Apparatus for cell culturing and related methods

Abstract

An apparatus for culturing cells includes a bioreactor with a fluid reservoir and a fixed bed, as well as a portion associated with the fluid reservoir and adapted for preventing cells from settling therein. The bioreactor may include a first vessel including the fluid reservoir and a second vessel with the fixed bed for the cell culture, which may be directly connected to the first vessel by a frangible connection to facilitate disassembly, such as for sampling. A conduit may be provided for connecting the first vessel to the second vessel, so as to establish fluid communication there between. A pump may also be provided for pumping fluid from the first vessel to the second vessel, such as though the conduit if present. Use of the apparatus as a screening tool for a large-scale bioreactor, in a kit, and related methods are also disclosed.

Description

[0001] APPARATUS FOR CELL CULTURING AND RELATED METHODS

[0002] This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63 / 668255, filed July 7, 2024, the disclosure of which is incorporated herein by reference.

[0003] TECHNICAL FIELD

[0004] This document relates generally to the cell culturing arts and, more particularly, to an apparatus for performing cell culturing and related methods.

[0005] BACKGROUND

[0006] Cell culturing usually involves the use of a fairly complex, large-scale bioreactor, which may in some instances have a fixed bed designed for adherent cell growth involving a large volume of culture medium. Setting up and operating such a bioreactor requires a significant amount of time and cost. This can be disadvantageous in certain situations, particularly when a desire exists to evaluate or screen a variety of operating conditions, such as the viability of a particular matrix material, cells, reagents, or the like for use in performing cell culturing (including for process development in connection with a design of experiment study, for example), or in situations where only small quantities of material are required or available. In large-scale bioreactors, sampling the cells cultured on the matrix material is also complicated, as it often risks breaching the often mandatory aseptic conditions unless significant precaution is taken, which further adds to the complexity, time, and cost of the procedure.

[0007] Accordingly, a need is identified for an apparatus that provides the ability to perform cell culturing in an easier, less costly manner, and which thus may be particularly useful as a tool for screening for the viability of certain conditions in culturing cells, for process development and / or optimization in connection with a design of experiment study, for example), or in situations where only small quantities of material are required. In some instances, the apparatus would be compact and portable to allow for convenient use in a variety of settings, including but not limited to clinical environments, without complex and costly set-up requirements associated with culturing cells in a bioreactor as part of a large scale bioprocessing system. This would especially allow for plural compact cell culturing apparatuses to be used simultaneously to evaluate certain operating conditions. The apparatus would also be made compact and the fixed bed when present or cell culture made readily accessible to allow for obtaining a sample of the cell culture made in an easy and inexpensive manner, as well as for use in connection with a variety of processes, including but not limited to cell attachment / inoculation, cell growth / proliferation, infection / transfection or differentiation and production of product of interest, and harvest of the product of interest or the productive cells. It would also be desirable to have a small fixed-bed chamber that can be easily operated in a simple cell culture incubator (e.g., a heating chamber controlling CO2 level in the headspace) without the use of complex and large foot-print bioreactor controllers.

[0008] SUMMARY

[0009] One object of the disclosure is to provide a cell culturing apparatus that provides the ability to perform cell culturing in an easier, less costly manner, and which thus is particularly useful as a tool for screening for the viability of certain conditions in culturing cells. The apparatus may be made compact and portable to allow for convenient use in a variety of settings, including but not limited to clinical environments, without complex and costly set-up requirements associated with culturing cells in a bioreactor as part of a large scale bioprocessing system. This smaller-scale arrangement allows the use of plural small-scale cell culturing apparatuses to evaluate certain operating conditions simultaneously, or to perform simultaneous bioprocessing using several such apparatuses in a manner normally achieved by a single, large-scale bioreactor. The simplicity of the apparatus in construction and small size may also provide the ability to obtain a sample of the fixed bed and / or cell culture in an easy and inexpensive manner.

[0010] In one embodiment, an apparatus for culturing cells is provided. The apparatus comprises a bioreactor including a fluid reservoir and a fixed bed for the cell culture. The bioreactor includes a portion associated with the fluid reservoir adapted for preventing cells from settling therein.

[0011] In one embodiment, the bioreactor comprises a first vessel including the fluid reservoir and a second vessel including the fixed bed. The apparatus may further include a conduit for connecting the first vessel to the second vessel. The portion may comprise a frusto-conical shape for preventing cells from settling therein, and may be provides as a lower portion of the first vessel, a lower portion of the second vessel, or as lower portions of both the first and second vessels.

[0012] In this or another embodiment, the second vessel comprises a tubing open at both ends. A first end of the tubing is connected to the first vessel. A second end of the tubing is connected to the conduit.

[0013] The first and second vessels may be connected to form a common interior chamber. The first vessel may be detachably connected to the second vessel. In one particular version, the first vessel has a volume of about 50 ml or less. The apparatus may also include a pump for pumping fluid from an outlet of the first vessel to an inlet of the second vessel. The first vessel may be adapted to provide a waterfall flow of fluid therein. The apparatus may also include a support for supporting the first and second vessels in a generally vertical orientation. The first and second vessels may be configured for placement in an incubator.

[0014] According to a further aspect of the disclosure, an apparatus for culturing cells includes a first vessel including a fluid reservoir and a second vessel including a fixed bed for the cell culture. The second vessel is detachably connected directly to the first vessel. A conduit connects the first vessel to the second vessel, and a pump is provided for pumping fluid though the conduit.

[0015] In one embodiment, the first vessel comprises a frusto-conical lower portion. In this or another embodiment, the second vessel includes a lower portion adapted for preventing cells from settling therein, which may be frusto-conical in shape. The second vessel may comprise a tubing open at both ends, with a first end being connected to the first vessel and a second end being connected to the conduit. The first and second vessels may form a common interior chamber, and the first vessel may have a volume of about 50 ml or less.

