls of the cell culture chambers of a cell culture vessel of the invention are substantially manufactured from PS. The plastic material used may optionally be surface coated to make it suitable for the cell type with which it is to be used. For example a plastic surface such as a PS surface may be treated by exposure to a plasma gas in order to increase the presence of oxygen-containing functional groups such as hydroxyl and carboxyl. The plastic material used may be optionally surface coated, for example with poly-D-lysine, collagen, laminin or mixtures thereof. In certain embodiments, it may be surface coated with a cell activating factor, a cell adhesion factor, a layer of cells or a cross-linking compound such as an immobilised antibody. The semipermeable barrier is preferably configured to permit selective passage of cells, for example to permit selective [passage of human immune system cells.

[0067] Construction of vessel - materials of semipermeable barrier

[0068] The semipermeable barrier may be made from any suitable material. In many embodiments of the second aspect of the invention, and many embodiments relating to other aspects of the invention, the semipermeable barrier may be made from one or more plastic materials. For example, it may comprise a thin film composite membrane, a polycarbonate membrane, or a polyamide membrane. Use of plastic material (especially a thermoplastic material) for both the semipermeable barrier and the walls of the cell culture chambers may advantageously permit the two components to be sealed together, for example by use of an adhesive, by solvent welding or by heat, friction, or ultrasonic welding. In other embodiments, the semipermeable barrier may comprise (optionally in combination with the one or more plastic material noted above) a biomolecule, such as a protein (such as collagen), or a polysaccharide (such as cellulose). In certain embodiments it may comprise a cell culture scaffold material or a hydrogel. According to certain embodiments, it may comprise hyaluronic acid (HA, for example a HA gel or a HA foam, alternatively it may comprise a bioactive glass foam. In other embodiments with may comprise an endothelial cell layer, an epithelial cell layer, an agarose gel, a polyacrylamide gel or an alternative hydrogel. Optionally a gel may comprise collagen, fibronectin or a mixture thereof. Certain materials from which the semipermeable barriers may be constructed permit the passage of cells because they are amenable to being altered by cells. For example, a semipermeable barrier which comprises protein may be susceptible to degradation by proteases (for example matrix metalloproteinases) and therefore capable of being permeable to cells, and optionally only to cells, which are capable of producing the correct protease. According to certain embodiments, robusticity of the semipermeable barrier may be provided by use of a solid plastic material having relatively large holes or gaps which is used to support a membrane, gel or other component of the semipermeable barrier having the required properties such as biocompatibility. The material may be chosen to provide the properties required as noted below. It may be fabricated by any suitable method. The material of the semipermeable barrier may optionally be surface coated to make it suitable for the cell type with which it is to be used. For example, a plastic surface such as a PS surface may be treated by exposure to a plasma gas in order to increase the presence of oxygen-containing functional groups such as hydroxyl and carboxyl. The semipermeable barrier may be optionally surface coated, for example with poly-D-lysine, collagen, laminin or mixtures thereof. In certain embodiments, it may be surface coated with a cell activating factor or a cross-linking compound such as an immobilised antibody. In certain embodiments, it may alternatively or additionally be coated with a layer of adherent cells.

[0069] Constructional features of the cell culture vessel

[0070] The cell culture vessels of the invention and for use in methods of the invention, may be constructed in various ways. Vessels of the invention comprise three general types (although hybrid vessels between the types are also covered by the invention). The first general type of cell culture vessels of the invention are modular cell culture vessels. A modular cell culture vessel comprises two or more (preferably three or more) modules, each module containing the side wall(s) of a subset of the total number of cell culture chambers (preferably each module contains the side wall of one of the cell culture chambers). One type of module is a module having one or more side walls, said side walls belonging to a single cell culture chamber, for example a generally cylindrical side wall, and a semipermeable barrier sealed to the lower edge of the side walls to close the end of the module (for example to close one end of the cylinder when the module is substantially cylindrical). The side walls of a module can optionally be moulded in a single piece (for example by injection moulding of a plastic material), and the semipermeable barrier can then be sealed to the side walls to close one end of the module. Another type of module is the module used to define the final cell culture chamber of the series of cell culture chambers in a cell culture vessel of the invention. This type of module comprises one or more side walls as described above and a rigid impermeable end closure across one of the ends of the space defined by the side walls. That end wall may be conveniently made from the same material as the side walls. It may, for example, be moulded together with the side walls as a single piece (for example by injection moulding of a plastic material). A cell culture vessel for use in methods of the invention can be assembled from three or more modules. Those three or more modules can be the same or different to each other. They may be assembled into a cell culture vessel by stacking the modules in a series of at least three modules. The lower module in the stack (or “column”) is typically of a type having a closed lower end without a semipermeable barrier. The open upper end of that module may be closed by the engagement of the semipermeable barrier on the end of the module stacked directly above it. If three modules are assembled by this stacking method, the bottom two modules in the stack will cooperate to define two lower cell culture chambers which are closed on the sides and at both ends. The top module of a stack may be open because there is no other module stacked above it. It may optionally be closed to form the upper cell culture chamber by means of a lid which may preferably be made of the same or similar material to the side walls of the modules. Thus a modular cell culture vessel may consist of three or more stacked modules and an optional lid, thus defining the three or more cell culture chambers of the cell culture vessel. It will be understood that such a vertically stacked arrangement of cell culture chambers will typically be used with the first cell culture chamber of the series at the top of the track and the final cell culture chamber of the series at the bottom of the stack. However, the opposite arrangement is also envisaged by the invention wherein the first cell culture chamber is at the bottom of the stack and the final cell culture chamber is at the top of the stack. Such an arrangement may be especially useful for cells which are motile (for example cells which are flagellated) and which can “swim” up through the stack of chambers. Alternatively, a modular cell culture vessel may consist of three or more modules and an optional lid which defines the three or more cell culture chambers in a horizonal arrangement with the cell culture chamber side by side horizontally.

[0071] Preferably the modules of a modular cell culture vessel are adapted to engage with each other so that in a stack of modules, the bottom module of the stack is engaged with an intermediate module above it, if there is more than one intermediate module, then each is engaged with the intermediate module above it, the top intermediate module is engaged with the top module of the stack and the open upper end of the top module of the stack is optionally closed by engagement with an optional lid. Effective engagement can be provided by any suitable engagement means, for example by provision of a screw thread, a tongue and groove or an interference fit. In certain embodiments, the modules are dimensioned and shaped, for example by the provision of mutually-engaging tongue and grooves, to achieve a firm interference fit between adjacent modules in a stacked series of modules. In such embodiments, the interference fit is substantially watertight. It is preferably such that modules may be separated from each other by pulling them apart. Accordingly, the engagement means of the modules may be arranged to engage with each other and disengage from each other by application of normal hand strength.

[0072] Alternatively, the cell culture vessel of the invention may be of a non-modular type. A non- modular type has the same basic configuration of cell culture chambers except that there are no separable modules. A non-modular type may optionally be made from a modular type by assembling modules and then fixing them together so that they are no longer separable. This may optionally be done by gluing them, or by welding them with friction, solvent, ultrasonic or heat welding. Alternatively, a non-modular cell culture device of the invention may be moulded as a single piece. Methods of the invention also encompasses, and relates to, hybrid cell culture vessels in which are partially but not completely separable into separate modules

[0073] An advantage of modular cell culture vessels include flexibility in that the modules may be combined in various orders in the series and configurations to suit a particular application from a limited stock of modules of a particular type (in particular modules having particular semipermeable barriers, modules comprising specific coatings and modules containing one or more particular cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants).

[0074] Another advantage of modular cell culture vessels is that because the modules are separable from each other they can be separated in order to allow access to the contents of the cell culture chambers. For example to allow access to change medium, introduce or withdraw cells, to take samples or for another purpose. Conversely, the possibility of access to the contents of the cell culture chambers may also increase the risk of contamination. According to certain embodiments relating to modular cell culture vessels, the modules are separatable, but additional access is provided into cell culture chambers by means of a pipette bypass as described elsewhere herein.

[0075] An advantage of a non-modular cell culture vessel is reduced risk of contamination and a reduced risk of the cell culture vessel coming apart into separate modules inadvertently, for example during transport.

[0076] A non-modular cell culture vessel because it cannot be disassembled to permit access to the contents of the cell culture chambers, is preferably provided with means of introducing and / or withdrawing cells and / or medium from the cell culture chambers. Such means may also optionally be provided in a modular cell culture chamber. Such means may include one or more access ports in the side wall of each cell culture chamber. For example it may be an injection port comprising a sealable rubber or silicone gasket through which material may be introduced or withdrawn by means of a hypodermic syringe. Alternatively, it may be a connection port for a tube, pipette tip, syringe nozzle or pump output / input through which material may be introduced or withdrawn. Use of a port, in modular and non-modular cell culture vessels provides the ability to introduce or withdrawn cells and / or medium during cell culture (without having to dismantle a modular cell culture vessel). This can be used to replenish medium, withdraw cells for use or testing, or change or supplement the chemical composition of the medium, for example by providing a temporally-changing chemical environment. Alternative means for introducing and / or withdrawing material from the cell culture chambers are described under the heading “pipette bypass”.

[0077] According to certain preferred embodiments, a cell culture vessel for use in a method according to the first aspect of the invention is a cell culture vessel according to the second aspect of the invention, that is to say, it is a cell culture vessel wherein each cell culture chamber is nestable within the next cell culture chamber of the series.

[0078] The third broad type of culture vessel described herein, which is suitable for use in accordance with methods of the invention and for producing products and treatments of the invention and also accords with the second aspect of the invention is a nestable cell culture vessel as described below. It is understood that features described herein in respect of cell culture vessels of the first or second type may also be applied to cell culture vessels of the third - nestable- type provided that cell culture vessels of the nestable type retain their nestable feature.

[0079] Nestable cell culture vessels

[0080] According to a second aspect, the invention provides a cell culture vessel comprising at least three cell culture chambers (for example 3 cell culture chambers or 4 cell culture chambers or five or more cell culture chambers) arranged in a series comprising a first cell culture chamber, one or more intermediate cell culture chambers and a final cell culture chamber, each chamber being separated from the previous and next cell culture chamber in the series by a semipermeable barrier, wherein each cell culture chamber is nestable within the next cell culture chamber of the series.

[0081] By nestable, it is understood that each of the first and intermediate cell culture chambers is received within the next cell culture chamber of the series. For example, when a cell culture vessel has three chambers - a first, intermediate and final chamber - the first cell culture chamber is able to be placed inside the intermediate cell culture chamber and the intermediate cell culture chamber may be placed inside the final cell culture chamber. This means that the final cell culture chamber holds the other cell culture chambers and may conveniently be lifted, carried and lidded whilst the other cell culture chambers are secure inside it. According to certain embodiments, each of the first and intermediate cell culture chambers is wholly received inside the final cell culture chamber when nested. According to other embodiments, the liquid receiving portion of each of the first and intermediate cell culture chambers is wholly received inside the final cell culture chamber when nested. For example, the volume of each of the first and intermediate cell culture chambers which receives cell culture medium in normal use is wholly received inside the volume of the final cell culture chamber. Optionally, each of the first and intermediate cell culture chambers will comprise side walls, an open top and a base over which comprises the semipermeable barrier. The final cell culture chamber will be similar but will have a solid enclosed base rather than a semipermeable barrier because it is the final cell culture chamber in the series. The side walls of the final cell culture chamber define a space large enough for the previous intermediate cell culture chamber in the series of cell culture chambers to be nested inside it. The side walls of the first intermediate cell culture chamber in the series of cell culture chambers define a space large enough for the first cell culture chamber to be placed inside it. Preferably the total enclosed volume (both the volume adapted to receive cell culture medium and any volume not adapted to receive cell culture medium) of each cell culture chamber in the series is larger than the enclosed volume of the previous cell culture chamber in the series.

[0082] Advantage of nested cell culture chambers

[0083] When the cell culture vessel is of the nestable type according to the second aspect of the invention, it is preferable that there is sufficient space in each of the cell culture chambers to provide for appropriate cell culture conditions (for example for each step of a method of the invention). It is also desirable that when in use with cell culture medium, that the semipermeable barriers separating each of the cell culture chambers in the series are in contact with medium on both sides. Such an arrangement avoids an “airgap” between the cell culture chambers which in certain embodiments would act as a barrier to cell migration and / or the diffusion of chemical agents such as chemoattractant between cell culture chambers. In the arrangement illustrated in figures 2 and 3 it may be technically possible but fiddly to avoid an airgap between adjacent cell culture chambers. In such an arrangement, the avoidance of an undesirable airgap may necessitate either the provision of ports to introduce medium and / or withdraw gas from an assembled cell culture vessel, or in the case of a demountable cell culture vessel of figures 2 and 3, the assembly process may require the careful filling of each of the intermediate and final cell culture chambers to the brim and then the fixing of the cell culture chamber above with sufficient skill to avoid spilling too much medium so as to allow the formation of an airgap. Such a method of assembly, whilst perfectly possible is not ideal because it requires a high level of technical skill and because it can lead to spillage of medium, which is wasteful, messy and might increase the chance of microbial contamination if nutritious medium remains on the outer surface of the cell culture chambers.

[0084] The nestable cell culture vessel of the invention in its second aspect mitigates these problems. It allows a cell culture chamber to be lowered into the next cell culture chamber in the series which contains cell culture medium in such a way that the external surface of the semipermeable barrier is in contact with the cell culture medium in the next cell culture chamber with a low chance of spilling of cell culture medium. That is because any displacement of cell culture medium in a cell culture chamber when the cell culture chamber earlier in the series is nested in it is amply accommodated by within the cell culture chamber because the later cell culture chamber in the series does not need to be full of medium. Alternatively, the cell culture vessel may be assembled “dry” with each of the cell culture chambers appropriately nested and then medium may be added to each of the cell culture chambers. A slight excess of cell culture medium may be used to ensure that both sides of the semipermeable barrier are in contact with the medium, any excess will harmlessly be displaced up the outside of the earlier cell culture chamber of the series but will remain fully enclosed within the later cell culture chamber in the series and thus enclosed (and in some embodiments fully enclosed) within the cell culture vessel as a whole (rather than cause a spillage) because of the nested arrangement.