[0016] According to a further aspect of the disclosure, an apparatus for culturing cells includes a vessel including a first portion forming a fluid reservoir, a second portion including a fixed bed for the cell culture, and a third tapered portion connecting the first portion to the second portion. In one embodiment, the first portion is an upper portion, the second portion is a lower portion, and the third portion is intermediate the upper and lower portions. A conduit connects an outlet of the vessel to an inlet of the vessel, and a pump is provided for pumping fluid though the conduit.

[0017] Still a further aspect of the disclosure relates to a cell culturing apparatus, comprising a tube including a fixed bed. The apparatus may further include a vessel connected to the tube by a conduit forming a closed loop, and a pump for circulating fluid through the conduit. A fastener may be provided for fastening the tube to the vessel. The tube including the fixed bed may be positioned in tandem with first and second segments of the conduit.

[0018] In any embodiment, the fixed bed may comprise at least one cell immobilization layer and at least one spacer layer. The fixed bed may comprise a structured fixed bed. In particular, the structured fixed bed may comprise a spiral fixed bed. The spiral fixed bed may comprise at least one layer of a non-woven material, and / or at least one spacer layer, which may comprise a woven or non-woven mesh. Likewise, the apparatus may include one or more ports for removing or adding substances to the apparatus, and a plurality of the apparatuses may be combined into a system for cell culturing. Still further, this disclosure relates to a screening tool for use in connection with a bioreactor including a first fixed bed for culturing cells. The tool comprises a first vessel including a fluid reservoir and a second vessel including a second fixed bed for the cell culture, the second fixed bed being representative of (e.g., a model of) the first fixed bed of the bioreactor. A conduit connecting the first vessel to the second vessel, and pump is provided for pumping fluid though the conduit.

[0019] Yet another aspect of the disclosure pertains to a system for culturing cells, comprising a first bioreactor including a first fixed bed for culturing cells, as well as a screening tool. The screening tool includes a first vessel having a fluid reservoir, a second vessel including a second fixed bed for culturing cells independent of the first fixed bed of the bioreactor, a conduit connecting the first vessel to the second vessel, and a pump for pumping fluid though the conduit.

[0020] Still another aspect of the disclosure relates to a kit for cell culturing. The kit includes a sealed vessel including a first portion forming a reservoir and a second portion including a cell culturing bed. The second portion is connected to, or adapted for connecting directly to, the first portion. The first portion and second portion may be connected by a frangible connection. The first portion may also be sealed and separate from the second portion, which is also sealed.

[0021] This disclosure also pertains to a method for use in connection with a bioreactor including a first fixed bed for culturing cells. The method comprises using a screening tool to perform cell culturing on a second fixed bed representative of the first fixed bed. The screening tool may include a first vessel including a fluid reservoir, a conduit forming a continuous loop for circulating fluid, and a pump for pumping fluid though the conduit, in which case the method comprises associating the second fixed bed with the first vessel. The associating step may comprise placing the second fixed bed in a second vessel in fluid communication with the first vessel.

[0022] BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0023] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0024] Figure 1 is a schematic view of a first embodiment of a cell culture apparatus according to the disclosure.

[0025] Figures 1A-1H illustrate examples of fixed bed arrangements for culturing cells. Figure 2 is an illustration of an embodiment of the apparatus of Figure 1. Figure 2A is a schematic cross-sectional view of part of the Figure 1 apparatus. Figure 3 is a schematic view of a second embodiment of a cell culture apparatus according to the disclosure.

[0026] Figure 4 illustrates one version of the apparatus of Figure 3.

[0027] Figure 5 illustrates another version of the apparatus of Figure 3.

[0028] Figure 6 depicts a combined reservoir and bioreactor forming part of the cell culturing apparatus of Figure 3.

[0029] Figure 6A is a cross-sectional view of the combined reservoir and bioreactor according to Figure 3.

[0030] Figure 7 is an illustration of another version of the apparatus of Figure 3. Figure 8 is an illustration of another version of the apparatus of Figure 3. Figure 9 is an illustration showing the apparatus of Figure 3 including a peristaltic pump.

[0031] Figure 10 is a schematic diagram of another version of the apparatus of Figure 3.

[0032] Figures 11 and 12 illustrate a kit for use in forming the apparatus for cell culturing.

[0033] Figure 13 illustrates a support for the apparatus.

[0034] Figure 14 illustrates a pumping arrangement for use in connection with a plurality of the cell culturing apparatuses according to the disclosure.

[0035] Figure 15 reflects experimental data obtained from use of the various apparatuses according to the disclosure.

[0036] DETAILED DESCRIPTION

[0037] Reference is now made to Figure 1, which illustrates one exemplary embodiment of a cell culturing apparatus 10 according to one aspect of the disclosure. As illustrated, the apparatus 10 may take the form of a bioreactor, which includes a first vessel 12 forming a reservoir for recirculating fluid to a second vessel 16. The second vessel 16 may be connected to the first vessel 12, such as by a conduit C, which may form a continuous loop between an inlet 16a and an outlet 16 of the second vessel 16. The first vessel 12 may be sealed, and associated with one or more ports 14 (including possibly one serving as an inlet and one as an outlet and / or one serving as a vent, such as for passive gas diffusion or active gassing). The one or more ports 14 may include a filter of the sterile or aseptic variety, such that an entirely closed arrangement results in order to maintain sterile conditions. In order to move fluid through the apparatus 10, a pump P may also be provided. For example, the pump P may be associated with the conduit C for circulating fluid between the first and second vessels 12, 16. The pump P may for example comprise a peristaltic pump for engaging the flexible tubing forming the conduit C, but could also comprise any other form of device for causing fluid to flow through the conduit, whether actively or passively operated (e.g., a syringe or similar manually operated device to inject fluid under pressure to the conduit C, which could include cells for culturing or other substances useful in connection with culturing cells or analyzing cell cultures).

[0038] The second vessel 16 may comprise a matrix material for cell culturing, which may take the form of a fixed bed 18 for performing cell culture (whether adherent or in suspension). The fixed bed 18 may comprise any substrate for achieving cell growth or cell immobilization, and may comprise, for example, a fixed bed (such as a structured fixed bed, which means that the bed is formed of an easily replicated, generally homogeneous, substantially fixed structure, and thus is not randomly oriented or unstructured, yet, as can be appreciated, could take a variety of sizes or shapes while meeting this qualification). The material of the fixed bed 18 may be woven, non-woven, a fiber matrix, a laminate, a monolith, a stack, or may take other forms (such as a packed bed or one comprising microcarriers). The fixed bed 18 may be fabricated in a variety of ways, such as for instance packing, rolling, lamination, or 3-D printing.

[0039] The fixed bed 18 may be formed of one or more of various polymer materials, including but not limited to polyethylene and polyethylene terephthalate, in particular. In one embodiment, as shown in a top view in Figure 1A, the fixed bed 18 comprises a woven or non-woven material 18a arranged in at least two layers on and within which cells grow, and may comprise a mesh (in either woven or nonwoven form). These layers are thus considered cell immobilization layers. In an embodiment, the fixed bed 18 may comprise a scaffold. In an embodiment, the fixed bed or the interior surfaces of the second vessel 16 may be treated, coated, functionalized, etc. In any embodiment, at least a portion of the fixed bed 18 is hydrophilic. Any material suitable for layer 18a may also be used for layer 18b. Figure 1A shows one embodiment of a matrix material for use as a structured fixed bed 18 and, in particular, a wound, concentric or spiral bed. In some embodiments, one or more cell immobilization layers 18a may be adjacent to one or more optional spacers, which may take the form of spacer layers 18b. These layers 18b may comprise a woven or non-woven mesh structure. In some embodiments, the layering may optionally be repeated several times to achieve a stacked or layered configuration. The spacer(s) need not comprise a layer of material, and may take other forms, such as one or more projections that may extend from or otherwise exist between the cell immobilization layers 18a in order to create a gap or space there between.

[0040] The mesh structure included in spacer layers 18b forms a tortuous path for cells (see cells L in Figure IB suspended or entrapped in the material of the immobilization layer 18a), and a cell culture may form part of any invention claimed herein) and fluid to flow through a channel thus created when layered between two immobilization layers 18a. In this arrangement, and despite the tortuous path, fluid flow may be primarily in the axial direction, as indicated by arrow A in Figure 1C, but may also extend along or through the layers 18b. Homogeneity of the cells is maintained within the structured fixed bed 18 as a result of using this type of arrangement.

[0041] As shown in Figure 1A, the fixed bed 18 may be spirally or concentrically rolled along an axis or core, which may be solid, partially filled, or hollow, as shown. In some embodiments, the layers of the structured fixed bed are firmly wound, but could be loosely wound. In some embodiments, thickness of each of the layers 18a, 18b may be between 0.1 and 5 mm, 0.1 and 10 mm, or .001mm and 15mm, but other ranges may be applicable depending on the circumstances of use. The fixed bed 18 may also be fabricated (e.g. wound) without an internal core.

[0042] In some embodiments, other structures can be used which form such tortuous paths. For example, Figure IE, which is a cross-sectional view of a woven substrate, shows that the one or more cell immobilization layers 18a may be adapted to form a structured fixed bed 18. The one or more layers 18a provide a tortuous channel of flow (arrow B) from a linear or regular inflow (arrow A) without using additional spacer layers (but such may be used, if desired). This may be achieved, for example, by providing a layer of woven fibers or filaments F that disrupt the flow, or by using a non-woven sheet that limits flow in the direction perpendicular to the non-woven sheet, structure or layer.

[0043] Figure IF shows that such a result may be achieved using a woven or non-woven material as the cell immobilization layer 18a. This may be achieved by forming this layer 18a as a reticulated arrangement (such as by 3-D printing, molding, or stamping) with openings or projections through which liquid may pass and return again, thus forming the tortuous channels that again promote homogeneity and also serve to further shear or divide any bubbles present in the liquid. This function may again be achieved with or without added spacer layers being present.

[0044] The orientation of the structured fixed bed 18 may be other than as shown in a Figure 1A where the flow is arranged vertically (bottom to top, in the example provided). For example, as shown in Figure 1G, the structured fixed bed 18 may be formed of one or more horizontally or vertically arranged material layers, with fluid flow arranged from an inlet end to an outlet end via a conduit C. The one or more layers may comprise a woven or reticulated material, as per Figures IE and IF, but as illustrated in Figure 1G, may comprise one or more cell immobilization layers 18a (three shown, but any number may be present) sandwiched by adjacent spacer layers 18b (vertical spacing exaggerated for purposes of illustration). The flow is thus arranged from side-to-side (left to right or right to left), with the material layer(s) (spacer or otherwise) providing for the channels for creating the tortuous flow (arrows B) from a linear or regular inflow (arrow A). Additional spacer layers may extend between the cell immobilization layers 18a, if desired. The fluid may flow through the layer(s) from top to bottom rather than along the layer(s) when substrate choice permits or allows for such flow.