[0085] Suspension of cell culture chambers

[0086] According to preferred embodiments of the cell culture vessels of the second aspect of the invention, each of the first and intermediate cell culture chambers may optionally be nestable within the next cell culture chamber of the series in such a way that they are suspendable within the next cell culture chamber so that their a gap between the base of the each of the cell culture chamber and the base of the next cell culture chamber in the series. This gap corresponds to the space, to be filled with cell culture medium in use, for culture of the cells to take place. By “suspendable” it is understood that when a cell culture chamber is nested within the next cell culture chamber in the series, although it remains fully enclosed within the next cell culture chamber, it is held at a distance (for example 2, 5, 10 or 20 mm) from the base of the next cell culture chamber. There are various alternative constructional configurations which can be used to achieve a gap between nested cell culture chambers. For example, there may be provided “spacers” between adjacent cell culture chambers (provided separately or as part of either of the adjacent cell culture chambers) to keep them apart. In certain preferred embodiments however, the gap is achieved by suspension of an earlier cell culture chamber in the series within the next cell culture chamber of the series. Typically, this suspension can be achieved by fixing one or more points of the interior surface of the sidewall of a later cell culture chamber in a series to one or more points of the exterior surface of the sidewall of an earlier cell culture chamber in the series. Accordingly such fixation of the sidewalls prevent the inner nested cell culture chamber from falling completely into the outer nested cell culture chamber and thus presence a gap. Such fixing may be permanent or temporary. For example in some embodiments the nested cell culture chambers may be permanently fixed (for example glued or welded) to each other). In other, generally preferred embodiments the cell culture chambers are not permanently fixed to each other so that the cell culture vessel may be disassembled. In such embodiments, the suspension may be achieved via temporary cooperation between features present in the inside wall of one cell culture chamber and the outside wall of the cell culture chamber situated within it. For example, this cooperation may be between lugs and / or detents moulded into (or affixed to) the walls of the cell culture chambers.

[0087] According to certain embodiments, each of the first and intermediate cell culture chambers may not be merely suspendable within the next cell culture chamber in the series, but rather they are suspended within the next cell culture chamber of the series.

[0088] Adjustable suspension

[0089] According to preferred embodiments of cell culture vessels of the second aspect of the invention, the suspension (and suspendability) of each of the first and intermediate cell culture chambers within the next cell culture chamber of the series may be adjustable. That is to say, the gap between adjacent cell culture chambers may be adjustable. This may be done in order to adapt the vessel to variable volumes of medium in each cell culture chamber. It is understood that when a cell culture chamber is nested within the next cell culture chamber in the series, although it remains fully enclosed within the next cell culture chamber, it can optionally be suspended at a variable distance from the base of the next cell culture chamber. There are various alternative constructional configurations which can be used to achieve such a variable gap between nested cell culture chambers. In one configuration, this may be achieved by providing lugs and / or detents on each of the cell culture chambers such that said lugs and detents are arranged to cooperate with each other in two or more different combinations, each different combination corresponding to a different suspension height of a cell culture chamber within the next cell culture chamber of the series. Each different combination of cooperating lugs and / or / detents may correspond to a different sized gap between the base of adjacent cell culture chambers and allow methods of using the cell culture vessel to accommodate an adjustable cell culture medium volumes in each of the chambers whilst ensuring that both sides of the semipermeable barrier separating adjacent cell culture chambers remains in contact with cell culture medium.

[0090] In one configuration, each of the first and intermediate cell culture chambers are provided on the outer surface of their walls with protruding lugs which are configured to engage with a first set of one or more detents (for example one or more slots) provided in the inner surface of the next cell culture chamber in the series. Those detents (for example slots) may be dimensioned to receive the corresponding lugs to a first specific depth corresponding to a specific suspension height and specific gap size. Each of the first and intermediate cell culture chambers may also be provided on the out surface of their walls with protruding lugs which are configured to engage with a second set of one or more detents (for example one or more slots) provided in the inner surface of the next cell culture chamber in the series. The second set of one or more detents may be dimensioned to receive the corresponding lugs to a second specific depth corresponding to a specific suspension height and specific gap size. In some configurations the lugs may be suitable for engaging with either set of detents. In other configurations, a separate set of lugs may be provided for engaging with each set of detents. More than two sets of detents (for example more than two sets of slots) may optionally be provided, each corresponding to a different suspension height and gap size. In some embodiments, the set of detents which will cooperate with corresponding lugs may be selected by rotating the cell culture chamber about a vertical axis, to align a set of lugs and a set of detents corresponding to a desired suspension height. It is generally preferred, in order that the suspension be reasonably stable that each set of lugs and each set of detents comprised two or more lugs or detents. For example each set of lugs and each set of detents may corresponding to, respectively, to two lugs and two detents which are disposed on the side walls of the respective cell culture chambers approximately opposite each other. In certain preferred embodiments the arrangement of lugs and detents may be such that the user is able to select three different suspension heights of each of the first and intermediate cell culture chambers.

[0091] Alternatives to adjustable suspension

[0092] One optional alternative to adjusting the volume of each cell culture chamber between adjacent semipermeable barriers is rather than using adjustable suspension to use adjustable spacer elements (“spacers”). For example, different spacer elements may be used to achieve different spacing. In its simplest form, the absence of a spacer element results in a first spacing and the addition of a spacer element results in a second, wider, spacing. The use of one or more spacer elements, may be especially preferred in the multichamber plate versions of the cell culture vessels described below, because a single spacer may be used to space all of the individual sets of nested chambers in a multichamber plate version of the cell culture vessel of the invention in a single simple operation, by means of the spacer elements(s) holding the plates apart by their edges.

[0093] General shape of the cell culture vessel.

[0094] Various shape may be used for the nestable cell culture vessels of the invention. According to certain embodiments, it is preferred that the base (and where present lid) of the final cell culture chamber is substantially square. By making the bases of the final cell culture chamber substantially square the whole cell culture vessel will have a substantially square base (because the other cell culture chambers nest inside the final cell culture chamber). This is a space efficient shape and allows multiple cell culture chambers to be efficiently extracted, for example within an incubator. A rectangular base, whilst being similarly spaceefficient, is less desirable because it has fewer folds of rotational symmetry compared with a square which may be used to provide different engagements combinations of lugs and detents and different suspension heights. The inner (first and intermediate) cell culture chambers may also have substantially square bases, or they may be substantially circular at their base, or they may have a base which is a squircle.

[0095] Transit position Cell culture vessels of certain optional embodiment of the second aspect of the invention which have adjustable suspension of the first and intermediate cell culture chambers, may be provided with a “transit position” whereby rattling of the first and intermediate cell culture chambers within the next cell culture chamber in the series is minimised by the provision of a suspension height which places the first and intermediate cell culture chambers as high as they can be whilst still remaining nested within the next cell culture chamber of the series. In such a position a lid may also be provided which limits upward movement of the nested first and intermediate cell culture chambers. The lid may optionally be kept in place by a clip-on design, by provision of a fastener or by the provision of an outer wrapping for example an outer plastic shrink wrap or an elastic band.

[0096] Pipette bypass

[0097] According to certain preferred embodiment of the nested cell culture vessels of the second aspect of the invention, each of the first and intermediate cell culture chambers are dimensioned so that they nest reasonably snuggly within the next cell culture chamber in the series. That ensures that space if used efficiently and reduced rattling of the chambers within the vessel which might disadvantageously lead to displacement of the inner chambers. However, there may be a desire to access the cell culture chambers which have an earlier cell culture chamber nested in them without having to dismantle the cell culture chamber, in order to for example add or withdraw cells or medium. Therefore, each of the first and intermediate cell culture chambers may be dimensioned to provide a route for a pipette tip to pass into the next cell culture chamber of the series. This “pipette bypass” may optionally be provided by in involution of the wall of each of the first and intermediate cell culture chamber which together with the wall of the next cell culture chamber in the series defines a channel through which a pipette to may be received in order to access said next cell culture chamber in the series. Optionally, the pipette bypass may also be capable to engaging with a tool for lifting a nested cell culture chamber out of the next cell culture chamber in the series into which it is nested. In the multi chambered plate embodiment of the invention discussed further below, the pipette bypass may comprise a hole in the plate between adjacent cell culture chambers (of the same position in multiple series of cell culture chambers). It may optionally additionally comprise an involution of the side wall of cell culture chambers.

[0098] Gas exchange means Living cells respire. Typically this involves the conversion of a carbon source into carbon dioxide in the presence of oxygen. This means that a means of gaseous exchange into and out of the cell culture chambers may need to be provided. Alternatively, a means of gaseous exchange is not provided, either because the cell culture vessel is to be used for a short period of time such that the gases dissolved in the medium in the cell culture chambers is sufficient to support short term cell respiration, or because the cell culture medium is to be exchanged such that oxygenated medium replaces medium containing dissolved carbon dioxide or because the cell culture is an anaerobic cell culture.

[0099] Accordingly, cell culture vessels of the invention, in all its aspects, may optionally include gas exchange means in the side walls of one or more of the cell culture chambers (and / or in any lid as may be present). A gas exchange means may optionally include an area of gas permeable material in the side wall and / or lid. For example, all or part of the walls of one or more of the cell culture chambers may comprise a gas permeable material such as polydimethylsiloxane (PDMS). In respect of cell culture chambers corresponding to the second aspect of the invention, the provision of such gas exchange means may not be necessary because each of the first and intermediate cell culture chambers may be suspended in the next cell culture chamber of the series sufficient loosely for gas exchange to take place past it. Any optional lid of the cell culture chamber may also be sufficiently loose to permit gas exchange past it. In some configurations it may be possible to temporarily tighten the lid, for example when transporting the cell culture vessel, and also loosen the lid to allow gas exchange, for example which the cell culture chamber has been positioned in an incubator.

[0100] Number of cell culture chambers

[0101] It has been found that providing a cell culture device having three cell culture chambers arranged in a series is for many applications sufficient. However in some embodiments, additional cell culture chambers may be advantageous and accordingly in alternative embodiments of all aspects of the invention, there may be provided 4, 5 or 6 or more cell culture chambers arranged in a series. In many embodiments, increasing the number of cell culture chambers beyond 5 or 6 brings diminishing returns. Therefore in certain embodiments the preferred number of cell culture chambers in a series of cell culture chambers of a cell culture vessel of the invention (and a cell culture vessel relating to other aspects of the invention) is from 3 to 5 or from 3 to 6.

[0102] Relative arrangement of cell culture chambers According to all aspects of the invention, the cell culture chambers of the invention are arranged in a series. Some embodiments of cell culture vessels of the second aspect of the invention and some cell culture vessels for use in methods according to the first aspect of the invention, require cell culture chambers to be arranged in a stack (or “column”), such that each cell culture chamber is placed above or below the next cell culture chamber in the series. Preferably, the first cell culture chamber in the series is at the top of the stack, with each subsequent cell culture chamber in the series located below the first cell culture chamber and the final cell culture chamber in the series is the bottom cell culture chamber in the series. With such an arrangement, methods of the invention, for example methods according to the first aspect of the invention, typically comprise adding a source of cells to the first cell culture chamber which is located at the top of the stack of cell culture chambers, and the final cell culture chamber from which cells are withdrawn is located at the bottom of the stack of cell culture chambers. Cells in culture experience gravity which means that they have a tendency to sink in medium to the bottom of a cell culture chamber. This means that arranging the series of cell culture chambers from top to bottom is most suited to most embodiments. There are, however, certain highly motile cell types (such as flagellated algae and mammalian spermatozoa) which are able to “swim”. For such cell types it may be applicable for the cell culture chambers to be arranged in a stacked series from bottom to top. That may especially be the case when the cell culture chamber is used in a method of selection according to a physiologically-relevant parameter such as high motility. According to the arrangement of the nested cell culture chambers of the invention in its second aspect, the cell culture chambers are nested such that the first cell culture chamber of the series is nested inside the other cell culture chambers in the series and each of the intermediate cell culture chambers are nested inside the next cell culture chamber of the series. The final cell culture chamber of the series is not nested inside a further cell culture chamber and as such defined the outer dimensions of the cell culture vessel. Methods of the invention also encompass those which use a cell culture vessel wherein the cell culture chambers are not necessarily nested, for example wherein the cell culture vessel has one of the alternative configurations described herein.

[0103] Semipermeable barrier features

[0104] According to the invention, there are at least two semipermeable barriers between adjacent cell culture chambers in the series of cell culture chambers in a cell culture vessel of the invention in all aspects. According to certain embodiments of the invention the semipermeable barrier is a semipermeable membrane. Semipermeable barriers of the invention are preferably permeable to at least one cell type. Optionally, they are impermeable to at least one other cell type. Selectivity for cell types to which the semipermeable barrier is permeable may be based partly or wholly on cell size. Additionally, or alternatively, selectivity for cell types to which the semipermeable barrier is permeable may be based solely or partly on cell mobility, such that the barrier may be more permeable to a mobile cell than a non-mobile cell, for example a cell type which is activated and mobile may be able to actively squeeze through the semipermeable barrier more effectively than a cell type which is less mobile. According to other embodiments, the semipermeable barrier is not selective for any one of the cell types that the cell culture vessel is used with. As such cell type selectivity in migration between adjacent cell culture chambers may be achieved solely by as a result of the chemicals present in each of the chambers. For example compounds which selectively activate increased mobility of a cell type or which selectively attract one or more cell types. According to methods of the invention, cells may be activated or otherwise have their phenotype altered in a cell culture chamber. In some embodiments that change of phenotype may comprise adoption of a more mobile phenotype which is better able to cross a semipermeable barrier into the next cell culture chamber in the series. In certain embodiments, the semipermeable barrier thus acts as a selective barrier for cells which have or which have not been activated or otherwise successfully had their phenotype changed. In certain embodiments, the semipermeable barrier is permeable to cells which are able to modify it. For example a semipermeable barrier comprising protein may be permeable to cells expressing an appropriate proteinase.