[0045] In another possible embodiment, and with reference to Figure 1H, the structured fixed bed 18 comprises a three-dimensional (3D) monolithic matrix in the form of a scaffold or lattice formed of multiple interconnected units or objects S, which have surfaces for cell adhesion. Preferably, the matrix includes a tortuous path for fluid and cells to flow there through when in use. In these or other embodiments, the matrix may be in the form of a 3D array, lattice, scaffolding, or sponge. In all cases, the matrix may be single use in nature to avoid the cost and complexities involved in cleaning according to bioprocessing standards. This may involve the use of removable and reversible connections, such as by way of snap fit or hose barb connections. In one embodiment, the matrix forming the fixed bed 18 is edible, and may also be dissolvable. The fixed bed 18 may be a cartridge that is inserted into the vessel 16, and / or may prefabricated or coupled to one or more ends of the vessel 16.

[0046] In one particular embodiment of the cell culture apparatus 10, the arrangement is such that it is of a small scale relative to a conventional cell culturing apparatus, which may accommodate many liters of fluid. For instance, the first vessel 12 may be sized to accommodate a volume of fluid of approximately 5 ml to 10L, but in some specific versions has a volume of 50 ml or less. Likewise, the second vessel 16 may be sized to accommodate a fixed bed 18 having a surface area of approximately 5 to 10,000 cm2, but in some specific versions has a surface area of approximately 250 cm2 or less.

[0047] An example of a fixed bed 18 for use in the cell culture apparatus 10, and the second vessel 16 in particular, is shown in Figures 2 and 2A. The fixed bed 18 in this example comprises at least two layers of material, and is designed to model the fixed bed of a large-scale bioreactor (e.g., one that accommodates a volume of fluid greater than 10L). These layers may include one or more sheets of a non-woven material 18a for culturing cells, and optionally one or more sheets of spacer 18b, which may comprise a non-woven or woven mesh material. The layers 18a, 18b may be arranged concentrically within the associated vessel 16. While two layers 18a, 18b are shown, it can be appreciated that only a single wound sheet for culturing cells may be used to form the fixed bed 18.

[0048] With reference to Figures 3 and 4, a further embodiment of the cell culture apparatus 100 in the form of a bioreactor is disclosed herein. Similar to the first embodiment, the apparatus 100 forming the bioreactor includes a first vessel 112 or a compartment within a vessel having more than one compartment, forms a reservoir for recirculating fluid to a second vessel 116 connected thereto by a conduit C forming a continuous loop, which may include one or more connectors along the conduit or at locations where it is connected to the vessels 112, 116. In this or any other embodiment, the first vessel 112 may be sealed, such as by providing a seal or closure, and associated with a vent 114, which may be of the sterile or aseptic variety, such that an entirely closed arrangement results in order to maintain sterile conditions. In any case, the cells may be introduced to the first vessel 112 along with the culture media, such as by pre-dilution.

[0049] A pump P may also be provided for circulating fluid between the first and second vessels 112, 116, which may for example comprise a peristaltic pump for engaging flexible tubing forming the conduit C. The fluid flow may be in either direction. The first and second vessels 112, 116 may also be combined into a single vessel. The first vessel 112 may be prefilled with media and cells and exist as a cartridge that is accessed when coupled to the second vessel 116. The fixed bed 118 can be dropped in through the first vessel 112 if one part, or inserted into the second vessel 116 (optionally as a cartridge) and coupled to first vessel 112 if two parts.

[0050] In this embodiment, the first vessel 112 includes an anti-settling feature to prevent cells from becoming trapped therein. In the illustrated version, this is achieved by providing the bioreactor and, in particular, the first vessel 112, with a portion specially adapted to achieve this result. In the illustrated embodiment, this portion of the vessel 112 is in a non-vertical orientation when the bioreactor is oriented in a typical upright configuration, and comprises a tapered or frusto -coni cal lower portion 112a upon which cells are prevented from settling as a result of the tapering. As illustrated, the first vessel 112 along this portion 112a may be connected to the outlet for delivering fluid to the downstream second vessel 116, which includes the cell culture matrix (which may again take the form of a fixed bed 118 and, in particular, a spiral fixed bed, which may be of the form shown in Figure 1A). As can also be appreciated, in this design, the fixed bed 118 is lower than the frusto- conical lower portion 112a of the first vessel 112. While considered optional, this arrangement prevents cells from settling in the associated chamber, and also prevents bubbles in the fixed bed 118, since bubbles rise and thus are not directed to it

[0051] Turning now to Figures 5, 6, and 6A, a further embodiment of the apparatus for cell culture 200 forming a bioreactor is shown. This version is similar to that of Figures 3 and 4, but uses a single vessel 202 as the bioreactor instead of distinct vessels for forming a reservoir and for cell culturing. Specifically, this single vessel 202 includes a first portion 202a connected to a conduit C and thus forming a fluid inlet, and a second portion 202b forming an outlet, which is also connected to the conduit C for forming a continuous loop (through which fluid may be circulated via a pump, not shown). The first portion 202a is shown above the second portion 202b, but the situation could be reversed, as could the direction fluid flow from what is shown. Between the portions 202a, 202b is a tapered or frusto-conical portion 202c. This portion 202c of the bioreactor, which thus serves to funnel fluid from the chamber formed by the upper portion 202a to the lower portion 202b, and also prevents cells from settling or unintentionally being trapped in the vessel 202. It can also be appreciated that the lower portion 202b of the vessel 202 may likewise include a tapered or frusto-conical portion 202d leaving to the outlet to the conduit C. A closure, valve, or other form of fluid restrictor may optionally be associated with the vessel 202 in order to increase the residence time of the fluid in the vessel in order to facilitate achieving cell adherence, if necessary or desired, and such could also be regulated via control of the fluid flow using the pump P.