[0105] According to methods of the invention, particularly in its first aspect, a cell culture chamber may contain a different mixture of one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants to an adjacent cell culture chamber in the series. Because the semipermeable barrier between adjacent cell culture chambers is preferably permeable to at least one cell type, and because cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants are typically smaller than cells, there is potential for the one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants in one cell culture chamber to diffuse across the semipermeable barrier to an adjacent cell culture chamber. This is potentially both advantageous and disadvantageous. In the case of chemoattractant some diffusion across the semipermeable barrier is advantageous because it establishes a concentration gradient of chemoattractant up which cells may migrate - if no chemoattractant “leaked” out of the cell culture chamber containing it, cells in the adjacent chamber would not detect it and it would be unable to function as a chemoattractant. However, rapid and complete mixing of chemical constituents between adjacent cell culture chambers in a series is typically undesirable both because it destroys any concentration gradient and because it prevents the chemical content of adjacent cell culture chambers from being different which may be essential for methods of using cell culture vessels of the invention, or methods of the invention which require differences in chemical constituents between adjacent cell culture chambers. In preferred embodiments of the invention, the semipermeable barrier is such that it limits large scale convective mixing of chemical constituents between adjacent cell culture chambers, whilst not being completely impermeable to chemical constituents (for example by diffusion). For example, for a chemokine such as chemokine (C-C motif) ligand 19 (CCL19), the semipermeable barrier and the cell culture vessel of the invention may be such that permeability (as measured by exchange of CCL19) between two adjacent cell culture chambers in a series is less than 5% at 37 °C per 24 hours under conditions in which the cell culture device is kept still (the 5% representing the proportion of total CCL19 measured in the cell culture chamber subsequent to the semipermeable barrier). Alternatively, in a similar test, the permeability between adjacent cell culture chambers in a series may be configured so as to provide exchange of between 1% to 5%, less than 1% or between 1% and 10%, or between 5% and 25% of a chemical such as CCL19.

[0106] Alternatively or additionally, different chemical signals may be maintained in adjacent chambers, by immobilizing (with an optional linker moiety) the one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants within a cell culture chamber. For example, by immobilizing to the inner surface of a wall of the one or more cell culture chambers, or to an insert optionally provided in the one or more cell attractants may optionally be immobilised such that they elute into solution over time. For example, at least 1%, at least 2% at least 5% or at least 10% (for example between 1% and 5%, between 2% and 10% or between 1% and 10%), of an immobilised cell growth factor, cell activating factor, cell differentiation factor, cytokine or cell attractant may elute into standard Dulbecco culture medium during incubation at 37 °C. According to certain embodiments, cell attractant factors may be provided in solution or linked to a substrate in such a way so as they elute into solution over time, cell activating factors may be similar provided, or because activation need only take place in the one or more intermediate cell culture chambers and unlike a cell attractant factor there is no requirement for a concentration gradient of attractant to be established, cell activating factors may optionally be provided immobilised to an inner surface or other solid substrate in the one or more intermediate cell culture chambers. Optionally, a cell activating factor may additionally be provided in (or immobilized within) the final cell culture chamber of the cell culture vessel. Such an arrangement may be useful in ensuring that activated cells which migrate from the intermediate cell culture chamber to the final cell culture chamber following activation retain their activated state. This may be especially useful if their activated state correlates with utility (for example clinical utility).

[0107] According to certain embodiments, the semipermeable barrier between adjacent cell culture chambers in the series may comprise a membrane or layer which is perforated by holes having a diameter of between 1 pm and 200 pm, for example between 1 pm and 100, or between 2 pm and 100 pm, or between 2 pm and 50 pm, or between 3 pm and 40 pm, or between 4 pm and 30 pm, or between 5 pm and 20 pm, or between 6 pm and 18 pm, or between 7 pm and 14 pm, or between 8 pm and 12 pm. The holes, in certain embodiments may be substantially circular. Alternative, when they are not substantially circular, the preferred dimension are such that the hole would exclude the passage of an inflexible sphere having a diameter of between 1 pm and 200 pm, for example between 1 pm and 100, or between 2 pm and 100 pm, or between 2 pm and 50 pm, or between 3 pm and 40 pm, or between 4 pm and 30 pm, or between 5 pm and 20 pm, or between 6 pm and 18 pm, or between 7 pm and 14 pm, or between 8 pm and 12 pm.

[0108] According to certain embodiments, the at least two semipermeable barriers of the cell culture vessel may be substantially identical to each other. According to other embodiments, they may be different from each other.

[0109] Multichamber plate embodiments of cell culture vessels

[0110] The present invention encompasses cell culture vessels (and related uses etc) wherein the cell culture vessel comprises multiple series of cell culture chambers. For example, at least 2, 4, 6, 12, 16, 18, 24, 48, 96, 384 or 1536 series of cell culture chambers may be arranged in a multichamber plate format and provided on one or more plates. Preferably all of the first cell culture chambers of each one of multiple series of cell culture chambers are provided on a first plate. Preferably all of the intermediate cell culture chambers having the same position in the multiple series of cell culture chambers are provided on a single further plate. Preferably all of the final cell culture chambers in multiple series of cell culture chambers are provided on a single plate. In certain embodiments 2, 4, 6, 12, 16, 18, 24, 48, 96, 384 or 1536 series of cell culture chambers may be arranged in a multichamber plate format. Such an arrangement allows multiple experiments or assets to be run side by side and may increase efficiency, for example by allowing multichamber pipetting, multichamber imaging or robotic handling. It may also result in an efficient use of plastic, cells, medium and incubator space.

[0111] According to this embodiment, multiple first cell culture chamber of multiple series may be provided in a plate format, optionally moulded from a single piece of plastic material. The multiple cell culture chambers at each subsequent position in parallel series may also be provided in a plate format, optionally moulded from a single piece of plastic material. The multiple final cell culture chambers in each of the series of cell culture chambers may also be provided in a plate format, optionally moulded from a single piece of plastic material.

[0112] Optionally, the plate format complies with one or more requirement of the Society for Laboratory Automation and Screening standards (and as such the plates may be referred to herein as “SLAS Standard plates”). For example it may comply with a footprint of 127.76 mm x 85.48 mm ± 0.5 mm and / or the 2:3 rectangular footprint. It may comply with bottom flange geometry to enable it to stack with SLAS standard microwell plates. The plate format may optionally comprise a corner notch (AKA chamfer) at the Al position. Plates having 96 of each chamber in a series position, may optionally have a well to well spacing of 9mm. Other plate formats may be scaled appropriately.

[0113] Cell culture vessels of the invention having multiple series of cell culture vessels may optionally provide multiple pipette bypasses to permit individual access to the final and intermediate cell culture chambers in each series. Each of the first and intermediate cell culture chambers at a specific position in the series may be provided on a separate plate. The nesting of the chambers within the next chamber in the series may be achieved by stacking the separate plates, so for example a plate comprising the multiple first cell culture chambers of multiple series may be stacked on a plate comprising the multiple intermediate cell culture chambers of multiple series to achieve nesting, and that plate may be stacked on a plate comprising the multiple intermediate cell culture chambers of multiple series.

[0114] The invention also provides in a further aspect a SLAS standard plate comprising an array of cell culture chambers each having at their lower end a semipermeable barrier (for example as described elsewhere) and each cell culture chamber having adjacent to it at least one pipette bypass. For example 1 or 2 or 3 or 4 pipette bypasses. Each pipette bypass permits access to a later cell culture chamber in the series without requiring the one or more plates incorporating the earlier chambers in the series to be removed. Preferable, the pipette bypasses are arranged with the same spacing relative to each other as the spacing of the multiple cell culture chambers on the plate. This permits standard liquid handling equipment, for example fluid handling robots or multi-tip pipettes to be used to access multiple cell culture chambers through multiple pipette bypasses simultaneously with a simple locational offset from the position in which they would be capable to accessing multiple cell culture chambers on a plate (or multiple wells in a prior at SLAS multiwell plate). Pipette bypasses may pass through a single plate or multiple plates. For example, pipette bypasses for accessing intermediate the cell culture chambers which directly follow in the series of cell culture chambers through the plate carrying the first cell culture chambers in the series will only pass through the single plate (that comprising the first cell culture chambers in the series). Pipette bypasses from accessing the next cell culture chambers in the series will pass through both of the overlying plates. This may be achieved by arranging holes in those plates which align with each other to form the pipette bypasses.

[0115] It is understood that the SLAS standard plates comprising an array of cell culture chambers each having at their lower end a semipermeable barrier (for example as described elsewhere) and each cell culture chamber having adjacent to it at least one pipette bypass according to the invention may optionally comprise cell culture chambers having one or more further features as described herein in reference to the cell culture chambers of cell culture vessels of the invention. For example, the semipermeable barriers may be as described elsewhere herein. SLAS standard plates according to the invention may be used in conjunction with an appropriate standard (prior art) multiwell plate as the plate comprising multiple final cell culture chambers or it may be used in conjunction with a specially designed bottom plate comprising multiple final cell culture chambers. SLAS standard plates of the invention may optionally be used in any of the methods of the invention. Because of their provision for parallelism, the use of SLAS standard plates in methods of the invention may be especially applicable to high throughput screening methods, for example high-throughput chemical compound or drug candidate screening.

[0116] Optional stand-off features In embodiments of the invention wherein the cell culture chambers are nestable and the cell culture vessel is disasemblable. The cell culture vessel optionally comprises constructional features to assist in the disassembly (“de-nesting”) of nested cell culture vessels. In embodiments where the cell culture chambers are part of multichamber plates, such constructional features may assist in separation of plates.

[0117] The cell culture vessels of the invention may in certain embodiments be designed to work with cell culture medium in contact with both sides of the semipermeable barriers separating adjacent cell culture chambers. The nesting of such cell culture chambers or filling of previously nested cell culture chambers with medium, may lead to the formation of a film of cell culture medium between surfaces of adjacent cell culture chambers in a series or in a multi chamber plate format, the formation of a film of cell culture medium between adjacent multichamber plates. Evaporation, condensation and / or capillary forces may lead to such films of cell culture medium impeding separation of multichamber plates or de-nesting of nested cell culture chambers. Similar impediments may be encountered to removing a lid in certain embodiments. Accordingly, the surfaces of adjacent cell culture chambers in a series and / or the surfaces of adjacent multichamber plate son a series may optionally be provided with “stand offs” to assist in their separation, especially when their surfaces are wet or have previously been wet. Stand offs maybe moulded or embossed onto surfaces which would otherwise be in contact and will result in the total contact area between those surfaces being reduced. According to certain embodiments the stand offs are between 0.1 and 1.0 mm in height, for example between 0.2 and 0.8 mm in height, for example about 0.5 mm in height. In certain embodiments, they may comprise raised moulded or embossed dots on the surface. In other embodiments they comprise mounded or embossed number letters or other indica, for example those identifying the chambers in a multi chambered plate. In multichamber plate embodiments, the stand offs may be provided around the edge of a plate. They may comprise the number and lettering of the grid arrangement of chambers. By increasing separation between adjacent chambers in a series they may also assist in promoting gas exchange.

[0118] Optional features of cell culture chambers

[0119] According to an embodiment of the of the invention in any of its aspects, the cell culture vessel may optionally include, for example within one or more of the cell culture chambers, a cell growth support structure, for example a collagen matrix, or another biodegradable polymer. According to certain embodiments, the one or more cell culture chambers may alternatively or additionally further comprise a population of cells contained therein, for example a population of mammalian cells, a population of human cells or a population of human immune cells.

[0120] According to certain embodiments, the one or more cell culture chambers in a series may alternatively or additionally comprise a cell culture medium contained therein.

[0121] According to certain embodiments relating to the culture of immune system cells, the one or more cell culture chambers in a series may alternatively or additionally comprise one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants contained therein, one or more of which may be optionally immobilized onto an internal wall of the one or more cell culture chambers or optionally immobilised onto a solid particle such as a plastic bead. Alternatively, one or more of which may be immobilised onto a scaffold, gel or extracellular matrix component. In such embodiments, the intermediate cell culture chamber preferably contains a first chemokine, the final cell culture chamber contains a second chemokine and the intermediate cell culture chamber contains a cell activating agent (which may optionally be immobilised within the cell culture chamber). Preferably, the first chemokine and the second chemokine are different. Preferably, the first chemokine is attractive to naive (non-activated) immune cells, and the second chemokine is attractive to activated immune cells. Optionally the second chemokine is not attractive to naive (non-activated) immune cells, and the first chemokine is not attractive to activated immune cells. In certain embodiments, the activating agent is cross linking antibodies immobilised to a surface or other solid substrate within the intermediate cell culture chamber.

[0122] Culture medium

[0123] Embodiments of the second aspect of the invention provide a cell culture vessel with the optional presence of a cell culture medium in one or more of the cell culture chambers. Other embodiments of the invention may relate to the use of, or products obtained from, a cell culture vessel of the invention, wherein there is optionally present in one or more of the cell culture chambers of the cell culture vessel cell culture medium. Preferably, a cell culture medium is a liquid cell culture medium. Typically such a cell culture medium will comprise, in an aqueous base, a carbon source such as glucose, salts, for example physiologically- compatible salts at physiological concentration, a source of amino acids and nitrogen (for example a beef digestate or yeast extract). Other optional ingredients include antibiotics, antifungal compounds, glutamate, serum (for example foetal calf serum) and indicators.

[0124] Cell types

[0125] The invention in all of its aspects relates to cell culture. According to certain embodiments, the cells are bacterial cells (including, for example, cyanobacteria), according to alternative embodiments, the cells are fungal or yeast cells, according to alternative embodiments, the cells are algal cells. According to alternative embodiments the cells are plant cells. According to alternative embodiments, the cells are protist cells. Preferably the cells are animal cells, more preferably mammalian cells. In many embodiments they are human cells, but they may also be cells of a non-human animal, especially an of economic or social importance, such as a farm animal, a companion animal, a sporting animal, or a research animal. Specific animals of importance include cattle, sheep, horses, goats, dogs, cats, rodents (including rats, mice, rabbits), camels, pigs, buffaloes, yaks, reindeer, grey asses, white asses, elephants, llamas, and alpacas.

[0126] Cells according to certain embodiments may be primary cells, in other embodiments they may be cell lines. In certain embodiments, they may comprise cells removed from a subject in diagnostic biopsy.

[0127] Cells may be of any cell type including immune cells, such as lymphocytes (including B- cells, plasma cells, memory B-cells, T-cells, memory T cells, cytotoxic T-cells, helper T- cells, natural killer cells, and natural killer T cells), monocytes (including macrophages, histiocytes, Kupffer cells, Langerhans cells, and dendritic cells), and granulocytes (including neutrophils, eosinophils, basophils and mast cells). Cells may optionally be genetically- engineered or otherwise manipulated cells. They may, in certain embodiments be CAR-T cells. Cells may be tumour cells, biopsy cells, or cells in a tissue sample. For example they may be a sample of blood cells, or a sample of red cell depleted blood (for example a Buffy coat) or a sample of leukocytes, for example a sample of peripheral blood mononuclear cells (PBMCs). In certain embodiments the cells may comprise stem cells, for example haematopoietic stem cells. In certain embodiments, the cells may comprise cells removed from the marrow of a bone of a subject.

[0128] According to embodiments of the second aspect of the invention wherein the cell culture vessel comprises cells in more than one of the cell culture chambers, the species and ultimate origin of the cells in each cell culture chamber is preferably the same. However, the phenotype of the cells in each cell culture chamber is preferably different. For example, the range of cell phenotypes of cells in a cell culture chamber later in the series of cell culture chambers may optionally be narrower than the range of cell phenotypes in the proceeding cell culture chamber of the series of cell culture chambers. Such an outcome would, in certain embodiments, be the result of selective migration of cells from the earlier cell culture chamber to the later cell culture chamber in the series. Alternatively, it may be because of differential amplification of cells having a subset of cell phenotypes.

[0129] In certain embodiments, the cells in a later cell culture chamber in the series of cell culture chambers are more highly differentiated than cells in a later cell culture chamber in the series of cell culture chambers. In some embodiments, cells in a later cell culture chamber in a series of cell culture chambers have a phenotype which is absent in the cells in an earlier cell culture chamber in the series of cell culture chambers. For example, they may have acquired a novel pattern of cell marker expression, for example a novel pattern of cell surface differentiation markers, or a novel pattern of cell surface receptors (for example a different pattern of chemokine receptor expression). Preferably, the cells will have changed their expression of chemokine receptors such that the chemokines to which they are attracted have changed.

[0130] In certain embodiments, additionally or alternatively, the number of cells in a later cell culture chamber in the series of cell culture chambers is greater than the number of cells in an earlier cell culture chamber in the series of cell culture chambers.

[0131] In certain embodiments, the cells in a later cell culture chamber in the series of cell culture chambers are more highly differentiated than cells in an earlier cell culture chamber in the series of cell culture chambers.

[0132] Scalability

[0133] An advantage of the present invention is that it is scalable. The cell culture vessel of the invention may be made and used in various sizes and volumes. In certain embodiments the cell culture chambers have a maximum dimension of between 1 mm and 1000 mm, for example between 1 mm and 500 mm, for example between 1 mm and 250mm, for example between 1 mm and 100 mm, for example between 2 mm and 100 mm, for example between 5 mm and 100 mm, for example between 10 mm and 100 mm, for example between 20 mm and 50mm or between 10 mm and 20 mm or between 5 mm and 15 mm. In certain embodiments the cell culture chambers are substantially cylindrical and have a diameter of between 1 mm and 1000 mm, for example between 1 mm and 500 mm, for example between 1 mm and 250mm, for example between 1 mm and 100 mm, for example between 2 mm and 100 mm, for example between 5 mm and 100 mm, for example between 10 mm and 100 mm, for example between 20 mm and 50mm or between 10 mm and 20 mm or between 5 mm and 15 mm. In embodiments wherein the cell culture chambers are substantially cylindrical they may have a height of between 1 mm and 500 mm, for example between 1 mm and 250 mm, for example between 1 mm and 200 mm, for example between 2 mm and 200 mm, for example between 2 mm and 150 mm or between 5 mm and 200 mm, or between 5 mm and 100 mm, for example between 5 mm and 50 mm, for example between 10 mm and 50 mm, for example between 10 mm and 20 mm.

[0134] In certain embodiments, the volume of each cell culture chamber is up to 20 L, for example up to 10 L for example up to 5 L, for example up to 2 L, for example up to 1 L, for example up to 500 mL, for example up to 200 mL, for example up to 100 mL, for example up to 50 mL, for example up to 20 mL, for example up to 10 mL, for example up to 5 ml. The minimum volume of each cell culture chamber , in certain embodiments is 0.5 mL, for example 1 mL, for example 2 mL, for example 5 mL, for example 10 mL, for example 20 ml, for example 50 mL, for example 100 mL, for example 200 mL, for example 500 ml, for example 1 L, for example 2 L, for example 5 L.

[0135] It should be noted that all of the dimensions given above are internal dimensions, because of the thickness of the walls, external dimensions will be slightly greater, for example 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm or 8 mm greater.

[0136] Methods of the inventions

[0137] According to a first aspect, the invention provides an in vitro method of producing a population of cells comprising the steps of:

[0138] A, introducing a source of cells into the first cell culture chamber of an in vitro cell culture vessel comprising three cell culture chambers arranged in a series, each chamber being separated from the next chamber in the series by a semipermeable barrier,

[0139] B, contacting the cells in the first cell culture chamber to a first composition of one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants so as to promote migration of at least a subset of cells from the first cell culture chamber into an intermediate cell culture chamber of the series, C, contacting cells in the intermediate cell culture chamber of the series to a further composition of one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants so as to promote migration of at least a subset of cells from the intermediate cell culture chamber to the final cell culture chambers in the series,

[0140] D, optionally withdrawing cells from the final cell culture chamber in the series.

[0141] According to certain embodiments relating to the culture of immune system cells, the first composition comprises a first chemokine and the second composition comprises a different, second chemokine. Preferably, the first composition is present in the intermediate cell culture chamber and is able to diffuse into the first cell culture chamber, and preferably, the second composition is present in the final cell culture chamber and is able to diffuse into the intermediate cell culture chamber. Additionally, the intermediate cell culture chamber preferably contains one or more cell activating factors which are anchored onto an internal wall of the intermediate cell culture chamber or optionally immobilised onto a solid substrate such as a plastic bead, present in the intermediate cell culture chamber. Preferably, the first chemokine and the second chemokine are different. Preferably, the first chemokine is attractive to naive (non-activated) immune cells, and the second chemokine is attractive to activated immune cells. Optionally the second chemokine is not attractive to naive (nonactivated) immune cells, and the first chemokine is not attractive to activated immune cells. In certain embodiments, the activating agent is cross linking antibodies immobilised to a surface or other solid substrate within the intermediate cell culture chamber.

[0142] Cell culture vessel for use in methods of the invention

[0143] According to preferred embodiments of methods according to the first aspect of the invention, may use a cell culture vessel according to a second aspect of the invention or an SLAS plate according to the invention. Conversely, a cell culture vessel according to the second aspect of the invention may optionally be adapted for use in a method according to the first aspect of the invention.

[0144] Populations of cells

[0145] Cells for use in methods of the invention, may comprise any of the cell types noted above, or any mixture thereof.

[0146] Source of cells Step A of a method of the first aspect of the invention requires a source of cells According to certain embodiments, that source is a biopsy (for example a cancer biopsy). According to other embodiments, that source is a blood sample. According to other embodiments the source is a white cell fraction of blood. According to other embodiments, that source is a bone marrow aspirate. According to other embodiments that source is PMBCs. The cells comprising the source of cells in step A of a method of the invention, may optionally be pre- processed. For example, in certain embodiments, they may be disaggregated. This may be especially important if the cell source is solid tissue biopsy. Disaggregation may optionally include physical disaggregation such as passing the solid tissue through a screen.

[0147] Additionally or alternatively, disaggregation may optionally include enzymatic treatment, for example with a protease to digest extracellular matrix.

[0148] According to other embodiments, pre-processing may include dilution, washing and / or suspension. It may optionally include some preliminary separation stage. For example, if the source of cells is whole blood, it may be pre-processed (for example by settling or centrifugation, such as density gradient centrifugation) to remove a substantial proportion of the red cells. In certain embodiments, pre-processing may include a period of cell culture. In such methods, the source of cells of step A of a method of the invention may be a cell culture, for example a culture of transfected cells.

[0149] According to preferred embodiments, the source of cells is a relatively mixed population of cell types whereas the population of cells produced by the method and optionally withdrawn from the final cell culture chamber in the series is a relatively pure population of cells of a substantially single cell type (for example at least 90% of a single cell type such as 90% T- cells, 90% B-cells, 90% NK cells, 90% NK T-cells, 90 % dendritic cells). In other embodiments a single cell type is enriched by a factor or 2, 3, 4, 5, 6, 7, 8, 10, 20, 40, 60 or 100 between the starting source of cells and the cells in the final cell culture chamber of the cell culture vessel.

[0150] Use of non-adherent cells

[0151] According to certain embodiments, the source of cells comprises non-adherent cells. According to certain embodiments the source of cells include adherent cells. Whilst it can be difficult to completely categorise cells into an adherent versus non-adherent category, in certain embodiments, the source of cells comprises cells that are at least partly mobile, meaning that they are either non-adherent, or not so strongly adherent to the inside of surfaces of the cell culture chambers or to each other that they are unable to migrate through the semipermeable barrier to the next cell culture chamber of the series.

[0152] Introduction of source of cells

[0153] According to certain embodiments, the source of cells may be introduced into the first cell culture chamber by removing a lid of the first cell culture chamber, introducing the cells, for example in a suspension by pipette, and replacing the lid of the first cell culture chamber. In other embodiments, for example in embodiments, wherein the first cell culture chamber does not have a removable lid. The source of cells may be introduced via a port, for example an injection port, for example in a suspension by means of a syringe.

[0154] Culture medium

[0155] In use, for example in methods according to the first aspect of the invention, the cell culture chambers preferably contain a cell culture medium. Various cell culture media are available, a person skilled in the art will be able to choose a suitable cell culture medium. Further optional features of cell culture media are disclosed above. In certain embodiments, at least all cell culture chambers subsequent to the first cell culture chamber in the series (and optionally all cell culture chambers in the series) are completely full of media. This provides an advantage when the cell culture chambers of the series are stacked into a column because it means that both sides of the semipermeable barriers between adjacent cell culture chambers are ”wef ’ and that there is no air gap for cells to drop through when migrating from one cell culture chamber to the other, nor any problem in retaining medium in upper cell culture chambers without it flowing into the air-gap in the cell culture chambers below. In methods which use a modular cell culture vessel of the invention, one method of ensuring that a cell culture chamber is full of medium is to assemble it from modules wherein both modules are immersed in cell culture medium before assembly and assembly takes place with the modules submerged in cell culture medium. Alternatively a module can be filled to the brim with medium and then closed (for example by engagement of another module above). In non- modular cell culture vessels, complete filling of the cell culture chambers can be achieved by using ports in the cell culture chambers to introduce medium whilst using the same or a different port to withdraw air.

[0156] First contacting of cells According to a method of the first aspect of the invention in step B, cells in the first cell culture chamber are contacted with a first composition of one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants so as to promote migration of at least a subset of cells from the first cell culture chamber into an intermediate cell culture chamber of the series. Preferably said contacting takes place in cell culture medium. According to certain embodiments migration is promoted by increasing the total number of cells (via cell expansion and or cell survival) such that because the total number of cells is increased, the total number of cells that migrate will also increase. In other embodiments, migration is increased by changing the phenotype of the cells, for example by making them adopt a more migratory phenotype. Said contacting of cells with the first composition is typically achieved by providing one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants in the culture medium in the first cell culture chamber. The one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants may be provided in the culture medium before the cells are introduced into the cell culture chamber (step A of the method of the first aspect of the invention), or they may be provided later by supplementing the cell culture medium after the cells are present with one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants, for example via injection ports.

[0157] According to certain embodiments the contacting of the cells with one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants is achieved by providing the one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants by introducing the one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants into the first cell culture chamber. However, in certain preferred embodiments, the one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants are initially provided in the intermediate cell culture chamber. The contacting of the cells in the first cell culture chamber to a first composition of one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants so as to promote migration of at least a subset of cells from the first cell culture chamber into an intermediate cell culture chamber of the series (as required by step B of methods of the first aspect of the invention) is achieved because at least a proportion of the one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants initially introduced into the intermediate cell culture chamber cross the semipermeable barrier between the first cell culture chamber and the intermediate cell culture chamber and contact cells in the first cell culture chamber. Further optional features of the nucleic acids may be as described below under the heading “nucleic acids”.