[0052] Figures 7 and 8 depict a different version of the above-mentioned embodiment of the apparatus 200 shown in Figures 5, 6, and 6A. In this version, the lower portion 202b of the vessel 202 is formed by a tube or tubular structure, such as an open- ended, hollow piece of tubing 216, which includes the cell culture matrix, such as a fixed bed 218. This tubing 216 may be oversized relative to the tubing forming the conduit C, and may be connected to the vessel 202 by suitable fasteners 220, such as for instance cable ties, but other forms of connection could be used (e.g, a snap fit, bayonet, screw thread, twist fit, press fit, frangible connector, or any other manner of forming a secure, fluid tight connection).

[0053] This arrangement facilitates detachment of the lower portion 202b of the vessel 202 (which may also be connected by a detachable connection) and recovery of the fixed bed 218, such as for sampling purposes. The vessel 202 may include a port 214, such as a vent, and may receive other conduits 216 for delivering agents to the vessel, such as nutrients, pH control, gases (such as air), or the like, or for withdrawing samples. Cell culture parameters such as pH and dissolved oxygen may alternatively be passively controlled by the diffusion of air / CO2 gas from an incubator in which the apparatus 200 may be placed. Figure 9 illustrates the apparatus 200 used in connection with a peristaltic pump P for causing fluid flow through the conduit C. Apparatuses 10, 100 may also be used in such an arrangement. The apparatuses 10, 100, 200 including the pump P if present may also be placed within an incubator.

[0054] Figure 10 is a schematic illustration of the apparatus 200, and illustrates in particular the "waterfall" by which fluid enters via an inlet the reservoir formed by vessel 202 via conduit C. This figure also shows that the conduit C also include one or more accessories 220, such as media addition or sampling ports 220 for the addition or withdrawal of fluid, and similar ports 216 may be provided in connection with the vessel 202. The conduit C may also include as an accessory 220 one or more flow through probes for sensing parameters, such as pH, temperature, glucose, lactate, or others. The fluid flow may be along the conduit C in either direction.

[0055] This disclosure also pertains to a kit that may be used to form the apparatus 10, 100, 200. In one example, the kit may comprise a vessel, such as vessel 202, which may be pre-loaded with fluid, a fixed bed material, and cells, as shown in Figure 11. To form the apparatus 200 from the kit, this vessel 202 may then be connected to a conduit (not shown), and associated with the various ports 214, 216, to form the apparatus 200. The connection may be established, for example, by puncturing seals sealing the vessel 202, including any fluid and cells if present therein, and then connecting the conduit. The vessel 202 may be made breakable or frangible to allow for gaining access to the interior, such as for sampling the cell culture once processing is completed.

[0056] As further illustrated in Figure 12, the vessel 202 may comprise two or more portions adapted to connect to each other. For instance, the upper portion 202a of the vessel 202 forming the reservoir, which may include fluid and / or cells, may be connected to the lower portion 202b, which includes a cell culturing bed, such as a fixed bed 218. The connection may be of any type that achieves the desired fluid tight arrangement, and may be releasable (e.g., frangible) for later detachment of the lower portion 202b including the fixed bed 218, such as for sampling purposes in an easy and efficient manner. The lower portion 202b may be preloaded with cells and media before being coupled to the upper portion 202a, and together the vessel 202 may form a cartridge.

[0057] Figure 13 illustrates a stand 300 for supporting the apparatus 200. The stand 300 may comprise a block of material, such as foam or a similar dimensionally stable material, with a receiver, such as a channel 302, sized and shaped for receiving and holding the vessel 202. The channel 302 may also include an open lower end for allowing passage of the conduit C. The chamber lies above the fixed bed, and the stand 300 is designed to maintain the apparatus 200 in a generally upright or vertical orientation. The stand 300 and / or channel 302 may be adapted for thermally regulating (e.g. providing heat or cooling to) the apparatus 200, and may also be used in connection with apparatuses 10, 100.

[0058] Figure 14 illustrates that a single pump P may be used in connection with a plurality of conduits C, each of which may be associated with an apparatus 10, 100, 200. A single conduit C may also be associated with more than one vessel to form the apparatus 10, 100, 200, such that bioprocessing may occur in parallel.

[0059] Figure 15 represents data from various working examples using an apparatus according to the present disclosure to perform cell culturing. Design A refers to the apparatus 10, and designs C and D refer to the apparatuses 100, 200.

[0060] In one particular version, the upper portion 202a of the vessel 202 may have an inner diameter of 1 to 10 centimeters, and in one example approximately 32 mm. The lower portion 202b may have an inner diameter ranging from about 0.5 to 15 centimeters, such as approximately 10 mm. The overall height of the vessel 202 may be about 3 to 80 centimeters, such as 140 mm, of which the lower portion 202b may comprise approximately 1 to 25 centimeters, or approximately 33 mm in one example. The angle of the taper of the frusto-conical portion 202c may form an acute angle relative to the horizontal axis. In one particular example, the angle of the taper a is about 60 degrees (see Figure 6B), but may range from approximately 15 to approximately 85 degrees in other examples.

[0061] In one possible implementation, the apparatus 10, 100, 200 disclosed herein may also be considered as a screening tool for use in evaluating the viability of a cell culturing arrangement. Thus, for example, in the case of a bioreactor with a particular type of fixed bed, the second vessel 16, 116, 216 may be provided with a similar, but smaller version of the same fixed bed, in order to screen it for operability under certain conditions (which may be similar to those present in the bioreactor). Such a bioreactor in combination with the apparatus 10, 100, 200 may thus be considered together to form a system for culturing cells, and may also be used in connection with a method of culturing cells.

[0062] As can be further appreciated, the compact, portable nature of the apparatus 10, 100, 200 allows for ready use in parallel to concurrently screen operation under different bioprocessing conditions. For example, a first apparatus may include a first version of a fixed bed, such as one formed of a particular material, having a particular arrangement of layers, or like conditions, and a second apparatus may include a different version, having a different arrangement of layers, formed of the same or a different material, and operated under like conditions. The ability to concurrently screen different configurations using the apparatus greatly reduces the time and expense associated with the use of such a fixed bed in a large-scale bioreactor.