[0158] Subsequent contacting of cells

[0159] According to a method of the first aspect of the invention, in step C, cells in the intermediate cell culture chamber are contacted with a further composition of one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants so as to promote migration of at least a subset of cells from the first cell culture chamber into an intermediate cell culture chamber of the series. Preferably said contacting takes place in cell culture medium. According to certain embodiments migration is promoted by increasing the total number of cells (via cell expansion and or cell survival) such that because the total number of cells is increased, the total number of cells that migrate will also increase. In other embodiments, migration is increased by changing the phenotype of the cells, for example by making them adopt a more migratory phenotype. Said contacting of cells with the further composition is typically achieved by providing one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants in the culture medium in the intermediate cell culture chamber. The one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants may be provided in the intermediate culture medium before the cells have migrated into the intermediate cell culture chamber, or they may be provided later by supplementing the cell culture medium after the cells are present with one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants. In some embodiments, they may have been added via a pipette bypass in the previous cell culture chamber.

[0160] According to certain embodiments the contacting of the cells with one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants in step C of a method according to the first embodiment of the invention is achieved by providing the one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants by introducing the one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants into the second cell culture chamber. However, in certain preferred embodiments, the one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants are initially provided in the final cell culture chamber. The contacting of the cells in the intermediate cell culture chamber to a further composition of one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants so as to promote migration of at least a subset of cells from the intermediate cell culture chamber into the final cell culture chamber of the series (as required by step C of methods of the first aspect of the invention) is achieved because at least a proportion of one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants initially introduced into the final cell culture chamber cross the semipermeable barrier between the intermediate cell culture chamber and the intermediate cell culture chamber and contact cells in the first cell culture chamber.

[0161] Optionally the final cell culture chamber may comprise a final composition of one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants. According to certain embodiments, the final composition has in common one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants with the further composition. According to certain embodiments, the final composition comprises one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants which are different to those in the further composition. According to certain embodiments, the final composition comprises one or more cell attractants which attract cells from the intermediate cell culture chamber to the final cell culture chamber.

[0162] According to certain embodiments of methods of the invention, the further composition comprises one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants to which cells in the intermediate cell culture chamber are responsive, but to which cells in the first cell culture chamber are less responsive. Additionally or alternatively, the final composition comprises one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants to which cells in the final cell culture chamber are responsive, but to which cells in the intermediate cell culture chamber are less responsive. According to certain embodiments, the first composition and the further composition are different. According to certain embodiments, the first composition and the second composition have in common one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants, together with one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants which are different between the compositions. According to certain embodiments, the further composition comprises one or more cell attractants which attract cells from the first cell culture chamber to the intermediate cell culture chamber.

[0163] According to certain embodiments of methods of the invention, the cell culture vessel used in the invention has medium in all three cell culture chambers. The content of the medium in each of the cell culture chambers is optionally different to both other cell culture chambers for example by virtue of a different composition of one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants.

[0164] According to certain embodiments of the first aspect of the invention, the first composition of one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants is introduced into the intermediate cell culture chamber of the vessel and the further composition of one or more cell growth factors, cell activating factors, cell differentiation factors, nucleic acids, cytokines and cell attractants is introduced into the final cell culture chamber. Optionally, these compositions are introduced prior to the source of cells being introduced into the first cell culture chamber of the cell culture vessel. Further optional features of the nucleic acids may be as described below under the heading “nucleic acids”. Alternatively or additionally, these compositions may be introduced after the cells have been introduced.

[0165] According to certain preferred embodiments of the method according to the first aspect of the invention, there is provided a method of producing a population of cells (especially a population of activated cells such as activated immune system effector cells such as activated T-cells, and especially a population which is substantially pure (for example 90% pure) population of a single cell type such as T-cells), said method optionally using a cell culture vessel of the invention having 3 cell culture chambers, and said method comprising the steps of A, introducing a relatively mixed population of cells into the first cell culture chamber of a cell culture vessel; comprising three cell culture chambers arranged in a series, each chamber being separated from the next chamber in the series by a semipermeable barrier,

[0166] Bl, contacting the cells in the first cell culture chamber to a first composition comprising one or more cell attractant factors (for example one or more chemokines), said contacting of cells in the first cell culture chamber to the first composition being preferably achieved by providing the first composition in the intermediate cell culture chamber in the series such that at least a proportion of the one or more cell attractant factors crosses the semipermeable barrier between the first cell culture chamber and the intermediate cell culture chamber, wherein the one or more cell attractant factors in the first composition attracts a subset of cell types to migrate from the first cell culture chamber to the intermediate cell culture chamber, said subset of cells expressing receptors for the one or more cell attractant factors in the first composition (for example one or more chemokines in the first composition may attract a subset of the cells from the first to the intermediate cell culture chamber due to said subset of cells expressing one or more receptors for the one or more chemokines, wherein those one or more receptors are not expressed on cells which are not attracted into the intermediate cell culture chamber),

[0167] B2, contacting cells in the intermediate cell culture chamber with one or more cellular activators in order to cause them to adopt an activated phenotype, wherein the activated phenotype includes up-regulated expression of receptors for one or more cell attractant factors (for example one or more chemokines) in a second composition, and optionally downregulation of expression of receptors for one or more cell attractant factors (for example one or more chemokines) in the first composition,

[0168] C, contacting cells in the intermediate cell culture chamber to the second composition comprising one or more cell attractant factors (for example one or more chemokines), said contacting of cells in the intermediate cell culture chamber to the second composition being preferably achieved by providing the second composition in the final cell culture chamber in the series such that at least a proportion of the one or more cell attractant factors of the second composition crosses the semipermeable barrier between the intermediate cell culture chamber and the final cell culture chamber, wherein the cell attractant factor in the second composition is able to attract activated cells to migrate from the intermediate cell culture chamber to the final cell culture chamber, D, optionally withdrawing cells from the final cell culture chamber of the series, characterised in that the second composition is attractive to cells which have been activated (and is preferably not attractive to cells which have not been activated), and the first composition is attractive to cells which have not been activated (and is preferably not attractive to cells which have been activated).

[0169] Nucleic acids

[0170] The first and subsequent contacting steps are described above as optionally comprising contact with a composition comprising a nucleic acid. In certain embodiments such a nucleic acid may be DNA. Alternatively it may be RNA. Or it may be a DNA or RNA derivative or a mixture of one of more of DNA, RNA or derivative of either thereof. Nucleic acid derivative include those produced by chemical medication (for example methylation) and or by sequence optimisation, for example to introduce an advantageous secondary or tertiary structure. Derivatisation may include the addition of moieties to the sequence or at either or both terminus. It may optionally include circularisation. RNA may optionally include mRNA, siRNA, miRNA, gene editing guide RNA and saRNA. DNA may optionally include DNA flanked by nucleic acid elements to promote genome integration or replication. In certain embodiments it may be naked RNA, naked DNA or a naked RNA or RNA derivative. In other embodiments the nucleic acid may be provided as part of a vector for example a viral vector, lipid nanoparticle vector, micellular vector, plasmid vector, cosmid vector, gene editing construct (for example a CRISPR / Cas gene editing construct) or artificial chromosome. The nucleic acid sequence may include a peptide coding sequence, or an interfering or activating sequence. It may optionally include an integration sequence. It may optionally include a promotor sequence. Optionally, the nucleic acid may be used to change the genotype and / or phenotype of the cells. For example it may cause the cell to become activated. It may cause the cell to express a new phenotype, for example a new cell surface marker or cell surface receptor (for example a receptor for cellular activation or chemotaxis or differentiation, or a T-cell receptor and / or T-cell coreceptor). It may cause a cell to multiply and / or differentiate. It may cause the cell to adopt a more migratory phenotype. In certain embodiments the nucleic acid, for example the nucleic acid in a composition present in an intermediate cell culture chamber may cause cellular transfection. It may create an immune effector cell, for example a CAR-T cell.

[0171] Further optional culture in the final cell culture chamber Between steps C and D of methods of the invention, the cells present in the final cell culture chamber may optionally be cultured for a period of time. The final cell culture chamber may optionally be supplied with medium, cytokines or growth factors to facilitate that. The base of the final cell culture chamber may optionally be adapted to facilitate desired cell growth and / or differentiation. For example, the base of the final cell culture chamber may optionally be adapted to encourage the formation of organoids from the cells present in the final cell culture chamber. For example, for the base may be textured in such a fashion to promote the formation of organoids.

[0172] Withdrawal from final cell culture chamber

[0173] According to methods of the first aspect of the invention, step D includes the optional feature of withdrawing cells from the final cell culture chamber of the series. According to certain embodiments step D includes withdrawing the cells from the final cell culture chamber by disassembling the cell culture vessel, for example a modular cell culture vessel. According to other embodiments, the cells may be withdrawn in step D via a port in the final cell culture chamber, for example by a syringe. According to other embodiments, the cells may be withdrawn in step D via a pipette bypass.

[0174] Additional optional features of method

[0175] If is to be understood that in addition to steps A, B, C and D, which take place in sequential order, methods of the invention may optionally include additional steps before steps A, B, C and D and / or after steps A, B, C or D and / or after steps A, B, C and D. For example additional steps may include obtaining the source of cells (including one of the pre-treatment steps disclosed above). Additional intermediate steps may optionally include selecting or manipulating the cells (for example by transfection or other genetic manipulation). Additional subsequent steps may optionally include further cell sorting, purification or characterisation, and / or formulating the cells for use (for example for medical administration), and / or administering the cells.

[0176] First and further compositions

[0177] According to certain embodiments of the method according to the first aspect of the invention, the first composition includes one or more cell migration promoting agents, for example one or more chemoattractants. Where the method of the invention is used with immune system cells (for example wherein the source of cells is blood or a fraction thereof, PBMCs, spleen cells, bone marrow aspirate or a subset of immune cells) the first composition may optionally include one or more (or all) of CCL-19, CCL-21, CCL-19 and CCL-21.

[0178] According to certain embodiments of the method according to the first aspect of the invention, the first composition additionally includes one or more cell activating agents, for example one or more cell activating factors. Where the method of the invention is used with immune system cells (for example wherein the source of cells is blood or a fraction thereof, PBMCs, spleen cells, bone marrow aspirate or a subset of immune cells) the first composition may optionally additionally include one or more (or all) of the following immune system cell activators: anti-CD3 antibodies or derivatives, anti-CD28 antibodies or derivatives.

[0179] According to certain embodiments of the method according to the first aspect of the invention (and optionally in addition to the features of the first composition as described immediately above) the further composition additionally includes one or more cell attractant agents, in particular one or more cell attractant agents which are cell attractant factors for activated cells (but optionally are less potent cell attractant factors for non-activated cells). Where the method of the invention is used with immune system cells (for example wherein the source of cells is blood or a fraction thereof, PBMCs, spleen cells, bone marrow aspirate or a subset of immune cells) the further composition may optionally additionally include CXCL-10.

[0180] According to certain embodiments of the invention, especially where the method of the invention is used with immune system cells (for example wherein the source of cells is blood or a fraction thereof, PBMCs, spleen cells, bone marrow aspirate or a subset of immune cells), all of the cell culture chambers contain cell culture medium which is supplemented with one or both of IL-2 and interferon-a in order to promote immune cell survival. Alternatively, or additionally, the cell culture chambers may be supplemented with one or both of 11-15 and IL-7 in order to promote immune cell survival.

[0181] According to certain embodiments of the invention, the method of the first aspect is a method of producing activated immune effector cells (such as T-cells, NK cells, B-cells and dendritic cells) by a two stage process in which cells are attracted from the first cell culture chamber to the second chamber of a cell culture vessel by a first chemoattractant (for example in response to signalling through CCR7 on the cell surface), the cells are activated in the second cell culture chamber of the cell culture vessel, this activating causes a change in chemokine receptor expression (for example an up-regulation of CXCR3, and / or a down regulation of CCR7, or both an up-regulation of CXCR3, and a down regulation of CCR7), the cells are then attracted to the final cell culture chamber in response a second chemoattractant for example in response to signalling through CXCR3. Preferably, the first and second chemoattractant are different. Optionally, the first chemoattractant is CCL-19 and / or CCL-2. Optionally, the second chemoattractant is CXCL-10.

[0182] Population of cells

[0183] According to a third aspect, the invention provides a population of cells produced in accordance with a method of the first aspect of the invention or produced by culturing a source of cells in a cell culture vessel according to the second aspect of the invention.

[0184] According to certain embodiments, the population of cells is a population of animal cells, for example a population of mammalian cells, for example a population of human cells. According to certain embodiments, the population of cells is a population of immune cells (for example a population of human immune cells). It may be a population of activated B- cells (for example a population of genetically engineered B-cells), a population of activated T-cells (for example a population of genetically engineered T-cells), a population of activated NK cells (for example a population of genetically engineered NK cells), or a population of activated dendritic cells (for example a population of genetically engineered dendritic cells)

[0185] Preferably, the population of cells is at least 80%, for example at least 90 or 95 or 98 % pure. That is to say the population of cells, may in certain embodiments be a pure population of activated B-cells (for example a pure population of genetically engineered B-cells), a pure population of activated T-cells (for example a pure population of genetically engineered T- cells), a pure population of activated NK cells (for example a pure population of genetically engineered NK cells), or a pure population of activated dendritic cells (for example a pure population of genetically engineered dendritic cells). According to certain embodiments the population of cells may be a pure population of CAR-T cells.

[0186] According to certain embodiments, the population of cells comprises T-cells having enhanced secretion of TNFa.

[0187] Method of cell therapy

[0188] According to a fourth aspect, the invention provides a method of cell therapy comprising administering to a subject in need thereof, a population of cells according to a third aspect of the invention. Said administering may optionally include infusing, injecting, or otherwise administering, the population of cells to the subject. According to certain embodiments, the method of cell therapy is a method of cellular immunotherapy. According to certain embodiments, the method is a method of treating an immune deficiency. According to certain embodiments, the method is a method of cancer immunotherapy. According to certain embodiments, the method is a method of corneal or retinal repair. According to certain embodiments, the method is a method of replacing missing or dysfunctional neurons (for example in Parkinson’s disease). According to certain embodiments, the method is a method of wound repair (for example wound repair or bum repair). According to certain embodiments the method is a method of treating diabetes. According to certain embodiments, the method is a method of prolonging survival of transplanted organs.

[0189] Route of administration and dosage

[0190] A population of cells of the invention may be administered by any suitable route and in any suitable dosage. For example the cells may be implanted (for example into a defective tissue such a pancreas which is depleted of beta cells), injected or infused. In certain embodiments a population of cells according to the invention is used to see a cell growth scaffold which is then implanted into the subject.

[0191] Subject to be administered to

[0192] Preferably the subject is a mammal. Preferably, the subject is human. Alternatively, the subject may be a non-human animal.

[0193] Medicaments

[0194] According to a fifth aspect of the invention, there is provided a population of cells of the third aspect of the invention for use as a medicament. Optionally, the medicament may be for use in treating a subject as described above. Optionally, the medicament may be for use in a method of cell therapy as described above.

[0195] The invention also provides use of a population of cells for the third aspect of the invention in the manufacture of a medicament for the treatment of a medical condition, for example in the treatment of a medical condition disclosed herein.

[0196] The invention also provides use of a population of cells for the third aspect of the invention in the manufacture of a medicament for the cell therapy, for example for cell therapy as described above.

[0197] Use in cell therapy According to a sixth aspect of the invention, there is provided a population of cells of the third aspect of the invention for use in cell therapy. Optionally, the cell therapy may be as described above.

[0198] Use in CAR-T therapy

[0199] According to certain embodiments, there is provided methods of CAR-T therapy comprising administering a population of cells according to the third aspect of the invention. There is also provided a population of cells according to the third aspect of the invention for use in CAR-T therapy. In such embodiments, the source of cells may be a source of genetically engineered T-cells (such as T-cells homologous to the subject to be treated by CAR-T therapy). Those T-cells may be attracted by means of a first chemokine from the first cell culture chamber to the intermediate cell culture chamber, they may then be activated in the intermediate cell culture chamber, said activation may optionally change the cellular expression of cytokine receptors such that a second chemokine (preferably different from the first chemokine) may then be used to attract the activated cells (and only the activated cells) into the final cell culture chamber. Alternatively, the method of the invention use as a source of cells T-cells isolated from a subject’s blood or grown from T-cell precursors, optionally isolated from a subject’s blood. The method of the invention may be employed to transduce the T-cells with a CAR (chimeric antigen receptor) in order to make CAR-T cells. In such methods, the first contacting step, for example in the first cell culture chamber in the series, may optionally comprise contacting the cells (for example a subjects T-cells) with an agent inducing proliferation of the cells (for example IL-2 and / or anti-CD3 antibodies and / or anti- CD3 / CD28 antibodies). A subsequent contacting step (for example in the next cell culture chamber of the series) may comprise contacting the cells with a composition comprising a nucleic acid so as to cause transduction of with a gene encoding an engineered CAR, for example a nucleic acid in a viral vector such as an integrating gammaretroviral vector or a lentiviral vector. Alternatively, a subsequent contacting step may comprise contacting the cells with a composition comprising a nucleic acid present in a non-viral gene editing construct such as a CRIAPR / Cas gene editing tool in order to integrate an engineered CAR gene into a specific site in the genome of the T-cell. Optionally, there may be subsequent steps of further explanation or selection or activation of the CAR-T cells. The invention encompasses methods of CAR-T therapy comprising administering these CAR-T cells.

[0200] Non-limiting Examples Example 1. Constructional features of cell culture vessel

[0201] An implementation of a cell culture vessel suitable for use in methods of the invention (1) is shown in Figure 1. The first (top) and intermediate (middle) cell culture chambers (10, 11) were made from a modified 5pm transwell insert. In short, this is a small plastic column with an open top and a diameter of 10mm, the wall of the cell culture chamber is 1mm thick giving an internal diameter of 8mm. Each column is 7mm tall and is made from polystyrene. The first and intermediate cell culture chambers have a polycarbonate membrane sealed (20, 21) into the bottom. The final (bottom) cell culture chamber (12) was similar in dimensions but unlike the other chambers had a solid bottom. A tongue 1mm wide and 1mm deep was tooled into the bottom of the first (and intermediate cell culture chambers. A groove 1mm wide and 1mm deep was tooled into the top of the intermediate and final cell culture chambers. This arrangement permitted the cell culture vessel (1) to be assembled and disassembled. A circular stand 11mm in diameter and 3mm in height was also created and placed inside a standard 24 well tissue culture plate (not shown in figure 1). A first composition comprising CCL19, CCL21, anti-CD3 and anti-CD28 was placed in the intermediate cell culture chamber (11). The further composition comprising CXCL10, CCL2, CCL5, anti-CD3 and anti-CD28 was placed in the final cell culture chamber (12). The bottom (final) cell culture chamber (12) also contained medium such that the medium in the bottom cell culture chamber (12) covered the bottom of the membrane (21) above it. Medium was then added to the middle cell culture chamber (11). The top cell culture chamber (10) was added to the middle cell culture chamber (11) so that the medium in the middle cell culture chamber (11) covered the bottom of the membrane (20) of the top cell culture chamber (10). A mixture of cells, from human PBMC or mouse spleen, in tissue culture medium was added to the top cell culture chamber. All of these cell sources comprise a mixture of cell types and shown diagrammatically in figure 1 by means of a diversity of shading. The lid of the 24 well plate was added, and the completed cell culture vessel (1) was left for 3-4 days for the cells to migrate through the cell culture chambers (10, 11) into the bottom cell culture chamber (12). Cells were collected at the end of the incubation period and counted and analysed.

[0202] In the top (first) cell culture chamber (10) murine spleens or human PBMC were added in complete RPMI media supplemented with 10% foetal bovine serum, penicillin and streptomycin and glutamine as is standard for tissue culture. Interleukin-2 was added. IL-2 is a cytokine that prolongs the survival of immune cells such as T cells, NK cells and B cells. This mixture is used because it supports the viability of the cells of interest.

[0203] In the middle (intermediate) cell culture chamber (11) complete medium as described above was supplemented with IL-2 and CCL-19 and CCL-21. CCL-19 and CCL-21 cause the migration of naive T cells, NK cell, B cells and dendritic cells that are present in murine spleen and human PBMC. The cells that migrate in response to CCL-19 and CCL-21 express the chemokine receptor CCR7.

[0204] In the middle (intermediate) cell culture chamber (11) the medium also contained antibodies against the T cell surface protein CD3 and CD28. When these antibodies bind to CD3 and CD28 on the surface of T cells, which is only present on T cells, it causes the activation of T cells. Activated T cells change the surface expression of chemokine receptors. CCR7 is downregulated and CXCR3 is upregulated.

[0205] In the bottom (final) cell culture chamber (12) complete medium as described above was supplemented with IL-2 and CXCL-10. CXCL-10 causes the migration of activated T cells. In the bottom cell culture chamber (12) the medium also contained antibodies against the T cell surface protein CD3 and CD28 to cause the continued activation and expansion of T cells.

[0206] As can be seen in figure 1, the cells in the first cell culture chamber (10) of the cell culture vessel (1) are a mixed population (shown diagrammatically by diverse shading). Only cells which are responsive to the chemokines CCL19 and / or CCL21 migrate into the intermediate cell culture chamber (11) as shown by a more limited range of shading of cells shown diagrammatically in that chamber. Anti-CD3 and anti-CD28 only activates T-cells in the intermediate cell culture chamber (11). The chemokines CXL10, CCL2 and CCL5 are only able to attract into the final cell culture chamber (12) cells which have been activated because activation changes the expression of chemokine receptors. Because only T-are activated, only T-cells migrate into the final cell culture chamber 12 as shown diagrammatically, by a single shading type.

[0207] Figure 2 shows diagrammatically a modular cell culture vessel (1) of an embodiment of the invention in a disassembled form. It can be seen that the side walls of the first and intermediate cell culture chambers of the cell culture vessel (10, 11) are provided with a semipermeable membrane on their lower edge (20 and 21 respectively). The final cell culture chamber (12) has a solid base (22). Lid (30) may be placed on the top of the vessel to enclose the first cell culture chamber (10). It can be seen that when in the disassembled form, access is allowed into each of the cell culture chambers (10, 11, 12).

[0208] Figure 3, shows in closeup one possible method for assembling the modules of a modular cell culture vessel of the invention. As can be seen in figure 3, mutually engaging grooves are provided in the lower edge of one module (10 or 11) and the upper edge of another module (11 or 12) below it. The modules can be engaged by pushing them together, remain in place by an interference fit, and can be disengaged by pulling them apart.

[0209] Where in the foregoing description, integers or elements are mentioned which have known, obvious or foreseeable equivalents, then such equivalents are herein incorporated as if individually set forth. Reference should be made to the claims for determining the true scope of the present invention, which should be construed so as to encompass any such equivalents. It will also be appreciated by the reader that integers or features of the invention that are described as preferable, advantageous, convenient or the like are optional and do not limit the scope of the independent claims. Moreover, it is to be understood that such optional integers or features, whilst of possible benefit in some embodiments of the invention, may not be desirable, and may therefore be absent, in other embodiments.

[0210] Example 2 - Generation of T-cells from murine splenocytes

[0211] A cell culture vessel as described in Example 1 (and referred to herein as “CoED” (column of expansion and differentiation) was produced and used to compare T cells grown from whole splenocytes in a static cell culture well or using a “CoED” device comprising of a first cell culture chamber, an intermediate cell culture chamber and a final cell culture chamber. Each chamber is separated by a membrane with 5 or 8pm diameter pores that are only permeable to cells that can migrate. In this experiment splenocytes are added above the membrane into the first cell culture chamber and at the end of the experiment T-cells are collected below the membrane in the final cell culture chamber.

[0212] The intermediate cell culture chamber was pre-coated with I g / ml anti-mCD3 and 5 g / ml anti-mCD28 antibodies to stimulate T-cell expansion and differentiation. Then the final cell culture chamber was filled with medium containing 300ng / ml IP-10 / CXCL-10 to promote the migration of CXCR3+activated T-cells. In some experiments, 3 g / ml CCL-25 (TECK) and / or lOOng / ml CCL-22 were added in the final cell culture chamber to promote the migration of CCR9+and CCR4+cells respectively. The modular cell culture vessel was assembled by placing the intermediate cell culture on top of the final cell culture chamber and medium containing 200ng / ml CCL-19 and 200ng / ml CCL-21 was added to the intermediate cell culture chamber to recruit naive Tc-cells from the first cell culture chamber containing the splenocytes. In some experiments no chemokine was added.

[0213] The first cell culture chamber was then placed on the intermediate cell culture chamber and 2 x 106splenocytes were added to the first cell culture chamber in complete medium. All medium was standard RPMI with 10% FBS, glutamine, pen / strep and 20U / ml IL-2 added. For controls splenocytes were cultured at 2 x 106cells / ml with Ipg / ml anti-mCD3 and 5pg / ml anti-mCD28 antibodies and the same mix of chemokines in 24 wells plates.

[0214] Cells were then cultured for 3-5 days. Cells were harvested from wells or the final cell culture chamber of the CoED and stained for flow cytometry with Aqua-BV510 viability dye, and antibodies against CD4-FITC, CD8-PECF594, CD44-EF450, CD62L-BV605, CD3-AF700, CXCR3-PE and CCR7-APC to assess phenotype and viability or Aqua-BV510 viability dye, CD4-BV605, CD8-PECF594, CD44-EF450, CD3-AF700, IFNy-PECy7, TNFa-PE, Perforin- APC and Granzyme B-FITC. Marilyn CD4 Tccells were additionally stained with anti- CCR9-PE. Data was analysed using FloJo software and the fractions of cells expressing the various markers was calculated and is shown in figures 4 to 7.

[0215] Results from naive murine splenocytes.

[0216] The first study demonstrated that T cells migrated all the way through the cell culture vessel and could be collected in the final cell culture chamber. Surprisingly, when compared to cells grown in wells the T cells that were collected from the cell culture vessel had a higher fraction of live cells, CD4 Tccells and CD8 Tccells and a lower fraction of B cells (figure 4 A). When the phenotypes of the CD4 and CD8 T-cells were analysed, it was discovered that both Tccell types had a higher proportion of effector memory phenotype cells and a lower proportion of central memory phenotype cells (figure 4B and C).

[0217] To ensure that migration of Tccells through the cell culture vessel was due to active migration in response to chemokines the fraction of cells in the final cell culture chamber was compared with and without chemokines. There was a very significant reduction in Tccells harvested in the final cell culture chamber when no chemokines were used which demonstrates that chemotaxis is the primary reason that Tccells migrate through the cell culture vessel (figure 5). Given the differences in the phenotypes of Tccells expanded from splenocytes in a culture vessel of the invention when compared to cells cultured in a well plate it was decided to investigate the functional markers of tumour cell killing and migration of the Tc-cells. Surprisingly, Tccells grown in a cell culture vessel of the invention had higher levels of the tumour killing proteins perforin and tumour necrosis factora (figures 6A and 6C). Moreover, CD4 Tccells generated in the cell culture vessel of the invention expressed significantly higher levels of IFNy, CCR7 and CXCR3 suggesting that they will be more effective at migrating into tissues in response to inflammation (figure 6B and 6D).