[0063] As can also be appreciated, the ability to readily access and remove a small scale fixed bed 18, 118, 218 from the associated vessel 16, 116, 216 allows for easy and efficient sampling, including when used in the form of a recoverable or removable cartridge. Any cover or lid sealing the vessel 16, 116 may simply be removed to gain access to the fixed bed 18, 118, or in the case of vessel in the form of a tube 216, it can simply be detached from vessel 202, such as by twisting or otherwise breaking any connection formed. The small scale arrangement of the apparatus 10, 100, 200 also allows for use in an incubator or other small space under which sterile conditions are easier to maintain, which may avoid the need for sealing any of the vessels used.

[0064] While use of the apparatus 10, 100, 200 in a small scale version is proposed, it should be appreciated that this disclosure is not limited in scope to using any particular size of vessel in the apparatus. For example, the in the second and third embodiments of Figures 3, 4, 5, 6, and 7, the vessel 116, 216 including the fixed bed 118, 218 may be of any size, and in all cases, the vessel may take any shape or configuration desired to perform cell culturing.

[0065] In any embodiment, the apparatus 10, 100, 200 may comprise a mixer, such as a static structure (e.g., baffles or protrusions) to create turbulence, or a dynamic one (e.g., a magnetic stir bar) for mixing. The mixer may be located in the chamber forming the reservoir, such as for instance the first vessel 112 in the apparatus 100. The apparatus 10, 100, 200 may be fabricated of inert cell culture material, including plastic (PC, PET, PETG) but also glass or stainless steel. Preferably, the apparatus 10, 100, 200 is fabricated from one or more plastics from the group comprising polycarbonate (PC), polyethylene terephthalate (PET), and polyethylene terephthalate glycol (PETG). The apparatus 10, 100, 200 may be sterilized by autoclaving, gamma-irradiation or any other sterilization method. The apparatus 10, 100, 200 may be single use, reusable, or a combination of single use and reusable components.

[0066] The apparatus may be operated in perfusion mode or batch mode (with media exchange, which may be performed under a laminar flow hood). The apparatus may also be used in connection with the transfection of cells.

[0067] In summary, the present disclosure proposes an apparatus for cell-culturing offering numerous benefits and advantages, as listed herein, and possibly others yet to be discovered. The apparatus may employ a variety of different cell culture matrices, such as a fixed bed, a structured fixed bed, a packed bed, stacks, etc. The fixed bed used may be a relevant model of a large-scale fixed bed, and thus may serve as a scaled down version of a larger fixed bed reactor (similar in terms of structure and compaction of the fixed bed). The apparatus may be easy, small, and simple to operate, and may include ports for sampling and for filling and emptying. The apparatus may be easily dismantled at the end of processing to recover the fixed bed and the cells, such as for quality control. Optional probes and sensors may be provided for monitoring process conditions. Media circulation may be achieved using a simple peristaltic pump, to thus maintain a low shear environment. The apparatus may be arranged to achieve waterfall circulation for gas exchange, and may include a vent for gas exchange. The apparatus may have a vessel with a frusto-conical shape and having the fixed bed placed in a lower portion of the reservoir to prevent cell settling and having bubbles inside the circulation loop and the fixed bed. Given its compact size, several of the apparatuses may be easily used in parallel, and may be operated in batch or perfusion mode. The amount of media inside the apparatus may be easily adjusted. The amount and size of the fixed bed may also be readily adjusted depending on the purpose of a particular experimental protocol. Aseptic operation under a laminar air flow may be employed, then the apparatus may be operated inside a CO2 incubator (such as like a simple T-Flask, shake-flask with just a simple pump), using passive temperature control and passive pH control thought the presence of CO2, as well as passive oxygen control thought the vent air diffusion. The apparatus would be compatible with all the cell culture steps, such as inoculation, growth, infection, transfection, production, and harvest.

[0068] Summarizing, this disclosure covers any or all of the following items in any ordered combination:

[0069] 1. An apparatus for culturing cells, comprising: a bioreactor including a fluid reservoir and a fixed bed for culturing cells, the bioreactor including a portion, such as a non-vertical wall portion when the bioreactor is oriented vertically, adapted for preventing cells from settling therein.

[0070] 2. The apparatus of item 1, wherein the bioreactor comprises a first vessel including the fluid reservoir and a second vessel including the fixed bed.

[0071] 3. The apparatus of item 2, further including a conduit for connecting the first vessel to the second vessel.

[0072] 4. The apparatus of item 2 or item 3, wherein the portion comprises a frusto- conical shape for preventing cells from settling therein.

[0073] 5. The apparatus of any of items 2-4, wherein the portion comprises a lower portion of the first vessel. 6. The apparatus of any of items 2-4, wherein the portion comprises a lower portion of the second vessel.

[0074] 7. The apparatus of any of items 2-4, wherein the second vessel comprises a tubing open at both ends, a first end being connected to the first vessel and a second end being connected to the conduit.

[0075] 8. The apparatus of any of items 2-5, wherein the first and second vessels are connected to form a common interior chamber.

[0076] 9. The apparatus of any of items 2-8, wherein the first vessel is detachably connected to the second vessel.

[0077] 10. The apparatus of any of items 2-9, wherein the first vessel has a volume of about 50 ml or less.

[0078] 11. The apparatus of any of items 2-10, further including a pump for pumping fluid from an outlet of the first vessel to an inlet of the second vessel.

[0079] 12. The apparatus of any of items 2-11, wherein the first vessel is adapted to provide a waterfall flow of fluid therein.

[0080] 13. The apparatus of any of items 2-12, further including a support for supporting the first and second vessels in a generally vertical orientation.

[0081] 14. The apparatus of any of items 2-13, wherein the first and second vessels are configured for placement in an incubator.

[0082] 15. An apparatus for culturing cells, comprising: a first vessel including a fluid reservoir; a second vessel including a fixed bed for the cell culture, the second vessel being detachably connected directly to the first vessel; a conduit connecting the first vessel to the second vessel; and a pump for pumping fluid though the conduit.

[0083] 16. The apparatus of item 15, wherein the first vessel comprises a frusto-conical lower portion.

[0084] 17. The apparatus of item 15 or item 16, wherein the second vessel includes a lower portion adapted for preventing cells from settling therein.

[0085] 18. The apparatus of item 17, wherein the lower portion of the second vessel is frusto-conical.

[0086] 19. The apparatus of any of items 15-18, wherein the second vessel comprises a tubing open at both ends, a first end being connected to the first vessel and a second end being connected to the conduit.

[0087] 20. The apparatus of any of items 15-19, wherein the first and second vessels form a common interior chamber.

[0088] 21. The apparatus of any of items 15-20, wherein the first vessel has a volume of about 50 ml or less. 22. An apparatus for culturing cells, comprising: a vessel including a first portion forming a fluid reservoir, a second portion including a fixed bed for the cell culture, and a third tapered portion connecting the first portion to the second portion.

[0089] 23. The apparatus of claim 22, wherein the first portion is an upper portion, the second portion is a lower portion, and the third portion is intermediate the upper and lower portions.

[0090] 24. The apparatus of any of items 22-23, further including: a conduit connecting an outlet of the vessel to an inlet of the vessel; and a pump for pumping fluid though the conduit.

[0091] 25. A cell culturing apparatus, comprising a tube including a fixed bed.

[0092] 26. The apparatus of item 25, further including a vessel connected to the tube by a conduit forming a closed loop, and a pump for circulating fluid through the conduit.

[0093] 27. The apparatus of item 26, further including a fastener for fastening the tube to the vessel.

[0094] 28. The apparatus of item 26, wherein the tube is positioned in tandem with first and second segments of the conduit.

[0095] 29. The apparatus of any of items 1-28, wherein the fixed bed comprises at least one cell immobilization layer and at least one spacer layer.

[0096] 30. The apparatus of any of items 1-29, wherein the fixed bed comprises a structured fixed bed.

[0097] 31. The apparatus of item 30, wherein the structured fixed bed comprises a spiral fixed bed.

[0098] 32. The apparatus of item 31, wherein the spiral fixed bed comprises at least one layer of a non-woven material.

[0099] 33. The apparatus of item 32, wherein the spiral fixed bed comprises at least one spacer layer.

[0100] 34. The apparatus of item 33, wherein the at least one spacer layer comprises a mesh.

[0101] 35. The apparatus of any of items 1-34, further including one or more ports for removing or adding substances to the apparatus.

[0102] 36. A system for culturing cells comprising a plurality of the apparatuses of any of claims 1-35.

[0103] 37. bioreactor including a first fixed bed for culturing cells, comprising : a first vessel including a fluid reservoir; a second vessel including a second fixed bed for the cell culture, the second fixed bed being representative of the first fixed bed of the bioreactor; a conduit connecting the first vessel to the second vessel; and a pump for pumping fluid though the conduit.

[0104] 38. A system for culturing cells, comprising: a first bioreactor including a first fixed bed for culturing cells; and a screening tool, comprising: a first vessel including a fluid reservoir; a second vessel including a second fixed bed for culturing cells independent of the first fixed bed of the bioreactor; a conduit connecting the first vessel to the second vessel; and a pump for pumping fluid though the conduit.

[0105] 39. A kit for cell culturing, comprising: a sealed vessel including a first portion forming a reservoir and a second portion including a cell culturing bed, the second portion being connected to, or adapted for connecting directly to, the first portion.

[0106] 40. The kit according to item 39, wherein the first portion and the second portion are connected by a frangible connection.

[0107] 41. The kit according to item 39 or item 40, wherein the first portion is sealed and separate from the second portion, which is also sealed.

[0108] 42. A method for use in connection with a bioreactor including a first fixed bed for culturing cells, comprising: using a screening tool to perform cell culturing on a second fixed bed representative of the first fixed bed.

[0109] 43. The method of item 42, wherein the screening tool comprises: a first vessel including a fluid reservoir; a conduit forming a continuous loop for circulating fluid; and a pump for pumping fluid though the conduit; and the method comprises associating the second fixed bed with the first vessel.

[0110] 44. The method of item 42 or item 43, wherein the associating step comprises placing the second fixed bed in a second vessel in fluid communication with the first vessel.

[0111] As used herein, the following terms have the following meanings:

[0112] "A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

[0113] "About," "substantially," or "approximately," as used herein referring to a measurable value, such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / - 20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0114] "Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

[0115] The disclosures of the following documents are incorporated herein by reference: International Patent Application PCT / EP2020 / 084317; U.S. Provisional Patent Application Ser. No. 63 / 153,082, filed Feb. 24, 2021; International Patent Application Ser. No. PCT / EP2020 / 084317, filed Dec. 2, 2020; U.S. Provisional Patent Application Ser. No. 62 / 942,345, filed Dec. 2, 2019; U.S. Provisional Patent Application Ser. No. 63 / 004,706, filed Apr. 3, 2020; U.S. Provisional Patent Application Ser. Nos. 62 / 758,152, 62 / 733,375, and 62 / 608,261; U.S. Patent Application Publication No. 2018 / 0282678; International Patent Application PCT / EP2018 / 076354; U.S. Provisional Patent Application 62 / 711,070; and U.S. Provisional Patent Application 62 / 725,545.