[0218] Results from transgenic Marilyn murine splenocytes

[0219] The experiment was repeated using the same methodology, with splenocytes from Marilyn transgenic mice. Marilyn mice are transgenic for a class II restricted TCR receptor and only have CD4 Tccells. Marilyn CD4 Tccells express high levels of CCR9 so in this experiment CCL-25 was added to the chemokine combination in the final cell culture chamber to improve migration of CCR9 expressing CD4 Tccells.

[0220] The experiment demonstrated that Marilyn CD4 Tc cells which had migrated through the cell culture vessel were like the CD4 Tccells of the previous experiment because they had significantly higher expression of cytokines, IFNy and TNFa and the chemokine receptor CXCR3. Marilyn T cells showed lower expression of CCR7 than non-transgenic CD4 Tccells. The Marilyn T cells were also tested for expression of IL-2 and the chemokine receptor CCR9 which where both increased when compared to cells cultured in wells under the same conditions. Additionally, Marilyn T cells produced in a cell culture vessel of the invention had significantly lower expression of the inhibitory ligand CTLA-4 and of the naive T cell marker CCR7. Marilyn Tccells generated in a cell culture vessel of the invention with the addition of CCL-25 expressed high levels of CCR9, the cognate ligand for CCL-25 (figure 7).

[0221] Example 3, Tc cells generated from human PBMC

[0222] This experiment demonstrates that methods and devices of the invention can be used with human cells and is an adaptation of the murine experiments described above using the same cell culture vessel.

[0223] Human whole blood was sourced from NHS Blood Transfusion Service. Peripheral blood mononuclear cells (PBMC) were purified from blood using a Ficoll density gradient. The lymphocyte enriched PBMC was either cultured in a standard 24 well based culture or in a cell culture device of the invention. The intermediate cell culture chamber of the cell culture vessel was filled with media containing 25ng / ml CCL-19 and 25ng / ml CCL-21 to recruit naive T cells (Tc) from the first cell culture chamber into which PBMC were introduced. The final cell culture chamber was pre-coated with 300pl of PBS containing 30ng / ml anti-CD3 and 30ng / ml anti-CD28 antibodies to stimulate Tccell expansion and differentiation. The final cell culture chamber contained a mixture of lOng / ml CCL2 and / or lOng / ml CCL5 and / or lOOng / ml CXCL-10 to promote the migration of CXCR3+, CCR2+and / or CCR5+Tc cells depending on the selected mixture of cytokines used. 2 x 106PBMC in 400pl was added to the top first cell culture chamber of the assembled “CoED” cell culture vessel. For controls Tc cells were cultured at 1 x 106cells / ml in RIO with lOOU / ml IL-2 and 30ng / ml anti-CD3 and 30ng / ml anti-CD28 and the same mix of chemokines in 24 wells plates.

[0224] In a separate experiment using 4 individual PBMC donors (n=4), T cells from wells or the final cell culture chamber of a cell culture vessel of the invention were tested for their ability to migrate in response to chemokine IP- 10 (aka CXCL-10) in a transwell based chemotaxis assay. 2 x 105Cells were added to the top of the transwell and then the number of cells that migrated to the bottom were counted after 2 hours.

[0225] Cells were harvested from the wells or the layers of the CoED, counted and then analysed by flow cytometry for the expression of chemokine receptors, CXCR3, CCR2, CCR5 and CCR7 and cytokines IFNy and TNFa as well as the lineage markers CD45RA, CD45RO and CCR7.

[0226] Results

[0227] On day 3, cells from the first cell culture chambers, intermediate cell culture chambers and final cell culture chambers of the cell culture vessel, and from control wells were harvested, counted, and then stained for flow cytometry as described. Surprisingly, there was a significantly higher proportion of live cells, and effector and naive Tc cells in the cell culture vessel final cell culture chambers than there was in the wells suggesting that the migration through the column was refining the Tccell population (Figure 8A and B). Additionally, there were fewer contaminating B cells in the final cell culture chambers of the cell culture vessel suggesting that migratory stimuli had selected Tccells over B cells. Moreover, B cells made up 5.22% of cells in the lower ED of the column compared to just 0.75% in the collection chamber of the column (Figure 9). This suggests that whilst B cells migrated to the intermediate cell culture chamber in response to CCL19 and CCL21, they did not migrate to the final cell culture chamber in response to CXCL10, this corresponds to the expected chemokine receptor expression of naive B cells which are CCR7+CXCR3' and the expected unchanging phenotype of B cells in response to conditions in the intermediate cell culture chamber. Like results in murine Tccells, human Tccells generated using the cell culture vessel of the invention also expressed higher levels of TNFa, a key cytokine required for tumour cell killing (Figure 6, C).

[0228] Tccells generated in wells or in a cell culture device of the invention were compared for their ability to migrate towards IP-10, results suggest that there is a trend towards Tccells generated in the cell culture vessel of the invention migrating more effectively towards IP- 10 than cells cultured in wells (Figure 8, D).

[0229] Conclusions

[0230] The results above suggest that the cell culture vessel of the invention purifies and expands Tccells from human PBMC, and naive and transgenic murine splenocytes. The data presented above demonstrated that Tccells migrate all the way through the cell culture vessel of the invention and become positively selected for a Teff and Tncell phenotype. The combination of effective migration and enhanced secretion of TNFa means that these Tccells are potentially more potent at killing tumour cells.

[0231] Example 4 - An embodiment of a nestable cell culture vessel

[0232] An embodiment of a nestable cell culture vessel of the second aspect of the invention is described below with reference to figures 10 to 16.

[0233] Figure 10 shows a general view of a disassembled cell culture vessel (1) of the second aspect of the invention. Although shown in disassembled form, the parts are dimensioned so that the first cell culture chamber (10) is able to be fully nested within the intermediate cell culture chamber (11) and the intermediate cell culture chamber is able to be fully nested within the final cell culture chamber (12). When in a fully nested arrangement, lid (30) is able to close the whole cell culture vessel. The final cell culture vessel (12) is provided, in its inner sidewalls with slots (52, 52’). Further slots are provided on the inner surfaces of the side walls not visible in this view. The intermediate cell culture chamber (11) is provided with lugs (61, 61’) on the outer surface of its side wall. Slot 52 and the slot in the opposite side wall not shown are provided to a first depth. Slot 52’ and the slot in the opposite side wall not shown are provided to a second deeper depth, When intermediate cell culture chamber 11 is nested within the final cell culture chamber 12, the user has a choice of allowing lugs (61, 61’) to engage with slot 52 (and its corresponding oppositive slot) or slot 52’ (and its corresponding opposite slot) , thereby achieving a choice of heights of suspension of intermediate cell culture chamber 11 in final cell culture chamber 12. This choice accommodates different volumes of cell culture medium in final cell culture chamber 12. A similar arrangement is used with the first cell culture chamber (10) wherein lugs (71, 71’) may be engaged with slot 62 (and opposite slot not shown) or slot 62’ (and opposite slot not shown) to achieve different suspension heights to accommodate differing volumes of cell culture medium. Also shown in figure 10 is a pipette bypass (75) via which a pipette tip may be used to access the contents of intermediate chamber (11) whilst first cell culture chamber (10) remains nested within it. A similar pipette bypass (65) is also provided on the intermediate cell culture chamber.

[0234] Figure 11 shows the same embodiment as figure 10 from a different viewpoint. Also shown in figure 11 is the position of (transparent) semipermeable membranes (20, 21) between adjacent cell culture chambers.

[0235] Figure 12 shows in more detail, an intermediate cell culture chamber (11). In addition to the lugs (61, 61’) and slots (62, 62’), pipette bypass (65) is shown. It can also be seen more clearly in this figure than in figures 10 and 11, that the slots (62, 62’) in this chamber provide 3 different suspension heights. Each slot provides a part of a first depth (which in this example is the same as between slot 62 and 62’) and a part of a second depth (which differs in this example between slot 62 and 62’). The deepest suspension of the first cell culture chamber is provided when the lugs (71, 71’) of that cell culture chamber (10) engage with the deepest part of slot 62 and the deepest part of the corresponding slot opposite not shown in this view). An intermediate level of suspension is provided when the lugs engage with the deepest part of slot 62’ and highest level of suspension is provided when the lugs (71, 71’) engage with the upper part of the slots. The height of suspension can be chosen by rotating the first cell culture chamber (10) about a vertical axis before nesting it into the intermediate cell culture chamber. In figure 12 it can be seen that the intermediate cell culture chamber is provided with indicator dots (•, ••, •••) moulded into the surface of the cell culture chamber (11) by aligning those indicator dots with a marker on the first cell culture chamber (10) a user can rote the first cell culture chamber by the correct amount to select the required suspension height. Figure 13 shows in more detail a first cell culture chamber (10) showing the position of lugs (70., 70’). The first cell culture chamber lacks slots of receive lugs because it is the first cell culture chamber and there is no earlier cell culture chamber in the series to nest within it. The pipette bypass (75) is shown. It provides a passageway past the cell culture chamber (10) into the next cell culture chamber (11) in the series. In this embodiment the pipette bypass also functions as a marker which can be aligned with indicator dots (•, •••) moulded into the surface of the cell culture chamber (11). In this embodiment the pipette bypass (75) and also the pipette bypass (65) of the intermediate cell culture chamber (11) may be engaged with a tool in order to assist with lifting the chamber out of the chamber into which it is nested.

[0236] Figures 14 and 15 illustrate in cross section a cell culture vessel of an embodiment of a second aspect of the invention. The same cell culture vessel is illustrated in both figures. The figures differ in that different suspension heights have been chosen for the inner cell culture chambers by selecting different lugs and slot combinations. It can be seen that the suspension heights of the first cell culture chamber (10) and the intermediate cell culture chamber (11) result in different amounts of space within each of those chambers for containing medium. By allowing such an adjustment, both sides of the semipermeable barriers (membrane 20, 21) between adjacent cell culture chambers be kept in contact with medium present in the cell culture chambers in differing volumes.

[0237] Figure 15 shows a configuration in which if used for transit (for example of empty cell culture vessels being delivered prior to use) would result in the possibility of cell culture chambers 10 and 11 sliding up and down within cell culture chamber 12. That may result in damage. To mitigate this risk, the cell culture vessels shown in figures 14 and 15 are configures to have a transit position as illustrated in figure 14 wherein both chambers 11 and 12 are in the highest position whilst still being nested and where any upward movement is limited by the presence of a lid. The lid can be held in place by being of a clip-on design or by shrink wrapping the entire cell culture vessel. Movement and damage is thereby minimised.

[0238] Figure 16 shows in plan a further embodiment of a cell culture vessel of the invention. As can be seen the cell culture chambers are each squircular in plan. Pipette bypasses 65 and 75 are shown. Slots 52, 52’, 52”, 52’”, 62, 62’, 62”, 62’” are shown having two parts of different depths. Lugs (61, 61’, 71; 71’) are able offset so as to be able to engage with either part of the slots.

[0239] Whilst the present invention has been described and illustrated with reference to particular embodiments, it will be appreciated by those of ordinary skill in the art that the invention lends itself to many different variations not specifically illustrated herein.

[0240] Example 5 - An example of a multichamber culture vessels of the invention and SLAS standard plates of the invention.

[0241] According to certain embodiments of the invention in its second aspect, multiple cell culture chambers may be provided in multichamber plates. Nesting of cell culture chambers may be achieved in those embodiments by stacking the multichamber plates.

[0242] Figure 17 shows an assembled cell culture vessel of the invention which comprises a stack of three multichamber plates and a lid (130). The stacked plates consists of top plate (110) which comprises multiple first cell culture chambers (10, not shown in this view) of multiple series of cell culture chambers, an intermediate plate (111) which comprises multiple intermediate cell culture chambers of multiple series (11, not shown in this view), and a bottom plate (112) which comprises multiple final cell culture chambers of multiple series (12, not shown in this view).

[0243] Figure 18 shows the same cell culture vessel as illustrated in figure 17 in a separated unstacked view. In this arrangement the detains of each plate can be seen. It can be seen that the plate (110) comprising the first cell culture chambers (10, a single example only of which has been labelled) and the intermediate plate (111) comprising the intermediate cell culture chambers (11, a single example only of which has been labelled), comprise holes which may form pipette bypasses. A set of pipette bypasses allowing access to the intermediate cell culture chambers comprise an array of holes (75, a single example only of which has been labelled) through the first plate (110). A set of pipette bypasses allowing access to the final cell culture chambers comprise an array of holes (66, a single example only of which has been labelled); 65, a single example only of which has been labelled) through, respectively, the first plate (110) and the intermediate plate (111). Those holes are positioned so that when the plates are stacked the holes align to form the pipette bypass. The plates (110, 111) comprising the first and intermediate cell culture chambers may be regarded as embodiments of SLAS standard plates of that aspect of the invention. Figure 19 shows a cross-section though a set of stacked plates similar (but as explained below not identical) to that illustrated in figure 17. It can be seen from this illustration that when the first plate (110) is stacked on the intermediate plate (111) and that in turn is stacked on the final plate (112), the first cell culture chambers of each series (10, a single example only of which has been labelled), nest within the intermediate cell culture chambers (11, a single example only of which has been labelled), and the intermediate cell culture chambers of each series (11) nest within the final cell culture chambers of each series (12, a single example only of which has been labelled). In this embodiment, the extent of the nesting of both the first and intermediate cell culture chambers within the cell culture chambers next in the series, is restricted by the presence of spacer (190) which holds the plates apart somewhat, The extent of nesting an therefore the volume of each of the intermate and final cell culture chambers can be adjusted by removing the space (190) or replacing the spacer with a differently-dimensioned spacer (190). The arrangement shown in figure 17 lacks such a spacer. It is illustrated in figures 24 and 25 discussed below.