[0116] While embodiments of the present apparatus have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the protection under the applicable law and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS1. An apparatus for culturing cells, comprising: a bioreactor including a fluid reservoir and a fixed bed for culturing cells, the bioreactor including a portion associated with the fluid reservoir adapted for preventing cells from settling therein.

2. The apparatus of claim 1, wherein the bioreactor comprises a first vessel including the fluid reservoir and a second vessel including the fixed bed.

3. The apparatus of claim 2, further including a conduit for connecting the first vessel to the second vessel.

4. The apparatus of claim 2, wherein the portion comprises a frusto-conical shape for preventing cells from settling therein.

5. The apparatus of claim 2, wherein the portion comprises a lower portion of the first vessel.

6. The apparatus of claim 2, wherein the portion comprises a lower portion of the second vessel.

7. The apparatus of claim 3, wherein the second vessel comprises a tubing open at both ends, a first end being connected to the first vessel and a second end being connected to the conduit.

8. The apparatus of claim 2, wherein the first and second vessels are connected to form a common interior chamber.

9. The apparatus of claim 2, wherein the first vessel is detachably connected to the second vessel.

10. The apparatus of claim 2, wherein the first vessel has a volume of about 50 ml or less.

11. The apparatus of claim 2, further including a pump for pumping fluid from an outlet of the first vessel to an inlet of the second vessel.

12. The apparatus of claim 2, wherein the first vessel is adapted to provide a waterfall flow of fluid therein.

13. The apparatus of claim 2, further including a support for supporting the first and second vessels in a generally vertical orientation.

14. The apparatus of claim 2, wherein the first and second vessels are adapted for placement within an incubator.

15. An apparatus for culturing cells, comprising: a first vessel including a fluid reservoir; a second vessel including a fixed bed for the cell culture, the second vessel being detachably connected directly to the first vessel; and a conduit connecting the first vessel to the second vessel; and a pump for pumping fluid though the conduit.

16. The apparatus of claim 15, wherein the first vessel comprises a frusto-conical lower portion.

17. The apparatus of claim 15, wherein the second vessel includes a lower portion adapted for preventing cells from settling therein.

18. The apparatus of claim 17, wherein the lower portion of the second vessel is frusto-conical.

19. The apparatus of claim 18, wherein the second vessel comprises a tubing open at both ends, a first end being connected to the first vessel and a second end being connected to the conduit.

20. The apparatus of claim 15, wherein the first and second vessels form a common interior chamber.

21. The apparatus of claim 15, wherein the first vessel has a volume of about 50 ml or less.

22. An apparatus for culturing cells, comprising: a vessel including a first portion forming a fluid reservoir, a second portion including a fixed bed for the cell culture, and a third tapered portion connecting the first portion to the second portion.

23. The apparatus of claim 22, wherein the first portion is an upper portion, the second portion is a lower portion, and the third portion is intermediate the upper and lower portions.

24. The apparatus of claim 22, further including: a conduit connecting an outlet of the vessel to an inlet of the vessel; and a pump for pumping fluid though the conduit.

25. A cell culturing apparatus, comprising a tube including a fixed bed.

26. The apparatus of claim 25, further including a vessel connected to the tube by a conduit forming a closed loop, and a pump for circulating fluid through the conduit.

27. The apparatus of claim 26, further including a fastener for fastening the tube to the vessel.

28. The apparatus of claim 26, wherein the tube is positioned in tandem with first and second segments of the conduit.

29. The apparatus of any of claims 1-28, wherein the fixed bed comprises at least one cell immobilization layer and at least one spacer layer.

30. The apparatus of any of claims 1-28, wherein the fixed bed comprises a structured fixed bed.

31. The apparatus of claim 30, wherein the structured fixed bed comprises a spiral fixed bed.

32. The apparatus of claim 31, wherein the spiral fixed bed comprises at least one layer of a non-woven material.

33. The apparatus of claim 32, wherein the spiral fixed bed comprises at least one spacer layer.

34. The apparatus of claim 33, wherein the at least one spacer layer comprises a mesh.

35. The apparatus of any of claims 1-28, further including one or more ports for removing or adding substances to the apparatus.

36. A system comprising a plurality of the apparatuses of any of claims 1-28.

37. A screening tool for use in connection with a bioreactor including a first fixed bed for culturing cells, comprising: a first vessel including a fluid reservoir; a second vessel including a second fixed bed for the cell culture, the second fixed bed being representative of the first fixed bed of the bioreactor; a conduit connecting the first vessel to the second vessel; and a pump for pumping fluid though the conduit.

38. A system for culturing cells, comprising: a first bioreactor including a first fixed bed for culturing cells; and a screening tool, comprising: a first vessel including a fluid reservoir; a second vessel including a second fixed bed for culturing cells independent of the first fixed bed of the bioreactor; a conduit connecting the first vessel to the second vessel; and a pump for pumping fluid though the conduit.

39. A kit for cell culturing, comprising: a sealed vessel including a first portion forming a reservoir and a second portion including a cell culturing bed, the second portion being connected to, or adapted for connecting directly to, the first portion.

40. The kit according to claim 39, wherein the first portion and the second portion are connected by a frangible connection.

41. The kit according to claim 40, wherein the first portion is sealed and separate from the second portion, which is also sealed.

42. A method for use in connection with a bioreactor including a first fixed bed for culturing cells, comprising: using a screening tool to perform cell culturing on a second fixed bed representative of the first fixed bed.

43. The method of claim 42, wherein the screening tool comprises: a first vessel including a fluid reservoir; a conduit forming a continuous loop for circulating fluid; and a pump for pumping fluid though the conduit; and the method comprises associating the second fixed bed with the first vessel.

44. The method of claim 43, wherein the associating step comprises placing the second fixed bed in a second vessel in fluid communication with the first vessel.

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