[0244] Figures 20 and 21 illustrate the first plate (110). In addition to comprising an array of 24 first cell culture chamber (10, only one example of which is labelled), the plate also comprises an array of holes (75, only one example of which is labelled) forming pipette bypasses into the intermediate cell culture chambers of an intermediate plate onto which this first plate (110) is stacked. It also comprises an array of holes (66, only one example of which is labelled) which form pipette bypasses into the final cell culture chambers when this first plate (110) is stacked into an intermediate plate and a final plate (not shown in this view) in cooperation with an array of holes (65 in figure 22) in the intermediate plate (111 in figure 22).

[0245] Figure 21 shows a lower view of the first plate (110) and shows the location of the semipermeable barriers (20, only one example of which is labelled) closing the ends of each of the first cell culture chambers and that pipette bypasses (75, one one example of which is labelled) in this embodiment is not merely an array of holes, but that the side wall of the cell culture chambers is dimensioned to provide a channel.

[0246] Figure 22 illustrates the intermediate plate (111). In addition to comprising an array of 24 intermediate cell culture chambers (11, only one example of which is labelled), the plate also comprises an array of holes (65, only one example of which is labelled) which in cooperation with the array of holes (66) in the first plate form pipette bypasses into the final cell culture chambers. Figure 23 illustrates the final plate (112). It comprises an array of 24 final cell culture chambers (12, only one example of which is labelled). This plate also has a stacking flange (180) which can be used to stack multiple sets of plates.

[0247] Figures 24, 25 and 26 illustrate how plates (110, 111, 112) and a lid (130) of the invention may be stacked into a cell culture vessel with the addition of a spacer (190). The spacer (190) is essentially a hollow ring which cooperates with the edges of the stacked plates and holds them apart. Differently-dimensioned spacers (190) may be used to achieve different separation of the plates and thus vary the extent of nesting of each of the first and intermediate cell culture chambers within the next cell culture chamber in the series.

[0248] According to certain embodiments, the spacer (190) may comprise a lower edge dimensioned to engage with the edge of a plate (110) comprising multiple final cell culture chambers and an upper edge having two concentric portions, the inner portion being dimensioned to engage with one or more intermediate plates (111) and the outer portion being dimensioned to engage with the upper plate (112).

[0249] Figures 27 and 28 illustrate an embodiment of the plates featuring “stand offs” (200, only some examples of which are shown). Figure 27 illustrates a portion of a first plate and figure 28 illustrates a final plate. Equivalent features may also be provided on intermediate plates. As can be seen, stand offs ate provided around the edge of the plates and serve to reduce the area of contact between the plate (or lid) stacked above it and to make separation earlier especially if the area of contact is wet or has previously been wet. The stand offs may optionally be raised dots or raised moulded letters or numbers, for example those identifying the wells in a grid arrangement.

[0250] Where in the foregoing description, integers or elements are mentioned which have known, obvious or foreseeable equivalents, then such equivalents are herein incorporated as if individually set forth. Reference should be made to the claims for determining the true scope of the present invention, which should be construed so as to encompass any such equivalents. It will also be appreciated by the reader that integers or features of the invention that are described as preferable, advantageous, convenient or the like are optional and do not limit the scope of the independent claims. Moreover, it is to be understood that such optional integers or features, whilst of possible benefit in some embodiments of the invention, may not be desirable, and may therefore be absent, in other embodiments.

Claims

Claims1. An in vitro method of producing a population of cells comprising the steps of:A, introducing a source of cells into the first cell culture chamber of an in vitro cell culture vessel comprising three cell culture chambers arranged in a series, each chamber being separated from the next chamber in the series by a semipermeable barrier,B, contacting the cells in the first cell culture chamber to a first composition of one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines, and cell attractants so as to promote migration of at least a subset of cells from the first cell culture chamber into an intermediate cell culture chamber of the series,C, contacting cells in the intermediate cell culture chamber of the series to a further composition of one or more cell growth factors, cell activating factors, cell differentiation factors, cytokines and cell attractants so as to promote migration of at least a subset of cells from the intermediate cell culture chamber to the final cell culture chambers in the series,D, optionally withdrawing cells from the final cell culture chamber in the series.

2. A method according to claim 1 in which the first composition comprises one or more cell attractant factors (for example one or more chemokines) and the second composition comprises one or more cell attractant factors (for example on or more chemokines) wherein the first composition and the second composition are different from each other in respect of the cellular receptors needed for the respective cell attractant factors to show attractive activity.

3. A method according to claim 1 or claim 2, in which the first composition is provided in the intermediate cell culture chamber and the second composition is provided in the final cell culture chamber.

4. A method according to of claims 4 in which step A comprises introducing a relatively mixed population of cells into the first cell culture chamber of a cell culture vessel.

5. A method according to claim 3 or claim 4, wherein step B further comprises activating a subset of cells in the intermediate cell culture chamber, said activation causing the activated cells to change their expression of receptors for cell attraction factors (for example to change their expression of chemokine receptors).

6. A method according to claim 5, wherein activation causes the activated cells to upregulate their ability to respond to the cell attractant factor(s) in the second composition and optionally further causes the activated cells to downregulate their ability to respond to the cell attractant factor(s) in the first composition.,7. A method according to any of claims 1 to 6, wherein the source of cells is a mixed population of immune system cells (for example a white cell fraction of blood, PBMCs, spleen cells, bone marrow aspirate cells, immune system cells isolated from a tumour site.

8. A method according to any of claims 1 to 7, wherein the source of cells comprise CAR-T cells.

9. A method according to any of claims 1 to 8, which is a method of producing a population of activated immune effector cells, for example activated NK cells, activated B- cell, activated T-cells, activated NK T-cells, or activated dendritic cells.

10. A method according 1 to 9, wherein the in vitro cell culture vessel comprising at least three cell culture chambers arranged in a series comprising a first cell culture chamber, one or more intermediate cell culture chambers and a final cell culture chamber, each chamber being separated from the previous and next chamber in the series by a semipermeable barrier, and wherein the semipermeable barriers are configured to limit mixing of a cytokine between chambers whilst permitting migration therethrough of cells.

11. A method according to claim 10 wherein the in vitro cell culture vessel consists of three cell culture chambers.

12. A method according to claim 10 or claim 11 wherein the in vitro cell culture vessel comprise injection moulded polystyrene.

13. A method according to any of claims 10 to 12, wherein the in vitro cell culture vessel comprises at least three cell culture chambers are stacked on each other.

14. A method according to any of claims 10 to 13, wherein the in vitro cell culture vessel comprises at least three cell culture chambers which are separable from each other to permit access into them.

15. A method according to any of claims 1 to 13 wherein the in vitro cell culture vessel comprises at least three cell culture chambers which are not separable from each other.

16. A method according to any of claims 10 to 15, wherein the in vitro cell culture vessel comprises a wall of at least one of the cell culture chambers which is provided with an access port through which fluid may be withdrawn and / or introduced.

17. A method according to any of claims 10 to 16, wherein the in vitro cell culture vessel comprises at least one of the cell culture chambers which is provided with one or more sensors to detect one or more of - temperature, liquid level, pH, oxygenation, nutrient levels.

18. A method according to any of claims 10 to 17, wherein the in vitro cell culture vessel comprises at least three cell culture chambers which are substantially cylindrical.

19. A method according to any of claims 10 to 18, wherein the in vitro cell culture chambers each have a volume of between 1 ml and 10000 ml, for example between 1 ml and 500 ml.

20. A method according to any of claims 10 to 19, wherein the semipermeable barrier is a semipermeable membrane having pores with a maximum dimension of between 1 pm and 30 pm.

21. A method according to any of claims 10 to 20, wherein the medium in the one or more intermediate cell culture chambers comprises a cell attractant factor (such a chemokine) and the medium in the final cell culture chamber comprises a different cell attractant factor (such as a different chemokine), and the medium in the one or more intermediate cell culture chambers comprises a cell activating factor.

22. A method according to any of claims 10 to 21 wherein the in vitro cell culture vessel comprises a number of modules which may be disassembled.

23. A method according to any of claims 10 to 22 wherein the in vitro cell culture vessel further comprises access ports in the walls of at least one or the cell culture chambers suitable for introducing and withdrawing cell culture medium24. A cell culture vessel comprising at least three cell culture chambers arranged in a series comprising a first cell culture chamber, one or more intermediate cell culture chambers and a final cell culture chamber, each chamber being separated from the previous and next chamber in the series by a semipermeable barrier wherein the semipermeable barriers are configured to limit mixing of a cytokine between chambers whilst permitting migrationtherethrough of cells, wherein each cell culture chamber is nestable within the next cell culture chamber of the series.

25. A cell culture vessel comprising according to claim 24 consisting of three cell culture chambers.

26. A cell culture vessel according to any of claims 24 to 25 comprising injection moulded polystyrene.

27. A cell culture vessel according to any of claims 24 to 26, wherein the at least three cell culture chambers are separable from each other to permit access into them.

28. A cell culture vessel according to any of claims 24 to 26, wherein the at least three cell culture chambers are not separable from each other.

29. A cell culture vessel according to any of claims 24 to 26, wherein the wall of at least one of the cell culture chambers is provided with an access port through which fluid may be withdrawn and / or introduced.

30. A cell culture vessel according to any of claims 24 to 27, wherein at least one of the cell culture chambers is provided with one or more sensors to detect one or more of - temperature, liquid level, pH, oxygenation, nutrient levels.

31. A cell culture vessel according to any of claims 24 to 28, wherein the at least three cell culture chambers are substantially cylindrical.

32. A cell culture vessel according to any of claims 24 to 29, wherein the cell culture chambers each have a volume of between 1 ml and 2000 ml, for example between 1 ml and 500 ml.

33. A cell culture vessel according to any of claims 24 to 30, the semipermeable barrier is a semipermeable membrane having pores with a maximum dimension of between 4 pm and 30 pm.

34. A cell culture vessel according to any of claims 24 to 31, wherein each semipermeable barrier is provided in the base of a cell culture chamber and each of the first and intermediate cell culture chambers are nestable within the next cell culture chamber in the series by being configured to be suspendable within the next cell culture chamber of the series in such a way that there is a gap between the base of each of the first and intermediate cell culture chambers and the base of the next cell culture chamber of the series.

35. A cell culture vessel according to claim 34, wherein the gaps are adjustable.

36. A cell culture vessel according to claim 35, wherein the gaps are adjustable by providing lugs and / or detents on each of the cell culture chambers such that said lugs and detents are arranged to cooperate in two or more different combinations, each different combination resulting in suspension of each of the first and intermediate cell culture chambers at different height within the next cell culture chamber in the series.

37. A cell culture vessel according to claim 36, wherein the lugs and / or detents are configured such that the management of different combinations of lugs and / or detents can be selected by rotation of each of the first and intermediate cell culture chambers relative to the next cell culture chamber in the series.

38. A cell culture vessel according to claim 37, further comprising a lid configured to close the final cell culture chamber and fully enclose the other cell culture chambers nested inside the final cell culture chamber.

39. A cell culture vessel according to claim 38, wherein the lugs and / or detents are arranged such that each of the first and intermediate cell culture chambers may be nested inside the final cell culture chamber in such a way that when the lid closes the final cell culture chamber, they abut the lid and reduce any rattling of each the other first and intermediate cell culture chambers within the next cell culture chambers of the series.

40. A cell culture vessel according to any of claims 24 to 39, further comprising cell culture medium in each of the cell culture chambers.

41. A cell culture vessel according to claim 40, wherein the cell culture medium is in contact with both sides of the semipermeable barriers separating each of the cell culture chambers.

42. A cell culture vessel according to claim 40 or 41, further comprising a cell culture.

43. A cell culture vessel according to claim 40, 41 or 4, wherein the medium in the one or more intermediate cell culture chambers comprises a cell attractant factor (such a chemokine) and the medium in the final cell culture chamber comprises a different cell attractant factor (such as a different chemokine), and the medium in the one or more intermediate cell culture chambers comprises a cell activating factor.

44. A cell culture vessel according to any of claims 24 to 43, further comprising access ports in the walls of at least one or the cell culture chambers suitable for introducing and withdrawing cell culture medium.

45. A cell culture vessel according to any of claims 24 to 44, wherein each of the first and intermediate cell culture chambers is adapted to provide a route for a pipette tip to bypass each of said first and intermediate cell culture chamber in order to introduce or withdraw cell culture medium to / from the next cell culture chamber in the series.

46. An SLAS standard plate comprising an array of cell culture chambers each having at their lower end a semipermeable barrier and each cell culture chamber having adjacent to it at least one pipette bypass.

47. A method of producing a population of cells according to any of claims 1 to 23, comprising use of a cell culture chamber according to any of claims 24 to 45 or an SLAS standard plate.

48. A population of cells produced in accordance with a method of any of claims 19 to 27, and / or produced by culturing a source of cells in a cell culture vessel according to any of claims 24 to 45 or an SLAS standard plate according to claim 46.

49. A population of cells according to claim 48, which is a population of CAR-T cells.

50. A population of cells according to claim 48, which is a population of activated T- cells.

51. A method of cell therapy comprising administering to a subject in need thereof, a population of cells according to any of claims 48 to 50.

52. A population of cells according to any of claims 48 to 50 for use as a medicament.

53. A population of cells according to any of claims 48 to 50 for use in cell therapy.

54. A population of cells according to any of claims 48 to 50 for use in cancer immunotherapy.

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